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CONTENTS OF No. 221, N.S., DECEMBER, 1910.

MEMOIRS: PAGE
The Early Development of the Marsupialia, with Special Reference
to the Native Cat (Dasyurus viverrinus). (Contributions
to the Embryology of the Marsupialia, IV.) By J. P. Hitt,
D.Sc., Jodrell Professor of Zoology and Comparative Anatomy,
University of London, University College. (With Plates 1-9
and 2 Text-figures) : : ; 1
Notes on a Deep-Sea Bebinroid, ear en ene shiplei
(n.g. et n. sp.), with Remarks on the species Hamingia ijimai
Ikeda. By Iwasi Ikepa. (With Plate 10) . : . 185
A Study of the Blood of certain Coleoptera: Dytiscus margi-
nalis and Hydrophilus piceus. By J. O. WaxkeELin
Barratt, M.D., D.Sc., and GEorGcE ARNOLD, M.Sc., from the
Cancer Research Laboratory, University of Liverpool. (With
Plate 11) ; . ; . . 149

CONTENTS OF: No.) 222, N-S., FEBRUARY, 1911.

MEMOIRS :

On the Morphology of the Cranial Muscles in Some Vertebrates.
By F. H. Eperworts, M.D., D.Sc., Professor of Medicine,
University of Bristol. (With 100 ‘Text-figures) 2 Leu

A Monograph of the Tape-worms of the Sub-family Aeripollntinee.
being a Revision of the Genus Stilesia, and an Account of the
Histology of Avitellina centripunctata (Riv.). By Lewis
Henry Goucu, Ph.D. (With Plates 12-14 and 6 Text-figures) 317

Brief Notes on the Structure and Development of Spirocheta
anodonte Keysselitz. By W.Crcit bosanquer, M.D. (With
Plate 15) ; . 887

1V CONTENTS.

CONTENTS OF No. 223, N.S., APRIL, 1911.

MEMOIRS:

Contributions to the Cytology of the Bacteria. By C. Ciirrorp
Dose tt, Fellow of Trinity College, Cambridge; Lecturer at the
Imperial College of Science and Technology, London. (With
Plates 16-19 and 1 Text-figure)

On Cristispira Veneris nov. spec., and the Affinities end Classi-
fication of Spirochets. By C. Ciirrorp Doser ty, Fellow of
Trinity College, Cambridge ; Lecturer at the Imperial College of
Science and Technology, London. (With Plate 20 and 2 Text-
figures)

On the Resonant aaa Seracnare of the och oplore of
Hydroides uncinatus (Eupomatus). By CRESSWELL
SHearerR, M.A., Trinity College, Cambridge. (With Plates
21-23 and 29 Text-fizures) é

Studies in the Experimental rage sis of Sex By G8orrrey
SmirH, M.A., Fellow of New College, Oxford

CONTENTS OF No. 224, N.S., JUNE, 1911.

MEMOIRS

Cortical Cell Lamination of the Hemispheres of Papio Hama-
dryas. By E. H. J. Scuusrer, M.A., D.Sec., Fellow of New
College, Oxford. (With Plates 24-30) .

On Some Stages in the Life-History of Leptomonas musce
domestic, with some remarks on the Relationships of the
Flagellate Parasites of Insects. By J. 8. Dunxkerty. (With
Plate 31) :

On Merlia normani, a Sponge w ith a Silresous and Caleareons
Skeleton. By R. Kirxparrick. (With Plates 32-88 and 5
Text-figures)

Tirte, INDEX, AND CONTENTS.

PAGE

507

543

591

6138

645

THE HARLY DEVELOPMENT OF THE MARSUPIALIA. |

The Early Development of the Marsupialia, with
Special Reference to the Native Cat (Dasy-
urus Viverrinus).

(Contributions to the Embryology of the Marsupialia, IV.)

By
J. P. Hill, D.Se.,

Jodrell Professor of Zoology and Comparative Anatomy, University
of London, University College.

With Plates 1-9 and 2 Text-figs.

TABLE OF CONTENTS.

AGE

INTRODUCTION. : . : ‘ 2
CHAPTER I.—CRITICAL Racine OF PREVIOUS OBSERVATIONS

ON THE EARLY DEVELOPMENT OF MARSUPIALIA . : 5

CHAPTER I].—THE Ovum or DASYURUS : : nl

1. Structure of Ovarian Ovum . ; Bis gb

2. Maturation and Ovulation . : rel

3. Secondary Egg-membranes . 3 sla eo

4. Uterine Ovum . 5 eS

CHAPTER IIJJ.—CLEAVAGE AND Ronaatone OF Shicnaasen te:

x Cleavage . , : bo ESS

. Formation of Blnctocyet ‘ HSS

CHAPTER LY. ee OF BLASTOCYST AND Rane
TION OF THE EMBRYONAL ECTODERM AND THE ENTODERM 495

1. Growth of Blastocyst : 43
2. Differentiation of the Embryonal Ectoder m aaa
the Entoderm . 52
3. Establishment of the MEnwire Embr eel Area 65
4, Summary 72
CHAPTER V.—SOME EARLY Sraczs OF ‘PRRAMELES AND
Macropus. . - : inka

voL. 56, part 1.—NEW SERIES. 1

2 J.'P. HILL:

PAGE
CHAPTER VI.—GENERAL SUMMARY AND CONCLUSIONS Oe
CHAPTER VII.—THE EARLY ONTOGENY OF THE MAMMALIA
IN THE LIGHT OF THE FOREGOING OBSERVATIONS a oe
1. The Early Development of the Prototheria . 86
2. The Early Development of the Metatheria and
Eutheria : A ‘ - 96
3. The Entypie Condition of the Eutherian
Blastocyst : : : . A
ADDENDUM , ; ; ; : > 12il
List OF REFERENCES 122
EXPLANATION OF PLATES . 3 ; ‘ . 125

INTRODUCTION.

“Tn mammalian embryology very many surprises are yet in store for
us’ (Hubrecht, ’08).

Tue present contribution contains an account of the prin-
cipal results and conclusions at which I have arrived after a
somewhat protracted and much interrupted study of an
extensive collection of early developmental stages of Marsu-
pials, ranging from the fertilised egg to the blastocyst in which
the two primary germ layers are definitely established. I
believe I am now able to give for the first time an account of
early Marsupial ontogeny, based on the examination of an
adequate material, and both consistent in itself and with what
we know of the early development in the other two Mamma-
lian sub-classes. The material at my disposal was obtained
during my tenure of office in the University of Sydney, and
with the aid of grants from the Royal Society and of a George
Heriot Research Fellowship. It represents the proceeds of
some eight years’ collecting, and comprises a fairly complete
series of stages of the native cat (Dasyurus viverrinus),
together with a few early stages of other Marsupials, notably
Perameles and Macropus.

Dasyurus proved in many ways a convenient subject for
embryological purposes. It can readily be trapped in many
districts in New South Wales; it lives and breeds fairly well
in captivity, and though always somewhat intractable, it can,
owing to its size, be easily handled, and so may be subjected

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 3

if necessary to daily examination.! But it has this great dis-
advantage, which it apparently shares with other Marsupials,
that a very variable period intervenes between coitus and
ovulation. As a consequence, the obtaining of any desired
cleavage or early blastocyst stage is largely a matter of
chance.” It is true that the changes which take place in the
pouch, in correlation with ovulation and the events connected
therewith, do afford in the case of late pregnant females some
indication of the stage of development likely to be met with,
but these changes are at first of too indefinite a character to
be of much service beyond indicating that ovulation may have
taken place.

Dasyurus breeds but once a year, the breeding season
extending over the winter months—May to August. One
remarkable feature in the reproduction of Dasyurus, to which
I have directed attention in a previous paper (Hill, ’00), may
be again referred to here, and that is the fact that there is no
correlation between the number of ova shed during ovulation
and the accommodation available in the pouch. The normal
number of teats present in the latter is six, though the
presence of one or two supernumerary teats is not uncommon ;
the number of ova shed at one period is, as a rule, far m
excess of the teat number. I have, for example, several
records of the occurrence of from twenty to twenty-five eggs,
two of twenty-eight, one of thirty, and one of as many as
thirty-five! (twenty-three normal blastocysts and twelve

1 Perameles, on the other hand, though quite common in many parts
of the State, is by no means such a convenient type. It is much less
easily trapped than Dasyurus, does not live nearly so well in captivity,
and is particularly difficult to handle. I have to thank Mr. D. G. Stead,
now of the Department of Fisheries, Sydney, for first directing my
attention to the breeding habits of Dasyurus, and also for providing
me with the first female from which I obtained segmenting eggs.

2 For example, I obtained unsegmented ova from the uteri, four, five,
six, seven and eight days after coitus, 2-celled eggs six and seven
days after, 4-celled eggs eleven and eighteen days after. In one case
the young were born eight days after the last observed act of coitus,
in another sixteen days after, and in yet another twenty days after.

4, J. P. HILL.

abnormal). ‘here can be little doubt that Dasyurus, lke
various other Marsupials (e.g. Perameles, Macropus, etc.),
has suffered a progressive reduction in the number of young
reared, but even making due allowance for that, the excess
in production of ova over requirements would still be remark-
able enough. Whether this over-production is to be correlated
in any way with the occurrence of abnormalities during early
development or not, the fact remains that cleavage abnor-
malities are quite frequently met with in Dasyurus.

Technique.—As fixatives, I have employed for ovaries
the fluids of Hermann, Flemming, Ohlmacher, and Zenker ;
for ova and early blastocysts, Hermann, Flemming, Perenyi,
and especially picro-nitro-osmic acid (picro-nitric acid [Mayer]
96 c.c., 1 per cent. osmic acid 2 c.c., glac. acetic acid 2 c.c.) ;
for later blastocysts, the last-named fluid especially, also
picro-corrosive-acetic and corrosive-acetic.

To facilitate the handling of ova and early blastocysts
during embedding, I found it convenient to attach each
specimen separately to a small square of pig’s foetal membrane
by means of a dilute solution of photoxylin (1 to2 per cent.).
Orientation of the specimen was then easily effected during
final embedding, under the low power of the microscope. The
larger blastocysts were double-embedded in photoxylin and
paraffin, the cavity of the blastocyst being tensely filled with
the photoxylin solution by means of a hypodermic syringe
fitted with a fine needle.

For the staining of sections, Heidenhain’s iron-hema-
toxylin method proved the most satisfactory, and was almost
exclusively employed. Kntire portions of the blastocyst wall
were stained either with Ehrlich’s or Delafield’s hematoxylin.

I am much indebted to Mr. L. Schaeffer, of the Anatomical
Department of the University of Sydney, and to Mr. F.
Pittock, of the Zoological Department, University College,
for invaluable assistance in the preparation of the photo-
micrographs reproduced on Plates 1-5, and also to Mr. A.
Cronin, of Sydney, and Miss M. Rhodes, for the drawings
from their respective pencils reproduced on Plates 6 and 7.

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 5)

To Miss V. Sheffield I am indebted for the original of fig. 63.
To my friend Dr. F. P. Sandes, Sydney, I am indebted for
kind help in the revision of certain parts of the manuscript.

CaHapter [.—Cerrrica, Review or Previous OBSERVATIONS ON
THE Harty DEVELOPMENT OF THE MARSUPIALIA.

Apart from the very brief abstract of a short paper on the
development of Dasyurus, which I read before Section D of
the British Association in 1908 (included in Dr. Ashworth’s
Report, ‘Nature,’ vol. lxxviii), our knowledge of the processes
of cleavage and germ-layer formation in the Marsupialia is
based (1) on the well-known observations of the late Emil
Selenka (86) on the development of the Virginian opossum
(Didelphys marsupialis), published in 1886 as Heft 4 of
his classical ‘ Studien’; and (2) on those of W. H. Caldwell
(87) on the uterine ovum, and cleavage process in the native
bear (Phascolarctus cinereus).

Selenka’s account of the mode of origin of the germ-layers
in Didelphys differs widely, as the sequel will show, from my
description of the same in Dasyurus. Now Didelphys and
Dasyurus are two marsupials, admittedly allied by the closest
structural ties, and we should therefore not expect & priori
that they would differ fundamentally in the details of their
early ontogeny, however much they might diverge in respect
of the details of their embryonal nutritional arrangements.

Furthermore, we might reasonably hope, in view of the
generally admitted relationships of the Marsupialia, that a
knowledge of their early development would aid us in the
interpretation of that of Eutheria, or, at least, that their
early developmental phenomena would be readily comparable
with those of Eutheria. It cannot be said that Selenka’s
observations realise either of these expectations. “ Which-
ever view is taken of Selenka’s description of the opossum,”
writes Assheton (98, p. 254), ‘‘many obvious difficulties
remain for the solution of which no satisfactory suggestion
can as yet be offered.”

(or)

Joe EU:

As concerns my own observations, I venture to think it is
possible to bring them into line with what we know of the
early ontogeny in the other two mammalian sub-classes, and
I have attempted to do so in the concluding chapter of this
paper, with what success the reader can judge, whilst as regards
the divergence between Selenka’s results and my own, I am
perfectly convinced that the explanation thereof is to be
found in the fact that the whole of Selenka’s early material
was derived from but two pregnant females, and that much
of it consequently consisted of eggs which had failed to
develop normally. From the one female, killed 5 days
after coition, he obtained one egg in the 2-celled stage,
one with about twenty cells and nine unfertilised ova. From
the second, killed 5 days 8 hours after coition, he obtained
“ ausser zwei tauben, 14 befruchtete Hier néimlich je ein Ki mit
4, 8, 42, 68 Zellen, eine junge und eine iltere Gastrula mit
noch dicker Eiweisschicht und endiich acht auch gleichen
Entwickelungsstufe stehende weit gréssere Keimblasen, deren
Wand noch grosstentheils einschichtig war” (’86, p. 112).
Selenka recognised that the last-mentioned blastocyst “ die

> since he found

normale Entwickelungsphiise repriisentiren,’
as arule that all the embryos from one uterus were in the same
developmental stage. Nevertheless he proceeded to describe
the segmenting eggs and the two “ gastrule ” which lagged so
far behind the blastocysts, as if they were perfectly normal
developmental stages. He does, indeed, question whether or
not the 42-celled stage is normal, but decides in the affirma-
tive, “denn wenn ich von zwei Aweifelhaften Fallen absehe,
so habe ich niemals Hier aus den ersten Tag aufgefunden,
welche auf irgend welche Anomalie der Entwickelung
hinwiesen.” ''his, however, can hardly be accepted as a
satisfactory reason for his conclusion, since apart from the
other eggs of the same batch, he had but the two eggs from
the other female for comparison, viz. the 2-celled egg (and
even that is, in my view, not quite normal), and the 20-celled
egg, which is stated to have suffered in preparation. With
the exception of the two eggs just mentioned, all the crucial

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 7

early stages (ranging from the 4-celled stage to the completed
blastocyst), on whose examination Selenka based his account
of germ-layer formation in Didelphys, would thus appear to
have been derived from a single female.!| No wonder it is
impossible to reconcile his description either with what we
know of germ-layer formation in the Prototheria and Eutheria
or with my account of the same in Dasyurus.

My own experience with the latter has shown me that no
reliance whatever is to be placed on segmenting eggs or
blastocysts which exhibit marked retardation in their stage
of development as compared with others from the same
uterus, and also that batches of eggs or blastocysts in which
there is marked variation in the stage of development attained
should likewise be rejected. Abnormalities in the process of
cleavage and of blastocyst formation are by no means un-
common in Dasyurus, and during the earlier stages of my
own work I spent much time and labour on the investigation
of just such abnormal material as that on which Selenka, no
doubt unwittingly, but I feel bound to add, with an utter
disregard for caution, based his account of the early develop-
ment of Didelphys.

I propose now, before passing to my own observations, to
give a short critical account of Selenka’s observations, my
comments being enclosed in square brackets.

The uterine ovum of Didelphys is enclosed by (1) a rela-
tively thin “ granulosamembran,” formed by the transforma-
tion of a layer of follicular cells [really the shell-membrane,
first correctly interpreted by Caldwell (87) and formed in the
Fallopian tube]; (2) a laminated layer of albumen, semitrans-
parent; (3) a zona radiata, not always recognisable [in my
experience invariably distinct |.

Cleavage begins in the uterus, is holoblastic, and at first
equal. <A 2-celled stage is figured (Taf. xvii, fig. 3) [uot
quite normal as regards the relations of the blastomeres], and
also a 4-celled stage [normal in appearance except for the

1 The collection of my own early material of Dasyurus has involved
the slaughter of over seven dozen females.

8 5; 0P; BIL:

enormous thickness of the albumen layer], in which the four
equal-sized blastomeres are radially arranged round a cleavage
cavity and are conical in form, their upper ends being more
pointed, their lower ends thicker and richer in yolk-material.
The nucleus of each is excentric, being situated nearer the
upper pole. [This description is applicable word for word to
the 4-celled stage of Dasyurus. |

An 8-celled stage (fig. 6) is next described, seven of the
blastomeres being equal in size and one being smaller. They
are arranged somewhat irregularly in two circles. [This stage
I regard as abnormal both in respect of the arrangement of
the blastomeres and the occurrence of irregularity amongst
them.] Selenka (p. 119) thought it probable that the third
cleavage planes cut the first two at right angles and divided
each of the first four blastomeres into a smaller ectodermal
cell and a larger more granular entodermal, but states that
he was unable to establish this owing to the opacity of the
albumen-layer. [My observations show that it is the fourth
cleavage in Dasyurus, not the third, which is equatorial, un-
equal, and qualitative, and that even then the cells formed are
not ectodermal and entodermal insignificance. ‘lhe albumen
is normally never opaque. |

A 20-celled stage is mentioned, but not described, since it
suffered in preparation. It is said to have a large entoderm
cell in the cleavage cavity. [A statement of very doubtful
value, since the blastomeres were admittedly pressed together
and probably displaced by the shrunken egg-membranes. |

The next stage described is a spherical “gastrula” (Taf. xvii,
figs. 7,8), composed of forty-two cells with an open “ blasto-
pore” at the vegetative pole, a smaller opening at the animal
pole, and a large “ ur-entoderm ” cell in the cleavage-cavity,
just inside the “ blastopore.”” The wall of the “ gastrula”
consists of cells graduated in size; those in the region of the
blastopore are the largest and richest in deutoplasm, those at
the opposite pole are the smallest and most transparent. [This
is a very characteristic stage in the formation of the blastocyst,
with which I am quite familar in Dasyurus. Selenka’s speci-

THE EARLY DEVELOPMEN'T OF THE MARSUPIALIA. 8)

men, judging from Dasyurus, is normal as regards the consti-
tution of its wall and the occurrence of an opening at each pole.
The lower opening, however, has no blastoporic significance,
but, like the upper, owes its presence to the mode of formation
of the blastocyst-wall by the spreading of the blastomeres
towards the poles of the sphere formed by the egg-envelopes.
Selenka’s blastopore simply marks the last point of closure.
This specimen I hold to be abnormal from the presence of
the so-called ‘‘urentoderm” cell in its interior. I figure
(Pl. 3, fig. 37) a section of a fairly comparable and un-
doubtedly abnormal blastocyst of Dasyurus in which there is
also present in the blastocyst cavity a large free cell. Here
this latter is unquestionably a blastomere of the lower hemi-
sphere, which, having failed to divide, has become enclosed
by the spreading of its neighbours. Selenka’s “ urentoderm-
zelle” I regard as a similarly displaced blastomere. |

A 68-celled “ gastrula” (figs. 9 and 10) is next described.
It is essentially similar to the preceding, only the “ blasto-
pore”’ has closed.

The succeeding stage (fig. 11) is a somewhat older
“oastrula,” in which gastrulation is said to be still in
progress, since over the lower pole, in the region of the now
closed blastopore, it is no longer possible to say which cells
belong to the ectoderm, which to the entoderm. ‘I'he latter
layer is described as being several cells thick in the blastoporic
region, and as in course of spreading round inside the ecto-
dermal wall of the“ gastrula” towards the upper oranimal
pole. [This specimen is undoubtedly abnormal, at all events
there is no comparable stage in Dasyurus. It is difficult to
obtain a clear idea of Selenka’s conception of the mode of origin
of the germ-layers, but he evidently held (cf. pp. 116 and 119)
that the large yolk-rich cells of the lower (‘ blastoporic Za)
pole constitute the anlage of the entoderm, and that they
become inturned at the “ blastopore ” and proliferate to form
the definitive entoderm, which then gradually extends round to
the animal pole, in contact with the inner surface of the wall
of the gastrula, that wall forming the ectoderm. He appa-

10 J. P. <ALL.

rently did not regard the ‘ urentodermzelle ” as the sole
progenitor of the entoderm, but simply as an entoderm-cell
precociously inturned from the ‘ blastoporic ” margin.

This view of Selenka, however, lands us in the predicament
of having to regard the embryonal area as differentiating over
the vegetative hemisphere, since in the next stage the
‘“blastopore”’ is described as being situated excentrically in
that area. Either Selenka’s determination of the poles in the
42-celled blastocyst is wrong, or the entoderm does not
originate as he describes it. My own observations force me
to accept the latter alternative. In his paper Selenka gets
over the difficulty very easily by altering the orientation of
his figures. On ‘Taf. xvii, the figures of sections of blasto-
cysts are so placed that the “ blastopore ”’ is below, next the
bottom of the plate. These figures I hold to be correctly
orientated. On Taf. xviii, the figures are inverted, so that
the ‘ blastopore ” is above; as the result the animal pole of
fig. 11, ‘Taf. xvii, becomes the vegetative pole of the stage
next described (fig. 2, Taf. xviii). ]

The stage just referred to, described as an “ eif6rmige

> is represented in a drawing made from the fresh

gastrula,’
specimen as lying quite free in a large perivitelline space
enclosed by a very thick layer of albumen, outside which is
the “granulosa-membran.” In section (fig. 2) a mass of
entoderm is seen to reach the surface at one pole (marked 61.)
uppermost in the figure, whilst other entodermal cells are
shown spreading from this towards the lower pole. ‘The
ectoderm of the wall is represented as composed of definitely
cubical cells. [The presence of a large perivitelline space,
by itself stamps this specimen as not normal. ‘The sectional
figure must be schematic. |

The last of Selenka’s early stages to which reference need
be made here is formed by eight “ gastrule” (blastocysts),
reckoned as ten hours after the commencement of cleavage
[a reckoning I consider of no value] (Taf. xviii, figs. 3 and 4).
‘The embryonal area is now distinguishable by the larger size
of its ectodermal cells. The entoderm is unilaminar, and has

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 11

extended beyond the limits of the embryonal area. The
> is said to be marked in all by a
mass of coagulum attached to the wall, and in three by a

position of the ‘ blastopore ’

definite opening as well. It is situated excentrically in the
embryonal area. [Except for the “blastopore” and the
presence of a thick layer of albumen, this blastocyst stage is
quite comparable with the corresponding one in Dasyurus;
the latter, however, is considerably larger. Of Selenka’s
early material, I think it is these blastocysts alone which had
any chance of giving origin to normal embryos. |

W. H. Caldwell, who, as Balfour student, visited Australia
in 1883-4, obtained a very rich collection of early marsupial
material, of which, unfortunately, no adequate account has
ever been published. He gave, however, in his introductory
paper on the ‘ Embryology of the Monotremata and Marsu-
pialia’ (’87), an account of the structure of the ovum, both
ovarian and uterine, in Phascolarctus, and he showed that
the ovum during its passage down the Fallopian tube becomes

ce

enclosed outside the albumen layer in ‘‘a thin transparent
membrane, ‘0015 mm. thick,’’ which he homologised with the
shell-membrane of the monotreme egg. This important dis-
covery of the existence of a shell-membrane in the Marsu-
pialia I can fully confirm. Iam, however, unable to accept
his interpretation of the internal structure of the ovum
of Phascolarctus, or his remarkable statement that cleavage
in that form is of the meroblastic type. Cleavage is not
described in detail, nor is any account given of the mode
of origin of the germ-layers.

Cuarter I].—Tue Ovum or Dasyorus.

1. Structure of the Ovarian Ovum.

The full-grown ovarian ovum of Dasyurus (PI. etion sl!)
appears as a rounded, or more usually, ovalish cell, the
diameter of which varies in section in ten eggs measured
from °28 x °126 mm. to °27 x -26 mm. (average, ‘24 mm.),
and is therefore large relatively to the ova of Eutheria. It

1 J eke ELLA

is enclosed by a thin, but very definite refractive membrane
or zona (vitelline membrane of Caldwell) of an approximate
thickness of *002 mm. (fig. 1, z.p.), on which the cells of the
discus proligerus (fig. 1, d.p.) directly abut, a differentiated
corona radiata and syncytial layer being absent. It appears
to be identical in its relations and optical characters with the
membrane investing the monotreme ovum, and never shows
in section any trace of radial striations (though I believe I
have detected an extremely faint appearance of such in the
fresh zona), or of the extension into it of protoplasmic
processes from the adjacent cells of the discus proligerus,
such as Caldwell figures in the case of the ovum of Phas-
colarctus (cf. his Pl. 29, fig. 5). Within the zona the
peripheral cytoplasm of the ovum is differentiated to form an
exceedingly thin but distinct bounding layer or egg-membrane
(vitelline membrane, sensu stricto).

The cytoplasmic body of the ovum exhibits a very obvious
and striking differentiation into two regions in correspondence
with the presence in it of two definitely localised varieties of
deutoplasmic material, respectively granular and fluid. Peri-
pherally it consists of a relatively narrow cytoplasmic zone of
practically uniform width, dense and finely granular in
appearance owing to the presence in it of numerous particles
of deutoplasmic nature. This we may distinguish as the
formative zone (fig. 1, f.z.). In it lies embedded the large
vesicular nucleus (about ‘06 x ‘03 mm. in diam.). Centrally
and forming the main bulk of the ovum is a mass of greatly
vacuolated cytoplasm presenting the appearance of a clear
wide-meshed reticulum. Its framework is coarser peripherally
where it passes over without definite limit into the formative
zone, with which it is structurally identical, but much finer
and wider-meshed centrally, so fine, indeed, that it almost
invariably breaks down under the action of fixatives, and
appears in sections as an irregular space, perhaps crossed by
a few fine interlacing strands (fig. 1, d.z.). The meshes of
this reticulum are occupied by a clear fluid which must be
held to constitute the central deutoplasm of the egg. We

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 13.

may accordingly designate this central reticular area as the
deutoplasmic zone.

If we pass now from the full-grown to the ripe ovarian
ovum (Pl. 1, figs. 2 and 3), 1. e. an ovum in which either the
first polar spindle has appeared or the first polar body has
already been separated off, it at once becomes evident that
important changes have occurred in the disposition and
relative proportions of the two constituent regions of the ege-
cytoplasm. The full-grown ovum is of the centrolecithal
type, the central deutoplasmic zone forming its main bulk
and being completely surrounded by the thin formative zone.
The ripe ovum, on the other hand, exhibits an obvious and
unmistakable polarity, and is of the telolecithal type, as the
following facts show. The cytoplasmic body evidently con-
sists of the same two regions as form that of the full-grown
ovum, but here the dense formative region now forms its
main bulk, and no longer surrounds the clear deutoplasmic
region as a uniform peripheral layer. It has not only
increased considerably in amount as compared with that of
the full-grown ege, and at the expense apparently of the more
peripheral coarser portion of the deutoplasmic zone, but it
has undergone polar segregation, with the result that it now
occupies rather more than one hemisphere of the egg as a
dense finely granular mass, with vacuoles of varying size
sparsely scattered through it (figs. 2 and 3, fiz). It
accordingly defines one of the ovular poles. ‘The opposite
pole is just as markedly characterised by the presence imme-
diately below it of a more or less rounded clear mass,
eccentrically situated, and composed of an extremely fine
cytoplasmic reticulum with wide fluid-filled meshes. It is
completely surrounded by formative cytoplasm (though over
the polar region the enclosing layer is so extremely thin that
it here almost reaches the surface), and its cytoplasmic frame-
work is perfectly continuous with the same, the line of
junction of the two being abrupt and well defined. So
delicate, however, is this framework that it breaks down
more or less completely under the action of fixatives of such

14 Te PS SW

excellence even as the fluids of Flemming and Hermann,
and thus in sections usually all that represent it are a few
irregular cytoplasmic strands crossing a large, sharply defined
clear space (figs. 2 and 3, d.z.). The mass in question has
thus all the characters of the deutoplasmic zone of the full-
grown ovum, and it must undoubtedly be held to represent
the central portion of that which has not been utilised in the
upbuilding of the formative cytoplasm, and which has been
forced to take up an excentric position immediately below
the polar region of one hemisphere, owing to the increase of
the formative cytoplasm and its segregation in the other
hemisphere.

The ripe ovum of Dasyurus thus possesses a polarity which
in its way is equally as striking as that of the Monotreme
ego. ‘Towards the one pole the main mass of the ovum is
composed of dense, slightly vacuolated formative cytoplasm,
in which the polar spindle is situated peripherally, but nearer
the equator than the formative pole. Toward the opposite
pole and practically reaching the surface is a rounded mass
of greatly vacuolated deutoplasmic cytoplasm. Roughly,
the formative cytoplasm constitutes about two-thirds of the
bulk of the ripe egg, the deutoplasmic the remaining third.
Such being the structure of the ripe ovarian egg, if we
classify it at all, we must place it, it seems to me, with eggs
of the telolecithal type. My view of the significance of this
marked polar differentiation of the constituent materials of
the ripe ovum of Dasyurus I shall presently indicate. Mean-
time I would lay special emphasis on the fact that the
eccentric mass of deutoplasmic cytoplasm represents material,
surplus deutoplasmic material which has not been utilised in
the upbuilding of the formative cytoplasm.

The fact of the occurrence in the Kutherian ovum of a
polar differentiation of its constituent materials is now
definitely established, thanks especially to the valuable
researches of Prof. O. Van der Stricht and his pupils—H.
Lams and the late J. Doorme. In this connection I wish to
refer here in some detail to the extremely interesting obser-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 15

vations of Van der Stricht [’03, 705] on the structure and
polarity of the ovum of the bat (Vesperugo noctula), since
these observations are in essential agreement with my own
on the ovum of Dasyurus, and enable ine to affirm that the
polar differentiation herein recorded for the first time for the
Marsupial ovum is attained as the result of vitellogenetic
processes, which essentially correspond with those of the ovum
of the bat. Van der Stricht, as is well known, has made a
special study of the process of vitellogenesis in the Kutherian
ovum, and is, indeed, at the present time the foremost
authority on this particular subject, so that his views are
worthy of all respect.

Study of the odcyte of Vesperugo during the period of
growth shows, according to Van der Stricht, that “a un
moment donné du développement du jeune ceuf, les boyaux
et amas vitellogénes [derived, according to him, from ‘ une
couche vitellogéne, mitochondriale, present in the young
odcyte in the first stage of growth] disparaissent au profit du
vitellus, dont la structure pseudo-alvéolaire s’accentue
eraduellement.” The full-grown odcyte at the stage just
prior to the appearance of the first polar spindle is charac-
terised by the presence of this “ pseudo-alveolar structure ”’
throughout the extent of its cytoplasmic body. The alveoli
or vacuoles are of variable size, are filled by a clear liquid,
and “correspondent incontestablement au deutoplasma de
Voeuf. A ce stade du développement de l’odcyte, ce vitellus
nutritif, auquel s’ajoutent bientot des granulations graisseuses,
est répandu uniformément dans toutes les profondeurs du
cytoplasme. Nulle part on ne constate une zone deutoplas-
mique distincte d’une zone de vitellus plastique.” In
Dasyurus the stage in vitellogenesis which almost exactly
corresponds with that of the full-grown odcyte of Vesperugo
just described is seen in odcytes not quite full-grown. In
fig. 4 is shown an odcyte of Dasyurus (26 x °20 mm. in
diameter), in which the same pseudo-alveolar structure as
described by Van der Stricht for the Vesperugo odcyte is
perfectly distinct. Here, however, fatty particles are not

16 J. P.- HILM.

apparent, and the peripheral portion of the cytoplasm tends
to be free from vacuoles. In Dasyurus the formation of these
deutoplasmic vacuoles begins in odcytes about *2 mm. or less
in diameter. This characteristic ‘‘ pseudo-alveolar ”’ stage is
followed in both Vesperugo and Dasyurus by one in which
there is recognisable in the cytoplasmic body of the ovum a
differentiation into a dense peripheral zone and a central
vacuolated area. In Vesperugo this stage is attained about
the time of appearance of the first polar spindle, whilst in
Dasyurus it is attained somewhat earlier, always prior to the
formation of the latter. So close is the agreement between
the two forms that Van der Stricht’s description of the bat’s
egg at the time of appearance of the first polar spindle might
equally well be applied to the full-grown ovum of Dasyurus.
He writes [’03, p. 43]: “Vers Pépoque de Vapparition du
premier fuseau de maturation, le vitellus prend un autre
aspect. La partie centrale deutoplasmique conserve une
structure pseudo-alvéolaire, mais dans le voisinage immédiat
du premier fuseau et dans toute l’étendue de la couche
périphérique du protoplasme, apparait une mince zone de
vitellus compact et dense, plus ou moins homogene ow les
vésicules claires font défaut. . . . A ce moment, on
distingue dans l’odcyte de V. noctula une zone centrale
trés étendue, riche en deutoplasme et une zone corticale
trés mince, riche en vitellus plastique.’ This centro-
lecithal phase, as we may term it, is followed in Vesperugo
during fertilisation and the separation of the second polar
body by a telolecithal phase characterised by a distinct
polarity. ‘La zone de vitellus plastique s’épaissit encore,
mais surtout a un pole de l’ceuf, a celui opposé au pdle ot se
détachent les deux globules polaires. Ce péle, ot: s’accumule
graduellement le vitellus formateur, mérite le nom de pdle
animal. Il est opposé au pdle d’expulsion des globules
polaires, vers lequel est refoulé le deutoplasme, et qui se
comporte désormais comme le pdle végétatif. Pendant que
les deux pronucléus male et femelle se forment, le vitellus
plastique augmente graduellement en abondance au péle

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 17

animal, tandis qwil diminue au péle végétatif, et le deuto-
plasme, parsemé d’un plus grand nombre de boules graisseuses,
constitue une masse sphérique excentrique, voisine des deux
globules polaires” (Van der Stricht, ’03, pp. 44-45). It is
evident, then, that the fertilised ovum of Vesperugo exhibits a
polarity comparable with that of the ripe ovarian ovum of
Dasyurus, and that the vitellogenetic processes in the ova of
these two widely separated forms proceed along lines almost
identical, at all events so far as their broad outlines are con-
cerned. In both we find during growth a_ progressive
vacuolisation of the egg-cytoplasm consequent on the elabora-
tion of a deutoplasmic fluid. In both, the “pseudo-alveolar ”
condition so engendered is followed by one in which there
is recognisable a differentiation into a peripheral ‘‘ forma-

> zone rich in deutoplasmic granules, and a central

tive’
“deutoplasmic”’ zone rich in fluid yolk, and finally in both
there occurs a segregation of the granular “ formative” and
fluid yolk-constituents to opposite regions of the egg, with
resulting attainment of a definite polarity. In view of the
close general agreement in the vitellogenetic processes, and in
the constitution of the ova in Vesperugo and Dasyurus, it
might be expected that the poles would accurately correspond,
but such is not the case if Van der Stricht’s determination of
the poles in the ovum of Vesperugo is correct. In the latter,
according to Van der Stricht, the deutoplasm is located at
that pole from which the polar bodies are given off; at the
opposite pole the “ plastic ” vitellus accumulates, and close to
it the two pronuclei unite and the first cleavage spindle is
formed. Accordingly Van der Stricht concludes that ‘‘le
premier pole correspond au pole végétatif, le second au pole
animal des ceufs & deutoplasme polaire (O. Hertwig).” In
Dasyurus, on the other hand, I am perfectly convinced (and
adequate reason for my conviction will be forthcoming in the
course of my description of the processes of cleavage and
germ-layer formation) that the pole of the ripe ovum in
relation to the mass of deutoplasmic cytoplasm is not the
vegetative pole, but represents morphologically the upper or

VoL. 96, PART 1.—NEW SERIES. 2

18 TRAE Pes 8

animal pole of the egg, the opposite pole in relation to which
the formative cytoplasm is situated being the lower or
vegetative. The deutoplasmic cytoplasm thus les in the
upper hemisphere, whilst the formative cytoplasm occupies
the lower. If Van der Stricht’s determination of the poles of
the ovum of Vesperugo be accepted, then we must conclude
that the poles of the Dasyurus ovum are exactly reversed as
compared with those of the bat’s egg. In this connection it
may be recalled that Lams and Doorme [’07] have demon-
strated the occurrence in Cavia of an actual reversal of the
original polarity of the ovum, prior to the beginning of
cleavage. These facts may well give us pause before we
proceed to attach other than a purely secondary significance
to the exact location of the formative and deutoplasmic con-
stituents in the Metatherian and HKutherian ovum. But
besides this apparent difference in the location of the deuto-
plasmic constituents of the ova of Dasyurus and Vesperugo,
there exists yet another which concerns the fate of these con-
stituents in the respective eggs. In Vesperugo, Van der
Stricht shows that the “deutoplasm” remains an integral
part of the egg, and retains its polar distribution in the
blastomeres up to at least the 4-celled stage. In Dasyurus,
on the other hand, the fate of the dentoplasmic mass is a very
different, and, indeed, a very remarkable one. It does not
remain an integral part of the segmenting egg as in Vesperugo,
but prior to the completion of the first cleavage furrow it
becomes bodily separated off, apparently by a process of
abstriction, from the formative cytoplasm as a clear rounded
mass which takes no further direct part in the developmental
processes. As soon as its elimination is effected, the remainder
of the cytoplasmic body of the ovum, formed of the formative
cytoplasm alone, divides into the first two equal-sized blasto-
meres, the first cleavage plane being coincident with the polar
diameter and at right angles to the plane of separation of the
deutoplasmic mass, or “ yolk-body ” as we may term it (PI. 2,
fies. 14-16, 19, y.b.), so that it is this formative zone of the
' Vide, however, “ Addendum ” (p. 121).

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 19

ovum which is alone concerned in the production of the
embryo and its foetal membranes.

We have but to recall the conclusion already reached that
the clear vacuolated zone at the upper pole of the ripe ovum
of Dasyurus consists of surplus material, mainly in the form of
fluid of deutoplasmic nature which has not been utilised in
the upbuilding of the formative cytoplasm, and the signifi-
cance of this remarkable and, so far as the Mammalian ovum
is concerned, absolutely unique occurrence becomes at once
manifest. We have to do here with an actual elimination of
surplus deutoplasmic material by the Marsupial ovum—a phe-
nomenon only paralleled elsewhere, so far as I.am aware, and
even then but distantly, by the curious temporary separation
of the so-called yolk-lobe which occurs during the cleavage
of the yolk-laden eges of certain Molluscs (Nassa, Ilyanassa,
Modiolaria, Aplysia, Dentalium) and Annelids (Myzostoma,
Cheetopterus). In these forms cleavage of the ovum into the
first two blastomeres is accompanied by the separation of a
portion of the ovular substance in the form of a non-nucleated
mass or so-called yolk-lobe. This latter, which has been shown
to be connected with the formation of determined organ-
anlagen, reunites with one of the two blastomeres, and then
the same process of abstriction and reunion recurs at the
second cleavage.” We have here evidently a purely adaptive
phenomenon, the object of which no doubt is to permit of the
total cleavage of the yolk-laden ovum on what are. presumably
the old ancestral lines, and I believe a comparable explanation
will be found applicable to the elimination of surplus yolk-
material by the Marsupial ovum.

As regards the significance of the occurrence of the deuto-
plasmic zone in the ovum of Dasyurus, holding the views that
I do as to the phylogeny of the Marsupialia (viz. that the
Metatheria and Eutheria are the divergent branches of a

1 Vide “Addendum” (p. 121), in which reference is made to the dis-

covery by Prof. Van der Stricht of the elimination of deutoplasm in
the ovum of Vesperugo.

2 Cf. Korschelt u. Heider, ‘Lehrbuch d. vergl. Entwicklungs-
geschichte,’ Lief. 5, p. 107, 1909.

20 Jy 12) VSDNaE

common stock, itself of Prototherian derivation), and bearing
in mind the occurrence of an undoubted representative of the
shell round the Marsupial ovum, I venture to see in the fluid-
material of the deutoplasmic zone the partial and vestigial
equivalent of the yolk-mass of the monotreme egg. In other
words, I would regard the deutoplasmic fluid as the product
of an abortive attempt at the formation of such a solid yolk-
mass. The objection will no doubt be forthcoming that this
interpretation cannot possibly be correct since the supposed
equivalent of the yolk-mass in the Dasyure ovum is located,
on my own showing, at the wrong pole—at the upper instead
of at the lower. But its precise location does not seem to me
to be a matter to which we need attach any great importance,
since it has doubtless been adaptively determined in correla-
tion with the special character of the cleavage process.

The belief that the minute yolk-poor ovum of the Eutheria
is no pure primarily holoblastic one, but that it has only
secondarily arrived at the total type of cleavage as the result
of the all but complete loss of the yolk ancestrally present in
it, consequent on the substitution of the intra-uterine mode of
development for the old oviparous habit, is now widely held
amongst Mammalian embryologists. Hubrecht, however, is
an exception, wedded as he is to a belief in the direct deriva-
tion of the Hutheria from Protetrapodous ancestors with yolk-
poor, holoblastic eggs. Whether the interpretation I have put
forward, viz. that the non-formative or deutoplasmic zone of
the Dasyure ovum is the reduced and partial equivalent of the
yolk-mass of the Monotreme egg, be accepted or not, I venture
to think that my discovery of an actual elimination of deuto-
plasmic material by the Marsupial ovum affords a striking
confirmation of the truth of the prevailing conception as to
the phylogeny of the Eutherian ovum, and I further venture
to think that the facts I have brought forward in the preceding
pages justify us in regarding the ripe ovarian ovum of
Dasyurus as being potentially of the yolk-laden, telolecithal
type, and the uterine ovum, by bodily casting out the super-
fluous part of its deutoplasm, as becoming at the same time

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. a

secondarily homolecithal and secondarily holoblastic. The
Marsupial ovum presents itself to my mind as the victim of
tendencies conditioned by its ancestry, and in particular it
appears as if its inherited tendency to elaborate yolk had not
yet been brought into accurate correlation with the other
changes (reduction in size, intra-uterine development), which
it has undergone in the course of phylogeny. As the conse-
quence it manufactures more yolk than it can utilise, and so
finds itself under the necessity of getting rid of the surplus.
Whether or not a comparable elimination of deutoplasmic
material occurs in the ova of other Marsupials, future investiga-
tion must decide. I should be quite prepared to find variation
in this regard, correlated perhaps with the size of the egg.
In the case of Phascolarctus, Caldwell gives the diameter ot
the ovum as ‘17 mm., and his figure of a (horizontal ?) section
of the uterine ovum (here produced as text-fig. 1, p. 27) shows
a differentiation of the cytoplasmic body of that into vacuo-
lated and granular zones quite comparable with that of the
Dasyure ovum. From the few measurements of ova of other
marsupials that I have been able to make, it would appear
that the ovum of Trichosurus approximates in size to that of
Dasyurus, whilst that of Perameles and probably also that
of Macropus are smaller. From Selenka’s figure I have
calculated that the ovum of Didelphys measures about *13 x
‘12 mm. in diameter. In the smaller ova it is quite likely
that yolk-formation may not proceed so far as in the relatively
large ovum of Dasyurus.

2. Maturation and Ovulation.

The details of the maturation process have not been fully
worked out, owing to lack of material. As in the Eutheria
(Sobotta, Van der Stricht, Lams and Doorme, and others),
the first polar body is separated off in the ovary, the second
apparently in the upper part of the Fallopian tube where
entrance of the sperm takes place. The first polar figure
(late anaphase observed, fig. 5) lies in the formative cyto-

a J, oP, eile

plasm, close below and at right angles to the zona. Its exact
site is subject to some slight variation, and is best described
as adjacent to the equatorial region of the egg, sometimes
nearer the lower pole, more usually, perhaps, nearer the
upper. Centrosomes and polar radiations were not observed.
The heterotypical chromosomes (gemini) have the form of
somewhat irregular, more or less angular granules. I have
not been able to determine their number. The figure is
barrel-shaped, and almost as broad as long, measuring
015 x ‘013mm. The first polar body (fig. 6, p.b1.) 1s small
relatively to the size of the egg, its diameters varying round
‘03 x ‘Ol mm., and its shape is that of a flattened bi-convex
disc. In uterine eges there is some evidence pointing to the
probability of its having undergone division.

‘he second polar spindle (figs. 3 and 7) lies immediately
subjacent to the first polar body in the fully ripe ovarian
ovum. It is shorter than the first, measuring ‘013 mm., and
much narrower. The second polar body measures about
‘O15 x ‘Ol mm. in diameter, and is thus smaller than the first.
I have only seen the second polar body in uterine ova, and
therefore can only presume that it is separated off in the
upper part of the Fallopian tube, subsequently to the pene-
tration of the sperm, as in Hutheria.

Ovulation takes place irrespective of whether copulation has
occurred or not, and it is a fact worthy of record that, even
if the ova be not fertilised, the pouch and mammary glands
undergo the same series of growth changes as are charac-
teristic of, at all events, the earlier stages of normal
pregnancy.

The follicular cells of the discus proligerus investing the
ovum are already in the ripe follicle in a state of disruption,
and I believe they separate completely from the ovum at the
moment of dehiscence, so that, except for the zona, the ova
are quite naked when they enter the tube. Ihave no evidence
of the existence outside the zona of a layer of proalbumen
such as Caldwell describes round the ovum of Phascolarctus.
Apparently the ova are shed almost simultaneously, and they

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 23

must pass with considerable rapidity down the tubes to the
uteri where cleavage begins, for I have only once found a
tubal ovum, and that one had evidently been retarded for
some reason, and was polyspermic.

3. The Secondary Hgg-membranes: Albumen and
Shell-membrane.

During the passage of the ovum down the tube it is
fertilised, and becomes enclosed externally to the zona by
two secondary layers formed as secretions by the cells of the
oviducal lining. First of all, the ovam becomes surrounded by
a transparent to semi-transparent laminated layer of albumen,
‘015 to ‘022 mm. in thickness, composed of numerous very
delicate concentric Jamelle, and having, normally, numbers
of sperms imbedded in it (figs. 8-11, alb., sp.). Then outside
the albumen layer there is laid down a definite, but at first
very thin, double-contoured membrane (figs. 8 and 10, s.m.),
which, following Caldwell, I have no hesitation in homo-
logising with the shell-membrane of the Monotreme ege.
Caldwell in 1887 described and figured a definite membrane
enclosing the uterime ovum of Phascolarctus, externally to,
and quite distinct from the albumen, which he interpreted
as the representative of the shell-membrane of the Mono-
tremata, but owing apparently to the fact that Selenka
altogether failed to recognise its true nature in Didelphys,
since he regarded it as a derivative of the follicular epithe-
lium, and termed it the “ granulosa-membran,” this highly
significant discovery of Caldwell has been largely ignored.
Such a membrane is constantly present and easily recognisable
in all the Marsupials (Dasyurus, Perameles, Trichosurus,
Macropus, Petrogale, Phascologale, Acrobates, Phascolarctus,
Bettongia), of which I have had the opportunity of studying
early developmental stages. It is laid down in the Fallopian
tube, is perfectly distinct from the albumen, and increases in
thickness in the uterus, and if it has not the significance
which Caldwell has suggested, then I must leave it to those

24 te P. LG.

who decline to accept Caldwell’s interpretation to put forward
an alternative one, since I am unable to do so.

The shell-membrane of Dasyurus (PI. 1, figs. 8-11; Pl. 2,
fies. 17, 18, s.m.) is a transparent, perfectly homogeneous
layer, highly refractive in character and of a faint yellowish
tint. When fully formed it possesses firm, resistant properties,
recalling those of chitin, and is doubtless composed of a
keratin base. It is distinguishable at once from the albumen
by its optical characters and staining reactions, so that there
is not the slightest justification for the supposition that it
may represent simply the specially differentiated outermost
portion of that layer. In ova which have just passed into the
uterus (fig. 10) the shell-membrane is extremely delicate, its
thickness being only about ‘0016 mm., but even before cleavage
begins it has increased to ‘002 mm. (fig. 12); in the 2-celled
stage (fig. 18) it has reached ‘005 mm., in the 4-celled stage
(fig. 22) -0072 mm., whilst in the 16-celled stage (figs. 24-26)
it has practically attained its maximum thickness, viz., ‘(0075—
‘008 mm. Caldwell’s measurements in the case of Phasco-
larctus agree closely with the above (shell of unsegmented
ovum from the uterus, 0015 mm. thick, that of the °5 mm.
“ovum,” ‘Ol mm.). Its presence renders the thorough
penetration of ova and early blastocysts with paraffin a
capricious and frequently troublesome operation, and its
resistant shell-like nature becomes only too obvious in the
process of section-cutting, since it cracks with the utmost
readiness (cf. Pl. 3, figs. 32, 37).

The occurrence of a shell-membrane round the Marsupial
ovum is a feature of considerable phyletic significance, as I
need hardly point out. It shows us that the ancestors of the
Metatheria must have been oviparous, or must themselves
have come from an oviparous stock, which there is no valid
reason for supposing was other than Prototherian in its
characters. It also renders untenable the views of Hubrecht to
the effect that the Metatheria are the descendants of Eutheria,
whilst the Kutheria themselves have been directly derived
from some presumed viviparous group of hypothetical Prote-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 25

trapods, unless we are to suppose that the Metatheria are
even now on the way to acquire secondarily the oviparous
habit, much in the same way as the Monotremes, according
to Hubrecht, have long since succeeded in doing.

The occurrence of a shell-membrane round the Marsupial
ovum has also an important ontogenetic significance in rela-
tion to the mode of formation of the blastocyst, as I shall
endeavour presently to show.

4. The Uterine Ovum.

The unsegmented ovum from the uterus (figs. 8-13)
consists of the following parts:

(1) The shell-membrane externally, ‘0016-002 mm. in
thickness.

(2) The laminated layer of albumen, ‘015-022 mm. or
more in thickness.

(8) The zona, about ‘0016 mm. in thickness.

(4) The perivitelline space, between the zona and the
ovum, occupied by a clear fluid which coagulates under the
action of certain fixatives, e. ¢. Hermann’s fluid (fig. 11, p.s.),
and which has diffused in from the uterus. ‘The minute
polar bodies lie in this space, usually nearer the upper pole
than the lower.

(5) The ovum proper.

The entire egg is spherical in form, and varies in diameter
in the fresh state from about *3 mm. to ‘36 mm. (average
about “32 mm.).

The ovum itself is ovoidal, its polar diameter always slightly
exceeding the equatorial. Its average diametrical measure-
ments in the fresh state run about °25 x -24 mm., though |
have records of ova measuring as much as ‘3 x °29 mm., and
I find that there is an undoubted slight variation in the size
of the ova of even one and the same batch, as well as in those
from different females.

The uterine ovum exhibits the same marked polarity as

26 J. P. HILL.

—

characterises the ripe ovarian ovum (the upper pole being
marked by the vacuolated deutoplasmic zone (figs. 8-11, d.z.),
and so far as its cytoplasmic body is concerned it shows no
essential difference from that.

Examined fresh in normal salt solution, the formative cyto-
plasm forming the bulk of the ovum appears dense, finely
granular, and of a very faint lightish-brown tint, its opacity
being such that the two pronuclei situated in its central region
can just be made out. In section, this central region is dis-
tinguishable from the peripheral zone by its uniform, more
finely granular character and by the absence of the fluid-filled
vacuolar spaces which are generally present in the latter
figs. 10 and 12). The deutoplasmic zone at the upper pole,
which is only partially visible in the entire egg owing to the way
in which it is enclosed by the formative cytoplasm (figs. 8, 9,
d.z.), presents a characteristically clear or semi-transparent
vacuolated appearance in the fresh state, but may have em-
bedded in it a small dense mass (fig. 8, cf. also figs. 1] and 14),
evidently formed by the transformation of a portion of its fluid
constitutent into the solid state, and so to be regarded as com-
parable with a bit of formative cytoplasm.

In most of the unsegmented uterine ova at my disposal the
male and female pronuclei have attained approximately the
same size and lie in proximity in the central more homo-
geneous region of the formative cytoplasm (figs. 10-12). The
transformation of the sperm-head into the male pronucleus
probably takes place during the passage of the ovum down
the tube, and was not observed, and I am as yet uncertain
whether the pronuclei unite to form a single cleavage nucleus
or give origin directly to the chromosomes of the first cleavage
figure.

Caldwell figures (’87, Pl. 30, fig. 5) a section through the
uterine ovum of Phascolarctus which I reproduce here as
Text-fig. 1,in order to facilitate comparison with my figs. 1land
12, with which it shows an essential agreement, apart from
the presence of follicular cells in the albumen which I have
never observed in Dasyurus, and making allowance for the

THE EARLY DEVELOPMENT. OF THE MARSUPIALIA. aE

difference in sectional plane. The figure is stated to represent
“the seventeenth section of a vertical longitudinal series of
thirty-five sections through the segmenting ovum, containing
two nuclei, taken from the uterus and measuring ‘17 mm. in
diameter.” Caldwell has, I think, fallen into several errors
in his interpretation of the structural features seen in this

TrxtT-FIG. 1.

Section of uterine ovum of Phascolaretus cinereus.
(After Caldwell.)

figure. In the first place, the sectional plane appears to me
not to be vertical as in my own figs. 11 and 12, but horizontal,
and to have passed through the lower portion of the deuto-
plasmic zone, shown in the figure as a central markedly
vacuolated area. Then there is no evidence to be derived
from the figure in support of the description of the ovum as
segmenting. The part inside the zona (vm.) labelled y' and
described as “ protoplasm with finest yolk-granules,” I would

28 J. P. HILL.

interpret simply as coagulum in the perivitelline space, whilst
the so-called “segmentation nuclei” (7, 3) situated in it are
probably the polar bodies or their derivatives. The part
labelled v2, and designated ‘‘ white yolk,’ I would regard as
the ovum itself. It exhibits an obvious differentiation into a
central vacuolated area and a peripheral, dense, granular
zone with scattered vacuoles, and I think there can be little
doubt but that the former corresponds to the deutoplasmic
zone of the Dasyure ovum, the latter to the formative zone.
It is these errors of interpretation apparently which misled
Caldwell into making the statement, now widely quoted in
the text-books, that cleavage in Phascolarctus is of the
meroblastic type.

CuHapter III.—CLeavaGE AND ForRMATION OF THE BLASTOCYST.

1. Cleavage.

Cleavage begins in the uterus as in Didelphys, Phasco-
larectus, and no doubt Marsupials in general. ‘The first ex-
ternally visible step towards it consists, as already described,
in the elimination by abstriction of the deutoplasmic zone at
the upper pole. The yolk-body so formed appears as a
definitely limited, clear, rounded mass which lies in contact
with the slightly concave upper surface of the formative
remainder of the ovum. It is quite colourless and transparent
except for the frequent occurrence in it of a small, more or
less irregular opaque mass, representing probably a condensa-
tion product of its fluid material (cf. Pl. , figs. 8, 14, y.b.).
Consisting as it does of a very delicate cytoplasmic reticulum
with fluid-filled meshes it is extremely fragile, and is seen to
advantage only in fresh material (figs. 14 and 19, y.b.). It
takes no direct part in the later developmental processes»
though during the formation of the blastocyst it becomes
enclosed in the blastocyst cavity and finally undergoes dis-
integration therein, its substance becoming added to the fluid
which fills the same, so that it may be said, in this indirect
way, to fulfil, after all, its original nutritional destiny. Separa-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 29

tion of the yolk-body is rapidly followed by the completion
of the division of the formative remainder of the ovum into
the first two blastomeres, the plane of division being co-
incident with the polar diameter or egg-axis and at right
angles to the plane of separation of the yolk-body (PI. 2, fig.
14). I obtained relatively little material between the stage
of the unsegmented ovum with two equal-sized pronuclei seen
in fig. 12 and the 2-celled stage (fig. 14), both of which
are well represented in my material, so that it would appear
that the separation of the yolk-body and the division of the
formative remainder of the ovum are effected with considerable
rapidity. Fig. 15 shows, however, a section of an un-
segmented ovum in which the chromosomes of the metaphase
of the first cleavage figure are visible in the central region
of the formative cytoplasm, but situated, it is worthy of note,
nearer the future upper pole than the lower pole. The deuto-
plasmic zone (d.z.) still forms an integral part of the egg, and
there is no sign of commencing abstriction. I have also
sections of ova in a still more advanced stage of the first
cleavage, in which the daughter-nuclei have but recently been
constituted and are still quite minute, and the cleavage furrow
is well marked on the surface of the egg. In these ova the
yolk-body is already separated, so that we may conclude with
a fair degree of certainty that its elimination about coincides
with the first appearance of the cleavage furrow.

Figs. 14-16 show the 2-celled stage, respectively in side,
lower polar, and end views. ‘The blastomeres are of approxi-
mately equal size and otherwise quite similar. Selenka also
found the same to be the case in Didelphys, though in the
single specimen of the 2-celled stage he had for examination
(Taf. xvii, fig. 3) the blastomeres are displaced and somewhat
shrunken. Each blastomere has much the shape of a hemi-
sphere from which a wedge-shaped segment has been sliced
off, a form readily accounted for when we take account of the
effect of the elimination of the deutoplasmic zone. After
that event, the formative remainder of the ovum has the
form of a sphere from which a somewhat bi-convex lens-

30 J. Re ED:

shaped piece has been gouged out at the upper pole.
Consequently, when it divides along its polar diameter, the
resulting blastomeres will have the form of hemispheres with
obliquely truncated upper surfaces or ends, which will be pro-
portionately thicker than the lower ends. In correlation
therewith we find the nucleus of each blastomere situated
slightly excentrically, rather nearer the upper than the lower
pole (fig. 18). The rounded yolk-body lies partly enclosed
between the upper truncated surfaces of the blastomeres.

Two-celled eges are shown in vertical section in figs. 17
and 18. The cytoplasm of the blastomeres exInbits a well-
marked differentiation into two zones corresponding to that
already seen in the formative cytoplasm of the unsegmented
egg, only much more accentuated, viz. a dense, fine-grained
perinuclear zone, and a less dense, more vacuolated peripheral
zone, in which there is present a coarse, irregular network of
deeply staining strands, recalling the framework of mito-
chondrial origin described by Van der Stricht (04, ’05) in
the human ovum and that of Vesperugo. We have here in
this differentiation of the cytoplasm, evidence of the occur-
rence of an intense metabolic activity which has resulted ina
marked increase in the amount of deutoplasmic material
present in the blastomeres as compared with that found in
the ovarian egg or even in the unsegmented uterine egg.
The blastomeres consequently present a somewhat dense
opaque appearance when examined in the fresh state, their
nuclei being partially obscured from view. Amongst the
Eutheria, various observers (Sobotta, Van der Stricht, Lams
and Doorme) have described a similar increase in the deuto-
plasmic contents of the egg after its passage into the
Fallopian tube or uterus.

The second cleavage plane is also vertical and at right
angles to the first. The resulting four equal-sized blastomeres
viewed from the side (PI. 2, fig. 19) are seen to be ovalish in
outline, their lower ends being slightly narrower and more
pointed than their upper ends, which diverge somewhat to
enclose the lower part of the yolk-body. Seen from one of

THE EARLY DEVELOPMENT OF 'THE MARSUPIALIA, 51

the poles, in optical section (figs. 20, 21), they appear tri-
angular with rounded corners and centrally directed apices.
The space occupying the polar diameter, which they enclose
is the cleavage cavity. ‘lhe blastomeres are now somewhat
less opaque than those of the 2-celled stage, so that their
nuclei, excentrically situated nearer their upper ends and
enclosed in the central granular zone of the cytoplasm, can
now be fairly distinctly made out in the fresh egg.

The arrangement of the blastomeres at this stage is
exceedingly characteristic, and is identical with that of the
blastomeres in the corresponding stage of Amphioxus or the
frog, but is quite different from that normal for the 4-celled
stage of the Eutheria. They he disposed radially or meri-
dionally around the polar diameter, occupied by the cleavage
cavity, their thicker upper ends partially surrounding the
yolk-body. Selenka figures a precisely similar arrangement
in his 4-celled stage of Didelphys, so that we may conclude
it holds good for the Marsupials in general.

Whilst, then, in Marsupials the first two cleavage planes are
vertical or meridional, and at right angles to each other, and
the first four blastomeres are arranged radially around the
polar diameter (radial type of cleavage), in the Eutheria
such is never the case, at all events normally, so far as is
known. In the Eutheria the first tour blastomeres form, or
tend to form, a definite cross-shaped group, as the result
apparently of the independent division of the first two blasto-
meres in two different planes at right angles to each other,
the division planes being meridional in the one, equatorial
in the other.' This pronounced difference in the spatial
relations of the first four blastomeres in the Metatheria and
Kutheria is a feature of the very greatest interest and im-
portance, since it is correlated with and in part conditions
the marked dissimilarity which we meet with in the later
developmental occurrences in the two groups, in particular
in the mode of formation of the blastocyst in the two.

1 Compare in this connection Assheton’s remarks (’09, pp. 232-233),
which have appeared since this chapter was written.

32 Je Ps Ee

Moreover, so far as the Eutheria are concerned, it affords us,
I believe, a striking and hitherto unrecognised example of a
phenomenon to which Lillie (’99) has directed attention, viz.
adaptation in cleavage.

Fig. 22 shows a horizontal section through the 4-celled
stage, and fig. 23 a vertical section of the same. The blasto-
meres in their cytoplasmic characters essentially resemble
those of the 2-celled stage, but the peripheral deutoplasmic
network is here more strongly developed, and it is especially
worthy of note that it is more marked towards the lower
poles of the blastomeres (fig. 23), as also appears to be the
case in the 2-celled stage. The shell-membrane measures in
thickness ‘0072 mm.

The next succeeding (third) cleavages are again meri-
dional, each of the four blastomeres becoming subdivided
vertically into two, not necessarily synchronously. Fig. 53.
Pl. 6, shows a side view, and fig. 54a view from the lower
pole of a 6-celled egg, two of the blastomeres of the 4-celled
stage having divided before the other two. The _ blasto-
meres have moved apart, and now form an open ring
approximately equatorial in position, and surrounding the
central cleavage space, the upper opening of which is
occupied by the yolk-body. I have failed to obtain a
perfectly normal 8-celled stage, nevertheless the evidence
clearly shows that the first three cleavage generations in
Dasyurus are meridional and equal, and that the resulting
eight equal-sized blastomeres form an equatorial ring in
contact with the inner surface of the sphere formed by the
zona and shell-membrane.

Whilst, then, the first three cleavage generations are
meridional and equal, the succeeding divisions (fourth cleavage
generation), on the contrary, are equatorial and unequal, each
of the eight blastomeres becoming divided into a smaller,
more transparent upper cell, with relatively little deutoplasm,
and a larger, more opaque lower cell with more abundant
deutoplasmic contents. In this way there is formed an
exceedingly characteristic 16-celled stage, consisting of two

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 33

superimposed rings, each of eight cells. The upper ring of
smaller and clearer cells partially encloses the yolk body, and
is situated entirely in the upper hemisphere of the sphere
formed by the egg-envelopes. The lower ring of larger, more
opaque cells lies approximately in the equatorial region of the
said sphere. This 16-celled stage is figured in fig. 55, Pl. 6,
as seen from the side, and in fig. 56 as seen from the upper
pole, both frgures being taken from a spirit egg ‘37 mm. in
diameter. The marked differences in the cells of the two
rings are well brought out in the micro-photographs reproduced
as figs. 24, 25, and 26, Pl. 2. Figs. 24 and 25 represent
horizontal sections of an egg *58 mm. in diameter, the former
showing the eight cells of the lower ring, and the latter the
eight cells of the upperring. Fig. 26 shows a vertical section
through an egg also of a diameter of *38 mm., but with
seventeen cells, one of the original eight cells of the upper ring
having divided and one being in process of division. The section
passes through the yolk-body (y.b.), which is seen as a faintly
outlined structure lying in contact with the zona between the
two cells of the upper ring (fic.).

The shell-membrane in eggs of this 16-celled stage has
attained a thickness of ‘(0075 mm., and the albumen layer has
been almost completely absorbed, so that the zona now lies
practically in apposition with the shell-membrane, the two
together forming a firm resistant sphere, to the inner surface
of which the blastomeres are closely applied. ‘The separation
between the zona and shell-membrane seen in the figures is
largely, if not wholly, artificial.

The average measurements of the cells of the two rings in
the ‘38 mm. egg, figured in figs. 24 and 25, are as follows :

Upper ring cells. Lower ring cells.
Diameter . (06 x ‘058mm. . ‘09 x ‘064mm.
Vertical height °095 mm. fel Lonmin
Nucleus . . ‘0165 mm. 2 O2imimn:

These measurements demonstrate at a glance the distinct
difference in size which exists between the cells of the two
rings, whilst the cytoplasmic differences between them are

VOL. 56, PART 1.—NEW SERIES. 3

34, dig) 125 Jetillinl iy,

equally evident from an inspection of the micro-photographs,
fis. 24-26. In the larger cells of the lower ring (fig. 24,
tr.ect.) the nucleus (rich in chromatin and nucleolated)
is surrounded by a perinuclear zone of clearer, coarsely
vacuolar cytoplasm, outside of which is a densely eranular
deutoplasmic zone, which extends to within a short distance
of the periphery of the cell-body. In the smaller cells of the
upper ring (fig. 25, fc.) the cytoplasm is coarsely reticular,
with a tendency to compactness round the nucleus, and its con-
tained deutoplasmic material is spare in amount as compared
with that of the lower cells, being mainly located in a quite
narrow peripheral zone. The upper cells thus appear relatively
clear as compared with the dense, opaque-looking lower cells
(fig. 26).

It becomes evident, then, that we have to do here, in this
fourth cleavage generation, with an unequal qualitative
division of the cytoplasm of the blastomeres of the 8-celled
stage. Just such a division as this we should expect if the
deutoplasmic material were mainly aggregated towards the
lower poles of the dividing cells. The evidence shows that
this is actually the case. In the 2-celled and especially in the
4-celled egos we have already seen that the deutoplasmic
network is already most strongly developed towards the lower
poles of the blastomeres. This polar concentration of the
deutoplasm reaches its maximum in blastomeres of the 8-
celled stage, and confers on these an obvious polarity.
Although I failed to obtain normal examples of the latter stage,
I have fortunately been able to observe the characters of the
blastomeres in sections of eggs with twelve, thirteen, and
fourteen cells respectively.

In the 12-celled egg (PI. 6, fig. 57), measuring *38 mm.
in diameter, four of the eight original blastomeres are still
undivided; the remaining four have undergone division
unequally and qualitatively, one but recently, so that 4 +
(4 x 2) = 12. The undivided blastomeres are large (average
diameter, ‘11 x ‘076 mm.) and ovoidal in form, their lower
ends being thicker than their upper, and they exhibit a well-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 35

marked polarity. The nucleus les excentrically in the
upper half of the cell, just above the equator, and is sur-
rounded by a finely granular zone of cytoplasm, outside which
is a thin irregular ring of deutoplasmic material. The cyto-
plasm of the apical part of the cell is clear and relatively free
from deutoplasm; that of the lower half, on the other hand,
is so rich in deutoplasm as to appear quite dense and opaque.
The conclusion is therefore justified that the blastomeres of
the 8-celled stage possess a definite polarity, which has been
acquired as the result of the progressive concentration of
deutoplasmic material at their vegetative poles during the
cleavage process. Division, in the equatorial plane, of cells
so constituted must necessarily be unequal and qualitative, so
far at least as the cytoplasm is concerned,

In the 15-celled stage three of the original eight blasto-
meres are in process of division, and five have already divided
unequally and qualitatively, so that 8 + (5 x 2) = 13, andin
the 14-celled stage two of the original blastomeres are in
division and six have already divided: 2 + (6 x 2) = 14.

The significance to be attached to this characteristic unequal
and qualitative division of the blastomeres of the 8-celled stage
to form two superimposed cell-rings, markedly differentiated
from each other, we shall presently consider. Meantime I
may categorically state the conclusions I have reached in
regard thereto. ‘The wall of the blastocyst in Dasyurus is at
its first origin, and for some considerable time thereafter,
unilaminar throughout its entire extent, and I regard the
upper cell-ring of the 16-celled stage as giving origin to
the formative or embryonal region of the unilaminar wall,
the lower cell-ring as furnishing the extra-embryonal or non-
formative remainder of the same. I shall therefore refer to
the upper cell-ring. and its derivatives as formative or
embryonal, and to the lower cell-ring and its derivatives
as non-formative or extra-embryonal.

The formative or embryonal region furnishes the embryonal
ectoderm and the entire entoderm of the vesicle, and I accord-
ingly conclude that it is the homologue of the embryonal knot

36 ‘ J.P: “HILL

or inner cell-mass of the Eutherian blastocyst. The non-
formative or extra-embryonal region directly gives origin to
the outer extra-embryonal layer of the bilaminar blastocyst
wall, i.e. to that layer which in the Sauropsida and Proto-
theria is ordinarily termed the extra-embryonal ectoderm. I
regard it as such, and as the homologue of the so-called
trophoblast (or as I prefer to term it, the ‘‘ trophoblastic
ectoderm ” or “tropho-ectoderm ”’) of the Hutherian blasto-
cyst.

A word or two here before concluding this section by way
of summary, as to the condition of the enclosing egg-envelopes.
During the sojourn of the egg in the uterus the albumen is
gradually resorbed, and by about the 16-cell stage it has all
but completely disappeared, thus permitting the zona to come
into direct apposition with the inner surface of the shell-
membrane. The shell-membrane itself increases very con-
siderably in thickness during cleavage, and by the 16-celled
stage had practically reached its maximum, viz. ‘0075—
‘008 mm., i.e. it is nearly five times thicker than that of the
ovum which has just entered the uterus. The thickened
shell-membrane by itself is firm and resistant, and it becomes
still more so by the application of the zona to its inner surface,
the two together forming a spherical supporting case round
the segmenting ege, to the inner surface of which the blasto-
meres become closely applied.

The existence of such a firm supporting envelope round
the Marsupial egg is, in my view,-a feature of very great
ontogenetic significance, and one which must be taken into
account in any comparison of the early developmental occur-
rences in the Metatheria and Kutheria. As the sequel will
show, the mode of formation of the blastocyst in these two
sub-classes is fundamentally different, and in my opinion
the explanation of this difference is to be found in the
retention by the Metatheria of a relatively thick resistant
shell-membrane, and its complete disappearance amongst the
Kutheria.

Ss

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 37

2. Formation of the Blastocyst.

It is characteristic of the Marsupial that the cleavage-cells
proceed directly to form the wall of the blastocyst, without
the intervention of a morula stage, as in the Kutheria.

The fifth cleavages are meridional, each of the eight cells
of the two rings of the 16-celled stage becoming subdivided
vertically into two, so that there results a 32-celled stage
consisting of two rings, each composed of sixteen cells. As
might be expected, the smaller less yolk-rich cells of the
upper ring tend to divide more rapidly than the larger yolk-
laden cells of the lower ring, but the difference in the rate of
division of the two is only shght. I have, for example,
sections of a 17-celled stage (that already referred to, fig. 26)
consisting of nine formative cells (= 6 + [1 x 2] + 1 in
division) and eight non-formative cells, and also of a 31-celled
stage (Pl. 6, fig. 59, seen from lower pole; cf. also fig. 60,
showing a side view of another 31-celled egg, both eggs
*375 mm. in diameter), consisting of sixteen formative and
fifteen non-formative cells, of which one is in process of
division. But I have also preparations of 52-celled egos with
an equal number of formative and non-formative cells,
showing that the latter may make up their leeway, the former
resting meantime. On the other hand, the cells of the two
rings may divide more irregularly, as evidenced by a stage
of about forty-two cells, consisting approximately of twenty-
three formative cells (= 9+ [7 x 2]) and nineteen non-
formative (= 13 + [8 x 2]). Whatever the rate of division,
the important point is that the division planes are always
radial to the surface, so that all the resulting blasto-
meres retain a superficial position in contact with the inner
surface of the supporting sphere formed by the zona and
shell-membrane. In apposition with the continuous surface
afforded by that, the blastomeres, continuing to divide,
gradually spread round towards the poles, the descendants of
the upper or formative cell-ring gradually extending towards
the upper pole marked by the yolk-body, whilst those of the

38 J.P. HILL.

lower or non-formative cell-ring similarly spread towards the
lower pole. As the blastomeres divide and spread they
become smaller and more flattened, and gradually cohere
together, and so in this way they eventually give origin to a
complete unilaminar layer lining the inner surface of the
sphere formed by the egg-envelopes. It is this unilaminar
layer which constitutes the wall of the blastocyst.

The just completed blastocyst of Dasyurus is a spherical
fluid-filled vesicle measuring about ‘4 mm. in diameter (PI. 3,
figs. 27-29, Pl. 6, figs. 61, 62), and invested externally by
the thin zona and the shell-membrane (‘0075-0078 mm. in
thickness), ‘The albumen layer has completely disappeared,
and the shell-membrane, zona, and cellular wall are from
without inwards in intimate apposition. ‘The smallest com-
plete vesicles which I have examined measure ‘39 mm. in
diameter (figs. 27, 61), and in one of these I find the cellular
wall consists approximately of about 108 cells. In tour other
eggs of the same diameter and from the same female the wall
of the blastocyst is as yet incomplete at the lower pole (fig.
31, l.p.), and in these, rough counts of the cells yielded the
following respective numbers—89, 93, 121, 128. In another
also incomplete blastocyst of the same batch, ‘41 mm. in
diameter (fig. 32), the cellular wall consists of about 130 cells.
The largest complete blastocyst in this same batch measured
‘49 mm. in diameter, so that we havea range of variation in size
of the just completed blastocyst extending from *39 to °49 mm.

The umlaminar wall of the blastocyst consists of a con-
tinuous layer of more or less flattened polygonal cells (figs.
27-29, 61, 62) lying in intimate contact with the zona, itself
closely applied to the shell-membrane. Over the lower hemi-
sphere the non-formative cells are on the whole larger and
plumper than the formative cells of the upper hemisphere,
and in surface examination they appear somewhat denser
owing to the fact that they possess much more marked peri-
nuclear zones of dense cytoplasm than do the formative cells
(cf. fig. 63, representing a °6 mm. vesicle). In sections,
however, this latter difference is much less obvious, indeed,

THE EARLY DEVELOPMENT OF THE MARSUPIALTIA. 39

is hardly, if at all, detectable, so that one has to depend
partly on the relative thickness of the cells, partly, and,
indeed, mainly, on the yolk-body in determining which
hemisphere is which.

The blastocyst cavity is tensely filled by a coagulable fluid
derived from that poured into the uterine lumen through the
secretory activity of the uterine glands. Also situated in the
blastocyst cavity, in contact with the inner surface of the
wall in the region of the upper pole, is the spherical yolk-
body (fig. 29, y.b.). It becomes overgrown and enclosed in
the blastocyst cavity as the result of the completion of the
cellular wall over the upper polar region, much in the same
sort of way as the yolk in the meroblastic egg becomes
enclosed by the peripheral growth of the blastoderm. In the
majority of my sections of early blastocysts the yolk-body
has been dragged away from contact with the formative cells
through the coagulation of the albuminous blastocystic fluid,
and lies more or less remote from the wall enclosed by the
coagulum, except on the side next the upper hemisphere (fig.
31, y.b., c.g.) In two instances, one of which is shown in
fig. 32,1 find the yolk-body had become so firmly attached to
one of the formative cells that the coagulum formed during
fixation failed to detach it, and only succeeded in drawing it
out to a pear-shape.

The yolk-body, it may here be mentioned, persists for a
considerable time in the blastocyst cavity; I have found it
shrunken indeed, but still recognisable, in relation to the
embryonal area in vesicles 45-6 mm. in diameter. And
there may even appear within it peripherally, irregular strands
which stain deeply with iron-hematoxylin and which recall
those forming the peripheral deutoplasmic network of the early
blastomeres. Eventually, however, it seems to disappear, its
substance passing into the blastocystic fluid, so that, as already
remarked, it fulfils in this indirect way its original destiny.

Normally the cavity of the just completed blastocyst con-
tains no cellular elements whatever. In one otherwise
perfectly normal blastocyst (‘39 mm. diam.) I find present,

40 ye Pe SEE tie

however, a small spheroidal body ‘028 mm. in diameter,
composed of glassy-looking cytoplasm enclosing a central
deeply staining granule. ‘his I interpret as a cell or cell-
fragment which has been accidentally separated off from the
wall, and which has undergone degeneration. In later
blastocysts such cellular bodies exhibiting more or less
evident signs of degeneration are of fairly common occur-
rence. They are of no morphological significance.

Selenka’s ‘ Blastopore.’”—Normally the wall of the
blastocyst is first completed over the upper hemisphere, in
correspondence with the fact that the formative cells not
only divide somewhat more rapidly than the non-formative
but have a smaller extent of surface to cover, since the upper
cell-ring from which they are derived lies about midway
between the upper pole of the sphere formed by the egg-
envelopes and the equator of the same, whilst the lower cell-
ring from which the non-formative cells arise is approximately
equatorial in position. We thus meet with stages in the
formation of the blastocystic wall such as are represented in
surface view on PI. 3, fig. 30, and in section in figs. 31 and
32,in which the blastocystic cavity, prior to the completion
of the cellular wall over the lower polar region, is more or less
widely open below. There can be no doubt, I think, but that
this opening corresponds to that observed by Selenka in his
42-celled “gastrula” of Didelphys and regarded by him as
the blastopore, since he believed the entoderm arose from its
lips. My observations conclusively show that it has no
connection whatever with the entoderm, this layer arising
from the formative region of the upper hemisphere, aud that
ib is a mere temporary opening of no morphological signifi-
cance, blastoporic or other. Prior to the completion of
the wall at the upper pole a corresponding opening is tem-
porarily present there also. Both owe their existence to the
characteristic way in which the blastocyst wall is formed by
the spreading of the products of division of the two cell-rings
of the 16-celled stage towards opposite poles in contact with
the surface provided by the enclosing egg-envelopes.

THE EARLY DEVELOPMENT. OF THE MARSUPIALIA. 41

I have met with one specimen, an incomplete blastocyst
*39 mm. in diameter (bélonging to the same batch as the
other blastocysts referred to in this section!), in which the
lower hemisphere would appear to have been completed before
the upper, for the yolk-body lies in contact with the zona in
the region where the cellular wall is as yet absent, and that
the yolk-body has not been secondarily displaced is proved by
a micro-photograph of the specimen in my possession (taken
immediately after its transference to the fixing solution), in
which the yolk-body is seen to lie at the unclosed pole in
exactly the same position as in the sections.

In connection with this exceptional specimen, it may be
recalled that Selenka, in his 68-celled ‘‘ gastrula”’ of Didelphys
(fig. 10, Taf. xvii), figures the wall as complete at the lower
pole, the ‘‘ blastopore”’ having already closed, but as still in-
complete at the upper pole, there being present a small opening
leading into the blastocyst cavity. In the 42-celled “gastrula”’
(fig. 8, Taf. xvii) this same opening and the “ blastopore” as
well are present. ‘he occurrence of these openings at
opposite poles, and the general agreement in the constitution
of the blastocyst wall (larger, more yolk-rich cells at lower
pole, smaller, less yolk-rich cells at upper), in the corre-
sponding stages in Didelphys and Dasyurus justify the con-
clusion that the blastocyst of the former develops in the same
way as does that of the latter. It is worthy of remark,
however, that the just completed blastocyst of Didelphys
appears to be considerably smaller than that of Dasyurus.
Selenka unfortunately gives no measurements of his early
stages, but I have calculated from the figure, the magnification
of which is given, that the 68-celled blastocyst has a diameter
of about ‘137 mm. The corresponding stage of Dasyurus
measures about ‘39 mm., and is therefore nearly three times as
large.

1 This batch, from female 2 B, 16. vii .’01, comprised altogether
twenty-eight eggs, of which some eighteen were normal complete and
incomplete blastocysts (39-49 mm. in diameter) and ten abnormal, four
of these being unsegmented ova.

42 pace, EVER:

Selenka’s Urentodermzelle—wWhilst the 42- and 68-
celled blastocysts described by Selenka may be regarded
as normal so far as the occurrence of polar openings and
the constitution of their wall are concerned, I hold them to be
abnormal in respect of the presence in each of a single large
yolk-laden cell, regarded by Selenka as entodermal in signifi-
cance. It is well to point out that Selenka was not able
actually to determine the fate of this cell; he merely presumed
that it took part in the formation of the definitive entoderm.
No such cell occurs in normal blastocysts of Dasyurus at any
stage of development, and in my opinion Selenka’s “ urento-
dermzelle”’? is none other than a retarded and displaced
blastomere, i.e. a blastomere which has failed for some
reason to divide, and which has become secondarily enclosed
by the products of division of its fellows, and I am
strengthened in this interpretation by the occurrence in
an abnormal blastocyst of Dasyurus of just such a large
cell as that observed by Nelenka. ‘The vesicle in question
is one of the batch already referred to, and measured °597 mm.
in diameter. he cellular wall (fig. 37) isapparently normal,
but is incomplete at one spot, and the gap so left is occupied
by a large binucleated cell, rich in deutoplasm and measuring
"12 x :072 mm. (fig. 37, abn.). This cell corresponds in its
size and cytoplasmic characters with a non-formative blasto-
mere of about the 16-celled stage, and I regard it simply as
a blastomere which has failed to undergo normal division.
In another abnormal blastocyst (‘39 mm. diam.) from the
same batch, the cellular wall appears complete and normal,
but the blastocyst cavity contains a group of about sixteen
spherical cells averaging about ‘032 mm, in diameter, and in
yet another abnormal egg of the same diameter and batch
there is present an incomplete layer of flattened cells over
one hemisphere, and towards the opposite pole of the egg-
sphere there occurs a group of spherical cells of variable size
and some of them multinucleate. In this abnormal egg it
appears as if the formative cells had divided in fairly normal
fashion, whilst the non-formative cells had failed to do so.

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 45

Cuaprer [V.—GrowTH oF THE BLAsTocysTt AND DIFFERENTLA-
TION OF THE EMBRYONAL ECTODERM AND THE ENTODERM.

l. Growth of the Blastocyst.

In the preceding chapter we have seen that the cleavage
process in Dasyurus results in the formation of a small
spherical vesicle, about *+ mm. in diameter, which consists,
internally to the investment formed by the apposed zona and
shell-membrane, simply of a cellular wall, unilaminar through-
out its entire extent, and enclosing a_ fluid-filled cavity
normally devoid of any cellular elements. The stage of the
just completed blastocyst is followed by a period of active
growth of the same, and it is a noteworthy feature in the
development of Dasyurus that during this time the blastocyst
undergoes no essential structural change, but remains uni-
laminar until it has reached a diameter of from 4:5 to 5°75 mm.
Even during cleavage, the egg of Dasyurus increases in
diameter, partly owing to the thickening of the shell mem-
brane, partly, and, indeed, mainly, as the result of the accumu-
lation of uterine fluid under pressure within the egg-envelopes,
but the increase due to these causes combined is relatively
insignificant, being only about *Limm. As soon, however, as
the cellular wall of the blastocyst is completed, rapid growth
sets in, under the influence of the hydrostatic pressure of the
fluid, which tensely fills the blastocyst cavity, with the result
that the small relatively thick-walled blastocyst becomes
converted into a large extremely thin-walled vesicle, but
beyond becoming very attenuated, the cellular wall durmg
this period of active growth undergoes no essential change,
and retains its unilaminar character until the blastocyst, as
already mentioned, has reached a diameter of from 4°5 to 5°)
mm. In vesicles of about this size there become differentiated
from the formative ceils of the upper hemisphere the em-
bryonal ectoderm and the entoderm, and this latter layer then
gradually spreads round inside the non-formative (extra-
embryonal ectodermal) layer of the lower hemisphere so as to

44. Jee. eels

form a complete lining to the blastocyst, which thereby
becomes bilaminar. Such a marked enlargement of the blasto-
cyst prior to the differentiation of the embryonal ectoderm
and entoderm as is here described for Dasyurus does not
apparently oceur, so far as known, in other Marsupials : in
Perameles, for example, the embryonal ectoderm and the
entoderm are in process of differentiation in vesicles a little
over 1 mm. in diameter (v. p. 77), in Macropus these two layers
are already fully established in a vesicle only *8 mm. in
diameter (v. p. 79), and much the same holds good for Tricho-
surus and Petrogale. It is paralleled by the marked growth
which in the Monotremes follows the completion of the blasto-
cyst and which precedes the appearance of embryonal difter-
entiation. It must be remembered, however, that the growing
blastocyst in the Monotreme is bilaminar and not unilaminar
as in Dasyurus, owing tothe fact that the entoderm is estab-
lished as a complete layer at a very much earlier period than
is the case in the latter. I am nevertheless inclined to regard
the attainment by the Dasyurus blastocyst of a large size,
prior to the differentiation of the embryonal ectoderm and the
entoderm, as a more primitive condition than that found in
other Marsupials. The pronounced hypertrophy which the
uteri of Dasyurus undergo during the early stages of gesta-
tion, an hypertrophy which appears to be proportionately
greater than that met with in other forms,' is no doubt to be
correlated with the presence in them of such a considerable
number of actively growing blastocysts.

Selenka states (Heft 5, p. 180) that he examined seven
blastocysts of Dasyurus “? mm.” in diameter, taken from a
female fifteen days after copulation. He describes their
structure as follows: ‘“‘ Man unterscheidet (1) eine sehr
zarte aussere, homogene Haut (Granulosamembran), (2)

' For example, the uteri of a female (5, 18. vii. °01) from which I
obtained twenty-one normal vesicles, 4°5-6 mm. in diameter, with the
embryonal area definitely established, measured as follows : Left uterus,
4:5 x 47 x 14 em. (fourteen vesicles) ; right uterus, 4°5 x 4:2 x 1:45 em.
(seven vesicles and one shrivelled).

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 45

darunter ein Lager von Ektodermzellen, welche im Gebiete
des Embryonalschildes prismatich, am gegeniiberliegenden
Pole nahezu kubisch, im itbrigen abgeplattet erscheinen, (3)
ein inneres zusammenhangendes Lager von abgeflachten Ento-
dermzellen.”” This description, apart from the reference to
the thin shell-membrane, is entirely inapplicable to blastocysts
of Dasyurus of the mentioned size which I have studied.

I have examined a practically complete series of vesicles of
Dasyurus ranging from ‘4 mm. to 4 mm. in diameter and all of
them without exception are unilaminar.

Of vesicles under 1 mm. diameter I possess serial sections
of more than two dozen, ranging from ‘5 mm. to ‘8 mm. in
diameter, and obtained from three different females. These
differ structurally in no essential respect from the just com-
pleted blastocysts. A surface view of a blastocyst *6 mm. in
diameter is shown in fig. 63, Pl. 6; in this the difference in
the cytoplasmic characters of the cells of opposite hemispheres
is clearly brought out, the non-formative cells of the lower
hemisphere having much more marked perinuclear zones of
dense cytoplasm (deutoplasm) than the formative cells of the
upper hemisphere ; moreover, the former cells tend to be of
larger superficial extent than the latter. Fig. 34, Pl. 3,
represents a section of a blastocyst ‘57 mm. in diameter, and
fig. 55 a section of one *73 mm. in diameter. These blasto-
cysts differ in no essential way from the ‘43 mm. blastocyst
represented in fig. 53. As in the latter, the cellular wall is
unilaminar throughout, but both it and the shell-membrane
have undergone considerable attenuation. Moreover in these
blastocysts, apart from the clue afforded by the shrivelled
yolk-body, it is practically impossible to determine from the
sections which is morphologically the upper hemisphere and
which the lower. In fig. 36, from a °6 mm. blastocyst, on the
other hand, the cells of the hemisphere opposite the yolk-body
(y.b.) are larger than those of the hemisphere adjacent to
which that body is situated. In the ‘57 mm. blastocyst the
shell-membrane has a thickness of ‘0052 mm., in the ‘73 mm.
blastocyst it measures ‘0045 mm., and in a *84 mm. blastocyst

46 J, GES Eb:

‘0026 mm. The zona is now no longer recognisable as an
independent membrane. In blastocysts of this stage of
growth a variable number of small spherical cells or cell-
fragments are frequently met with in the blastocyst cavity,
usually lying in contact with the inner aspect of the cellular
wall (fig. 34, 7.c.). In some blastocysts such structures are
absent, in others one or two may be present, in yet others
numbers of them may occur. They may be definitely nucleated,
but this is exceptional; more usually they contain one or more
deeply staining granules (of chromatin?), or are devoid of
such. They are of no morphological importance, and I think
there can be no doubt that they represent cells or fragments
of cells which have been separated off from the cellular wall
during the process of active growth. ‘They are of common
occurrence in later blastocysts, and it is possible the so-called
“ yolk-balls ” observed by Selenka in Didelphys are of the
same nature,

If we pass now to vesicles from 1 to 3 or 3°5 mm. in
diameter, we find the wall still unilaminar, but considerably
more attenuated than it is in the blastocysts last referred to.
In a vesicle with a diameter of 1:24 mm. the shell-membrane
has a thickness of about ‘0015 mm., whilst the cellular wall
has a thickness of only ‘0045 mm. Ina 3°5 mm. vesicle the
shell-membrane measures about ‘0012 mm., whilst the cellular
wall ranges from ‘0018 to ‘0048 mm. in thickness. A small
portion of the wall of a vesicle, 24 mm. in diameter, is shown
in Pl. 6, fig. 64. In these later vesicles I have failed to detect,
either in surface examination of the vesicles in toto or in
sections, any regional differences between the cells indicative
of a differentiation of the wall into upper or formative, and
lower or non-formative, hemispheres. Everywhere the wall
is composed of flattened, extremely attenuated cells, polygonal
in surface view, and all apparently of the same character. It
might therefore be supposed that the polarity, which is recog-
nisable in early blastocysts, and which is dependent on the
pronounced differences existent between the cells of the
upper and lower rings of the 16-celled stage, is of no funda-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 47

mental importance, since it apparently becomes lost at an
early period during the growth of the blastocyst. Such an
assumption, however, would be very wide of the mark, as I
hope to demonstrate in the next section of this paper, and,
indeed, in view of the facts already set forth, is an altogether
improbable one.

Reappearance of Polar Differentiation in the
Blastocyst Wall.—Following on the period of what may
be termed the preliminary growth of the blastocyst, in the
course of which the original polar differentiation in the
blastocyst wall apparently becomes obliterated, is an
extremely interesting one, during which that differentiation
again becomes manifest. In view of the fact (1) that the
fourth cleavage in Dasyurus is of the nature of a qualitative
cytoplasmic division, and (2) that approximately one half or
rather less of the unilaminar vesicle wall is formed from the
eight smaller and less yolk-rich cells of the upper ring of the
16-celled stage, and its remainder from the eight larger
more yolk-rich cells of the lower ring, it thus becomes a
question of the first importance to determine if we can the
significance of that differentiation.

Amongst the Eutheria, it has been conclusively shown by
various observers (Van Beneden, Heape, Hubrecht, Assheton,
and others) that there occurs during cleavage an early
separation of the blastomeres into two more or less distinctly
differentiated groups, one of which eventually, by a process
of overgrowth, completely encloses the other. The peripheral
cell-group or layer forms the outer extra-embryonal layer of
the wall of the later blastocyst (the trophoblast of Hubrecht,
or trophoblastic ectoderm as I prefer to term it). It therefore
takes no direct part in the formation of the embryo, and may
be distinguished as non-formative. The enclosed cell-group,
termed the inner cell-mass or embryonal knot, gives rise, on
the other hand, to the embryonal ectoderm as well as to the
entire entoderm of the vesicle, and may accordingly be dis-
tinguished as formative. May it not be, then, that we have
here at the fourth cleavage in Dasyurus a separation of the

48 ee, UT

blastomeres into two determinate cell-groups, respectively
formative and non-formative in significance, entirely compar-
able with, and, indeed, even more distinct than that which
oceurs during cleavage in the Eutheria? I venture to think
that the evidence brought forward in this paper conclusively
justifies an answer in the affirmative to that question.

If we assume that the upper cell-ring of the 16-celled stage
in Dasyurus is formative in destiny and the lower cell-ring
non-formative, then we might naturally expect to find in the
unilaminar wall of the later blastocyst some differentiation
indicative of its origin from two distinct cell-groups, and
indicative at the same time of the future embryonal and
extra-embryonal regions. Now just such a differentiation
does, as a matter of fact, become evident in vesicles 3°65 to
4:5 mm. in diameter. We have already seen that the wall in
early blastocysts ‘4 to ‘8 mm. in diameter exhibits a well-
marked polar differentiation in correspondence with its mode
of origin from the differentiated cell-rings of the 16-celled
stage, its upper hemisphere or thereabouts consisting of
smaller cells, poor in deutoplasm, its remainder of larger
cells, rich in deutoplasm. In later blastocysts, 1-3 mm. or
more in diameter, it is no longer possible to recognise this
distinction—at all events I have failed to observe it—but if
we pass to blastocysts 4°5 mm. in diameter, in which the wall

is still unilaminar, we find on careful examination of the
entire vesicle under a low power that there is now present a
definite continuous line, which encircles the vesicle in the
equatorial region so as to divide its wall into two hemi-
spherical areas (PI. 4, fig. 38, 7.l.). If we remove and stain
a portion of the wall of such a vesicle, including this line,
and examine it microscopically (figs. 42-46), it becomes
apparent at once, from the disposition of the cells on either
side of the line, that we have to do with a sutural line or line
of junction produced by the meeting of two sets of cells,
which are pursuing their own independent courses of growth
and division. The cells never cross the demarcation line
from the one side to the other, but remain strictly confined

THE EARLY DEVELOPMENT: OF THE MARSUPIALIA. 49

to their own territory, so that we are justified in regarding
the vesicle wall as composed of two independently growing
zones. Now the existence of two such independent zones in
the unilaminar wall is, to my mind, only intelligible on the
view that they are the products of two originally distinct,
predetermined cell-groups, and if this be admitted, then I
think we are justified in concluding, in view of the facts
already set forth, that the two zones in question are derived,
the one from the upper cell-ring of the 16-celled stage, the
other from the lower ring; that, in other words, they repre-
sent respectively the upper and lower hemispheres of the
early blastocysts.

If, now, we find that the embryonal ectoderm and the ento-
derm arise from one of these two regions of the unilaminar
wall, whilst the other directly forms the outer extra-embryonal
layer of the later (bilaminar) vesicle, then we must designate
the former region as the upper or formative, and the latter as
the lower or non-formative. Further, bearing in mind the
characters of the cells of the two rings of the 16-celled stage,
I think we are justified in holding that the formative region
is derived from the ring of smaller, less yolk-rich cells, and
the non-formative region from the ring of larger, more yolk-
rich cells, even if it is impossible to demonstrate an actual
genetic continuity between the constituent cells of these two
rings and those forming the independently growing areas of
the later blastocyst. I have recently re-examined a series of
vesicles, measuring 1°5-1°8 mm. in diameter, obtained from a
female killed in 1906, and I have so far found it impossible,
either in the entire vesicle or in portions of the wall stained
and mounted on the flat, to distinguish between the cells over
opposite hemispheres. Thus the only actual guide we have
for the determination of the poles in such vesicles is the
yolk-body, and though the latter is liable to displacement, it
is worthy of record that I have several times found it in
relation to the formative area in vesicles 4°5-6 mm. in
diameter, but never in relation to the non-formative region.
This evidence is, therefore, so far as it goes, confirmatory of

VoL. 56, PART 1.—NEW SERIES. +

50 ees ORNL:

the conclusion reached above, viz. that the formative hemi-
sphere is derived from the smaller-celled ring of the 16-celled
stage. On that conclusion is based my interpretation of the
poles in the unsegmented ovum, and of the two cell-rings of
the 16-celled stage as respectively upper and lower.

Of vesicles over 1 mm. in diameter, the smallest in which I
have been able to detect the sutural line above referred to
measure 3°25 mm, in diameter. In three lots of vesicles, 3:5
mm. in diameter from three different females, I have failed to
recognise it, whilst in two other lots, respectively 3°75 mm.
(average) and 4 mm. in diameter, the line appears to be in
course of differentiation as in the 3°25 mm. vesicles. A
portion of the wall of one of the 3°5 mm. vesicles just referred
to is shown in Pl. 4, fig. 41, and a portion of the wall of the
3°25 mm. stage, including the sutural line, in fig. 42. Both
vesicles were fixed in the same fluid, viz. picro-nitro-osmic
acid. Comparison of the two figures reveals the existence, quite
apart from the presence of the junctional line in fig. 42, and its
absence in fig. 41, of certain more or less obvious differences
between them. In fig. 41 the cells are larger, and their cyto-
plasmic bodies are inconspicuous, being fairly homogeneous
and lightly staining. In fig. 42, on the contrary, the cell-
bodies are strongly marked, the cytoplasm being distinguish-
able into a lighter-staiming peripheral zone, and a much more
deeply staining perinuclear zone, showing evidence of intense
metabolic activity. This latter zone is more or less vacuolated,
and contains, besides larger lightly staining granules, numerous
smaller ones of varying size, stained brown by the osmic acid
of the fixative. In the 4 mm. vesicles the cells show precisely
the same characters; in the 3°75 mm. vesicles, which were
fixed in a picro-corrosive-acetic fluid, the granules are absent
from the cytoplasm, otherwise the cells are similar to those
of the other two. Mitotic figures are common. The sutural
line is recognisable in all three sets of vesicles (3°25, 3°75, and
4mm.) (fig. 42, 7./.), but I cannot be certain that it runs con-
tinuously round, and it appears to have a rather more sinuous
course than in later blastocysts. The cells of the two regions

THE BARLY DEVELOPMENT OF THE MARSUPIALIA. 51

of the blastocyst wall, separated by the sutural line, differ
somewhat in their characters. On one side of the line (fig.
42, tr.ect.) the ceils appear to be on the whole slightly larger,
and of more uniform size than they are on the other, and they
also stain somewhat more deeply. Comparison with later
blastocysts shows that the region of more uniform cells is
non-formative, that of less uniform, formative. At this stage,
however, the differences between the cells of the two regions
are as yet so little pronounced that it is practically impossible
in the absence of the sutural line to say to which hemisphere
an isolated piece of the wall should be referred.

I am inclined to regard the sutural line in these vesicles as
being in course of differentiation, and judging from the dis-
position of the cells on either side of it, I think its appearance
is to be correlated with the marked increase in the mitotic
activity of the cells of the two hemispheres which sets in in
vesicles of 3-4 mm. diameter. The preliminary increase in
size of the blastocyst up to about the 3 mm. stage might be
described as of a passive character, 1.e. 16 does not appear
to be effected as the result of the very active division of the
wall-cells, but is characterised rather by a minimum of mitotic
division and a maximum of increase in surface extent of the
cells, due to excessive stretching consequent on the rapid
imbibition of uterine fluid. Once, however, the requisite size
has been attained, the cells of the unilaminar wall commence
to divide actively, and doubtless as the outcome of that
wave of activity, the sutural line makes its appearance
between the two groups of independently growing cells.

On the inner surface of the blastocyst wall, especially in
the region of the formative hemisphere, there are present
in these vesicles numbers of small deeply staining cells of
spherical form, and containing osmicated granules similar
to those in the wall-cells. They may occur singly or in groups,
and appear to me to be of the same nature as the internal cells
of the earlier blastocyst. In addition to these cells, there are
present clusters of cytoplasmic spheres, staining similarly to
the spherical cells, and apparently of the nature of fragmenta-

DZ J. °P: HILL.

tion products formed either directly from the wall-cells or
from these internal cells.

2. Differentiation of the Embryonal Ectoderm and
the Entoderm.

After the preliminary growth in size of the blastocyst is
completed, the next most important step in the progressive
development of the latter is that just dealt with, involving
the appearance of the sutural line, with resulting re-establish-
ment of polar differentiation in the blastocyst wall. Following
on that, we have the extremely important period during
which the embryonal ectoderm and the entoderm become
definitely established.

For the investigation of the earlier phases of this critical
period I have had at my disposal a large number of
unilaminar blastocysts derived from three females, dis-
tinguished in my notebooks as (3, 25. vii. 01, with fifteen
vesicles of a maximum diameter of 4°56 mm.; 8.vii.’99, with
twelve vesicles, 4°5 mm. in diameter; and 6. vii. ’04, with
twenty-two vesicles, 4°5 and 5mm. in diameter. These three
lots of vesicles may for descriptive purposes be designated
as ’O1, 799, and ’04 respectively.

The ’O1 vesicles are distinctly less advanced than the
other two. The sutural line is now, at all events, definitely
continuous, and can readily be made out in the intact vesicle
with the aid of a low-power lens (Pl. 4, fig. 38, 7./.), but.
the differences between the cellular constituents of the two
hemispheres which it separates are much less obvious than
they are in the 799 and ’04 vesicles. Here, again, one
hemisphere forming half or perhaps rather more of the entire
vesicle is distinguished from the other by the greater uni-
formity and the slightly deeper staining character of its
constituent cells (figs. 43 and 44, tr. ect.). ‘his hemisphere,
subsequent stages show, is the lower or non-formative
hemisphere. It is characterised especially by the striking
uniformity in the size of its cells. Over the opposite hemi-
sphere, the upper or formative one (figs. 43 and 44, f.a.), the

THE EARLY DEVELOPMENT OF THE MARSUPTALIA. 53

cells are more variable in size, the nuclei thus appearing less
uniformly and less closely arranged, and they stain, on the
whole, somewhat less deeply than those of the lower hemi-
sphere. The non-formative cells are on the average smaller
than the largest of the formative cells, but they are more
uniform in size, and their nuclei thus lie at more regular
distances apart, and appear more closely packed. They are
also richer in deutoplasmic material, and so stain rather more
deeply than the formative cells. Sections show that the
cellular wall is unilaminar throughout its extent, and that,
whilst it is somewhat thicker than that of 3°5 mm. vesicles,
it is still very attenuated, its thickness, including the shell-
membrane, ranging from ‘004 to 008 mm. I have examined
a number of series of sections taken through portions of the
wall known to include the sutural line, and find it quite
impossible to locate the position of the latter; indeed, I
cannot certainly distinguish between the formative and non-
formative regions.

In the blastocyst cavity,.lying in contact with the inner
surface of the wall, and most abundant in the region of the
formative hemisphere, there are present numbers of deeply
staining spherical cells with relatively small nuclei similar to
those described in connection with the 3°25 mm. vesicles.
They occur singly or in groups, and may appear quite normal
or may show more or less evident signs of degeneration.
Their nuclei may stain deeply and homogeneously, or may be
represented by one or two deeply staining granules, vacuoles
may occur in their cytoplasm, and spherical cytoplasmic masses
of very variable size, with or without deeply staining granules
of chromatin, may occur along with them. In sections and
preparations of the wall of these and other 45 mm. vesicles
there are to be found, in both the formative and non-formative
hemispheres, small localised areas from which such spherical
cells are being proliferated off in numbers together. PI. 54,
fig. 47, from the formative hemisphere of an 704 vesicle shows
one of the most marked examples of such proliferative activity
that I have encountered. A similar but smaller proliferative

54. 72 7P. TLL.

area occurs on the non-formative hemisphere of the same
vesicle.

These spherical cells are, I am convinced, of no morpho-
logical importance, and are destined sooner or later to de-
generate. They have certainly nothing to do with the
entoderm, the parent-cells of that layer arising exclusively
from the formative hemisphere and not from cells such as
these, which are budded off from both hemispheres. The fact
that they are, in unilaminar vesicles, more numerous over the
formative hemisphere may perhaps be taken as an indication
of the greater mitotic activity of the formative as compared
with the non-formative cells.

The Primitive Entodermal Cells.—Following closely
on the stage represented by these ’01 blastocysts is the ex-
tremely important one constituted by the 99 and ’04 vesicles
before referred to. This stage is the crucial one in primary
germ-layer formation, and marks the transition from the uni-
laminar to the bilaminar condition, since in it the entodermal
cells are not only distinctly recognisable as constituents of the
formative region, but are to be seen both in actual process of
separation from the latter and as definitely internal cells, fre-
quently provided with, and even connected together by,
pseudopodial-like processes of their cell-bodies. Such cells
are already present in the ’01 vesicles (fig. 71), and probably
also in the blastocysts in which the sutural line first makes
its appearance, but are much less conspicuous than in these
older blastocysts.

The ?99 blastocysts are distinctly more advanced than the
’01 batch and are just a little earlier than the ’04 lot. The
former measured, as already mentioned, 4°5 mm. in diameter,
the latter 45 and 5 mm. (the majority being of the latter
size). In my notes on the intact 799 vesicles I find it stated
that one hemisphere, forming rather less than half of the
entire extent of the vesicle wall, appeared somewhat denser
than the other, the sutural line marking the division between
the two. I naturally inferred at the time that the denser
hemisphere corresponded to the embryonal region of the

~

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 5d

Eutherian blastocyst and the less dense to the extra-embry-
onal region of the same, but just the reverse proves to hold
true for the ’04 vesicles, the formative hemisphere in these
appearing less dense than the non-formative. I cannot now
test my former inference by direct observation since I do not
appear to have any of the ’99 vesicles left intact, but amongst
my in toto preparations of the vesicle wall I find one
labelled as from the “lower pole” which unmistakably
belongs to the formative hemisphere, hence I conclude that
the denser and slightly smaller region which I originally
regarded as formative is really non-formative, a conclusion
which brings the ’99 vesicles into agreemeut with the ’04
batch.

In these latter vesicles the sutural line and the two regions
of the wall can be quite readily made out on careful examina-
tion under a low power with transmitted light. ‘lhe one
region appears slightly denser (darker) and has more closely
arranged nuclei (i.e. 1s composed of smaller cells) than the
other. On the average this denser region appears to be
rather the less extensive of the two; the two regions may be
about equal ; on the other hand the denser may be the smaller.
Examination of stained preparations of the wall demonstrates
that the darker hemisphere is non-formative, the lighter,
formative. It would therefore seem that in certain of these
’04 vesicles the formative region has grown more rapidly than
the non-formative.

In stained preparations of the wall both of the ’99 and ’04
vesicles, the differences between the two hemispheres are now
so well marked that there is no difficulty in referring even an
isolated fragment to its proper region. The non-formative
hemisphere differs in no essential way from that of the ’01
vesicles, and as in these, is readily distinguishable from the
formative by the much greater uniformity in the size and
staining properties of its cells (fig. 45), as well as by the fact
that there are no primitive entodermal cells such as occur in
relation to the formative hemisphere, in connection with it.
Its constituent cells are on the average distinctly smaller than

56 ‘oP. y ai.

the largest of the formative ; their nuclei lie nearer each other,
with the result that in surface examination of the blastocyst
the non-formative region appears rather denser than the
formative. In in toto preparations of the wall the former
usually stains darker than the latter (fig. 45), but this is not
always the case; in fig. 46, from an ’04 vesicle, there is
practically no difference in this respect between the two
regions ; in yet others of my preparations of 799 vesicles the
formative region has stained more deeply than the non-
formative.

The formative hemisphere in the earlier blastocysts of this
particular developmental stage was described (ante, p. 51) as
differing from the non-formative in that its constituent cells
were much less uniform in character than those of the latter.
This same feature, but in much enhanced degree, characterises
the formative region of the vesicles under consideration, for it
can now be definitely stated that the latter region is consti-
tuted by cells of two distinct varieties, viz. (1) more lightly
staining cells which form the chief constituent of the forma-
tive region, its basis so to speak, and which are on the
average larger than those of the other variety, and (2), a less
numerous series of cells, distinctly smaller than the largest
cells of the former variety, and with denser, more granular and
more deeply staining cytoplasm, and frequently met with in
mitotic division (cf. Pl.6, fig. 65). The two varieties of cells are
intermingled promiscuously, the smaller cells occurring singly
and in groups but in a quite irregular fashion, so that here
and there we meet with patches of the wall composed exclu-
sively of the larger cells.

‘The evidence presently to be adduced shows that the larger
cells furnish the embryonal ectoderm, and that the smaller
cells give origin to the primitive entodermal cells from which
the definitive entoderm arises. The smaller cells may there-
fore be regarded as entodermal mother-cells. Whether these
latter cells are progressively formed from the larger cells
simply by division, orwhether the two varieties become
definitely differentiated from each other ata particular stage in

THE BARLY DEVELOPMENT OF THE MARSUPIALIA. 57

development, must for the present be left an open question. Of
the actual existence in the unilaminar formative region of these
°99 and ’04 blastocysts of two varieties of cells, respectively
ectodermal and entodermal in significance, there can be no
doubt. In preparations of the formative region, however,
whilst one can without hesitation identify certain cells as
being in all probability of ectodermal significance and others
as prospectively entodermal (cf. figs. 65, 66), it must be
admitted that one is often in doubt as to whether one is
dealing with small ectodermal cells or with genuine ento-
dermal mother-cells. It is, therefore, hardly to be wondered
at that I have not yet been able to satisfactorily determine
at what precise period the entodermal mother-cells first
become differentiated, though judging from the facts that
in the earliest vesicles in which the sutural line is recognis-
able one region of the wall already differs from the other in
the less uniform size of its constituent cells, and that internally
situated entodermal cells are already present in small numbers
in the ’01 vesicles (fig. 71), I incline to the belief that it
will probably be found to about coincide with the first
appearance of the sutural line. ‘To this question I may
perhaps be able to return at some future time.

In addition to the presence of these entodermal mother-
cells, which enter directly into its constitution, the formative
region of the ’99 and ’04 blastocysts is characterised by the
occurrence on its inner surface of definitely internal cells,
which generally agree with the former cells as regards size
and staining properties and are evidently related to them. It
is these internally situated cells which directly give origin to
the definitive entoderm of the later blastocysts, and one need,
therefore, have no hesitation in applying to them the designa-
tion of primitive entodermal cells. They are exclusively found
in relation to the formative hemisphere, and appear in in toto
preparations as flattened, darkly staining cells closely applied
to the inner surface of the unilaminar wall, and disposed quite
irregularly, singly, and in groups. ‘They vary greatly in
number in blastocysts of even the same batch, but on the

58 Je Py HILL.

whole are most abundant in the ’04 series, and they also
exhibit a remarkable range of variation in shape. They may
have a perfectly distinct oval or rounded outline (figs. 67, 71,
72), or, as is more frequently the case, they may lack a
determinate form and appear quite like amceboid cells owing
to their possession of cytoplasmic processes of markedly
pseudopodial-like character (fig. 69). Frequently, indeed,
the cells are connected together by the anastomosing of these
processes, so that we have formed in this way the beginnings
at least, of a cellular reticulum (figs. 68, 69, 70).

The question now arises, How do these primitive ento-
dermal cells originate from the small, darkly staining cells of
the unilaminar formative region designated in the foregoing
as the entodermal mother-cells ? I can find no evidence that
the primitive entodermal cells are formed by the division of
the mother-cells in planes tangential to the surface; on the
contrary, all the evidence shows that we have to do here with
an actual inward migration of the mother-cells, with or with-
out previous mitotic division, such inward migration being
the outcome of the assumption by the mother-cells, or their
division products, of amoeboid properties ; in other words, the
evidence shows that the formation of the entoderm is effected
here not by simple delamination (using that term in the sense in
which it was originally employed by Lankester), but by a pro-
cess involving the inward migration, with or without previous
division, of certain cells (entodermal mother-cells) of the uni-
laminar parent layer, a process comparable with that found in
certain Invertebrates (Hydroids) and distinguished by Metsch-
nikoff as “ gemischte Delamination.”

In this connection it has to be remembered that the cells of
the unilaminar wall of the blastocyst are under considerable
hydrostatic pressure, and, in correlation therewith, tend to
be tangentially flattened, though the flattening in this stage
is much less than in the earlier blastocysts. From a series of
measurements made from an ‘04 vesicle, I find that over the
formative region the ratio of the breadth to the thickness of
the cells varies from 6: 1 to 2: 1, and even to3:2. On the

THE KARLY DEVELOPMENT OF THE MARSUPIALIA. 59

whole cells of the type indicated by the ratio 6: 1 predominate,
and we should hardly expect to find such cells dividing tangen-
tially. In fact, the only undoubted examples of such division I
have met with occur in the single abnormal vesicle present in
the 04 batch. In this particular vesicle, which had a diameter
of 3 mm. and was thus smaller than the others, there was
present on what appeared to correspond to the formative
hemisphere of the normal blastocyst a well-defined and con-
spicuous ovalish patch, 1°23 x -99 mm.indiameter.! Sections
show that over this area the cells of the umlaminar wall are
much enlarged and more or less cubical in form, their thick-
ness varying from °012 to ‘019 mm. '‘I'hese cubical cells
exhibit distinct evidence of tangential division, both past and
in progress. But in normal vesicles, whilst mitotic figures are
quite commonly met with in the cells of the formative region
(in which, indeed, they are more numerous than in those of
the non-formative region), I have failed to find in my sections
after long-continued searching even a single spindle disposed
directly at right angles to the shell-membrane ; the mitotic
spindles le disposed either tangentially to the surface or
obliquely thereto.

For the determination of the mode of origin of the
primitive entodermal cells, it is absolutely necessary to
study both in toto preparations of the formative region,
1.e. small portions of the unilaminar wall stained and
mounted on the flat, and sections of the same. Sections alone
are, on the whole, distinctly disappointing so far as the
question under discussion is concerned, and, indeed, give one
an altogether inadequate idea of the primitive entodermal cells
themselves, seeing that practically all one can make out is that

1 Curiously enough, amongst the °99 vesicles there also occurred
a single small one, likewise 3mm. in diameter, and with a thickened
patch 1:28 x 1mm. in diameter, quite similar in its character to that
described in the text. I am as yet uncertain whether the thickened
area in these two vesicles represents the whole of the formative hemi-
sphere of normal blastocysts or only a hypertrophied part of the same,
or whether, indeed, it may not represent the retarded non-formative
hemisphere.

60 J. P.- HILL.

there are present, in close apposition with the inner surface of
the umilaminar wall, small, darkly staining cells, apparently
quite isolated from each other and usually of flattened form
(figs. 73, 74, 76, ent.). One has only to glance at a well-
stained in toto preparation of the formative region (cf.
fig. 70) to realise how inadequate such a description of the
primitive entoderm cells really is.

Sections nevertheless do yield valuable information on
certain points. Besides affording the negative evidence of
the absence of tangential divisions and the positive evidence
that the primitive entodermal cells are actually internal (figs.
73, 74, 76), they show that growth of the wall in thickness
has already set in, and that it is most marked over the
formative region, though the thickness attained by the cells
is as yet very unequal (figs. 75-76). Measurements taken
from an ?04 vesicle show that over the non-formative region
(fig. 77) the cells vary in thickness from ‘006 to ‘009 mm.,
whilst over the formative region the range of variation is
greater, viz. from ‘006 to ‘(013 mm., so that we may conclude
that the latter region is on the average thicker than the
former (cf. figs. 73-76, with fig. 77 depicting a small portion
of the non-formative region). It is still impossible to deter-
mine the position of the sutural line, even in sections of
fragments of the wall known to contain it.

The entodermal mother-cells are not very readily recog-
nisable in sections. In fig. 75, however, which is drawn
from an accurately transverse section through the formative
region of an ’O04 vesicle, there is depicted what is undoubtedly
an entodermal mother-cell (ent.). The interesting point
about this particular cell is that its cell-body, whilst still
intercalated between the adjoining cells of the unilaminar
wall, has extended inwards so as to directly underlie one of
the wall-cells. Division of such a cell as this would neces-
sarily result in the production of an internally situated cell
with all the relations of one of the primitive entodermal type.
The inwardly projecting spheroidal cell situated immediately
to the left (in the figure) of the one just referred to, I also

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 61

regard as an entodermal mother-cell. Cells of this type are
not infrequently met with in sections; they usually stain
somewhat deeply, and are often found in mitosis.

The evidence obtainable from the study of in toto pre-
parations conclusively proves that some at all events of the
primitive entodermal cells are actually derived from the ento-
dermal mother-cells much in the way suggested above, whilst
others of the primitive entodermal cells are directly formed
from mother-cells which bodily migrate inwards.

Fig. 65, Pl. 6, represents a small portion of the formative
region of an ’04 vesicle viewed from the inner surface. In
the centre of the figure, surrounded by the larger, lighter
staining (ectodermal) cells of the wall, is a smaller cell in the
telophases of division, the cytoplasm of which is granular and
stains deeply. That cell unmistakably forms a constituent of
the unilaminar wall. I regard it as an entodermal mother-
cell. Fig. 66 shows another cell of the same character in the
anaphases of division, which hkewise forms a constituent of
the unilaminar wall, but which differs from the corresponding
cell in fig. 65 in that its cytoplasmic body has extended out
on one side (lower in the figure), so as to directly underlie
part of an adjacent ectodermal cell. In other words we have
here a surface view of the condition represented in section in
fig. 75, only the entodermal mother-cell depicted therein is not
actually in process of division. Fig. 67, taken from the same
preparation as fig. 65, shows what I take to be the end result
of the division of such a cell as is represented in the two
preceding figures. Here we see two small deeply staining
cells towards the centre of the figure, which from their dis-
position and agreement in size and cytological characters
are manifestly sister-cells, and the products of division of
just such an entodermal mother-cell as is represented in fig.
65, or, better, fig. 66. The one cell (upper in the figure) is
more angular in form and manifestly still hes in the uni-
laminar wall ; the other (lower in the figure) is ovalish in form
and is no longer a constituent of the unilaminar wall, but is
on the contrary a free cell, definitely internal both to the

62 reitp) ML

—

latter and to its sister-cell. It is, in fact, a primitive ento-
dermal cell, as comparison with fig. 68 proves, and that it has
been formed by the division of a mother-cell situated in the
unilaminar wall can hardly, I think, be doubted. _ Its sister-
cell, which is still a constituent of the wall, would presumably
have migrated inwards some time later.

It is to be noted that the primitive entodermal cell referred
to above and those depicted in figs. 71 and 72 are definitely
contoured, ovalish and rounded cells, entirely devoid of pro-
cesses. In these respects they differ markedly from the ento-
dermal cells shown in figs. 68-70, which are very variable in
form owing to their possession of more or less elongated
pseudopodial-like processes. It might therefore be inferred
that the formation of these processes only takes place after
the entodermal cells have become definitely internal. Such
an inference, however, would be incorrect, for I have abundant
evidence showing that such processes may be given off from
the entodermal mother-cells whilst they are still constituents
of the wall. In in toto preparations, it is often difficult to
determine with certainty whether a particular entodermal cell
still enters into the constitution of the unilaminar wall or not.
In the portion of the formative region of a ’04 vesicle depicted
in fig. 70, however, I am satisfied that all the entodermal
cells therein shown (they are readily distinguishable by their
smaller size and more deeply staining character) are, with the
possible exception of the one on the extreme right, at least
partially intercalated between the larger ectodermal cells of
the wall. Some of them are entirely situated in the wall ;
others have extended inwards in varying degree so as to
partially underlie the ectodermal cells. It is these latter
entodermal cells in particular which exhibit the cytoplasmic
processes above referred to. As the figure shows, these pro-
cesses have all the characters of pseudopodia; they vary in
size, form, and number from cell to cell, individual processes
may be reticulate and their finer prolongations may anasto-
mose with those of others, and they are formed of cytoplasm,
less dense and rather less deeply staining than that of the

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 63

cell-bodies from which they arise. Attention may be specially
directed to the cell towards the left of the figure (marked ent.).
Here we have an entodermal cell whose cytoplasmic body is
evidently still partially intercalated between the cells of the wall,
but which is, at the same time, prolonged inwards (towards
the left) so as to underlie the adjoining ectodermal cell.
From this inward prolongation there are given off two slender
processes, one short and tapering, the other very much
longer ; this latter, after becoming very attenuated, gradually
widens to form an irregular fan-shaped expansion, sucker-
hke in appearance, and produced into several slender
threads, which is situated adjacent to the nucleus of
the ectodermal cell on the extreme left. Then from the
right side of the same cell there is given off a small inwardly
projecting bulbous lobe which may weil be the start of just
such another process as arises from the left side. Processes
of the peculiar sucker-lhke type just described, formed of a
slender elongated stem and a distal expanded extremity from
which delicate filamentous prolongations are given off, are
abundantly met with in preparations, and strikingly recall the
pseudopodia of various Rhizopoda. They are seen in con-
nection with other entodermal cells in fig. 70, and with many
of those in fig. 68. I regard them as veritable pseudopodia.
Towards the right side of fig. 70 the two entodermal cells
there situated stand in direct protoplasmic continuity by
means of two slender connecting threads, whilst the upper of
these two cells is again joined by a very fine process to the
irregular pseudopodial expansion which arises from one of
the two entodermal cells situated nearer the middle of the
figure, and that same expansion is directly connected with the
second of the two entodermal cells just mentioned, so that we
have here established the beginning of a cell-network, prior
to the complete emancipation of its constituent entodermal
elements from the unilaminar wall. We have, then, clear
evidence that the entodermal elements in Dasyurus, prior to
their separation from the unilaminar formative region are
capable of exhibiting amceboid activity, since not only may

64. 1 pe) eect 401

they send lobose prolongations of their cytoplasmic bodies
inwards below the adjacent ectodermal cells, but they may
emit more or less elongated processes of indubitable pseudo-
podial character, which similarly he in contact with the inner
surface of the wall-cells. Furthermore, we have evidence
that these pseudopodial processes may anastomose with each
other so as to initiate the formation of an entodermal reticulum,
whilst the cells from which they arise are still constituents of
the unilaminar wall—an especially noteworthy phenomenon.
Certain of the primitive entodermal cells, as we have seen,
are at first devoid of such processes, but since they all
eventually form part of a continuous reticulum, it is evident
that the entodermal elements are capable of emitting pseudo-
podial processes as well after as before their separation from
the formative region.

Finally, in view of the fact that the entodermal mother-cells
depicted in fig. 70 are not actually in process of division, and
therein differ from those of figs. 65 and 66, we may conclude
that the formation of the primitive entodermal cells is effected
either with or without the previous division of the mother-cells.

if we admit, as I think on the evidence we must admit,
that the entodermal cells in Dasyurus are endowed with
amoeboid properties, then we are relieved of any further
difficulty in regard to the mechanism of their inward migration
from the unilaminar wall. Doubtless, in the case of those
entodermal mother-cells which do not undergo division, the
precocious formation of the above-described pseudopodial
processes which spread out from the cells like so many
suckers considerably facilitates their direct detachment from
amongst the cells of the wall. In the case of those primitive
entodermal cells which originate as the direct products of
division of the mother-cells, it no doubt depends on a variety
of circumstances (e.g. actual form of the dividing cell,
direction of the spindle, etc.) whether they exhibit amceboid
activity precociously (i.e. before their actual separation), or
only at a later period.

The entoderm varies considerably in its degree of diffe-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 65

rentiation in different vesicles of this stage, and even in
different parts of the formative region of one and the same
vesicle. In some vesicles there are relatively few primitive
entodermal cells, in others they are much more abundant.
Fig. 68, from the formative region of an ’04 vesicle, shows a
typical patch of them and illustrates very well the highest
stage of differentiation which they attain in these vesicles. The
entodermal cells therein depicted all appear to be definitely
internal, and it is especially worthy of note that the portion
of the unilaminar wall in relation to them is composed exclu-
sively of the larger, lighter staining cells. It is these cells
which directly form the embryonal ectoderm of the blastocysts
next to be described. The entodermal cells are obviously
amceboid in character (observe especially the cells near the
middle of the figure), and are in active process of linking
themselves together into a cellular reticulum. In fig. 69 is
shown a small portion of the formative region of another ’04
vesicle. A single entodermal mother-cell in process of
division occurs in position in the unilaminar wall, which is
otherwise composed of ectodermal cells, whilst internally there
are present three entodermal cells, already linked together by
their pseudopodial processes. ‘The two lowermost cells afford
especially striking examples of amceboid activity, the elongated
pseudopodial process of the cell on the left terminating in a
well-marked reticulation in definite continuity with the corre-
sponding, but shorter and thicker process of the cell on the

right.

3. Establishment of the Definitive Embryonal
Area.

Following directly on the stage represented by the ’04
blastocysts described in the preceding section is one desig-
nated in my list as 5, 18.vii. Ol and referred to here as 5, ’Ol.
It comprises twenty-two blastocysts obtained from a female
killed fifteen days after coition and all normal, with the
exception of one which was shrivelled, and all in precisely

~

VOL, 56, PART 1.—NEW SERIES, 5)

66 J. P.- HILL.

the same stage of development. They measured from 4°5 to
6 mm. in diameter.

In this stage the formative region of the preceding. blasto-
cysts has become transformed into the definitive embryonal
area (embryonic shield, Hubrecht) as the result of the com-
pletion of that process of inward migration of the entodermal
mother-cells which we saw in progress in the vesicles last
described, and the consequent establishment of the entoderm
as a continuous cell-layer underlying and independent of. the
embryonal ectoderm constituted by the larger passive cells of
the original unilaminar formative layer.

In the entire blastocyst (Pl. 4, fig. 39) the embryonal area
is quite obvious to the naked eye as the more opaque, hemi-
spherical region, forming rather less than half the entire
extent of the vesicle wall; the larger remainder of the same
is formed by the much more transparent, non-formative or
extra-embryonal region. Sections of the entire blastocyst
show (1) that the embryonal area is bilaminar over its entire
extent, its outer layer consisting of embryonal ectoderm,
already somewhat thickened, its much thinner inner layer
consisting of entoderm, partly still in the form of a cellular
reticulum, and (2) that the extra-embryonal region is still
unilaminar throughout and composed of a relatively thin
layer of flattened cells (extra-embryonal or trophoblastic ecto-
derm, trophoblast [Hubrecht])1! (PI. 8, fig.78). The entoderm
is co-extensive at this stage with the embryonal ectoderm,
and terminates in a wavy, irregularly thickened, free edge
(Pl. 5, fig. 49), which over most of its extent either directly
underlies or extends very slightly beyond the line of junction
between the embryonal and extra-embryonal ectoderm. The
junctional line is thus not very easily seen. In fig. 48, however,

1 In consonance with my conviction that this layer is homologous
both with the so-called trophoblast of Eutheria and the extra-embryonal
ectoderm of Prototheria, and in view of the theoretical signification
which Hubrecht now insists should be attached to the term “ tropho-
blast.” and which I am wholly unable to accept, I venture to suggest as
an alternative name for this layer that of “ tropho-ectoderm. ’

THE EARLY DEVELOPMENT OF THE MARSUPIALIA, 67

a small portion of the line shows with sufficient distinctness, I
think, to demonstrate its ‘identity with that of the preceding
stage.

The vesicle wall in all my sections of this stage appears
to be somewhat thinner than that of the ’04 blastocysts, but
apart from this apparently variationai difference the present
blastocysts are almost exactly intermediate between the latter
and those next to be described.

The embryonal ectoderm (fig. 78, emb. ect.) appears in
section fairly uniformly thickened, though its cells are still of
the flattened type. In surface view in in toto preparations
(cf. fig. 48), they exhibit the same polygonal form and lightly
staining qualities as the larger cells of the formative region
of the ’04 blastocysts, which we have already identified as
prospective embryonal ectodermal cells. The junctional line
between the embryonal ectoderm and the extra-embryonal is
now for the first time readily distinguishable in sections
(fig. 78). The extra-embryonal ectoderm (tropho-ectoderm)
(Pl. 5, figs. 48 and 49, Pl. 8, fig. 78, tr. ect.) differs in no
essential respect from the corresponding layer in the ’04.
blastocysts.

The entoderm in these blastocysts is exceedingly closely
adherent to the inner surface of the embryonal ectoderm and
cannot be removed therefrom by artificial means. It varies
slightly in its character in different vesicles and in different
parts of its extent in the same vesicle. Mostly it appears as
a continuous thin cell-layer (figs. 49 and 78, ent.), but here and
there patches occur in which the cells form a reticulum quite
similar to that shown in fig. 68 of the preceding stage.

The next stage (designated in my list as 8.vi.01), and the
last of Dasyurus that need be described in the present com-
munication, comprises eleven vesicles (5—5°5 mm. in diameter),
in which the embryonal area is conspicuous and distinctly in
advance of that of the preceding vesicles, but is still devoid
of any trace of embryonal differentiation (Pl. 4, fig. 40;
PIS: 79):

The embryonal area is hemispherical in form (its greatest

68 J.P, HIG:

diameter varying from 3°5 to 4 mm.) in all except two of the
blastocysts, in which it is elongate, with longer and shorter
diameters. It occupies about a third or less of the entire
extent of the vesicle wall, and thus appears relatively smaller
than that of the preceding (5, 01) vesicles. The entoderm now
extends for a distance of about 1 mm. beyond the limits of
the area, so that in the entire vesicle (fig. 40) three zones
differing in opacity are distinguishable, viz. the dense hemi-
spherical zone at the upper pole, constituted by the embryonal
area; below that, a less dense, narrow annular zone, formed of
extra-embryonal ectoderm and the underlying peripheral
extension of the entoderm; and finally, the still less dense
hemispherical area, forming the lower hemisphere of the
blastocyst and constituted solely by extra-embryonal ecto-
derm. ‘Thus approximately the upper half of the blastocyst
is bilaminar, the lower half unilaminar. Sections show that
the embryonal ectoderm (fig. 79, emb. ect.) is now a quite
thick layer of approximately cubical cells, whilst the extra-
embryonal ectoderm (tr. ect.) is formed of relatively thin
flattened cells. The line of junction between the two is per-
fectly obvious, both in sections (fig. 79) and in surface view
(Pl. 5, fig. 50). The embryonal ectodermal cells, though
much thicker than the extra-embryonal, are of less superficial
extent; their nuclei therefore lie closer together than those
of the Jatter, moreover they are larger, stain more deeply, and
are more frequently found in division, all of which facts
testify to the much greater growth-activity of the embryonal
as compared with the extra-embryonal ectoderm at this stage
of development (cf. fig. 50, emb. ect. and tr. ect.; in the prepara-
tion from which this micro-photograph was made the entoderm
underlying the embryonal ectoderm has been removed, whilst
it is still partially present over the extra-embryonal ectoderm).

The entoderm (fig. 79, ent.) over the region of the em-
bryonal area is readily separable as a quite thin membrane,
and is then seen to consist of squamous cells, polygonal in
outline, and either in direct apposition by their edges or con-
nected together by minute cytoplasmic processes. Beyond the

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 69

embryonal area, however, its peripheral extension below the
extra-embryonal ectoderm is much less easily separable in the
intact condition (cf. fig. 50), because of its greater delicacy
due to the fact that it has here largely the form of a cellular
reticulum. In this extra-embryonal region the entodermal
cells are frequently found in mitosis. It would appear, then,
that the entoderm is first laid down in the region of the em-
bryonal area as a cellular reticulum, which later becomes
transformed into a continuous cell-membrane, and that its
peripheral extension over the inner surface of the extra-
embryonal ectoderm is the result of the growth and activity
of its own constituent cells.

This peripheral growth continues until there is formed
eventually a complete entodermal lining to the blastocyst
cavity. ‘’he rate of growth appears to be somewhat variable.
In a series of primitive streak vesicles (6-6°75 mm. in diameter)
the lower third of the wall is, I find, still unilaminar. In
another series of vesicles of the same developmental stage
(4-5-6 mm. in diameter) a unilaminar area is present at the
Jower pole, varying from 1 x 5 mm. in diameter to as much
as 4 mm. Lven in vesicles 7-7°5 mm. in diameter a uni-
laminar patch may still occur at the lower pole, but in vesicles
85 mm. in diameter (stage of flat embryo) the entodermal
lining appears always to be complete.

The Origin of the Entoderm in Kutheria,— The
remarkable facts relative to the origin of the entoderm in
Dasyurus which I have been able to place on record in the
preceding pages, thanks to the large size attained by the
blastocyst prior to the differentiation of the formative germ-
layers and to the circumstance that the formative cells are
not arranged, as they are in Eutheria, in the form of a more
or less compact cell-mass, but constitute a thin unilaminar
cell-layer of relatively great extent which can easily be cut
up with scissors, and which, after staining and mounting on
the flat can be examined under the highest powers, throw, it
seems to me, a new and unexpected light on the mammalian
entoderm, and at the same time help to fill the considerable

70 7. oP, SAG.

gap which has hitherto existed in our knowledge of its early
ontogenesis. Although the mode of origin of the entoderm
in Dasyurus would appear, in the present state of our know-
ledge, to find its closest parallel, not amongst vertebrates, but
in certain invertebrates (cf. the mode of origin of the ento-
dermal cells from the wall of the blastula in Hydra as
described by Brauer!), the observations of Assheton (94)
on the early history of the entoderm in the rabbit, when
viewed in the light of the foregoing, seem to me to afford
ground for the belief that phenomena comparable with those
here recorded for Dasyurus will eventually be recognised as
occurring also in Kutheria,

Hubrecht (’08), in his recent treatise on early Mammalian
ontogeny, deals very briefly with the question of the origin
of the entoderm in the latter group, merely stating that
“from the inner cell-mass arises by delamination a separate
lower layer which we designate as the entoderm of the
embryo. These entoderm cells wander in radial direction
along the inner surface of the trophoblast, which in many
cases 1s thus soon transformed into a didermic structure.

When the entoderm has separated off by delamina-
tion from the embryonic knob, the remaining cells of the
latter form the ‘embryonic ectoderm,’ which is thus situated
between the entoderm and the trophoblast.”

Assheton, in the paper just referred to, has given a careful
account of the first appearance of the entodermal cells in the
rabbit, and of what he believes to be the mode of their
peripheral extension below the trophoblastic wall of the
blastocyst. He shows that the inner cell-mass, at first
spherical, gradually, as the blastocyst enlarges, flattens out
below the “ covering layer” of the trophoblast until it. forms
an approximately circular plate “nowhere more than two
cells thick.” During the process of flattening, cells are seen
to jut out from the periphery of the mass; these eventualiy
separate, and appear as rounded cells scattered irregularly
over the inner surface of the trophoblast and “extending

1 * Zeitschr. f. wiss. Zool.,’ Bd. lii, 1891,

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 71

over an arc of about 60° from the upper pole in all directions.”
These “straggling” cells, as Assheton terms them, as well as
the innermost cells of the now flattened inner cell-mass, are
regarded as hypoblastic and the outermost cells of the same
as epiblastic (embryonic epiblast). “The hypoblast, as a
perfectly definite layer, is formed by the time the blasto-
dermic vesicle measures *5 mm, in diameter, that is, about the
102nd hour after coition. It is not, however, as yet by any
means a continuous membrane ; it is a network or fenestrated
membrane. For this reason, in. section it appears to be
represented by isolated cells lying beneath the embryonic
disc (v. fig. 29, Hy.)” (cf. Dasyurus). In considering the
question how the peripherally situated (“straggling ”’) ento-
dermai cells, which are undoubtedly derived trom the inner
cell-mass, “‘ apparently wander round the inside of the blasto-
dermic vesicle,’ he reaches the conclusion that this is not the
result of amceboid activity or growth “in the sense of migra-
tion” on the part of these cells, but ‘is only an apparent
growth round produced by the more rapid growth of a
zone of the [trephoblastic] wall of the vesicle immediately
surrounding the embryonic disc, in which zone the marginal
cells of the inner mass le.’ He is unable to find any
evidence of the production of pseudopodial processes by
these peripheral entodermal cells, the majority of them
appearing at first to be quite isolated from each other and
approximately spherical. “Certain of the cells here and
there are connected by threads of protoplasm, but this, I
think, is not a sign of pseudopodic activity, but merely
indicates the final stage in division between the two cells.”
By the sixth day the hypoblast of the embryonic disc has
assumed the form of a continuous membrane, composed of
completely flattened cells, whilst the peripheral hypoblast
cells have become more numerous, and ‘“‘many of them,
possibly all of them, are now undoubtedly connected by more
or less fine protoplasmic threads.” Such, in brief, is
Assheton’s account of the early history of the entoderm in
the rabbit; it presents obvious points of agreement with wy

72 ; 7. iP; “HOLL:

own for Dasyurus, and I venture to think the agreement is
even greater than would appear from Assheton’s conclusions.
In adopting the view that the more. active growth of the
region of the blastocyst wall immediately surrounding the
inner cell-mass is the sole causal agent in effecting the separa-
tion and peripheral spreading of the entodermal cells, I cannot
but feel, in view of his ewn description and figures and of my
own results, that he has attributed a much too exclusive import-
ance to that phenomenon and a much too passive role to the
entodermal cells themselves. In Dasyurus the inward migra-
tion and the later peripheral spreading of the entodermal
cells is effected without any such marked unequal. growth of
the blastocyst wall as occurs, according to Assheton, in the
rabbit, as the direct outcome of their own inherent activity,
and I believe the possession of a like activity characterises
the entodermal cells of the rabbit. The evidence of Assheton’s
own fig. 40, which shows in surface view a portion of the
vesicle wall with the peripheral entodermal cells in relation
thereto, and which should be compared with my figs. 68 and
69, conclusively demonstrates, to my mind, the possession by
these cells of amceboid properties, and thus support is
afforded for the belief that the separation of the entodermal
cells from the formative cell group (inner cell-mass) is here
also the expression of an actual migration. Whether or not
the strands of protoplasm which Assheton (’08, 09) describes
as present in the sheep, pig, ferret, and goat, connecting the
inner lining of the inner mass to the wall of the blastocyst,
and which he interprets as tending “‘ to show that the inner
lining of the inner mass is of common origin with the wall of
the blastocyst,” are of any significance in the present connec-
tion, I cannot certainly determine.

4, Summary.
: ; ;
The results and conclusions set forth in the preceding
pages of this chapter may be summarised as follows:
* As ale
(1) The unilaminar wall of the blastocyst of Dasyurus con-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 73
sists of two regions distinct in origin and in destiny, viz. an
upper or formative region, derived from the upper cell-ring
of the 16-celled stage, and destined to furnish the em-
bryonal ectoderm and the entoderm and a lower or non-
formative region derived from the lower cell-ring of the
mentioned stage, and destined to form directly the extra-
embryonal or trophoblastic ectoderm (tropho-ectoderm) of the
bilaminar vesicle.

(2) The formative region, unlike the non-formative, is
constituted by cells of two varieties, viz.: (i) a more
numerous series of larger, lighter-staining cells destined
to form the embryonal ectoderm, and (11) a less numerous
series of smaller, more granular, and more deeply staining
cells, destined to give origin to the entoderm and hence
distinguishable as the entodermal mother-cells.

(3) The entodermal mother-cells, either without or subse-
quently to division, bodily migrate inwards from amongst the
larger cells of the unilaminar wall and so come to he in
contact with the inner surface of the latter. ‘hey thus give
origin to the primitive entodermal cells from which the
definitive entoderm arises. ‘The larger passive cells, which
alone form the unilaminar wall after the inward migration of
the entodermal cells is completed, constitute the embryonal
ectoderm.

(4) he entodermal cells as well before as after their
migration from the unilaminar wall are capable of exhibiting
amceboid activity and of emitting pseudopodial processes, by
the anastomosing of which there is eventually formed a
cellular entodermal reticulum underlying, and at first co-
extensive with, the embryonal ectoderm.

(5) The definitive entoderm thus owes its character as a
connected cell-layer primarily to the formation of secondary
anastomoses between the pseudopodial processes emitted by
the primitive entodermal cells (or entodermal mother-
cells).

(6) The assumption by the entodermal cells of amoeboid
properties whilst they are still constituents of the unilaminar

74 Bs TGs

wall affords an intelligible explanation of the mechanism of
their inward migration.

(7) The entoderm is first laid down below the formative or
embryonal region of the blastocyst; thence it extends gradu-
ally by its own growth round the inner surface of the uni-
laminar non-formative region so as to form eventually a
complete entodermal lining to the blastocyst cavity. In this
way the blastocyst wall becomes bilaminar throughout.

(8) The bilaminar blastocyst consists of two regions, respec-
tively embryonal and extra-embryonal. ‘The embryonal
region (embryonal area) is constituted by an outer layer of
embryonal ectoderm and the underlying portion of the ento-
derm, and the extra-embryonal, of the extra-embryonal or
trophoblastic ectoderm (tropho-ectoderm), which is separated
from the embryonal by a well-marked junctional line, together
with the underlying portion of the entoderm, which is per-
feetly continuous with that below the embryonal ectoderm.

(9) The formative or embryonal region of the blastocyst
in Dasyurus is from the first freely exposed, and at no time
during the developmental period dealt with in this paper
does there exist any cellular layer externally to it, i.e. a
covering layer of trophoblast (Deckschicht, Rauber’s layer)
is absent and there is no entypy of the primary germ-layers

(ene p. tit).

CHarrer V.—Somre Harty Sraces or PERAMELES AND
Macropus.

The early material of Perameles and Macropus at my
disposal comprises only a small number of stages, but is of
special importance, since it enables me to demonstrate that
so far as these particular stages are concerned, the early
developmental phenomena in these forms are essentially the
same as in Dasyurus, and thus affords ground for the belief
that there is one common type of early development through-
out the series of the Marsupialia. Moreover, it is of interest
since it reveals the existence of what might be termed

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 75

specific differences in the early development of these Marsu-
pials, especially in regard to the time of appearance of the
entoderm. In Dasyurus, it will be remembered, the primitive
entoderm cells first become definitely recognisable as inter-
nally situated cells in vesicles 4°5 mm, in diameter. In
Perameles they occur in vesicles just over 1 mm. in diameter,
while in Macropus they are already present in a blastocyst
only *35 mm. in diameter, so that it would appear that the
entoderm is differentiated much earlier in the higher, more
specialised types than in the more generalised forms. ‘This
difference in time of appearance of the entoderm is perhaps
to be correlated with a difference in size of the ovarian ova
in the three genera mentioned.

1. Perameles.

The earliest material of Perameles I possess consists of two
egos of P. obesula, which | owe to the skill and enthusiasm
of my friend Mr. S. J. M. Moreau, of Sydney. Egg a
measures ‘23 mm. in diameter, and egg B, ‘24 x °23 mm.
The former consists of thirty-two cells, the latter of thirty. In
both the shell-membrane has partially collapsed, but the general
plan of arrangement of the blastomeres can still fairly readily
be made out. Fig. 51, Pl. 8, represents a micro-photograph
of a section of ege B, the better of the two. It shows the
shell-membrane (nearly °005 mm. thick) externally, con-
siderable remains of the albumen between that and the
deeply stained zona, and then, closely applied to the inner
surface of the latter, the blastomeres arranged in the form of
an inverted fl, so as to enclose a central space, open below
as the figure stands. This latter opening extends through
the series, and it seems probable that there was a corres-
ponding one opposite to it in the intact egg. Evidently we
have here a stage in the formation of the blastocyst, in which
the blastomeres are in course of spreading towards one or
both of the poles of the sphere formed by the egg-envelopes,

76 7p. VE

just as happens in the corresponding stage of Dasyurus (cf.
fig. 51 with fig. 31, though the latter represents a somewhat
older stage in Dasyurus). ‘lhe blastocyst-wall here appears
relatively more extensive than in the 32-celled stage of
Dasyurus, an apparent difference which may perhaps be ac-
counted for by the difference in size of the respective eggs
(24 mm. as compared with ‘36 mm.). The blastomeres situated
adjacent to the opening and those on the right side of the
figure tend to be more flattened and of greater super-
ficial extent than the remainder, but I can recognise no
difference in the cytological characters of the cells. The
space or cleavage cavity enclosed by the blastomeres is partly
occupied by a granular coagulum, and towards the opening
there is present a lightly staining reticular mass, which
recalls the yolk-body of Dasyurus, though I am not prepared
to affirm that it is of that significance. The fixation of the
specimen is not quite perfect.

My next stage of Perameles is constituted by a blastocyst
of P.nasuta, for which Iam again indebted to Mr. Moreau
measuring in the preserved condition *29 x ‘26mm. Fig. 52,
Pl. 3, shows a section of this blastocyst. Structurally,
it corresponds in all essential respects with the “43 mm.
blastocyst of Dasyurus, figured on the same plate (fig. 33).
The blastocyst wall is complete and unilaminar throughout.
It is distinguishable into two regions, a more extensive region
over which the cells are large and flattened and a less extensive,
composed of smaller but thicker cells (left side of fig. 52).
In the early blastocysts of Dasyurus, it may be recalled, the
evidence showed that the region of more flattened cells is
formative in significance, that of more bulky cells, non-forma-
tive. It is possible the same holds good for this Perameles
blastocyst. On the other hand, the structural condition of
the stage next to be described rather supports the view that
the smaller region, composed of plumper cells, is in this case
formative. ‘l'hat view seems to me the more probable of the
two, but there is a considerable difference in size between the
present blastocyst and those next available, so that it is

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. YAP

impossible to decide this point with certainty. The blasto-
cyst cavity is partly occupied by coagulum. ‘There are no
cells present in it, but the question of the presence of a yolk-
body must remain open. ‘The shell-membrane (‘0045 mm, in
thickness) and zona are in close apposition.

Following this early blastocyst, I have three vesicles of
P. nasuta, two of them measuring 1:3 mm. in diameter,
the other 1:1 mm. In their stage of development they
agree pretty closely with the 4-5-5 mm. vesicles of Dasyurus,
referred to in the preceding pages under the designation
6, ?04, the entoderm being in process of differentiation. The
formative region was readily distinguishable in the intact
vesicles as a darker patch occupying about three eighths of
the surface extent of the wall. in section (PI. 8, figs. 80, 81)
it is characterised by its greater thickness as compared with
the non-formative or trophoblastic region, and by the
presence below it of numbers of primitive entodermal cells.
Compared with the corresponding stage in Dasyurus, the
chief difference consists in the relatively much greater thick-
ness of the cells of the formative region in the Perameles
vesicle. ‘lhe latter cells are here already more or less defi-
nitely cubical in shape, their thickness varying from ‘09
mm. to ‘015 mm., and altogether they form a layer of amuch
more uniformly thickened character than that of the 6, ’04
vesicles of Dasyurus. The trophoblastic ectoderm (figs. 80,
81, tr. ect.) is composed of somewhat flattened cells, varying
in thickness from ‘005 to ‘008 mm.

The primitive entodermal cells (figs. 80, 81, ent.) are
present below the formative region in fair abundance, more
especially around the periphery of the same, which may thus
appear somewhat thickened (fig. 81). ‘The cells vary in size
from ‘01 x ‘007 mm. to 024 x -009 mm.,and they stain on the
whole somewhat more deeply than the formative cells, to
whose under-surface they are closely applied. They occur
singly and in groups. Mitotic figures are frequently met
with in the cells of the formative area (observe the obliquely
disposed figure in one of the formative cells in fig. 81), and

78 Teepe elie

they also occur in the primitive entodermal cells. Hxamina-
tion of the sections leaves no doubt in one’s mind as to the
source of the entodermal cells. They are undoubtedly derived
from the formative region of the vesicle wall. The shell-
membrane has a thickness of about ‘0027 mm.

2 Maeno pits,

Of Macropus the earliest stage I have examined is a blasto-
cyst of M. ruficollis, 25 x -21 mm. in diameter. It is not
in a quite perfect state of preservation, but is in a sufficiently
good condition to enable me to say that the wall is complete
and unilaminar throughout, just as in the *29 x ‘26 mm.
blastocyst of Perameles. The shell-membrane has a thickness
of about ‘005 mm., and there are still remains of the albumen
between it and the zona.

My next stage (figs. 82-85) is a blastocyst of the same
species, “35 mm. in diameter. It unfortunately suffered in
preparation, but practically the whole of the formative area
of the blastocyst wall and part of the trophoblastic ectoderm
are comprised in the sections (Pl. 9, fig. 82), so that it is still
possible to make out its chief structural features. In its stage
of development this blastocyst closely agrees with the last
described blastocysts of Perameles. The formative area of
the wall is perfectly distinct in the sections because of its
greater thickness and the presence below it of the primitive
entodermal cells. It attains its greatest thickness (027 mm.)
peripherally, whilst it is thinnest centrally (‘006 mm.), so that,
taken as a whole, it is not quite such a uniformly thickened
layer as is that of the Perameles blastocysts. Primitive ento-
dermal cells are present below it, but not in great abundance
(figs. 82, 84, 85, ent.). In fig. 83, a formative cell is seen in
division, the axis of the spindle being oblique to the surface.
The trophoblastic ectoderm (figs. 82, 83, tr. ect.) is composed
of the usual flattened cells, and varies in thickness from
005 to ‘0067 mm.

In the blastocyst cavity, adjacent to the trophoblastic

a

THE EARLY DEVELOPMEN'T OF THE MARSUPIALIA. 79

ectoderm on the left side of fig. 82, there is visible a small
spherical cell similar to the degenerate cells met with in
blastocysts of Dasyurus.

My last stage of M. ruficollis comprises an excellently
preserved blastocyst, measuring ‘8 mm. in diameter, in which
the embryonal ectoderm and the entoderm are definitely
established. It thus corresponds to the 8, ’01 stage of
Dasyurus (blastocysts 5-5°5 mm. diameter). The embryonal
area is circular and measures *468 mm. in diameter. Its
constituent cells are cubicai and from ‘008 to 013 mm. in
thickness, whilst the trophoblastic ectoderm is formed of
flattened cells, °(006 mm. in thickness. The entoderm is
present as a continuous layer of attenuated cells below the
embryonal ectoderm, and it probably also forms a continuous
layer below the trophoblastic ectoderm. Entodermal cells are
certainly present over the lower polar region of the vesicle,
but itis difficult to be certain from the sections whether or not
they form a perfectly continuous layer. The shell membrane
has a thickness of ‘0026 mm.

I have a corresponding blastocyst of Petrogale peni-
cillata ‘915 mm. in diameter, with an oval, embryonal area
525 x ‘45 mm. in diameter, and a later blastocyst of M.
ruficollis 1:46 mm. in diameter, with a circular embryonal
area ‘O/7 mm. in diameter.

Cuaprer VI.—GeneraL Summary AND ConcLuUSIONS.

The observations recorded in the preceding pages and the
conclusions deducible therefrom may be summarised as
follows :

(A) Ovum.—The uterine ovum of Dasyurus is characterised
(1) by its large size relatively to those of HKutheria; (2) by
the presence externally to the zona of a layer of albumen and
a shell-membrane, both laid down in the Fallopian tube and
homologous with the corresponding structures in the Mono-
treme ovum, the shell-membrane, like the shell of the latter,
increasing in thickness in the uterus; (3) by its marked

80 J. Ps. HlbL.

polarity, its lower two thirds consisting of formative cyto-
plasm, dense and finely granular in appearance, owing to the
presence of fairly uniformly distributed deutoplasmic material,
and containing the two pronuclei, its upper third being
relatively clear and transparent, consisting as it does of a
delicate reticulum of non-formative cytoplasm, the meshes of
which are oceupied by a clear deutoplasmic fluid. Study of
the process of vitellogenesis in ovarian ova demonstrates that
this fluid represents surplus deutoplasmic material which has
not been utilised in the upbuilding of the formative region of
the ovum.

The fate of the clear non-formative portion of the ovum is
avery remarkable one. Prior to the completion of the first
cleavage, it is separated off from the formative remainder of
the ovum as a spherical mass or yolk-body, which takes no
direct part in development, though it becomes enclosed in the
blastocyst cavity on completion of the blastocyst wall at the
upper pole. Its contained deutoplasmic fluid is to be regarded
as the product of an abortive attempt at the formation of a
solid yolk-mass, such as is found in the Monotreme ovum.
By its elimination the potentially yolk-laden telolecithal ovum
becomes converted into a secondarily homolecithal, holoblastic
one. All the evidence is held to support the conclusion that
the Marsupials are descended from oviparous ancestors with
ineroblastic ova.

(sp) Cleavage.—Cleavage begins in the uterus, is total, and
at first equal and of the radial type. The first two cleavage
planes are meridional and at right angles to each other.
The resulting four equal-sized blastomeres lie disposed radially
around the polar diameter like those of the Monotreme (not
in pairs at right angles to each other as in Kutheria), and
enclose » segmentation cavity open above and below, their
upper ends partially surrounding the yolk-body. The third
cleavage planes are again meridional, each of the four blasto-
meres becoming subdivided equally into two. The resulting
eight cells form an equatorial ring in contact with the inner
surface of the sphere formed by the egg-envelopes. They

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 81

contain deutoplasmic material, which is, however, located
mainly in their lower halves. ‘The ensuing fourth cleavages
are equatorial, and in correlation with the just-mentioned
disposition of the deutoplasm, are unequal and qualitative,
each of the eight blastomeres becoming subdivided into an
upper smaller and clearer cell, with relatively little deuto-
plasm fairly uniformly dispersed through the cytoplasm, and
a lower larger, more opaque cell with much deutoplasm,
mainly located in a broad zone in the outer portion of the
cell-body. A 16-celled stage is thus produced in which the
blastomeres are characteristically arranged in two super-
imposed rings, each of eight cells, an upper of smaller, clearer
cells next the yolk-body, and a lower of larger, denser cells.
The former is destined to give origin to the formative or
embryonal region of the blastocyst wall, the latter to the
non-formative or extra-embryonal region of the same.

(c) Formation of the Blastocyst.—There is in the
Marsupial no morula stage as in Hutheria, the blastomeres
proceeding directly to form the wall of the blastocyst. ‘The
cells of the two rings of the 16-celled stage divide at first
meridionally and then also equatorially, the division planes
being always vertical to the surface. The daughter-blasto-
meres so produced, continuing to divide in the same fashion,
gradually spread towards opposite poles in contact with
the inner surface of the firm sphere formed by the zona and
the thickened shell-membrane. Eventually they form a com-
plete cellular lining to the said sphere and it is this which
constitutes the wall of the blastocyst. The latter is accord-
ingly unilaminar at its first origin, and it remains so in
Dasyurus until it has attained, as the result of active growth
accompanied by the imbibition of fluid from the uterus, a
diameter of 4-5 mm. It consists of two parts or regions,
distinct in origin and in destiny, and clearly marked off from
each other in later blastocysts by a definite junctional line
approximately equatorial in position, viz. an upper, embryonal
or formative region derived from the upper cell-ring of the
16-celled stage, and a lower, extra-embryonal or non-

VOL. 56, PART 1.—NEW SERIES, 6

82 j. P>. AGG:

formative region derived from the lower cell-ring of the same
stage.

(p) Later History of the Two Regions of the Blasto-
cyst Wall (for details see pp. 72-74).— From the embryonal
region are derived the embryonal ectoderm and the entire
entoderm of the vesicle. I conclude, therefore, that it is the
homologue of the inner cell-mass or embryonal knot of the
Eutherian blastocyst. ‘The extra-embryonal region directly
furnishes the outer extra-embryonal layer of the vesicle wall,
i.e. the outer layer of the omphalopleure and chorion of later
stages. Assuming, as the facts of comparative anatomy and
palwontology entirely justify us in doing, that the Mammals
are monophyletic and of reptilian origin, and further assuming
that the foetal membranes are homologous structures through-
out the Amniotan series (also in my view a _ perfectly
justifiable assumption)’, then the homologies of this extra-
embryonal region of the Marsupial blastocyt are not far to
seek. It is clearly the homologue of the extra-embryonal
ectoderm of the Sauropsidan and Monotreme egg, and the
homologue also of the outer enveloping layer of the Kutherian
blastocyst, to which Hubrecht has given the special name of
“ trophoblast.”?. In my view the trophoblast is none other
than extra-embryonal ectoderm which in the viviparous
mammals, in correlation with the intra-uterine mode of
development, has acquired a special significance for the
nutrition of the embryo.

These, then, are my conclusions, and to me they seem on
general grounds perfectly obvious, viz.: (1) that the em-
bryonal or formative region of the unilaminar Marsupial
blastocyst is the homologue of the inner cell-mass or

1 How Assheton can maintain (09, p. 266) “that the amnion of the
rabbit is not more homologous to the amnion of the Sauropsidan than
the horny teeth of Ornithorhynchus are homologous to the true teeth
of the mammal or reptile, which they have supplanted,’ how he can
hold this view and yet proceed to utilise the presence of the amnion as
one of the leading characters distinguishing the Amniota from the’

Anamnia, I fail to comprehend. Surely the presence of a series of
purely analogous structures in a group is of no classificatory value.

85

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THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 895

embryonal knot of the Eutherian blastocyst ; and (2) that the
extra-embryonal or non-formative region of the same is the
homologue of the extra-embryonal ectoderm of the Saurop-
sida and Monotremata and of the trophoblast of the
Eutheria.

As regards conclusion (1) there is not likely to be much
difference of opinion, but as regards (2), whilst perhaps the
majority of embryologists support the obvious, not to say
commou-place view which I here advocate, it seems certain
that it will prove neither obvious nor acceptable to those
mammahan embryologists (I refer specifically to my friends
Professor A. A. W. Hubrecht and Mr. R. Assheton) who, with
only Selenka’s account of early Marsupial ontogeny before
them, have formulated other and quite divergent views as to
the morphological nature of the outer enveloping layer of the
Kutherian blastocyst. It is therefore necessary to discuss
this question further, though I would fain express my convic-
tion that had the observations recorded in this paper been
earlier available, much vain speculation as to the phylogeny
of the trophoblast might possibly have been avoided.

CHaprer VII.—l'ne Earty Ontogeny or THE MAMMALIA IN
THE LigHr oF THE FOREGOING OBSERVATIONS.

In entering on a discussion of the bearings of the results
of my study of the early development of Marsupials on
current interpretations of early Mammalian ontogeny, and
especially of the homologies ot the germ-layers, I desire at
the outset to emphasise my conviction that, specialised
though the Marsupials undoubtedly are in certain features of
their anatomy, e. g. their dentition, genital ducts, and mam-
mary apparatus, the observations recorded in the preceding
pages of this paper afford not the slightest ground for the
supposition that their early ontogeny is also of an aberrant
type, devoid of significance from the point of view of that of
other mammals. On the contrary, | hope to demonstrate
that the Marsupial type of early development not only readily

&6 , J. P. HILL.

falls into line with that of Eutheria, and with what we know
of the early development of the Prototheria, but furnishes
us with the key to the correct interpretation of that extra-
ordinarily specialised developmental stage, the Hutherian
blastocyst. In particular I hope to show that the description
which I have been able to give of the mode of formation of
the Marsupial blastocyst, bridges in the most satisfactory
fashion the great gap which has till now existed in our
knowledge of the way in which the transition from the
Monotrematous to the Eutherian type of development has
been effected.

1. The Early Development of the Monotremata.

Our knowledge of the early development of the oviparous
mammals is admittedly still far from complete. Nevertheless
it is not so absolutely fragmentary that it can be passed over
in any general discussion of early mammalian ontogeny, and
I certainly cannot agree with the opinion of Assheton (’08,
p- 227) that from it “we gain very little help towards the
elucidation of Eutherian development.’ On the contrary, I
think that the combined observations of Semon (794), and
Wilson and Hill (07) shed most valuable light on the early
ontogenetic phenomena in both the Metatheria and Kutheria,
I propose therefore to give here a very brief resumé of the
chief results of these observers,! and at the same time to
indicate how the knowledge of early Monotreme ontogeny
we possess, limited though it be, does help us to a_ better
understanding of the phenomena to which I have just
referred.

The ovum, as is well known from the observations of
Caldwell (87), is Reptilian in its character in all but size.
It is yolk-laden and telolecithal, the yolk consisting of
discrete yolk-spheres, and it is enclosed outside the zona
(vitelline membrane) by a layer of albumen and a definite shell.

‘ In so doing I have largely utilised the phraseology of Wilson and
Hill’s paper (07).

THE EARLY DEVELOPMENT. OF THE MARSUPIALIA. 87

At the moment of entering the oviduct it has a diameter of
3°5-4 mm. (2°5-3 mm. according to Caldwell), and is therefore
small relatively to that of a reptile of the same size as the
adult Monotreme, but large relatively to those of other
mammals, being about twelve times larger than that of
Dasyurus, and about eighteen times larger than that of the
rabbit.

Cleavage is meroblastic. The first two cleavage planes are
at right angles to each other, as in the Marsupial, and divide
the germinal disc into four approximately equal-sized cells
(Semon, ‘laf. ix, fig. 30). Hach of these then becomes sub-
divided by a meridional furrow into two, so that an 8-celled
stage is produced, the blastomeres being arranged symmetri-
cally, or almost symmetrically, on either side of a median line,
perhaps corresponding to the primary furrow (Wilson and Hill,
p. 37, text-figs. land 2). Imagine the yolk removed and the
blastomeres arranged radially, and we have at once the open
ring-shaped 8-celled stage of Dasyurus. The details of the
succeeding cleavages are unknown. Semon has described a
stage of about twenty-four cells (Semon, Taf. ix, fig.31),in which
the latter formed a one-layered circular plate with no evidence
of bilateral symmetry, and this is succeeded by a stage also
figured by Semon (figs. 82 and 33, cf. also Wilson and Hill,
Pl. 2, fig. 2), in which the blastoderm has become several
cells thick, though it has not yet increased in surface extent.
It is bi-convex lens-shaped in section, its lower surface being
sharply limited from the underlying white yolk. No nuclei
are recognisable in the latter, either in this or any subsequent
stage, nor is there ever any trace of a syncytial germ-wall,
features in which the Monotreme egg differs from the
Sauropsidan.

The next available stage, represented by an egg of Ornitho-
rhynchus, described by Wilson and Hill (’07, p. 38, Pl. 2, fig.
4), and by an egg of Echidna, described by Semon (94, p. 69,
figs. 22 and 33), is separated by a considerable gap from the
preceding, and most unfortunately so, since it belongs to the
period of commencing formation of the germ-layers. The

88 eT eePs! SELLE

cellular lens-shaped blastoderm of the preceding stage has
now extended in the peripheral direction so as to enclose
about the upper half of the yolk-mass, and in so doing it has
assumed the form, almost exclusively, of a unilaminar thin
cell-membrane, composed of flattened cells and closely applied
to the inner surface of the zona. At the embryonic pole,
however, in the region of the white yolk-bed, there are
present in the Ornithorhynchus egg a few plump cells,
immediately subjacent to the unilaminar blastoderm, but
separate and distinct from it, whilst in the Echidna egg
Semon’s figure (fig. 33), which is perhaps somewhat schematic,
shows a group of scattered cells, similar to those in the
Ornithorhynchus egg but placed considerably deeper in the
white yolk-bed. Unfortunately we have no definite evidence
as to the significance of these internally situated cells. One
of two possible interpretations may be assigned to them.
Hither they represent the last remaining deeply placed cells
of the blastodise of the preceding stage, which have not yet
become intercalated in the unilaminar blastodermic membrane
believed by Semon to be the condition attained in eggs of
about this stage of development, or they are cells which have
been proliferated off from this unilaminar blastoderm, to
constitute the parent cells of the future yolk-entoderm. As
regards Hchidna, Semon expresses a definite enough opinion ;
he holds that these deeply placed cells actually arise by a
somewhat diffuse proliferation or ingrowth from a localised
depressed area of the blastoderm at the embryonic pole, and
that they give origin to yolk-entoderm. This interpretation
of Semon seems probable enough in view of the mode of origin
of the entoderm in the Metatheria and Eutheria. Moreover
in the next available stage, an egg of Ornithorhynchus, just
over 6 mm. in diameter, described by Wilson and Hill, the
blastoderm is already bilaminar throughout its extent, so that
we might very well expect to find the beginnings of the ento-
derm in the somewhat younger eggs.

In the 6 mm. egg just referred to, the peripheral portion of
the unilaminar blastoderm of the preceding stage has grown

i

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 89

so as to enclose the entire yolk-mass in a complete ectodermal
envelope, whilst internally to that a complete lining of yolk-
entoderm has become established. As the result of these
changes, and of the imbibition of fluid from the uterus, the
solid yolk-laden egg has become converted into a relatively
thin-walled vesicle or blastocyst, possessed of a bilaminar
wall surrounding the partly fluid vitelline contents of the egg.
Throughout the greater part of its extent the structure of the
vesicle wall is very simple. It consists externally of an
extremely attenuated ectodermal cell - membrane closely
adherent to the deep surface of the vitelline membrane
(zona), and within that of a layer of yolk-entoderm, composed
of large swollen cells, containing each a vesicular nucleus,
and a number of yolk-spheres of varying size. Over a small
area, overlying the white yolk-bed, however, the ectodermal
layer of the wall presents a different character to that
described above. Its constituent cells are here not flattened
and attenuated, but irregularly cuboidal in form and much
more closely packed together; moreover they stand in pro-
liferative continuity with a subjacent mass of cells, also in
process of division. ‘The irregular superficial layer and this
latter mass together form a thickened lenticular cake, 3) mm.
in greatest diameter, projecting towards the white yolk-bed
but separated from it by the yolk-entoderm, which retains
its character as a continuous cell-membrane. ‘his differen-
tiated, thickened area of the wall, situated as it is at the upper
pole of the egg, as marked by the white yolk-bed, must be
held to represent a part of the future embryonal region.
Wilson and Hill incline to regard it as in some degree tlie
equivalent of the “primitive plate” of Reptiles and as the
initial stage in the formation of the primitive knot of later
eggs. This question, however, does not closely concern us
here: the point I wish to emphasise is the relative inactivity
of the cells composing the embryonal region of the blastoderm
in the Monotreme as compared with the marked activity dis-
played by those constituting the peripheral (extra-embryonal)
region of the same. It is these latter cells which by their

90 Ten. SEMI.

rapid growth complete the envelopment of the yolk-mass and
so constitute the lower hemisphere of the blastocyst.

The bilaminar blastocyst of the Monotreme, formed in the
manner indicated above, is entirely comparable with the
Marsupial blastocyst of the same developmental stage. ‘There
are differences in detail certainly (e.g. in the characters,
time of formation, and rate of spreading of the entoderm,
in the mode of formation of the blastocyst cavity and in its
contents, in the apparent absence in the Monotreme of any
well-marked line of division between the embryonal and extra-
embryonal regions of the ectoderm, in the relatively earlier
appearance of differentiation in the embryonal region in the
Monotreme as compared with the Marsupial), but the agree-
ments are obvious and fundamental; in particular, I would
emphasise the fact that in both the embryonal region is
superficial and freely exposed, and forms part of the blasto-
cyst wall just as that of the reptile forms part of the general
blastoderm. Moreover, should future observations confirm
the view of Semon that the primitive entodermal cells of the
Monotreme are proliferated off from the embryonal region of
the unilaminar blastoderm, then we should be justified in
directly comparing the latter with the unilaminar wall of the
Marsupial blastocyst, and in regarding it also as consisting
of two differentiated regions, viz. a formative or embryonal
region, overlying the white volk-bed, and giving origin to
the embryonal ectoderm and the yolk-entoderm, and a non-
formative region which rapidly overgrows the yolk-mass so
as to eventually completely enclose it, just as does the less
rapidly growing extra-embryonal ectoderm of the Saurop-
sidan blastoderm.' Meantime I see no reason for doubting
that this rapidly growing peripheral portion of the unilaminar
blastoderm of the Monotreme is anything else than extra-
embryonal ectoderm homogenous with that of the reptile.
Indeed, lam not aware that any embryologist except Hubrecht
thinks otherwise. Kven Assheton is, I believe, content to

' We should further be justified in concluding that the entoderm is
similar in its mode of origin in all three mammalian sub-classes.

THE EARLY DEVELOPMENT OF ‘THE MARSUPIALIA. 91

regard the outer layer of the Monotreme blastocyst as
ectodermal. Hubrecht’s view is that the primitive entodermal
cells of Semon give origin, not to yolk-entoderm, but to the
equivalent of the embryonal knot of Kutheria, whilst the
unilaminar blastodermic membrane itself is a larval layer
—the trophoblast—that portion of it overlying the internally
situated cells representing the covering layer (Rauber’s layer)
of the Kutherian blastocyst. ‘‘For this view,” remarks
Assheton [’09, p. 253), “I can see no reason derivable from
actual specimens described and figured by those four authors”
(Caldwell, Semon, Wilson and Hill), with which criticism I
am in entire agreement, as also with the following statement,
which, so far as the Metatheria are concerned, is based on
my own results: “ Neither in the Prototheria [n | or the
Metatheria is there really any tangible evidence of a tropho-
blast occurring as a covering layer over the definitive epiblast
as in Hutheria” (p. 234).

In connection with the peripheral growth of the unilaminar
blastoderm in the Monotreme, it is of interest to observe that
this takes place, not apparently in intimate contact with the
surface of the solid yolk, as is the case with the growing
margin of the extra-embryonal ectoderm in the Sauropsidan
ege, but rather in contact with the inner surface of the
thickened zona, perhaps as the result of the accumulation in
the perivitelline space of fluid which has diffused into the latter
from the uterus. In other words, the peripheral growth of
the extra-embryonal ectoderm to enclose the yolk-mass appears
to take place here in precisely the same way as the spreading
of the non-formative cells in Dasyurus to complete the lower
pole of the blastocyst. In my view the latter phenomenon
is none other than a recapitulation of the former; on the
other hand, I regard the spreading of the formative cells in
Dasyurus towards the upper pole as a purely secondary
feature, conditioned by the loss of the yolk-mass aud the
attainment of the holoblastic type of cleavage.

If it be admitted that the outer extra-embryonal layer of
the Monotreme blastocyst is homogenous with the extra-

92 eae, alli

embryonal ectoderm of the Reptile, then it seems to me there
is no escape from the conclusion that these layers are also
homogenous with the non-formative region of the unilaminar
Marsupial blastocyst. I need only point out here that the
chief destiny of each of the mentioned layers, and I might
also add that of the outer enveloping layer of the Mutherian
blastocyst (the so-called trophoblast), is one and the same,
viz. to form the outer layer of the chorion (false amnion,
serous membrane) and omphalopleure (unsplit yolk-sac wall,
Hill [’97]),! and that to deny their homogeny to each other
implies the non-homogeny of these membranes and the amnion
in the Amniotan series, and consequently renders the group
name Amniota void of all morphological meaning.

‘The rapidity with which the enclosure of the yolk-mass
is effected, and the relative tardiness of differentiation in the
embryonal region are features which sharply distinguish the
early ontogeny of the Monotremes from that of the Sauropsida,
and which, in my view, are of the very greatest importance,
since they afford the key to a correct understanding of the
peculiar coenogenetic modifications observable in the early
ontogeny of the Metatheria and Eutheria. To appreciate the
significance of these features it is necessary to take account
of the great difference which exists between the Sauropsidan
and Monotreme ovum in regard to size, as well as of the very
different conditions under which the early development goes
on in the two groups. ‘The Sauropsidan ege is large enough
to contain within its own confines the amount of yolk neces-
sary for the production of a young one complete in all its
parts and capable of leading an independent existence
immediately it leaves the shell. Furthermore, it is also large

' In certain Amniotes the layers in question appear also to participate
in the formation of the inner lining of the amnion (amniotic ectoderm)
(cf. Assheton [09], pp. 248-9), but this does not affect the statement in
the text. In the Sauropsida and Monotremata I think I am correct in
saying that no sharp distinction is recognisable between the embryonal
and extra-embryonal regions of the ectoderm, hence it is difficult, if not
impossible, to determine with certainty their relative participation in
the formation of the amniotic ectoderm.

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 93

enough to provide room for the development of an embryo
without any secondary growth in size after it leaves the ovary.
Moreover we have to remember that after it has become
enclosed in the shell, it remains but a short time in the ovidnet
and receives little or no additional nutrient material from the
oviducal walls. The yolk-mass in any case retains its solid
character; there is no necessity for its rapid enclosure, and
so enclosure is effected slowly, contemporaneously with the
differentiation of the embryo.

In the Monotreme the conditions are altogether different.
The ripe ovarian ovum when it enters the oviduct has a
diameter of about 3°5 to 4 mm., and is thus considerably
smaller than that of a Reptile of the same size as the adult
Monotreme. The amount of yolk which it is capable of con-
taining is not anything like sufficient to last the embryo
throughout the developmental period, and, moreover, it does
not provide the space essential for the development of an
embryo on the ancestral Reptilian lines. As Assheton (’98,
p- 251) has pointed out, “the difference in size between
the fertilised ovum of a reptile or bird or of a mammal
is very great; but the difference in size between the
embryo of, say, a bird with one pair of mesoblastic
somites and of a mammal of the same age is comparatively
small. This means that nearly the same space is required
for the production of the mammalian embryo as of the
Sauropsidan, and has to be provided.” In the Monotreme
not only is additional room necessary, but also additional
nutrient material, sufficient with that already present in the
ego to last the embryo throughout the period of incubation.
Both are acquired contemporaneously during the sojourn of
the egg in the uterine portion of the oviduct, wherein the egg
increases greatly in size. When it enters the uterus, the
Monotreme egg has a diameter, inclusive of its membranes, of
about 4-5 mm.; when it is laid, it measures in Ornitho-
rhynchus, in its greatest diameter, 16-19 mm., and somewhat
less in the case of Echidna. Prior to the enclosure of the yolk
the increase in diameter, due to the accumulation of fluid in

94. Tee Ps EL ube

the perivitelline space and between the zona and shell, is but
slight. But as soon as the yolk becomes surrounded by a
complete cellular membrane, i.e. as soon as the egg has
become converted into a thin-walled blastocyst, rapid growth
sets in, accompanied by the active imbibition of the nutrient
fluid, which is ponred into the uterine lumen as the result of
the secretory activity of the abundantly developed uterine
glands. The fluid absorbed not only keeps the blastocyst
turgid, but it brings about the more or less complete dis-
integration of the yolk-mass, its constituent spherules
becoming disseminated in the fluid contents of the blastocyst
eavity. Although a distinct and continuous subgerminal
cavity, such as appears beneath the embryonal region of the
Sauropsidan blastoderm, does not occur in the Monotreme
ego, vacuolar spaces filled with fluid develop in the white
yolk-bed underlying the site of the germinal dise and appear
to represent it. As Wilson and Hill remark (’03, p. 317),
“ one can, without hesitation, homologise the interior of the
vesicle with the subgerminal cavity of a Sauropsidan egg,
extended so as to include by liquefaction the whole of the
yolk itself.” In the Marsupial the blastocyst cavity has a quite
different origin, since it represents the persistent segmentation
cavity, whilst in the Eutheria the same cavity is secondarily
formed by the confluence of intra- or inter-cellular vacuolar
spaces, but no one, so far as I know, has ever ventured to
assert that, because of this difference in mode of origin, the
blastocyst cavity in the series of the Mammalia is a non-
homogenous formation.

To return to the matter under discussion, it appears to me
that the necessity which has arisen, consequent on the reduc-
tion in size of the ovum, for rapid growth of the same in
order to provide room for the development of an embryo and
for the storage of nutrient material furnished by the maternal
uterus, affords a satisfactory explanation of the much more
marked activity of the extra-embryonal region of the blasto-
derm as compared with the embryonal, which is such a striking
feature in the early ontogeny of the Monotremes, and not

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 95

only of them, but, as Assheton has pointed out (’98, p. 251),
of the higher mammals as well (cf. the process of epiboly and
the inertness at first displayed by the formative cells of
the embryonal knot as compared with the activity of the non-
formative or tropho-ectodermal cells), an activity which
results in the rapid completion of that characteristically
mammalian developmental stage—the blastocyst or blasto-
dermic vesicle.

The necessity for the early formation of such a stage,
capable of rapidly growing in a nutrient fluid medium
provided by the mother, has profoundly influenced the early
ontogeny in all three mammalian subclasses, and naturally
most of all that of the Eutheria, in which reduction of the
ovum, both as regards size and secondary envelopes, has
reached the maximum. And I think there can be little
doubt but that it is this necessity which has induced that
early separation of the blastomeres into two categories,
respectively formative and non-formative in significance,
which has long been recognised as occurring in Kutheria, and
which I have shown also occurs amongst the Metatheria.
This early separation of the blastomeres into two distinct
groups is not recognisable in the Sauropsida, and the idea
that it is in some way connected with the loss of yolk which
the mammalian ovum has suffered in the course of phylogeny,
was first put forward, I believe, by Jenkinson. In his paper
on the germinal layers of Vertebrata (’06, p. 51) he writes:
“« Segmentation therefore is followed in the Placentalia by
the separation of the elements of the trophoblast from those
destined to give rise to the embryo and the remainder of its
foetal membranes, and this ‘precocious segregation’
seems to have occurred phylogenetically during
the gradual loss of yolk which the egg of these
mammals has undergone.’ Whether or not such a
“precocious segregation” has already become fixed in the
Monotremes, future investigation must decide (cf. ante, p.90).

The loss of yolk, with resulting reduction in size which the
Monotreme ovum has suffered in the course of phylogeny, we

96 J; (Pst:

must assume to have taken place gradually and in correlation
with the longer retention of the egg in the oviduct, the
elaboration of the uterine portion of the same as an actively
secretory organ, and the evolution of the mammary apparatus,
The Monotremes thus render concrete to us one of the first
great steps in mammalian evolution so far as developmental
processes are concerned, viz. the substitution for intra-ovular
yolk of nutrient material furnished directly by the mother to
the developing egg or embryo. We see in them the begin-
nings of that process of substitution of uterine for ovarian
nutriment which reaches its culmination in the Eutheria with
their microscopic yolk-poor ova and long intra-uterine period
of development. The Marsupials show us in Dasyurus an
interesting intervening stage so far as the ovum is concerned,
in that this, though greatly reduced as compared with that
of the Monotreme, still retains somewhat of its old tendencies
and elaborates more yolk-material than it can conveniently
utilise, with the result that it has to eliminate the surplus
before cleavage begins. But as concerns their utilisation of
intra-uterine nutriment, they have specialised along their
own lines, and instead of exhausting the possibilities implied
by the presence of that, they have extensively elaborated
the mammary apparatus for the nutrition of the young, born
in a relatively immature state, after a short period of intra-
uterine life (cf. Wilson and Hill [’97, p. 580]).

In view of the fact that the young Monotreme enjoys three
developmental periods, viz. intra-uterine, incubatory, and
lactatory, the question might be worthy of consideration
whether it may not be that the Marsupial has merged the
incubatory period in the lactatory, the Eutherian the same in
the intra-uterine.

2. The Early Development of the Metatheria and
Eutheria.

It will have become evident from the foregoing that the
Metatherian mode of early development is to be regarded as

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 97
but a slightly modified version of the Prototherian, such
differences as exist between them being interpretable as cceno-
genetic modifications, induced in the Metatherian by the
practically complete substitution of uterine nutriment for
intra-ovular yolk, a substitution which has resulted in the
attainment by the marsupial ovum of the holoblastic type of
cleavage. In the present section I hope to demonstrate how
the early ontogeny of the Metatheria enables us to interpret
that of the Eutheria in terms of that of the Prototheria.

If we proceed to compare the early development in the
Metatheria and Kutheria, we encounter, from the 4-celled
stage onwards, such obvious and profound differences in the
mode of formation of the blastocyst, and in the relations of
its constituent parts, that the differences seem at first sight
to far outweigh the resemblances. Nevertheless, apart from
their common possession of the same holoblastic mode of
cleavage, there exists one most striking and fundamental
agreement between the two in the fact that in both there
occurs, sooner or later during the cleavage process, a separa-
tion of the blastomeres into two distinct, pre-determined cell-
groups, whose individual destinies are very different, but
apparently identical in the two subclasses. In the Marsupial,
as typified by Dasyurus, the fourth cleavages are, as we have
seen, unequal and qualitative, and result in the separation of
two differentiated groups of blastomeres, arranged in two
superimposed rings, viz. an upper ring of eight smaller, less
yolk-rich cells, and a lower of eight larger, more yolk-rich
cells. The evidence justifies the conclusion that the former
gives origin directly to the formative or embryonal region of
the vesicle wall, the latter to the non-formative or extra-
embryonal region.

Amongst the Eutheria the evidence is no lessclear. It has
been conclusively shown by various observers (Van Beneden,
Duval, Assheton, Hubrecht, Heape, and others) that, sooner
or later, there occurs a separation of the blastomeres into two
distinct groups, one of which eventually encloses the other
completely. The two groups may be clearly distinguishable

vou. 06, PART 1.—NEW SERIES. 7

Once.

al oat 0
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Stace »

Diagrams illustrating the mode of formation of the blastocyst
in Metatheria (A—D) and Eutheria (1-3). b.c. Blastocyst cavity.
i.c.m. Inner cell-mass. pr.amu.c. Primitive amniotic cavity.
r.l. Rauber’s layer. s.c. Segmentation cavity. For other
reference letters see explanation of plates (p. 125).

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 99

in early cleavage stages, owing to differences in the characters
and staining reactions of their cells, and in such cases there
is definite evidence of the occurrence of a process of overgrowth
or epiboly, whereby one group gradually grows round and
completely envelops the other, so that in the completed
morula a distinction may be drawn between a central cell-
mass and a peripheral or enveloping layer (rabbit, Van
Beneden ; sheep, Assheton). In other cases, where it has
been impossible to recognise the existence of these two
distinct cell-groups in the cleavage stages, we nevertheless
find, either in the completed morula or in the blastocyst, that
amore or less sharp distinction may be drawn between an
enveloping layer of cells and an internally situated cell-mass
(inner cell-mass).

E. van Beneden, in his classical paper on the development
of the rabbit, published in 1875, was the first to recognise
definitely the existence of two categories of cells in the
segmenting egg of the Kutherian mammal. In this form he
showed how in the morula stage a cap of lighter blastomeres
gradually grows round and envelops a mass of more opaque
cells by a process of overgrowth or epiboly. In his more
recent and extremely valuable paper on the development of
Vespertilio (799), he again demonstrated the existence of two
groups of blastomeres as wellin the segmenting egg as in the
completed morula, but failed to find evidence of epiboly in all
cases. Nevertheless he holds fast to the opinion which he
expressed in 1875: “ Que la segmentation s’accompagne, chez
les Mammiféres placentaires, d’un enveloppement progressif
@Vune partie des blastoméres par une couche cellulaire, qui
commence a se différencier dés le début du développement,”
and states that “dans tous les ceufs arrivés a la fin de la
segmentation et dans ceux qui montraient le début de la
cavité Blastodermique j’ai constamment rencontré une couche
périphérique complete, entourant de toutes parts un amas
cellulaire interne, bien séparé de la couche enveloppante.”
The latter layer he regards as corresponding to the extra-
embryonal ectoderm of the Sauropsida, and points out that

100 J, Ps BL:

“chez tous les Chordés les premiers blastoméres qui se
differencient et qui avoisinent le péle animal de lceuf sont
des éléments épiblastiqnes. C’est par la couche cellulaire qui
résulte de la segmentation ultérieure de ces premiers blasto-
méres épiblastiques que se fait, chez les Sauropsides, Penve-
loppement du vitellus. . Dans l’ceuf réduit a n’étre plus
qu’une sphére microscopique, |’épibolie a pu s’achever dés la
fin de la segmentation, voire méme avant Pachévement de ce
phénoméne.” The “amas cellulaire interne” (embryonal
knot, inner cell mass), Van Beneden shows, differentiates
secondarily into “un lécithophore et un bouton embryon-
naire.’ The former is the entoderm of other authors, the
latter the formative or embryonal ectoderm. Hubrecht, in
the forms studied by him (Sorex, ‘Tupaia, Tarsius') finds
a corresponding differentiation. In Tupaia he describes the
morula stage as consisting of a single central lightly staiming
cell, which he regards as the parent cell of the inner cell-mass
of later stages, and of a more darkly staining peripheral layer
which forms the unilaminar wall of the blastocyst. Here,
then, the parent cells of the two cell-groups would appear to
be separated at the first cleavage. Hubrecht, hke Van
Beneden, holds that the inner cell-mass furnishes the
embryonal ectoderm and the entire entoderm of the blastocyst.
‘he peripheral layer he has termed the trophoblast (88, p.
511), and in his paper on the placentation of the hedgehog
(89, p. 298) he defines the term as follows: “I propose to
confer this name to the epiblast of the blastocyst as far as it
has a direct nutritive significance, as indicated by proliferating
processes, by immediate contact with maternal tissue, maternal
blood, or secreted material. The epiblast of the germinal
area—the formative epiblast—and that which will take part
in the formation of the inner lining of the amnion cavity 1s,
ipso facto, excluded from the definition.” ‘Thus the name

' In Erinaceus the entoderm, from Hubrecht’s observations, appears
to be precociously differentiated, prior to the separation of the embryonal
ectoderm from the overlying trophoblast, but the details of the early
‘development in this form are as yet only incompletely known.

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 101

trophoblast was originally employed by Hubrecht as a con-
venient term designatory of what he at the time regarded as
the extra-embryonal ectoderm of the mammalian blastocyst.
In the course of his speculations on the origin of this layer,
however, he has reached the conclusion that it is really of the
nature of “a larval envelope, an Embryonalhiille” (?08, p. 15),
inherited by the mammals, not from the reptiles (which have
no direct phylogenetic relationship to the latter), but from
their remote invertebrate ancestors (“‘vermiform predecessors
of ccelenterate pedigree, provided with an ectodermal larval
investment [ Larvenhiille ] ”’).

Assheton, again, although he was unable to convince him-
self (94) of the correctness of van Beneden’s account of the
occurrence of a process of epiboly in the segmenting eggs of
the rabbit, finds in the sheep (’98) that a differentiation into
two groups of cells is recognisable “ perhaps as early as the
eight segment stage,” and that one of the groups gradually
envelops the other. ‘ Let it be noted,” he writes (’98, p. 227),
“that we have now to face the fact, based on actual sections,
that there is in certain mammals a clear separation of
segments at an early stage into two groups, one of which
eventually completely surrounds the other,’ and instances
Van Beneden’s observations on the rabbit (of the correctness
of which he, however, failed to satisfy himself, as noted above),
Duval’s observations on the bat, Hubrecht’s on Tupaia, and
his own on the sheep. Assheton thinks this phenomenon
“must surely have some most profound significance,”
but finds himself unable to accept the interpretations of
either Van Beneden or Hubrecht, and puts forward yet
another view, “ based on the appearance of some segmenting
eggs of the sheep ” (08, p. 233), “that in cases where this
differentiation does clearly occur, it is a division into epiblast
and hypoblast, the latter being the external layer” (98, p. 227).
Assheton thus differs from all other observers in holding that
the inner cell-mass or embryonal knot of the HKutherian
blastocyst gives origin solely to the formative or embryonal
ectoderm, and | believe I am correct in stating that he also

102 J.P. Albu

differs from all other observers in holding that the outer
enveloping layer of the same is entodermal.!

The fact, then, of the occurrence amongst Eutheria of a
“precocious segregation ” of the blastomeres into two distinct
groups, one of which eventually surrounds the other com-
pletely, is not in dispute, though authorities differ widely in
the interpretation they place upon it. In the Eutherian
blastocyst stage, the enveloping layer forms the outer uni-
laminar wall of the vesicle, and encloses the blastocyst cavity
as well as the other internally situated group. This latter
typically appears as a rounded cell-mass, attached at one spot
to the inner surface of the enveloping layer, but more or less
distinctly marked off from it. It is generally termed the
inner cell-mass or embryonal knot (“amas cellulaire interne ”’
of Van Beneden). For the enveloping layer Hubrecht’s name
of “trophoblast ” is now generally employed, even by those
who refuse to adopt the speculative views with which its
originator has most unfortunately, as I think, enshrouded this
convenient term.

I have demonstrated the occurrence of an apparently com-
parable ‘precocious segregation”? of the blastomeres into
two distinct groups in one member of the Metatheria which
there is no reason to regard as an aberrant type, and I have
shown beyond all shadow of doubt that from the one group,
which constitutes what I have termed the formative region
of the unilaminar vesicle-wall, there arise the embryonal
ectoderm and the entire entoderm of the vesicle, both em-
bryonal and extra-embryonal, and that the other group, which
constitutes the non-formative region of the vesicle-wall,
directly furniskes the extra-embryonal ectoderm, i.e. the
ectoderm of the omphalopleure and chorion.*

' Assheton states (08, p. 233, cf. also ‘98, p. 220) that his interpreta-
tion ‘“‘owes much also to the theoretical conclusions of Minot and
Robinson.” However that may be, both Minot and Robinson in their
most recent writings continue to speak of the chorionic ectoderm,

2» Whether or not it participates in the formation of the amniotic
ectoderm future investigation must decide.

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 103

As regards Eutheria, we have seen that Van Beneden and
Hubrecht, though their views in other respects are widely
divergent, both agree that the inner cell-mass of the blasto-
cyst furnishes the embryonal ectoderm (as well as the amniotic
ectoderm wholly or in part) and the entire entoderm of the
vesicle. That, in fact, is the view of Mammalian embryologists
generally (Duval and Assheton excepted),! and if we may
assume it to be correct, then it would appear that the later
history of the formative region of the Marsupial blastocyst
and that of the inner cell-mass of the Eutherian are identical.
That being so, and’ bearing in mind that both have been
shown, at all events in certain Mammals, to have an identical
origin as a group of precociously segregated blastomeres,” I
can come to no other conclusion than that they are homo-
genous formations. If that be accepted, then this fact by itself
renders highly probable the view that the so-called tropho-
blast of the Eutherian blastocyst is homogenous with the
non-formative region of the Metatherian vesicle, and when
we reflect that both have precisely the same structural and
topographical (not to meution functional) relations in later
stages, inasmuch as they constitute the ectoderm of the chorion
and omphalopleure (with or without participation in the
formation of the amniotic ectoderm), and that both have a
similar origin in those Mammals in which a precocious segre-
gation of the blastomeres has been recognised, their exact

‘The view of Duval [95], based on the study of Vespertilio, that the
inner cell-mass gives rise solely to entoderm, and that the enveloping
layer furnishes not only the extra-embryonal but also the embryonal
ectoderm, is shown by Van Beneden’s observations on the same form to
be devoid of any basis of fact. | Assheton’s views are referred to below
(p. 110).

> The fact that the phenomenon of the “ precocious segregation ” of
the blastomeres into two groups with determinate destinies has already
become fixed in the Marsupial lends additional weight to the view of
Van Beneden that such a segregation will eventually be recognised as
occurring in all Eutheria without exception. Without it, it is difficult
to understand how the entypic condition, characteristic of the blasto-
cysts of all known Eutheria, is attained, unless by differentiation in
situ, which seems to me highly improbable.

104 Ji; ee MERU

homology need no longer be doubted. In the preceding section
of this paper (ante, pp. 91, 92) 1 have shown reason for the
conclusion that the non-formative region of the Marsupial
blastocyst is the homologue of the extra-embryonal ectoderm
of the Monotreme and Reptile, and if that conclusion be
accepted it follows that the outer enveloping layer of the
Kutherian blastocyst, the so-called trophoblast of Hubrecht,
is none other than extra-embryonal ectoderm, as maintained
by Van Beneden, Keibel, Bonnet, Jenkinson, Lee, MacBride
and others, the homologue of that of Reptilia.

IT am therefore wholly unable to accept the highly specula-
tive conclusions of Hubrecht, set forth with such brilliancy
in a comparatively recent number of this Journal (’08), as
to the significance and phylogeny of this layer. ‘hese con-
clusions, on the basis of which he has proceeded to formulate
such far-reaching and, indeed, revolutionary ideas not only
on questions embryological, but on those pertaining to the
phylogeny and classification of vertebrates, have already
been critically considered by Assheton (’09) and MacBride
(09), also in the pages of this Journal, and found wanting,
and they are, to my mind, quite irreconcilable with the facts
I have brought to light in regard to the early development
of Marsupials. I yield to no one in my admiration for the
epoch-making work of Hubrecht on the early ontogeny and
placentation of the Mammalia, and I heartily associate
myself with the eulogium thereanent so admirably expressed
by Assheton in the critique just referred to (p. 274), but
I am bound to confess that as concerns his views on the
phylogeny of this layer, which he has termed the “ tropho-
blast,” he seems to me to have forsaken the fertile field of
legitimate hypothesis for the barren waste of unprofitable
speculation, and to have erected therein an imposing edifice on
the very slenderest of foundations.

Before I proceed to justify this, my estimate of Hubrecht’s
views on the phylogeny of the trophoblast, let me first set
forth his conception so far as I understand it. He starts
with the assumption that the vertebrates (with the exception

THE HWARLY DEVELOPMENT OF THE MARSUPIALIA. 105

of Amphioxus, the Cyclostomes, and the Elasmobranchs) are
descended from ‘“vermiform predecessors of ccelenterate
pedigree” possessed of free-swimming larvee, in which there
was present a complete larval membrane of ectodermal deriva-
tion, and of the same order of differentiation “as the outer
larval layer which in certain Nemertines, Gephyreans, and other
worms often serves as a temporary envelope that is stripped
off when the animal attains to a certain stage of development.”
When, for oviparity and larval development, viviparity and
embryonic development became established in the Prote-
trapodous successors of the ancestral vermiform stock, the
larval membrane did not disappear. On the contrary, it is
assumed that it merely changed “its protective or locomotor
function into an adhesive one,’ and so, development now
taking place in utero, it is quite easy to understand how the
larval membrane could gradually become transformed into
a trophic vesicle, containing the embryo as before, and
functional in the reception of nutriment from the walls of
the maternal uterus. ‘he final stages in the evolution of
this trophic vesicle constituted by the old larval membrane
are met with amongst the mammals, since in them it
became vascularised so as to constitute a “yet more
thorough system of nourishment at the expense of the
maternal circulatory system.” Such, then, is the phylogeny
of the trophoblast according to Hubrecht. The Hutherian
mammals, which it is held trace their descent straight back to
some very early Protetrapodous stock, viviparous in habit and
with small yolk-poor, holoblastic eggs, exhibit the tropho-
blast in its most perfect condition. Hubrecht therefore starts
with them, and attempts to demonstrate the existence of a
larval membrane, or remnants of such, externally to the
embryonal ectoderm in all vertebrates with the exceptions
already mentioned. There is no question of its existence in
the Meta- and Eutherian mammals. “ We may,” writes
Hubrecht (’08, p. 12), . . . ‘“‘insist upon the fact that
. . . all Didelphia and Monodelphia hitherto investi-
gated show at a very early moment the didermic stage out of

106 J: P. HILL.

which the embryo will be built up enclosed in a cellular
vesicle (the trophoblast), of which no part ever enters into
the embryonic organisation.” ‘lhe common possession by the
Metatheria and Eutheria of a larval membrane is after all
only what might be expected, “since after Hill’s (97)
investigations, we must assume that the didelphian mammals
are not descended from Ornithodelphia but from monodelphian
placental ancestors.” As concerns the Prototheria, although
they cannot in any sense be regarded as directly ancestral to
the other mammals, we nevertheless find the trophoblastic
vesicle ‘comparatively distinct.” ‘In many reptiles and
birds,” however, it is “distinguished with great difficulty
from the embryonic shield,” and this is explained by the
fact that the Sauropsida which are assumed to have taken
their origin from the same Protetrapodous stock as the
mammals but along an entirely independent line, have
secondarily acquired, like the Prototheria, the oviparous
habit, with its concomitants, a yolk-laden egg anda shell, and
this latter acquisition has naturally tended ‘to relegate any
outer larval layer to the pension list”? (09, p. 5). ‘* Con-
cerning the yolk accumulation in the Sauropsidan ege, there
is no trouble at all to suppose that the vesicular blastocyst
of an early viviparous ancestor had gradually become yolk-
laden. The contrary assumption, found in the handbooks,
that the mammalian ege, while totally losing its yolk, has
yet preserved the identical developmental features as the
Sauropsid, is in reality much more difficult to reconcile with
sound evolutionary principles” (’09, p. 5).

Amongst the lower Vertebrates the larval membrane is
clearly enough recognisable in the so-called Deckschicht of
the Teleostomes, Dipnoans, and Amphibians. It is frankly
admitted that Amphioxus, the Cyclostomes, and the Elasmo-
branchs “show in their early development no traces of a
Deckschicht” (larval layer, trophoblast), but there is no
difficulty about this, since it is easy enough to suppose, in
view of other characters, that ‘ the Selachians may very well
have descended from ancestors without any outer larval layer ”

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 107

(08, p. 151), and “for Cyclostomes the same reasoning holds
good” (p. 162).

The trophoblast, then, is conceived of by Hubrecht as a
larval membrane of ectodermal derivation, which invests the
embryonal anlage in all Vertebrates with the exceptions
mentioned, which is subject to secondary reduction, and which
is homologous throughout the series. As I understand the
couception, what is ordinarily called extra-embryonal ecto-
derm in the Sauropsida is not trophoblast, otherwise Hubrecht
could hardly write—‘‘in reptiles and birds traces of the
larval layer have in late years been unmistakably noticed”
(09, p. 5); nevertheless what other writers have termed
embryonal and extra-embryonal ectoderm in the Prototheria
is claimed by Hubrecht as trophoblast (at all events that is
my interpretation of his statement that a trophoblastic vesicle
is present in these forms), and yet some years ago Hubrecht
(04, p. 10) found it difficult “to understand that the name
has been misunderstood both by embryologists and gyneco-
logists.” My own feeling is that the more recent develop-
ments in his views have tended to obscure rather than to
clarify our ideas as to the trophoblast, especially if we must
now hold that the chorion or serosa of the Sauropsida is not
homologous with that of the Prototheria, which necessarily
follows if the extra-embryonal ectoderm of the Sauropsidan is
not the same thing as that of the Monotreme.

Assuming that we have formed a correct conception of the
trophoblast as a larval membrane, and bearing in mind that it
is best developed in the Metatheria and Kutheria, since these
alone amongst higher Vertebrates have retained unaltered
the viviparous habits of their Protetrapodous ancestors, let us
see what basis in fact there is for the statement of Hubrecht
(08, p. 68) that “before the ectoderm and the entoderm
have become differentiated from each other there is in
mammals a distinct larval cell-layer surrounding (as soon as
cleavage of the ege has attained the morula stage) the
mother-cells of the embryonic tissues.” Now that statement
as it stands, I have no hesitation in characterising as entirely

108 Ja Pe: ELLE

misleading, inasmuch as it is applicable not to the Mammalia
as a whole, but, so far as it refers to matters of undisputed
fact, to one only of the three mammalian subclasses, viz. the
Eutheria. So far as the latter are concerned, practically all
observers, as we have seen, are agreed that there is present
during at least the early stages of development a complete
outer layer of cells which encloses the embryonal anlage
or inner cell-mass (that portion of it immediately overlying
the latter being termed the “ Deckschicht” or ‘ Rauber’s
layer”). It is, of course, this enveloping layer or tropho-
blast which Hubrecht interprets as a larval membrane.
It fulfils the conditions, and were the Eutheria the only
Vertebrates known to us, the idea might be plausible
enough.

Turning now to the Metatheria, and remembering that these,
according to Hubrecht, are descended from the Eutheria, we
should naturally expect to find the supposed larval membrane
fully developed, with all its ancestral relations ; and so we do
if we are content to accept Hubrecht’s interpretation of
Selenka’s results and figures in the case of Didelphys. The
“urentodermzelle ” of Selenka is for Hubrecht “ undoubtedly
the mother-cell of the embryonic knob,’ the ectoderm of
Selenka is manifestly the trophoblast—a complete larval
layer. It is no doubt unfortunate that Hubrecht had to rely
on the work of Selenka as his source of information on the
early development of Marsupials, but it must be remembered
that he reads his own views into Selenka’s figures. On the
basis of my own observations on the early ontogeny of Mar-
supials, | have no hesitation in affirming that a larval mem-
brane, in the sense of Hubrecht, does not exist in any of the
forms (Dasyurus, Perameles, Macropus) studied by me. The
observations recorded in the preceding pages of this paper
demonstrate, in the case of Dasyurus without the possibility
of doubt, the entire absence of any cellular layer external
to the formative region of the blastocyst, i.e. in a position
corresponding to that occupied by Rauber’s layer in Kutheria,
whilst in the case of Perameles and Macropus, they yield not

et

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 109

the slightest evidence for the existence of any such layer.
The formative region -of the Marsupial blastocyst, which is
undoubtedly the homologue of the inner cell mass of the
Eutheria, forms from the first part of the unilaminar blasto-
cyst wall, and is freely exposed. The remainder of the latter
is constituted by a layer of non-formative cells, the destiny
of which is the’same as that of the so-called trophoblast of
the Eutheria. I have therefore ventured to suggest that they
are one and the same. If, then, the trophoblast is really a
larval membrane, we must assume, in the case of the Mar-
supial, either that its “ Deckschicht” portion has been com-
pletely suppressed (but why it should have been I fail to
understand, unless, perhaps, it is a result of the secondary
acquisition by the Marsupials of a shell-membrane, these
mammals being even now on the way to secondarily assume
the oviparous habit !), or that the non-formative region of the
Marsupials is not the homologue of the trophoblast, in which
case the Marsupials must be held to have entirely lost the larval
membrane, since there is no other layer present which could
possibly represent it. ‘These considerations may well give us
pause before we calmly accept Hubrecht’s conception of the
trophoblast as a larval membrane present in all mammals
without exception.

Coming now to the Prototheria, we find, according to
Hubrecht, ‘the trophoblastic vesicle . . . yet compara-

” and so it is if we accept the interpretation of

tively distinct,
Hubrecht of the observations and figures of Semon, Wilson
and Hill. The unilaminar blastoderm of these authors is
unmistakably the trophoblast. he cells situated internally
to that in the region of the white yolk-bed are not ento-
dermal, as suggested by Semon, but constitute for Hubrecht
“the mother cells of the embryonic knob.’ I need only quote
again the opmion of Assheton thereanent and express my
agreement therewith ; he writes (’09, p. 233) ; “ For this view
I can see no reason derivable from actual specimens described
and figured by those four authors” (Caldwell, Semon, Wilson
and Hill). It would appear, then, that the assumption of

110 7) GP. Al

Hubrecht of the presence of a larval membrane of the nature
postulated in the Prototheria and Metatheria is devoid of
foundation in fact, so that there but remains the question of
the significance of the outer enveloping layer of the Kutherian
blastocyst. As regards that, I venture to think that the
alternative interpretation of E. van Beneden and _ other
investigators, which I have attempted to develop in the
pages of this paper, affords a simpler and more satisfying
explanation of its significance and phylogeny than that
advocated by Prof. Hubrecht, an interpretation, moreover,
which is more in accordance, not only with all the known
facts, but “ with sound evolutionary principles ” and with the
conclusions arrived at by the great majority of comparative
anatomists and paleontologists as to the origin and inter-
relationships of the Mammalia.

And I also venture to think that what has just been said
holds true with reference to the views advocated by Mr.
Assheton. These views owed their origin to certain appear-
ances which he found in some segmenting ova of the sheep
(but, be it noted, not in all those he examined), and he has
attempted to re-interpret not only his own earlier observations,
but those of other workers on the early ontogeny of the Hutheria
in the light of his newer faith, and not only so, he holds that it
is also possible to apply that in the interpretation of the early
ontogeny of Marsupials (v. 708, p. 235, and ’09, p. 229). He
maintains that the inner cell-mass of Eutheria is purely ecto-
dermal, and that the enveloping trophoblast layer of the blasto-
cyst arises in common with the entodermal lining of the same
and is therefore also entodermal. ‘‘ On the theory I advocate,”
he writes (’09, p. 235), “the trophoblast is of Eutherian
mammalian origin only and is not homologous to any form of
envelope outside the group of Eutherian mammals.” These
views of Assheton are not only at variance with those of all
other investigators who have worked at the early ontogeny of
Kutheria, but they are quite irreconcilable with my observa-
tions on the development of Dasyurus herein recorded. I claim
to have shown in that Marsupial that the formative region, the

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. ital

homologue of the inner cell-mass, gives origin not only to the
embryonal ectoderm, but to the entire entoderm, whilst the
non-formative region, whose homology to the trophoblast of
Eutheria is admitted by Assheton, arises quite independently
of the entoderm and a long time before the latter makes its
appearance. There is, then, in Dasyurus no question of a
common origin of the entoderm and the non-formative or
trophoblastic region of the blastocyst wall. And exception
may be taken to Assheton’s views on quite other grounds
(e. 2. the question of the homologies of the foetal membranes
in the series of the Amniota),as he himself is well aware, and
as Jenkinson (’00) has also emphasised. I feel, however, I can
leave further discussion of Assheton’s views until such time
as my observations on Dasyurus are shown to be erroneous or
inapplicable to other Marsupials.

3. The Entypic Condition of the Hutherian
Blastocyst.

If, now, on the basis of the homologies I have ventured to
advocate in the preceding pages, we proceed to compare the
Metatherian with the Eutherian blastocyst, we have to note
that, whereas in the latter the extra-embryonal or tropho-
blastic ectoderm alone forms the blastocyst wall in early
stages and completely encloses the embryonal knot, in the
former, the homologous parts, viz. the non-formative or extra-
embryonal and the formative or embryonal regions, both
enter into the constitution of the unilaminar blastocyst
wall, there being no such enclosure of the one by the
other as occurs in the Eutherian blastocyst (Text-fig. 2, p. 98).
It is characteristic of the Marsupial as of the Monotreme that
the embryonal region is from the first superficial and freely
exposed. It is spread out as a cellular layer and simply
forms part of the blastocyst wall or blastoderm. It is equally
characteristic of the Eutherian that the homologous part, the
embryonal knot, has at first the form of a compact mass,
which is completely enclosed by the trophoblastic ectoderm.

j 2 ieee SUA GE

The latter alone constitutes the unilaminar wall of the
blastocyst and has the embryonal knot adherent at one spot
to its inner surface. The formative cells which compose
the knot thus take at first no part in the constitution of
the outer wall of the blastocyst, and may or may not
do so in later stages according as the covering layer of the
trophoblast (the Deckschicht or Rauber’s layer) is tran-
sitory or permanent. This peculiar developmental con-
dition, characterised by the internal position of the formative
or embryonal cells within the blastocyst cavity, has been
termed by Selenka (00) “entypy” (Hntypie des Keim-
feldes).1 It is a phenomenon exclusively found in the
Eutheria and characteristic of them alone, amongst the
mammals. In the Marsupial, as in the Monotreme, the
formative cells are freely exposed, and constitute from the first
part of the blastocyst wall just as those of the Sauropsida form
a part of the general blastoderm. Limited as entypy thus
appears to be to the higher mammals, the probability is that
we have to do here with a purely secondary, adaptive feature.

If we proceed to inquire what is the significance of this
remarkable difference in the early developmental phenomena
of the lower and higher mammals, it seems to me that we have
to take account, in the first place, of the differences in the
structure of their respective eggs, and especially we have to
bear in mind that the Hutherian ovum is considerably more
specialised than even the Metatherian. It is on the average
smaller than the latter, i.e. it has suffered in the course of
phylogeny still further reduction in size, and has lost, to an
even greater extent than the Marsupial ovum, the store of food-
yolk ancestrally present init. Moreover, it has suffered a still
further reduction in respect of its secondary egg-membranes.
The Metatherian ovum still retains in its’ shell-membrane a

1“ Unter Entypie des Keimfeldes méchte ich daher verstanden
wissen: Die nicht durch Bildung typischer Amnionfalten geschehende,
sondern durch eine schon wahrend der Gastrulation erfolgende Absch-
niirung des Keimfeldes ins Innere der Hiblasenhiille (Chorion) ” (’00,
p. 203).

THE EARLY DEVELOPMENT OF -THE MARSUPIALIA. 1135

vestigial representative of the shell of the presumed oviparous
common ancestor of the Metatheria and Eutheria. The
Eutherian ovum, on the other hand, has lost all trace of the
shell in correlation with its more complete adaptation tothe con-
ditions of intra-uterine development. The albumen layer is
variable in its occurrence, being present in some (e.g. rabbit)
and absent in others (e.g. pig, Assheton), whilst the zona
itself, though always present, 1s variable both as to its thick-
ness and the length of time it persists.

Strangely enough, although the prevaling opinion amongst
mammalian embryologists is that the Eutherian ovum has
been derived phylogenetically from an egg of the same telo-
lecithal and shell-bearing type as is found in the Monotremes,
no one, so far as I am aware, has ever taken the shell into
account, and ventured to consider in what way its total dis-
appearance from an ovum already greatly reduced in size,
might affect the course of the early developmental phenomena.
That is what I propose to do here, for in my view it is just in
the complete loss of the shell by the Eutherian ovum that we
find the key to the explanation of those remarkable differences
which are observable between the early ontogeny of the
Eutheria and Metatheria, and which culminate in the entypic
condition so distinctive of the former. The acquisition of a
shell by the Proamniota conditioned the appearance of the
amnion. ‘The loss of the shellin the Eutheria conditioned the
occurrence in their ontogeny of entypy.

As we have seen, the mammalian ovum, already in the
Monotremes greatly reduced in size as compared with that of
reptiles, and quite minute in the Metatheria and Hutheria,
contains within itself neither the cubic capacity nor the food
material necessary for the production of an embryo on the
ancestral reptilian lines. We accordingly find that the
primary object of the first developmental processes in the
mammals has come to be the formation of a vesicle with a
complete cellular wall, capable of absorbing nutrient fluid from
the maternal uterus and of growing rapidly, so as to provide
the space necessary for embryonal differentiation.

VOL. 56, PART 1.—NEW SERIES. 8

114 j. 2. HILL.

In the Monotremes this vesicular stage is rapidly and
directly attained as the result, firstly, of the rearrangement
of the blastomeres of the cleavage-disc to form a unilaminar
blastodermic membrane overlying the solid yolk, and, secondly,
of the rapid extension of the peripheral (extra-embryonal)
region of the same, in contact with the inner surface of the
firm sphere furnished by the egg-envelopes. During the
completion of the blastocyst embryonal differentiation remains
in abeyance, and practically does not start until after growth
of the blastocyst is well initiated.

In the Marsupial, notwithstanding the fact that the ovum
has become secondarily holoblastic, the mode of formation
of the blastocyst is essentially that of the Monotreme.
Cleavage is of the radial type, and owing to the persistence
of the shell, which with the zona forms a firm resistant
sphere enclosing the egg, the radially arranged blasto-
meres are able to assume the form of an open ring and to
proceed directly to the formation of the unilaminar wall of
the blastocyst. The enclosing sphere provides the necessary
firm surface over which the products of division of the upper
and lower cell-rings of the 16-celled stage can respectively
spread towards opposite poles, so as to directly constitute the
formative and non-formative regions of the blastocyst wall.
In my opinion it is the persistence of the resistant shell-
membrane round the ovum which conditions the occurrence
in the Marsupial of this direct method of blastocyst formation.
As in the Monotreme, so here also embryonal differentiation
commences only after the blastocyst has grown considerably
in size.

In the Eutheria, on the other hand, in the absence of the
shell-membrane, not only is the mode of formation of the
blastocyst quite different to that in the Marsupial, but
the relations of the constituent parts of the completed
structure also differ markedly from those of the homo-
genous parts in the latter. The cleavage process here leads
only indirectly to the formation of the blastocyst, and must be
held to be ceenogenetically modified as compared with that of

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 115

lower mammals. In the cross-shaped arrangement of the
blastomeres in the 4-celled stage, in the occurrence of a
definite morula-stage and of the entypic condition, we have
features in which the early ontogeny of the Eutheria differs
fundamentally from that of the Metatheria. They are inti-
mately correlated the one with the other, and are met with in
all Kutheria, so far as known, but do not occur either in the
Prototheria or the Metatheria, so that we must regard them
as secondary features which were acquired by the primitive
Eutheria under the influence of some common causal factor
or factors, subsequent to their divergence from the ancestral
stock common to them and tothe Metatheria. Now the cross-
shaped 4-celled stage and the morula-stage are undoubtedly
to be looked upon simply as cleavage adaptations of prospective
significance in regard to the entypic condition, so that the
problem reduces itself to this—how came these adaptations
to be induced in the first instance? In view of the facts that
in the Metatheria, in the presence of the shell-membrane, the
formation of the blastocyst is the direct outcome of the cleavage
process, and is effected along the old ancestral lines without
any enclosure of the formative cells by the non-formative,
whilst in the Eutheria, in the absence of the shell-mem-
brane, blastocyst formation results only indirectly from the
cleavage-process, is effected in a way quite different from
that characteristic of the Metatheria, and involves the
complete enclosure of the formative by the non-formative
cells, I venture to suggest that the cleavage adaptations
which result in the entypic condition were acquired in the first
instance as the direct outcome of the total loss by the already
greatly reduced Kutherian ovum of the shell-membrane.!
This view necessarily implies that the presence of a thick
zona such as occurs round the ovum in certain Eutheria is
secondary, and what we know of this membrane in existing
Eutheria is at all events not adverse to that conclusion.

1 This suggestion I first put forward in a course of leciures on the
early ontogeny and placentation of the Mammalia delivered at the
University of Sydney in 1904.

116 Ji. 0. SEL

Amongst the Marsupials the zona is quite thin (about ‘0016
mm. in Dasyurus), presumptive evidence that it was also thin
in the ancestral stock from which the Meta- and Kutheria
diverged, whilst amongst the Eutheria themselves the zona,
as Robinson (’03) has pointed out, is not only of very varying
thickness, but persists round the ovum for a very varying
period in different species. It appears to be thinnest in the
mouse (‘001 mm.), in most Eutheria it is considerably thicker
(Ol mm., bat, dog, rabbit, deer), whilst in Cavia it reaches
a thickness of as much as ‘02 mm. In those forms in which
the blastocyst early becomes embedded in, or attached to, the
mucosa, the zona naturally disappears early. In the rat,
mouse and guinea-pig it disappears before the blastocyst is
formed. Hubrecht failed to find it in the 2-celled egge of
Tupaia, and it was already absent in the 4-celled stage of
Macacus nemestrinus, discovered by Selenka and de-
scribed by Hubrecht. On the other hand, it may persist for
a much longer period, up to the time of appearance of the
primitive streak (rabbit, dog, ferret). These facts suffi-
ciently demonstrate the variability of the zona in the EKutherian
series, and its early disappearance in certain forms before the
completion of the blastocyst stage shows that it can have no
supporting function in regard to that.

Postulating, then, the disappearance of the shell-membrane
and the presence of a relatively thin, non-resistant zona (with
perhaps a layer of albumen) round the minute yolk-poor ovam
of the primitive Eutherian, and remembering that the ovum
starts with certain inherited tendencies, the most immediate
and pressing of which is to produce a blastocyst comprising
two differentiated groups of cells, the problem is how, in the
absence of the old supporting sphere constituted by the egg-
envelopes, can such a vesicular stage be most easily and
most expeditiously attained ? The Eutherian solution as we see
it in operation to-day is really a very simple one, and withal a
noteworthy instance of adaptation in cleavage (Lillie, ’99).
In the absence of any firm supporting membrane round the
egg, and the consequent impossibility of the blastomeres pro-

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 117

ceeding at once to form the blastocyst wall, they are under
the necessity of keeping together, and to this end cleavage
has become adapted. For the ancestral radial arrangement
of the blastomeres in the 4-celled stage, characteristic of the
Monotreme and Marsupial, there has been substituted a
cross-shaped grouping into two pairs, and, as the outcome of
this adaptive alteration in the cleavage planes, there results
from the subsequent divisions, not an open cell-ring, as in the
Marsupial, but a compact cell-group or morula. In this we
again encounter precisely the same differentiation of the
blastomeres into two categories, respectively formative
(embryonal) and non-formative (trophoblastic) in significance,
as is found in the 16-celled stage of the Marsupial, but, since
the two groups of cells are here massed together, and in the
absence of any firm enclosing sphere, cannot spread inde-
pendently so as to form directly the wall of the blastocyst,
there has arisen the necessity for yet other adaptive modifi-
cations. Attention has already been directed to the tardiness
of differentiation in the embryonal region of the Mouotreme
and Marsupial blastocyst, and here in the minute Eutherian
morula we find what is, perhaps, to be looked upon as a
further adaptive exaggeration of this same feature in the
inertness which is at first displayed by the formative cells,
and which is in marked contrast with the activity shown by
the non-formative ectodermal cells.!. It is these latter, it

' The inertness of the formative cell-mass is accounted for by Assheton
(98, p. 251) as follows: “ Now, as the epiblast plays the more prominent
part in the formation of the bulk of the embryo during the earliest
stages, it clearly would be useless for the embryonic part to exhibit
much energy of growth until the old conditions {in particular sufficient
room for embryonal differentiation | were to a certain extent regained;
hence the lethargy exhibited by the embryonic epiblast in mammals
during the first week of development. No feature of the early stages of
the mammalian embryo is more striking than this inertness of the
embryonic epiblast —or, as I should now prefer to call it, simply epiblast
—during the first few days.” Assheton, it should be remembered, holds
that the inner cell-mass of Hutheria furnishes only the embryonal
ectoderm.

118 J. P. -HIUL.

should be recollected, which exhibit the greatest growth-
energy during the formation of the blastocyst in the Mono-
treme and Marsupial, and so their greater activity in the
Kutherian morula is only what might be expected. Dividing
more rapidly than the formative cells, they gradually grow
round the latter, and eventually form a complete outer layer
enveloping the inert formative cell-group. This process of over-
growth or epiboly is entirely comparable in its effect with the
spreading of the extra-embryonal region of the unilaminar
blastodermic membrane in the Monotreme to enclose the yolk-
mass, and with that of the non-formative cells in the Marsupial
to complete the lower hemisphere of the blastocyst, growth
round an inert central cell-mass being here substituted for
growth over the inner surface of a resistant sphere constituted
by the egg-envelopes, such as occurs during the formation of
the blastocyst in the Monotreme and Marsupial. Just as the
first objective of the cleavage process in the latter is to effect
the completion of the cellular wall of the blastocyst, so here
the same objective recurs, and is attained in the simplest
possible way in the new circumstances, viz. by the rapid en-
velopment of the formative by the non-formative cells. Thus
at the end of the cleavage process in the Eutherian we have
formed a solid entypic morula in which an inner mass of
formative cells is completely surrounded by an outer envelop-
ing layer of non-formative or tropho-ectodermal cells, homo-
genous with the extra-embryonal ectoderm of the Sauropsidan
and Monotreme and the non-formative region of the uni-
laminar blastocyst of the Marsupial. Conversion of the solid
morula into a hollow blastocyst capable of imbibing fluid
from the uterus and of growing rapidly now follows. Intra-
or intercellular vacuoles appear below the inner cell-mass, by
the confluence of which the blastocyst cavity is established,
and the inner cell-mass becomes separated from the envelop-
ing layer of tropho-ectoderm, except over a small area where
the two remain in contact.

The complete enclosure of the formative cells of the inner
cell-mass by the non-formative ectodermal cells of the

THE EKARLY DEVELOPMENT OF THE MARSUPIALIA. 119

enveloping layer which produces this peculiar entypic condi-
tion in the Eutherian’ blastocyst, I would interpret, then, as
a purely adaptive phenomenon, which in the given circum-
stances effects in the simplest possible way the early completion
of the blastocyst wall, and whose origin is to be traced to
that reduction in size and in its envelopes which the Eutherian
ovum has suffered in the course of phylogeny, in adaptation
to the conditions of intra-uterine development. In particular,
starting with a shell-bearing ovum, already minute and
undergoing its development in utero, I see in the loss of
the shell such as has occurred in the Kutheria an intelligible
explanation of the first origin of those adaptations which
culminate in the condition of entypy. Iam therefore wholly
unable to accept the view of Hubrecht (708, p. 78), that “what
Selenka has designated by the name of Entypie is—from
our point of view—no secondary phenomenon, but one
which repeats very primitive features of separation between
embryonic ectoderm and larval envelope in invertebrate
ancestors.”

I see no reason for supposing that the intimate relationship
which is early established in many Eutheria between the
trophoblastic ectoderm and the uterine mucosa has had any-
thing to do with the origination of the entypic condition. In
my view such intimate relationship involving the complete
enclosure of the blastocyst in the mucosa only came to be
established secondarily, after entypy had become the rule.
On the other hand, the peculiar modifications of the entypic
condition met with in rodents with “inversion” (e.g. rat,
mouse, guinea-pig) are undoubtedly to be correlated, as Van
Beneden also believed (’99, p. 332), with the remarkably early
and complete enclosure or implantation of the germ in the
mucosa such as occurs in these and other Eutheria. Similar
views are expressed by Selenka in one of his last contributions
to mammalian embryology. He writes (’00, p. 205)—“ Dass
die Entypie des Keimfeldes und die Blattinversion begiinstigt
wird durch die friihzeitige Verwachsung der Hiblase mit dem
Uterus, ist nicht in Abrede zu stellen. Aber da dieser

120 J.P. HILL.

Prozess auch in solchen Hiblasen der Saugetiere vorkommen
kann, die tiberhaupt nicht, oder erst spater mit dem Uterus
verwachsen, so kann die Keimfeld-Entypie zwar durch die
friihe Verwachsune veranlasst, aber nicht ausschhesslich
hervorgerufen werden.’ He goes on to remark that—“ Die
Vorbedingungenu zur Entypie miissen in der Struktur der ver-
wachsenden Wiblase gesucht werden,’ and expresses his
agreement with the views of Van Beneden as to the signifi-
cance to be attributed to the early cleavage phenomena in
Kutheria.

The attitude of the illustrious Belgian embryologist whose
loss we have so recently to deplore, towards this problem is
clearly set forth in the last memoir which issued from his
hand. ‘Je suis de ceux,” he wrote (’99, p. 332), “qui pensent
que toute ’embryologie des Mammiféres placentaires temoigne
qwils dérivent d’animaux qui, comme les Sauropsides et les
Monotrémes, produisaient des ceufs méroblastiques. Je ne
puis & aucun point de vue me rallier aux idées contraires formu-
lées et détendues par Hubrecht. L’hypothése de Hubrecht
se heurte a des difficultés morphologiques et physiologiques
insurmontables: elle laisse inexpliquée l’existence, chez les
Mammitéres placentaires, d’une vésicule ombilicale et d’une
foule de caractéres communs a tous les Amniotes et distinctifs
de ces animaux.” Holding this view of the origin of the
Eutheria, Van Beneden based his interpretation of their early
ontogenetic phenomena on the belief that ‘la reduction pro-
gressive du volume de l’ceuf d’une part, le fait de son
développement intrauterin de Vautre ont du avoir une in-
fluence prépondérante sur Jes premiers processus évolutifs.”

Balfour, in |is classical treatise, had already some eighteen
years earlier expressed precisely the same view. ‘The
features of the development of the placental Mammalia,” he
wrote (‘Mem. Edn.,’ vol. ii, p. 289), “receive their most
satisfactory explanation on the hypothesis that their aucestors
were provided with a large-yolked ovum like that of Saurop-
sida. ‘The food-yolk must be supposed to have ceased to be
developed on the establishment of a maternal nutrition through

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. Lt

the uterus. . . . The embryonic evidence of the common
origin of Mammalia and Sauropsida, both as concerns the
formation of the layers and of the embryonic membranes is
as clear as it can be.”

That view of the derivation of the Mammalia receives, I
venture to think, striking confirmation from the observations
and conclusions set forth in the preceding pages of this
memoir, and from it as a basis all attempts at a phylogenetic
interpretation of the early ontogenetic phenomena in the
Mammalia must, I am convinced, take their origin. Such an
attempt I have essayed in the foregoing pages, with what
success the reader must judge.

ADDENDUM.

The memoir of Prof. O. Van der Stricht, entitled “ La struc-
ture de l’ceuf des Mammiféres (Chauve-souris, Vesperugo
noctula): Troisitme Partie” (‘Mem. de VPAcad. roy. de
Belgique,’ 2nd ser., t. ii, 1909), came into my hands only
after my own paper had reached its final form, and therefore
too late for notice in the body of the text. In this extremely
valuable contribution, Van der Stricht gives a detailed
account of the growth, maturation, fertilisation, and early
cleavage-stages of the ovum of Vesperugo, illustrated by a
superb series of drawings and photo-micrographs. All I can
do here, however, is to direct attention to that section of the
paper entitled ‘‘Phénoménes de deutoplasmolyse au pole
végétatif de Poeuf” (pp. 92-96), in which the author describes
the occurrence in the bat’s ovuin of just such a process of
elimination. of surplus deutoplasmic material as I have
recorded for Dasyurus. Van der Stricht’s interpretation of
this phenomenon agrees, I am glad to find, with my own.
He writes (pp. 92-93): ‘Ce deutoplasme rudimentaire, a
peine ébauché dans l’ovule des Mammitéres, parait étre
encore trop abondant dans l’ceuf de Chauve-souris, car ces
materiaux de réserve, en partie inutiles, sont partiellement
éliminés, expulsés de la cellule.”

122 J.P. HELE.

To this process of elimination of surplus deutoplasm he
applies the name “deutoplasmolyse,’ and states that “Ce
phénoméne consiste dans l’apparition de lobules vitellins
multiples, en nombre trés variable, a la surface du vitellus au
niveau du pdle végétatif. Ces bourgeons a peu pres tous de
méme grandeur, les uns étant cependant un pen plus volumi-
neux que les autres, apparaissent dans le voisinage des globules
polaires et présentent la structure du deutoplasme. Ils sont
formés de vacuoles claires, a ’intérieur desquelles on apercoit
parfois de petits grains vitellins, dont il 4 été question plus
haut. . . . Ce processus de deutoplasmolyse devient
manifeste surtout aprés l’expulsion du second globule polaire,
pendant la période de la fécondation. I] peut étre trés
accentué, au stade du premier fuseau de segmentation et au
début de la segmentation de Pceuf, notamment sur des ovules
divisés en deux et en quatre (figs. 59, 61, 62, d).”’. It would
therefore appear that, whilst in Dasyurus the surplus deuto-
plasm is eliminated always prior to the completion of the
first cleavage and in the form of a single relatively large
spherical mass, in Vesperugo it is cast off generally, though
not invariably, before cleavage begins, and in the form of a
number of small separate lobules.

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‘08. ———— “The Blastocyst of Capra, with Some Remarks upon the
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‘09. ——— “Professor Hubrecht’s Paper on the Early Ontogenetic
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“04.
08.

00.

06.

°O1.

°02.

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 123

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de Anat. et de la Physiol.,’ t. xxxi.

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“Onthe Fetal Membranes, Placentation and Parturition of
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“ Studies in Mammalian Embryology: (1) The Placentation
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“Furchung und Keimblattbildung bei Tarsius Spectrum,”

‘Verhand. Kon. Akad. v. Wetensch. Amsterdam,’ vol. viii.

“ The Trophoblast,” ‘ Anat. Anz.,’ Bd. xxv.

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Bearing on our Interpretation of the Phylogeny of the Verte-

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“The Foetal Membranes of the Vertebrates,” ‘ Proc.
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Jenkinson, J. W.—* A Re-investigation of the Early Stages of the
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“ Remarks on the Germinal Layers of Vertebrates and on
the Significance of Germinal Layers in General,” ‘Mem. and
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Keibel, F.—‘* Die Gastrulation und die Keimblatthbildung der
Wirbeltiere,” ‘Ergebnisse der Anatomie und Entwickelungs-
geschichte’ (Merkel u. Bonnet), Bd. x.

“Die Entwickelung der Rehes bis zur Anlage des Meso-

blast,” ‘ Arch. fiir Anat. u. Physiol. Anat. Abth.’

124 je ae. Le

‘07. Lams, H., and Doorme, J.—‘ Nouvelles recherches sur la Matura-
tion et la Fécondation de l’ceuf des Mammiféres,”’ ‘ Arch de Biol.,’
1 ap-6-680

‘03. Lee, T. G.—‘* Implantation of the Ovum in Spermophilus
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99. Lillie, F. R.—* Adaptation in Cleavage,’ ‘ Biol. Lect. Wood’s
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09. MacBride, E. W.—* The Formation of the Layers in Amphioxus
and its bearing on the Interpretation of the Early Ontogenetic
Processes in other Vertebrates,” *‘ Quart. Journ. Mier. Sci.,’ vol. 54.

03. Robinson, A.—** Lectures on the Early Stages in the Development
of Mammalian Ova and on the Formation of the Placenta in
Different Groups of Mammals,” ‘Journ. of Anat. and Physiol.,’
vol. xxxviil.

86. Selenka, E.—* Studien ther Entwickelungsgeschichte der Thiere,’
IV (1 and 2), * Das Opossum (Didelphys virginiana), Wies-
baden.

‘91. ——— * Beutelfuchs und Kanguruhratte; zur Entstehungs-
geschichte der Amnion der Kantjil (Tragulus javanicus) ;
Affen Ost-Indiens,” ‘ Studien tther Entw. der Tiere, H. 5, Erste
Halfte.

°00. ——— ‘Studien wher Entw. der Tiere,” H. 8, Menschenaffen.
“TIT, Entwickelung des Gibbon (Hylobates und Siamanga),”
Wiesbaden: C. W. Kreidel.

"94. Semon, R.—* Zur Entwickelungsgeschichte der Monotremen,”
‘Zool. Forschungsreisen im Australien, ete.,’ Bd. ii, Lief 1.

95. Sobotta, J. * Die Befruchtung und Furchung des Eies der Maus,”
‘ Arch. fiir Mikr. Anat.,’ Bd. xlv.

7). Van Beneden, E.—* La Maturation de l’ceuf, la fécondation et les
premicres phases du développement embryonnaire des Mammi-
feres d’aprés les recherches faites sur le Lapin,” ‘ Bull. de Acad.
roy. des sciences, des lettres, et des beauxarts de Belgique,’ t. xl.

*80 ——— “ Recherches sur Pembryologie des Mammiféres, la forma-
tion des feuillets chez le Lapin,” ‘ Arch. de Biologie,’ t. i.

‘99 ——— “Recherches sur les premiers Stades du développement du
Murin (Vespertilio murinus),” ‘Anat. Anz.,’ Bd. xvi.

03 Van der Stricht, O.—*‘ La Structure et la Polarité de l'ceuf de
Chauve-Souris (V. noctula),” ‘Comptes rendus de I’ Association
des Anatomistes, V* Session, Liége.’

‘04. ——— * La Structure de l’euf des Mammitéres. Premiére partie,
L’oocyte au stade de Vaccroissement,” ‘Arch. de Biologie,’
t. Xxi,

THE EARLY DEVELOPMENT OF THE MARSUPIANLIA. 125

05 Van der Stricht, O.—‘ La Structure de lwuf des Mammifeéres.
Deuxieme partie, Structure de lceuf ovarique de la femme,” ‘ Bull.
de l’ Acad. Roy. de Médicine de Belgique, Séance du 24 Juin, 1905.

‘97 Wilson, J. T., and Hill, J. P.—* Observations upon the Develop-
ment and Succession of the Teeth in Perameles; together with
a Contribution to the Discussion of the Homologies of the Teeth
in Marsupial Animals,” ‘ Quart. Journ. Micr. Sci.,’ vol. xxxix.

03 — “Primitive Knot and Early Gastrulation Cavity co-
existing with independent Primitive Streak in Ornithorhynchus,”
‘Proc. Roy. So0c.,, vol. bexi.

07 * Observations on the Development of Ornithorhyn-

chus,” ‘ Phil. Trans. Roy. Soc.,’ Series B, vol excix.

EXPLANATION OF PLATES 1-9,

Illustrating Prof. J. P. Hill’s paper on “The Early Develop-
ment of the Marsupialia, with Special Reference to the
Native Cat (Dasyurus viverrinus).”

[All figures are from specimens of Dasyurus, unless otherwise indi-
cated. Drawings were executed with the aid of Zeiss’s camera lucida,
except figs. 61-63, which were drawn from photographs. |

List oF COMMON REFERENCE LETTERS.

Abn. Abnormal blastomere, fig. 37. alb. Albumen. cg. Coagulum.
d. p. Discus proligerus. d.z. Deutoplasmic zone. emb. a. Embryonal
area. emb.ect. Embryonal ectoderm. ent. Entoderm. ff. ep. Follicular
epithelium. f. a. Formative area of blastocyst wall. jf. c. Formative
cell. f. 2. Formative zone. 7. c. Internal cell, fig. 34. 1. ent. Limit of
extension of entoderm. J/. p. Incomplete area of blastocyst wall at lower
pole. p. b'. First polar body. p. b'. s. First polar spindle. p. b®. s.
Second polar spindle. p. s. Perivitelline space. s. m. Shell-membrane.
sp. Sperm in albumen. #7. ect. Non-formative or trophoblastic ecto-
derm (tropho-ectoderm). y. b. Yolk-body. z. p. Zona.

PLATE 1.

Fig. 1—Photo-micrograph (x 150 diameters) of the full-grown
ovarian ovum, ‘27 X *26 mm. diameter. The central deutoplasmic
zone (d. z.) and the peripheral formative zone (f. z.), in which the

126 Fo OP, ME

vesicular nucleus (‘05 x °03 mm. diameter) is situated, are clearly dis-
tinguishable. The zona (z. p.) measures ‘0021-0025 mm. in thickness.
Outside it are the follicular epithelial cells of the discus proligerus
(d. p.), which is thickened on the upper side of the figure, where it
becomes continuous with the membrana granulosa. (D.viv., 21. vii.
04, 42. Hermann’s fluid and iron-hematoxylin.)

Fig. 2.—Photo-micrograph (x 150) of ripe ovarian ovum (in which
first polar body is separated and second polar spindle is present, though
neither is visible in figure), "29 X ‘23 mm. maximum diameter. Follicle
14 x 1:1 mm. diameter. The ovum exhibits an obvious polarity.
Deutoplasmic zone (d. z.) in upper hemisphere; formative zone (/. 2.)
forming lower. (D. viv., 14, 26. vii. 02, =4,. Flemming’s fluid and
iron-hematoxylin.)

Fig. 3.—Photo-micrograph (x 150) of ripe ovarian ovum (‘28 x ‘24
mm. diameter) with first polar body (p. b!.) and second polar spindle.
First polar body, 026-03 x ‘Ol mm. Second polar spindle, ‘013 mm.
in length. (D.viv., 14, 26. vii. ’02, 2%. Flemming’s fluid and iron-

4—3°
hematoxylin.)

Fig. 4.—Photo-micrograph (x 256) of ovarian ovum in process of
growth (‘“pseudo-alveolar” stage). Ovum, ‘26 x ‘20 mm. diameter.
Zona, °0017—002 mm. in thickness. (D. viv., 14, 26. vii. ‘02, 4.
Hermann, iron-hematoxylin.)

Fig. 5.—Photo-micrograph (x 1250) of peripheral region of ripe
ovarian ovum (‘28 xX ‘126 mm. diameter) with first polar spindle (‘015
Xe Ol Sumi) sD evatives oo). will. 02: . Ohlmacher’s fluid, iron-hema-
toxylin.)

2-8

Fig. 6.—Photo-micrograph (x 1250) of peripheral region of ripe
ovarian ovum (‘26 x ‘18 mm.), showing first polar body (p. b'.) (‘03 x
006 mm.). (D. viv., 14, 26. vii. 02, +5. Flemming, iron-hematoxylin.)

Fig. 7.—Photomicrograph (x 1250) of peripheral region of ovum, fig.
3, showing portion of first polar body (p. 6'.), and the second polar
spindle. The dark body lying between p. b'. and the surface of the

ovum is a displaced red blood-corpuscle.

Figs. 8 and 9.—Photo-micrographs (x about 84) of unsegmented ova,
respectively ‘53 mm. and 35 mm. in diameter, from the uterus, taken
immediately after their transference to the fixing fluid (picro-nitro-
osmie acid), showing the shell-membrane (s. m.), laminated albumen
(alb.), with sperms (sp.), the zona (z. p.), perivitelline space (p. s.), and
the body of the ovum, with its formative (f. z.), and deutoplasmic (d. z.)
Zones 1(Devilsve, Lo, 19). yi. -O1:)

Fig. 10.—Photo-micrograph (x 150) of section of unsegmented ovum
almost immediately after its passage into the uterus, showing the very

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 127

thin shell-membrane externally (s.m.) (about ‘0016 mm. in thickness),
the albumen (a/b.), zona (z.’p.).and the dentoplasmie (d. z.) and formative
(f. z.) zones of its cytoplasmic body. The male pronucleus is visible in
the formative zone. Diameter of entire egg about ‘29 mm. (D. viv.,
15,19. vii. ’01, 3. Picro-nitro-osmic and iron-hzematoxylin.)

Fig. 11.—Photo-micrograph (x 150) of section of unsegmented ovum
from the uterus, slightly older than that of fig. 10. Diameter of entire
egg in fresh state 34—35.mm., of the ovum proper *5 X ‘28 mm.; thick-
ness of shell, ‘0024 mm. In the figure the female pronucleus is visible
near the centre of the formative zone (f.z.), and the male pronucleus
lies a little above it and to the right. The perivitelline space ( p. s.)
is partially occupied by coagulum. (D. viv., 21.v.°03,%. Hermann,
iron-hematoxylin.)

PLATE 2.

Fig. 12.—Photo-micrograph (x 150) of an unsegmented ovum from
the uterus, of the same batch as that of fig. 11, and 54 mm. in diameter.
The two pronuclei are visible in the central region of the formative
zone.

Fig. 13.—Photo-micrograph (x 350) of uterine ovum. Stage of first
cleavage spindle. Diameter, 315 mm. (D. viv., 1, 15.vii.’01, §.
Picro-nitro-osmic, iron-hematoxylin.)

Fig. 14.—Photo-micrograph (x about 78) of egg in the 2-celled stage,
taken immediately after its transference to the fixing fluid. Lateral
view. y.b. Yolk body. Diameter of entire egg about ‘34mm. (D. viv.,
1,15 .vii.’01. Picro-nitro-osmic.)

Fig. 15.—Photo-micrograph (x about 78) of another 2-celled egg,
seen from lower pole. Diameter, °35 mm. (D. viv., 4 B, 23. vi. ’02.
Perenyi’s fluid.)

Fig. 16.—Photo-micrograph (x about 78) of another 2-celled egg,
of the same batch as preceding. End view, showing one of the two
blastomeres and the yolk-body (y. b.).

Fig. 17.—Photo-micrograph (x 150) of vertical section of 2-celled
egg, ‘34 mm. in diameter, showing the shell-membrane (‘0064 mm. thick),
traces only of the albumen, the zona (z. p.), and the two blastomeres (the
left one measuring, from the sections, ‘16 x ‘18 x ‘10 mm., its nucleus
031 x ‘027 mm.; the right one, ‘16 x ‘19 x ‘09 mm., its nucleus, -03 x
‘028 mm.). Note the differentiation in their cytoplasmic bodies.
(D. viv., 6, 21. vii .’01, §. Picro-nitro-osmic and iron-hematoxylin.)

Fig. 18.—Photo-micrograph (x 150) of vertical section of 2-celled
egg, ‘32 mm. in diameter, with shell-membrane ‘005 mm. thick, showing

oD?
the two blastomeres, and enclosed between their upper ends the yolk-

128 TP Ls

body (y. b.). (D. viv., Wa Seecvan 2 OL, =. Picro-nitro-osmic, iron-haema-
toxylin.)

Figs. 19 and 20.—Photo-micrographs (x about 70) of 4-celled eggs
taken immediately after transference to Perenyi’s fluid. Fig. 19, side
view, showing yolk-hody (y.b.); fig. 20, polar view. Diameter of entire
egg about 35 mm. (D. viv., 14B,18.vi. 02. Perenyi.)

Fig. 21—Photo-micrograph (x about 70) of another 4-celled egg,
from the same batch as the preceding, seen from lower pole.

Fig. 22.—Photo-micrograph (x 150) of section of 4-celled egg of
same batch as those of figs. 19 and 20. The two right and the two
left blastomeres respectively form pairs, so that the plane of the first
cleavage is parallel with the sides of the plate, that of the second with
the top and bottom of the same. The two left blastomeres are still
connected by a narrow cytoplasmic bridge. Thickness of shell,
‘0072 mm.

Fig. 23.—Photo-micrograph (x 150) of a vertical section through
a 4-celled egg, 35 mm. in diameter, showing two of the blastomeres

and a small portion of the yolk-body (y.b.). Note, as in fig. 22, the
marked differentiation in the cytoplasm of the blastomeres. (D. viv.,
4,27. vi.’O1. Picro-nitro-osmic, iron-hxematoxylin.)

Figs. 24 and 25.—Photo-micrographs (x 140) of horizontal sections
through a 16-celled egg, “38 mm. diameter, fig. 24 showing the eight
larger, more yolk-rich cells of the lower (non-formative) ring, and fig. 25
the eight smaller, less yolk-rich cells of the upper (formative) ring.
Shell -0075 mm. in thickness, yolk-hbody (not included in the figures)
‘11 x (10 mm. in diameter. (D. viv., 3B, 26.vi.’01; 15, 8 and §.
Picro-nitro-osmic and iron-haematoxylin.)

Fig. 26.—Photo-micrograph (x 140) of a vertical section of an egg
of the same batch and size as that. represented in figs. 24 and 25, but
with seventeen cells—formative = 9 (6 + [1 x 2] + 1) in division ;
non-formative = 8. Two of the formative cells (f. ¢.) of the upper ring

are seen enclosing between them the faintly marked yolk-body (y. b.),
and below them two of the much more opaque non-formative cells
(tr. ect.) of the lower ring.

PLATE 3.

Fig. 27.—Photo-micrograph (x about 76) of the just completed
blastocyst, 39 mm. in diameter. From a spirit specimen. The dark
spherical mass (cg.) in the blastocyst cavity is simply coagulum, pro-
duced by the action of the fixative (picro-nitro-osmic acid) on the
albuminous fluid which fills the blastocyst cavity. (D. viv., 2 B,
16. vii. 01.)

THE FARLY DEVELOPMENT OF THE MARSUPIALIA. 129

Fig. 28.—Photo-micrograph (x about 76) of a blastocyst of the same
batch as the preceding, ‘45 mm. in diameter. From a spirit specimen.
eg. Coagulum.

Fig. 29.—Photo-micrograph (x about 75) of another blastocyst,
‘45 mm. diameter, of the same batch as the preceding, but taken
immediately after transference to the fixative. Viewed from the upper
pole. y.b. Yolk-body seen through the unilaminar wall.

Fig. 30.—Photo-micrograph (x about 75) of a blastocyst of the same
batch as the preceding, about ‘39 mm. in diameter, in which the cellular
wall has not yet been completed over the lower polar region.

Fig. 31.—Photo-micrograph (x 140) of a section of a_ blastocyst,
°39 mm. diameter, of the same batch as the preceding and at precisely
the same developmental stage, the cellular wall having yet to be com-
pleted over the lower polar region (/.p.). In the blastocyst cavity is
seen the yolk-body (y.b.) partially surrounded by a mass of coagulum
(cg.). (D. viv., 2B, 16.vii.’01, m. = 39, 2. Picro-nitro-osmic and
iron-hematoxylin.)

Fig. 32.—Photo-micrograph (x 140) of another blastocyst, “41 mm.
in diameter, of the same batch as the preceding, also with the cellular
wall still absent over the lower polar region. Shell-membrane ‘0075 mm.
in thickness. y. b. Yolk-body. ¢.g. Coagulum. The cellular wall
comprises about 150 cells.

Fig. 33.—Photo-micrograph (x 140) of a blastocyst of the same batch
as the preceding, with a complete unilaminar cellular wall. y.b. Yolk-
body, in contact with inner surface of wall, in the region of the upper
pole.

Fig. 54.—Photo-micrograph (x 100) of a section of a blastocyst
‘57mm.in diameter. ¢.c. Internalcell. (D.viv.,29. vi. ’04, 1°. Picro-
nitro-osmic.)

Fig. 35.—Photo-micrograph (x 100) of a section of a blastocyst, °73
mm. diameter, of the same batch as the preceding, shell, -0045 mm.
thick.

Fig. 36.—Photo-micrograph (x 100) of a section of a blastocyst °66
mm. diameter, of the same batch as the preceding. Lower hemisphere
opposite yolk-body (y. b.) formed of larger cells than upper. Hermann
fixation.

Fig. 37.—Photo-micrograph (x 140) of section of an abnormal
vesicle, ‘397 mm. diameter of the same batch as the normal vesicles
represented in figs. 27-33. abn. large binucleate cell, regarded as a
blastomere of the lower hemisphere which has failed to divide in normal
fashion, cf. text, p. 42.

VOL. 50, PART 1—NEW SERIES. 9

130 Te APS) AAD ade

PLATE 4.

Fig. 38.—Photo-micrograph (x 10) of entire blastocyst 4°5 mm. dia-
meter to show the junctional line (j. /.) between formative and non-
formative regions. From a spirit specimen. (D.viv., 6, 25. vii. ‘01.
Picro-nitro-osmic.)

Fig. 39.—Photo-micrograph (x about 10) of an entire blastocyst,
4:5 mm. diameter with distinct embryonal area (emb.a.). (D. viv., 5,
TS Asya: (O12)

Fig. 40.—Photo-micrograph (x 10) of entire blastocyst about 5 mm.
diameter showing embryonal area (emb. a.), peripheral limit of ento-
derm (/. ent.), and the still unilaminar region of the wall (tr. ect.). (D-
Vives Oo iviie 2.)

Fig. 41.—Photo-micrograph (x 150) of an in toto preparation of the
wall of a blastocyst of 3°55 mm. diameter. (D. viv., 16, 21. vii . 01.)

Fig. 42.—Photo-micrograph (x 150) of an in toto preparation of the
wall of a blastocyst of 3°25 mm. diameter. 7. /. Junctional line between
the formative (f. a.) and non-formative (tr. ect.) regions of the wall.
(D. viv., 24. vii. 01.)

Figs. 43 and 44.—Photo-micrographs (xX 150) of in toto preparations
of the wall of 45 mm. blastocyst showing the junctional line between
the formative (f. a.) and non-formative (tf. ect.) regions. (D. viv.,
B, 25 .vii.’OL. Picro-nitro-osmic and Ehrlich’s hematoxylin )

Fig. 45.—Photo-micrograph (x 150) of a corresponding preparation
of the wall of a more advanced 45 mm. blastocyst (°99 stage), in which
the two regions of the wall are now clearly distinguishable. (D.viv.,
8.7.°99. Picro-nitro-osmic, Ehrlich’s hematoxylin.)

Fig. 46.—Photo-micrograph (x 150) of a corresponding preparation
of a slightly more advanced blastocyst (04 stage). (D.viv.,6.7. 704.
Picro-nitro-osmic, Ehrlich’s hematoxylin.)

PLATE 5.

Fig. 47.—Photo-micrograph (x 150) of an in toto preparation of the
formative region of a 6.7.04 blastocyst, showing the proliferation
of spherical internal cells referred to in the text, p. 53.

Fig. 48.—Photo-micrograph (x 150) of an in toto preparation of the
wall of a vesicle of the same batch as that represented in fig. 39, in
which a small part of the junctional line between the embryonal ecto-
derm and the extra-embryonal (tr. ect.) is visible, the free edge of the
entoderm (evt.) not having reachedit. (D.viv.,5, 18. vii. ’01. Picro-
nitro-osmic, Ehrlich’s hzmatoxylin.) ;

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 131

Fig. 49.—Photo-micrograph (x 150) of a corresponding preparation
of a vesicle of the same batch as the preceding, in which the wavy and
irregularly thickened free edge of the entoderm (ent.) practically
coincides with the junctional line and so conceals it from view.

Fig. 50.—Photo-micrograph (x 150) of an in toto preparation of a
vesicle (8 . vi . ‘01 batch) viewed from the inner surface as in the corres-
ponding preceding figures. The entoderm in the region of the embryonal
area has been removed,so that one sees the inner surface of the embryonal
ectoderm (emb. ect.); it is still in situ, though not in a quite intact con-
dition over the adjoining portion of extra-embryonal ectoderm. The
entoderm has not yet extended over the region indicated by the reference
line to tr. ect., so that here the extra-embryonal ectoderm is clearly
visible. The junctional line is apparent. (D.viv.,8.vi.’01. Picro-
nitro-osmic. Ehrlich’s hematoxylin.)

Fig. 51 (Plate 5) —Photo-micrograph (x 310) of a section of a 30-
celled egg of Perameles obesula; egg b, :24 x ‘23 mm. diameter,
showing the unilaminar layer formed by the blastomeres.

Fig. 52 (Plate 3).—Photo-micrograph (x 240) of a section of a
blastocyst of P. nasuta ‘29 x ‘26 mm. diameter, showing the shell-
membrane (s.i.), zona (z.p.), and the unilaminar cellular wall. The
portion of the latter adjacent to the reference lines is composed of
smaller but thicker cells than the remainder.

PLATE 6.

Figs. 53 and 54.—Drawings ( x 84) of a 6-celled egg ‘34 mm. diameter,
fig. 53 showing a side view and fig. 54 a view from the lower pole.
Observe the characteristic ring-shaped arrangement of the blastomeres.
y. b. Yolk-body, the shell-membrane, albumen layer with sperms in-
cluded, and the zona are readily distinguishable. Outlines drawn with
the aid of the camera lucida immediately after transference of the egg
to the fixing fluid. (D. viv., 22,16. vii. ’01.)

Figs. 55 and 56.—Drawings (x about 88) of a 16-celled egg (about 37
mm. diameter) as seen from the side and lower pole respectively, from
the same batch as the eggs represented in figs. 24, 25, and 26. The charac-
teristic arrangement of the blastomeres in two superimposed, open
rings (each of eight cells) and the difference in size between the cells of
the two rings are evident. The irregular body (c.g.) seen in the cleavage
cavity in fig. 56 is a mass of coagulum. Drawn from a spirit specimen.
The albumen layer as represented in fig. 56 is too thick. (D. viv.,
3 B, 26. vi. ’O1.)

Figs. 57 and 58.—Drawings (xX about 85) of a 12-celled egg (38 mm.
diameter) as seen from the side and lower pole respectively. Four of

eye jap Eb,

the blastomeres of the 8-celled stage have already divided (4+ 4 x 2)
=12. From a spirit specimen and from same batch as preceding.

Fig. 59.—Drawing (xX about 88) of a 31-celled egg (°375 mm. diameter)
as seen from the lower pole. From a spirit specimen and from the same
batch as the preceding. The irregular body in the blastocyst cavity is
formed by coagulum. Formative cells = 16; non-formative = 14+ 1
in division.

Fig. 60.—Drawing ( X about 88) of another 51-celled egg (375 diameter)
from the same batch as the preceding. Side view.

Fig. 61.—Drawing (x 100) of an entire blastocyst (39 mm. diameter)

from the same batch as those shown in figs. 27-29.

Fig. 62.—Drawing (x about 80) of an entire blastocyst (4 mm.
diameter) from the same batch as the preceding.

Fig. 63.—Drawing (xX 80 of an entire blastocyst (‘6 mm. diameter)
made from a photograph taken directly after transference of the speci-
men to the fixing fluid. Cells of lower hemisphere with much more
marked perinuclear areas of dense cytoplasm than those of the upper.
Devive.2, Ub vai - 701.)

Fig. 64.—Section of the wall of a blastocyst, 2.4 mm. diameter
630). Dewi... 7.- vi. OL.)

Figs. 65, 66, 67.—Drawings (x 630) of small portions of in toto
preparations of the formative region of 6 . 7 . ‘04 blastocysts to demon-
strate the mode of origin of the primitive entodermal cells (ent., fig. 67).
Fig. 65 shows a dividing entodermal mother-cell in position in the
unilaminar wall, surrounded by larger lighter staining cells (prospective
embryonal ectodermal cells). In fig. 66 is seen a corresponding cell, a
portion of whose cell-body has extended inwards so as to underlie
(overlie in figure) one of the ectodermal cells of the wall. In fig. 67
are seen two entodermal cells, evidently sister-cells, the products of the
division of such a cell as is seen in figs. 65 or 66. One of them (the
upper) is still a constituent of the unilaminar wall, the other (ent.) is a
primitive entodermal cell, definitely internal. (D. viv.,6.7.°04, Picro-
nitro-osmic, Ehrlich’s hematoxylin.)

PLATE. 7.

Figs. 68, 69, 70.—Drawings (x 630) of portions of preparations
similar to the above. For description see text. (D. viv., 6, 7, ‘04.)

Fig. 71—Drawing (x about 630) of a portion of an in toto pre-
paration of the formative region of an 01 blastocyst showing two
primitive entodermal cells, one of them in division. (D. viv., B,
25. vii. °O1. Picro-nitro-osmic and Ehrlich.)

THE EARLY DEVELOPMENT OF THE MARSUPIALIA. 133

Fig. 72.—Drawing (x 630) corresponding to the above, from the
formative region of a 6.7 . ‘04 blastocyst, also showing two primitive
entodermal cells, evidently sister-cells.

PLATE 8.

Figs. 73, 74, 76.—Sections of the formative region of 6.7 . 04 blasto-
cysts, showing the attenuated shell-membrane, the unilaminar wall, and
in close contact with the inner surface of the latter, the primitive ento-
dermal cells (ent.) (x 630).

Fig. 75.—Section corresponding to the above, showing an entodermal
mother-cell (ent.), part of whose cell-body underlies the adjacent ecto-
dermal cell of the wall. The spheroidal inwardly projecting cell on the
left is probably also an entodermal mother-cell (x 630).

Fig. 77.—Section (x 630) of the non-formative region of a 6.7 . ‘04
blastocyst.

Fig. 78.—Section (x 630) of the embryonal area, and the adjoining
portion of the still unilaminar extra-embryonal region of a blastocyst of
the 5.01 stage. emb. ect. Embryonal ectoderm. ent. Entoderm. tr.
ect, Extra-embryonal ectoderm (tropho-ectoderm). The position of the
junctional line is readily recognisable. (D. viv., 5,18. vii.’01. Picro-
nitro-osmic and Delafield’s hematoxylin.)

Fig. 79.—Section (x 630) through the corresponding regions in an
8. vi. Ul blastocyst. Note the thickening of the embryonal ectoderm
(emb. ect.), and the peripheral extension of the entoderm (ent.) below
the tropho-ectoderm. (D. viv., 8. vi. ‘01. Picro-nitro-osmic and
Delafield. )

Fig. 80.—Section (x 600) through the formative (embryonal) region
of a blastocyst of P. nasuta, 13 mm. in diameter. It is thicker than
that of the Dasyure blastocyst at the corresponding stage of develop-
ment ; the primitive entodermal cells are well marked.

Fig. 81.—Section (x 600) corresponding to the above from another
13 mm. blastocyst of P. nasuta, of the same batch as the preceding,
but apparently very slightly earlier, the entodermal cells being still in
process of separating from the unilaminar wall. ent. Entoderm. tr. ect.
Tropho-ectoderm.

PLATE 9:

Fig. 82.—Section (x about 430) of a section of a blastocyst of M.
ruficollis 35 mm. in diameter, showing the major portion of the
formative region (f. a.) and a small portion of the non-formative (tr. ect.).

134 Je, PstI.

The shell-membrane varies in thickness in the sections from -005 mm.
over the former region to ‘003 mm. over the latter.

Figs. 83, 84, 85. —Drawings (x 630) of small portions of the formative
n fig. 83 of the adjoining portion of the non-formative) region of
the above blastocyst of M. ruficollis more highly magnified. ené.
Primitive entodermal cells. Note in fig. 83 a cell of the wall in division,
the axis of the spindle being oblique to the surface.

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NOTES ON A DEEP-SEA ECHIUROID. 135

Notes on a Deep-sea Echiuroid, Acantho-
hamingia shiplei (n. g. et n. sp.), with Re-
marks on the Species Hamingia ijimai,
Ikeda.

By
Dr. Iwaji Ikeda.

With Plate 10.

In July, 1909, I obtained an Echiuroid animal through the
kindness of Mr. Aoki, of the Marine Biological Station at
Misaki, Japan. As at that time the date of my departure
from Japan was near at hand I was compelled to bring with
me the specimen, which remained untouched until the end of
October, when I entered the Zoological Laboratory of the
University of Cambridge. After some weeks’ work in that
laboratory I discovered that this single specimen was a very
curious and undescribed species belonging to the Bonellide,
and apparently to the genus Hamingia. Doubt was, how-
ever, thrown upon relationship of the new formto Hamingia
by two remarkable features, the presence (1) of the abnor-
mally numerous ventral hooks and (ii) of the skin-papille.
‘The absence of the two structures just mentioned represents
the most essential generic characteristics of Hamingia, dis-
tinguishing the genus from the rest of the Bonellida,
Bonellia and Protobonellia.! Seeing many close rela-
tionships between the present species and Hamingia 1jimai,”

' Tkeda, I., “* Notes on a New Deep-sea Echiuroid, Protobonellia
mitsukurii,” ‘ Annot. Zool. Jap.,’ vol. vi, Part IV, 1908.

> Ikeda, IL, “On a New Echiuroid (Hamingia ijimai) from the
Sagami Bay,” ‘ Annot. Zool. Jap.,’ vol vii, Part I, 1908.

136 IWAJL IKEDA.

both in its general ‘‘ facies”? and its anatomy, I suspected
that the type-specimen of H. ijimai might possess the two
structures mentioned above, and I asked Mr. 8. Takahashi,
my colleague in the College of Hiroshima, to re-examine the
type-specimen. His reply was that H. ijimai has hooks
similar in nature to those of the present species, but no
skin-papille at all. Thus informed, I have decided to propose
the new genus Acanthohamingia for the reception of both
the present species and that which I formerly described as
Hamingia ijimai. The question of classification will be
more fully discussed later. The following contribution,
though brief and incomplete in many points, is produced
with the hope of adding something to our present knowledge
of the group of Bonellide. Before going further, I wish to
express my deep gratitude to Professor J. Stanley Gardiner,
F.R.S., and Mr. A. E. Shipley, F.R.S., through whose great
kindness and generosity I have been enabled to produce the
present study, being supplied with every necessary con-
venience of the laboratory. I also feel greatly indebted to
Mr. 8. Takahashi for his kindness in supplying me with the
prompt information for which I troubled him.

ACANTHOHAMINGIA SHIPLEI (n.g. etn. Sp.).

This unique specimen was taken, in January of 1909, from
the basin (the Okinosé) of the Sagami Bay at a depth of
400 fathoms, the same locality from which Protobonellia
mitsukurii and Hamingai ijimai had been procured.
Fig. 1, Plate 10, represents the animal in the preserved state
and the natural size, the ventral side being turned upwards.
As is shown in the figure, the skin of the body proper was
seriously broken, on the left side, by a hook of the long lne
with which the animal was caught. Through the wound
thus caused some loops, mostly torn, of the coiled intestine
protruded. he fact last mentioned has made it difficult or
almost impossible rightly to trace and identify different parts
of the entangled intestinal loops. The general measurements
of the animal are as follows:

NOTES ON A DEEP-SEA ECHIUROID. 137

Length of proboscis (measuredas fully stretched) 85 mm.
Maximum breadth of ee ; : a) 40
Length of body proper. ‘ ; . 62 mm.
Maximum breadth of body proper . 22

As regards the colour of the animal in the fresh state,
nothing could be ascertained beyond Mr. Aoki’s statement
that it was of a pale yellow colour; a faint tinge of this
remained after preservation.

To refer more fully to the external morphology of the
animal, the proboscis is of the usual shape, resembling that
of Hamingia ijimai. Itis deeply and widely grooved on
the ventral side along its whole length. Near the anterior
end it becomes abruptly narrowed and ends rather pointedly
(fig. 2). Towards the mouth the free margins come together,
partly overlapping each other, so as to form a funnel-shaped
passage directly leading to the mouth. ‘The whole surface of
the organ is smooth, but with many minute wrinkles.

Both extremities of the body proper are rounded. The
integument, which is rather thin and somewhat transparent
in the most swollen part of the body, is rather rough owing
to the presence partly of dense accumulation of skin-folds in
both terminal regions and partly of scattered papillary bodies
over the remaining parts. The skin-folds, which are about
O'1 mm. thick, are arranged in irregular transverse rows
something like the leaves of a book. In fig. 1 some con-
spicuous folds (or more properly, grooves separating the folds)
are represented. Arising close to the mouth there is a light
and narrow mid-ventral grooveabout 10 mm. long, which begins
anteriorly with a narrow, streak-like fissure and terminates
posteriorly in a comparatively broad depression. ‘This groove
is in my opinion of the same nature as that formerly described
by me in Hamingia ijimai. It might be named the genital
groove, since in both species it is practically concurrent with
the genital aperture, which is situated at the posterior angle
of the groove.

No special papilla around the genital aperture, such as

138 IWAJI IKEDA.

occur in Hamingia arctica,!

are found in the genital
groove.

The papillary bodies are scattered almost uniformly over
the non-wrinkled surface, but they are more densely crowded
at places where the skin is more contracted. When viewed
from the surface they appear as small opaque patches of
irregular shape and various size, which are separated from
each other mostly by numerous light, transverse furrows,
varying somewhat in length and in their course (fig. 3).
Most of them are nearly flat, but some of larger size
(measuring 0°3-0-4 mm. in diameter) are slightly elevated
above the skin-surface. Each structure (fig. 4) looks, if
seen by transmitted light, like a thick accumulation of very
small refringent granules heaped up into several smaller
irregular-shaped masses. Some of the granules are seen to
be of the same nature as those found dispersed throughout
the general surface of the skin. In the wrinkled regions,
also, the papillary bodies are constantly present, and are
crowded on to the very ridges of the transverse skin-folds.
The only difference which exists between them and the dis-
persed papillee is that they are a little smaller in size and
more irregular in shape.

The structure of the skin resembles that described by
Horst? in Hamingia arctica (=gracilis), that is, the
skin is composed of six distinct layers—the epidermis, the
cutis, the outer circular, the middle longitudinal, the inner
oblique muscles, and the peritoneal epithelium. But the
present species shows some points of peculiarity with regard
to the histology of the cutis and the epidermis. The cutis
(fig. 5, cew.), which is the thickest of all the dermal layers,
consists essentially of a gelatinous matrix, which is traversed
by very numerous and highly delicate fibrils (fb.) running

' Danielsen, D. C., and Koren, J., ‘The Norwegian North Expedition,
1876-1878; Gephyrea (Zoology), 1881, Christiania.’

> Horst, R., ‘Die Gephyrea gesammelt wahrend der zwei ersten
Fahrten des ‘ Willem Barent, ”’ * Niederland. Arch. fiir Zool.,’ suppl.,
Bd. 42, 1881.

NOLES ON A DEEP-SEA ECHIUROID. 139
radially across the whole thickness. Besides these fibrils,
the cutis contains small, mostly round, and rather sparsely
dispersed spaces (lac.), each of which encloses a very small
cell (c.). Although owing to the imperfect preservation I
have been unable to make out exactly the minute structure
of these corpuscle-like cells, yet, as far as my preparations
show, the latter are indubitably nucleated, and are provided
with a few slender processes reaching the sides of the
enclosing capsule. These cells are no doubt similar to the
cutis-corpuscles known in the cutis of Hamingia arctica
(Danielsen and Koren, and Horst); in this case, however,
the cells are not embedded in any special cavity. I am not
able at present to decide whether these lacuna-spaces are or
are not largely artefacts due to strong contraction of the
corpuscles themselves.

The epidermis is comparatively thin, consisting of a single
layer of cells, cubical or subcylindrical in shape, and of
unicellular glands of various sizes. Rather curiously, the
cuticle, which is clearly differentiatedin Hamingia arctica,
is not found as a distinct layer or, at least, cannot be distin-
guished from the epidermal! epithelium. Whereas in smooth
parts of the skin the gland-cells are in general small and only
sparsely distributed, in the papille they (p. gl.) are so con-
spicuously large and so closely aggregated that the real state
of their arrangement is often hardly perceptible. ‘The
papillary glands are very variable in size and shape and show
different structures, probably according to states of the
secretory activity of the cells ; hence the cell contents, which
stain deeply with hematoxylin, may be seen as a compact
mass, or aS an aggregation of minute granules, or as a more
hightly stained mass showing a reticular structure.

No pigment-granules are recognisable in any of dermal
tissues.

In connection with the skin, the ventral hooks may be
described. They are unusually numerous, that is, eight
instead of two. As in Bonellia miyajimai,! hitherto the

‘Ikeda, L., “On Three New and Remarkable Species of Echiuroids

140 IWAJI IKEDA.

only case in which this multiplicity of the ventral hooks has
been recorded in the Echiuroids, the hooks are comparatively
small (about 0°1 mm. in height), and are crowded together,
without showing any regular arrangement, at a spot nearly in
the middle of the genital groove (fig. 6). They are almost
transparent, light brown in colour, and have a slightly curved
sharp apex. Another point of peculiarity is shown by the
fact that the hooks, when examined from the inside of the
skin, are not borne on the usual muscular cushion provided
with radiating muscles, as in the case with all known Echiu-
roids. They are simply rooted in the thickness of the
epidermis, not even a slight bulge of skin being present. It
was the discovery of these hooks that made me feel the neces-
sity of a re-examination of the type specimen of Hamingia
ijimai, which has many points of agreement with the present
species. As I remarked in the introductory paragraphs, Mr.
Takahashi examined the type specimen for me and found out
ten small hooks in the middle of the genital groove. Fig. 7
is a Sketch of the hooks of Hamingia ijimai drawn by Mr.
Takahashi. Here we see the ten hooks directed posteriorly
and crowded irregularly. Judging from this figure, which
was drawn by means of Abbe’s Zeichenapparat, it will be
seen that these hooks of Hamingia ijimai are straighter
and larger than those of the present species. As these hooks
in both species are minute and embedded in the narrow
streak-like groove, | unfortunately overlooked them when I
was examining the type specimen of Hamingia ijimai.

Referring to the internal anatomy, the animal shows some
noteworthy characteristics.

As some loops of the alimentary canal were, as before
stated, severed off, the real state of connection of these torn
loops with others still remaining in the natural position, and
further, the actual and relative lengths of the three tracts
(fore-, mid-, and hind-guts) of the whole canal, could not be
made out. As far as the general characters of the canal are

(Bonellia miyajimai, Thalassema tenioides, and T. elegans),”
‘Journ. Coll. Sci. Imp. Univ., Tokyo, Japan,’ vol. xxi, art. 8, 1907.

NOTES ON A DEEP-SEA ECHIUROID. 141

concerned, the present species shows no striking characteristic
points as compared with the known allied forms of the
Bonellide. This similarity is especially remarkable if the
present species is compared with Hamingia ijimai, the one
interesting point of difference being the reversed or posterior
(in this species) instead of anterior position of the junction
between the fore and the hind guts. ‘This posterior shifting
of the junction of the two parts of the gut causes the extra-
ordinary elongation of the neuro-intestinal vessel.

On both sides of the posterior terminal part of the rectum
there are two bushy groups of anal glands. With regard to
the manner of branching of the organs, the present species
presents a remarkable point of identity to Hamingia ijimai,
for in both species the main tube or stem is multiplhed in
number. Fig. 8 represents the organ on the right side cut
short of all branches in order to bring forth more clearly the
relation which the organ bears to the rectum. ‘There four
larger and smaller main stems are seen clustered together at
their roots, which arise from the side walls of the rectum
almost independently from one another. ‘That stem standing
out most dorsally (hindmost in the figure) is the stoutest,
and gives off several secondary branches. ‘The organ on the
left side is essentially of the same nature as that just described,
the only difference being that the dorsal largest stem arises
more decidedly apart from the others. Each of these main
stems gives rise to numerous branches (from primary to
tertiary), to which comparatively large funnels are attached,
about three to eight to each terminal branchlet. All of the
primary as well as many of the larger secondary branches are
attached in their mid-way respectively by a thick fixing
muscle arising from the inside of the skin.

The vascular system of the body proper consists, as in
many other species of Echiuroids, of the ventral vessel, the
dorsal vessel, and the neuro-intestinal vessel. The ventral
vessel (fig. 9, v.v.) is supported by a conspicuously wide
mesentery (m.s.), arising from, and running along, the whole
length of the nerve-cord (v..). Reaching the posterior end

142 IWAJI IKEDA.

of the body, the ventral vessel and the mesentery do not
end with the nerve-cord, but run a little further over the
ventral surface of the rectum, very near the anus (see
fig. 8), so that they recall a feature somewhat resembling the
rectal mesentery known in some forms of the genus
Thalassema.

About 15 mm. behind the external aperture of the ovi-
duct (0. d., fig. 9) the ventral vessel gives off the neuro-
intestinal vessel (2.7. v7.), which is remarkably long, being
0-70 mm. ‘The extraordinary length of the vessel is, as
referred to before, correlated to the reversed posterior
position of the junction of the fore-gut with the mid-gut,
to which the vessel under consideration finds its first
attachment. A short way off (about 5 mm.) from this
attachment the vessel becomes split into two long branches
20 mm. long, which run parallel to, but entirely apart from,
the intestine. They are, however, connected to the colla-
teral intestinal (c. 7.) by means of a series of numerous
delicate muscle-fibres, which frequently branch towards the
vessels and end mostly with a small nodule-like swelling firmly
adhering to the surface of the vessels (see fig. 9). It is greatly
to be regretted that these two vessels could not be traced com-
pletely owing to the destruction of part of the mid-gut.

It is not less interesting to note that the dorsal vessel
does not arise, in the usual way, from the mid-gut, where
the neuro-intestinal branches are attached, but it originates
from a part of the fore-gut about 50 mm. anterior to the
beginning of the collateral intestine (see d.v., fig. 9). Under
these conditions, and since the hinder portion of the fore-
eut passes close to the pharynx (ph.), the dorsal vessel
has to run a very short way (about 10 mm.) to reach the
pharynx. At the point where it reaches the gut, the dorsal
vessel is seen to pass over to two villi-like ridges lying
side by side and directed posteriorly. No doubt these
structures are a part of the so-called heart, which in the
present case is not seen as such. Very probably the heart
may be present as a diffuse sinus-like space in the gut-walls,

NOTES ON A DEEP-SEA ECHIUROID. 143

extending between the roots both of the dorsal and the neuro-
intestinal vessels.

The single oviduct (od., fig. 9) is situated on the right side
of, and very close to, the ventral nerve-cord (v. 7.) Itis a
comparatively small tubular sac, measuring about 16 mm.
in length, and consists of four parts; the narrow and highly
muscular neck, the swollen glandular part, the thin-walled
reservoir, and the stalked funnel. As is the case with
Hamingia ijimai, the funnel, which is fimbriated in its
margin, springs from the very beginning of the reservoir.
There were no egg-cells, either in the interior of the reser-
voir or in the glandular part. The female gonad also was
not found either on the ventral vessel or at any other place.

This concludes the account of the anatomy of the female.
Lastly, a few words must be devoted to the parasitic males,
four of which were picked out of the glandular part of the
oviduct of the female. With regard to the three whole males,
one was broken to pieces while being removed ‘They are
3:8-4-2 mm. long and about 0°15 mm. thick (at the broadest
part). The anterior end is slightly broader than the posterior.
The whole surface is uniformly covered with cilia. There
are no ventral hooks or any other sort of spines. Thus it is
found that these males possess very nearly the same external
characters as those of the males of Hamingia ijimai. So
also in their internal anatomy both males of the two species
seem to be almost identical. Only points of slight difference
can be mentioned; these are:

(1) In the present species the body-cavity of the tail-region
extends a little further posteriorly than it does in Hamingia
ijimal.

(2) he alimentary canal is complete in the present species,
while in H. ijimai it consists of many discontinuous pieces.

(3) The sperm-reservoir in the present species is a little
longer than that of H. ijimat.

But none of these features seem to be of decisive specific
characters. We may naturally expect a close morphological
similarity between two such forms as H. ijimai and A.

144. IWAJI IKEDA.

shiplei, for here are two causes promoting similarity,
namely, the close specific relationship on one hand, and the
similar degenerative processes due to parasitism on the other.

The description so far given sufficiently indicates that the
present species is a member of the family Bonellidz and is more
closely related to the genus Hamingia than to Bonellia or
Protobonellia. Italso plainly indicates that in the present
species several important generic characteristics of Hamingia
as diagnosed by Danielsen and Koren! are absent. Thus in
the female of Hamingia the ventral hooks and the skin-
papille are absent, the anal glands are of the ordinary number,
or two, and, in the male,” the ventral hooks are present. The
rest of the generic characteristics of Hamingia, for instance,
the shape of the proboscis, the texture of the skin, the number
of the oviducts, and the sexual dimorphism, are not peculiar
to the genus, since some or nearly all of them may be recog-
nised in the genera Bonellia and Protobonellia. ‘Thus
compared, it becomes obvious that the present species does
not belong to the genus Hamingia. The multipled con-
dition of the anal glands, and the fact that the ventral hooks
lack a muscular sheath as well as radial muscles, are certainly
two interesting characteristics which accurately distinguish
the present species from every known Bonellian Kchiuroid
except Hamingia ijimai. The latter species is that with
which I made the erroneous generic identification, chiefly
owing to having overlooked the presence of the small hooks
in the female form. ‘lhe hooks which are now found in the
two species in the same condition seem to be strikingly
different from those known in other Echiuroids in one
important point, that is, they are in an extremely abnormal,
and, very probably, degenerative state of existence. In
Bonellia miyajimai, which has hitherto represented the
single case known of the acanthous abnormality, the abnor-

1 Vide note on p. 138.
2 The male was not known to Danielsen and Koren, but was discovered
and described by Sir Ray Lankester, ‘Ann. and Mag. Nat. Hist.,’ 1883,

xl, pp. 37-45.

NOTES ON A DEEP-SEA ECHIUROID. 145

mality seems hardly to imply a degeneration, as it causes no
essential change in the structure of the whole hook-apparatus,
except in the number of hooks. But it seems to me very
probable that even this kind of abnormality actually indicates
a certain phase antecedent to the total degeneration of the
hooks as known in Bonellia misakiensis! or in the genus
Hamingia, if we take into consideration the present case in
which the abnormality in number is coupled with the entire
absence of the muscular apparatus. If considered from the
point of view of the ventral hooks only, the two species, which
are similarly characterised, may be looked upon as if they
were an intermediate form between Protobonellia and
Hamingia, both of which have the ordinary proboscis. But
such a view as the above cannot be maintained if we take
into consideration the anomalous anal glands possessed by
the two species, because such a multiplied condition of the
organs could not be regarded as an intermediate characteristic.
Besides, we see in the embryology of the group Echiuroids
that the anal glands arise at first as two paired in-growths of
the ectoderm near the larval anus.

The facts and considerations stated above point to the
conclusion that the present species and that which I described
formerly as Hamingia ijimai are to be classed as a genus
distinct from any already existing in the Echiuroids. I pro-
pose to call the new genus Acanthohamingia and the two
allied species respectively as Ac. ijimaiand Ac. shiplei,
the new species being named in honour of Mr, A. E. Shipley,
F.RS.

The new genus may be diagnosed as follows :

ACANTHOHAMINGIA.

A sexually dimorphic Echiuroid.
Female.—The shape of the proboscis is much like that of
Thalassema; the skin is thin and delicate in texture, with

' Tkeda, I., “ The Gephyrea of Japan,” ‘ Journ. Coll. Sci. Imp. Univ.
Tokyo, Japan,’ vol. xx, art. 4, 1904.

VOL. 56, PART 1.—NEW SERIES. 10

146 IWAJI IKEDA.

or without papille, which are but poorly developed; the
genital opening lies in a narrow longitudinal groove of the
skin (the genital groove), in which also lie crowded numerous
ventral hooks wanting the muscular sheath and radial
muscles; the anal glands are more than two in number and
branch off several times before ending in funnels ; the oviduct
is one and unpaired, with a stalked funnel.

Male.—The body is long and slender, the whole surface
being uniformly ciliated. No ventral hook is present; the
spermatic duct is long and wide, with a single funnel opening
to the body-cavity.

The two species are briefly described as follows :

ACANTHOHAMINGIA SHIPLEI, nN. S.p.

A deep-sea Hamingia-like Hehiuroid. The proboscis
is long and narrow, and ends with a rather abruptly pointed
tip. The skin is thin and partly semi-transparent, and’ is
covered with small papilla: poorly developed. In the middle
part of the genital groove are rooted and crowded the small
and numerous hooks with a curved and pointed apex. The
neuro-intestinal vessel is disproportionately long ; the dorsal
and neuro-intestinal vessels arise from the gut at two widely
separated places. ‘The anal glands consist of four main
stems on one side. The single oviduct and the parasitic
males are of the same form and structure as those of

Ac. ijimai.

ACANTHOHAMINGIA IJIMAI, IKEDA.
Synonym: Hamingia ijimai, Ikeda.

A deep-sea Hchiuroid having nearly the same external
feature as that of the preceding species. The proboscis ends
with a rounded margin. The skin is thin, semi-transparent,
and devoid of any sort of papille. The ventral hooks, which
are crowded in the middle of the genital groove, are less
curved and larger than in Ac. shiplei. The anal glands

NOTES ON A DEEP-SEA ECHIUROID. 147

consist of three main stems on one side, arising widely apart
from each other and from the rectum. The single oviduct is
of the same shape and position as that of Ac. shiplei. The
males have a Nematode-like shape. The whole surface is
uniformly ciliated. No ventral hook is present. The
spermatic duct is long and wide, ending with a single
funnel.

ZOOLOGICAL LABORATORY,
UNIVERSITY OF CAMBRIDGE;
May, 1910.

EXPLANATION OF PLATE 10,

Illustrating Dr. Iwaji Ikeda’s “Notes on a Deep-sea Echiu-
roid, Acanthohamingia shiplei (n. g. et n.sp.), with
Remarks on the Species Hamingia ijimai, Ikeda.”

Fig. 1.—Ventral view of the animal ; natural size.

Fig. 2.—Ventral view of the proboscis tip; magnified about five
times.

Fig. 3.—Surface view of the skin in the middle part of the body, to
show the arrangement of the papille ; magnified about fifteen times.

Fig. 4.—A magnified view of a papilla of a larger size.

Fig. 5.—Transverse, slightly obliquely cut section of the skin, passing
through a papilla: seen with oc. 2 and ob. D (Zeiss) ; ¢., corpuscular
cell in a Jacunar space (lac.) ; ¢. m., circular muscles ; c2., cutis ; ep., epi-
dermal epithelium ; fb., fibrils in the cutis; p. gl., papillary gland-cells.

Fig. 6.—Ventral hooks in the genital groove; seen with oc. 2 and
ob. A (Zeiss).

Fig. 7.—Ventral hooks of Ac. ijimai; seen with oc. 1 and ob. AA
(Zeiss).

Fig. 8.—Rectum near the anus with the roots of the anal glands (on
the right-hand side) and the nerve-cord (n.) and ventral vessel (v. v.) ;
magnified about ten times.

Fig. 9—A sketch showing the oviduct (od.) and the blood-vessels in
situ; very slightly enlarged ; c.i., collateral intestine ; d.v., dorsal
vessel; f.g., fore-gut; m.g., mid-gut; m.s., mesentery supporting the
ventral vessel; 1.7. v.,neuro-intestinal vessel; ph., pharynx; v.2., nerve-
cord; v.v., ventral vessel.

Huth Litht, London.

Iwadi Ikeja,del.

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 149

A Study of the Blood of certain Coleoptera:
Dytiscus marginalis and Hydrophilus
piceus.

By
J. O. Wakelin Barratt, M.D., D.Sc.,
and
George Arnold, M.Sc.,

From the Cancer Research Laboratory (Mrs. Sutton Timmis Memorial),
University of Liverpool.

With Plate 11.

SYNOPSIS OF CONTENTS.

PAGE

Introduction. . . 149
Mode of Collecting Blood! 151
General Characters of the Blood ee Dytise us ane Hydro-

philus ‘ : se ol
Characters of the Blood- iene of Daeiee us : . 152
Characters of the Blood-plasma of Hydrophilus . . 155
Characters of the Blood-cells ; : . 158
Comparison with Mammalian Blood : A162
Literature P : : , = 163
Explanation of Plate : : . 164

InrRopUCTION.

THE present investigation had its origin in a study of the
cell changes occurring in malignant growths, during the
course of which attention was directed towards the presence
of wandering cells in such growths. It appeared likely that
light would be thrown upon the morphology and life-history
of the wandering cells of the higher vertebrates by comparison
with the free cells of the blood of various invertebrates. To
this end the present investigation, which is confined to
Coleoptera, was undertaken. ‘The work, however, extended
beyond the limits originally assigned, for it became necessary,

150 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

partly in order to prepare an isotonic fluid for the blood-cells
studied, and partly in order to determine the nature of the
medium in which they lived, to examine the fluid part of the
blood also.

The literature of the subject is scattered and appears to be
very scanty, so that further research in the light of the
more recent development of methods of investigation seemed
very desirable.

As early as 1864 Landois (1) studied the blood of insects,
noting the colour, smell, and reaction, and ascertaining the
presence of iron in the serum. He did not, however, give a
definite classification of the blood-cells, though he states that
division takes place by the nucleus usually splitting into two
parts.

The morphology of the formed elements in Molluscs and
Arthropods was further studied by Cattaneo (2, 1889) and
Wagner (3, 1890).

Cuénot (4, 1891) gave a voluminous but not very illu-
minating contribution to the literature of the blood of
invertebrates. This author observed that the blood of
Hydrophilus piceus is at first pale yellow, and when ex-
posed to the air becomes altered resembling caramel ; neither
uranidin, lutein nor fibrin is present ; the albuminoid present,
which coagulates at 60°—61°, is called heemopheine. ‘The blood
of Blaps, which is also pale yellow, and on oxidation becomes
quickly ochreous yellow, contains an albuminoid which is
regarded as identical with hamopheine.

An important observation in respect of the Coleoptera was
made by Durham (5, 1892), who ascertained that the blood-
cells of Dytiscus exhibited phagocytosis, readily ingesting
particles of Indian ink.

Reference may here be made to a much more exhaustive
examination of the coelomic Huid of Lumbricus by Lim Boon
Keng (6, 1896). This author found that the coelomic fluid
had a specific gravity of 1:007 to 1:009, and was of alkaline
reaction ; it also contained crystals, pigment and microbes,
and held cells in suspension, some of which exhibited phagocy-

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 151]

tosis. The latter were divided into—small non-granular, large
hyaline, small granular, large granular and chloragogen cells,
and also spindle cells.

Some interesting observations were made by Benham (7,
1901) on the ccelomic fluid of Acanthodrilids, which was
found to undergo a sort of coagulation on standing, becoming
white, sticky, and slimy. The cellular elements of the
cceelomic fluid are divisible according to Benham into four
groups; amcebocytes (granular cells), eleocytes (containing
fatty globules), lamprocytes (containing granules), and lino-
cytes (containing threads).

Hollande (8, 1909) divides the cellular elements of Coleoptera
into three groups: lymphocytes, granular leucocytes, and
leucocytes with spherules.

The Coleoptera selected for the present investigation have
been Hydrophilus piceus (Linn.) and Dytiscus margin.

alis (Linn.).

Mope or CoLiectinc Bioop.

In order to obtain blood from Hydrophilus and Dytiscus
the following procedure was adopted. The wing cases were
litted up and pinned aside in a paraffin dish. ‘The wings
were then divided with scissors, so as to display the dorsal
segments of the abdomen. One of the dorsal segments was
next opened at the side and a flap of chitin cut off after being
previously freed from adherent connective tissue. The blood
which was seen lying in the body cavity between the viscera
was then removed drop by drop by means of a capillary tube-
If this is carefully done it should be possible to withdraw
blood without damaging any organ or setting free any cells
derived from the body tissues.

GENERAL CHARACTERS OF 'HE BLoop oF DyTIscus AND
HYDROPHILUS.

The average amount of blood obtainable from Hydro-
philus piceus was ‘32.¢.c. The amount of blood obtained

£52 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

from five specimens (in April) was found to measure 1°6 c.c.
(average amount ‘32 ¢.c. from each) ; later in the same month
three specimens yielded °45 c.c. (average amount ‘14 c.c. from
each) ; on another occasion (in July) ‘26 c.c. per specimen was
obtained.

From Dytiscus the average amount obtainable was ‘10
c.c. As affording some idea of the range observable the
following data may be given: ‘42 c.c. obtained from three
specimens in February (average amount ‘14 c.c. from each) ;
375 c.c. obtamed from six specimens also in February
(average amount ‘065 c.c. from each) ; 1°5 ¢.c. obtained from
twelve specimens in March (average amount ‘108 ¢.c. from
each) ; 1°65 c.c. obtained from seventeen specimens in April
(average amount ‘10 c.c, from each).

The blood was found on centrifugalisation to consist partly
of fluid and partly of suspended material. The latter was
variable in different animals, but was relatively small both in
Hydrophilus and in Dytiscus, amounting in the observa-
tions made to about 1 per cent. (by volume) of the blood.

The suspended material consisted partly of cells, partly of
free granules. ‘The latter are described in detail below in
connection with the blood-plasma; the former are taken in
the succeeding section. The cells formed a relatively small
amount of the precipitate obtained on centrifugalisation, but
owing to the circumstance that the two constituents of the
precipitate cannot be separated, no quantitative comparison
of the two could be made.

CHARACTERS OF THE Broop-Prasma or Dyriscus MARGINALIS.

Colour and Spectroscopic Appearance.—The blood-
plasma immediately after removal was, in a layer four
millimetres thick, of a deep amber colour, subsequently
changing at the surface of contact with the air to dark brown,
almost black (well seen when the blood was kept in a narrow
pipette, the upper layer of liquid becoming deeply coloured,
while that below, where access of air was prevented, remained

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 153

unchanged). Since the blood darkened on exposure to air,
or rather to oxygen, it follows that it contained exceedingly
little dissolved oxygen in the straw-yellow condition which it
exhibited in the living body. On spectroscopic examination
of a layer six millimetres thick the portion of the spectrum
lying to the blue end of the green was completely cut off and
the green itself in part absorbed, but the red of the spectrum
was little altered. When the blood had become darkened
the spectrum became dim but no absorption bands were seen.
The brownish-black colour which the blood assumed on ex-
posure to air could not be removed by adding ammonium
sulphide.

Odour.—The blood immediately after collection had a
sweet smell somewhat resembling malt extract, but was also
distinctly offensive. A faint odour of free ammonia was
recognisable. On adding sodium hydrate and boiling, the
issuing vapour readily turned neutral litmus paper blue, thus
affording additional evidence of the presence of ammonia or
an ammonium salt (it will be seen below that carbon dioxide
was present in the blood-plasma).

Specific Gravity.—This ranged, in the specimens ex-
amined, from 1°025 to 1°027.

Reaction.—The blood when examined immediately after
collection was always found to be alkaline to litmus paper.

Basicity and Acidity.—Observations were made im-
mediately after removal of the blood from beneath thoracic or
abdominal tergites, great care being taken to avoid injury to
viscera. In every case it was found that the blood, which
was strongly alkaline to litmus on removal, still remained

, - N
alkaline on adding an equal volume of 30 HCl; on adding an
5)

equal volume of = HCl it became faintly alkaline ; on adding

an equal volume of a HCl it became acid to litmus paper.
The basicity of the blood-plasma (which is in part due to
ammonium carbonate) is therefore slightly greater than 1s

154 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

N
represented by a 30 solution of hydrochloric acid. As the

blood-plasma is alkaline to litmus its basicity cannot be
determined by adding potassium hydrate.
Coagulation.—No spontaneous coagulation of the blood
of Dytiscus marginalis occurred on standing.
Composition.—Vhe blood-plasma was found to contain
6°6 per cent. to 10°4 per cent. of solids, dried at 110° C. (1
c.c. of blood was taken for estimation of total solids).
On rendering the blood slightly acid with acetic acid and

then heating, it became solid. When the blood was diluted

for)
with three parts of distilled water or *85 per cent. solution of
sodium chloride, made slightly acid with a | per cent. solution
of acetic acid and boiled, a brown precipitate formed. After
centrifugalisation the supernatant liquid was found to remain
turbid (this being apparently due to the presence of ammonium
salt in the plasma), so that complete separation of the proteid
from the non-proteid solids of the plasma could not by this
means be effected. By weighing the brown precipitate after
drying at 110° C. it was found that the former was not less
than 1°3 per cent.; this figure has little value, however, since
the supernatant liquid contained proteid forming a gelatinous
mass as evaporation proceeded.

On adding blood to a large excess of distilled water turbidity
appeared, followed by the formation of a white precipitate,
showing the presence of globulin.

The dried solids of the plasma contained about 9 per cent.
of ash, which was of a brownish-white earthy aspect. Owing
to the small amount available further analysis of the ash was
not possible.

Osmotic Pressure.—The freezing-point of the blood-
plasma determined by Beckmann’s method (1°3 ¢.c. of blood-

plasma were employed) was — ‘77° C., corresponding to an
; M
undissociated 5, solution.
sv

Granular Material.—This consisted of granules 1 pu to
‘2 w in diameter, exhibiting Brownian movement and in part

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 155

precipitated on centrifugalisation. In addition numerous
ultra-microscopic particles of much smaller size were recog-
nisable on strong illumination against a dark background.
The former granules when a film of blood was prepared by
Leishman’s method (alcohol fixation, staining with methylene-
blue-eosin) stained blue.

Gases Dissolved in Blood.—1°65 c.c. of blood-plasma
(obtained from seventeen Dytisci) were placed in con-
nection with a Toepler pump and ‘14 c.c. of gases extracted.
On exposing this to the action of a 10 per cent. solution of
caustic potash the volume was reduced by ‘11 c.c. On
adding a 50 per cent. solution of caustic potash containing
2°3 per cent. of pyrogallol a very slight diminution of volume,
too small to determine, occurred, and ‘03 c.c. of gas remained
behind, representing nitrogen and argon. The percentage
amounts of dissolved gases were therefore :

Carbon dioxide . ; . 6°7 per cent.

Using ; «Q

Nitrogen 1°8 =
Total ; 2 Ore

CHARACTERS OF THE Brioop-PLasmMA oF HypropHitus PICEUs.

Colour and Spectroscopic Appearance.—Immedi-
ately after collection the blood was, in a layer four millimetres
thick, of a straw-yellow colour. Subsequently it became
dark brown, the change first appearing at the upper surface,
in contact with the air. When kept in hydrogen the blood
remained for several hours of a pale yellow colour, whence it
follows that the darkening is due to absorption of oxygen.
The tension of dissolved oxygen in the blood must, therefore,
be very low. On spectroscopic examination of the blood in a
layer 18 mm. thick a general darkening of the spectrum was
observed, the extent of the spectrum diminishing towards
both the red and the blue, but no absorption bands were
visible. When darkening of the blood occurred on standing

156 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

still further obscuration of the spectrum took place, but no
absorption bands appeared.

Odour.—The blood had a faint offensive odour resembling
decaying grass. No distinct odour of free ammonia could be
detected, but on adding the blood (collected from the hving
insect a few minutes before use) to a solution of caustic
potash (previously ascertained to be free from ammonia) and
boiling, the issuing steam readily turned neutral litmus paper
blue, showing the presence of an ammonium salt.

Specific Gravity.—This was found to be 1°012 (only one
specimen was examined).

Reaction.—The blood examined immediately after col-
lection was alkaline to litmus paper.

Basicity and Acidity.—The blood was tested imme-
diately after collection, great care being taken to avoid injury
to viscera during collection. The reaction remained slightly

5 G . 5 tb
alkaline to litmus when mixed with an equal volume of 50
5

HCl; when an equal volume of s HCl was added the reaction

: N
became neutral to litmus; when an equal volume of 30 HCl
was added the reaction became acid. The basicity of the

N
blood-plasma is therefore represented by a 40 solution of

hydrochloric acid. Since the blood had an alkaline reaction
its acidity could not be determined by the addition of caustic
potash. It is obvious that the basicity was, as in the case of
the blood-plasma of Dytiscus, in part due to the presence of
ammonium carbonate, already referred to.

Coagulation.—No spontaneous coagulation of the blood
occurred on standing.

Composition.—The blood-plasma contained 11:6 per
cent. of solid matter (43 c.c. of plasma was taken for the
estimation of total solids).

The plasma contained proteid coagulable on acidifying
with acetic acid and boiling, but as was the case with that of

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 157

Dytiscus, complete, precipitation did not occur, so that a
quantitative estimation of the amount of coagulable proteid
was not possible.

On diluting the blood with ten times its volume of distilled
water a copious white precipitate formed, showing the pre-
sence of globulin.

The dried solids of the plasma contained about 3 per cent.
of ash of a white, porous, earthy aspect. Owing to the small
amount of ash obtainable no determination of its composition
could be made.

Osmotic Pressure.—The freezing-point of the blood-
plasma, determined by Beckmann’s method (1 c¢.c. of fluid
was employed), was —*647° C., corresponding to an undisso-

solution.

)

\
ciated =

-

Granular Material.—This consisted of small particles,
exhibiting Brownian movement, *2 uw to 2 mw, in diameter,
the former being the more numerous. In addition ultra-
microscopic particles less than *2 4 in diameter could be
seen on strong illumination on a dark background. The
granules increased in number on standing; some of the
larger granules may have been derived from the disintegra-
tion of the blood-cells. The granules, in films fixed by
Flemming’s solution, stained by basic dyes.

Gases Dissolved in Blood-plasma.—By means of a
Toepler pump the dissolved gases contained in 1°6 c.c. of
blood-plasma (obtained from five Hydrophili) were collected
and were found to measure ‘09 c.c. After the absorption of
carbon dioxide by caustic potash the volume of gas was
reduced to ‘05 c.c. Very little further reduction could be
obtained by the action of pyrogallol in strongly alkaline
solution. The percentage of dissolved gases was therefore:

Carbon dioxide . : . 93°8 per cent.
Nitrogen. : : : £\ SES Ma

Total ; i : RS e7i

158 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

It will be noticed that no dissolved or loosely combined
oxygen was obtained from the blood-plasma of Dytiscus
and Hydrophilus. When oxygen was absorbed in vitro
the blood-plasma became darkly coloured. It follows, there-
fore, that as long as the blood-plasma remains straw-yellow
coloured the absence of dissolved oxygen may be inferred.
No data are, however, available to indicate the means
by which darkening of the circulating fluid is avoided
in the living insect. The blood appears to serve solely asa
nutritive medium. | ‘The tissue-cells, it may be observed, are
in direct relationship to the finest ramifications of the
tracheal vessels (9), which penetraté to all parts of the body
of these insects. From the tracheal vessels the tissue-cells
appear to derive their supply of oxygen directly, not being
dependent on the mediation of the blood-plasma as in
mammals and in animals living exclusively in water.

THE CHARACTERS OF THE BLOOD-CELLS.

The blood-cells were studied in films fixed in Flemming’s
strong solution, without previous drying, and also in dry films.
In addition, Flemming’s solution was added to the blood, and
the formed elements, after centrifugalisation, embedded and
cut in paraffin.

The stains chiefly used were Heidenhain’s iron-alum heema-
toxylin, Breinl’s methylenblue-saffranin-orange G. triple stain
and basic fuchsin-methylenblue-orange G. triple stain. Intra
vitam methylenblue staining was also employed.

In Dytiscus marginalis and Hydrophilus piceus
the blood consists of flocculent suspended material made up of
fine granules, about | » to *2 w in diameter, and of cells. ‘hese
latter are of two kinds—(1) phagocytes, and (2) small round-
cells! The number of cells counted varied from 120 to 500
per cubic millimetre in Dytisecus, and from 1030 to 4440
per cubic millimetre in Hydrophilus.

The phagocytic cells are usually spindle-shaped when seen

1 We have not observed blood-platelets in the plasma.

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 159

on edge, and round, with two polar prolongations, when viewed
from above. They measure in both Dytiscus and Hydro-
philus from 17 4 by 19 to 154 by 30. In Dytiscus the
cytoplasm of these cells is coarser and more largely vacuolated
than in Hydrophilus. The nucleus in Dytiscus has
a definite membrane and the chromatin is diffusely and
irregularly distributed. Faint strands of linin connect
together the chromatin masses. Generally only one nucleolus
is present (see figs. 1-4). In Hydrophilus a well-defined
nuclear membraue is also present, but otherwise the nucleus
is strikingly different in appearance to that of Dytiscus, for
instead of being distributed in unequal masses, as in the latter
insect, the chromatin occurs in the form of about twenty-five
to thirty nearly equal-sized aggregations, and these generally
appear to be split in one direction, giving the appearance of
twin masses of chromatin.’ The linin is inconspicuous (see
figs. 11-13). When these cells have ingested foreign particles
from the plasma they change their shape, gradually drawing
in their polar extensions and becoming more or less round
(see figs. 6-9 and 12-14). Both in the fresh and well-fixed
blood of Dytiscus and Hydrophilus it can be seen that
the majority of the phagocytes which contain no food-particles
or recent food-vacuoles in their cytoplasm possess the polar
prolongations. At all times the phagocytes may exhibit
short and thin pseudopodia extruded from various parts of
the cytoplasm, but the polar extensions, although of a more
permanent nature, are themselves only pseudopodia, and are
distinctive of that phase in the life of the cell in which no
ingestion and digestion occur.

The other kind of cell found in the blood is a small cell,
with large nucleus and very little cytoplasm (see figs. 10 and
18). These cells, for want of a more convenient term, we
designate as small round-cells. Asin the case of the phagocytes,

‘ This arrangement of the chromatin in twin groups is apparently
characteristic of the somatic cells of Hydrophilus. It can be seen,
for instance, in the Malpighian tube cells, in the cells of the glands of
the mid-gut, and in the spermatogonia.

160 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

the cytoplasm of these cells is coarser in Dytiseus than in
Hydrophilus. Small round-cells are present in the blood
in much smaller number than are phagocytic cells, varying
from one in fifty to one in thirty of the total number in
Dytiscus, and amounting to one in fifty or less in Hydro-
paalws.

In the phagocytic cells, a series of interesting changes
follow the ingestion of solid particles, which may now be
described in some detail.

In Dytiscus the ingestive activity of these cells is very
great. ‘Thus, if a solution of Indian ink be injected into the
abdominal cavity, it can be seen that after a few hours most
of the phagocytes have particles of the ink in their cytoplasm,
as is illustrated by fig. 4 (four and a half hours after injec-
tion). As digestion proceeds a clear area appears round
each particle, becoming a well-defined vacuole later on.
(see figs. 4, 6 and 8). <A part of the ingested matter is not
digested, being eventually ejected into the plasma. The
vacuoles slowly contract until they become indistinguishable
in the schaumplasma. The nucleus undergoes the following
changes during the digestion of ingested matter. The
chromatin becomes more plentiful (cf. figs. 6 and 7 with 1 and
4) ; the nuclear membrane invaginates here and there and
eventually the nucleus becomes multilobulate (see fig. 8), some
cells being even polynuclear (see fig. 9).

In Hydrophilus the changes which take place in the
cytoplasm after the ingestion of solid particles are similar to
those of Dytiscus. The nucleus undergoes only a slight
change, the chromatin becoming more diffuse and the linin
strands more apparent (cf. fig. 12 with figs. 13 and 14).
Lobulation of the nucleus does not occur. As in Dytiscus
some of the granules, representing probably indigestible
portions of the ingesta, are extruded from the cytoplasm and
discharged into the surrounding blood-plasma. ‘The staining
reaction of the solid ingesta changes, for in the cytoplasm at
first these take the acid stain, later they are stained by a
mixture of the acid and basic stains, and ultimately, at the

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 161

time of extrusion, the undigested residue becomes increasingly
basic in its staming reaction.

On examining with dark background illumination a drop of
blood immediately after removal, both forms of cells, phago-
cytes and small round cells, which are observed to be very
granular, are seen to exhibit very numerous fine pseudopodia,
about *2 in diameter and of length occasionally surpassing
that of the cell. No amoeboid movements are seen, but after
a time the granules of the protoplasm exhibit Brownian
movement, which ultimately ceases when death has occurred,
without the cell, however, becoming vacuolated as do human
leucocytes. At the time of death the pseudopodia have
become somewhat indistinct, their situation being indicated
by granules apparently derived from the protoplasm.

The mode of division of the phagocytes does not always
appear to be the same. In Dytiscus only amitotic division
was seen (fig. 5), and that comparatively rarely. In Hydro-
philus, however, mitotic divisions were fairly common (see
figs. 15 and 16), but no amitotic forms were met with. — It is
quite possible, however, that both forms of division occur in
the two genera examined, but we have nevertheless been
unable to observe either mitotic division in Dytiscus, or
amitotic division in Hydrophilus. We have not been able
to observe cell-division in the small round-cells.!

The origin of the free cells in the blood of insects has been
attributed to various sources by different writers. Cuénot
(10) and Balbiani (10) derive the leucocytes from the peri-
cardial cells. Schaffer (10) derives at least some of these cells
from the fat-body cells, but Kowalewsky (10) denies any
leucocyte formation to either the pericardial or fat-body cells,
asserting that the blood-cells arise from special nests or
islands of tissue near the heart.

1 Hollande (loc. cit.),in dealing with other Coleoptera, Coccinella,
Mysia and Epilachnia, describes phagocytes similar to, but consider-
ably smaller than, those above described. These reproduce by mitosis.
The other class of cells which he described in those species, viz. the * cel-
lules & sphérules,” is entirely absent in Dytiscus and Hydrophilus.

VOL. 56, PART 1.—NEW SERIES. 11

162 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

Our own observations do not permit us to make any con-
fident statement as to the origin of the blood-cells from any
of the tissues of the body. It appears to us, however, that
the evidence adduced in favour of the above suggested modes
of origin is altogether inadequate, for both the pericardial and
fat-body cells are totally different in aspect from the blood-
cells. hus the pericardial cells of Dytiscus and Hydro-
philus are considerably larger than the largest blood-cell,and
their nuclei are relatively smaller, while their cytoplasm is
very abundant. Again, if the blood-cells arose from any of
the above sources, transitional forms wonld be present ; these
we have failed to observe.

On the other hand, whatever other mode of origin of the
blood-cells may exist, it is clear, since division figures occur
in Hydrophilus and Dytiscus (mitotic and amitotic), that
the supply of these cells is kept up, in part at any rate, by
multiplication in the blood-plasma.

ComMPARISON WitH MamMALIAN BLoop.

In the above investigation we have applied the term “blood”
to designate the circulating fluid of Coleoptera. This fluid
presents, however, several important points of difference from
the blood of the higher mammalia. It will, therefore, be of
advantage briefly to contrast the characters of these two fluids
so far as the present limitations of knowledge permit a com-
parison to be made.

The blood of Dytiseus and Hydrophilus resembles mam-
malian blood in so far as it consists of an albuminous fluid
containing cells. The fluid part resembles mammalian blood-
plasma in containing proteid, coagulable by heat. Whether
more than one form of heat-coagulable proteid is present can-
not as yet be stated, but it may be observed that a globulin
precipitable by dilution with distilled water, and therefore
held in solution by the saline constituents of the fluid, is also
present as in mammalian blood. The cellular elements of
mammalian blood are represented in the blood of Dytiscus

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 163

and Hydrophilus by phagocytes and small round-cells. In
the phagocytes of Dytiscus fragmentation of the nucleus
occvrs, which is comparable to that seen in polynuclear
leucocytes.

Turning now to points of difference, the low osmotic
pressure of the blood of Dytiscus and Hydrophilus, repre-

sented by a a to (undissociated) solution, contrasts with
20 v4,

that of mammalian blood, represented by — (dissociated) solu-
/

tion. A more striking point of difference is the absence of
cells containing hemoglobin in the blood of the former,
which is also free from dissolved hemoglobin. Moreover, the
white cells in Dytiscus and Hydrophilus are scanty,
relatively to those of mammalian blocd; no oxyphile granules
can be recognised in the phagocytes when fixed and stained
by Leishman’s method, nor are platelets present. Mitoses are
not uncommon in the phagocytic cells of Hydrophilus. In
both Dytiscus and Hydrophilus no spontaneous coagula-
tion of the blood occurs on standing. In both insects oxygena-
tion appears to occur by direct transfer to the tissues of
oxygen supplied by the tracheal vessels, the blood-plasma not
serving asa medium of exchange.

LITERATURE.

1. Landois, M.—* Beobachtungen iiber das Blut der Insekten,” ‘ Zeit.
f. wiss. Zool.,’ 1864, Bd. xl, S. 55.
2. Cattaneo.‘ Sulla morphologia delle cellule amceboidi dei Mol-
luschi e Artropodi,” * Boll. Sci. Pavia,’ 1889, v. 11.
3. Wagner, W. A.—‘* Uber die Form der kérperlichen Elemente des
Blutes bei Arthropoden, etc.,” ‘Biol. Centralbl,’ 1890, Bd. x,
S. 428.
. Cuénot, L.—* Etudes sur le sang et les Glandes Lymphatiques dans
la Serie Animale,” ‘ Archives de Zoologie exp.,’ 1891, t. 9, p. 18.
5. Durham, M. E.—‘* On Wandering Cells, more especially in regard
to Exeretory Functions,” ‘Quart. Journ. Mier. Sci.,’ 1892, vol. 33,
p. 81.

pS

164 J. O. WAKELIN BARRATT AND GEORGE ARNOLD.

6. Lim Boom Keng.—‘*On the Celomic Fluid of Lumbricus
terrestris in reference to a Protective Mechanism,” ‘ Philo-
sophical Trans. of the Roy. Soe. of Lond.,’ 1896, B, vol. elxxxvi,
Deseo:

7. Benham, W. Blaxland.—* The Celomic Fluid in Acanthodrilids,”
‘Quart. Journ. Micr. Sci.,’ 1901, vol. 44, p. 565.

8. Hollande, A. C. R.—* Contribution a étude du sang des Coléop-
teres,” ‘Archives de Zool. expérimentale et générale, 1909, t. 2.
Dail

9. Cf. Leydig, ‘Untersuchungen zur Anatomie und Histologie der
Tiere,’ 1884, and * Zelle und Gewebe,’ 1885; C. v. Wistinghausen,
* Ueber Tracheenendigungen in den Sericterien der Raupen,”
‘ Zeit. f. wiss. Zool.,’ 1890, vol. xlix, p. 965, Also Packard, * Text-
book of Entomology,’ 1898, p. 435.

10. Quoted by Packard, loc. cit., pp. 419-423.

HXPLANATION OF PLATE 11,

Illustrating the paper by Dr. J. O. Wakelin Barratt and Mr.
George Arnold entitled “A Study of the Blood of certain
Coleoptera: Dytiscus marginalis and Hydrophilus
piceus.”

[Lhe drawings are made using a 2 mm. apochromatic oil-immersion
abjective with 8 and 18 compensating oculars, are drawn without the
aid of a projection apparatus, and are represented as stained by
methylene-blue and orange G. Magnification 1250 diameters.

Figs. 1-9.—Dytiscus. Phagocytic cells are represented in figs. 1-8 ;
small round-cells in fig. 10.

Figs. 1-3.—Phagocytie cells in the active condition with pseudopodia.
Figs. 1 and 3 viewed from above; fig. 2 seen on edge. Nucleus entire,
chromatin inconspicuous. Cytoplasm contains no food-vacuoles in
figs. 1 and 3. '

Fig. 4.—Phagocytic cell with recently ingested particles of Indian
ink in the cytoplasm. Vacuoles have not yet formed round the particles.

Fig. 5.—Phagocytic cell exhibiting amitotic division, nearly com-
pleted.

Figs. 6 and 7.—Phagocytic cells exhibiting active digestion. In
fig. 6 the ingested matter is in close contact with, and depressing, the

A STUDY OF THE BLOOD OF CERTAIN COLEOPTERA. 165

nucleus; the remains of a food-vacuole are seen below. In fig. 7 the
process is more advanced; vacuoles surround two of the ingested
particles, which are being digested.

Fig. 8.—Phagocyte after digestion. Vacuoles empty. The nucleus
has become lobulate.

Fig. 9.—Phagocyte in the resting condition, showing numerous small
pseudopodia. Nucleus fragmented.

Fig. 10.—Small round-cells. The characteristic feature of these cells
is the small amount of cytoplasm, which forms a thin layer round the
nucleus.

Figs. 11-18—Hydrophilus. Phagocytic cells are represented in
figs. 11-17; small round-cells in fig. 18.

Fig. 11.—Phagocytic cell, with pseudopodia, seen on edge. A small
particle surrounded by a vacuole lies in one pole.

Figs. 12-14.—Phagocytic cells with ingested particles. After a time
these particles become enclosed in vacuoles, and, as digestion proceeds,
lose their staining reaction.

*, 15.—Phagocytic cell. Polar view of mitotic division-figure.
Fig. 16.—Phagocytic cell. Equatorial view of mitotic division-figures.
Fig. 17.—Large phagocyte with abundant cytoplasm containing

numerous flaky granules.

Fig. 18.—Small round-cells. The characteristic feature of these cells
is the scanty cytoplasm surrounding the nucleus.

nu. Nucleolus. jf. Ingested material.

VoL. 56, part 1.—NEW SERIES. Pe

‘ai [Tt 3

see ATO GAY 7
GbE NS AN
Ch. “OULU. LVM. CAAALL.

- G. Arnold, del. . a a ‘Huth Lith? Dondoa.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 167

On the Morphology of the Cranial Muscles in
~ Some Vertebrates.

By

F. H. Edgeworth, M.D., D.Se.,
Professor of Medicine, University of Bristol.

With 100 Text-figures.

In the following paper I have described and compared
the development of some of the cranial muscles in Scyl-
lium canicula, Squalus acanthias, Acipenser sturio,
Lepidosteus osseus, Amia calva, Salmo fario, Cera-
todus Forsteri, Triton cristatus, Rana temporaria,
Alytes obstetricans, Bufo lentiginosus, Pelobates
fuscus, Chrysemys marginata, Lacerta agilis, chick,
rabbit, and pig.

The object has been to determine as far as possible the
morphology of the muscles, and so to deduce a morphological
classification of the motor nuclei of the cranial nerves, more
especially in mammals and man.

A subsidiary object has been to ascertain what evidence 1s
afforded by these muscles in regard to the speculations ot
zoologists on the phylogenetic relations of the various verte-
brate groups. .

The paper is a sequel to one previously published on some of
the cranial muscles of Sauropsida.

The adult anatomy of the muscles has been,described by
Vetter, Fiirbringer, Pollard, McMurrich, Allis, Jaquet,
Driiner, Ecker and Gaupp, Mivart, Krause, and others referred
to in the paper. In the majority of cases observation ot the
development of the muscles merely serves to support deduc-
tions as to their homologies already made from consideration

VOL. 96, PART 2.—NEW SERIES. 15

168 F. H. EDGEWORTH.

of their adult forms, but in some cases it suggests corrections.

In the case of Lepidosteus only the hypobranchial muscles
have been described—by Fiirbinger, and the names for other
muscles have been as faras possible those used by Vetter and
Allis in Ganoids and Teleostei. The cranial muscles of
Polypterus have been described by Pollard, and the hypo-
branchial muscles by Firbringer. ‘hey did not state the
species examined. Those of Polypterus senegalus (speci-
mens 74 to 94 cm. long) differ in a few particulars from
the descriptions given by those authors. The cranial muscles
of Salmo fario have not hitherto been described, but the
researches of Vetter in other Teleostei made identification
and nomenclature possible.

Van Wijhe described the early stages of the development of
the cranial muscles of Scylhum; and Miss Platt those of
Necturus; observations otherwise have been limited to the
development of individual muscles or muscle-groups.

The nomenclature employed by previous writers has, in
general, been followed. In cases where different names have
been applied to homologous muscles in related animals a
choice has been made, and this has necessitated some
changes.

The paper is divided into the following sections: (1) The
segmentation of the head; (2) mandibular muscles; (3)
hyoid muscles; (4) eye muscles of the rabbit; (5) branchial
muscles ; (6) cesophageal, laryngeal, and pharyngeal muscles ;
(7) muscles derived from trunk myotomes passing to the
upper ends of the branchial bars; (8) hypobranchial spinal
muscles; (9) lingual muscles; (10) some phylogenetic
speculations; (11) on Fiirbringer’s theory of the skull; (12)
a suggested morphological classification of the motor centres
of the mid- and hind-brain in man.

THe SEGMENTATION OF THE HEAD.

In the body region of Scyllium embryos it is found that
there is an unsegmented part below enclosing the ccelom, and

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 169

a segmented part above, the somite, which subsequently
separates from the dorsal edge of the coelom and develops
into myotome and sclerotome.

In the head the conditions in the mandibular and hyoid
segments are different from those in the branchial segments ;
in the latter the lateral plates of mesoderm are at first con-
tinuous ventrally with the wall of the pericardium or cephalic
ccelom! (Text-fig. 2); in the mandibular and hyoid segments

Trxt-FIGc. 1.2 TEXT-FIG. 2.

ZN

Aas \

| on } i \
\ j \ i { XN

I]

\ E Deo 4
iu]
Yau Bas, cosh cal

Text-figs. 1 and 2.—Scyllium, embryo 7 mm., transverse sections.
Text-fig. 1 is the more anterior.

the lateral plates of mesoderm are continuous ventrally with
the walls of the mandibular and hyoid sections of the cephalic
celom. These differ from the branchial sections in that no
communication from side to side takes place (‘Text-fig. 1).
In the rabbit, however (Text-fig. 76), the cephalic ccelom is
continuous from side to side in the mandibular and hyoid
segments, just as it is in the branchial segments.

1 The latter name is perhaps preferable, as probably the heart origin-
ally lay behind the branchial region.

2 For reference letters on Text-figures see p. 514.

L770 F. H. EDGEWORTH.

The lateral plate of the hyoid segment extends upwards
and forwards lateral to the alimentary canal between the first
and second gill-clefts ; its upper end in 6} and 7 mm. embryos
(= stages H and I of Balfour) is continuous above with the
“fourth myotome” of van Wijhe.

Van Wijhe says that the “ fourth myotome” in stage J is
separated from the lateral plate and is very rudimentary,
also that it atrophies towards the end of that period ; further,
that ‘bis in dieselbe Hoéhe aber mehr lateral verlangen sich
in spateren Stadien die Wande der jetzigen Hyoidhohle.
Mit dieser Verlingerung darf.das vierte Myotome nicht
verwechselt werden.” ‘The Anlage of the rectus externus,
regarded by van Wijheas the third myotome, was stated to be
continuous in stage I with the solid cell mass in the hyoid
arch, and in stage J to be no longer so.

In the embryos of 64 and 7 mm. in length (= stages H and I)
examined, it was found that the Anlage of the rectus externus
was continuous behind with the upper end of the epithelium-
lined cavity in the hyoid arch, 1. e. with the fourth myotome
of van Wijhe. In embryos of 9 mm. (Text-fig. 3) and 10mm.
it was found that the epithelial walls of the hyoid cavity had
come together, so that the cavity had disappeared, that the
now solid cell column had extended upwards, and that the
Anlage of the rectus externus was continuous behind with
this cell column some little distance from its upper end, at a
site corresponding with the original upper end of the hyoid
cavity. No trace of a separated “fourth myotome” was
seen. It is therefore possible, on the analogy of what takes
place in the trunk, to regard the whole of the ‘lateral plate ”
and ‘fourth myotome ”—which do not become separate—as
together forming the hyoid myotome.

This theory is supported by the difficulty of finding any
structure in the body region which is homologous with the
lateral plates of the head. Ziegler was of opinion that they
were homologous with the ‘ Urwirbelkommunikation”’ (of
Rabl), the ‘‘ Ursegmentsteil” (of Felix). But it is scarcely
possible that epithelial structures of the head, which develop

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 171

into muscles, can be homologous with epithelial structures in
the body, which are the “ Mutterboden fur den verschiedenen
Harnkanilchen ” (Felix). Nor do the lateral plates of the
head appear to be homologous with the “ Seitenplatten” (of
Felix) in the body, for this term denotes the epithelium
lining the ccelom.

The Anlage of the rectus externus may be regarded as a

TERXT-FIG. 3.

Scyllium, embryo 9 mm., transverse section. The line attached
to the mark * shows the point of junction of the Anlage of the
external rectus with the hyoid myotome.
very early anterior prolongation of the upper end of the
hyoid myotome.

Ziegler was of opinion that the eye-muscles gave no
evidence of the primary segmentation of the head, but attri-
buted the Anlage of the rectus externus to the mandibular
segment.

The Anlage of the obliquus superior was regarded by van
Wijhe as the second myotome of the head. On the analogy
of the rectus externus it may be looked upon as simply a
forward projection of the upper end of the mandibular

172 F. H. EDGEWORTH.

myotome. Both these Anlagen are anterior projections from
the upper ends of their respective myotomes, to add to the
musculature of the eye, which is primarily formed from the
pre-mandibular segment.

As above stated, the lateral plate of the mandibular
segment, which is serially homologous with that in the hyoid
segment, may be regarded as the myotome of that segment.

In the case of the five branchial segments, the epithelium-
lined cavities—the lateral plates of van Wijhe—are con-
tinuous below with the cephalic ccelom. Above, they are

TEXT-FIG. 4.

Scyllium, embryo 14 mm., transverse section.

stated by van Wijhe to be continuous with myotomes—lus
sixth, seventh, eighth, and ninth. ‘These myotomes were
stated to separate from the lateral plates, and to undergo
various changes, the fifth atrophying, the sixth becoming
very rudimentary, and the seventh, eighth, and ninth forming
“Vom Schiidel zum Schultergiirtel ziehende Muskeln nebst
dem vordersten Theile des sterno-hyoideus.” In regard to
this asserted continuity between the myotomes and lateral
plates, it is noteworthy that Ziegler says it is ‘‘schwierig den
unten Ursprung des Ursegments mit dem oben sichbaren
Myotom in Verbindung zu bringen, und dies ist bis jezt
keinem einzigen Forscher in der richtigen Weise gelungen.”

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 173

It might be expected that if the lateral plates are the
splanchnic elements of myotomes above, their relation to the
latter would be constant. But this is not so; for instance,
in the figure given by Ziegler of a 6°5 mm. Torpedo embryo,
two lateral plates (those of the second and third branchial
arches) lie beneath three myotomes—his fifth, sixth, and
seventh (= sixth, seventh, and eighth of van Wijhe), and the
lateral plate of the fourth branchial arch lies below his
eighth. In Ceratodus, according to Greil, the lateral plates
of the first three branchial segments are continuous above
with one (the first) myotome, and those of the fourth and
fifth branchial segments are continuous above with one
myotome (the second). In Rana esculenta, with five to
six somites (Corning, ’99, Taf. ix, figs. 7 and 11), the lateral
plates of the first and second branchial segments le in front
of the first myotome. '‘l’o this may be added that in rabbit
embryos 5 mm. long (Text-fig. 78) the first branchial lateral
plate hes in front of the first trunk myotome, and the second
and third branchial lateral plates (as yet not separated) lie
beneath the first trunk myotome.

Secondly, it might be expected that the lateral plate would
in all animals be at first continuous with a myotome above,
but in Necturus embryos (Text-figs. 51-53) and rabbit
embryos (Text-fig. 78) it is not possible to see any continuity.

Both these poimts have been emphasised by Agar in a
recent criticism of the theory of van Wijhe. Agar’s theory is
that the fourth somite of van Wijhe represents the condensed
somatic portion of the hinder palingenetic head somites, and
he points out that some observers have described more than
one somite in this situation, e. g. Brauss, two in Spinax, and
Miss Platt, three in Acanthias. The difficulty in accepting
this theory is that, as above stated, this fourth somite is, in
Scyllium, contimuous with the lateral plate of the hyoid
segment.

Ziegler throws doubt on the existence of van Wijhe’s fifth
myotome—the one which lies above the first branchial seg-
ment—on the ground that in Torpedo its cavity is no more

174: F. H. EDGEWORTH.

TEXT-FIG. 5.

Text-figs. 5 and 6.—Scyllium, embryo 16 mm., transverse sec-
tions. Text-fig. 5 is more anterior. In the sections the right
side is slightly anterior to the left.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 175

than acleft, and that it quickly becomes rudimentary. He
concludes that it is‘only a small cavity in the mesenchyme
and of no theoretic importance. It is difficult to share this
opinion, for the corresponding structure in Scyllium is lined by
epithelial cells and closely resembles the next following
myotome (‘l'ext-figs. 1 and 2).

Ziegler counts three myotomes in Torpedo (his fifth, sixth,
and seventh) corresponding to three lateral plates, regards
the vagus as a “zusammengesetzte Nervencomplex” corre-
sponding to three segments, and is of opinion that it is in
correspondence with this that the next following myotome
(his eighth = van Wijhe’s ninth) is the foremost to have any
nerve-roots, an anterior one only. ‘The difficulties in accept-
ing such a view are: first, as stated above, the want of antero-
lateral correspondence between the myotomes and _ lateral
plates ; and secondly, in Scyllium, and probably also in 'Tor-
pedo at a later stage, a fifth branchial lateral plate is formed,
7. e. there would be an overlapping of the territories of the
yagus and spinal roots.

Greil’s views as to the nature of the mesoderm of the head
of Ceratodus are very different from the foregoing. He holds
that the musculature of the branchial region is derived from
downgrowths of the first two myotomes, that in the first
three branchial arches being derived from the first myotome,
that in the fourth and fifth arches from the second myotome.
The cells forming these downgrowths can be distinguished
from the immediately subjacent lateral plates by the shape of
their nuclei and by the later absorption of their yolk-granules.
The downgrowths increase in vertical depth from behind
forwards. ‘The lateral plates of the branchial] arches degene-
rate Into connective tissue. The dorsal portions of the first
three arches, i. e., those which are formed from the first
myotome, develop dorsally into the levatores arcum branchi-
alum and ventrally into the second and third interbranchi-
ales and the ceratohyoideus; that of the fourth arch into the
fourth levator and fourth interbranchialis; that of the fifth
arch anteriorly into the dorso-laryngeus and the fifth inter-

176 F, H. EDGEWORTH.

branchialis, and posteriorly into the fifth levator and levator
scapule. The musculature of the hyoid and mandibular
arches cannot be regarded as derivations either of a somite
or of a lateral plate; it never takes on lateral-plate
characteristics.

A difficulty in accepting this theory is that it does not seem
to be of general applicability in Vertebrates. Thus, as stated
above, in the rabbit the lateral plate of the first branchial
arch is in front of the first trunk myotome, and those of the
second and third arches lie below the first trunk myotome ;
and there is a gap between the dorsal edges of the lateral
plates of the second and third arches and the ventral edge ot
the first myotome. A difference in the shape of the cell-
nuclei and in the rate of absorption of yolk-granules does not
appear to be of sufficient importance to justify a separation
into upper and lower portions of a structure which in other
animals bas been called lateral plate. As far as I have been
able to observe, the ventral end of the lateral plate, taken in
its usual sense, does not degenerate into connective tissue,
but becomes converted into muscles.

These difficulties lead to the following theory of the seg-
mentation of the head. It is probable that it originally con-
sisted of five segments only—the premandibular, mandibular,
hyoid, first branchial, second branchial—each having a myo-
tome, which, in the case of the four latter, contains a slit-like,
epithelium-lined cavity continuous with the cephalic ccelom
below. ‘To each yotome passed a nerve—the IlIrd, Vth,
Vilth, [Xth, and Xth.!. The gill-clefts are intersegmental.
New segments were added, one by one, behind the second
branchial, the head extended back into the body-region, and

' According to Neal (98) only one encephalomere corresponds to the
vagus—a fact which, if the encephalomeres have segmental, or rather
inter-segmental, worth, agrees with the theory that the vagus is not a
“zusammengesetzte Nervencomplex,’ but primarily, as regards its
motor branches, of one segment only—the second branchial—and that
additional branches were developed as the number of gill-clefts and
branchial segments was added to.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 177

the added myotomes necessarily lie beneath the anterior body
myotomes, with their upper ends at varying distances from the
ventral surface of the latter, with which they may or may
not agree in antero-posterior extent. In Rana esculenta,
with five to six somites, the most anterior trunk myotome
lies above the third branchial, i.e. the first added myotome ;
a little later there is a relative shifting forward of the trunk
myotomes, so that the first trunk myotome comes to he above
the first branchial myotome (see Corning’s figures, ‘l'af. 1x,
figs. 7, 11, and 26). In Scyllium this overlapping is, second-
arily, antedated in development, so that the first branchial
myotome never lies in front of the first trunk myotome. In
the rabbit it does so.

‘he backward growth of the head into the body by this
process of metameric increase leads to the non-development
of the coelomic portion of the anterior trunk-somites. In
Scyllium, for instance, the first four trunk-sonites have no
coelomic portions.

In the head, as in the body, each myotome is at first an
epithelium-lined cavity, which is continuous below with the
celom. ‘The differences between the myotomes of the body
and those of the head are: (1) Whereas in the body the
myotomes extend dorsally to the mid-dorsal line, and, subse-
quently, ventrally outside the somatopleure wall of the ccelom
to the mid-ventral line, neither of these secondary phenomena
takes place in the head; (2) whereas the ccelom is large in
the body and contains the alimentary canal and other viscera,
it is small in the head and lies entirely ventral to the alimen-
tary canal; (3) in correlation with this the myotomes of the
body lie, at first, dorso-lateral to the alimentary canal, those
of the head lie dorso-lateral and lateral to it; (4) the sclero-
tome elements of the body-myotomes are formed by ventro-
medial outgrowths, those of the head from scattered cells
given off from the premandibular, mandibular, and hyoid
myotomes. These differences are intimately associated with
the development of gill-clefts and a cranium in the head, of
viscera and a vertebral column in the body.

178 F. H. EDGEWORTH.

If, following Fiirbringer, it be supposed that primitively
the myotomes lay exclusively lateral to the chorda dorsalis, it
would follow that they have taken different paths of develop-
ment in the head and body, resulting in the conditions above
stated.

According to the generally accepted theory, certain
Selachii, e. g. Heptanchus, are the most primitive of gnatho-
stome Vertebrates, in that they have the greatest number of
branchial segments; and the lessened number of branchial
segments in T'eleostomi, Amphibia, and Dipnoi is due to a
disappearance of the hinder ones. If, however, it be supposed
that the original number of branchial segments was two, 1. e.
first and second, and that these were added to by a process of
metameric increase, the interesting question arises as to the
least number present in these Vertebrate groups, for this may
be supposed to have been possessed by some primitive form.
The Amphibia have four branchial segments, Dipnoiand most
Teleostomi five (though Polypterus has only four), and most
Elasmobranchs five, though they may have as many as seven.
It may therefore be supposed that the original number
present in Amphibia was added to in the other groups. ‘This
harmonises with the conclusion, stated later, that the condition
of the muscles of the head in Amphibia is more primitive than
in Dipnoi, Teleostomi, and Klasmobranchs.

The lessened number present in Sauropsida and Mammalia
may be supposed to have resulted from reduction of the hinder
of these four branchial segments. In the later stages of
Reptiles, previously investigated, only two branchial myotomes
were seen, but the early stages of Chrysemys marginata
show four. In Gallus only two are present, even in the early
stages. In Lepus only the first three are developed.

The cephalic ccelom disappears in the mandibular and
hyoid segments early in development, and its walls develop
into the intermandibularis and interhyoideus, which are at
first continuous with the mandibular and hyoid myotomes.
The lower ends of the branchial myotomes separate from the
wall of the branchial portion of the cephalic ccelom, and they

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 179

develop into the branchial muscles. | No muscles are directly
formed from the walls of the branchial portion of the cephalic
coelom, which subsequently retreats from the head.

MaAnprispuLaR MUSCLES.

Seyllium.—On the formation of the palato-quadrate, in
16 mm. embryos, the mandibular myotome lies outside of and
across the palatine process, and then separates into an upper

Led

TrxtT-FIG. 7.

Scyllium, embryo 17 mm., transverse section. The right side of
the section is slightly anterior to the left.
levator maxillee superioris and lower adductor mandibule,
the process beginning in 20 mm. embryos (l'ext-fig. 11). The
separation of these two muscles by the palato-quadrate is com-
plete, and this is also the case in Acanthias, where, acccord-
ing to Marion, the hinder portion of the levator maxille superi-
oris—forming a separate first dorsal constrictor—is inserted
into the lower jaw. This must consequently be the result of
secondary downgrowth. The upper edge of the adductor
mandibule gains, anteriorly, an additional origin from the
suborbital cartilage in 530 mm. embryos (Text-fig. 17), and
this anterior portion of the adductor separates, in 40 mm.

180 F. H. EDGEWORTH.

embryos, forming the levator labii superioris (or add. (3) of
Vetter. In 45 mm. embryos the add. y of Vetter is begin-
ning to be delaminated from the outer face of the adductor,
and the hindmost fibres of the adductor have grown down
into the intermandibularis, forming a band similar to that
described in Acanthias by Vetter.

The intermandibularis (Cs, of Vetter, Cgmv of Ruge) is
formed from the ventral part of the mandibular cavity,

Trxt-FiGc. 8.

bru Wy.

if Sa ee Dice
COV. VoapeRs 8.
Scyllium, embryo 17 mm., transverse section.

which, as mentioned above, does not meet its fellow in the
mid-ventral line, but passes backwards ventro-median to the
ventral end of the hyoid cavity to open into the front end of
the cephalic coelom. It results from this that there is no
developmental stage in which the intermandibularis lies
altogether in front of the interhyoideus. It gradually extends
backwards, underlying the interhyoideus, so that in 23 mm.
embryos its hinder edge lies posterior to the ventral ends of
the ceratohyals (‘Text-fig. 12).

The nictating muscles of Scyllium (Ridewood) consist of a
levator palpebr nictitantis, retractor palpebree superioris,

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 181

and constrictor spiraculi—all innervated by the Vth. Ride-
wood supposed that the first-named was differentiated from the
same tract as the levator maxille superioris and that the
second belonged to the same category, whilst the constrictor
spiraculi appeared to belong to a purely dermal system of
muscles.

According to Harman, the musculature of the eyelids of
Mustelus arises “ from two original sources—one a superficial
dermal layer, the other a portion of a deeper dermal muscle
layer,’ the former originating from “a mass which appears

TEXT-FIG. 9.

Ante ov

Scyllium, embryo 20 mm., longitudinal horizontal section through
first branchial segment on left side.

first in the region of the branchial bar which is the second
after the spiracle,”’ the latter from a mass which separates
into ‘a maxillary mass and a spiracular mass.” Harman
also stated that “the progressive growth of the spiracle
muscles in the service of the face can be traced to their full
development in the facial muscles of man.”

In Scyllium the Anlage of the nictating muscles can be seen
in 30 mm. embryos (Text-figs. 16, 17) asa mass of cells being
proliferated from the outer surface of the upper end of the
levator maxillz superioris. In 40 mm. embryos the mass
has become partially divided into a deeper and a more
superficial layer—the former is the Anlage of the levator

TEXT-FIG. 10.

F. H. EDGEWORTH

Text-figs. 10 and 11.—Sceyllium, embryo 20 x
is the more dorsal.

nm,

Text-fig. 10

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 183

palpebree nictitantis, the latter that of the retractor palpebra
superioris. In 45 mm. embryos, the latter sends down
behind the spiracle an offshoot which develops into the con-
strictor spiraculi. All these muscles in Scyllium are thus
developed from an Anlage budded off from the levator
maxille superioris. It may be added that the facial muscles
of mammals are hyoid in origin, formed from the upgrowing
interhyoideus (pp. 221 and 222), and consequently are not
homologous with the eyelid muscles of Seyllium.

The myotome of the mandibular segment in T'eleostoman
embryos lies at first across and outside the palatine process of
the quadrate (Text-fig. 23) and then divides into parts above
and below this. ‘he division takes place in Acipenser in
7; mm., in Lepidosteus in 84 mm., in Amia in 64 mm., and
in Salmo in 5 mm. embryos. The part above the palatine
process is the levator maxille superioris, the part below the
adductor mandibule. From the upper end of the levator
maxiliz superioris of Lepidosteus, Amia (Text-fig. 28), and
Salmo, is given off the dilatator operculi, which extends back-
wards below the first gill-cleft into the opercular fold, and the
remainder forms the levator arcus palatini, which is inserted
into the palato-quadrate (Text-fig. 28). In Acipenser the
levator maxille superioris does not divide into levator arcus
palatini and dilatator operculi; it grows backwards, without
having had any temporary insertion into the palato-quadrate,
and becomes inserted into the hyomandibula, forming the
protractor hyomandibularis (Text-figs. 18, 19, 20).

The dilatator operculi is partially inserted into the hyo-
mandibula in Lepidosteus, and wholly in Polypterus, ? species,
described by Pollard,'but in Poly pterus senegalus it passes
backwards in the outer wall of the spiracle and is inserted
into skin only (Text-figs. 35,36). The adductor mandibule, at
first passing from the palato-quadrate to Meckel’s cartilage,

' Protractor hyomandibularis of Pollard, but not homologous with
the protractor hyomandibularis of Acipenser, as he describes a levator

maxillze superioris (i.e. levator arcus palatini), and this is also present
in Polypterus senegalus.

VOL. 56, PART 2.—NEW SERIES. 14

Text-figs. 12

¥. H. BDGEWORTH.

Tpxt-Fia. 12.

cau bronchi | Meare

cov yal 13.

and 13.—Scyllium, embryo 23 mm. ‘Text-fig. 12
is the more anterior.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 185

undergoes various changes in the specimens examined. In
Acipenser embryos up to the length of 11 mm. it remains
undivided, but in the adult (Vetter) it has additionally spread
up to the skull. InSalmo it additionally spreads backwards
to the hyomandibula. In Lepidosteus, Amia (T'ext-figs. 28, 34),
and probably (vide infra) Polypterus, the adductor mandi-
bulee divides into internal and external portions. In Lepi-
dosteus (Text-figs.26, 27) the internal portion extends upwards
outside the palato-quadrate to gain an additional origin from
the cranial wall; whilst the external portion, keeping its
original origin, arises from the external surface and upper
end of the quadrate outside the insertion of the levator arcus
palatini. In Amia! the internal portion (in 11 mm. embryos)
sends forwards a projection from its upper end which forms
the muscle connected with the olfactory chamber (LAP, of
MecMurrich, LMS, of Allis); in 14 mm. embryos the re-
mainder of the internal portion extends upwards above the
level of the palato-quadrate and divides into three parts
(LAP,, 3,4, 0f McMurrich, LMS8,,1,, of Allis). The external
portion of the adductor additionally extends backwards to
the hyomandibula in 9 mm. embryos, and divides into the
parts described, as parts of the adductor, by Allis.

In Polypterus, Pollard described the adductor as consisting
of three parts, the pterygoid, temporal, and masseter’; of

1The adult condition of the mandibular muscles of Amia has been
described by McMurrich and by Allis. McMurrich stated that they
consist of a levator arcus palatini and an adductor mandibule, and
that the former is divided into five parts—from behind forwards
LAP,, 2. 3, 4 53 Of these LAP, is inserted into the metapterygoid with
some of its hindmost fibres into the operculum, LAP.,, ;, , join the
adductor, and LAP, is in connection with the olfactory chamber.
Allis separated LAP, into a dilatator operculi and a levator arcus
palatini, whilst LAP,, ;, ,. ; he called the second, first, third and fourth
divisions of the levator maxille superioris. He suggested that LAP, and,
(LMS, and ,) are derived from add. 6 of Selachians, and that (his)
levator arcus palatini and dilatator operculi are the homologue of add.
y of Selachians. From the embryological findings mentioned above it
would appear that a new nomenclature for LAP,, ,, ,, ; (= LMS,, ;. 5, »)
is needed—in terms of an internal adductor.

2 Pollard thought that the pterygoid and temporal were the homologue

186 F. H. EDGEWORTH.

these the pterygoid and temporal are together the homologue
of the internal adductor, and the masseter the homologue of
the external adductor, of Lepidosteus and Amia.

TrExt-Fic. 14.

Awlerroru

Externe ok

Trxt-Fic. 15.

ate QOVU.an.

Gru

&

Text-figs. 14 and 15.—Scyllium, embryo 28 mm., longitudinal
horizontal sections. Text-fig. 14 is the more dorsal, through
second, third, and fourth branchial segments on left side.

of Add. B of Selachians, but this is not possible. In Text-figs. 35 and 56 I
have denoted his “ masseter” by the term “external adductor,” and his
pterygoid and temporal might be called parts of the internal adductor.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 187

The intermandibularis of Teleostoman embryos forms, at
first, with its fellow, a transverse muscle attached laterally to
Meckel’s cartilage (Text-figs. 25, 24, 28).

In Salmo it does not extend backwards, but does so in
Acipenser, Lepidosteus, Amia and Polypterus, and partially
underlies the fore part of the interhyoideus. In Salmo,
Polypterus,! and Lepidosteus, it remains single, in Acipenser
and Amia it divides into anterior and posterior parts. In

Trxt-Fic. 16.

Cov.mand 16

Scylium, embryo 30 mm., transverse section.

Acipenser the intermandibularis anterior (Cs, of Vetter) is
attached laterally to Meckel’s cartilage, and the intermandi-
bularis posterior (Cs, and Cs, of Vetter) spreads upwards,
laterally, towards the skull.2 In Amia both intermandi-
bularis anterior® and posterior+ are attached laterally to

1 Intermaxillaris anterior of Pollard.

> All these parts are innervated by the Vth (Allis).

3 Intermandibularis of Allis.

* Superficial or inferior portion of geniohyoid of Allis ; the muscle has,
however, no genetic relation to the superior portion of the geniohyoid
(called in this paper “ hyomaxillaris”’?) which is developed in the hyoid
segment.

188 F. H. EDGEWORTH.

Meckel’s cartilage (the process of separation into anterior and
posterior portion beginning in 9} mm. embryos and being
completed in 14 mm. embryos).

In Ceratodus the myotome of the mandibular segment
spreads upwards lateral to the Gasserion ganglion ('l’ext-fig.
39), and separates from the lateral half of the intermandibularis
between stages 40 and 42 (of Semon). It divides into outer
and inner portions—pterygoid! and temporal*—the former of
which, in stage 48 (Text-fig. 46), arises from the trabecular
wall, and the latter from the anterior and outer surface of the
quadrate. The intermandibularis® joins its fellow in a median
raphé and becomes attached laterally to Meckel’s cartilage ;
its posterior edge extending backwards underhes the fore
part of the interhyoideus (Text-figs. 41, 45).

In Necturus (Miss Platt) the mesothelium of the mandi-
bular arch (here interpreted as “myotome”’) divides into an
internal part, the temporal (here called, following Driiner, the
“ pterygoid”), andan external part, the masseter. In Triton
the myotome of the mandibular segment also divides into an
internal and an external part ; the upper end of the internal,
pterygoid, part extends up to the side of the skull; the
external part, at first arising from the suspensorium only,
divides into an outer portion, the masseter, which keeps this
origin, and an inner portion, the temporal, which extends up
to the auditory capsule.

The intermandibularis of Necturus* remains single, its
posterior edge underlhes the anterior interhyoideus (‘Text-fig.
55); in Triton the intermandibularis (in larve between the
lengths of 84 and 10 mm.) divides into anterior and posterior
parts,” the latter of which partially underlies the inter-
hyoideus.

1 Pterygoid of Jaquet.

* Adductor mandibule seu digastricus of Jaquet.

* Camy of Ruge; mylohyoideus pars anterior of Jaquet.

4 Mylohyoideus anterior of Mivart and Miss Platt.

* Intermaxillaris anterior and posterior of Wiedersheim; inter-
mandibularis anterior and posterior of Driiner.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 189

The myotome of the mandibular segment of Rana separates
from the lateral half of the intermandibularis in 5 mm.
embryos; it extends backwards in 7 mm. embryos, dividing
into internal and external portions (Text-fig. 58). The myo-
tome thus comes to he in a nearly horizontal position internal
to the muscles developed in the hyoid segment. The internal
portion develops into the pterygoid muscle, the external into
the temporal, sub-temporal, extra-temporal, and masseter

Trxt-FIG. 17.

)

peasy, S uboul c

N.

17.
Scyllium, embryo 30 mm., longitudinal vertical section.

(Text-figs. 59, 60). The masseter and extra-temporal arise
from the internal surface of the processus muscularis of the
palatoquadrate bar. The anterior end of the pterygoid shifts
outwards beneath the anterior ends of the other muscles and is
inserted into the outer end of Meckel’s cartilage. The tem-
poral is inserted into the inner end of Meckel’s cartilage ; the
masseter is inserted into Meckel’s cartilage a little distance
from its outer end; the subtemporal is inserted, by two
tendons, into Meckel’s cartilage and the superior labial car-
tilage ; the extra-temporal divides into two portions, one of

Trxt-Fic. 18.

Text-figs. 18 and 19.—Acipenser, embryo 8mm. Text-fig. 18
is the more anterior.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 191

which joins the temporal (‘Text-fig. 60) and the other the sub-
temporal.

The muscles of Alytes, Bufo lentiginosus, and Pelo-
bates! are similar to those of Rana (Text-fig. 63), except that
the extra-temporal is inserted only into the superior labial
cartilage,

The Anlage of the levator bulbi is given off from the upper
surface of the hinder part of the temporal in 9 mm. larve ;
its outer end becomes inserted into the skin and upper edge
of the palato-quadrate bar; it remains relatively undeveloped
until late in metamorphosis. On the development of the lower
eyelid a slip is separated from the levator bulbi, forming the
depressor palpebree inferioris.

At metamorphosis, on the atrophy of the superior labial
cartilage the sub-temporal and extra-temporal fuse with the
temporal, and the muscles become more vertical in position
on the rotation of the palato-quadrate bar.

The Anlage of the intermandibularis of Rana divides in
7mm. embryos into three parts—the submentalis, the man-
dibulo-labialis, and the submaxillaris. The submentalis
develops later than the other two muscles ; in 12 mm. embryos
it forms a mass of small round cells lying beneath and
extending backwards from the inferior labial cartilages, and
at the beginning of metamorphosis forms a layer of trans-
versely directed muscle-fibres connecting together the infe-
rior surfaces of the inferior labial cartilages (Text-fig. 60).
The mandibulo-labialis, arising from the inner aspect of the
transversely directed Meckel’s cartilage, passes down external
to the genio-hyoid and is partially inserted into skin, partially
interlaces with the muscle of the opposite side (‘Text-fig. 60).

The submaxillaris arises from the under surface of Meckel’s
cartilage. The conditions in larve of Bufo lentiginosus

1 This account differs from that of Schultze, in that the subtemporal
is stated to be inserted into Meckel’s cartilage as well as into the
superior labial cartilage, and in the description of an extra-temporal.
The results were obtained from serial sections of larvee, 10, 18, 22, and
30 mm. long.

192 Fr. H. EDGEWORTH.

Trxt-Fic. 20.

hypobr apm An.

al.

Text-figs. 20 and 21.—Acipenser, embryo 83 mm. Text-fig. 20
is the more anterior. The right side of the sections is slightly
anterior to the left.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 193

are similar to those of Rana; in Alytes the submaxillaris arises,
like the mandibulo-labialis, from the inner aspect of Meckel’s
cartilage, so that the two muscles are much more continuous
than is the case in Rana, Bufo, and Pelobates. The con-
dition in 10 mm. larve of Pelobates is similar to that of
12 mm. larve of Rana; in 13 mm. larve the mandibulo-
labialis has spread additionally into the upper lip, the condition
described by Schultze. He states that the submentalis is
attached to the inner aspect of Meckei’s cartilage, but up to
the stage of 30 mm. it is attached, as in Rana, Bufo, and Alytes,
to the inferior labial cartilages, as a very minute transverse
muscle,

At metamorphosis in Rana, the attachment to the skin of
the mandibulo-labialis is lost, and the muscle forms one sheet
with the submaxillaris.!

Observations on the development of the mandibular
muscles have been made by Reuter in pig-embryos, and
in regard to the tensor tympani by Futamura in human
embryos. Reuter stated that the mandibular muscles are
first visible in pig embryos measuring 16 mm. in “ Nacken-
Steisslange’’* in the form of an inverted Y, the two limbs of
which he on either side of the lower jaw. The temporal
develops from the upper limb, the masseter from the lower
external limb, and the two pterygoids from the lower internal
limb. No mention is made of the tensor tympani or the
palatine muscles. According to Futamura the tensor tympani
and tensor veli palatini form a ‘‘ ganz einheithchen Muskel”
in human embryos of seven weeks. This Anlage and the
levator veli palatini are developed about the branches of the
palatine nerves from a ‘‘ Muskelblastemgewebe” which
“deutlichen Zusammenhang mit dem tiefen Teil der Platys-
maanlage erkennen lasst.” ‘“ Die Nervendste fiir diese

1 Submaxillaris of Ecker and Gaup.

> This stage is an advanced one, as the figures show that the ossification
of the lower Jaw has begun. The Anlage of the mandibular muscles
was quite evident in a pig embryo of 8 mm. crown-rump measurement,
from which Text-fig. 98 was taken.

194. F. H. EDGEWORTH.

Muskeln lassen sich leicht vom Facialis hervorfolgen.”?!

He also states that in pig embryos the levator veli palatini
and M. uvule develop as in man from ‘Gewebe des
Platysma colli das von der vorderen Seite des Oberkiefer-
fortsatzes nach seiner medialen Seite zieht.”

In 2 mm. embryos of the rabbit the cells which will form
the myotome of the mandibular segment cannot be differen-
tiated from the other cells occupying the segment. In
3 mm. embryos (Text-fig. 76) the myotome is visible, and the
walls of the mandibular section of the cephalic ccelom are
beginning to come together, forming the intermandibularis,
The myotome separates from the lateral edge of the inter-
mandibularis in 7 mm. embryos. In 13 mm. embryos it has
partially separated into external and internal portions, which
form the two limbs of a A-shaped mass, the apex of which
lies just below the Gasserian ganglion (‘lext-figs. 94, 95) ; the
external portion is the Anlage of the temporal masseter and
external pterygoid muscles; it extends up to the skull in
16 mm. embryos, the external pterygoid is cut off from the
internal surface of the lower end of the temporal. The
internal portion separates into internal pterygoid and tensor
tympani. ‘The intermandibularis forms the mylohyoid of the
adult; it is covered over, in 10 mm. embryos, by the forward
growing interhyoideus.

The Homologies of the Mandibular Muscles.—Com-
parison of the various ways in which the myotome of the mandi-
bular segment develops shows that they may be reduced to two
types: (1) That in which the myotome does not divide into
upper and lower portions—Ceratodus, Necturus, Triton, Rana,
Alytes, Bufo lentiginosus, Pelobates, Lepus. (2) That in
which the myotome divides into portions above and below
the palato-quadrate, into levator maxille superioris and

adductor mandibule—Scyllium, Acipenser, Lepidosteus, Aimia,
Salmo, Sauropsida.
Driiner supposed that a portion homologous with the
1 Beevor and Horsley showed, however, that no movement of the palate
is produced in the monkey on intra-cranial stimulation of the VIIth.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 195

levator maxille superioris of Selachians disappears in
Amphibia.! ‘There’ is, however, no trace of this in the on-
togeny of Amphibia. According to Gaupp the pterygoid
process of Amphibia presents features which lead to the

TExT-FIG. 22.

aes

Acipenser, embryo 8imm. The left side of the section is
slightly anterior to the right.

suggestion that it is in process of “ Riickbildung.” If this
be so, and if the pterygoid process of Amphibia be homo-
logous with that of Selachians—a matter which Gaupp says

1 The levator maxille superioris ‘ist wohl mit der Verwandlung der
Streptostylie in die Monimostylie der Urodela verloren gegangen.”

196 F. H. EDGEWORTH.

is not certain—it might be supposed that a muscle strip
which formerly divided into upper and lower portions now by
some atavistic process no longer does so. On the other hand,
the fact that, in all the animals of the second class, the myo-
tome, undivided, lies at first across and unattached to the
palato-quadrate, i.e. shows a condition which is the perma-
nent one in Amphibia and Ceratodus, suggests that the con-

TrxtT-FIG. 22a.

Acipenser, embryo 9mm.

dition in Amphibia, Ceratodus, and Mammalia is the primary
one, and that the one present in Selachii, Teleostomi, and
Sauropsida is a secondary one. It would follow that the
palatine or pterygoid process of the quadrate was not primarily
a process for attachment of muscles nor an upper jaw.
Fiirbringer divided Vertebrates into two classes with
regard to the connection of the quadrate with the skull—
those with movable quadrates (streptostylic), and those with
immovable quadrates (monimostylic). The latter condition,

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 197

he thought, was secondary to the first. ‘ Die Monimostylie
alleemein von der Streptostylie ableitet.”’

he development of the mandibular muscles in the Sau-
ropsida suggests that in them there are two streptostylic con-
ditions—a primary streptostylic pterygo-quadrate in birds,
and a secondary streptostylic quadrate in Lacerta vera,

Trxt-Fig. 23.

Lepidosteus, embryo 8 mm., transverse section.

Rhiptoglossa, and Ophidia, and that the monimostylic con-
dition of Chelonia, Crocodilia, and Rhyncocephalia was
developed—and probably independently—from a primitive
streptostylic pterygo-quadrate which has been preserved in
Birds (loc. cit.).

The development of the mandibular muscles in Amphibia
and Ceratodus affords no evidence that the monimostylic con-
dition there present has been derived from a streptostylic
one, and a fixed quadrate would appear to be a necessary

198 F. H. EDGEWORTH.

correlative of an undivided mandibular myotome, to form a
point d’appui for the lower jaw.

It would follow that the streptostylic condition present in
Selachians, Teleostomi, and Sauropsidan embryos is one
which developed in correlation with a division of the myotome
into upper and lower parts, inserted into and arising from
the palatine process of the quadrate.

Text-FIG. 24.

fli ,

WY
Wy

%
yh

We
ay

24.

Lepidosteus, embryo 12 mm., transverse section.

In Ceratodus, Amphibia, and Lepus, where the mandi-
bular myotome does not become divided into upper and lower
parts, it separates into internal and external portions. In the
Anuran larvee the outer division divides into parts, some of
which have a temporary insertion into the superior labial
cartilage, and the whole myotome assumes a uearly hori-
zontal position in correlation with that of the palato-quadrate
bar; at metamorphosis both bar and muscles rotate into a
more vertical position. In the rabbit the inner division sepa-
rates into the internal pterygoid and the tensor tympani

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 199

muscles, the outer division into the temporal, masseter, and
external pterygoid.

Secondary changes take place in the levator maxillx
superioris and adductor mandibule in all the animals inves-
tigated; no one preserves them as such. In Scyllium the

Taxr-nire. 2a.

by wt in

we Ons vera v —

a = rg
“

\\ ae \ /
VY x NG y Uf /
Nee 6
S

Lepidosteus, embryo 14 mm., transverse section.

Anlage of the nictating muscles is proliferated from the
levator maxille superioris, and add. (§ and add. y are sepa-
rated from the adductor. In Teleostomi the levator maxille
superioris either forms a protractor hyomandibularis or
divides into a dilatator operaculi and levator arcus palatint ;
and the adductor may either remain single as in Salmo, or
divide into external and internal portions, of which either the
internal (Lepidosteus), or both (Amia, Polypterus), or ? the
VOL. 56, PART 2.—NEW SERIES. 15

TExT-FIG. 26.

1 mferma n a.
I y

TEXT-FIG. 27.

rveck ext

hyahyoud sub

|
[es Ri ood
nae 27.

Text-figs. 26 and 27.—Lepidosteus, embryo 19 mm. Text-fig. 26
yi s
is the more anterior.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 201

external (Acipenser), grows up to the skull. In Sauropsidan
embryos the depressor palpebre inferioris is given off from
the anterior margin of the levator maxille superioris, which
becomes inserted into the palato-quadrate—this is preserved
in birds, whereas in reptiles various changes, modifications

Trxt-rieG. 28:

1 dilatetere

\

Text-figs. 28-33.—Amia, embryo83 mm. Text-fig. 28 is the most
anterior,

or atrophy, occur; and the adductor mandibule divides into
external and internal portions, of which the former grows up
to the skull, whilst the primitive origin of the latter was
probably to the palato-quadrate and the hind end of the
palato-pterygoid bar—this is preserved in Chelonia, but is
variously modified in other groups (loc. cit.).

A comparison of the various forms of the intermandibularis

202 F. H. EDGEWORTH.

shows that its primitive condition is that of a transverse
sheet passing from one ramus of the lower jaw to the other.
This exists only in Salmo. In Necterus, Triton, Ceratodus,
Scyllium, Acanthias, Polypterus, Lepidosteus, and Amia
it extends backwards, underlying the fore part of the inter-

TrExt-Fic. 29.

hyo max An

eS ey as bx ee
ae SSS = Be ea huohy ont 2 9.
ze gen Rayead.

hyoideus, and in Amia and Triton it divides into anterior
and posterior portions. In Anuran larve it divides into
submentalis, mandibulo-labialis and submaxillaris, of which
the first has a special relationship to the inferior labial
cartilages. In Sauropsida it forms a continuous sheet with
the interhyoideus and C,vd. In Lepus it is overlapped by
the forward-growing interhyoideus.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 203

The intermandibularis, in correlation with its development
in the mandibular segment, is usually innervated by the Vth
cranial nerve. Vetter, however, found that in Scyllium and
Prionodon the portion immediately behind the symphysis of
the jaws was innervated by the Vth, and the greater portion

Trxt-Fie. 30.

iy igs —_%*
AR ety

ra \ \ ‘ee
Ae \ lex by iP

%, a

Vee
ae

EB ca GE

=

= fuyohy sub.
COr.Ryoid ger hyped Ryphay unt: 30.

of the muscle by the VIIth, and that in Acanthias, Heptan-
chus, and Scymnus the whole of the muscle was innervated
by the VIIth. He concluded that in the former the greater
part, and in the latter the whole, of the intermandibularis
(Csv,) had disappeared, and had been replaced by the inter-
hyoideus (Csv,), which had gained a secondary insertion into
the lower jaw. But this opinion, which was founded on adult

204. F. H. EDGEWORTH.

anatomy only, is at variance with the phenomena of develop-
ment ; both in Scyllium and Acanthias a well-marked inter-
mandibularis is formed in the mandibular segment, and
spreads back below the interhyoideus and fusing with it behind
the hyoid bar. Its partial or total innervation by the VIIth
must consequently be a secondary phenomenon.

TEXT-FIG. 31.

The intermandibularis of Ceratodus is also innervated by
the VIIth (Ruge), and its hinder part in Triton (Driiner).

Ruge held that what is here called the intermandibularis is
a facial muscle, and that its innervation from the Vth is
secondary, but in Ceratodus, as in all the vertebrates examined,
itis developed in the mandibular segment. Ruge’s theory
was based on the idea that “ Hs liegt auch nicht der geringste

»

TEXT-FIG. 3

oe

Oo,

TEXT-FIG.

206 F. H. EDGEWORTH.

Grund vor um an der Ursprunglichkeit der Einrichtungen bei
den Notidaniden zu zweifeln.’ Study of the comparative
embryology of the cranial muscles, however, leads to consider-
able doubt on this matter.

Hyor Muosctzs.

In Scyllium the ventral end of the hyoid myotome becomes
continuous with the lateral edge of the future interhyoideus
in 14mm. embryos. In 16 mm. embryos the formation of
the hyoid bar begins by aggregation of the mesoblast cells,
forming a pro-cartilaginous tract lateral to the alimentary
canal, and the myotome is at first partly continuous with the
interhyoideus, partly inserted into the upper end of the bar
(‘Text-figs. 5 and 6), forming a levator hyoidei. In 17 mm.
embryos the hyoid bar extends upwards towards the auditory
capsule (‘l'ext-fig. 7), partly covered by the myotome, which is
inserted into its lateral surface (C,hd of Ruge). It is only
later, in embryos between the lengths of 23 and 30 mm., that
the hyoid bar separates into ceratohyal and hyomandibula, as
in Acanthias (Gaupp). The continuity of the myotome and
the interhyoideus becomes lost, and the lateral edge of the
latter is inserted into the ceratohyal. In 23 mm. embryos
(‘Text-figs. 12,13, cf. Text-figs. 10 and 11) backward extension
of the myotome and interhycideus takes place, so that a con-
tinuous dorso-ventral sheet (C,vd of Ruge) is formed behind
the hyoid bar. Later on, in 40 mm. embryos, the myotome
extends forwards, completely covering the hyomandibular
cartilage, and its anterior edge is inserted into the quadrate.

In the Teleostomi the relations of the fore part of the
hyoid myotome (retractor or adductor mandibulz) to the
hyomandibular cartilage are different from those existing in
Selachii. The retractor of Acipenser is inserted into its
hinder edge, and of Polypterus into its inner surface, and the
adductor of Lepidosteus, Amia, and Salmo is inserted into
its inner surface. Further, the VIIth nerve (hyoid branch
of VIIth im Polypterus) winds round the cartilage in

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 207

Acipenser and EOL DEaE, pierces it in Lepidosteus, Amia,
and Salmo.

The development is not yet known in Polypterus. In the
first stages, hitherto described, of Acipenser ruthenus
(Parker), Lepidosteus (Parker), and Salmo trutta (Stohr),
the hyomandibula is stated to abut against the auditory
capsule. Rutherford! states that in the brown trout a down-
growth of no great size, from the periotic capsule at the edge

>

TEXT-FIG. 34.

alii 4 n/

i Wate (2
Mr BI

Amia, embryo 10mm., transverse section.

of the fenestra ovalis, joins with the symplecticum in front of
the VIIth nerve, and finally unites with the primitive hyo-
mandibula.

In 8mm. embryos of Acipenser the hyoid bar, in a pro-
cartilaginous condition and unsegmented, does not extend up
to the auditory capsule. The VIIth nerve passes over the
upper end of the bar, and then downwards outside it (Text-
fig. 19). In 8} mm. embryos the hyoid bar extends up towards

' The paper is as yet only published in abstract.

208 Fr. H. EDGEWORTH.

qer. byork,

>

TrExT-FIG. 36.

Cons. obec

nro, vent i
3hypoln. hypo 3 Frans varus vent ji
Cor hyoid . 36.
Text-figs. 35-37.—Polypterus, larva 75cm. Text-fig. 35 is the

most anterior.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 209

the auditory capsule and in front of, and outside, the VIIth
nerve, which now winds round it (Text-figs. 20 and 21). The
hyo-mandibular cartilage is formed in part from the upper
portion of the bar present in 8 mm. embryos, and in part
from the upward extension. ‘lhe hyoid muscles in 8 mm.
embryos consist of a hyoid myotome, the anterior part of
which is inserted into the upper end of the hyoid bar (‘T'ext-

TExT-FIG. 37.

l<

~_ Gab
OS vel Ayn ct pane
7 eee
B ns A y \ Lear fn iV
2 é oN Ip z lew be tii

fig. 19), forming a levator hyoidei, and the posterior part of
which forms a dorso-ventral sheet—homologous with C,vd of
Selachians—continuous with the posterior part of the inter-
hyoideus (Text-fig. 21), whilst the anterior part of the inter-
hyoideus is inserted laterally imto the hyoid bar.

The sequence of events in the other Teleostomi examined
is similar to that occurring in Acipenser, the upgrowth of
the hyoid bar to the auditory capsule taking place in 8 mm.
embryos of Lepidosteus, 64 mm. embryos of Amia, and 55 mm.
embryos of Salmo fario. In no case was any downgrowth

210 F. H. EDGEWORTH.

from the periotic capsule found. In Lepidosteus, Amia, and
Salmo, the VIIth nerve, at first winding round the hyoid
bar, subsequently pierces the hyomandibula owing to chon-
drification spreading round it ; the more primitive condition
is preserved in Acipenser and Polypterus.

The adult condition of the hyoid muscles in these 'leleostomi
is not quite uniform. In all the dorso-ventral sheet C,vd
divides into dorsal and ventral portions. In Polypterus the
anterior and posterior portions of the myotome do not separate
from each other, but form one muscle, the retractor hyomandi-
bularis et opercularis. In the others separation takes place ;
the anterior part, i.e. the original levator hyoidei, forms a
retractor hyomandibularis in Acipenser, and an adductor
hyomandibularis in Lepidosteus, Amia, and Salmo. ‘The
posterior part, i.e. the upper part of C,vd, forms a M.
opercularis in Acipenser and Lepidosteus, an adductor and
levator operculi in Amia and Salmo. In 9} mm. embryos of
Salmo the adductor mandibularis additionally spreads forwards,
forming the adductor arcus palatini.

The fore part of the interhyoideus of Acipenser forms the
hyoideus inferior (Cs; of Vetter), the hinder part, i.e. the
lower part of C,vd, forms a constrictor operculi (Cs; and Cs,
of Vetter). In Polypterus the condition is similar.! In Lepi-
dosteus, Amia, and Salmo, the fore part forms the hyoideus
inferior; the hinder part becomes attached laterally to the
hyoid bar (only partially so in Lepidosteus), and forms the
hyoideus superior. The median raphé of these muscles is
preserved in Acipenser, Lepidosteus, and Polypterus; in
Salmo and Amia it is lost, and the hyoideus inferior becomes
attached to the hypohyals of the same and opposite side.

In 84 mm. embryos of Amia the Anlage of the hyomaxillaris”
muscle becomes separated from the upper edge of the hyo-
hyoideus inferior (‘Text-fig. 29) ; it grows forward to Meckel’s

' Intermaxillaris posterior and mantle muscle of Pollard.

2 Superior deeper portion of the genio-hyoid of Allis. <A different
terminology is used in this paper, as the word * genio-hyoid” is generally
used to denote the anterior element of the hypobranchial spinal muscles.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. PA

Trxt-FIG. 38.

Text-figs. 38 and 39.—Ceratodus, embryo stage 40; transverse
sections. Text-fig. 38 is the more anterior; the left side of
the sections is slightly anterior to the right.

SA FE. H. EDGEWORTH.

cartilage dorsal to the intermandibularis posterior. A similar
muscle is formed in Salmo ; it grows forward ventral to the
inter-mandibularis. In 9} mm. embryos of Lepidosteus and
9mm. embryos of Acipenser (Text-fig. 22 a) a similar Anlage
is formed from the dorsal edge of the hyoideus inferior or Cs,
and develops into the hyomanxillaris ligament.' In Polypterus,
? species, Pollard described, but did not name, a small muscle

Text-FieG. 40.

Text-fig. 40.—Ceratodus, stage 40, longitudinal horizontal section.

“at the angle of the jaw in the substance of the lgament
which binds the hyomandibula quadrate and stylohyal,”
innervated by the opercular branch of the hyoid branch of
the VIIth. In Polypterus senegalus only a ligament is
present.

In Ceratodus, between the stages of 38 and 40, the hyoid
myotome and interhyoideus spread backward in the opercular

= Mandibulo-hyoid ligament of van Wijhe.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 213

fold (Text-fig. 40), and form in the latter stage (T'ext-fig. 58)
anteriorly a levator hyoidei inserted into the upper end of the
hyoid bar, and an interhyoideus, and posteriorly a continuous
ventro-dorsal sheet C,vd in the operculum. In stage 42 the
Anlage of the hyomaxillaris ligament is cut off from the
upper edge of the interhyoideus and spreads forwards to the
hind end of Meckel’s cartilage (Text-fig. 41). In stage 42
(Text-fig. 41) the hyoid bar extends upwards and inwards
towards the under surface of the pro-cartilaginous tract con-
necting the parachordal plate with the auditory capsule,
forming the hyomandibula, the original hyoid bar forming
the ceratohyal and hypohyal.

The upper part of the originally pro-cartilaginous hyoman-
dibula chondrifies ; the lower forms a fibrous tract connecting
its outer end with the upper end of the ceratohyal (Text-fig.
50). In stage 48 a downgrowth occurs from the outer edge
of the anditory capsule, external to the hyomandibular branch
of the VIIth (Text-fig. 47),and becomes separated, forming a
cartilage abutting against the outer end of the hyomandibula
(Text-fig. 50), and a second more dorsally situated piece is
subsequently cut off from the auditory capsule (‘l'ext-fig. 50).!
The insertion of the levator hyoidei into the upper end of the
ceratohyal is preserved in the oldest embryo examined, but
is not present in the adult (Ruge) ; it is, however, retained in
Protopterus (vide description by Ruge).

In Necturus Miss Platt stated that the “hyoid mesothelial
tissue”? (here interpreted as “ myotome”) divided into a
dorsal digastricus, which ‘is ultimately connected with the
posterior extremity of the mandibular bar by a long tendon”

! This description of the hyomandibula coincides with and amplifies
that of K. Firbringer. The cartilage (or cartilages) cut off from the
auditory capsule is probably, from its relation to the hyomandibular
branch of the VIIth, that described by Huxley, Ridewood, Ruge, and
Sewertzoff as the “hyomandibula.”” From the descriptions given it
would appear probable that no part of a true hyomandibula is preserved
in the adult, and that (vide Ridewood) the cartilages cut off from the
auditory capsule are variable.

214, F. H. EDGEWORTH.

Text-Figc. 41.

TExt-FIG. 42.

42.

Text-figs. 41-45.—Ceratodus, embryo stage 42; transverse sec-
tions. Text-fig. 41 is the most anterior; the left side of the
sections is slightly anterior to the right side.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 215

and a ventral ceratohyoideus externus. Hxamination of
sections of 144 mm. embryos shows that there is a stage in
which the future digastricus (the “ cephalo-dorso-mandi-
bularis ” of Driiner) forms a levator hyoidei, inserted into the
upper end of the hyoid bar, whilst below the cerato-hyoideus
externus the Anlage of the hyomaxillaris ligament is formed
(Text-fig. 55).

TExT-FIG. 43.

Cyd.

It is of interest to note that in Siren (Driiner) there is a
levator hyoidei, a slip of the cephalo-dorso-mandibularis,
i.e. 1n that animal the primary insertion of the muscle is not
wholly lost.

The interhyoideus' of Necturus spreads backward behind
the hyoid bar in the opercular fold, and this posterior part,
at first entirely ventral, spreads upwards laterally external to
the cerato-hyoideus externus and depressor mandibule. There
is a Similar development of the interhyoideus in T'riton, and

' Posterior mylohyoid of Mivart and Miss Platt.
VOL. 596, PART 2.—NEW SERIES. 16

216 F. H. EDGEWORTH.

the hinder part gains an attachment to the first branchial
bar.!

Schultze described the hyoid muscles of the larve of
Pelobates fuscus as consisting of an orbito-hyoideus, sus-
pensorio-hyoideus, cerato-hyo-angularis, quadrato-angularis,

Trxt-FIG. 44.

Text-figs. 44 and 45.—Ceratodus, embryo stage 46 ; longitudinal
horizontal sections. Text-fig. 44 is the more dorsal; the left
side of the sections is slightly dorsal to the right side.

and suspensorio-angularis. In Rana the myotome of the
hyoid segment separates from the interhyoideus in 6 mm.
embryos. It divides in 7 mm. embryos into an upper and lower
portion (Text-fig. 58); the former develops into the orbito-
hyoideus, which passes from the processus muscularis of the

! First interbranchial of Driiner.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 217

palato-quadrate to the ceratohyal; the latter, which is the
hyomaxillaris, grows forwards to the outer end of Meckel’s
cartilage and divides into the cerato-hyo-angularis, suspen-
sorio-angularis, and the quadrato-angularis (Text-fig. 59). In
Bufo lentiginosus the upper portion of the myotome forms
only an orbito-hyoideus, as in Rana; in Pelobates and Alytes it
forms an orbito-hyoideus and suspensorio-hyoideus. In Pelo-

TrxtT-Fig. 45.

\ e
— 7 Eu nanud,
\

\

quUoduouanve®

2
f
&
os
je

0)

yt!

?

bates and Bufo lentiginosus the hyo-maxillaris divides
into three muscles as in Rana, in Alytes into two only, the
cerato-hyo-angularis and quadrato-angularis (‘l'ext-fig. 63).
In Rana, at metamorphosis, the upper end of the orbito-
hyoideus extends upwards on the atrophy of the processus
muscularis of the palato-quadrate; subsequently, on rotation
backward of the palato-quadrate, the lower end of the orbito-

! The coraco-mandibularis in this drawing is unfortunately not named.

ils) F. H. EDGEWORTH.

Tpxt-FIG. 46.

oT WN
y B06 AS

hyonvar lug

rv hyalc

ass 46

=. Sa ty

rats vert.it

COL. youd 47.

Text-figs. 46-48.— Ceratodus, embryo stage 48. Text-fig. 46 is
the most anterior.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 219

hyoideus gains a new insertion to the hind end of the lower
jaw. The cerato-hyo-angularis, suspensorio-angularis, and
quadrato-angularis assume a more vertical position on the
rotation of the palato-quadrate, and form the inner portion,
whilst the original orbito-hyoideus forms the outer portion,
of the depressor mandibule.

The interhyoideus of the larve of Rana! forms a transverse
band connecting together the two ceratohyals (Text-fig. 57),
and this is also the case in Alytes. In Pelobates and Buto

TExt-FiG. 48.

lentiginosus it extends backwards below the branchial
cavity, and this hinder part forms the sub-branchialis (of
Schultze), and the diaphragmato-branchialis (of Schultze) is
separated from the median edge of this muscle.

The digastric muscle? of Mammals is either monogastric
(digastricus spurius), inserted into the jaw, or digastric
(digastricus verus), with a tendon between the two bellies
which may or may not be connected with the hyoid bone.

1 Subhyoideus of Ecker and Gaup, and of Schultze.
* This bare outline only is given, as the matter has been so thoroughly
discussed by Bijvoet.

220 F. H. EDGEWORTH.

The anterior belly of a digastricus verus is innervated by the
Vth, the posterior belly by the VIIth. A _ digastricus
spurius has a deeper or superficial tendinous inscription,
and is also innervated by both Vth and VIIth.

Anatomists who have investigated the adult conditions of
the muscle have expressed various views as to its origin;
some, e.@. His and Chaine, thought that the muscle was
developed from one mass; others, e. g. Humphrey, Gegen-
bauer, Riige, Fiirbringer, Buijvoet, have held that it was
developed from two masses.

Bijvoet, the latest investigator of the subject, was of opinion
that the condition present im Monotremes—a M. depressor
mandibule anterior, and a M. stylohyoideus—is the “ Aus-
gangsform,” that from this was developed a digastricus
verus, and that a digastricus spurius was a secondary con-
dition. He was also of opinion that a M. intermandibularis
separated into a M. mylohyoideus and a M. depressor
mandibule anterior. The only descriptions of the develop-
ment of the digastric muscle which have hitherto been given
are those by Futamura of human and pig embryos. He says
that the common Anlage of the digastricus, stylohyoideus,
and stapedius is visible in twenty-seven to thirty day human

embryos as a ‘

‘medialwiarts sich anhiiufende dicht gedringte
Muskelblastemgewebe,” which is continuous laterally with the
platysma Anlage, the whole forming asingle mass. In embryos
a little older the platysma and digastric Anlagen separate. In
six weeks’ embryos the digastric Anlage passes forward to the
anterior border of the lowerjaw. The mylohyoid nerve at this
date passes to the mylohyoid muscle only; it does not innervate
the anterior belly of the digastric until the age of seven weeks.
The stylohyoid and stapedius begin to separate from the
posterior belly of the digastric at the age of six weeks. ‘The
digastric is thus solely a facial muscle, theanterior part of which
receives a secondary innervation from the trigeminal nerve.

Bijvoet doubted this account given by Futamura, as it
appeared to him to disagree with the results of comparative
anatomy.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 221

Futamura’s account of the development of the facial and
platysma muscles is ‘that their Anlage separates, as stated
above, from the outer aspect of the common facial Anlage ;
in thirty-one to thirty-four day embryos it spreads aborally
almost to the shoulder region, forming the platysma colli, and
in six-week embryos also forwards, in two directions sepa-
rated by the outer ear, forming a platysma occipitalis and a
facial portion. The latter passes forwards over the edge of
the lower jaw to the forehead, eyelid, upper lip, and temporal
region, separating into a deep and a superficial layer.

In 5 mm. embryos of the rabbit the hyoid myotome is con-
tinuous below with the epithelium lining the hyoid section of
the cephalic ccelom (Text-fig. 77). In 4 mm. embryos the
wails of the cephalic coelom have come together, and the
Anlage of the interhyoideus is formed from its epithelial wall ;
each half is continuous laterally with the lower end of the
myotome (‘lext-fig. 81). In 7} mm. embryos the myotome is
partially separated into an upper and a lower part ; the former
is the Anlage of the posterior digastric, stylohyoid, and stape-
dius, the latter that of the anterior digastric (Text-fig. 89). In
embryos between 8 and 9 mm. the hyoid bar is formed,! and
in its whole extent simultaneously, so that there is no stage
in which the Anlage of the posterior digastric, stylohyoid, and
stapedius is inserted into the upper end of a hyoid bar which
has not yet extended up to the auditory capsule. In 9 mm.
embryos (‘lext-figs. 92 and 93) the lateral edge of the inter-

1 The * body of the hyoid” in the rabbit is formed from the ventral
ends of the hyoid and first branchial bars. No basihyal nor basi-
branchial is developed. In 9 and 15 mm. embryos a column of cells—
the remains of the glosso-thyroid duct—extends downwards from the
foramen cecum in the middle line between the hyoid and first branchial
bars. Antero-posterior fusion of the ventralends of these bars to form
the body of the hyoid, and the commencement of formation of a joint
between the body and the first branchial cornua, take place in embryos
between the stages of 13 and 17 mm. Chondrification takes place later
in the “body” than in the hyoid and first branchial cornua. These
findings confirm the suggestions of Parsons; in the rabbit, however,
there is no basihyal.

22D F. H. EDGEWORTH.

hyoideus has separated from and extended upwards outside the
anterior digastric, which has grown a little forwards. In 10
mm. embryos the anterior end of the anterior digastric has
reached Meckel’s cartilage, the interhyoideus has extended
forwards, and also backwards in the neck forming the platysma
colli, and the stapedius has separated from the Anlage of the
posterior digastric and stylohyoid. In 15 mm. embryos the
Anlage of the posterior digastric and stylohyoid has separated
into those muscles, and the lateral edge of the fore part of the
interhyoideus (Text-fig. 96) has extended upwards and for-

TEXT-FIG, 49.

Ne
pos
6

Sr

X

8

3
eS

7

:
:

rw J
SANS?
Cs
a
<
ee
—

49.

Text-fig. 49.—Ceratodus, embryo 15 mm.; portion of transverse
section taken between the fifth branchial segment and the lung.
wards forming the platysma occipitalis and platysma faciel.

The posterior digastric forms a tendon (vide Krause).

There is thus in the rabbit a stage of development prior to
the first described in man by Futamura, in which the Anlage
of the platysma faciei, platysma occipitalis, and platysma
colliis astructure homologous with the interhyoideus of lower
forms. The anterior digastric is homologous with the hyo-
maxillaris of some Amphibia and ‘lTeleostomi.

The Homologies of the Hyoid Muscles.—In Alytes,
Bufo, Rana, Pelobates, and Lepus, the lower end of the hyoid
myotome becomes separated from the part above and forms
a longitudinal muscle

the hyo-maxillaris—connecting the
hyoid bar with the lower jaw. An homologous muscle is

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 223

formed in Amia and Salmo, but differs in that it is separated
from the upper end of the hyoideus inferior. The first men-
tioned method of formation is probably the primitive one,
for serially homologous muscles, interarcuales ventrales, are
formed from the lower ends of branchial myotomes where
there are no muscles homologous with the interhyoideus ;
the method of formation in these Teleostomi is probably
related to the secondary position of the adductor hyomandi-
bularis internal to the upper part of the hyoid bar. In
Urodela (Necturus, Triton), Ceratodus, Lepidosteus, and

TExt-FIG. 50.

Text-fig. 50.—Ceratodus, advanced embryo, length ¥; portion of
transverse section, to show relations of the hyomandibular
cartilage.

Acipenser a corresponding Anlage develops into a hyo-
maxillaris ligament. In Selachi (Scyllium, Acanthias) and
in Sauropsida no hyo-maxillaris Anlage is formed.

The developmental history of the hyo-maxillaris suggests
that its primitive condition was that of a longitudinal muscle
passing from the hyoid bar to the hind end of Meckel’s carti-
lage, but this is not preserved in any of the animals investi-
gated. In T’eleostomi (Amia and Salmo) the front end of the
muscle extends forwards along Meckel’s cartilage.

In Chimeera there is a similar muscle (hyoideus inferior of
Vetter) extending from the hyoid bar along the lower jaw.

224 F. H. EDGEWORTH.

In Rana, at metamorphosis, the muscle becomes more
vertical in position, and forms, with the orbito-hyoideus, the
depressor mandibule ; this stage is preceded by a larval one,
in which the muscle, having a longitudinal direction, is in part
attached to the palato-quadrate bar. In Mammals the hyo-
maxillaris (anterior digastric) may lose its attachment to the
hyoid bar and form part of a digastricus verus with a tendon
not attached to the hyoid bar, or part of a digastricus spurius.

‘The part of the hyoid myotome above the hyo-maxillaris
in cases where this is formed, or above this and the cerato-
hyoideus externus (in the Urodela), or the whole myotome,
where either or both of these muscles is not formed, is at first
wholly or partially (Scyllium) inserted into the upper end of
a hyoid bar, which has, or has not yet, extended up to the
auditory capsule, forming a levator hyoidei. This holds for
Scyllium, Acipenser, Lepidosteus, Amia, Salmo, Ceratodus,
Necturus, Triton, Rana, and other Anuran larvee, Chrysemys,
and Gallus. In the Amphibia the hyoid bar remains in this
primitive condition, in the other groups it extends upwards
to the auditory capsule.

According to Kingsbury and Reed, the columella auris of
Urodela is the homologue of the hyomandibula of other
forms. If this be so, its dorsal relation to the seventh nerve
would suggest that, it is homologous with the hyomandibula
of 'leleostei, where a similar relation holds, rather than with
that of Selachii, Ceratodus, and Sauropsida, which hes ventral
to the VIIth.

Against such homology, however, it might be urged that in
Scyllium, Acipenser, Lepidosteus, Amia, Salmo, Ceratodus,
Chrysemys, and Gallus (Geoffry Smith), the hyomandibula
is formed by a secondary segmentation occurring in a hyoid
bar, which extends continuously up to the auditory capsule,
whereas this is not stated by Kingsbury and Reed to occur in
Urodela.

A levator hyoidei—attached to the upper end of a hyoid
bar—is not preserved to adult life in any of the animals
examined. In Urodela and Sauropsida it extends downwards

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 225

TEXxtT-FIG. 5].

cebh cool

52.

Text-figs. 51 and 52.—Necturus, embryo 12mm. Text-fig. 51
is the more anterior.

226 F. H. EDGEWORTH.

to Meckel’s cartilage, forming the depressor mandibule. In
Rana this also occurs at metamorphosis, the muscle forming
the outer part of the depressor mandibule. In Scyllium the
levator hyoidei, on the upgrowth of the hyoid bar, is inserted
into its outer surface, and in very late stages of development
extends forwards to the quadrate. In Teleostomi, on the up-
growth of the hyoid bar, the levator hyoidei becomes inserted
into its posterior edge or inner surface, and in Salmo
additionally spreads forward to the palato-quadrate. In
Ceratodus the insertion of the levator hyoidei to the upper
end of the ceratohyal is lost, but itis preserved in Protopterus
(vide description by Ruge). In Lepus there is no stage in
development in which there is a levator hyoidei inserted into
the upper end of a hyoid bar, for this is formed simultaneously
in its whole length, but the portion of the myotome above the
Anlage of the hyo-maxillaris (anterior digastric) 1s a mass,
which is homologous with the levator hyoidei of other forms,
and lies outside the hyoid bar; it subsequently divides into
stapedius, stylohyoideus and digastricus posterior. It is
homologous with the M. styloideus of Monotremes. The
upper end of the levator hyoidei is attached to the auditory
capsule ; this attachment, in Anura, is preceded by a larval
stage, in which the upper end of the muscle takes origin
from the processus muscularis of the palato-quadrate bar.
The primary form of the interhyoideus—formed from the
walls of the obliterated hyoid section of the cephalic cocelom,
and serially homologous with the intermandibularis—would
appear to have been a transverse band connecting the two
hyoid bars, but in all forms examined, other than Rana and
Alytes, it extends backwards. In Urodela, Anura, and
rabbit there is a backward extension of the interhyoideus
only, but in Selachii, Teleostomi, Ceratodus, and Sauropsida,
of both myotome and interhyoideus, forming a continuous
ventro-dorsal sheet (C,vd of Ruge) behind the hyoid bar,
‘he backward extension of the interhyoideus, or of this and
of the myotome, takes place in the opercular fold in Urodela,
Anura, Ceratodus, and Teleostomi; in part in the opercular

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 227

Text-FIG. 53.

Sey

aa
0% 6
re

e

De:

Text-figs. 55 and 54.—Necturus, embryo 13 mm., transverse
section. Text-fig. 53 through the fourth branchial segment,
Text-fig. 54 a little more posterior.

228 F. H. EDGEWORTH.

fold (only existing ventrally) in Sauropsida and rabbit ; and
in Scyllinm entirely in the wall of the head, no opercular fold,
even as an atrophying Anlage, being developed.

The backward extension of the interhyoideus forms the
hinder part of the interhyoideus of Urodela, and the sub-
branchialis of Anura. In the rabbit the attachment of the
interhyoideus to the hyoid bar is lost, and it spreads down
the neck, forming the platysma, and also upwards and
forwards, forming the superficial occipital and facial muscles.

C,vd persists as a continuous ventro-dorsal sheet im
Scyllium, Ceratodus, and Sauropsida ; in the latter group, in
correlation with the atrophy of the gill-clefts, it spreads
backwards in the neck, forming the constrictor colli; in
Teleostomi it separates into dorsal and ventral portions, the
former developing into the opercular muscle or muscles, the
latter into a constrictor operculh (Acipenser and Polypterus),
or hyohyoideus superior (Lepidosteus, Amia, Salmo).

The hinder part of the interhyoideus of Urodela and the
platysma of the rabbit imitate, to some extent, the constrictor
colli of Sauropsida, but this is due to a dorsal extension of
their lateral edges over the hyoid myotome after their
formation from the interhyoideus only. |

It will be shown, later, that the probable primitive con-
dition of the muscles of each branchial arch was, a M. levator,
a M. marginalis, a M. interarcualis, and a M. transversus
ventrahs. ‘The levator hyoidei is serially homologous with
the first, and the hyo-maxillaris with the third of these. The
cerato-hyoideus externus is serially homologous with the Mm.
marginales and their homologues the Anlagen of the gill-
muscles, and may possibly be derived from a hyoidean M.
marginalis, but there is no trace of such a muscle in other
groups. ‘There are no homologues of the transversi ventrales
in the mandibular and hyoid segments.

The primitive condition of the mandibular and hyoid
muscles was probably one in which the former lay wholly in
front of the latter. This condition exists in the embryo but
does not persist ; during development, in correlation with the

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 229

TExT-FIG. 55.

Text-figs. 55 and 56.—Necturus, embryo 145 mm., transverse
sections. Text-fig. 55 through the hyoid segment, Text-fig. 56
through the fourth branchiai segment.

230 F. H. EDGRWORTH.

increase in size of the jaws, there is a gradual overlapping of
the hyoid bar by the mandibular skeletal structures, with
associated secondary changes in the muscles. ‘Thus, the
levator hyoides shifts its insertion from the hyoid bar to the
hind end of Meckel’s cartilage in Amphibia and Sauropsida,
and the intermandibularis partially underlies the interhyoi-
deus in Selachi, Teleostomi, Ceratodus, and Urodela. It is
probably in connection with this overlapping that the hyo-
maxillaris Anlage becomes a lgament in Urodela, Cera-
todus, and some ‘eleostomi, and is not developed in
Selachii and Sauropsida.

Kye Muscies or THE Rassit.

The development of the external ocular muscles in 'Teleo-
stoman and Amphibian embryos was not followed.

In pig embryos of 10 mm. maximum and 6 mm. Nacken-
lange, Reuter found the Anlage of the eye muscles as an

“als Anhau-

“untrennbarer Zellcomplex,”’ which, he held,
fung von morphologisch urspriinglich Keimzellen aus dem
Mesenchym hervorgeht.”

In3 mm. embryos of the rabbit the premandibular Anlage
is developed, on each side, as a hollow outgrowth of the fore
end of the alimentary canal (Text-fig. 75). This outgrowth
becomes solid by proliferation of its walls as it is constricted
off, and in 54 mm. embryos (Text-fig. 79) it forms a solid mass
connected with its fellow bya solid string of cells lying across
the fore end of the alimentary canal just below the anterior
down-turned end of the notochord. The connecting string
of cells has disappeared in 44 mm. embryos. The premandi-
bular Anlage forms a crescent-shaped mass in 4 mm. embryos
(Text-fig. 84); and from this the recti superior, inferior,
internus, and obliquus inferior are formed (‘T'ext-figs. 96, 97).
The obliquus superior is formed from the anterior extremity
of the upper end of the mandibular myotome in 31 mm.
embryos (Text-fig. 78). The development of the abducens
Anlage is doubtful; in 34 mm. embryos it is visible lying

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 231

behind the premandibular Anlage and internal to the man-
dibular myotome (Text-fig. 79). In 2} mm. embryos it
apparently is represented by a group of cells lying above
the first gill-cleft and continuous behind with cells lying in
the hyoid segment—cells which in 3 mm. embryos have diffe-
rentiated into myotome and surrounding mesoblast. It is
probable, therefore, that the abducens Anlage is formed from

the mesoblast of the hyoid segment before the myotome is

unlechyod *

57.

Rana, embryo 6mm., longitudinal vertical section.

formed. The abducens Anlage begins to separate into rectus
externus and retractor oculi in 15 mm. embryos (Text-fig. 96).

These observations suggest that perhaps in pig embryos of
a younger age than those investigated by Reuter a similar
series of events may occur. In the later stages of develop-
ment there is also a difference between the pig and the
rabbit ; in the former, according to Reuter, the retractor oculi
is formed from all four recti, in the latter it is formed from
the abducens Anlage only, as is usual in Vertebrates.

VOL. 56, PART 2.—NEW SERIES. NZ)

232 F. H. EDGEWORTH.

BRANCHIAL MUSCLES.

The homologies between the branchial muscles of various
vertebrates are obscured by the absence of uniformity in
nomenclature. The word “interbranchial ” has been apphed
to many quite different muscles. It was used by Vetter to
denote the layer of muscle-fibres which hes external to the
branchial bars and internal to the superficial constrictor in
Selachii. It was subsequently employed by Bronn and by
Schultze to denote longitudinal muscles between the ventral
ends of branchial bars in Amphibia; by Driiner, to denote
transverse muscles in Amphibia; by K. Fiirbringer and by
Greil to denote slender vertical muscles in Ceratodus.

In this paper the word “interbranchial” is used exclusively
in the Selachii in the sense of Vetter; longitudinal muscles
between the ventral ends of branchial bars are called ‘ inter-
arcuales ventrales”? (vide infra); transverse muscles are
called ‘‘transversi ventrales,’? and the vertical muscles of
Ceratodus are called Mm. marginales, as they are exactly
homologous with the Mm. marginales of Anuran_ larve
described by Schultze.

Fiirbringer included two sets of muscles under the term
“ hypobranchial spinal musculature” '—(1) Those which are
sometimes called ventral longitudinal muscles, e. g. coraco-
mandibularis, coraco-hyoideus, genio-hyoideus, sterno-
hyoideus; (2) the coraco-branchiales of Klasmobranchs,
Teleostomi, and Dipnoi.

Investigation of the development of these muscles, how-
ever, shows that whereas the ventral longitudinal muscles
are derived from trunk-myotomes, the coraco-branchiales
are derived from the ventral end of one or more branchial
myotomes, i.e. are of cranial origin. ‘lhe above-mentioned
terminology would therefore appear to be in need of revision,

‘

1 The name “hypoglossal musculature” employed by Neal and by
Corning has nearly, but not quite, the same meaning as Fiirbringer’s
“ hypobranchial spinal musculature,” e. g., it would include the cerato-
hyoideus (interarcualis ventralis I) of Sphenodon.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 233

and in this paper longitudinal muscles derived from trank-
myotomes are called’ “ hypobrauchial spinal muscles,” and
longitudinal muscles derived from branchial myotomes are
called “‘ hypobranchial cranial muscles.”

The Anlagen, which in some Vertebrates grow backwards
and form coraco-branchiales, are homologous with Anlagen
in other Vertebrates, which form muscles passing between
the ventral ends of the branchial bars. Many different

TEXT-FIG. 58.

58.

Rana, embryo 7mm., transverse section.

names have been given to the latter muscles, e.g. inter-
branchiales s. constrictores arcuum branchialium (Bronn),
constrictores arcuum (Mivart), interbranchiales (Schultze),
subarcuales (Driiner).

Vetter called certain muscles in the branchial arches of
Acipenser “‘interarcuales ventrales,” and gave the name of
“obhqui ventrales” to exactly homologous muscles in
Teleostei. One or other term is superfluous, and following
Allis, that of “obliqui ventrales” is used in this paper for
the Teleostomi generally. ‘he name ‘‘interarcuales ventrales”

234 F. H. EDGEWORTH.

is used for the longitudinal muscles passing between the
ventral ends ofthe branchial bars. |

The foremost ‘‘interarcualis ventralis” passing from the
first branchial to the hyoid bar has received many names,
e. @. ceratoidien latéral on petit ceratoidien (Cuvier), inter-
cornalis (Owen), cerato-hyoideus internus (Ftirbringer, Miss
Platt, Driimer), cerato-hyoideus (Osawa), hyoideus lateralis
(Bendz), kerato-hyoideus (Ellenberger and Baum), inter-
hyoideus (Dubois), kerato-thyro-hyoideus (Jaquet). The
name “branchio-hyoideus” was apphed to this muscle in
Sphenodon (loc. cit.), and is employed in the case of other
animals, when it occurs, in this paper.

In Seyllium, van Wijhe described the coraco-branchialis
and coraco-mandibularis as developing from the walls of the
obliterated anterior prolongation of the pericardium, and
from “ Nebenzweige,” given off from the “ Unterenden der
Wiinde der Visceralbogenhéhlen.”

It will be shown later (p. 268) that the coraco-mandibularis
and coraco-hyoideus are developed from the fourth to eighth
trunk-myotomes. The five branchial myotomes form at first
epithelium-lined cavities continuous below with the cephalic
ccelom (Text-fig. 2). They separate from the cephalic ccelom
in 14 mm. embryos, and their cavities become obliterated
(Text-fig. 4). In 17 mm. embryos the lower ends of the
branchial myotomes grow backwards, and become cut off
from the remainder of the myotomes, forming coraco-
branchiales (Text-figs. 8,10). The (now) ventral ends of the
myotomes grow downwards outside the Anlagen of the
coraco-branchiales forming the ventral ends of the constrictor
(Text-fig. 15). The upper ends of the myotomes, in embryos
between the lengths of 17 and 20 mm., increase in antero-
posterior extent (Text-fig. 14), and, fusing together, extend
backward as the trapezius' to the shoulder-girdle. Below

1 It is stated by van Wijhe that the seventh, eighth and ninth
myotomes give rise to “ Vom Schadel zum Schultergurtel ziehende
Muskeln nebst dem vordersten Theile des sterno-hyoideus,”’ but the
trapezius is not specifically mentioned by him.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 235

the Anlagen of the trapezius each branchial myotome forms
a transversely broad plate in the branchial septum (Text-fig.
9). The part internal to the branchial bar forms the adductor
(Text-fig. 15) ; the part external to the bar forms—next the
bar, dorsally the arcualis dorsalis! (Text-fig. 14), and below
that the interbranchial, whilst the external edge forms the
constrictor superficialis.

In Acipenser the lower ends of the branchial myotomes

TrxtT-FIG. 59.

Rana, larva 12 min., transverse section.

separate from the cephalic ccelom, and in 84 mm. embryos
(Text-fig. 22) grow downwards, except in the fourth branchial
segment,” forming the Anlagen of the coraco-branchiales,
and also downwards and inwards dorsal to the cephalic ccelom,
forming the lower parts of the obliqui ventrales. The ventral
end of the lower part of obliquus ventralis I becomes attached

' Employing Firbringer’s terminology (vide p. 266).

> In the adult forms examined by Vetter the coraco-branchialis

IV was absent, and it was not developed in the embryos examined.
According to Firbringer it is present.

236 F. H. EDGEWORTH.

to the ceratohyal, and those of the obliqui ventrales I] and
III to the corresponding hypobranchials, whilst the 1Vth and
Vth meet their fellows in the median line, in the case of the
IVth also becoming attached to the basibranchial. The
parts of the first three branchial myotomes above the
Anlagen of the coraco-branchiales form adductors internal
to the branchial bars, and the upper portions of the obliqui
ventrales external to the branchial bars; in the case of the
fourth myotome only the upper portion of obliquus ventralis

TEXT-FIG. 60.

2x tralemb

gubtemb GP
exhaleup

hes
Subrrax : a 2
/ subment gen hyord.
/
‘Yen.gloss 60.
Rana, larva with hind legs moderately developed, transverse

section.

IV. The uppermost portions of the first four branchial myo-
tomes form levatores arcuum branchialium; in 8 mm.
embryos the first is attached to the auditory capsule, the
second, third, and fourth lie outside the trunk myotomes
(Text-fig. 22) ; in 1l mm. embryos the upper ends of the third
and fourth have also shifted to the auditory capsule with addi-
tional attachments to the second pharyngo-branchial, and the
upper end of the second has become attached to the second
pharyngo-branchial ; all four are inserted to the correspond-
ing epibranchials. he trapezius is given off from the fourth

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 237

levator in 8} mm. embryos (Text-fig. 22), and grows back to
the shoulder-girdle ; in 11 mm. embryos its fore part has dis-
appeared, and the anterior end of the part remaining is
attached to the skin; in the adult it is absent (Vetter). ‘The
portion of the fifth branchial myotome above the coraco-bran-
chialis V forms a muscle attached above the fourth epi-
branchial and below to the fifth cerato-branchial—the fifth
levator of Vetter.

In Amia the lower end of the fifth branchial myotome forms
in 81 mm. embryos (T'ext-figs. 31, 32) the lateral half of a
transversus ventralis V and a coraco-branchialis V, as in
Acipenser ; the lower end of the fourth branchial myotome
forms (J'exi-fig. 30) the lateral half of a transversus ventralis
IV and the Anlage of the interarcualis ventralis IV, which
grows forward to the third branchial bar. Neither coraco-bran-
chiales! nor interarcuales ventrales are formed in the first
three myotomes ; the lower ends of the myotomes grow
downwards and inwards, forming the ventral portions of the
obliqui ventrales I, If, and III. The portion of the fourth
myotome, next above the Anlagen of the interarcualis ven-
tralis [V and transversus ventralis IV, forms the obliquus
ventralis of that arch, which is serially homologous with the
dorsal portions of the obliqui ventrales of the first three
arches. In 15 mm. embryos the hind end of the interarcualis
ventralis IV grows backward to the fifth bar, and in
19 mm. embryos its front portion divides longitudinally into
two (Allis), so that there are formed two longitudinal muscles
extending from the third bar to the fourth and fifth respec-
tively; both are innervated by the nerve to the fourth arch
(Allis).

Allis homologised these longitudinal muscles with the
lower portions of the obliqui ventrales of the first three
arches, but their development shows that the latter are homo-

1 Firbringer described a coraco-branchialis I], but it is not des-
cribed by Allis or MeMurrich, and was not present in the embryos
examined.

238 F. H. EDGEWORTH.

logous with the transversi ventrales of the fourth and fifth
arches.

The coraco-branchialis V divides, in 14 mm. embryos, into
pharyngo-clavicularis internus and externus.

The upper ends of the first branchial myotomes form leva-
tores arcuum branchialium ; the first two broaden transversely
(Text-fig. 29) and divide into external and internal portions.
The first and second externi and third and fourth levatores

Trext-FIG. 61.

!

Mord |

Rana, larva with large hind legs, transverse section.

become attached to the first, second, third, and fourth epibran-
chials, the first internus to the second pharyngo-branchial, and
second internus ! to the third pharyngo-branchial constituent
of the superior pharyngeal bone? of Allis (os pharyngeum
superior of v. Wijhe, Pharyngealplatte of Wuiedersheim).
All take their origin from the auditory capsule. The

! Protractor laryngis of Wiedersheim.

* The os pharyngeum superior of Amia and Lepidosteus (Text-fig.
25) is formed by the union of the pharyngo-branchials of the third and
fourth arches—hbearing out the theory of v. Wijhe.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 239

trapezius is formed from the fourth levator, and persists ;
it is the muscle described by Allis as the “fifth externus”
levator, “found in 40 mm. fishes as a part of the fourth
levator.” The portion of the fifth branchial myotome above
the coraco-branchialis V does not divide into levator and
(dorsal portion of) obliquus ventralis ; it forms two muscles—
the second obliquus dorsalis and second adductor of Allis,
passing from the fourth epi- and cerato-branchial to the fifth
cerato-branchial.

The development of the branchial muscles of Lepidosteus
is similar to that of Amia, the only exceptions being (1) the
coraco-branchialis V does not divide into pharyngo-clavi-
cularis externus and internus (‘l'ext-fig. 25)'; (2) the inter-
arcualis ventralis IV is not developed.

The differences between the branchial muscles of Salmo
and Amia are that in the former (1) the interarcualis ven-
tralis IV retains the primitive condition of a longitudinal
muscle between the fourth and third bars, and does not, as
in Amia, secondarily extend back to the fifth bar. (2) The
third levator arcuum branchialium, as well as the first two,
divides into external and internal portions, of which the
externus is inserted into the third epibranchial, and the
internus into the fourth pharyngo-branchial. (3) The portion
of the fifth myotome above the coraco-branchialis V forms
one muscle only (obliquus dorsalis of Vetter), passing from
the fourth epibranchial to the fifth cerato-branchial. (4) No
adductor is formed in the fourth arch.

It is noteworthy that the trapezius persists in Salmo, as in
Menidia (Herrick) ; in Esox, Cyprinus, and Perca it is absent
in the adult (Vetter). In some Teleostei there are a greater
number of interarcuales ventrales present than in Salmo,
e.g. in Cyprinus an interarcualis ventralis I, and in Esox
an interarcualis ventralis II] are additionally present (Vetter).

The muscles attached to the ventral ends of the branchial
bars of Polypterus, apparently, are very different in various

* According to Fiirbringer, “ Lepidosteus hat kein coraco-branchialis
mehr.”

240 EF. H. EDGEWORTH.

species. Fiirbringer stated that in Polypterus, ? species, there
are four coraco-branchiales attached to the four branchial bars.
Pollard did not describe these ; he stated that in Polypterus,
? species, the coraco-hyoideus sends additionally a long
tendon to the lower end of the first cerato-branchial, and also
that there is a muscle belonging to the system of the coraco-
arcuales, which, arising from the fourth, i.e. last cerato-
branchial, passes horizontally forwards and affixes itself to
the lower ends of the second and first cerato-branchiales. It
is apparently supphed by the united first and second spinal

TEXT-FIG. 62:

sub bal: cot

62.

Rana, larva with fully formed hind legs, transverse section.

nerves. ‘There is also ‘‘a flat muscle of small size, which
takes its origin from the last cerato-branchial. It loses
itself in the skin near the anterior edge of the dermal clavicle.
Its innervation was not traced.”

In Polypterus senegalus (larve 7} to 9} em. long)
there is a pharyngo-clavicularis externus and internus (=
coraco-branchialis IV) attached anteriorly to the fourth
cerato-branchial, and passing downwards through the coraco-
hyoideus to the shoulder-girdle (Text-fig. 37). In front of
this is a longitudinal muscle passing from the fourth to the
second cerato-branchial, and innervated by the nerve to the

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 241

third arch; this, on comparison with the interarcualis ventralis
IV of Amia, is probably an interarcualis ventralis [11, which
has additionally extended back to the fourth bar. In front
of this are the interarcuales ventrales Il and I, the former
passing from the second to the first cerato-branchial, and the
latter from the cerato-branchial to the ceratohyal (Text-figs.
39 and 36).

In Polypterus senegalus there are transversi ventrales
III and IV (Text-figs. 36, 37); the median edges of the
former are attached to the basibranchial; the latter in its
anterior part forms a transverse muscle, and in its posterior
part enters into relation with the rima glottidis, forming the
dilatator of Wiedersheim. He called the fore part of the
muscle M. adductor are. branch., but adductors, in the sense
of Vetter, are not present in Polypterus senegalus, and
the whole muscle is a transversus ventralis of the fourth arch.

In Polypterus, ? species, Pollard described four “ inter-
arcuales ventrales ” (i.e. in the terminology of this paper,
“ obliqui ventrales”’), one to each branchial bar. In Poly-
pterus senegalus these muscles are not present in the first

bd

and second branchial segments; in the third and fourth
segments their dorsal portions are present in the form of very
minute muscles, the lower ends of which are attached to the
cerato-branchiales (‘Text-fig. 37). Pollard described four leva-
tores arcuum branchialium inserted into the upper ends of the
cerato-branchials. In Polypterus senegalus the first is
inserted into the first pharyngo- and epi-branchial, the second
and third into the respective pharyngo-branchials, and the
fourth, which has an additional head from the third pharyngo-
branchial, into the fourth cerato-branchial. According to
Pollard, there is no trapezius corresponding to that of
Selachians, but he mentions that a muscular slip—presumably
of the fourth levator—continues on beyond the last (fourth)
cerato-branchial, and is inserted into the skin-lgaments in
front of the shoulder-girdle. In Polypterus senegalus
there is a trapezius arising in common with the fourth
levator and passing back to the shoulder-girdle (‘T'ext-fig. 37).

242 F. H. EDGEWORTH.

The development of the branchial muscles of Ceratodus, as
given by Greil, is summarised above (pp. 175 and 176). In the
specimens examined the lower ends of the branchial myotomes
separate from the lateral wall of the cephalic ccelom in stage
42 (Text-figs. 42,45). In stage 46 the lower end of the first
branchial myotome grows forward to the hypohyal (Text-fig.
44), forming the interarcualis ventralis I's. branchio-hyoideus ;

TExtT-FIG. 63.

hyogless submwor. 63.

Alytes, larva 12 mmm., transverse section.

in the second, third, and fifth branchial segments the lower ends
of the myotomes grow downwards, forming coraco-branchiales
II (Text-fig. 47), [1 and V, and also downwards and inwards,
forming the (lateral halves of the) transversi ventrales

' Cerato-hyoideus internus of Fiir bringer; cerato-hyoideus of Greil;
M. grand abducteur du premier are branchial of Jaquet, who states
that the hind end of the muscle is attached to the first and second
branchial bars.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 243

TJ,' I11,' and V*; in the fourth branchial segment only a
coraco-branchialis IV is formed. This condition—of an
interarcualis ventralis I and coraco-branchiales II, III, IV,
and V—persists till stage 65; at a later stage the hinder end
of the interarcualis ventralis I grows backwards, forming,
in the specimen examined, a longitudinal muscle, which is
attached posteriorly to the fifth bar, and also a coraco-bran-
chialis I. From this and the descriptions given of the adult
by M. Fiirbringer and by Jaquet, it may be inferred that
the hind end of the interarcualis ventralis | always grows
back, forming a coraco-branchialis I, and may or may not
also grow back to a more posterior branchial bar.

The portions of the branchial myotomes immediately above
the interarcualis ventralis I and coraco-branchiales II, III,
and TV form Mm. marginales.* No adductors are developed.
The upper ends of the first four branchial myotomes and the
whole of the fifth branchial myotome above the coraco-bran-
chialis V (no fifth M. marginalis being developed) form
levatores arcuum branchialium? (‘Text-fig. 48).

The trapezius’ is proliferated from the outer side of the fifth
levator in stage 48 (Text-fig. 48).

In Necturus (Miss Platt), in embryos of 124 mm. there is
an outgrowth from the ventral part of the glosso-pharyngeal
muscle—the beginning of the cerato-hyoideus internus; and
there are three constrictors arcuum, the first growing forwards
from the mesothelium of the first vagus arch where this joins

' M. chiasmique of Jaquet; second and third Mm. interbranchiales
of Greil.

? The posterior margin of the transversus ventralis V in stage 63
underlies the lung.

3M. branchialis of Jaquet; first, second, third, and fourth Mm.
interbranchiales of K. Fiirbringer ; fourth and fifth Mim. interbranchiales
of Greil. In the adult, according to Jaquet. these muscles are attached
dorsally to the upper ends of the branchial bars, according to K.
Fiirbringer to the skull.

4 Cranio-branchiales of Jaquet; levatores arcuum branchialium of
Greil.

° M. scapulo-branchialis of Jaquet; levator scapuiz of Greil.

24.4 KF. H. EDGEWORTH.

the wall of the pericardium, the second and third arising as a
single muscle from the wall of the pericardium in the region
where the mesothelium of the second vagus arch unites with
the pericardial wall. Above these muscles are found the gill-
muscles, and dorsally the three levatores arcuum.

This would mean, according to the theory which was
suggested above, that the interarcuales ventrales I, II, and
III are formed from the ventral ends of the first, second,
and third branchial myotomes, the Anlagen of the gill-
muscles above these, and the three levatores from the upper-
most portions.

There are three other branchial.muscles in Necturus which
were not mentioned by Miss Platt—the transversus ven-
tralis [V,! fourth? levator arcuum, and the trapezius.’ In 12
mm. embryos there is present a fourth branchial myotome
serially homologous with the first, second, and third (‘Text-figs.
51, 52). In 13 mm. embryos this has separated from the
cephalic coelom and divided into a fourth levator and lateral
half of a transversus ventralis IV (Text-fig. 55); im 143 mm.
embryos the lower half of the transversus ventralis IV has
spread inwards dorsal to the cephalic ccelom and below the
developing larynx to meet its fellow in the middle line (‘lext-
fig. 56). No interarcualis ventralis [V is developed. ‘T'rans-
versi ventrales are not developed in the first three arches.®
The trapezius is proliferated from the outer surface of the
fourth levator in 16 mm. embryos.

In Triton cristatus the events are similar; an interarcu-
alis ventralis 1V is developed, in correlation with the formation
of the fourth branchial bar. The interarcuales ventrales II,
III, and IV become divided into the muscles called sub-

' The fourth pharyngo-branchialis of Wilder; the hyo-pharyngeus of
Goppert.
* The fourth levator and trapezius were described by Mivart; the
latter, in the terminology of Fiirbringer, is a dorso-scapularis.

* This confirms the opinion of Goppert that his hyopharyngeus is
not formed by fusion of transversi ventrales III and IV, but is only a
transversus ventralis IV.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 245

arcuales recti and obliqui by Driiner. The trapezius is a
capiti-dorso-scapularis; it is formed by proliferation from the
outer surface of the fourth levator in 8} mm. (just hatched)
larvee.

Driiner described in Urodela a first, third, fourth, and fifth
transversus ventralis (called by him ‘interbranchial”’) in the
territory of the first, third, fourth, and (an atrophied) fifth
branchial arches. He also stated that the first is formed by
a secondary attachment of the ventral facial muscles to the
first branchial arch—this, which is not a true transversus, is
described above (p.215). In Necturusand Triton cristatus
a transversus ventralis III is not formed; there is only a
IVth. The question whether the laryngei represent a Vth
is discussed below. ‘The transversi ventrales were included
by Driiner in the ventral head muscles, but they are not
serially homologous with the intermandibularis and inter-
hyoideus, which are developed from the walls of the cephalic
ccelom in the mandibular and hyoid segments, whereas the
transversi ventrales are formed by downgrowths of the
branchial myotomes dorsal to the cephalic ccelom.

In Rana temporaria the ventral ends of the four
branchial myotomes separate from the parts above in 63 mm.
embryos, and form the Anlagen of the four interarcuales
ventrales, and in the second, third, and fourth segments the
Anlagen of the transversi ventrales. In the first three seg-
ments the middle portions of the myotomes form the Mm.
marginales and the upper parts the levators. The portion
of the fourth myotome above the interarcualis ventralis forms
the fourth levator, no M. marginalis being developed.

The median ends of transversi ventrales IT and III become
attached to the posterior surface of a ventral projection of
the first basibranchial (second copula), and their lateral
edges to the processus branchialis. The lateral edges of
transversus ventralis [V become attached to the fourth cerato-
branchial (Text-fig. 61), and their median edges meet in a
central raphé, which underlies the fore part of the larynx.
There are similar muscles in larve of Alytes, Bufo lenti-

246 F. H. EDGEWORTH:.

ginosus, and Pelobates,'. In Rana, at the end of the meta-
morphosis, the transversi ventrales II] and III disappear,
whilst transversus ventralis IV persists.

Wilder was of opinion that transversus ventralis IV
(constrictor laryngis, hyopharyngeus of Goppert, Veren-
gerer des Aditus laryngis of Henle) was a derivative of the
intrinsic ring, 1.e. of the sphincter laryngis. Géppert, on
the other hand, thought that it was homologous with the
hyopharyngeus of Urodela, only differing in that it fails
in the larva to be attached to the fourth bar. This homology
of Géppert is confirmed by the development of the muscles.
In the Anuran larvee examined the muscle was attached to
the fourth bar.

The Anlagen of the interarcuales ventrales develop into
longitudinal muscles, each extending from the bar of its
segment of origin to the next anterior one. In 9 mm. larvee
the interarcualis ventralis I s. branchio-hyoideus divides
longitudinally into two parts, one of which connects the first
branchial bar to the ceratohyal, the other forms with the
interarcualis ventralis IT a muscle extending from the second
branchial bar to the ceratohyal. A similar development of
the interarcualis ventralis I takes place in Bufo lenti-
ginosus, Alytes, and Pelobates.?

The Mm. marginales of Alytes, Bufo, Rana, and Pelobates
(vide Schultze), run along the external edges of the corres-
ponding branchial bars ; their dorsal ends are attached to the
external surfaces of the upper ends (below the insertions of the
levators) of their respective bars. The ventral end of the first
is attached to the second bar—to the processus branchialis of
Schultze, which is formed from the second bar, the ventral

' The transversi ventrales II and III are collectively termed “* basi-
hyobranchialis ” by Schultze, in Pelobates.

» Schultze did not describe the muscle passing from the first
branchial bar to the ceratohyal in older larvee of Pelobates, but it per-
sists up to the stage of 30 mm. The interarcuales II and III he
collectively terms the “interbranchial” ; and the muscle passing from
the second bar to the ceratohyal the “‘ cerato-hyo-branchialis.”

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 247

ends of the second and third to the third bar, just behind
its junction with the processus branchialis. In Rana, at the
end of metamorphosis, the cerato-branchial portions of the
branchial bars disappear and the Mm. marginales also. The
upper end of the first levator becomes attached in part to
the palato-pterygoid bar and in part to the periotic capsule,
the second, third, and fourth to the periotic capsule only
(Text-fig. 61). This is also the case in Alytes, Bufo lenti-
ginosus, and Pelobates.!

According to Wilder, in Rana clamitans the fourth
levator is formed during metamorphosis by division of the
dorso-laryngis into the fourth levator, and dilatator laryngis ;
and this is stated to hold generally in Anura. In Rana
temporaria, however, the fourth levator is formed in 7mm.
embryos, and the dorso-laryngeus not until 74 mm., and the
two muscles have no genetic connection ; and both muscles

are present in larve of Pelobates, Alytes, and Bufo lenti-
ginosus, of 10, 12$,and 10 mm. respectively, i.e.long before
metamorphosis.

In Rana, late in metamorphosis, the partial origin of the
first levator from the palato-pterygoid bar is given up, and,
on the atrophy of the cerato-branchials and Mm. marginales,
all four leyators extend downwards, and their lower ends
become attached to the body and processus posterior medius
of the hyoid bar.

In 12 mm. larvee of Rana a downgrowth of the lower end
of the fourth levator takes place, forming the diaphragmato-
branchialis lateralis? (of Schultze). Its upper end becomes
attached to the fourth bar, its lower end to the diaphragm.
It is innervated by the Xth. It is also formed in Alytes,

' Schultze, in older larvee of Pelobates, described all four levatores as
arising from the palato-quadrate bar.

* Schultze gave the name “diaphragmato-branchialis medialis” to
the muscle called * sterno-hyoid ” in this paper. He did not describe
the innervation or development of the larval muscles of Pelobates ; his
names are purely descriptive, and do not imply that he thought that the
two muscles * diaphragmato-branchialis”’ and “ medialis’? have any
genetic connection.

VOL. 56, PART 2.—NEW SERIES. 18

248 F. H. EDGEWORTH.

Bufo lentiginosus, and Pelobates, in Jarve of about the
same length. In Rana it disappears late in metamorphosis.
The trapezius (capiti-scapularis, of Fiirbringer; cucullaris,
of Ecker and Gaupp) is formed early in metamorphosis from
cells proliferated from the outer surface of the fourth levator
(Text-fig. 61).

In 6 mm. embryos of Chrysemys marginata there are
four branchial myotomes; in8 mm. embryos (T'ext-figs. 66-69)
the middle portion of the first is very slender and that of the
second has disappeared, and the middle and lower portions
of the third and fourth have disappeared ; the upper end of
the fourth has extended back a little in the neck, the upper
end of the third has extended back to that of the fourth, the
upper end of the second isa separate structure, and the upper
end of the first is still connected with the rest of the myotome.
In 12 mm. embryos the dorsal ends of the first and second
have each grown backwards into the next segment, and there
is thus formed a long column of cells which has grown still
further backwards into the neck, forming a trapezius—the
capiti-plastralis of Furbringer; the middle portion of the
first and the lower end of the second myotomes! have dis-
appeared, whilst the lower end of the first forms the inter-
arcualis ventralis I, which, extending from the first branchial
bar to Meckel’s cartilage, is the branchio-mandibularis.

In Lacerta agilis the dorsal edge of the primitive
trapezius extends upwards outside the trunk myotomes of the
neck (‘l'ext-figs. 70, 71), and in 20 mm. embryos it has divided
into dorsal and ventral portions, the capiti-dorso-clavicularis
and capiti-cleido-episternalis of Fiirbringer. ‘he former is
innervated solely by spinal nerves, the latter by the actes-
sorius vagi. Fiirbringer concluded from this innervation
that the capiti-dorso-clavicularis is a new formation, and that

1 The curious persistence for a time of the lower end of the second
branchial myotome, after disappearance of the middle portion of the
myotome, is in favour of the idea (loc. cit.) that ancestors of the
Sauropsida may have possessed an interarcualis ventralis Il, passing
from the second to the first branchial bar,

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 249

the whole muscle is a complex of muscle-metameres,! but this
inference is not borne out by study of its development.

In Gallus the upper ends of the first and second branchial
myotomes separate from the parts beneath on the fourth day
(Text-figs. 72, 73). They fuse together and extend back-
wards in the neck (Text-fig. 74) forming the trapezius

TEXT-FIG. 64.

hybobe, m : .
Ss An intefyord 64.
Text-figs. 64-69.—Chrysemys, embryo 8mm. Text-fig. 64 is the
most anterior; Text-figs. 64 and 65 are through the hyoid
segment, Text-fig. 66 through the first branchial, Text-fig. 67
through the second branchial, Text-fig. 68 through the third
branchial, and Text-fig. 69 through the fourth branchial seg-
ment.

(Cucullaris of Firbringer). The lower end of the first
branchial myotome forms the interarcualis ventralis I s.

1 “So entstand ein neugebildeter, dem ursprunglichen M. cucullaris
nur in seinen vordersten Theile homologer, in seinen Hauptmasse aber
blos initatorisch-homodynamer oder parhomologer Muskel.”

250 EF. H. EDGEWORTH.

branchio-mandibularis. ‘The middle portion of the first and
the whole of the second (below the Anlage of the trapezius)
branchial myotomes disappear.

In 5 mm. embryos of the rabbit the upper ends of the first,
second, and third branchial myotomes separate from the parts
below, the upper end of the third grows backward in the
neck, and the upper end of the second backward to join that

TExT-FIG. 65.

‘
| A
hyoud Vou

/

of the third (Text-figs. 85, 86, 87); in 6 mm. embryos the
upper end of the first has grown back to that of the second.
‘The hind end of the primitive trapezius, thus formed from the
upper ends of all three branchial myotomes, reaches the
anterior limb area in 7 mm. embryos (Text-fig. 88) ; its dorsal
edge extends upwards in 74 mm. embryos (Text-fig. 90), and
in 9 mm. embryos it has divided into the trapezius and

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 251

TEXT-FIG. 66.

ZAole .dow

fl CU GOU - Nu «

Ne Fi Siete

: Vhs eile \ = Rurole, sb. ny. A
= oe NEIZAN ee F “s———« e, mesh
es a fos A eee

66.

TEXT-FIG. 67.

dor aor

-X
o ae
2 Uv. tar, S
Sb
= Try.
2 filantan:

hybobr. sb. im. An.

67.

i.

H. EDGEWORTH.

TEXT-FIG. 68.

= hupobe >b kn Ar

ceph coed

68.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 253

sterno-mastoid of the adult. The portions of the branchial
myotomes below the Anlagen of the trapezius have dis-
appeared in 7 mm. embryos.

In the pig the lower end of the first branchial myotome
persists (Text-fig. 98), and forms the interarcualis ventralis I
s. branchio-hyoideus (‘Text-fig. 99). This muscle is also con-
stantly present in the dog,' and in Monotremes,’ and is occa-
sionally present in man.* In Monotremes there is also an
interarcualis ventralis III passing from the third to the
second branchial arch.*

On rHeE HoMmoLoGiEs BETWEEN THE BRANCHIAL MUSCLES OF
VARIOUS VERTEBRATES.

(1) The Hypobranchial Cranial Muscles.—The
lower ends of the branchial myotomes develop into longi-
tudinal muscles—interarcuales ventrales, and coraco bran-
chiales—in Selachii, Teleostomi, Dipnoi, Amphibia, and
Mammalha. ‘Their innervation varies. Those which remain
in their segment of origin, or nearly so, extending forward
to the ventral end of the next anterior branchial bar or
hyoid bar (or additionally and subsequently to the next pos-
terior bar, in Amia, and probably Polypterus senegalus)
are innervated by the corresponding branchial nerve, [Xth or
branch of Xth, or by this and the next anterior branch.
This holds for the interarecuales ventrales of Amia,
Polypterus senegalus, Salmo, Ceratodus, Amphibia, and
Mammalha. The interarcualis ventralis I of Sauropsida
(branchio-hyoideus or branchio-mandibularis) is an exception
to the rule; it is innervated by the XIIth.

A coraco-branchialis, or pharyngo-clavicularis externus and
internus, developed by backward growth from the last branchial
myotome, 1.e. fourth in Polypterus senegalus, fifth

' Kerato-hyoideus of Elenberger and Baum.
? Interhyoideus of Dubois.

% Kerato-thyro-hyoideus of Shattuck.
Interthyroideus of Dubois.

_

254, F. H. EDGEWORTH.

in Amia, Salmo, Hsox, Menidia, nay either retain its original
branchial innervation from the Xth, e.g. Amia (Allis),
Ksox (Vetter), Menidia (Herrick), Lepidosteus, Polypterus
senegalus, or be innervated by spino-occipital nerves, e. g.
Amieurus (Wright), Salmo (Harrison). When coraco-
branchiales are developed from all the branchial myotomes,
they are innervated by the spino-occipital nerves, e.g.
Selachu (Vetter, Fiirbringer), Acipenser (Vetter), Polypterus
? species (Fiirbringer), Ceratodus (Fiirbringer).

The coraco-branchiales muscles have been generally classed
with the hypobranchial spinal muscles, but investigation of
developmental stages shows that the ventral ends of
branchial myotomes may form longitudinal muscles, which
either grow forwards, forming interarcuales ventrales, or
backwards, forming coraco-branchiales, but not in both
directions. (There are two, probably three, exceptions to
the above rule; in Amia, at a late stage of development, the
hind end of the interarcualis ventralis 1V grows backward
to the fifth bar; the innervation in Polypterus sene-
galus suggests that the hind end of the interarcualis ven-
tralis III similarly grows back to the fourth bar; and in
Ceratodus the hind end of the interarcualis ventralis I, at a
late stage of development, grows back, forming the coraco-
branchialis I, and also, at least in some cases, to a more
posterior bar.) The first condition, that of interarcuales
ventrales, is the primary one, as shown by the correspondence
of cranial nerve innervation, with segment of origin. The
second condition, that of coraco-branchiales, is a secondary
one, in which a change of function to one very similar to
that of the coraco-hyoideus is correlated, though in varying
degree, with a change of innervation to one by the spino-
occipital nerves.

An approximation to what was, probably, the primitive
condition, is seen in Amphibia. This was a series of
interarcuales ventrales, each extending from the bar of its
segment of origin to the next anterior one. The hyo-
maxillaris, in the hyoid segment, is serially homologous with

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 255

the branchial interarcuales ventrales. There is no homologue
in the mandibular segment. These longitudinal muscles
possibly date from a period where there were no median
cartilages connecting the ventral ends of the hyoid and
branchial bars, and formed a rectus system of the head
serlally homologous with that of the body, though now
covered over by the latter, owing to its extension forwards
into the head.

Text-Fric. 70.

70.

Lacerta, embryo 8 mm., transverse section. The right side of
the section is a little anterior to the left.

(1) Transversi Ventrales, Mm. Marginales, and
Obliqui Ventrales——In Scyllium, Acanthias, Sauropsida,
rabbit, and pig, the hypobranchial cranial muscles are the most
ventral ones formed from the branchial wnyotomes; no trans-
versi ventrales are formed. ‘This is also the case in the first
branchial segment of Anuran tadpoles, the first, second, and
third of the Necturus and Triton, the first and fourth of
Ceratodus, the first and second of Polypterus senegalus.

256 FF. H. EDGEWORTH.

But in the second, third, and fourth branchial segments
of Anuran tadpoles, the fourth of Necturus and Triton, the
second, third, and fifth of Ceratodus, and in those segments
of Teleostomi in which hypobranchial cramial muscles are
formed, the lower ends of the branchial myotomes also grow
downwards and inwards above the cephalic ccelom, towards,
or to the middle line forming the (lateral halves of the)
transversi ventrales, or their homologues, the lower portions
of the obliqui ventrales. In branchial segments of ‘l'eleostom1,
where hypobranchial cranial muscles are not formed. i.e.
first four of Lepidosteus, first three of Amia and Salmo,
fourth of Acipenser, there is a similar downward and inward
growth of the ventral ends of the branchial myotomes, to
form the lower portions of the obliqui ventrales.

The hinder part of the transversus ventralis IV of Polyp-
terus and Amphibia, and of the transversus ventralis V
of Ceratodus, comes into intimate relations with the ventral
larynx, though in varying ways, underlying it in Amphibia
and Ceratodus, forming a dilatator in Polypterus.

The portions of the branchial myotomes next above the
Anlagen of the hypobranchial cranial muscles form the
Anlagen of the muscles of the external gills in the first
three seoments of Necturus and ‘Triton, and the Mm. mar-
ginales in the first three segments of Anuran larve and the
first four segments of Ceratodus. Homologous Anlagen form
the upper portions of the obliqui ventrales in ‘Teleostoman
embryos—of the first four segments of Acipenser, Lepi-
dosteus, Amia, Salmo, Polypterus (Pollard), and of the third
and fourth segments of Polypterus senegalus ; these may
or may not unite with the lower portions. In some segments
of Teleostomi, 1. e. first three of Acipenser, fourth of Lepi-
dosteus, Amia, and Salmo, adductors are formed from por-
tions of the myotomes lying internal to the branchial bars ;
they are not developed in Polypterus.

In Seyllium the portions of the branchial myotomes next
above the Anlagen of the coraco-branchiales form adductors
internal to the branchial bars, and the superficial con-

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 257

strictors, interbranchials, and arcuales dorsales external to
them. ‘The lower ends of the superficial constrictors extend
downwards external to the coraco-branchiales, but such down-
growths do not appear to be homologous with the trans-
versi ventrales or inferior portions of the obliqu ventrales
of Teleostomi, Ceratodus, and Amphibia.

In Sauropsida, rabbit, and pig embryos the portions of
the branchial myotomes next above the Anlagen of the hypo-
branchial cranial muscles, and also the lower ends where these
Anlagen are not formed, atrophy.

Levatores arcuum branchialum are developed from the
upper ends of the branchial myotomes in ‘l'eleostomi, Cera-
todus, and Amphibia, but are not developed in Scylhum,
Sauropsida, rabbit, and pig. The method of development of
the trapezius—apparently a homologous muscle throughout
these vertebrate groups—is intimately related to these differ-
ences. It is developed in Teleostomi and Amphibia from the
fourth, in Ceratodus from the fifth, levator, i.e. from the
penultimate or ultimate levator!; whereas in Scyllium,
Chrysemys, Gallus, and rabbit, it is formed from the upper
ends of the branchial myotomes—five in Scyllium, four in
Chrysemys, two in Gallus, and three in the rabbit.

In view of the facts that in Seyllium the subspinalis and
interbasales, developed from trunk-myotomes, are attached
to the pharyngo-branchials, and that the trapezius is inner-
vated only by the XIth—the most posterior of the vagus
roots—even though a constituent from the glossopharyngeal
(first branchial) segment takes part in its formation, it is
probable that the absence of levatores and associated method
of development of the trapezius in Scyllium, Sauropsida, and
rabbit are secondary phenomena, and that the primary con-
dition is a series of levatores formed from the uppermost
portions of the branchial myotomes. ‘This theory would also
afford an explanation of the curious fact that whereas the

' In Teleostoman embryos the trapezius is developed from the upper
edge of the levator, in Ceratodus and Amphibia from its external
surface.

258 F. H. EDGEWORTH.

general development of the myotomes takes place from belore
backwards, the separation of the upper ends of the branchial
myotomes, their backward growth, and fusion to form a
trapezius, in Seyllium, Chrysemys, Gallus, and Lepus, take
place from behind forwards—the process beginning in the
last branchial myotome.

Adductors of the branchial bars are formed in Scyllium
and in certain segments of some '‘l'’eleostomi, on the inner

Trxt-Fig. 71.

y
d i
a5 A
Ix Ba *
3 e
2 =
: =
= b
=
2
=: hyorm
x ;
F cCeu-hyc
x

coma col

shane oud.
He Ms 7E.
Lacerta, embryo 12 mm., transverse section.

side of the branchial bars. The observations of Balfour!
showed that the primary situation of the muscles is one
external to the bars, so that the non-development of adduc-
tors in Amphibia, Ceratodus, and Polypterus would appear to
represent a primitive condition.

It may be added that the adductors of the branchial bars
are not serially homologous with the adductor mandibule,
which is formed external to the mandibular arch.

' «Comparative Embryology,’ vol. ii, p. 471.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 259

The simplest and probably primary condition of the
muscles developed between the levatores above and the
hypobranchial cranial muscles below is a series of Mm.
marginales, as found in Anuran larve and Ceratodus. In
Teleostomi these may unite with the (lateral halves of the)
transversi ventrales. In Scylhum they undergo a quite
special development, which is not found in any other group.

The above comparisons suggest that the probable primitive
condition of each of the branchial myotomes was, from
above downwards, a levator, a M. marginales, an inter-
arcualis ventrales, and (the lateral half of a) transversus
ventralis.

(sophageal, Pharyngeal,and Laryngeal Muscles.

ce

—The term “ pharynx” is employed by writers in two senses,
either restricted in meaning tothe branchial region of the
alimentary canal, or including this and the next succeeding
portion. In this paper it is used in the first sense.

The cesophagus is surrounded by a circular muscle, the
constrictor, which is derived from cells given off from the
splanchnic layer of the ccelomic epithelium.

No circular muscles are present in the branchial region of
Selachii, Acipenser, and Ceratodus, but are present, in the
form of transversi dorsales, in T'eleostomi (Vetter), Amia
(Allis), and Lepidosteus (Wiedersheim), and are formed by
the constrictor of the cesophagus extending forwards,
dorsally, into the branchial region. In Polypterus
senegalus the fore part of the cesophageal constrictor
slightly overlaps the branchial region dorsally, but the
transversely directed fibres are not attached to any branchial
bar. In Lepidosteus and Amia! the conditions are com-
plicated by the presence of a dorsal larynx. The dorsal
larynx of Lepidosteus is formed, in 8 mm. embryos, as a
solid median upgrowth from the then solid cesophagus just
behind the branchial region. The cesophageal constrictor
(constrictor pharyngis of Wiedersheim) is formed from cells

' The adult condition of the dorsal larynx and its musculature has
been fully described by Wiedersheim.

260 F. H. EDGEWORTH.

given off from the coelomic epithelium ; it spreads upwards
round the cesophagus and dorsal larynx, forming the con-
strictor laryngis, and subsequently, in 95 mm. embryos,
spreads forward to the branchial region and becomes attached
to the os pharyngeum superior (of van Wijhe, the Pharyngeal-
platte of Wiedersheim) forming the transversus dorsalis and
first obliquus dorsalis. he dilatator laryngis is formed from
the dorsal part of the cesophageal constrictor (Text-fig. 32).
The development of the retractor and protractor laryngis is
described on pp. 258 and 267.

The development of the dorsal larynx of Amia is similar to
that of Lepidostens. The forward extension of the
cesophageal constrictor begins in 8} mm. embryos (Text-figs.
30, 31, 32); 1t forms the transversus anterior and posterior
and first obliquus dorsalis (of Allis). In Salmo fario the trans-
verse fibres become attached to the fifth cerato-branchial, the
fourth pharyngo- and epi-branchial and the third pharyngo-
branchial.

The ventral larynx and musculature of Polypterus have
been deseribed by Wiedersheim, who says that the muscles
consist of a dilatator and sphincter glottidis, the latter of
which is continuous below with a muscle-sheet surrounding
the lungs. <As stated above, the dilatator is the trans-
versus ventralis [V. The sphincter glottidis is continuous
above with the constrictor of the cesophagus, which is not
attached laterally to the trunk muscles.

The development of the ventral larynx of Ceratodus
has been described by Kellicott, and the adult condition
by Wiedersheim. It may be added that the posterior edge
of transversus ventralis V underlies the lung, and that, though
there is no dorso-laryngeus (Wiedersheim), the lateral edges
of the constrictor of the cesophagus are attached to the
lower ends of the trunk-myotomes (Text-fig. 49).

It was supposed by Gegenbauer, Wilder, Géppert, and
Driiner, that the cartilagines laryngei of Amphibia were
branchial bars, and that the laryngeal muscles were deriva-
tives of branchial muscles. Wilder stated that the dorso-

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 261

laryngei s. dorso-tracheales were serially homologous with
the levatores arcuum ‘branchialium (called by him ‘‘ dorso-
branchiales ”’), that the laryngei were homologous with the

transversi ventrales (called by him “‘

pharyngo-branchiales ”’),
and that these muscles and the laryngeal cartilages belonged
to the fifth branchial arch.

This theory of the nature of the laryngeal muscles was
formed on the evidence of adult anatomy only, without
knowledge of the method of development.

Wiedersheim stated that the fibro-cartilage present in
association with the ventral larynx of Protopterus might be
due to the remains of a sixth arch, ‘noch plausibler aber
erscheint es mir die betreffenden Knorpel auf einen in der
Raphé des pharyngealen Constrictors sich abspielenden
Chondrificationsprocess, das heisst auf Muskelwirkung

zuruckzufuhren”; also that “

a prior1 steht der Annahme
gewiss nichts in der Wege das sich auch bei Amphibien die
primitive Cartilago lateralis als eine Sehnenverknorpelung
bilden kann ohne das dabei phylogenetische Beziehung zu
den Kiemenbogen angenommen werden mussen.”’

In Necturus the larynx is formed as a median ventral
pouch of the pharynx in the hinder part of the fourth
branchial segment in 15 mm. embryos (Text-figs. 53,54). The
cells which form the constrictor of the cesopbagus are budded
off from a thickened portion of the splanchnic ccelomic
epithelium, and spread upwards round it (Text-fig. 54). A
similar production of splanchnic mesoblast from a thickened
portion of splanchnic coelomic epithelium takes place
in the fourth branchial segment (‘T'ext-fig. 52), and the
cells spread upwards round the developing larynx and
pharynx. In 145 mm. embryos this splanchnic meso-
derm spreads upwards on either side of the pharynx
internal to the fourth levatores arcuum branchialium, and
forms the Anlagen of the dorso-laryngei and of the pharyn-
geal constrictor (T’ext-fig. 56). The splanchnic mesoderm
ventral to the pharynx develops into the laryngei muscles,
and, next the larynx, the laryngeal cartilages.

262 F. H. EDGEWORTH.

A similar development of the laryngeal muscles takes
place in Triton and in Rana.

If the method of development of the laryngeal muscles be
compared with that of the branchial muscles it is seen that
they are very different. The branchial muscles are developed
from the branchial myotomes, the laryngeal muscles are
differentiations of an cesophageal or pharyngeal constrictor,
which is formed from cells given off by the ccelomic epi-
thelum. In Polypterus and Ceratodus the larynx is
developed just behind the branchial region, in Amphibia in
the last, i. e. fourth branchial, se2ment. In Polypterus the
cesophageal constrictor is not attached to the trunk-
myotomes; in Ceratodus it is attached, though no dorso-
laryngei are developed; in Amphibia dorso-laryngei are
developed. In Ceratodus there are no intrinsic laryngeal
muscles; in Polypterus there is a sphincter glottidis, which
remains in continuity with the constrictor of the cesophagus ;
in Amphibia there are laryngei or a sphincter laryngis,
which is not continuous with the constrictor of the pharynx.
In Polypterus and Ceratodus there are no skeletal structures ;
later than
the branchial bars and ventral, not lateral to the pharynx.

in Amphibia laryngeal cartilages are developed

These similarities and differences suggest that the conditions
found in Polypterus and Ceratodus preserve stages in the
phylogenetic development of the laryngeal structures of
Amphibia. They tend to confirm the theory of Wiedersheim.
The relations of the transversus ventralis of the last branchial
segment (fourth in Polypterus and Amphibia, fifth in Cera-
todus) to the laryngeal structures have been described above.

In 5 mm. embryos of Chrysemys the opening of the larynx
is in the fourth branchial segment ; the coelomic epithelum in
the third and fourth branchial segments is thickened and
proliferating mesoblast cells, which surround the pharynx
and developing larynx. In 8 mm. embryos the opening of
the larynx is in the third and fourth branchial segments ; in
the fourth a constrictor pharyngis is formed from the
splanchnic mesoblast ; in the third and fourth the Anlagen of

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 263

the sphincter laryngis and dilatator laryngis (Text-fig. 69) are
formed, the latter having a vertical position and passing up
outside the vagus. The dilatator has lost this position in
15 mm. embryos, and its hind end is attached to the cricoid.
This supports the theory of Géppert that the dilatator
laryngis of Sauropsida is the homologue of the dorso-laryn-
geus, and the sphincter the homologue of the sphincter and
laryngei of Amphibia.

In 3 mm. embryos of the rabbit, cells are proliferated from
the coelomic epithelium in the three branchial segments
(Text-fig. 80) ; they spread upwards internal to the branchial
aortic arches. In 5 mm. embryos they have spread a little
round the pharynx in the second and third branchial segments
(Text-figs. 85, 86), but in the first they exist only lateral to
it. In 6mm. embryos the splanchnic mesoblast forms a con-
tinuous sheet dorsal to the pharynx in the second and third
segments, but in the first itis still only lateral to it. In
7mm. embryos it is present there also, more probably as a
result of forward extension from the second branchial seg-
ment than of upward extension in the first segment. This
continuous splanchnic mesoderm sheet is continuous behind
with that round the cesophagus (Text-fig. 90), which is
formed in a similar way.

The stylo-pharyrgeus is formed in the splanchnic meso-
blast of the first branchial segment; it is first visible in
74 mm. embryos and gains an attachment to the hyoid bar
(Text-fig. 93). The pharyngeal constrictor is also formed in
the three branchial segments, though, perhaps, vide supra,
derived from cells of the second and third segments only.
The Anlage of the laryngeal muscles is also visible in 7} mm.
embryos in the third branchial segment. ‘The later develop-
ment of the laryngeal muscles, in man, has been traced by
Frazer, who says that they are developed in the third bran-
chial segment from the ventral part of the layer of cells
round the pharynx and larynx; of this laryngeal portion
“the formation of the arytenoid seems to convert the dorsal
hinder part into the crico-arytenoideus posticus, the upper

VOL. 56, PART 2.—NEW SERIES. 19

264. F. H. EDGEWORTH.

part into the arytenoideus, and the ventral part into the
thyro-arytenoideus and lateral crico-arytenoid.”” According
to Gbppert, the crico-arytenoideus posticus is the homologue
of the dorso-laryngeus of Amphibia, the other intrinsic
muscles representing the sphincter and laryngei dorsalis and
ventralis of Amphibia.

-
‘

TEXT-FIG.

Text-figs. 72, 73.—Gallus, embryo four days. Text-fig. 72 through
the first branchial, Text-fig. 73 through the second branchial
segment.

Frazer states that in man the thyroid cartilage is developed
solely in the second branchial segment, i. e. it is the second
branchial bar only, whereas in Echidna (G6ppert) a third
branchial bar is also formed.

The crico-thyroideus, according to Frazer, is developed

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 265

from the second branchial pharyngeal constrictor; this con-
firms the opinion of Fiirbringer.

The pharyngeal musculature extends forwards into the
mandibular segment in 15 mm. embryos (Text-fig. 94) ; in
14 mm. embryos the anterior end becomes separated, forming

the tensor palati, and in 16 mm. embryos the next succeeding
portion forms the levator palati.

TEXT-FIG. 73.

AW.

According to Futamura, as stated above, the Anlage of the
palatal muscles of man is at first one with that of the tensor
tympani, and is derived from the platysma faciei. I have
failed in verifying this in the rabbit; the platysma faciei in
that animal remains superficial, and the Anlage of the
palatal muscles when first visible was quite distinct from
that of the tensor tympani, which is cut off from the inner
lhmb of mandibular myotome.

266 F. H. EDGEWORTH.
These phenomena afford an explanation of the imnervation

of the palatal muscles by the XIth medullary.

Mousctes Derived From ‘l'runK-Myoromers PAssING TO 'THE
Urrer Enpbs or THE BrRAncHIAL Bars.

Vetter described a subspinalis and interarcuales muscles in

Trxt-FIG. 74.

Gallus, embryo 43 days, transverse section.

Selachii. Fiirbringer subsequently showed that the sub-
spinalis and upper interarcuales (which he re-named “ inter-
basales ””), were innervated by the spino-occipital nerves, and
he classed them together as “epibranchial spinal muscles.”
The lower interarcuales (second and third interarcuales of
Vetter), he called ‘ arcuales dorsales.”

Observation of the development of these muscles confirms
Fiirbringer’s theory of their nature. The subspinalis and

' Beevor and Horsley.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 267

first, second, and third! interbasales of Scyllium are developed
in 23 mm. embryos from the first, second, third, and fourth
spinal myotomes (‘I'ext-figs. 12, 13, 14), by growths from their
ventral edges internal to the ganglia of the ninth and tenth
nerves.

A retractor arcuum branchialinum dorsalis is found in
some Teleosteii (Vetter), e. ¢. Perca and Cyprinus, in Amia
(Allis, Wiedersheim?), and in Lepidosteus (Wiedersheim’),
but is not present in Polypterus (Pollard),or in Salmo fario.
In Amia the muscle, which is inserted into the third infra-
pharyngo-branchial, arises according to Allis from the third
and fourth, according to Wiedersheim from the seventh and
eighth, vertebral bodies. In Lepidosteus the muscle 1s
inserted into the third infrapharyngo-branchial, and arises
from the lateral surface of the third and fourth vertebral
bodies {Wiedersheim). The retractor was supposed by Allis
and by Wiedersheim to belong to the system of the levatores
arcuum branchialium.

‘he muscle is developed in Amia from downgrowths from
the lower surface and lower part of the internal surface of the
sixth to thirteenth trunk-myotomes in 84 mm. embryos (Text-
fig. 33); these downgrowths form a longitudinal muscle, the
anterior end of which grows forward to the third bar. Its
development in Lepidosteus is similar, taking place,in 12 mm.
embryos, from the third to ninth truank-myotomes.

HypospraNCHIAL SPINAL MuscLEs.?

In Scyilium, van Wijhe stated that the coraco-hyoideus
was developed from ventral prolongations ‘ sowohl des hin-
stersten Kopfmyotomes als die der vorderen Rumpfmyotome,”
and that the coraco-branchiales and coraco-mandibularis
were formed from the walis of the “ unpaaren vorderen

' The third interbasalis, developed in the embryo, is not described in
the adult by Firbringer.

> Retractor laryngis of Wiedersheim.

* This name is used in the sense stated above.

268 F. H. EDGEWORTH.

Verlingerung des Pericardiums.” Neal, in Squalus acan-
thias, found that the Anlage of the hypoglossus musculature
was formed from the fourth to the eighth post-otic myotomes
by buds which separate and come to le ventral to the
branchial basket; they do not fuse into a common cell mass,
but show their primary metamerism, the bud from the fourth
myotome coming to lie between the hyoid and mandibular
cartilages and forming ‘fin part the Anlage of the proper

? whilst “the four following myotomic buds

tongue muscles,
come to lie between the hyoid and procoracoid.”

I find that inScyllium the initial stages of the development
of the coraco-mandibularis and coraco-hyoideus are similar to
those of Squalus acanthias,as stated by Neal. This stage
is completed in 16mm. embryos, and is immediately followed
by one (17 mm.), in which the hind end of the primitive
genio-hyoideus, which does not become affixed to the hyoid
bar, grows backwards along the median edge of the coraco-
hyoideus towards the shoulder-girdle—formine the coraco-
mandibularis (Text-figs. 11, 12, 13).

The coraco-hyoideus of Salmo salar (Harrison) is
developed from ventral downgrowths of the second, third,
and fourth trank myotomes, which bend round the pharyngeal
region, and form a longitudinal column, the anterior edge of
which extends forwards to the hyoid bar. A similar develop-
ment of the hypobranchial spinal muscles takes place in
Acipenser, Lepidosteus, Amia, and Salmo, occurring in 8 mm.,
8imm., 7 mm., and 5 mm. embryos respectively, and in each
case from the second, third,and fourth trunk myotomes. In
Salmo fario and in Lepidosteus the forward growth of the
anterior end reaches the hyoid bar only,so that only a coraco-
hyoid is formed. In Acipenser and Amia it extends further,
to the symphysis, reaching this in 8} mm. embryos of
Acipenser (Text-figs. 21, 22), and in 8 mm. embryos in Amia.
The long column then divides at the level of the hyoid
bar into an anterior and a posterior group—the genio-hyoid!

' Branchio-mandibularis of Vetter and Allis.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 269

and coraco-hyoid.' The posterior end of the genio-hyoid grows
backwards (Text-figs. 28, 29, 30), and becomes attached, in
Acipenser to the third hypobranchial, and in Amia by two
tendons, to the second and third hypobranchials and to a
median aponeurosis between the two coraco-hyoidei(Y-shaped
tendon of Allis).

In Polypterus,? species, Pollard described the hypobranchial
spinal muscles as consisting of a branchio-mandibularis s.
genio-hyoideus extending from the symphysis of the lower
jaw to the first basi-branchial, and of a coraco-byoideus which
had an additional tendon attached to the first cerato-branchial.
Fiirbringer described the muscles as consisting of a coraco-
mandibularis extending from the symphysis to the shoulder-
girdle, and of a coraco-hyoideus.

In Polypterus senegalus (Tl'ext-figs. 35, 36, 37), the
muscles consist of a genio-hyoideus and a coraco-hyoideus ;
the former extends from the symphysis backwards to the level
of the third branchial bar, where it ends by being attached to
the third cerato-branchial and by a tendon which passes down-
wards and is attached to a little median ossicle lying between
the two coraco-hyoidei. The coraco-hyoideus extends from
the cerato-hyal backwards to the shoulder-girdle, and has no
tendon passing to the first cerato-branchial.

Greil stated that the “hypobranchial musculature” of
Ceratodus was developed from ventral downgrowths of the
third and fourth myotomes. He apparently included the
coraco-branchiales as well as the coraco-mandibularis and
coraco-hyoideus under this head, as the first-named were not
described as developing in the branchial region. It has been
stated above (p. 234) that the coraco-branchiales are developed
from the lower ends of the branchial myotomes. ‘The hypo-
branchial spinal muscle Anlage spreads forwards (‘Text-fig. 39)
reaching the anterior extremity of Meckel’s cartilage in
stage 43. The portion in front of the hyoid bar separates

1 Main portion of coraco-arcualis anterior (Vetter) in Acipenser ;
hyopectoralis (MceMurrich), sterno-hyoideus (Allis) in Amia; the term
used above is that of Fiirbringer.

270 F. H, EDGHEWORTH.

from that behind, and its hind end grows backwards below
the coraco-hyoideus (Text-figs. 45, 46,47) to the shoulder-
girdle, forming the coraco-mandibularis. ‘The portion behind
the hyoid bar forms the coraco-hyoideus ; in stage 65 it is
partially separated into the coraco-hyoideus and abdomino-
hyoideus of the adult, of which the latter is continuous with
the trunk muscles behind the shoulder-girdle.

In Necturus (Miss Platt) the hypobranchial spinal muscles
are developed from ventral downgrowths of the third, fourth,
and fifth post-otic somites, joined by a few scattered cells
from the second somite; the genio-hyoideus is formed from
the third, the sterno-hyoideus from the fourth and fifth.

In Triton there is a similar development from the third,
fourth, and fifth trunk myotomes in 65 mm. embryos.

The hypobranchial spinal muscles of Rana are developed
from downgrowths of the first and second trunk myotomes
in 6 mm. embryos (Text-fig.57), which bend round the bran-
chial region, forming a longitudinal column which reaches
the inferior labial cartilage in 8 mm. embryos. It divides
opposite the third branchial bar into genio-hyoid and sterno-
hyoid.t| The front end of the former is attached to the
inferior labial cartilage (Text-figs. 60, 62), and its hind end to
the hypobranchial plate as far back as the antero-posterior level
of the third branchial bar. In 12 mm. embryos the internal
portion of the genio-hyoid is proliferated from the median edge
of the original muscle (Text-fig. 59). At metamorphosis the
inferior labial cartilage forms the anterior end of the lower
jaw, andthe muscle so retains its primitive attachments. The
front end of the sterno-hyoid becomes attached to the third
cerato-branchial, and the muscle extends back to the dia-
phragm. Towards metamorphosis the shoulder-girdle is
developed and the sterno-hyoid becomes attached to it, and
a little later the omo-hyoid is separated from its external
edge.

In Alytes, Bufo lentiginosus and Pelobates there is no

' Genio-hypobranchialis and diaphragmato-branchialis medialis of
Schultze.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 271

proliferation of an internal portion of the genio-hyoid ; other-
wise the condition in the larvee is the same. In Alytes the hind
end of the genio-hyoid is attached to the third cerato-branchial.

In the rabbit the hypobranchial spinal muscles are formed
from downgrowths of the first three trunk myotomes in
4 mm. embryos (‘l'ext-fig. 82). These have separated in 44
mm. embryos, and form a longitudinal column which extends
forwards dorsal to the interbyoideus and intermandibularis,
reaching the anterior extremity of Meckel’s cartilage in
8 mm. embryos, and backwards, reaching the area of the
auterior limb in 7 mm. embryos. In 13 mm. embryos it
has divided into genio-hyoid and (primitive) sterno-hyoid,
the adjacent ends of which are attached to the first branchial
bar. In 17 mm. embryos the primitive sterno-hyoid has
divided into the sterno-hyoid, sterno-thyroid, thyrohyoid,
and omo-hyoid. ‘The first trunk myotome, from which the
most anterior of the downgrowths above mentioned takes
place, atrophies in 73 mm. embryos, the second and third in
9 mm. embryos. -

The Homologies of the Hypobranchial Spinal
Muscles.—In Amphibia, Sauropsida, and rabbit, the Anlage
of the hypobranchial spinal muscles divides into anterior and
posterior portions—the genio-hyoid and sterno-hyoid. The
former extends from the symphysis of the lower jaws to the
basi-branchial or some branchial bar, the latter extending
thence to the shoulder-girdle or sternum. ‘The division takes
place in the neighbourhood of the first branchial bar in
Urodela, Sauropsida, and rabbit; in Anuran larve it is at
the level of the third branchial bar.

In Seyllium, 'eleostomi, and Ceratodus, a similar division
of the Anlage of the hypobranchial spinal muscles takes place
at the level of the hyoid bar; the hind end of the anterior
portion, which does not gain any temporary insertion to the
hyoid bar, then grows backwards ventral or ventro-lateral to
the posterior portion (coraco-hyoideus) and becomes attached
to the first (Polypterus, ? species, described by Pollard), or
to the second and third (Amia), or third (Polypterus sene-

PA fe FE. H. EDGEWORTH.

galus, Acipenser) branchial bar, or to the shoulder-girdle,
forming a coraco-mandibularis (Scyllium, Ceratodus, Poly-
pterus ? species, described by Fiirbringer).

The anterior attachment of the genio-hyoid and coraco-
mandibularis is to the front end of Meckel’s cartilage except
in Anuran larve, where it is to the inferior labial cartilage.
In Acanthias, where there is an inferior labial cartilage
(Gaupp), the coraco-mandibularis is not attached to this but
to Meckel’s cartilage. In Callorrhynchus (Fiirbringer) there
is a coraco-premandibularis developed, attached anteriorly to
the inferior labial cartilage.

Fiirbringer homologised the gemo-hyoideus with the
coraco-inandibularis of Selachi, and supposed that the
former was derived from the latter, by giving up its attach-
ment to the shoulder-girdle, and gaining a new one to (more
rostally lying) portions of the byobranchial skeleton. Such
a deduction was a legitimate one from the evidence of adult
anatomy only, though the alternative was possible, and
the embryological history of the muscles shows that 1 is
this alternative which occurs; the condition in Teleostom1,
Klasmobranchs, and Ceratodus is a secondary one.

The method of development of the hypobranchial spinal
muscles in Scyllium lends additional interest to, and receives
corroboration from, some anatomical facts described by Vetter
and Fiirbringer. The degree of backward extension of the
coraco-mandibularis towards the shoulder-girdle varies, even
amongst the Selachi. ‘hus in Heptanchus and Scyllium it
does not reach the coracoid, whereas in Lemargus and
Prionodon it does. Further, the coraco-mandibularis is not
crossed by tendinous inscriptions, in this forming a marked
contrast to the coraco-hyoideus, alongside of which it les.
‘The only possible exception to this among the forms depicted
by Fiirbringer is Cestrastion, and this is probably an apparent
one only; it is possible that the tendinous inscription really
separates the coraco-hyoideus from the coraco-manibularis,
which only reaches the coracoid by its median edge.
Similarly, according to Fiirbringer, there are three tendinous

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 273

inscriptions in the cerato-hyoideus of Ceratodus, whilst
there is only one doubtful one in the coraco-mandibularis ;!
in Protopterus there are two in the coraco-hyoideus, none in

TEXT-FIG. 795.

Mand cup: on

75.

trand . aa. Ov.

am eae Untes urcid
abh _coel.

22+
ira
2 :

<n?
SON
ye

76.

vent COL.

Text-figs. 75-77.—Rabbit, embryo 3mm. Text-fig. 75 is through
the premandibular Anlage, Text-fig. 76 through the mandibular
seoment, Text-fig. 77 through the hyoid segment.

the coraco-mandibularis; in Polypterus ? species there are
two in the coraco-hyoideus, none in the coraco-mandibularis.

1 It was not apparent in stage 63 (vide Text-fig. 45).

274 FE. H. EDGEWORTH.

Again, there are two inscriptions in the coraco-hyoid of
Amia (Allis), and Acipenser (Vetter), none in the genio-
hyoid.

The non-development of a genio-hyoid, even as an atrophy-
ing Anlage, in Lepidosteus and Salmo would appear, on
comparison with other forms, to be a secondary condition.

The developmental history of the hypobranchial spinal
muscles suggests that their Anlage was primitively an
undivided column, extending forward to the symphysis of

TEXT-FIG. 77.

the jaws, an anterior prolongation of the rectus system of the
trunk into the head segments. his divided into genio-
hyoid and coraco-hyoid. In fishes the genio-hyoid secon-
darily extended backwards, overlapping the coraco-hyoid in
varying degrees.

The Anlage of the hypobranchial spinal muscles is formed
from downgrowths of several—two or more—trunk
myotomes, that from the foremost myotome taking part in
its formation extending furthest forward, that from the next
succeeding it, and so on. ‘here is a certain correlation
between the number of trunk myotomes taking part in the

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 275

formation of the hypobranchial spinal muscles and_ the
number of trunk-segments included in the skull (vide table,
p- 299). In general, the less the number of trunk-segments
included in the skull, the greater tendency there is for
the most anterior trunk myotomes to take part in the
formation of the hypobranchial spinal muscles, and the less
the number of anterior trunk myotomes atrophying without
taking part in their formation. The table also shows that
the hypobranchial spinal muscles are derived from a variable
number of trunk myotomes. The number varies from two
(Rana) to five (Seylhum). Derivation from the smallest
number is probably the most primitive condition. The number
does not vary with that of the trunk-seements included in
the skull, nor with the number of head-segments.

LinauaL Mosc ies.

The researches of Gegenbaur and Kallius in Salamandrina
have shown that the genio-glossus is developed from the
gvenio-hyoideus and the sterno-glossus from the sterno-
hyoideus ; the former ends between the lingual glands which
form the fore part of the tongue, and is “also ein Driisen
Muskel” (Gegenbaur) ; the latter becomes attached to a plate
of dense connective tissue which probably develops at the site
of fusion of the primitive tongue with the glandular portion.
Kallius has also shown that in the Anura the genio- and hyo-
glossus are developed from the genio-hyoid. The genio-
glossus grows towards the subsequently glandular, precopular
field, and later, the hyo-glossus fibres cross those of the
oenio-glossus.

To this may be added that in Rana the genio- and hyo-
glossus are developed from the inner division of the genio-
hyoid, which (vide p. 270) is proliferated from the inner side
of the primitive muscle.

In 27 mm. larve of Alytes the genio-glossus ends free
beneath the mucous membrane of the precopular field, and
the hyo-glossus passes forward at first below and then lateral

276 F. H. EDGEWORTH.

to the long forward projecting basihyal (Text-fig. 63), and
is attached to it near its anterior end. In larve of Bufo,
Rana, and Pelobates the hyo-glossus is not attached to the
rudimentary basihyal.

The development of the lingual muscles of Lacerta
muralis has also been investigated by Kallius. The develop-
ment of the lingual muscles in other groups of Seuropsida
(loc. cit.) showed that the primitive condition of the lingual
muscles is a genio- and hyo-glossus, both developed from

TExtT-FIG. 78.

78.

Rabbit, embryo 3} mm., longitudinal vertical section.

the genio-hyoid, attached to the long basihyal, the former
to its front end, the latter to its side; and that extension
into the tongue is a secondary phenomenon.

The condition of the hyo-glossus of Alytes is of interest
when considered in relation to that of the lingual muscles of
Sauropsida. Gegenbaur was of opinion that ‘‘die Musku-
larisirung scheint vorwiegend im Dienste der Drusen zu
stehen.” An alternative hypothesis suggested by the con-
dition in Alytes larvee would be that the condition in Saurop-
sida is the primary one, and that their functions as glandular
muscles in Amphibia is correlated with the absence (e.g.

ho
~J
~J

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES.

TExtT-FIG. 79.

rand aor av

79.

Rabbit embryo 34mm. The upper part of the section is posterior
to the lower.

TExtT-FIG. 80.

91

Rabbit, embryo 5;mm. The right side of the section is a little
anterior to the left, which passes through the second branchial
myotome.

278 F. H. EDGEWORTH.

Triton) or rudimentary condition (e.g. Rana, Pelobates,
Bufo) of the basihyal.

In the rabbit the Anlagen of the lingual muscles are formed
from the anterior part of the Anlage of the hypobranchial
spinal muscles, i.e. from the future gemio-hyoid, in 9 mm.
embryos, that of the genio-glossus and lingualis by upward
erowth, that of the hyo-glossus and stylo-glossus by an out-

Trxt-FIG. 81.

! {
Ayala. INy

unferhyoud

Nand, seq

Vent aa Bl.

Text-figs. 81-83.—Rabbit, embryo 4mm. Text-fig. 81 is through
the hyoid segment, Text-fig. 82 through the fourth gill-cleft,
Text-fig. 83 through the third branchial gill-cleft.
erowth directed upwards and laterally (Text-fig. 91). In 13
mm. embryos these muscles have separated from the genio-
hyoid, and become distinct (Text-figs. 94, 95).

Some PHYLOGENETIC CONSIDERATIONS.

The probable phylogenetic relationships of the various
Vertebrate groups are determined by the total morphological

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES.

TEXT-FIG. 82.

Stee
: — hybobe, sb. mv Ar

83.
VOL. 56, PART 2.—NEW SERIES. 20

279

280 F. H. EDGEWORTH.

evidence available. The cranial muscles form one item only
of such evidence, but it is of interest to inquire how far their
morphology falls in with generally received opinion, and in
what direction it points in cases where opinions vary.

Such an inquiry is beset by the difficulties which arise
from—(a) secondary innervation of muscles; (B) develop-
ment of similar changes in various groups; (Cc) atrophy, and
non-development of muscles.

(A) Comparison of the development and innervation of the
cranial muscles shows that although, in general, a muscle is
innervated by the nerve corresponding to its segment of
origin, yet this is not invariably the case. Thus:

(1) The posterior part of the intermandibularis of Triton
is innervated by the VIIth (Driiner).1

(2) The intermandibularis of Selachians is, in part, e.g.
Scylhum (Vetter), or wholly, e.g. Acanthias (Vetter), inner-
vated by the VIIth.

(5) The intermandibularis anterior and posterior (the latter
called * inferior genio-hyoid” by Allis) of Amia are innervated
by both the Vth and VIIth (Allis).

(4) The hyo-maxillaris of Teleostomi, developed in the
hyoid segment, isin some, e.g. Menidia (Herrick), wholly in-
nervated by the VIIth; whereas in others, e.g. Hsox (Vetter),
Salmo, its hinder part is innervated by the VIIth and its fore
part by the Vth; and in Amia (Allis) it is innervated by the
Vth and VIIth.

(5) The anterior digastric of man, pig (Futamura), and
rabbit, developed in the hyoid segment, is innervated by the
Vth.

(6) The cerato-hyoideus externus of Urodela, developed in
the hyoid segment, is innervated either by the VIIth, e. g.
Necturus (Miss Platt), or by the [Xth, e. g. Triton (Driiner).

(7) The interarcuales ventrales of Urodela are innervated
both by the nerves corresponding to their segments of origin
and also by those of the next anterior segments (Driiner).

(8) In some Teleostomi, e. g. Polypterus senegalus,

' Roman numerals denote cranial nerves.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 281

Esox (Vetter), Menidia (Herrick), Amia (Allis), Lepidosteus>
the only coraco-branchialis present, developed from the most
posterior branchial segment (fourth or fifth), is innervated by
the Xth; whereas in others, e.g. Amieurus (Wright), Salmo
(Harrison), it is innervated by the spino-occipital nerves.

(9) The coraco-branchiales of Acipenser, Ceratodus, and
Seyllium, developed in branchial segments, are innervated
by spino-occipital nerves (Vetter, Fiirbringer). The spino-
occipital nerves also innervate the four coraco-branchiales of
Polypterus (?) species, described by Fiirbringer.

(10) The capito-dorso-clavicularis of Lacerta agilis,
developed from the primitive trapezius, i. e. from branchial
segments, is innervated by spinal nerves (Fiirbringer).

(11) The eucullaris, i.e. trapezius, of Gallus, developed
from branchial segments, is innervated both by the XIth
and by spinal nerves (Firbringer).

(12) The trapezius and sterno-mastoid of the rabbit,
developed from branchial segments, is innervated both by
the XIth and by spinal nerves.

(13) The retractor arcuum branchialium dorsalis of Amia
and Lepidosteus, developed from trunk myotomes, is inner-
vated by the Xth (Allis, Wiedersheim).

(14) The hinder part of the hypobranchial spinal muscles
of the rabbit, which are developed from the first three spinal
myotomes, are innervated by more posterior spinal nerves.

(15) The interarcualis ventralis I, i. e. branchio-hyoideus
or branchio-mandibularis of Sauropsida, is innervated by the
XIIth.

Fiirbringer held that ‘‘ Die Innervirung der Muskeln
durch bestimmte Nerven ist das wichstigte Moment fur die
Vergleichung.” In criticism of this theory, Cunningham
gave instances from the myology of the trunk and limbs in
which this criterion failed, and concluded that the nerve
supply is ‘‘ not an infallible guide” for determination of the
homology of a muscle. The above-cited observations show
that developmental phenomena should be taken into con-
sideration.

282 F. H. EDGEWORTH.

The first fourteen of the phenomena recorded appear to be
referable to a common cause ; if a muscle spreads into one or
more neighbouring segments, that portion tends to be inner-
vated by the corresponding nerve or nerves. The backward
extension of the origin of the XIth appears to be referable
to the same cause.

It is not yet known what happens within the central
nervous system—whether there is a corresponding migration
of motor neuroblasts or whether new ones are locally formed.

The cause of the phenomenon cited under (15) above is
much more obscure. ‘The muscle is the interarcualis ven-
tralis of the first branchial segment, and is homologous with
the similarly developed muscle of Amphibia, some Teleos-
tomi, and some Mammalia, and yet, unlike them, it is inner-
vated by spino-occipital nerves and not by the [Xth, just as
if it were a coraco-branchiahs I.

(ns) The possibility of the independent development of
similar secondary changes in the various groups arises in the
case of the hypobranchial spinal muscles, the hypobranchial
cranial muscles, the levatores arcuum branchialium, and
trapezius, the hyoid bar and related muscles, the adductor
mandibule.

In Ceratodus and in Scyllium the hind end of the genio-
hyoid secondarily extends backwards to the shoulder-girdle.
The question arises whether this feature is inherited from a
common ancestor. or whether it has been independently
acquired. In favour of the second view are the facts that
within the group of the Teleostomi all conditions exist
between that of a genio-hyoid which has shghtly extended
backwards and a coraco-mandibularis.

A similar question arises in regard to the formation of
coraco-branchiales in Ceratodus and Scyllium. Again, within
the group of the Teleostomi all variations exist between inter-
arcuales ventrales and their homologues, coraco-branchiales,

These secondary modifications in the hypobranchial-spinal
and hypobranchial-cranial muscles appear to be morpho-
logical expressions of an increased need of tying the

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 283

mandibulo-hyo-branchial skeleton to the shoulder-girdle,
and the change of function of the latter group of muscles to
one similar to that of the former tends to bring about a
secondary innervation from spinal nerves.

A similar question arises in connection with the presence
and absence of levatores arcuum branchialium. It has been
suggested above that their absence and the related method
of formation of the trapezius are secondary phenomena

TEXT-FIG. 84.

hubobr. $b.n. An

84.

Rabbit, embryo 4 mm., longitudinal vertical section.

(p. 257). If so, it is possible that this has been indepen-
dently acquired in Scyllium, Sauropsida, and rabbit.

In Seyllium and the Teleostomi a stage of development
occurs in which there is a short hyoid bar lke that of
Amphibia with a levator hyoidei, which is succeeded by one
in which the bar extends up to the periotic capsule. ‘The
relationship of the muscles and of the facial nerve to the
later formed portion of the bar are so different in Scyllum

284 F. H. EDGEWORTH.

and T'eleostomi that possibly the only common feature is the
above-mentioned first stage. In Ceratodus, Sauropsida, and
rabbit the hyoid myotome is external to the upper part of the
hyoid bar, as in Scyllium.

In Sauropsida and certain Teleostomi the adductor man-
dibule divides into internal and external portions, but in
Teleostomi there is no uniform upgrowth of the external

Trext-FIc. 85.

Fo
Yi
Va

Text-figs. 85-87.—Rabbit, embryo 5 mm. ; Text-fig. 85 is through
the first branchial segment, Text-fig. 86 through the third
branchial segment, Text-fig. 87 Just behind this.
portion to the skull as in Sauropsida. Both division and
upgrowth have been independent occurrences in these two
phyla.

c. Amongst the animals investigated there are but few in
which muscle-Anlagen are developed and then atrophy. The
Mm. marginales and interarcuales ventrales of the larva of
Rana, certain muscles of metamorphosing Urodela described
by Driiner, the levator maxille superioris of Chelone and

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 285

TEXT-FIG. 86.

TEXtT-FIG. 87.

286 FE. H. EDGEWORTH.

Alligator, and the genio-hyoid of Gallus, were the only ones
found. Otherwise if a muscle is not present in the adult it
is not formed during development.

There are certain instances in which comparative evidence
suggests that ancestors probably possessed muscles which are
now no longer developed, even as Anlagen. Such are the
genio-hyoid of Lepidosteus and Salmo, certain Mm. trans-
versi ventrales in Amphibia and Teleostomi, the first two
obliqui ventrales in Polypterus senegalus, the hyo-maxil-
laris inSelachii and Sauropsida, the levatores arcuum brauchia-
lium in Selachii, Sauropsida, and Mammalia.

Consideration of the changes which take place in the
Anlagen of the cranial muscles in the various Vertebrate
groups suggests that the most important are those occurring
in the myotome of the mandibular segment. In Amphibia
and Ceratodus it does not, whilst in Teleostomi, Selachii, and
Sauropsida it does divide into parts above and beiow the
palato-pterygoid or pterygoid process of the quadrate. It
has been stated above that the embryological phenomena
support the view that the second condition has been derived
from the first. In the rabbit the quadrate (incus) has no
pterygoid process, and the myotome—as in Amphibia and
Ceratodus—does not divide into upper and lower parts.

Changes take place in the Anuran tadpole, in the form of
the palato-quadrate bar and in certain muscles in association
with the development of a suctorial mouth, i.e. the back-
ward elongation of the mandibular muscles, the development
of a submentalis and mandibulo-labialis, the origin of the
orbito-hyoideus, or of this and the suspensorio-hyoideus, and
the partial origin of the first branchial levator from the
palato-quadrate bar, the division of the hyo-maxillaris and
attachment of one or two of its parts to the palato-quadrate
bar. As the condition before these events takes place is
very like that of an embryo of Ceratodus or an Urodelan,
it would appear probable that the changes are secondary
larval ones and not ancestral.!

' The difficult question as to the origin and nature of the larval

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 287

On the other hand, the existence of a hyo-maxillaris and of
Mm. marginales, the insertion of the orbito-hyoideus or of
this and the suspensorio-hyoideus to the cerato-hyal, and the
origin of the trapezius from the skull, are primitive features
which are not developed or soon modified in Urodelan
development.

In the Urodela the insertion of the levator hyoidei is
transferred, wholly or partially, from the hyoid bar to
Meckel’s cartillage early in development, and the hyo-
maxillaris Anlage forms a ligament. The development of
gill-muscles from Anlagen which are homologous with those
which give rise to the Mm. marginales of Anuran larve and
Ceratodus, and of a cerato-hyoideus externus, are features
peculiar to Urodela.

Ceratodus resembles Selachii and some Telecstomi, and
differs from Amphibia in the backward growth of the genio-
hyoid to the shoulder-girdle, and in the formation of coraco-
branchiales. Ceratodus resembles Selachii and Teleostoman
embryos, and differs from Amphibia in the backward growth
of both hyoid myotome and interhyoideus, resulting in the
formation of a continuous dorso-ventral sheet, C,vd, behind
the hyoid bar. Ceratodus resembles Teleostomi and Amphi-
bia, and differs from Selachii in the formation of levatores
arcuum branchialium and in the development of the trapezius
from a levator. Ceratodus resembles Amphibia, and differs
from Selachii and Teleostomi in the non-division of the
mandibular myotome into upper and lower portions. Cera-
todus resembles Anuran larve in the simple condition of the
Mm. marginales,and Urodela in the ligamentous condition of
the hyo-maxillaris.

According to K. Firbringer, “ Wenn wir somit keine
bestimmte Ordnung der Amphibia von den Dipnoern
ableiten konnen, so ergiebt sich daraus kein Kimwand
gegen eine Abstammung von den Dipnoern iiber-

condition of the suctorial mouth and jaws in Anuran larve was
discussed by Balfour and by Gaupp, though without reference to the
muscles.

288 F. H. EDGEWORTH.

9)

haupt. The development, however, in Ceratodus, of
a coraco-mandibularis, of coraco-branchiales, of a hyoman-
dibula, and of a dorso-ventral sheet C,vd behind the hyoid
bar, are all secondary to more primitive conditions present in
Amphibia.

Goodrich was of opinion that “the Dipnoi are probably a
specialised offshoot from the ‘Teleostoman stem which

TExt-FIG. 88.

88.

Rabbit, embryo 7 mm., longitudinal vertical section.

acquired an autostylic structure before the hyomandibula
had become very large and before the hyostylism had become
fully established.” The non-division of the mandibular
myotome and the persistence of the dorso-ventral sheet
C,vd are, however, more primitive features than exist in
Teleostomi; and in the embryo of Ceratodus there 1s
a hyomandibula, the relations of which are different from
those occurring in 'leleostomi.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 289

Graham Kerr’s opinion was that “the Teleostomes the
Dipnoans and the Amphibians have arisen in phylogeny from
a common stem . =F

Kellicott’s statements that ‘the resemblances in the
vascular system between Ceratodus (the most primitive of the
living Dipnoi) and the Amphibia, especially Urodela, are
numerous and fundamental and cannot be explained as
parallelisms,” and that ‘most of the Hlasmobranch
characters are parallelisms, some of them actually being
preceded by Amphibian conditions (e.g. the carotid arteries) ”
are also true of the cranial muscles.

Consideration of the common features in the cranial
muscles of Teleostoman embryos leads to the probability that
some remote ancestors possessed—a mandibular myotome
divided into upper and lower parts!; a levator hyoidei, which,
owing to the upgrowth of the hyoid bar to the periotic
capsule, was inserted into the inner or posterior surface of a
hyomandibula ; a dorso-ventral sheet in the opercular fold,
divided into a M. opercularis and a constrictor operculi; a
series of levatores arcuum branchialium ; a trapezius developed
from the fourth levator; a series of Mm. marginales not fused
with the transversi ventrales; a series of hypobranchial-
cranial muscles consisting of interarcuales ventrales and of a
coraco-branchialis attached to the last branchial bar ;
hypobranchial-spinal muscles, consisting of a coraco-hyoideus,
and of a genio-hyoid, the hind end of which had grown back
to some more posterior branchial bar overlapping the coraco-
hyoideus.

All these features, with five exceptions, may be supposed
to have characterised primitive Amphibia; and these excep-
tions, viz. division of the mandibular myotome, formation of
a M. opercularis, and of a coraco-branchialis, backward
growth of the genio-hyoid, upward extension of the hyoid

1 On the supposition that the protractor hyomandibularis of Aci-
penser is a case of atavism in its non-division into levator arcus
palatius and dilatator operculi, this division of the levator maxillze
superioris would have once characterised the whole group.

290 F. H. EDGEWORTH.

TEXT-FIG. 89,

Nia it

90.

Text-figs. 89 and 90.—Rabbit, embryo 73 mm.; Text-fig. 89 through
the hyoid segment, Text-fig. 90 through the neck,

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 291

bar to the periotic capsule—are, as shown by their develop-
ment, modifications of more primitive features existing in
Amphibia.

These phenomena may be considered as additional argu-
ments in favour of the theory of a descent of Teleostei, as
advocated by Assheton, from a proto-amphibian stock ; and
of Teleostomi in general, as advocated by Graham Kerr, from
a stem common to the Teleostomi, Dipnoi, and Amphibia.

In the condition of the cranial muscles Teleostei do not
show any closer resemblances to Amphibia than do other
groups of the Teleostomi.

The curious fact that the trapezius is developed from the
fourth levator arcuum branchialium in Acipenser, Lepidosteus,
Amia, and Salmo, though there are five branchial segments,
suggests that ancestors of the Teleostomi may have had, like
Amphibia, only four branchial segments, and that an increase
to five took place within the group.

In the possession of only four branchial segments, of
interarcuales ventrales I, II, and III, of obliquii ventrales
not fused with transversi ventrales, and of very primitive
laryngeal muscles, Polypterus senegalus shows closer
resemblances to’ Amphibia than do the other Teleostomi
examined.

The main characteristics of the cranial muscles of Selachii
are: (1) Division of the mandibular myotome into levator

maxille superioris and adductor mandibule; (2) great

or
backward extension of the intermandibularis below the
interhyoideus; (5) non-formation of an opercular fold;
(4) upgrowth of the hyoid bar internal to the hyoid myotome,
which, originally forming a levator hyoidei, becomes inserted
into its external surface (hyomandibula, or this and
ceratohyal); (5) non-formation of a hyo-maxillaris; (6)
extension backwards of hyoid myotome and interhyoideus
forming a dorso-ventral sheet C,vd behind the hyoid bar,
though not in an opercular fold; (7) non-formation of
levatores arcuum branchialum; (8) formation of a trapezius
from the upper ends of all the branchial myotomes; (9)

292 F. H. EDGEWORTH.

formation of subspinalis and interbasales from anterior trunk
myotomes; (10) formation of coraco-branchiales; (11)
formation of adductors from the portions of the branchial
myotomes which le internal to the branchial bars; (12)
formation of arcuales dorsales, interbranchials, and superficial
constrictors from the portions of the branchial myotomes
which lie external to the branchial bars; (15) non-formation
of transversi ventrales; (14) extension backward of the
genio-hyoid, forming a coraco-mandibularis. Of these
features, (3) (9) and (12) occur in Selachii and them only.

The great development of the branchial musculature,
external to the branchial bars, is correlated with the absence,
probably the loss, even in developmental stages, of an
opercular fold. It is of interest to note that in Chimera
(Vetter) (1) a hyo-maxillaris (hyoideus inferior) is present ;
(2) the dorso-ventral sheet C,vd is situated in an opercular
fold; (3) the branchial musculature, external to the bars,
consists of simple vertical muscles (‘‘interbranchials” of
Vetter), which are similar to the Mm. marginales of Anuran
larve and Ceratodus, and to the dorsal portions of the obhiqui
ventrales of Teleostomi.

According to Graham Kerr, “the Teleostomes, the
Dipnoans, and tne Amphibians have probably arisen in
phylogeny from a common stem, which would in turn
probably have diverged from the ancestral Selachian stock.”
Fiirbringer’s theories in regard to the hypobranchial muscles
and the neocranium, and Ruge’s respecting the facial
muscles, are also based on a similar theory.

Consideration of the morphology of the cranial muscles
leads to some doubt on this question. The embryology of
each group of cranial muscles, mandibular, hyoid, branchial,
hypobranchial-cramial, and hypobranchial-spinal, suggests
that the conditions found in Selachii are secondary to those
which may be supposed to have characterised Amphibian
ancestors—are modifications of a proto-amphibian type.
Certain of these modifications occur in other groups also: thus
division of the mandibular myotome into upper and lower

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 293

parts also occurs in Teleostomi and Sauropsida; backward
extension of both hyord myotome and interhyoideus to form
a dorso-ventral sheet also occurs in Ceratodus and ‘Teleo-
stomi (though in these, in an opercular fold) formation of

Trxt-Fia. 91.

Rypobr. sp. im An. 91.

Text-figs. 91-93.—Rabbit, embryo 9 mm.; Text-fig. 91 through
the mandibular segment, Text-figs. 92 and 93 through the
hyoid segment.

coraco-branchiales and of a coraco-mandibularis also occurs
in Ceratodus and some T'eleostomi; non-formation of levatores
arcuum branchialium, and the associated method of develop-
ment of the trapezius occurs in Sauropsida and rabbit.

294, F. H. EDGEWORTH.

The significance of such resemblances from a phylogenetic
point of view is doubtful, though probably the first two
named are by far the most important.

The ancestry of Mammals has been the subject of inquiry
and speculation for many years. ‘'l'wo theories have been

TrxT-FIG. 92.

held—one, that Mammals are descended from Sauropsida,
the other, that they are descended from Amphibia.

As regards the cranial muscles, Mammals resemble
Amphibia, and differ from Sauropsida in the following
particulars: non-division of the mandibular myotome into
dorsal and ventral parts, formation of a hyo-maxillaris
(anterior digastric), non-formation of a dorso-ventral sheet

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 295

Czvyd in the hyoid segment, innervation of the interarcualis
ventralis I (branchio-hyoideus) by the IXth.

On the other hand, Mammals resemble Sauropsida, and
differ from Amphibia, in the non-formation of levatores
arcuum branchialium, and the associated development of the

TEXT-FIG. 93.

inberkyoud

hupob le hn 93.

trapezius from the upper ends of all the branchial myotomes,
disappearance of the branchial myotomes (after formation of
trapezius and interarcuales ventrales from their upper and
lower ends), non-formation of transversi ventrales.

It has been suggested above in discussing individual groups
of muscles that all the first-named features are primary ones,
and that all the second-named features are secondary
phenomena. It is possible that secondary features may have

VOL. 56, PART 2.—NEW SERIES. ma

296 F. H. EDGEWORTH.

been independently acquired; thus the absence of levatores
arcuum branchialium and method of formation of the
trapezius also occurs in Selachi. ‘The morphology of the
cranial muscles is thus in favour of an Amphibian ancestry of
Mammals. In the attachment of the posterior digastric to
the hyoid bar, and not to the lower jaw, some Mammals

TEXT-FIG. 94.

hs \ .
Rey bal.+ {ers bol\,

Aw

94.

Text-figs. 94 and 95.—Rabbit, embryo 135 mm.; transverse sec-
tions through the mandibular segment. Text-fig. 94 is the
more anterior.

present a more primitive feature than is found in any adult
Amphibia. A descent from a proto-amphibian stock is thus
suggested.

The ancestry of Sauropsida has been the subject of but
few speculations. Fiirbringer was of opinion that “Die
strepto-stylen Pro-reptilia aber haben sich neben den strepto-
stylen Pro-mammalia auf tiefer stehenden streptostylen

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 297

Thieren entwickelt welche im Grade ihrer Ausbildung
amphibienartige Thiere gleichzusetzen sind. . . .’ Graham
Kerr’s opinion was that ‘the ancestors of the Amniota
probably diverged about one or several points from the
region of the stem common to Dipnoi and Amphibia.”

As regards the cranial muscles, the differences between

TEXT-FIG. 95.

Sauropsida and Amphibia have been mentioned above. In
the division of the mandibular myotome into upper and
lower portions, and in the formation of a ventro-dorsal sheet,
C,vd, in the hyoid segment, Sauropsida resemble Selachii and
Teleostomi, and also as regards C,vd, Dipnoi. The shifting
of insertion of the levator hyoidei from cerato-hyal to
Meckel’s cartilage and the morphologically primitive con-
dition of the hypobranchial spinal muscles are common to
both Sauropsida and Amphibia.

298 F. H. EDGEWORTA.

On FUrRBRINGER’S THEORY OF THE SKULL.

It is of interest to inquire whether the above suggestions
as to the phylogeny of various groups of Vertebrates receive
any support from the morphology of the skull.

According to Fiirbringer’s theory the portion of the cranium
in front of the exit of the vagus is the original cranium—the
paleocranium. The neocranium has been formed by the
addition of spinal skeletal elements, which originally were
free. This took place in several stages ; in the first a proto-
metamer neocranium is formed—present in Selachii and
Amphibia. The union of further additional elements brings
about the auximetamer condition of the neocranium, feund in
higher fishes and Amniota.

The added spinal nerves—spino-occipital nerves—can be

divided into two categories, the ‘

‘occipital,’ brought in with
the protometamer neocranium, and the “ oceipito-spinal,”
additionally added with the auximetamer neocranium. The
varying number of spino-occipital nerves is due to the varying
position of the cranio-vertebral junction.

The assimilated occipital nerves are indicated by the
terminal letters of the alphabet, the assimilated occipito-
spinal nerves by the initial letters. heir corresponding
myotomes are given corresponding (larger) letters. By this
method it is possible to express either or both of two possi-
bilities—the reduction of more anterior or the addition of
more posterior nerves.

The following table, which is taken mostly from Gaupp,
shows the results of the investigation of various vertebrates,
and a column has been added showing the number of
myotomes taking part in the formation of the hypo-
branchial muscles.

A spinal segment is typically indicated by a somite or
myotome, anterior nerve root, and posterior nerve root. The
researches of Fiirbringer and other observers have shown that
as segments are assimilated their nerve roots tend either not

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 299

|

| No. of |
trunk |
| segments
| taken up |
into the
skull.
/Anura—
| Pelobates (Sewertzoff) 3
| Rana (Miss Elliott) 3
Urodela—
| Siredon (Sewertzoff) 2
| Necturus (Miss Platt) 3
| ‘Triton =
Dipnoi—
Ceratodus (K. Fiirbringer) 5
| Protopterus (Agar) 3
| Lepidosiren (Agar) 3
‘Mammals—
| Sheep, calf (Froriep) 3
| Rabbit oe
\Reptiha—
| Ascalobates (Sewertzoft) 4,
| Lacerta (Hoffman) 5
Lacerta (Chiarugi and
Bemmelen ). 4.
‘Birds —
' T'innunculus (Suschkin ) 4
Gallus =
Teleostomi—
Amia (Schreiner)

Salmo salar (Wilcox) 5
Trutta fario (Wilcox) 5
Lepidosteus (Schreiner ) 7
Acipenser (Sewertzoff) 7

‘Selachii (Gaupp)— ii

Squalus acanthias

Seyllium canicula

Nature of
neocranium
according to
Fiirbringer’s
theory.

Protometamer

3?

Auximetamer
(Gaupp)

Protometamer

(Agar)
Ditto

Auximetamer

Protometamer

Taking part in the |

formation of hypo- |

branchial spinal
muscles.

Ist, 2nd.

2nd (few cells),
3rd, 4th, 5th
(Miss Platt).

2nd 38rd (Greil). |

2nd, 8rd, 4th
(Agar).

2nd, 38rd, 4th
(Agar).

Ist, 2nd, 3rd.

2nd, 3rd, 4th, 5th
( Hoffman)

lst (few cells),
2nd, 3rd, 4th, 5th.

2nd, 3rd, 4th.
2nd, 3rd, 4th
(Harrison).
2nd, 3rd, 4th.
2nd, 3rd, 4th.

4th, Sth, 6th, 7th,
8th (Neal).

4th, 5th, 6th, 7th,|

Sth.

to be developed, or after development to atrophy, and that

this takes place from before backwards,

The non-develop-

ment or atrophy affects dorsal more readily than ventral

roots.

Reduction, i.e. atrophy after development, of somites

300 F. H. EDGEWORTH.

or myotomes comes last. This general rule leads to hesitation
in accepting the existence of anterior nerve roots without
corresponding somites or myotomes as evidence of assimi-
lated seginents, e.g. deductions from the observations of
Chiarugi and Martin in Mammals.

The theory of Fiirbringer is based on the probability of the
primitive nature of the conditions found in Selachians. But
trom the foregoing table of the observed number of assimi-
lated spinal segments in various Vertebrates it would appear
that the descriptive adjectives applied to some neocrania are
not deserved. As determinated by the number of assimilated
spinal segments the Amphibian ueocranium is shorter than
that- of Selachians. It was therefore maintained that the
occipital region of Amphibians corresponds to a multiplum of
spinal segments. ‘The difficulty of doing so is emphasised by
the absence of any direct evidence in its favour. If the
muscles of the head in Amphibians and Selachians be com-
pared it is clear that the condition in the former is far more
primitive than in the latter, and that many cranial muscles
of Scylhum pass through what may be regarded as an
Amphibian stage during development ; and if the observed
facts in regard to the number of assimilated spinal segments
be taken sans parti pris the condition of the skull tells
the same tale. Fiirbringer states that the junction of the
skull and vertebral column is at the same place in Sauropsida
and Mammalia; hence the five occipital nerves in Reptilian
embryos are called v, w, x, y, 2; and the three in Mammals
Xx, y, Z, so that the last assimilated nerve is the same—z.
But in Mammals there appear to be only three assimilated
somites, in Reptiles four or five. The argument drawn from
the existence of a pro-atlas is probably of no great weight in
determining the limits of the skull and vertebral column, for
in Sphenodon (loc. cit.) that structure is the persisting costal
process of the last coalescing vertebra, and the same may be
true in Mammals without there being any but a serial
homology between these last coalescing vertebra.

The conclusion which might be drawn from the number of

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 301

TEXT-FIG. 96.
veh bulk vec ext

veck.sub. Len + ma. An.

oft, sw

\

rect ink bleuy,m.Ar. fae Setanta Lene bmp

veck inf

97.
Text-figs. 96 and 97.—Rabbit, embryo 13 mm., longitudinal vertical
sections. Text-fig. 96 is the more external.

302 F. AH. EDGEWORTH.

coalescing spinal segments in Amphibia, Sauropsida, and

Mammalia—viz. 2 or 3, 4 or 5, and 3—harmonises with the

evidence of the cranial muscles, in which there is a closer
similarity between Mammalia and Amphibia than between
Mammalia and Sauropsida.

A Svuacestep MorpHoLocicaAL CLASSIFICATION OF THE Moror
CENTRES OF THE Mip- anp Hinp-Brain in Man.

Gaskell divided the motor centres of the cranial nerves

TEXT-FIG. 98.

Kypotr spm An
hyoid my

premond

98.

Pig, embryo 8 mm., longitudinal vertical section.

into two categories: (1) Somatic, a continuation of the
anterior column of the spinal cord, innervating somatic
muscles—ITIrd (external ocular muscles), [Vth, VIth, VIIth
(part which arises from the VIth nucleus), XIIth. (2a)
Non-ganglionated splanchnic, a continuation of the
lateral column of the spinal cord, innervating voluntary
splanchnic muscles—Vth (motor descending root), Vth
(motor), VIIth, IXth, Xth, XIth (part which arises from
lateral horn). (28) Ganglionated splanchnic, a con-
tinuation of Clarke’s column—IIIrd (G. ciliare), VIIth (N.

MORPHOLOGY OF CRANIAL MUSCLES 1N SOME VERTEBRATES, 303

intermedius with gang. genic.), [Xth (gang. petros.), Xth,
XIth (gang. trunci vagi), XIIth (gang. hypoglossi).

This classification of the motor centres, as regards those of
voluntary muscles, followed v. Wijhe’s theory of the mor-
phology of the cranial muscles. It was also adopted by
Strong and by Herrick.

According to Streeter the motor nucleus of the Vth nerve
in man is developed in the lateral plate, and the nucleus
ambiguus of the VIIth, [Xth, and Xth in the basal plate.

TExT-FIG. 99.
eubmoy g syloqloss
\

tohupid, - Stethotyeud
a Many anda eae 99.

Pig, embryo 15 mm., portion of longitudinal vertical section.

The issuing fibres of the Vth pass straight outwards like
those of the dorsal efferent fibres of the IXth, Xth, and
XIth (medullary) ; whilst those of the VIIth, IXth, and
Xth, arising from the nucleus ambiguus, have a characteristic
curved path. The motor nucleus of the Vth is a hyper-
trophied representative of the dorsal motor nuclei of the
IXth, Xth, and XIth (medullary), or the latter is represented
in the mesencephalic root of the Vth.

Kappers showed that the original position of the VIIth,
IXth, and Xth motor nuclei is medio-dorsal, and that the

304 Fr. H. EDGEWORTH.

ventral position of the nucleus ambiguus is only found in
Mammals, where the importance of the ventral tegmentum
is increased by the pyramidal tract, whilst a part keeps its
original position near the mid-dorsal line because not very
much influenced by the long descending tracts of the frontal
parts of the brain.

It would result from a comparison of these researches that
the ventral position of the VIIth nucleus, and of the nucleus
ambiguus of the [Xth and Xth, is a secondary one, the curved
path of their issuing fibres representing a phylogenetic descent
of the whole or part of their nuclei; whilst the motor nucleus
of the Vth has preserved its original position. This position
is a dorso-median one. ‘lhe nucleus of the XIth spinal
occupies a more or less lateral position in the cervical cord,
but, as shown by the development of the muscles it inner-
vates, the nerve is a specialised branch of the Xth, the
nucleus of which has extended backwards into the spinal cord.

The following classification of the motor nuclei of the
cranial nerves is a repetition from a neurological point of
view of the theory which has been advanced above concern-
ing the morphology of the cranial muscles, and consequently
stands or falls with it.

Somatic, innervating muscles derived from the myotomes
of the cerebral and three anterior body segments; IIIrd
(external ocular muscles), IVth (superior oblique), VIth
(external rectus), Vth (temporal, masseter, pterygoids, tensor
tympani, anterior digastric), VIIth (posterior digastric, stylo-
hyoid, stapedius), IXth (interarcualis ventralis I s. branchio-
hyoideus, when present), Xth and XIth medullary (inter-
arcualis ventralis III s. interthyroideus, in Ornithodelphia),
XIth spinal (sterno-mastoid and trapezius), XIIth (hypo-
branchial spinal muscles, and lingual muscles derived from
the genio-hyoid). Splanchnic, innervating muscles derived
directly or indirectly from the walls of the cephalic ccelon,
i.e. part of motor nucleus of Vth, which innervates mylohyoid;
part of motor nucleus of VIIth, which innervates facial and
platysma muscles; part of motor nuclei of IXth, Xth, and

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 305

XIth medullary, which innervates tensor and levator palati,
palato-glossus, stylopharyngeus, pharyngeal constrictor, laryn-
veal muscles, crico-thyroid.

The primary cranial nerves are the IIIrd, Vth, VIIth,
TXth, and Xth; the Xth innervating in the rabbit two
myotomes (second and third branchial), the others one each.
The primary dorsal position of their motor nuclei (other
than that of the I[Ird), the dorso-lateral emergence of their
motor with their sensory fibres, and the relationship—
external—of the issuing nerves to the corresponding
myotomes, are related phenomena. If Balfour’s theory, that
the head and trunk became “ differentiated from each
other at a stage when mixed dorsal and sensory posterior
roots were the only roots present,’ be associated with
Fiirbringer’s theory that the myotomes_ primitively lay
exclusively lateral to the notochord, it would follow that in
the body region anterior nerve roots were secondarily
developed in correlation with the upgrowth of the myotomes
to the mid-dorsal line, and the posterior roots became exclu-
sively, or almost exclusively, sensory. In the head, where
this upgrowth does not take place, or to a very limited
extent, a more primitive condition persists both in the
position of the motor nuclei and the emergence of their
efferent fibres.

A further, probable, distinction between the somatic muscles
of the body and those of the head is that ganglionated muscle-
sensory nerve-fibres pass to the former but not to the latter.!

The position of the nucleus of the IIIrd nerve and the path
of its nerve-fibres may be associated with the loss of cutaneous
sensory fibres. Evidence of such loss and of a primitive dorso-
lateral emergence of its nerve-fibres is found in the observa-
tion of Neumeyer that in the twenty-nine and forty-three hours
old chick “ der Nerf vom dorsalen heile des Mittelhirns, also
in der Gegend der Ganglionleiste seinen Ursprung nehme,
sich also sekundér mit seinem definitiven Abgangsort
vereinige.”

' T hope to give the evidence for this in a future paper.

306 YT. H. EDGEWORTH.

The Anlagen of the superior oblique and external rectus
are developed from forward extensions of the upper ends of
the mandibular and hyoid myotomes, and the [Vth and VIth
nerves may be regarded as, phylogenetically, late formations.

There do not appear to be any investigations on the
existence of cell-groups in the Vth motor nucleus, which
might correspond to the somatic and splanchnic muscles
innervated. ‘he nucleus contains a centre for the anterior
digastric, but it is not known whether this migrates, during
development, from the facial nucleus, or whether it is locally
developed. The fibres of the Vth mesencephalic root join the
motor root (Cajal), but it does not appear certain what
structures it innervates.

The motor nucleus of the VIIth nerve consists, according
to van Gehuchten and Marinesco, of four cell groups, three
ventral and one dorsal: of these, the internal ventral is the
centre for the stapedius, the middle for the auricular muscles,
the external for the inferior facial muscles, and the dorsal
nucleus for the superior facial muscles (frontalis, corrugator
supercilil, and orbicularis palpebrarum). According to this
account there is no special cell-group for the posterior
digastric and stylohyoid, which seems unlikely. More
recently, Kosaka has stated that the dorsal cell group in the
fowl is the motor nucleus for the digastricus. ‘The subject
evidently needs further investigation.

The glosso-pharyngeal nucleus, according to v. Gehuchten,
consists of a ventral cell-group only ; according to Streeter
it has a dorsal nucleus as well asa nucleus ambiguus. In
the monkey (Beevor and Horsley) it innervates the stylo-
pharyngeus and (?) the middle constrictor of the pharynx.
It is not known whether there is a separate cell-group for the
branchio-hyoid in animals, e.g. pig, dog, where this muscle
exists.

The Xth and XIth medullary are primitively, in the rabbit,
the nerves of the second and third branchia! segments. The
Xth efferent fibres arise from dorsal and ventral motor nuclei,
those of the XIth medullary from a dorsal nucleus only (v.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 307

Gehuchten). As all the fibres of the XIth medullary join
the Xth, and all the fibres of the XIth spinal pass to the
trapezius and sterno-mastoid,! it is a little questionable whether
the old distinction of the two parts of the accessorius is worth
preserving. The term XIth or accessorius might well be
limited to what is now known as the XIth spinal. In a
Mammal like the rabbit, where the whole of the second and
third branchial myotomes (other than their dorsal ends
which take part in forming the trapezius and sterno-
mastoid) disappear during development, the Xth and XIth
medullary motor centres contain none of the original somatic
efferent fibres or cell-groups, and their new centres are those
innervating muscles derived from cells proliferated from the
wall of the cephalic cceelom. They also contain motor centres
for certain visceral muscles which are developed in the body
region.

The Xth and XIth medullary centres overlap antero-
posteriorly the hypoglossal nucleus, probably owing to their
backward extension into the first three segments of the
spinal cord.

The XIth spinal is, as emphasised by Fiirbringer, a true
cerebral and not a spinal nerve. It innervates a special
group of muscles which, in the rabbit, are derived from the
upper ends of the three branchial myotomes. Its nucleus
of origin is, from a phylogenetic point of view, a backward
extension into the spinal cord of the (dorsal) nucleus of the
XIth medullary, but it is not known what happens in
embryonic development.

The hypoglossal nucleus is the motor centre of the hypo-
branchial spinal muscles, of the rectus system, developed
from the first three body myotomes. Cell-groups corresponding
to the upper, atrophying portions of these myotomes have
been lost. It is not known whether the subdivision of the
nucleus into the parts with large and with moderate-sized
cells corresponds with individual muscles or muscle-groups.
The hinder part of the hypobranchial spinal muscles has a

1 In dog (loc. cit.) and man (Streeter).

308 rT. H. EDGEWORTH.

secondary innervation from cervical segments—first, second,
and third in man, first and second in the dog—but it is
not known whether this is due to backward migration or to
local development of motor neuroblasts.

On THE Size or tHE MeEpuLLATeD Nerve-Fipres Passina TO
Cranial. MuscuLes.

Gaskell stated that in the dog large fibres, 14-4 to 18 mu in
diameter, were present in the II Ird (external ocular muscles),
IVth, VIth, VIIth (destination not traced), and XIIth.
The corresponding muscles were considered to be somatic.
Nerve-fibres not exceeding 10°8 uw in diameter were found in
ViIlth (facial muscles), pharyngeal nerves, and recurrent
laryngeal; and the corresponding muscles were considered
to be splanchnic. Apparently he did not take the size of the
nerve-fibres as the sole criterion of the somatic or splanchnic
nature of a muscle, for the sterno-mastoid and trapezius
were considered to be splanchnic, though the nerve (spinal
XIth), showed the larger size of nerve-fibres. A further
analysis (loc. cit.) of the size of nerve-fibres passing to cranial
muscles in the dog shows that: (1) In any individual nerve,
fibres are found of all sizes up to the largest present ; (2) the
nerve-fibres taper very slightly as they pass from the central
nervous system to the muscles; (3) if comparison be made
between the maximum size of the nerve-fibres and the
morphological nature of the muscles to which they pass, the
following results appear: (A) Nerve-fibres of the greatest
size (17°6 « in diameter,! in some dogs only 16 «), are found
in the nerves of the external ocular muscles, temporal,
pterygoids, tensor tympani, digastric (both from Vth and
VIlth), stylo-hyoid, branchio-hyoid, trapezius, sterno-
inastoid, genio-hyoid, sterno-hyoid, sterno-thyroid, thyro -
hyoid, and omohyoid—all of which, according to the theory

' This is also the maximum size of the nerve-fibres in the anterior

roots of the non-limb portions of the spinal cord. In the limb areas it
is slightly greater.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 309

advanced above, are somatic in origin. (B) Nerve-fibres of a
less maximum diametér (12°8 uw, in some dogs only 11°2 1),
are found in the nerves of the mylohyoid, facial and platysma
muscles, palatal, pharyngeal,and laryngeal muscles and crico-
thyroid — all of which, according to the theory advanced
above, are splanchnic in origin; and also in the nerves of
the lingual muscles, which are developed from the genio-
hyoid—a somatic muscle.

Herrick stated that the nerve-fibres of the branchial
muscles of Menidia were characterised by their large size,
and supposed—on the theory that these muscles were of
splanchnic origin — that they had acquired this somatic
feature. On the theory advanced above, however, the bran-
chial muscles are somatic in origin.

The small size of the nerve-fibres of the lingual muscles is
curious, but the muscles, though somatic in origin, have
intimate relations to a splanchnic epithelium. This sugges-
tion is supported by the measurements of the nerve-fibres
passing to the genio-hyoid and lingual muscles of Lacerta
viridis and Testudo mauritania; in the former animal
the maximum diameters found are 11°6 and 9°6 4 respectively,
whereas in the latter animal both maxima are the same, viz.
75 pt.

I have, in conclusion, to express many thanks to Prot.
Salensky for embryos of Acipenser ; to Prof. Bashford Dean
for embryos and for the loan of sections of Ceratodus; to Prof.
Graham Kerr for specimens of Polypterus senegalus; and
to Prof. Fawcett for the loan of sections of the pig; also to
the last-named for much kindness shown to me during many
years in his laboratory.

LITERATURE.

Agar (‘08).—* The Development of the Skull and Visceral Arches in
Lepidosiren and Protopterus.” * Trans. Roy. Soc. Edin.,’ vol. xlv.
(‘08).—* The Development of the Anterior Mesoderm and Paired
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Roy. Soc. Edin.,’ vol. xlv.

310 F. H. EDGEWORTH.

Allis (97).—* The Cranial Muscles and Cranial and First Spinal Nerves
in Amia Calva,” *‘ Journ. of Morph.,’ vol. xii.

Assheton (07).—** The Development of Gymnarchus Niloticus”;
The work of J. 8. Budgett.

Beevor and Horsley (‘88).—‘* Note on Some of the Motor Functions of
Certain Cranial Nerves (Fifth, Seventh. Ninth, Tenth, Eleventh,
Twelfth) and of the First Three Cervical Nerves of the Monkey
(Macacus sinicus),” * Proc. Roy. Soc.’

Bijvoet (‘08)—* Zur vergleichenden Morphologie des Musculus digas-
tricus bei den Siiugethiere,”” * Zeitschr. f. Morph. u. Anthrop..,’
Bd. xi, Heft 2.

Corning (95).—** Ueber die Entwicklung der Zungenmuskulatur bei
Reptilien,” * Verh. d. Anat. Ges. im Basel,’ Bd. x.

——— (99).— Ueber einige Entwicklungsvorgiinge am Kopfe der
Anuren,”’ *‘ Morph. Jahrb., Bd. xxvii.

Cunningham (91).—* The Value of Nerve-Supply in the Determination
of Muscular Homologies and Anomalies,” ‘Journ. of Anat. and
Phys.,’ vol. xxv.

Driiner (03).—* Ueber die Muskulatur des Visceralskelets der Uro-
delen,”’ * Anat. Anzeig.,’ Bd. xxiii.

Edgeworth (‘99).—** On the Medullated Fibres of some of the Cranial
Nerves and of the Development of Certain Muscles of the Head,”
‘Journ. of Anat. and Phys., vol. xxxiv.

——— (07).—*“* The Development of the Head-muscles in Gallus
domesticus, and the Morphology of the Head-muscles in
Sauropsida,” ‘Quart. Journ. Mier. Sei.’ vol. 51.

Elliot, Miss ((07).—* Some Facts in the Later Development of the Frog,
Rana temporaria; Part 1: The Segments of the Occipital
Region of the Skull,” * Quart. Journ. Mier. Sci.,’ vol. 51.

Frazer (10)—‘* The Development of the Larynx,” ‘Journ. of Anat
and Phys., vol. xliv.

Felix (‘04).—‘“ Die Entwickelung des Harnapparatus,” Hertwig’s * Hand-
buch.’

Firbringer, M. (75).—‘* Beitrag. zur Kenntniss der Kehlkopfmus-
culatur, Jena.

——— (75).— “Zur vergleichenden Anatomie der Schultermuskeln,”
i Theil (73), ‘Jenaische Zeitschrift, Bd. vii; ii Thiel ('74)
Jenaische. Zeitschrift, Bd. viii; iii Theil (76), ‘Morph. Jahrb..’
Bd. i; iv Theil (00) ‘ Jenaische Zeitschrift. Bd. xxxiv.

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES, 511

Fiirbringer, M. (88).—‘ Untersuchungen zur Morphologie und System-
atik der Vogel, zugleich ein Beitrag zur Anatomie der Stiitz—
und Bewegungsorgane, Amsterdam.

(97).—* Ueber die Spino-occipitalen Nerven der Selachier und
Holocephalen und ihre vergleichende Morphologie,” * Festschr.
f. Gegenhbaur.’

('04).—* Zur Frage der Abstammung der Saugethiere,” Haeckel’s
‘ Festschr.’

Fiirbringer, K. ('04).—‘ Beitrage zur Morphologie der Skeletes der
Dipnoer, nebst Bemerkungen iiber die Pleuracanthiden Holo-
cephalen und Squaliden,” ‘ Jen. Denkschr.’

Futamura ('06).—‘* Ueber die Entwickelung der Facialis Muskulatur
des Menschen,” ‘ Anat. Hefte, Ad. xxx, Abt. i.

((07)—“Beitrige zur vergleichenden Entwickelungsgeschichte
der Facialis-muskulatur,” “Anat. Hefte,’ Bd. xxxii.

Gaskell (’86).—*‘ On the Structure, Distribution and Functions of the
Nerves which Innervate the Visceral and Vascular Systems,”
‘Journ. of Phys.,’ vol. vii.

-——— (’88).—* On the Relation between the Structure, Functions, Dis-
tribution and Origin of the Cranial Nerves, together with a
Theory of the Origin of the Nervous System of Vertebrata,”
‘ Journ. of Phys.,’ vol. x.

Gegenbaur ('94).—* Zur Phylogenese der Zunge,” ‘Morph. Jahrb.,
Bd. xxi.
van Gehuchten (98).—* Recherches sur Vorigine réelle des nerfs

craniens: ii, Le nerf facial,” ‘ Journ. de Neurol.’
Goodrich ('09)—* A Treatise on Zoology,’ edited by Sir E. Ray
Lankester : Part IX, Ist fascicle, “*‘ Cyclostomes and Fishes.”
Géppert ('94).—* Die Kehlkopfmuskulatur der Amphibien,” ‘Morph.
Jahrb.,’ Bd. xxii.
(98 and °00).—* Der Kehlkopf der Amphibien und Reptilien” :
i Theil, ‘ Morph. Jahrb., Bd. xxvi; ii Theil, Bd. xxviii.
‘01.—* Beitrage zur vergleichenden Anatomie des Kehlkopfes
und seiner Umgebung mit besonderer Berucksichtigung der
Monotremen,” Semon’s ‘ Zool. Forschungsreisen,’ Bd. iii, Lief. iv.

Greil ('07).—*‘ Ueber die Bildung des Kopfmesoderms bei Ceratodus
Forst,’ ‘ Anat. Anz.,’ August 10th.

('08).—* Entwickelungsgeschichte des Kopfes und des Blutgefass-
systems von Ceratodus Forsteri,’ Semon’s ‘ Forschungs-
reisen.”

VOL. 56, PART 2.—NEW SERIES. 22

Bue F. H. EDGEWORTH.

Harman (00).— The Palpebral and Oculo-motor Apparatus in Fishes,”
‘ Journ. of Anat. and Phys.,’ vol. xxxiv.

— ('07)—“ The Origin of the Facial Nerve,” ‘Brit. Med. Journ..,’
November 9th.

Harrison (°94).—* The Development of the Fins of Teleosts,” ‘The Johns
Hopkins’ Univ. Circulars,’ vols. xiii to xiv.

Herrick (°99).—* The Cranial and First Spinal Nerves of Menidia,”
‘Journ. Comp. Neur.,’ vol. ix.

Huxley (°76).—**On Ceratodus Forsteri, with Observations on the
Classification of Fishes.” ‘ Proc. Zool. Soe.’

Jaquet (°99).—* Contrib. a anat. comp. des systemes squelettaire at
musculaire de Chimera Collei, Callorrhynchus antaret.,
Spinax niger, Protopterus, Ceratodus, et Axolotl.” ‘ Archives
des Science. médical de Bucarest,’ 4° Memoire, tome iv.

Johnston (02)—* An Attempt to Define the Primitive Functional
Divisions of the Central Nervous System,” ‘Journ. Comp. Neur.,’
vol. xii.

Kallius (97).—*‘ Beitrige zur Entwickelunesgeschichte des Kehlkopfes,”’
‘ Anat. Hefte,’ Bd. ix.

—— (01).—* Beitrige zur Entwicklung der Zunge: i Theil, ‘Amphi-
bien und Reptilien,’” ‘ Anat. Hefte,’ Bd. xvi, ii Theil, Bd. xxviii.

Kappers (’08).—** Weitere Mitteilungen beziiglich der phylogenetischen
Verlagerung der motorischen Hirnnervenkerne, Der Bau des
autonomen Systemes,” ‘ Folia Neuro-biologica.’

Kellicott (‘05).—* The Development of the Vascular and Respiratory
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Kerr, Graham ('07).—** The Development of Polypterus senegalus,’
The work of J. 8. Budgett.

Kingsbury and Reed (’09)—‘* The Collumella auris in Amphibia,”
‘ Journ. of Morph.,’ vol. xx.

Kosaka (°07).—‘* Ueber die Facialiskerne des Huhns” (in Japanese),
reference in ‘ Folia Neuro-biologica,’ January, 1908.

Kosaka and Yagita ((07).—* Ueber der Ursprung des Herzvagus” (in
Japanese), reference in ‘ Folia Neuro-biologica,’ January, 1908.

Krause (’68).—‘ Die Anatomie des Kaninchen.’

Marinesco (’98).—‘* De l’Origine du Facial superieur,” ‘ Revue Neuro-
logique.

—— (’99).—** Nouvelles recherches sur lorigine du facial superieur et
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MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 313

Marion (’05).—* Mandibular and Pharyngeal Muscles of Acanthias and
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Mivart (69).—** Notes on the Myology of Menobranchus lateralis,”
‘Proc. Zool. Soe.’

MeMurrich (°85).—** The Cranial Muscles of Amia Calva L., with a
Consideration of the Relations of the Post-Occipital and Hypo-
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Neal ('97)—‘“ The Development of the Hypoglossal Musculature in
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—— ('82).—**On the Development of the Skull in Lepidosteus,”’
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Pollard (’92).—** On the Anatomy and Phylogenetic Position of Polyp-
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Reuter (97).—" Ueber die Entwickelung der Kaumuskulatur beim

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(97).—* Ueber die Entwickelung der Augenmuskulatur beim

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——. (94).—** On the 'Hyoid Arch of Ceratodus,” ‘Proce. Zool. Soe.’

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314 F. H. EDGEWORTH.

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thiere,”’ ‘ Jen. Zeitschr., Bd. 45.

EXPLANATION OF REFERENCE LETTERS ON THE
TEXT-FIGURES.
abd. An. Abducens Anlage. An. of sup. obl. Anlage of obliquus
superior. abd. hyoid. M. abdomino-hyoideus. add. mand. M. adductor
mandibule. add. mand. ext. M. adductor mandibule externus. add.
mand. int. M. adductor mandibule internus. ant. dig. M. digastricus

MORPHOLOGY OF CRANIAL MUSCLES IN SOME VERTEBRATES. 315

anterior. arcualis dors. M. arcualis dorsalis. au. temp. n. Auriculo-
temporal nerve. br. add. M. adductor arcus branchialis. br. aor. ar.
Branchial aorticarch. br. bar. Branchial bar. 67. my. Branchial myo-
tome. branch. hyoid. M. branchio-hyoideus. buce. cav. Buccal cavity.
ceph. cel. Cephalic celom. cer. br. Cerato-branchial cartilage. cer. hy.
ang. M. cerato-hyoideus angularis. cer. hyal. ce. Cerato-hyal cartilage.
jirst cerv. n. First cervical nerve. cel. Ccelom. cons. coll7. M. constrictor
colli. cons. opere. M. constrictor operculi. cor. branch. M. coraco-
branchialis. cor. hyotd. M. coraco-hyoideus. cor. mand. M. coraco-man-
dibularis. ¢,vd. Dorso-ventral muscular sheet inhyoid segment. dil. lary.
M. dilatator laryngis. dilat. operc. M. dilator operculi. dor. aor. Dorsal
aorta. dorso-lary. M. dorso-laryngeus. epibi. Epibranchial cartilage. ext.
aud. meat. External auditory meatus. extira-temp. M. extra-temporalis.
Gass. g. Gasserian ganglion. gen. glossus. M. genio-glossus. gen. glossus
and lingualis an. Anlage of M. gerio-glossus and lingualis. gen. hyotd.
Genio-hyoid. gill. m. An. Anlage of muscles of external gill. g.-c. Gill-
cleft. hy. ceph. cel. Hyoid section of cephalic celom. hyogloss. M. hyo-
glossus. hyogloss. and stylogloss. An. Anlage of M. hyoglossus and M.
styloglossus. hyohy. inf. M. hyo-hyoideus inferior. Hyohy. sup. M. hyo-
hyoideus superior. hyotd bar. Hyoidbar. hyotd my. Myotome of hyoid
segment. hyotd aor. ar. Hyoid aortic arch. hyomax. M. hyomaxillaris.
hyomax. ig. Hyomaxillaris ligament. hyomand. ¢. Hyomandibular carti-
lage. hypobr. ce. Hypobranchial cartilage. hypohyal. Hypohyal cartilage.
hypobr. sp. m. An. Anlage of hypobranchial spinal muscles. inf. lab.
cart. Inferior labial cartilage. cnterarc. vent. M. interarcualis ventralis.
interbas. M. interbasalis. cuterhyal. Interhyal cartilage. interhyoid.
M. interhyoideus. intermand. M. intermandibularis. lary. Larynx.
lev. br. M. levator arcus branchialis. lev. hyoid. M. levator hyoidei.
lev. lab. sup. An. Anlage of M. levator labii superioris. lev. max. sup.
M. levator maxille superioris. lev. pal. and tens. pal. An. Anlage of
levator and tensor palatini. lingualis. M. lingualis. M. marg. M.
marginalis. mand. aor. ar. Mandibular aortic arch. mand. ceph. cel.
Mandibular section of cephalic celom. mand. lab. M. mandibulo-labialis.
mand. my. Myotome of mandibular segment. mand. seg. Mandibular
segment. mass. M. massetericus. mylolyoid i. Mylohyoid nerve. Me.
Meckel’s cartilage. mental n. Mental nerve. N. Olfactory epithelium.
nictat. m. An. Anlage of nictating muscles. oblig. dors. M. obliquus
dorsalis. oblig. inf. M. obliquus inferior. obiig. sup. M. obliquus
superior. obliq. vent. M. obliquus ventralis. c@soph. const. Constrictor
of esophagus. oper. fld. Opercular fold. orb. hyotd. M. orbito hyoideus.
pal. pr. of quad. Palatine process of quadrate. pal. quad. Palato-
quadrate. pal. quad. Me. palato-quadrato-mandibular arch. phar.
Pharynx. phar. br. Pharyngo-branchial cartilage. phar. clav. ext. M.
pharyngo-clavicularis externus. phar. clav. int. M. pharyngo-clavicularis

316 F. H. EDGEWORTH.

internus. phar. m. Pharyngeal muscles. platys. colli. Platysma colli.
platysma fae. Platysma faciei. platys. occip. Platysma occipitalis.
premand. An. Anlage of premandibular muscles. post. diq., stylohy. and
stap. An. Anlage of posterior digastric stylohyoid and stapedius muscles.
proc. asc. Processus ascendens of qiadrate. proc. bas. Processus basalis
of quadrate. protr. hyom. M. protractor hyomandibularis. ptery. M.
pterygoideus. quad. Quadrate. quad. ang. M. quadrato-angularis. rec.
lary. n. Recurrent laryngeal nerve. rect. ewt. M. rectus externus. rect.
inf. M. rectus inferior. rect. int. M. rectus interior. rect. sup. M. rectus
superior. vetr. arc. br. M. retractor arcuum branchialium. ret. bulb.
M. retractor bulbi. retr. hyom. M. retractor hyomandibularis. — retr.
hyom. et operc. M. retractor hyomandibularis et opercularis. scap.
Seapula. sh. girdle. Shoulder girdle. spl. meso. Splanchnic mesoderm.
st. mast. M. sterno-mastoideus. sterno-hyoid. M. sterno-hyoideus. stylo-
gloss. M. styloglossus. stylophary. M.stylopharyngeus. submawx. M. sub-
maxillaris. submaw. g. submaxillary gland. swbment. M. submentalis.
suborb. c. Suborbital cartilage. subtemp. M. subtemporalis. susp. ang.
M. suspensorio-angularis. temp. M. temporalis. temp. and mass. An.
Anlage of M. temporalis external pterygoid masseter. tensor tymp. M.
tensor tympani. trach. Trachea. trap. M. trapezius. tr. my. Trunk
myotome. trans. vent. M. transversus ventralis. vent. aor. Ventral
aorta. Roman numerals. Cranial nerves.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 3817

A Monograph of the Tape-worms of the Sub-
family Avitellinine, being a Revision of the
Genus Stilesia, and an Account of the His-
tology of Avitellina centripunctata (Riv.).

By
Lewis Henry Gough, Ph.D.,

From the Zoological Laboratories of the Universities of Basel, Switzer-
land, and of Leeds; England.

With Plates 12-14 and 6 Text-figures.

‘He following paper was commenced at Leeds; it was
originally invended only to give an account of the anatomy
of Stilesia hepatica, Wollffhiigel, which has been very
imperfectly known until now, as much of the original
description is not only incorrect but actually misleading.
That section of this paper treating of Stilesia hepatica,
Wolffhiigel, was prepared at Leeds. I am much indebted to
Prof. Garstang for his hospitality in placing his laboratory
at my disposal and for the encouragement I received from him,
and desire to express my thanks to him for it here.

As I was spending the winter at Basel, Switzerland,
Prof. Zschokke kindly offered me a table in his laboratory,
and suggested extending the scope of the paper I had com-
menced at Leeds so as to cover all the known species of the
genus Stilesia; he also helped me to bring together the
material required in order to make the paper complete. Its
scope was again extended to include an account of the
histology of Avitellina (Tenia) centripunctata (Riv.),
on account of several histological peculiarities of the worm,
which seem to throw a new light on the problems connected

318 LEWIS HENRY GOUGH.

with the structure of the Cestodes, and because my material
was ina much better state of preservation than is usually seen.
Excepting the account of the histology and anatomy of
Stilesia hepatica, Wolffhiigel, the rest of this paper has
been prepared at Basel.

I feel great pleasure in expressing my thanks to Prof.
Zschokke for his hospitality and assistance.

My thanks are also due to Prof. Railhet, of Alfort, who
placed not only the original material of Stilesia vittata,
Railliet, at my disposal, but also more recent specimens of
both that species and of Stilesia globipunctata (Rivolta).
I must also thank Prof. Colin, of the Natural History Museum
of Berlin, for the loan of the type-specimens of Stilesia
hepatica, Wolffhiigel, and Prof. Fuhrmann, of Neuchatel,
for having kindly re-examined his specimens of Stilesia
sjOstedti at my request and for the loan of his type-
specimens, thus enabling me to fix its true systematic
position, and for procuring material of Dibothriocephalus
and 'Trizenophorus for me.

To my friend Dr. O. Huber I am indebted for the delinea-
tion of figs. 5, 6, 7, and 12, and desire to express my thanks
to him here.

I have divided this paper into two chapters. ‘The first
deals with the systematic revision of the genus Stilesia;
the second is an account of the histology of Avitellina
centripunctata (Riv.).

Revision oF THE GENUS SriLEstA, RAILuier.

The material employed in connection with this revision of
the genus is derived from the following sources :

Stilesia globipunctata (Rivolta).
(1) From the small intestine of a goat, collected in British
India by Leese, from Prof. Railliet’s collection (No. P196°).
(2) From the small intestine of a sheep, collected in
France, from Prof. Railliet’s collection (No. P191).

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 319

Stilesia vittata, Raiulliet.

(1) From the small intestines of a dromedary, collected at
Alfort, April 22nd, 1896, from Prof. Railliet’s collection
(No. F196). Type.

(2) From the small intestines of a dromedary, collected
at Alfort, May 27th, 1906, from Prof. Railliet’s collection
(No. P192).

(5) From the small intestine of a dromedary, collected in
British India by Leese, 1909, from Prof. Railliet’s collection
Nor P7135”):

Stilesia hepatica, Wolfthiigel.

(1) From the bile-ducts of sheep and goats, collected in
German Kast Africa, belonging to the Natural History
Museum, Berlin. ‘Type.

(2) From the bile-ducts of sheep, collected in the Lyden-
burg District, Transvaal, belonging to the Natural History
Museum, Berlin. Co-type.

(3) From the bile-ducts of sheep, collected at Pretoria,
Transvaal, 1909 (author’s collection).

Stilesia Sjéstedti, Fuhrmann.
(1) From the bile-ducts of Hippotragus equinus, col-
lected in North-east Rhodesia (author’s collection).
(2) From Tragelaphus silvaticus mernensis, collected
by the Sjostedt expedition (Fuhrmann’s type).

Avitellina centripunctata (Rivolta).

(1) From the small intestines of sheep, collected at Pretoria,
Transvaal, 1909 (author’s collection).

History oF tHE GENUS S1ILesta.

The genus Stilesia was proposed by Railliet (1893) to
include two species of tape-worm from the small intestines
of sheep, which had been described by Rivolta in 1874 as
Tenia globipunctata and Tenia centripunctata.

320 LEWIS HENRY GOUGH.

‘he best description of these two species available hitherto
was by Stiles (1893), who also revised the generic diagnosis,
basing his revision on the then known data, though evidently
not feeling quite sure as to the desirability of leaving both
species in one genus.

In 1896 Railliet described a new species, Stilesia vittata,
from the intestines of a dromedary ; he considered this species
to be very closely allied to Stilesia globipunctata (Riv.),
and, perhaps, only to be a variety.

In 1903 another new species, closely related to Stilesia
globipunctata (Riv.), was described by Wolffhiigel, from the
bile-ducts of sheep and goats in South and Kast Africa, as
Stilesia hepatica.

In 1906 Tempére briefly refers to Stilesia centripunc-
tata and figures its scolex, apparently only quoting from
Railliet (1893) and Neumann (1893).

In 1908 Gough states briefly that Stilesia hepatica,
Wolfthiigel, is usually not double-pored.

In 1909 Fuhrmann places Stilesia and Thysanosomain
a vew sub-family, the Thysanosomine.

In 1909 Fuhrmann describes a new species from Trage-
laphus sylvaticus mernensis, collected by Dr. Sjéstedt
on the Masai steppes, as Stilesia sjéstedti.

In 1909 Gough gives a full description of the anatomy of
Stilesia centripunctata (Rivolta), with remarks on
Stilesia hepatica, Wolffhiigel.

At present, therefore, the genus contains the following five
species :

Stilesia centripunctata (Rivolta), 1874; Stilesia
globipunctata (Rivolta), 1874; Stilesia vittata, Railliet,
1896; Stilesia hepatica, Wolffhiigel, 1903; Stilesia
sjOstedti, Fuhrmann, 1909.

The last four species agree very closely in their anatomy ;
the first differs from all the others in several important
respects of generic value. A new genus will therefore have
to be proposed for Stilesia centripunctata.

Stilesia globipunctata (Riv). is the type species of the

TAPE-WORMS OF THE SUB-FAMILY AVITELLININ”E. 321

genus Stilesia; this species and Stilesia vittata, Railliet,
were described as having irregularly alternating genital
pores. Stilesia hepatica, Wolffhigel, and Stilesia
sjOstedti, Fuhrmann, have been described as double-pored ;
they do not, however, differ from the type species in this
respect, all four being without doubt single-pored.

The generic diagnosis, as revised by Stiles (1895), reads :

“Stilesia, Railliet, 1893. Type species, S. g)obipunce-
tata (Riv.), Railliet, 18938. Head with four suckers, but no
hooks. Strobila thin and narrow. Genital pores irregularly
alternate. Segments broader than long. ‘I'wo distinct sets
of testicles present in each segment, one on each side, but no
testicles in the median line. Eggs very small and with but
one shell.

“The following points, which may prove to be of generic
value, have been established only for 8S. globipunctata:
Genital canals pass dorsally of nerve and ventral canal, but
ventrally of dorsal canal. Hgg-shell with two conical pro-
jections at opposite poles.

‘“ Habitat: Intestine of sheep. Development unknown.”

The generic description can now be amplified to some
extent and also altered in some respects.

Stilesia, Railhet, 1893. Type species, Stylesia globi-
punctata (Rivolta), Railliet, 1893. Head with four suckers,
but without hooks. Strobila thinand narrow. Genital pores
irregularly alternate. Segments broader than long. — 'I'wo
distinct sets of testicles present in each segment, one on each
side, but no testicles in the median line. Ovarium on the
pore side. No vitelline gland, no shell-gland. Uterus
double, finally void of eggs, which are contained in egg-
pouches (paruterine organ). ‘he genital canals pass dorsally
of the nerve and of the ventral canal, and ventrally of the
dorsal canal. Kegs with two envelopes. Habitat: Intestine
of sheep, goat, and dromedary, and bile-ducts of sheep, goat,
and South African wild antelopes (Africa, India, Italy,
France).

In the genus as thus restricted, only St. globipunctata

Si yre LEWIS HENRY GOUGH.

(Riv.), vittata, Railliet, hepatica, Wolffhiigel, and
sjéstedti, Fuhrmann, remain. For Tenia centripunce-
tata, Rivolta, a new genus must be erected, for which I
propose the name Avitellina (to denote the absence of a
vitelline gland).

Avitellina, nov. gen. ‘l'ypespecies, Avitellina centr1-
punctata (Rivolta). Head with four suckers, but without
hooks. Strobila thin and narrow. Segments broader than long,
flat in the proximal portion of the strobila, nearly cylindrical
in the posterior portion. Genital pores irregularly alternate.
Four distinct sets of testicles in each segment, one right and
one left of each longitudinal canal, but no testicles in the
middle field. Ovarium nearer the pore side; no vitelline
gland, no shell gland; a singleuterus. LHggs finally enclosed
in ege-pouches (paruterine organ). ‘The genital canals pass
dorsally of the nerve and longitudinal canals. Hees with two
envelopes. Habitat: Intestine of sheep, Africa, Italy.

The genera and the hitherto described species of Stilesia
and Avitellina can be recognised by the following key :

(1) Uterus single; a single paruterine organ; testicles in
four groups; the genital canals pass dorsally of the dorsal
canal, Avitellina, 4.

Uterus double; two paruterine organs; testicles in two
groups ; the genital canals pass ventrally of the dorsal canal.

Stilesia, 2.

(2) Testicles all lateral to the ventral canal. 3.

Testicles mostly median or dorsal to the ventral canal.

St. hepatica, Wolfthiigel.

(5) The vas deferens forms a dense packet of convolutions
(functionally a vesicula seminalis) between nerve and ventral
canal before reaching the cirrus pouch; inhabits the small
intestines of the dromedary. St. vittata, Railliet.

The vas deferens forms at the most three or four loose
convolutions between the nerve and the ventral canal before
reaching the cirrus pouch; inhabits the intestines of sheep
and goat. St. globipunctata (Rivolta).

(4) The vas deferens runs its entire length dorsal to the

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 323

testicles; length two to three metres ; inhabits the intestine
of sheep. Only known species: A. centripunctata

(Rivolta).

Srinesta HEPATICA, WoLLFHUGEL, 1903. Figs. 14-16;
Text-fig. 1.
Synonomy.

Stilesia hepatica, Wolffhiigel, 1903.

Stilesia hepatica, Wolffhiigel, Gough, 1908.

Stilesia sjostedti, Fuhrmann, 1909.

Literature.

Wolffhiigel—* Stilesia hepatica nov. spec. ein Bandwurm aus
den Gallengingen von Schafen und Ziegen Ostafrikas,” ‘ Berliner
Tierirzlichen Zeitschrift,’ 1903, No. 43.

Gough.—* Notes on South African Parasites,” *S.A.A.A.8.,° Grahams-
town, 1908.

Gough.—* Tne Anatomy of Stilesia centripunctata (Rivolta),”
‘The Veterinary Bacteriological Laboratories of the Transvaal,’ Pre-
toria, 1909.

Fuhrmann.—* Cestodes,” ‘Schwedische Expedition nach dem Kili-
mandjaro,’ 1909.

Habitat.—Bile-ducts of sheep, goats, and wild ruminants
in South, East, and Central Africa.

[Note.—Although Stilesia hepatica, Wolfthiigel, is not
the type species of the genus, I propose to consider it first,
as its anatomy is very much better known than that of
Stilesia globipunctata (Rivolta), the type species; the
anatomy of all known species of Stilesia is, as far as yet
worked out, very constant, only differing in minor points. As
a description at full length is necessary only for one of the
species, only the points in which the other three differ will be
found under their respective headings. |

Stilesia hepatica was described in 1905 by Wolffhiigel
as being double-pored, and as differing chiefly in that respect
from Stilesia globipunctata (Rivolta).

When working in the Transvaal I repeatedly had to deal
with a Stilesia infesting the bile-ducts of sheep, which I
identified with Stilesia hepatica, Wolfthiigel, although

324 LEWIS HENRY GOUGH.

all the specimens that passed through my hands were invari-
ably single-pored. In 1908, in a paper read before the South
African Association for the Advancement of Science, at
Grahamstown, C.C., I stated that Stilesia hepatica, Wolff-
hiigel, was single-pored, and that the original description
given by the author was at fault. Since then, by the kind-
ness of Prof. Colin, I have been able to examine the type
specimens of Stilesia hepatica, Wolffhtigel. There is no
possible doubt ; the type specimens are certainly single-pored,
with irregularly alternating pores. The anatomy of the worm
differs considerably also in other respects from the data given
by Wolffhiigel. Jn the following the anatomy of the worm
is given entirely on my own observations on fresh material,
supplemented by re-examination of the type.

The worm invariably inhabits the bile-ducts, never the
intestine. It occurs in sheep, goats, duiker (Cephalopus),
roan antelope (Hippotragus equinus), Hippotragus
sylvaticus mernensis, fide Fuhrmann, and various other
wild ruminants occurring in South, Kast, and Central Africa.
The scolex is almost invariably lodged in the peripheral
capillaries of the bile-ducts. The parasites are often present
in large numbers, dilating the bile-ducts; their presence
does not cause calcification of the ducts, as Distomum
hepaticum, L., does, but only a thickening of the tissues
of the ducts. They appear to do otherwise but little injury
to the host; almost all adult sheep in the Transvaal are
affected.

Stilesia hepatica, Wolffhiigel, is probably primarily
parasitic in wild ruminants, and can be supposed to have
adapted itself secondarily to sheep. The absence of
records of the conspicuous parasite from other parts of the
world, its occurrence in the wild antelopes, which are so
characteristic of the Kthiopian region, and the wide range
in its choice of hosts, would seem to speak for the probability
of its not being originally a parasite of sheep.

Stilesia hepatica, Wolffhiigel, is extremely contractile,
more so than most other cestodes I have handled hitherto.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 325

As one very rarely succeeds in extracting a worm entire
its total length is very difficult to estimate, but it is probably
between twenty and fifty centimetres. In life, when expanded,
it is thin, gelatinous in appearance, semi-transparent, the
edges of the strobila being serrated on account of the pro-
jection of the posterior angles of the segments. Against a
black surface, the middle field appears clear, the lateral fields
more or less opaque. In older segments in the posterior
portion of the strobila, the uteri and paruterine organs show
up as an opaque spot on each side of each proglottid; when
contracted, the worm is thick, with frilled edges, and more or
less opaque.

The scolex has four suckers, directed outwards and
forwards. Very frequently the head is followed by what
appears to be a thick “ neck,” 2mm. in length, as broad as,
or even slightly broader, than the scolex ; behind this “ neck ”
the strobila suddenly narrows to half the width. Hxami-
nations of the “neck” (in sections), however, reveals the
fact that it is composed of young segments, and consequently
belongs to the strobila and not to the scolex. The contrac-
tion of the first two millimetres of the strobila is of extremely
regular occurrence, so much so as to cause remark, when one,
as occasionally happens, comes across a worm not contracted
in this way. As the scolex is usually lodged in a capillary
of the bile-duct the swelling of the anterior portion of the
strobila can be of use to the worm as an aid to the suckers,
helping to anchor the worm by gripping the sides of the
duct. Wolffhiigel figures.a scolex in his paper, which he
states may belong to Stilesia hepatica; although the
scolex in question is not followed by the contraction of the
anterior portion of the strobila, I see no reason to doubt
its belonging to this species.

The swelling of the portion of the strobila directly posterior
to the scolex in Stilesia hepatica, Wolffhiigel, on
account of its probable function, can probably be compared
to the pseudo-scolices of Idiogenes and Fimbriaria, and
be considered as representing the first step towards the

326 LEWIS HENRY GOUGH.

acquisition of a psendo-scolex. A fundamental difference
is, however, that in Stilesia all segments must have passed
through the pseudo-neck during the course of their develop-
ment, whereas it is usually accepted that a true pseudo-scolex
is formed after the fertile segments have been produced, and
that the segments composing a pseudo-scolex remain sterile.
The habit of contracting the youngest segments appears to
be an old acquisition in the genus Stilesia; a scolex of
Stilesia globipunctata (Rivolta), is illustrated in fig. 12,
showing a similar contraction of the anterior portion of the
strobila, though in a less degree.

The segments are much shorter than wide, and about twice
as wide as thick. The width of the strobila varies from one
to two or three millimetres. The posterior segments are
longer than the anterior. The posterior margin of each
segment surrounds, collar-lke, the anterior end of the
following, except at the middle of the segment. Segmenta-
tion is quite distinct (without sectioning) at 2°8 mm. from
the scolex ; the genital anlagen appear already at 9 mm.

The genital pores open near the middle of the lateral
margin of the segment; they are single and irregularly
alternate.

The cuticula does not appear smooth (as that of Avitel-
lina centripunctata [Rivolta]), but is villous (in sections).

The longitudinal canals are both well developed. The
lumen of the dorsal canal does not become obliterated. The
ventral canal is situated lateral and ventral to the dorsal
canal. At the posterior end of each segment transverse
canals connect the ventral canals, forming a transverse ring,
the dorsal and ventral branches forming a few anastomoses
near the middle of the sezment. The transverse canals arise
from more than two, usually three or four openings in the
ventral canal on its median side, and two or three lateral to
the ventral canal; these last usually meet and form a lateral
loop. The histology of the transverse canals is the same as
of the ventral canal. They both have a thin membrane,
produced by flat epithelial cells, surrounding the canal ; these

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 327

cells in turn are in contact with the parenchyma, as described
lower down for the. excretory canals in the scolex of
Avitellina centripunctata (Rivolta).

The dorsal canals do not appear to be connected by trans-
verse commissures, nor to be connected to the ventral canals
or their transverse commissures.

The lumen of the ventral canals measures up to 58” by
504; the dorsal canals are about 304 by 25 in diameter.

According to the state of contraction, the course of the canals
can vary from being nearly rectilinear to very closely spiral.

Text-Fic. 1.

dc vd

Diagram of Stilesia hepatica. (For explanation of letters see list
of abbreviations at end of paper.)

Calcareous corpuscles are found in the medullary substance
of the scolex and at the posterior ends of the segments; their
diameter is 10 un.

The muscles of the strobila are not very strongly developed.
The longitudinal muscles are in two layers, the inner consist-
ing of bundles of about twelve, the outer of three or four
muscles. The transverse muscle is weak, as is also the dorso-
ventral muscle.

The sexuai organs differ greatly from Wolffhiigel’s descrip-
tion. In the first place each segment (of the type also!) has

VoL. 06, PART 2.—NEW SERIES. 23

328 LEWIS HENRY GOUGH.

but one pore, the pores being irregularly alternate. There is
also only a single ovary to each segment, not two; and finally
the arrangement of the testicles and vas deferens is quite
different from what Wolffhiigel described.

There are ten to twelve testicles on each side of the segment
(fig. 13 and Text-fig. 1) ; they lie dorsally between the ventral
and the dorsal canals, and dorsal to the ventral canal. Seen
from the dorsal side of a total mount, the testicles le in two
or three rows of about four or five (sometimes six) in a row.
On transverse sections (Text-fig. 1), one only sees a single row.
The diameter of a testicle is about 50 to 55. The vasa
efferentia arise on the dorsal side of the testicles, as do the
vasa deferentia. ‘he vas deferens of the testicles on the
right side of the proglottid runs from right to left, the vas
deferens collecting from the left group of testicles from left
to right; at the middle of the proglottid the left and mght
branches meet and join to form a common vas deferens which
bends ventrally, and, having passed into the depth of the
proglottid past the testicles, turns towards the pore side of the
seoment. It passes the dorsal canal ventrally, the ventral
canal and nerve dorsally. Before reaching the cirrus pouch
it forms a number of twists, whose function is that of a vesi-
cula seminalis; these lie above the ventral canal. The cirrus
pouch (fig. 16) is oblong, measuring 83 yu by 50 pw, the diameter
of the cirrus 165. Cirrus and vagina open into a wide and
deep genital cloaca, whose aperture is situated near the
middle of the segment.

The female organs also differ considerably from Wolff-
hiigel’s statements (fig. 15). There is but one ovarium, lying
on the pore side, between the ventral and dorsal canals,
nearer to the ventral than to the dorsal canal. ‘lhe uterus
is double, one uterus lying close to the ovarium, the other on
the other side of the proglottid in the corresponding position.
The two uteri are connected by a duct, the inter-uterine duct,
which, however, may be morphologically but the median
portion of the uterus. This duct crosses the ventral field
ventral to the dorsal canal. ‘The ovarium contains very few, at

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 329

the most fifty, eggs, measuring 14 4 in diameter; it atrophies
very rapidly after the appearance of the uterus. (Woiffhiigel’s
figure only shows the uteri, which have been erroneously
labelled ovarium by him.) ‘The ovarium measures 86 in
diameter.

The oviduct, the uterine duct and the canalis seminalis
meet a short distance from the receptaculum seminis, as in
Avitellina. There is no vitelline gland, nor shell-gland.
The function of the missing yolk-cells is exercised by abortive
eggs in the ovarium (ovarial nutritive cells, see p. 371), and
by cells derived from the uterine walls (uterine nutritive cells,
see p. 375). The eggs in the uterus are firmly embedded in the
uterine nutritive cells, as has also been observed by Fuhrmann
(1909), who already suggested that their function is probably
nutritive ; however, contrary to his supposition, the uterus is
originally hollow. The uterus measures 50 to 86; the eggs
are finally enclosed in paruterine organs similar to the par-
uterine organ described as occurring in Avitellina (see p.
375). The paruterine organ arises within the uterus ; each seg-
ment contains two paruterine organs, one within each uterus.
They measure 504 to 86 yu. ‘The uteri and later on the par-
uterine organs are connected by a band of fibrous tissue, which
covers the uterus anteriorly, passes through into the median
field and tapers off towards the middle of the segment, the two
halves of the band meeting inthe middle. heir course across
the segment is not quite direct, the middle portion drooping
towards the posterior end of the proglottid. The eggs are
enclosed in two envelopes, the outer of which invariably
appears wrinkled whilst the inner is always smooth and
rounded. ‘lhe inner envelope seems to possess a prolongation
at each pole (perhaps due to optical delusion and not existent ;
it is almost impossible to get rid of the outer envelope so as
to examine the inner properly). The eggs, measured over
the outer shell, are 26 4 long by 16 to 19 uw broad, the embryo
15 to 16m.

Wolffhiigel states the size of the eges as 26 x 164,
Fahrmann as 16 (evidently only the embryo) !

330 LEWIS HENRY GOUGH.

Caleareous bodies are frequent in this species, as also in
Stilesia globipunctata (Rivolta), and Stilesia vittata,
Railliet; they measure on an average 10, in diameter, and
are most frequent in the axis of the scolex, and at the posterior
end of the segments. No calcareous corpuscles were observed
in the type of Stilesia sjostedti, Wolffhiigel, but here, as
elsewhere, this is probably only due to individual variation.

STILESIA GLOBIPuUNCTATA (Rrvotta), 1874. Figs. 10, 11, 12.

Synonymy.

Tenia globipunctata, Rivolta, 1874.

Tenia ovipunctata, Rivolta, 1874.

Stilesia globipunctata (Rivolta), Railliet, 1893.
Stilesia globipunctata (Rivolta), Stiles, 1893.

Literature.

Rivolta.— Sopra alcune specie di Tenia della Pecora,’ Pisa, 1874.

Perroncito.—‘ I Parassiti dell’ Uomo e degli Animali Utili,” Milano,
1882.

Perroncito.— Trattato teorico-pratico sulle malattie piu communi
degli Animali domestici,” Torino, L886.

Railliet— Elements de Zoologie Médicale et Agricole,’ Paris, 1886.

Neumann.— Traité des Maladies parasitaires non-microbiennes des
Animaux domestiques,” Paris, 1888 (Ist edition).

Neumann.—* Observations sur les Ténias du Mouton,” ‘C. R. Soe.
Hist. Nat., Toulouse,’ 1891.

Neumann.— Traité,’ 2nd edit., 1892.

Stiles —* Bemerkungen iiber Parasiten 17. Uber die topographische
Anatomie des Gefiiss-systems in der Familie Tw niade,” * Centralblatt
fiir Parasitologie, 1895.

Stiles— Adult Cestodes of Cattle, Sheep. and allied Animals,’
Washington, 1893.

Railliet.— Elements, 2nd edit.

Railliet—* Sur quelques parasites du Dromadaire,” *C. R. Soe. Biol.,”
1896.

Perroncito.— Trattato teorico pratico,’ 2nd edit., 1902.

Wolffhiigel —“Stilesia hepatica nov. spec. ein Bandwurm aus
den Gallengingen von Schafen und Ziegen Ostafrikas,” * Berliner
Tierirzlichen Wochenschr., 1905, No. 45.

Fuhrmann.—* Cestodes,” * Wissenschaftliche Ergebnisse der Schwe-

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 331

dischen zoologischen Expedition nach dem Kilimandjaro. dem Meru
und den Umgebenden Masaisteppen deutsch Ostafrikas,’ Stockholm,
1909. :

Habitat.—Small intestine of sheep and goats. (Linstow’s
record from cattle in ‘Compendium der Helminthologie’ is <
printer’s error, as he is there citing Rivolta, who described
the worm from sheep.

Geographical Distribution.—Italy, Rivolta, 1874; India
(Giles fide Stiles, 1903—also specimens in Railliet’s collection
examined by the present author) ; France, Railliet, 1896.

For the following description I have had to rely consider-
ably on Stiles (1895), the material at my disposal being
rather badly macerated.

The worms are stated to be transparent, gelatinous in
appearance when living, resembling Stilesia hepatica,
Wolffhiigel, in this respect.

The length varies from 45 to 60 em., Railliet, 1896. The
widest segments are 2°5 m.m. broad, the anterior and posterior
being much narrower.

The scolex is square when viewed en face; it measures
0-768-0°9 mm. in diameter. ‘lhe suckers (fig. 12) are
directed anteriorly and diagonally ; their opening is round or
oval. The anterior portion of the strobila is sometimes con-
tracted, as is more frequently the case in Stilesia hepatica,
W olffhiigel.

The proglottids are always much broader than long, but in
the posterior portion of the strobila are comparatively longer
than in the middle of the worm. ‘The middle portion is very
frequently much contracted, the outline becoming crenate
and twisted.

There are four to seven testicles on each side, lying lateral
to the ventral canal, median to the nerve (fig. 10). The
ovarium lies on the pore side, just median to the ventral canal ;
the uterus is double, one lying dorsal to the ovarium, the
other close to the ventral canal of the other side of the body.
The vagina lies dorsal to the cirrus pouch; it crosses the
ventral canal dorsally ; median to the ventral canal it increases

ape LEWIS HENRY GOUGH.

in size and forms a receptaculum seminis. ‘The median end
of the receptaculum seminis forms two branches, one of which,
the oviduct, goes to the ovarium, the other, the uterine duct,
goes to the uterus. (Do these two ducts arise directly from
the receptaculum or from a canalis seminalis as in the other
members of the group?) The uteri of both sides are probably
connected as in St. hepatica, Wolffhiigel, by an interuterine
duct. The cirrus pouch is pyriform, 56 4 long by 40 « broad,
the cirrus 50 to 60 « long. Cirrus and vagina open into a
large and wide cloaca, which is directed diagonally lateral
and forwards, opening near the anterior angle of the segment.
Stiles observed the vas deferens to run from the cirrus-pouch
anteriorly of the testicles of the pore side, dorsally of the
ventral canal and female organs, ventrally of the dorsal canal,
then through the median field, lying anterior and dorsal to
the transverse canal; it is further stated to cross the dorsal
canal (of the opposite side) ventrally, the ventral canal
dorsally, and to be finally lost in the testicles. Should this last
be correct, it would be a totally different course to that of
the vas deferens in Stilesia hepatica, Wolffhiigel ; fresh
material will have to decide this point.

The ovary contains but few eggs; there is uo vitelline
eland, and no shell-gland. ‘The eggs enter the uterus ferti-
lised; in the uterus they are surrounded by nutritive cells,
as in St. hepatica, Wolffhiigel. The eggs are finally
enclosed in a paruterine organ. ‘They have two envelopes,
an outer wrinkled fusiform and an oval inner one; the inner
one is devoid of spines (fig. 11). The spines in Stiles’s
figure are probably the shrivelled outer envelope. The eggs
measure 56 ux 27 « over the outer, 27 wx 22 4 over the inner
envelope, 144 across the embryo.

The uteri of both sides, and later on the paruterine
organs, are in contact with a band of tibrous tissue, which
“extends partially around the uterus, crosses the dorsal canal
ventrally, and tapers off into a fine point, which runs through
the median field to meet,” and is continuous with, the corre-
sponding organ of the other side (Stiles, 1893, p. 78).

TAPE-WORMS OF THE SUB-FAMILY:AVITELLININE. 333

STILESIA vittaTa, Raiwiet, 1896. Figs. 8, 9.

Synonymy.
Stilesia vittata, Railliet, 1896.

Literature.
Railliet.—* Sur quelques parasites du Dromadaire,” * C. R. Soc. Biol.,’
1896, p. 491.

Habitat.—Small intestine of dromedary.

Geographical Distribution.—India. (Algiers? The
type was collected in Alfort in a dromedary that died there,
and a second batch was collected at the same place about
two weeks after the first.)

Stilesia vittata, Railliet, so closely resembles Stilesia
globipunctata (Rivolta), that Railliet, after describing the
species, states that it may be only a variety of Stilesia
globipunctata (Rivolta). However, certain constant
differences can be found, if one may rely on Stiles’s descrip-
tion of Stilesia globipunctata (Rivolta), and there
appears to me to be no reason to doubt the correctness of
that most accurate observer.

The worm has the same appearance (judging from for-
malin material) as Stilesia hepatica, Wolffhiigel, and as
Stilesia globipunctata (Rivolta). Its length is stated as
being 18 to 23.cm., its breadth Lmm.to 15mm. In shape
the scolex is similar to that of Stilesia globipunctata
(Rivolta) ; however, when viewed en face the breadth (latero-
lateral measurement) is somewhat greater than its thickness
(dorso-ventral measurement). Its length is shorter than the
breadth or thickness.

Three scolices measured were :

Broad . (1) 060mm. . (2) 054mm... (5) 0°55 mm.
Thick . (1) 0°525mm. . (2) 048mm. . (3) 048 mm.
Long = (Ll) 055mm... (2) O875 mm. . (3) 0:48 mm:

The shape of the proglottids is similar to that otf the other
two species, the posterior border of each segment overlapping
the anterior end of the next proglottid in the same way.

304 LEWIS HENRY GOUGH.

There are five to seven testicles on each side, lying lateral
to the ventral canal. ‘I'he entire course of the vas deferens
has not been made out, but the outer half of it runs ventral
to the dorsal canal, and dorsal to the ventral canal and
nerve. Between ventral canal and nerve the vas deferens
forms a number of very close and densely packed convolu-
tions, whose function is without doubt that of a vesicula
seminalis (fig. 8, v.s.). In a “ teased” specimen this packet
of convolutions comes away entire. It appears almost to be
enclosed in a membrane, but the material was too macerated
to make quite sure.

The cirrus pouch measures 75 in length; it opens into a
genital cloaca, which is directed laterally and anteriorly, and
opens near the anterior angle of the proglottid.

The position of the female organs and their arrangement
appears to be the same as in Stilesia globipunctata
(Rivolta), the band of fibrous tissue between the uteriis, how-
ever, somewhat more strongly developed.

The muscles are arranged in two layers, the inner being com-
posed of bundles of five to nine, the outer of only one to three.

The eggs have two envelopes, an outer shrivelled one and
an inner oval or rounded one. They measure 38 u x 24 4 over
the outer, 22 « over the inner envelope, the embryo measuring
about 14 (fig. 9). (Railliet’s measurements were 14-17 w x
15-17 4.) The inner envelope is not provided with spines of

any kind.

AVITELLINA CENTRIPUNCTATA (Rrvotra), 1874. Figs. 1 to 9,
17 to 55, 37 to 65. 'Text-fig. 2.
Synonymy.
Tenia centripunctata, Rivolta, 1874.
Stilesia centripunctata (Rivolta), Railliet, 1895.
Stilesia centripunctata (Rivolta), Stiles, 1893.
Stilesia centripunctata (Rivolta), Gough, 1909
Literature.
Rivolta.—‘ Sopra alcune specie di Tznia della Pecora,’ Pisa, 1874.
Perroncito.— I Parassiti del’uomo e degli Animali Utili,’ Milano,
1882.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 9339

Perroncito.— Trattato teorico-pratico sulle malattie pin communi
degli Animali domestici.” Torino, 1886.

Railliet.—* Elements de Zoologie Médicale et Agricole, Paris, 1886
(1st edition).

Neumann.—’ Traité des Maladies parasitaires non-microbiennes des
Animaux domestiques, Paris, 1888 (1st edition).
Neumann.—* Observations sur les Ténias du Mouton,” ‘ C. R. Soe.

Hist. Nat., Toulouse, 1891.

Neumann.— Traité,’ 1892 (2nd edition).

Railliet.— Elements.’ 1893 (2nd edition).

Perroncito.— Trattato teorico-pratico, 1902 (2nd edition).

Témpere.—* Parasites internes de homme et des Animaux domes-
tiques,” ‘ Micrographe Preparateur,’ vol. xiv, 1906, p. 27.

Gough.—* Notes on some South African Parasites,” *S.A.A.A.8.,’
Grahamstown, 1908.

Gough.—* The Anatomy of Stilesia centripunctata (Rivolta).”
‘The Veterinary Bacteriological Laboratories of the Transvaal, Pre-
toria, 1909.

Habitat.—Small intestine of sheep.

Geographical Distribution.—lItaly (Rivolta, 1874) ;
Algiers (Railliet, 1891); South Africa (Gough, 1908).

In life the worm has a gelatinous, semi-transparent appear-
ance. ~The strobila from 10 cm. from the scolex on appears
longitudinally ; there is a median opaque line, flanked on
either side by a very transparent line (caused by the enormous
ventral canals) ; laterally on each side the worm is again some-
what Jess transparent.

Avitellina centripunctata (Rivolta) reaches 202 cm.
to 285cm. in length. The greatest breadth is frequently,
but not always, near the scolex. The breadth varies from
1mm. to 3mm. (or even 4mm., Railliet). The anterior and
middle of the strobila is flat, the posterior end is round or
elliptical on section.

The scolex is large (fig. 7); in my specimens the suckers
are invariably directed diagonally outwards and forwards.
Railliet and Tempére, however, figure it with the suckers
directed anteriorly. ‘The scolex is usually, but not always,
broader than long; it measures from 1°5 mm. to 2°8 mm. broad

oOo ,
by 1:5 mm. to 3:1 mm. long.

336 LEWIS HENRY GOUGH.

The segments are always much broader than long, and
usually also much broader than thick (except in the posterior
portion of the strobila). The extreme brevity of the segments
causes the genitalia at male maturity all to lie in one plane,
single transverse sections 44 thick then often presenting the
whole anatomy, as in a diagram. When the paruterine
organ develops in the terminal portion of the strobila, the
anterior and posterior surfaces of the segments are no longer
flat, but are arched above the paruterine organ, bulging
thus into the segments nearest in front and behind, and
receiving depressions from the pressure of the paruterine
organs of the segments anterior and posterior to it (fig. 3).
Kxcept at the posterior end of the strobila, the hind end of a
proglottid does not surround the anterior end of the next.

The genital pores alternate irregularly ; they are very
slightly developed as compared to those of the Stilesiz.

Calcareous corpuscles are extremely rare; two only have
been observed in over one hundred series of sections.

The longitudinal muscles are apparently arranged in
bundles of twenty-four or more, and a few solitary muscles
are seen close to the subcuticula. The “ bundles” are, how-
ever, not distinct on horizontal sections (see p. 352). The
transverse and the dorso-ventral muscles are very weak.

The ventral canals are very strongly developed in the
strobila; their diameter varies from 72 4 at the apex of the
scolex to 160 x 240, at 70 cm. from the scolex. The dorsal
canal measures 72 4 at the apex, 32 u at the base of the scolex.
Its lumen is almost obliterated at 40 cm. from the scolex.

The course of the canals, in the scolex is described
further on.

The first traces of the genital organs are seen at 1 cm. from
the scolex. he testicles are recognisable at 12 em., the
ovarium appears at 40 cm., male sexual maturity is reached
at 70 cin.; at this stage the uterus begins to develop. The
paruterine organs commence to develop at 90 em.

There are three to six testicles on each side of each of the
ventral canals, leaving a great gap in the middle of the segment

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 337

without testicles (fig. 1 and Text-fig. 2). The testicles he
slightly dorsal to the transverse axis. ‘he vas deferens

)

(List of abbreviations at end of paper.)

TRXT-FIG. ¢

D

Diacram of Avitellina centripunctata.

crosses the dorsal side of the median field, quite close to the
transverse muscles, dorsal to the testicles, the nerve, the
longitudinal canals, and to the female genitalia. Between the

338 LEWIS HENRY GOUGH.

ventral canal of the pore side and the cirrus-pouch it becomes
distended with spermatozoa, and also slightly convoluted
(vesicula seminalis). The cirrus pouch les ventral or dorsal,
anterior or posterior, to the vagina (figs. 4 and 43). The end
of the cirrus is bent over and joins the vagina, it does not
appear to be introduced into, but fused to the end of the
vagina. There is a short and very narrow genital pore into
which the vagina opens. ‘The vagina runs straight to dorsal
of the nerve and of the testicles; passing the ventral canal
dorsally it widens median of the ventral canal (pore side) to
form a receptaculum seminis. From the receptaculum
seminis the canalis seminalis arises, which runs a short dis-
tance in the same direction as the axis of receptaculum would
if lengthened; then it branches, one branch, the oviduct,
turning ventrally towards the ovarium, the other, the uterine
duct, also turning ventrally, leads to the uterus. ‘The ovarium
is bean-shaped, or kidney-shaped ; it contains but few eggs.
There is no vitelline gland nor shell-gland. The eggs pass
through the oviduct into the uterine duct and then into the
uterus, fertilisation taking place during the transit. The
egos receive nourishment from certain cells in the ovary and
in the uterus (ovarial and uterime nutritive cells, pp. 371, 375).
The eggs are finally enclosed in a paruterine organ, which
arises within the uterus. Pads of fibrous tissue, lying anterior
to the uteri, serve as support to the paruterine organs, and
help to separate the genitalia of adjacent segments.

The eges are enclosed in two spherical envelopes ; the outer
measures 40 w, the inner 25 yu, the embryo 19 w (fig. 2).

THe Systematic PosrrioN OF THE GENERA STILESIA AND
AVITELLINA.

Fuhrmann (1908) placed Stilesia together with Thysa-
nosoma in a new sub-family which he called 'Thysano-
somine.

Now that the anatomy of the species of Stilesia and
Avitellina are so much better known than they used to be,

TAPE-WORMS OF THE SUB-FAMILY AVITELLININ”E. 339

it becomes necessary to review their position, and to see how
far they are related to Thysanosoma.

The points common fo the three genera Stilesia, Avitel-
linaand T'hysanosoma are: the marginal arrangement of
the testicles, the irregular alternation of the single genital
pores (which does not hold good for Thysanosoma, double-
pored specimens being frequently met with in South Africa)
and the possession of a paruterine organ. ‘They differ, how-
ever, in several very important points: Avitellina and
Stilesia do not possess either a shell-gland or a vitelline
gland ; their eggs receive nourishment from nutritive cells in
the ovarium and in the uterus.

The points in which the three genera agree are hardly of
sufficient importance to weigh very heavily; the position of
the testicles and of the genital pores is liable to vary con-
siderably within a sub-family ; the possession of a paruterine
organ can, as shown by Fuhrmann (1908), be acquired
independently by genera belonging to various sub-families.

The lack of a vitellogene gland and shell-gland and the
results of their absence are, however, quite sufficient to
separate the two genera from all other known cestodes. I
therefore propose to separate the genera Stilesia and
Avitellina from the Thysanosomine and to place them
in a new sub-family of the Anoplocephalide, calling the
new sub-family Avitellinine, after the genus Avitellina,
which is certainly the better known of the two genera.

Diagnosis of the Avitellininz.—Scolex without hooks
with four suckers. Segments short, genital pores irregularly
alternating, testicles in two or four groups, marginal, none in
the middle field. A single ovarium, no vitelline gland,
no shell gland; uterus single or double, eggs finally
enclosed in a paruterine organ. Eggs in ovary and uterus
surrounded (and nourished) by nutritive cells. Oncosphre
with two envelopes. Type genus, Avitellina, Gough, 1910.
All the known species inhabit Ruminants, development
unknown.

340 LEWIS HENRY GOUGH.

An Accounr oF THE HisTroLogy oF AVITELLINA CENTRIPUNC-
TaTaA (Rivonra).

Avitellina ceutripunctata (Rivolta) is on account of
the large size of its histological elements and the loosenesss
of their arrangement, exceptionally favourable for study.

The best results were obtained from worms fixed with
Zenker’s solution. I allow the solution to act for at least
six hours, then I transfer the specimens to running water for
twenty-four hours, after which they are carried through
alcohol 25 per cent. and 50 per cent., remaining in each for
at least three hours, being finally preserved in alcohol
75) per cent.

In order to obtain the worms as expanded as possible, I
usually hold them up with a pincette, allowing them to hang
free in the air; this almost instantly causes them to expand
on account of their own weight, when I suddenly plunge them
into the fluid, lifting them out at once; after letting them
hang again for a few seconds I finally deposit them in the
reagent. The worms treated in this way fix in a fairly
expanded condition, and are not contorted or twisted. It is
of great importance, however, to obtain the specimens alive,
and they ought not to be washed previous to fixing. It is
not necessary to use iodine to remove the last traces of subli-
mate from material treated in the manner described.

I have used sublimate, formalin, and silver nitrate, as well
as Zenker, but no other reagent I know is to be compared
with Zenker’s solution for fixing cestodes. It is specially
favourable for the study of the subcuticula and its connec-
tion with the muscles.

Staining was performed with Ehrlich’s hematoxylin and
orange G, which gives wonderfully clear pictures of the sub-
cuticula and muscles. For the study of the eggs, and the
changes taking place before maturation and fertilisation, I
recommend iron-hematoxylin and eosin. This combination
also presents the best results for the flame-cells and the
nephridial cells surrounding the dorsal canal, and also shows

TAPE-WORMS OF THE SUB-FAMILY AVITELLININA. 341

up the structure of the longitudinal muscles very distinctly.
I have also employed hematein Apathy and _ Delafield’s
hematoxylin with good results. In using the Delafield one
can obtain much the same results for the subcuticular cells as
with Ehrlich’s hematoxylin if, instead of differentiating
with acidulated alcohol and blueing with ammoniated alcohol,
one washes the specimens after staining in running water
only. The nuclear structures do not stain as cleanly, however,
as they do when using the stain in the ordinary way.

For specimens mounted in toto I use borax carmine;
most of my material has been stained with borax carmine
before cutting, being re-stained with hematoxylin after
sectioning. Ido not find that this spoils the final result ; on
the contrary, one often finds that the borax carmine helps to
differentiate the nucleoli from the chromatin bodies in the
nuclei.

My sections are invariably 4 thick, which appears to be
the best thickness for Avitellina material.

Almost all the drawings have been made with the Leitz oil-
immersion ;'; and ocular 2, and are reproduced as far as
possible at the same scale.

I have made sections of Twnia serrata, Goeze; Ano-
plocephala magna (Abilgaard); Dipylidium caninum,
L., and Stilesia hepatica, Wolffhiigel, in order to obtain
comparative material fixed, hardened, and stained in the same
way. It was unfortunately not possible to obtain Ligula

material.

Tue Coricuna. Figs. 17-21.

The cuticula consists of the usual two layers, which seem
to have been observed by all recent observers, namely, a thin
outer layer (Comidien Schicht, Minckert, 1906) and a
thick inner layer (Homogene Schicht, Minckert, 1906),
within which hes the extremely thin basal membrane
(Grenzstreifen, Minckert).

The outer layer, or comidial layer, stains very deeply with
hematoxylin; it does not appear to be provided with fine

342 LEWIS HENRY GOUGH.

hairs or other such structures. <A fine radial striation is,
however, readily observable in favourable sections (fig. 17).
The thickness of this layer is from lm to 15. The comi-
dial layer easily becomes detached, and is then sloughed.

The homogeneous layer is 3 4 thick on the average. Unlike
the outer layer, it hardly stains with hematoxylin, but takes
orange G or eosin readily. It appears (when fixed by
Zenker”) to be quite structureless, except on the suckers,
but impregnation with silver shows a definite structure also
elsewhere. I have not been able to find any structures which
can be compared to Minckert’s (1906) trophopores, tropho-
porelles, neurophyses, or neuropores in the homogeneous layer
of Avitellina centripunctata (Rivolta), nor any signs of
pores running from surface to surface through this layer.
However, in specimens impregnated with silver there are
minute black granules scattered through the homogeneous
layer, not quite evenly distributed, but more crowded towards
the basal membrane, more scattered towards the comidial
layer (fig. 18). I have observed a similar structure of the
euticula in Dipylidium caninum, L., fixed with Zenker,
stained with iron-hematoxylin, and counter-stained with
eosin ; here the homogeneous layer presents exactly the same
appearance as that of Avitellina when fixed with silver
nitrate. ‘he granules must therefore represent a finest
structure of the cuticula, and are probably not merely
artefacts (silver precipitates), as I was at first inclined to
believe.

Certain modifications of the homogeneous layer are, how-
ever, invariably present in the cuticula of the suckers. These
remain constant whether the specimen was fixed with Zenker,
silver nitrate, or formalin. The comidial layer presents no
contrasts to elsewhere, but the inner half of the homogeneous
layer appears spongy or reticulated (fig. 17). The reticula-
tions are formed by fibres running mostly at right angles to
the homogeneous stratum, and forming numerous anastomoses
amongst themselves. These fibres may be continuous with
the parenchyma fibres of the suckers, which they much

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. .343

resemble, especially in silvered specimens. The reticulations
formed by the fibres enclose cavities which are probably in
connection with each other. ‘This structure has only been
found in Avitellina; I have not observed anything similar
in the other species I have examined.

Within the homogeneous layer and separating it from the
subcuticular muscles hes an extremely thin membrane, the
basal membrane, which can usually be quite readily demon-
strated. The basal membrane is generally accepted as being
derived from the parenchyma.

The formation of the cuticula has recently been studied by
Young (1908) in Cysticercus pisiformis; according to
him, “ The cuticula of Cysticercus pisiformis is developed
from a groundwork of simple parenchyma fibrille by a
deposition among them of a cement substance. ‘There are
no specialised fibrille or cellular processes concerned in its
development. The fact that in its development the processes
of the subcuticular cells take part does not in any way
detract from the above statement, since primitively the sub-
cuticular cells themselves are undifferentiated parenchyma
cells.” Further (p. 288), “The cuticula is formed before
the differentiation of the subcuticular cells.”

Young’s opinions are totally opposed to Blochmann’s (1896)
and his followers, who consider the cuticula to be mainly a
product of the subcuticular cells.

It can, however, be proved from adult cestodes that the
cuticula is not derived from the subcuticula. There is no
doubt as to the presence of a cuticula consisting of comidial
and homogeneous layers on the surface of the suckers, yet
there are no subcuticular cells in the suckers. I have been
able to convince myself of their absence in Anoplocephala
magna (Abilgaard), Tenia serrata, Goeze, Dipylidium
caninum (L.), Stilesia hepatica Wolffhiigel. Young
(p. 225) also states, ‘The subcuticula cells are lacking in the
suckers.” In all these species the cuticula above the suckers
is similar to that elsewhere, differing somewhat in this respect
from Avitellina centripunctata (Rivolta). There being

VOL. 56, PART 2.—NEW SERIES. 24

544, LEWIS HENRY GOUGH.

no subeuticula in the suckers, the cuticula of the suckers
must arise independently of the subcuticula.

Unfortunately: neither Réssler (1902) nor Young (1908)
has made any observations concerning the development of
the suckers. Leuckart (‘Parasiten des Menschen,’ 2nd edition,
vol. 1, p. 445) states that their development commences
by the formation of four hemispherical depressions of the
cuticula in the substance of the “ Kopfzapfen,” representing
the cavities of the suckers, and that the radial muscles arise
out of the subcuticular cells. Glaser (1909) finds the suckers
arising somewhat differently, already before the subcuticula
is formed, at least he avoids using the term ‘ subcuticula.”
Leuckart considers that the fact of the development of
muscles out of the subcuticular cells of the suckers is evidence
that the subcuticula has nearer affinity to the musculature
than to the epidermoidal apparatus. In discussing the sub-
cuticula I will have to show that the subcuticula of the
proglottids stands in very close relationship to both the
dorso-ventral and to the transverse muscles; Leuckart’s
observation of the development of the radial muscles out of
subcuticular cells would contain nothing very remarkable, as
the subcuticular cells are actually muscle-elements.

If, as according to Blochmann, the cuticula is a product of
an epithelial layer, and the subcuticula is that layer, then no
cuticula can exist where there is no subcuticula. There is,
however, no subcuticula in the suckers, and yet the suckers
are clothed with a cuticula; the subcuticula cannot therefore
be the epithelium producing the cuticula.

Cuticula with no underlying subcuticula is also to be found
in older portions of the strobila, in segments where the
paruterine organ is fully developed; but here we have not to
deal with primary but with secondary conditions. In the
anterior portion of the strobila the dorsal and ventral surfaces
of the segments are more or less parallel to each other
(fig. 45) ; proceeding distally we find that the segments are
broader at their posterior than at their anterior end, the
strobila appearing serrated on longitudinal sections. Hxamina-

TAPE-WORMS OF THE SUB-FAMILY AVITELLININ®E. 9345

tion of a single section (fig. 21) shows that dorsally and
ventrally the following changes have taken place. The
original cuticula, together with the subcuticula, has shifted its
position from parallel to the longitudinal axis to one at right
angles to the long axis, pivotting on the posterior border of
the segment, the margin originally anterior having become
the outer margin. The space between the outer margin, the
anterior margin and the posterior border of the segment has
filled with parenchyma; the new surface from the anterior
border to the outer margin is clothed with a thin cuticula,
under which there is no subcuticula. This cuticula may have
been derived by stretching of the already present cuticula, or
it may be of new origin; the fact that the cuticula covering
the subcuticula now on the posterior surface is enormously
thickened (by contraction ?) would suggest that the cuticula
covering the new surface is of new origin. ‘The new cuticula
only measures 1°54 to 1:7 4 that on the posterior surface is
much folded, and measures up to 9. The subcuticular muscles
are also much more evident than usual on the posterior surface,
the whole giving the impression that the cuticula and muscles
have been compressed laterally (i.e. in the plane of the
surface of the cuticula), thus causing the increase in
thickness.

THe Suscuricuta. Figs. 19-21.

The subcuticula is present everywhere in a single layer
close under the cuticula, except in the suckers and places
mentioned above. It consists of a stratum of elongate cells,
standing vertically to the cuticula. Its component cells are
widely separated from each other, or densely congregated,
according to the state of contraction of the worm. In shape
they usually taper slowly towards the cuticula and are
rounded or tapering towards the parenchyma (figs. 19, 20).
Before reaching the cuticula several branches are usually
formed, so that each cell touches the cuticula in more places
than one. The outline of the cell is extremely distinct in
properly fixed and hardened material ; a thin membrane is

346 LEWIS HENRY GOUGH.

possibly present. ‘The plasma is finely granular, staming
readily. with hematoxylin. The nucleus is oval, clear,
surrounded by a distinct membrane, and contains a nucleolus
and usually four chromatin bodies. The nucleolus stains
differently to the chromatin bodies, my material being
mostly stained in toto with borax carmine, and stained in
section with EKhrlich’s haematoxylin, counter-stained with
orange G, the nucleolus staining reddish, the chromatin
blue. Whether conical or rounded towards the parenchyma,
each subcuticular cell gives off one or mere fibrillar processes,
which run inwards; the processes from several adjacent cells
usually converge and collectively join the dorso-ventral
muscles, where those muscles break through the longitudinal
muscle (fig. 20). The subcuticular cells on the extreme
lateral margin also behave in the same manner, only that they
join the transverse muscle. ‘Towards the lateral margins the
connection between the dorsal or ventral subcuticular cells
and the muscles is even more apparent, as the cells le
grouped around the base of the muscles, the subeuticular
elements belonging to any one of the outer dorso-ventral
muscles all lying lateral to their muscle, thus assuming a
direction oblique to the cuticula.

I have been able to verify this connection between sub-
cuticular cells and muscles in Dipylidium, Tenia,
Dibothriocephalus and Trizwnophorus. The sub-
cuticular cells consequently belong to the dorso-
ventral and transverse muscle systems. In well
preserved material it is possible to demonstrate this connec-
tion of muscles and subcuticula for every cell lying entirely
in the plane of the section.

The question arises, Why have such processes not been
observed before? In my opinion this is probably due to the
methods used for fixing and hardening, and to the necessity
to cut the sections in the plane of the cells. The subcuticular
cells appear to be very difficult to fix and harden satisfactorily,
Zenker’s solution being the only one which has given me
such resuits as yet. The silver impregnation method em-

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 347

ployed by Zernecke is probably not quite satisfactory, apart
from its capriciousness and unreliability.

In older proglottids the subcuticular cells atrophy, their
nuclei becoming smaller, their nucleolus and chromatin form-
ing apparently a single mass filling the entire nucleus ; and
the outlines of the cells themselves become indistinct (fig. 21).

Having shown that the subcuticula belongs to the dorso-
ventral and transverse muscle systems, and that the cuticula
of the suckers arises independently of a subcuticula, and
bearing in mind Young’s observation of the development of
the cuticula in Cysticercus prior to the differentiation of
the subcuticular cells, it will be seen that serious doubts must
again arise concerning the epithelial nature of the subcuticula.
Balss (1908) also opposes the theory that the subcuticula is
an ectodermal epithelium ; he considers both cuticula and sub-
cuticula to be of mesodermal origin, but he still admits that
the cuticula is a product of the subcuticula. Of course the
mere fact that the subcuticular celis form part of the dorso-
ventral and transverse muscles would not induce me to deny
their epithelial nature and their being the producers of the
cuticula ; remembering the epithelial muscles of hydroids, etc.,
it is mainly on account of the cuticula being formed in places
where there is no subcuticula.

Tse Muscuzs. Figs. 22-29.

The muscles in the strobila, scolex, and suckers can every-
where be divided into the two groups, subcuticular muscles
and parenchyma muscles.

The subcuticular muscles form two layers, whose fibres
run at right angles to each other; the outer of these is
situated very close to the cuticula, and runs horizontally, the
inner runs longitudinally.

The parenchyma muscles in the strobila form three distinct
muscle systems—the dorso-veutral, the transverse, and the
longitudinal muscle systems. ‘The first two are much
weaker than the last.

348 LEWIS HENRY GOUGH.

The muscles in the scolex are more complicated than those
in the strobila, as their course is modified by the suckers, and
their requirements (Liihe, 1894). The following systems of
muscles have been made out in connection with the suckers of
Avitellina centripunctata (Rivolta).

Commencing at the apex of the scolex, and proceeding
towards the strobila, we meet:

(1) A diagonal cross-system anterior to the terminal loops
of the excretory canals; it covers the entire anterior surface
of the scolex, passing from the front of the left ventral to
the front of the right dorsal sucker, and from the front of
the right ventral to the front of the left dorsal sucker.

(2) A second diagonal cross system, composed of four
bundles of muscles, each consisting of only a tew fibres, is
situated just posterior to the terminal loops of the ventral
canals; it runs from sucker to sucker in such a way as to
connect—

(a) The median face of the right ventral sucker with the
lateral face of the left dorsal sucker.

(s) The lateral face of the right ventral sucker with the
median face of the left dorsal sucker.

(c) The median face of the left ventral sucker with the
lateral face of the right dorsal sucker.

(p) The lateral face of the left ventral sucker with the
median face of the right dorsal sucker.

(3) An orthagonal cross-system, running dorso-ventral and
transversely, is situated just behind the second diagonal cross
system and between it and the great nerve commissures. It
connects ;

(a) The median faces of the two left suckers.

(B)

(c) The lateral faces of the two dorsal suckers.

(0)

The median faces of the two right suckers.

The lateral faces of the two ventral suckers.

(4) A second orthagonal cross-system is situated behind
the nerve commissure, near the base of the suckers. Its
insertions correspond to those of the previous orthagonal
system.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININ”[. 349

(5) Near their base the suckers are connected by a system
of muscles, which connects the two dorsal suckers with each
other and the two ventral suckers with each other, but does
not seem to connect the dorsal with the ventral suckers. Its
fibres connect :

(aA) The right sides of the two dorsal suckers,

(zs) The left sides of the two dorsal suckers.

(c) The right sides of the two ventral suckers.

(p) The left sides of the two ventral suckers.

The histological elements composing the parenchyma
muscles can be divided into the following groups:

(1) Bipolar myoblasts with terminal fibrillee.

(2) Bipolar myoblasts with lateral fibrillee.

(3) Elongate bipolar myoblasts lying axially within the
tubiform muscles of the longitudinal muscles.

The first group I have only found in the dorso-ventral
muscle; the second forms the transverse muscle, and occa-
sionally occurs in the dorso-ventral muscle ; the third occurs
in the longitudinal muscle.

(1) The bipolar myoblasts with terminal fibrille
occur in all parts of the dorso-ventral muscle, and are its
chief components. ‘The cells are spindle-shaped, with an
oval nucleus, whose long axis lies in the long axis of the cell
(figs. 22,23). The nucleus measures, on an average, 3°) yu X
4°25 «3; it contains a nucleolus and two or more round chro-
matin bodies. ‘he plasma stains deeply, and is sharply
defined at the margins, as though enclosed by a membrane.
Ateach end of these cells a fibrilla arises, which is several
times longer than the cell itself; the fibrille are extremely
thin, measuring only a fraction of a m in diameter, they run
in the general direction of the long axis of the cell; their
course is, however, usually not quite straight, but sinuous or
zig-zag. The fibrillae may, perhaps, only act as tendons, in
which case the cell would represent the contractile portion
of the combination, but it is more probable that the fibrilla
is itself contractile. These cells are usually found in rows,
their fibrillee lying in part apposed to each other, thus form-

350 LEWIS HENRY GOUGH.

ing the connection of the whole row to a muscle. The
fibrillee of the outermost myoblasts do not insert directly in
the cuticula, but are in contact with the fibrille arising from
the subcuticular cells.

Cells similar to these simplest myoblasts have been
seen and described by Schiefferdecker (1874) in Tenia
saginata, Kiichenm.; he considered them to be connective
elements; and by Hamann in Tenia (Mesocestoides)
lineata, Rud., who described them as elements of the
parenchyma.

(2) Bipolar myoblasts with lateral fibrille are, in
Avitellina, chiefly found in the transverse muscle. They
are the form of myoblast most frequently recorded by recent
authors (e. g. Pinter, 1881; Hamann, 1882; Kriimer, 1892 ;
Will, 18953; Zernecke, 1895; Réssler, 1902; Young, 1908)
in various genera and species. These muscle-cells differ from
those described above inasmuch as the fibrilla runs con-
tinuously over one surface of the cell and does not originate
only at the poles, otherwise they are very similar in appearance
and dimensions (figs. 24, 25). Their nuclei measure 3°5 1 x
4°25 41; their plasma is attached to the fibrilla, being widest
at the middle and drawn out to a point at each pole. The
plasma stains fairly deeply, and has a very distinct outline,
probably being enclosed by a membrane. ‘The fibrillee are
long, apparently homogeneous, and lie arranged parallel to
each other. At the margins the fibrille of the transverse
muscle spread fan-like towards the cuticula, as usual in other
cestodes. Some of them appear to connect with the most
lateral subcuticular cells. he fibrille are very thin, less
than lin diameter. Young (1908), describing such myo-
blasts, states that the myoblast is always connected to the
muscle by fibrille, even when the cell remains very close to
the muscle. I have not been able to verify this observation
in my material.

(3) The elongate bipolar myoblasts, lying axially
in the tubiform muscles of the longitudinal muscles, are
more difficult to observe than the other two varieties of

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 391

myoblast of Avitellina. The longitudinal muscles differ at
first sight from the dorso-ventral and transverse muscles
even under a low power, by the great development of the
muscle-fibres ; the myoblasts themselves are hard to find on
account of their great length, which reaches as much as 60
or more, and on account of the length of the muscle-fibres,
which can often be followed continuously through more than
nine segments. Owing to their elongation, it too frequently
happens that the myoblasts are not entirely contained in a
single section, and owing to the large number of fibres in the
longitudinal muscle it is hopeless to look for the continuation
of such myoblasts in the next section.

The muscles are thick, measuring up to 9» in diameter,
they are round or oval or even polygonal with rounded angles
on transverse sections. On thin transverse sections a large
number of the muscles, especially those of the outer layers,
appear annuliform, having a clear space in the middle.
Towards the inner layers of the transverse muscle, all the
fibres are found to be solid. Zernecke (1896) figures such
annuliform sections of the longitudinal muscles of Ligula,
and I have found similar muscles in Dipylidium. The
solid muscles are probably derived from tubiform muscles for
the following reason. In sections stained with iron-hema-
toxylin, if favourably differentiated, the solid longitudinal
muscles are seen to consist of a dark (black) staining core
surrounded by a light outer sheath. This peculiarity can be
observed in sections passing transversely to the muscles as
well as in such running in the direction of the muscle and
containing uncut muscles. As we will see later on, the tubi-
form muscles contain a portion of the axial myoblast in their
hollow. If the myoblast deposits or produces the muscle
substance, then the centre or axis of the muscle represents
the younger deposit, which again stains differently to the
outer portion. The fact that the annuliform sections very
frequently do not contain any portion of the myoblast is
probably due to’shrinkage or contraction during fixation.
If the solid muscles are the older ones, the outside position

302 LEWIS HENRY GOUGH.

of the annuliform muscles would indicate that the young
muscles develop on the outside of the transverse muscle.

The myoblasts are bipolar, spindle-shaped and very
elongate (figs. 26, 27) ; their nuclei resemble in most respects
the nuclei of the myoblasts of the other two systems, but
they are usually somewhat larger. Those measured averaged
4°25 p to 5 long by 2-5 to 4:25 broad. The plasma is
granular; a membrane appears to be present. ‘The ends of
the cells are inserted in the tubular ends of the muscles. In
some cases the two tubiform muscles appear to be connected
by a strip of muscle-tissue passing along one side of the
myoblast. Similar muscles were observed by Salensky (1874)
in Amphilina, although not re-found by either Hein (1904)
nor by Cohn (1904) in the same object.

The longitudinal muscles appear to me to be produced by
these cells, which in that case are true myoblasts, the con-
tractile substance being formed or deposited on the surface
of the cell, the body of the cell penetrating the axis of the
muscle for a long time; then, as the muscle develops, its
cavity becomes filled with muscle substance. In the end the
cell disappears from the axis of the muscle, and becomes
displaced so as to he laterally to the muscle. Nuclei lateral
to the muscles are frequently observed.

The muscle-fibres can be traced through a number of
segments. ‘Their course is not quite straight, but more or
less sinuous. ‘Text-fig. 3 is a photograph of a section cut
parallel to the longitudinal axis of the worm, and represents
a distance of about ten segments.

In transverse sections the muscles appear to be arranged
in bundles, whose elements are more crowded towards the
transverse muscle, more scattered towards the subcuticula.
However, I distrust the appearance of the bundles, since it is
impossible to re-discover them in horizontal sections (see Text-
fig. 5). It will be seen that where a few muscles do group
together, they receive fibres from the adjoining groups and
give off fibres.

The schematic ext-figs. 4, 5, 6 will help to make my

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 3535

meaning more clear. Fig. 4 shows the course of six muscles
composed of bundles of four; one sees the sections of the
dorso-ventral muscles separating the bundles. ext-figs. 5 and
6 have been drawn to show that it is possible to obtain
transverse sections giving the appearance of bundles, but that

TEXT-FIG. 3:

a?

Horizontal section through the longitudinal muscle. About nine or
ten seoments are included in the photograph.

in each case the bundles at the levels a, b, c, d are differently
composed. ‘Thus at level a, 1, 2, 3, 4 form the first, 5, 6, 7,
8 the second, 9, 10, 11, 12 the third bundle, ete. At level },
2,3, 4, 5 form the first, 6, 7, 8, 9 the second, 10, 11, 12, 13
the third bundle. By the time level e is reached not a single
fibre forming the first bundle is the same as at level a. The

ey)
Or
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LEWIS HENRY GOUGH.

TEXT-FIG. 4.

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‘ . ‘ .

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2 3.4.5.6.7 8.9.10 I. (2.1314.15.16 1718.19.20. 2122.23.26
The top row represents a transverse section, the lower part of the
figure a horizontal section. The figure shows the sections of
the dorso-ventral muscles separating the bundles. The
bundles are real.

TEXT-FIG. 5.

Ja" ie <0. cel[le os © ‘aifien woh ae aiff ois: viele) 0) 1% 2]

. e . ’ ) Ps to ee

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1.2.3. 4.5.6.7 8.9.10 1 12.13.)415.1617.1819.20.

The top row represents u transverse section, the lower part of the
figure a horizontal section. The figure shows the dorso-ventral
muscles passing through between the longitudinal fibres. At
the levels a, b, c, d, ete., one would find the muscles arranged
in bundles of four, as in Text-fig. 4. The arrangement in
bundles is only apparent.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININA. 35:

number of fibres in a bundle and the number of bundles have
remained the same, and if we were to judge entirely by
transverse sections, we would have to conclude that the
number of fibres in a bundle and the number of bundles is
constant.

The fibres of the longitudinal muscle are apparently
arranged in bundles of about twenty-four, with a few
solitary muscles scattered near the subcuticula.

The subcuticular muscles arise, according to the usual view

TEXT-FIG. 6.

- Oe if ei tates Pua tere ger Ged tosh

’

°
°
°
©

°

iow

Wi2e 3. 4. 9 516.7, 1G: 9 ION 12 13.14.15.16. 17

The figure represents a modification of the arrangement of the
muscle-fibres shown in Text-fig. 5. The arrangement in
bundles, as seen in transverse section, is only apparent.

as products of the “ Sommer-Landois ” cells, which are sup-
posed to be myoblasts. Young (1908) opposes this view, as
he finds the subcuticular muscles already formed before the
“Sommer-Landois” cells are differentiated. Young con-
sequently refuses to apply the term “myoblast” to them,
and calls them “neuro-muscular” cells, because they send
fibrillar processes into the muscles and because they are also
connected with the nerves.

The “Sommer-Landois” cells of Avitellina are best
examined in sections through the scolex. These cells send

356 LEWIS HENRY GOUGH.

one or two processes towards the cuticula to subcuticular
muscles, and also one or two processes into the depth of
the scolex to connect with the nerves (fig. 28). The nucleus
is large, round, measuring 5, in diameter; it contains a
nucleolus anda few chromatin bodies. The plasma is fibrillar
in appearance, several fibrille uniting to form the processes
leading to the subcuticular muscles or to the nerves. The
whole cell has much more resemblance to a nerve-element
than to a myoblast; it differs from the myoblasts of the paren-
chyma muscles in the obvious connection with the central
nervous system, in being connected to more than one muscle-
fibre, and in being multipolar instead of bipolar.

Young (1908) finds that the “Sommer-Landois’’ cells
originate from cells similar to those I have described as
occurring in the dorso-ventral muscle, and he lays importance
on the fact that the muscles are present before the ‘ myoblast”
differentiates. The nerve-connection was observed already
by Zernecke (1908) and Blochmann (1896) before Young. I
consider Young to have been quite justified in giving the
name ‘“‘neuro-muscular cell” to the Sommer-Landois cells, as
the cells are certainly quite as much nerve-elements as muscle-
elements.

The Muscles of the Suckers.—It is possible to distin-
guish between parenchyma and subcuticular muscles in the
suckers in the same way as in the rest of the body, the
myoblasts and ‘ neuro-muscular” or ‘ Sommer-Landois”
cells giving the clue to the group to which the muscles
belong.

The radial fibres belong to the parenchyma muscles, the
muscles just under the cuticula running parallel to the cuti-
cula to the subcuticular muscles.

In Avitellina the radial muscles have lost their myoblasts,
but in other forms examined by me (Tenia, Dipylidium,
Cittotenia) it has been possible to readily demonstrate
the presence of lateral myoblasts.

In Avitellina centripunctata (Rivolta), and in Ano-
plocephala magna (Abilgaard), there is a layer of cells

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 357

close to the cuticula of the suckers, separated from the cuti-
cula by the superficial ‘layer of muscles. ‘This layer of cells
does not represent the subcuticula, but rather must be homo-
logised with the “ Sommer-Landois” cells of the subcuticular
muscles of other parts of the worm. The cells are more or
less flattened, and lie in a plane parallel to the cuticula—sub-
cuticular cells are perpendicular to the cuticular ; these cells
are multipolar (fig. 29), each cell sending out several pro-
cesses, which either terminate in connection with muscle-
fibres, or connect with processes from similar adjacent cells.
The cells further stand in contact with the ganglion cells of
the sucker by means of neuro-fibrille coming from the nerve-
cells. ‘The nuclei of the neuro-muscular cells in the suckers
are oblong, small, measuring only 3°54 long. The points of
resemblance between these cells and the ‘ Sommer-Landois”’
cells are their connection with more than one muscle-fibre,
their multipolarity, their connection with the central nervous
system (of the sucker in this case).

Granted that these cells are really the homologa of the
“Sommer-Landois”’ cells of the rest of the body, then the
muscles they belong to must be subcuticular muscles, as
“ Sommer-Landois”’ cells have hitherto only been found con-
nected to subcuticular cells.

The arrangement of the muscles in the suckers follows
roughly the arrangement in the rest of the body; subcuti-
cular muscles, in two layers, occupy the space directly under
the cuticula, and are parallel to the cuticula; parenchyma
muscles run perpendicular to the cuticula, and, in deeper
layers, parallel to the cuticula.

The suckers are separated from the scolex by a delimiting
membrane; directly within this membrane are two sets of
fibres running parallel to it. The first set forms concentric
rings, as can be seen on sections touching the base of the
sucker ; the second set radiates, the centre of the radiation
being at approximately the same spot as the innermost of the
concentric rings. These two sets lie close to each other, and
remind one of the subcuticular muscles, which they possibly

598 LEWIS HENRY GOUGH.

are. Next to the subcuticula one also finds two sets of
muscles ; the outer is also composed of circular, concentric
muscles, the inner of radial fibres. These two sets are with-
out doubt subcuticular muscles; the ‘‘ Sommer-Landois” cells
mentioned above stand in contact with them. Between the
cuticula and delimiting membrane are the radial fibres; in
the middle of the thickness of the sucker there are other
muscles, running at right angles to the radial fibres, whose
course is possibly circular. These two last sets are without
doubt parenchyma muscles.

Around the margin of the opening of the suckers the sub-
cuticular circular muscles are much developed, forming a
kind of sphincter.

It is worthy of remark that the subcuticular muscles of the
suckers are much more developed in Avitellina and Ano-
plocephala than in Tenia and Dipylidium.

Tue Excrerory System. Figs. 5, 6, 80-87.

The excretory system consists of the usual two pairs of
longitudinal canals, the secondary transverse commissures,
and of the flame cells and their capillaries.

In the strobila the ventral canal is enormously developed ;
it lies lateral to the dorsal canal, whose lumen almost
atrophies in the posterior portion of the strobila.

Transverse commissures connect the ventral canals; they
are not very readily observed in Avitellina centripunc-
tata (Rivolta), but are wide and conspicuous in Stilesia
hepatica, Wolffhiigel, Stilesia vittata, Railhet, and
Stilesia globipunctata (Rivolta), where they form a more
or less complicated network at the posterior end of each
segment.

The flame cells are very frequent in the medullary zone of
Avitellina centripunctata (Rivolta), but extremely rare
exterior to the transverse muscle. This is worth nctice, as in
other cestodes (Tenia serrata, Goeze, Dipylidium
caninum [L.], and Ligula [fide Blochmann]) they seem
to be very frequent among the subcuticular cells.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 359

The course of the canals in the scolex is somewhat compli-
cated, but can be reduced to quite a simple scheme. If it is
remembered that the right and left halves only communicate
by a single loop situated at the apex of the scolex, and that
the dorsal and ventral canals of each side, after mounting
through the scolex, meet and join each other just at the
apical connecting loop, it will be seen that the whole can be
reduced to the scheme given by v. Janicki for Inermicepsifer
in a paper to be published shortly. The actual plan of the
canals in the scolex is, however, complicated by the fact that
the ventral canal forms a large number of loops and turns
before reaching the apex. The course of the dorsal canals,
on the other hand, is quite straight.

Before commencing the description of the course of the
ventral canal it is necessary to explain the terminology to be
used. I must distinguish between dorsal and ventral suckers,
also between right and left suckers; the median face and the
lateral face, the outer angle formed by two suckers and the
inner angle formed by two suckers, and the base and apex of
a sucker.

Dorsal and ventral require no further explanation ; the
median face of a sucker is that side which faces a line divid-
ing the right dorsal and ventral suckers from the left dorsal
and ventral suckers ; similarly the lateral face is that side
which looks towards the line separating the two dorsal from
the two ventral suckers. The outer angle formed by two
suckers is the angular space between the suckers near the
subcuticula, the inner angle the corresponding space or angle
opening on the medullary zone of the scolex. The base is the
convex end, turned towards the strobila, and the apex the end
of the sucker containing the mouth of the sucker (see diagram).

It has already been stated that the ventral canal in the
proglottids lies lateral to the dcrsal canal. Following the
canals from the strobila into the scolex, we find that their
relative positions change close to the scolex, the ventral
canals lying internal to the dorsal canals.

The left ventral canal (fig. 5), on entering the scolex, turns

VOL. 56, PART 2.—NEW SERIES. 25

360 LEWIS HENRY GOUGH.

at right angles to its course, running horizontally towards
the base of the dorsal sucker, then rising vertically it
proceeds up the lateral surface of the sucker to the level of
the bottom of the hollow of the sucker; it then turns over
and proceeds downwards parallel to its ascent. Arriving near
the base of the sucker it gives off a short branch, which runs
towards the median outer surface of the sucker and terminates
at the level of the bottom of the hollow of the sucker. The
main canal re-crosses the base of the scolex horizontally,
internal to and parallel to its first horizontal portion, but
external to the dorsal canal, until it reaches the base of the
left ventral sucker. It then ascends the median side of the
left ventral sucker, almost to the mouth of the sucker, then
turns again, running parallel to its former course until the
base of the sucker is once more reached ; it then passes around
the external surface of the base of the sucker, forming a few
spiral loops, until the outer lateral angle of the left suckers
is reached. It then ascends the outer lateral angle, giving off
a short blind branch towards the dorsal sucker at the level
of the nerve commissure. Arrived near the summit of the
scolex, it bends over and runs down the inner lateral angle of
the left sucker until the level of the cross nerve commissure
is reached ; here it bends inwards, and then upwards past the
nerve commissure, running ventral to and parallel to the
dorsal canal until the apex of the scolex is reached. Here it
bends over dorsally and fuses with (or is continued into) the
dorsal canal. At the point of junction of the two canals the
commissure connecting the right and left halves of the
excretory canal system inserts. ‘The dorsal canal is straight
throughout its entire length.

The right ventral canal runs in a similar manner, at first
ventral to and parallel to the right dorsal canal (fig. 6).
Near the base of the suckers it also bends over dorsally, and,
passing the dorsal canal externally, runs horizontally to the
base of the right dorsal sucker, following the outer face
of the sucker, which it ascends almost to its mouth,
veering during this ascent more and more to the right.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 361

Having ascended thus far, it again turns and descends
parallel to its ascent ;-on reaching the base of the sucker it
re-crosses the base of the scolex exterior to the dorsal canal,
but interior to its first horizontal stretch. Arrived at the
median internal side of the base of the right ventral sucker,
it ascends the median ventral side of the sucker to the height
of its mouth, then turns back parallel to the ascending
portion of the canal. Passing around the base of the right
ventral sucker internally, it ascends the outer angle of the
two right suckers, sending off two blind branches towards
the outer surfaces of the suckers at the level of the nerve
commissure, ‘I'he main canal continues its course parallel to
the long axis of the worm to near the apex of the scolex;
then it bends over, descends the inner angle of the right
suckers until the nerve commissure is reached, when it bends
over again, proceeding past the nerve commissure ventral to
and parallel to the dorsal canal. Arrived at the summit of the
scolex, it unites with the dorsal canal and with the connect-
ing commissure in the same way as the left canal does. he
right dorsal canal is straight throughout, agreeing in this
respect with the left dorsal canal.

The “blind” branches given off by the ventral canals I
have not been able to follow further than stated; of course,
they may be much longer than their observed length. Bearing
in mind the difficulty of tracing the transverse commissures in
the proglottids of Avite!lina centripunctata (Rivolta) for
any length (I had not seen them at all in 1909), I certainly
hesitate to say that a ring-canal is not formed; these branches
may represent portions of a ring-canal, which, in that case,
would be a transverse canal in the scolex homologous to the
transverse commissures in the proglottids.

It is probable that the various loops change their course
considerably according to the state of contraction of the
scolex. Stilesia hepatica, Wolffhiigel, also invariably
shows a similar dorsal bend of the ventral canal at the base
of the scolex ; the entire course, however, | have not been
able to study.

362 LEWIS HENRY GOUGH.

The ventral and dorsal canals are equal in diameter at the
apex of the scolex, where they both measure 72 4; proceed-
ing away from the apex, or from the scolex, the ventral canal
increases in diameter and the dorsal canal decreases. At the
base of the scolex the ventral canal measures 128 «1 in diameter,
the dorsal 32 4. At 10 cm. from the scolex the ventral canal
was found to be 324 by 80y, the dorsal l6u by 32m; at
20 cm. the ventral canal measured 96 4, the dorsal 28 w by
20 w; at 30 cm. the ventral had increased to 112 by 72 p,
the dorsal decreased to 4. After this the dorsal canal
becomes almost obliterated, whilst the ventral canal goes on
increasing until it measures 160 « by 240, this size being
reached at 70 cm. from the scolex.

The dimensions quoted show that the ventral canal is not
always round on section ; its shape varies considerably accord-
ing to the state of contraction of the proglottid from round
or oval to angular, square or polygonal sections being quite
frequent in histologically perfect material. The ventral canal
often occupies almost the entire thickness of the medullary
substance, leaving scarcely enough space for the genital canals
to pass.

The structure of the excretory canals varies according to
the portion of the worm examined. A histological difference
can also be observed between the dorsal and ventral canals in
the strobila.

The longitudinal canals possess a fine membrane, which can
occasionally be seen to be finely striated at right angles to the
long axis of the canal. This membrane is the product of a
layer of epithelial cells, which separate it from the parenchyma.
These cells are best studied in the scolex ; they are, as Bugge
(1902) found in Moniezia expan sa (Rud.), flat cells; their
plasma is collected somewhat more above the nuclei than
elsewhere, and their margins are not observable. Seen
from the side, one observes that the layer is not equally
thick everywhere, being thicker near the nuclei, thinner
towards the margins. EHxactly the same structure can be
observed in Stilesia hepatica, Wolffhiigel, where the

TAPE-WORMS OF THE SUB-FAMILY AVITELLININAE. 363

transverse canals present the same structure as the longi-
tudinal canals.

I have not observed any special musculature of either the
dorsal or the ventral canals in Avitellina.

In the strobila certain changes take place in the epithelial
layer of the canals. The nuclei of the epithelial cells sink
into the surrounding parenchyma, the membrane remaining
connected to the epithelial cells by a fibrillar structure standing
vertical to the surface of the canal (fig. 30). This arrange-
ment is especially noticeable in the dorsal canal, but is also
present in the ventral canal. Bugge (1898) figures the same
radial arrangement of the fibres without comment.

Fig. 51 shows the dorsal canal at about 25 em. from the
scolex. The lumen has almost disappeared, being only 1 or
2h wide. ‘he membrane has consequently thickened con-
siderably, and is seen to consist of two layers, a thin dark-
staining inner and a thick light-staining outer layer. The
outer layer is followed by a darker layer with radial structure,
whose fibres appear to be continuous with the radial fibres
belonging to the epithelial cells ; the epithelial cells have sunk
considerably deep into the surrounding parenchyma. ‘Their
plasma shows a fibrillar structure, arranged radially where in
contact with the canal, and less definitely around the nucleus.
The plasma stains very lightly with orange G, but takes
eosin deeply. ‘The nucleolus is large, its membrane stains
distinctly ; it contains a nucleolus and one or two chromatin
bodies. At first the cells appear to lie close to the radial
fibres; then they grow in length and their nuclei move further
away trom the canal; the portion of the cell surrounding the
nucleus becomes rounded or retort-shaped, the plasma
becoming less dense as it recedes from the canal. A con-
nection by its fibrillee with the canal is always to be made
out. In appearance these cells suggest glands, which swell
and increase in size as they come into function. Their con-
nection with the dorsal canal is always evident; the radial
fibres surrounding the canal certainly belong to them, not to
the parenchyma. In cells which have reached their full

364 LEWIS HENRY GOUGH.

development the radial fibres are seen to enter the cell and to
be continuous with the fibrillar plasma. I consider these
modified epithenal cells of the dorsal canals to be nephro-
cytes ; they do not appear to have attracted the attention of
other observers as yet. As no flame-cells arise from the dorsal
canals they must represent the terminal nephridial element of
the dorsal canals.

In the ventral canals a similar change takes place. The
epithelial cells also sink into the surrounding parenchyma
(fig. 80), remaining connected with the membrane by radial
fibrille. They do not, however, become directly modified into
gland-cells, but indirectly give rise to the flame-cells and their
capillaries. Figs. 32, 33, and 36 show the development of the
flame-cells. The first stage is represented by fig. 82; here
we see a single epithelial cell sinking into the parenchyma,
away from the membrane of the ventral canal, but remaining
attached to the canal. The next change apparently takes
place very rapidly, but without doubt it consists in the multi-
plecation of the cell, a row of five nuclei, as shown on fig. 33,
being produced. ‘hese five nuclei belong to four developing
flame-cells and their capillary cell. A group of young flame-
cells is to be seen on the same figure. ‘The development of
the flame-cells in Avitellina thus goes very much on the
lines described by Bugee (1902), only here we have four
flame-cells in a group instead of only three, the capillary cell
in each case originating in the same way. As Bugge, I have
only found developing flame-cells quite close to the ventral
canal, never at any distance from it. Hxamination of Tenia
serrata gives the same results, only in this species the flame-
cells are in groups of three, plus one capillary cell (see
fig. 36).

I cannot agree with Young (1908) that the flame-cells
develop from parenchyma cells, and that “ the capillary cell
is at least a parenchyma cell of separate origin from the
flame-cells, and that the capillary is formed at first as a
passage in parenchyma strands, to become modified later into
a definite tube with a specially modified wall.’ On the other

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 365

hand, every stage of the development of the flame-cells
observed by me in Avitellina has served to verify Bugge’s
statements.

The fully developed flame-cells do not present any remark-
able differences from those figured and described by other
authors, except that they almost regularly contain a small
corpuscle in the plasma, whose nature I have not been able to
identify (wv). The cells always occur in groups of four, most
frequently in the medullary substance, very rarely in the
transverse muscle, hardly ever exterior to the muscle. They
consist of a cell (figs. 34 and 57) with granular plasma,
usually with irregular, star-shaped outline; the nucleus
contains a nucleolus and a varying number of chromatin
bodies. The flame usually stands vertically to the cell, more
rarely tangentially; the flame is often quite close to the
nucleus, but it is sometimes separated from the main portion
of the cell and the nucleus by a longer or shorter ‘ neck” of
plasma. The flame arises from a basal plate, a meniscus-
shaped body with its concave side turned towards the flame,
its convex side embedded in the cell. The flame itself consists
of cilia. ‘he funnel is widest a little way from its base;
near its middle it is suddenly thickened, a ring of the
substance of the funnel projecting into and somewhat con-
stricting the lumen. he capillary usually runs straight in
the same direction as the funnel for about once or twice the
length of the funnel, and then bends suddenly to one side;
after a short distance it bends again to resume its original
direction. The capillaries can often be followed for a con-
siderable distance, and frequently present a very devious
course. It is interesting to note that the flame-cells invari-
ably lie at right angles to the longitudinal axis of the worm,
so that they fall within the piane of a transverse section.
‘They are fairly frequent in the scolex as well as in the strobila,
but do not occur in the suckers.

The flame-cells have long been recognised to be unicellular
glands (Pinter, Lang, Bugge, Blochmann), and the capillaries
to be their ducts ; both the flame-cells and the nephrocytes

366 LEWIS HENRY GOUGH.

of the dorsal canal have a common origin—the epithelial cells
of the excretory system.

Tae Nervous System. Figs. 38-40.

The central nervous system of the strobila consists of a
nerve lying lateral to the ventral canal on either side of the
strobila. From this main nerve branches are given off in
every segment, which are, however, very difficult to observe.

The nerves on entering the scolex first bend inwards,
passing well into the central space enclosed by the suckers.
At the level of the bottom of the hollow of the suckers four
ganglia are found, two lying in the inner lateral angles formed
by the suckers, two in the inner median angles. These four
ganglia are connected by transverse commissures, so that the
lateral ganglion of each side is connected to both the dorsal
and the ventral median ganglia; the ganglia are further con-
nected by a transverse commissure, connecting the two lateral
ganglia, and a dorso-ventral commissure, connecting the two
median ganglia. The transverse and the dorso-ventral com-
missures fuse in the middle of the scolex, where they cross
each other and form a central ganglion. Hach of the four
peripheral ganglia gives off two nerves, which proceed at first
anteriorly and then bend over and unite with the nerve of the
neighbouring ganglion, the eight nerves thus forming four
loops connecting the four gangha. Anterior to these is a
second central nerve-plate, poorer in ganglion cells, whose
connection with the rest of the nervous system has not been
made out.

The histology of the nervous system presents on the whole
the usual structures. The lateral nerves in the strobila
consists chiefly of neurofibrille, arranged parallel to each
other on longitudinal sections, having a reticulated appear-
ance on transverse sections. ‘he neurofibrille are accom-
panied by scattered glia cells, which are chiefly found on the
surface of the nerve. Ganglion cells were very rarely observed
in the nerves of the strobila.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 367

In the ganglia of the scolex nerve-cells are very prominent,
their large size and deep staining (with Ehrlich’s hema-
toxylin) making them most conspicuous. Multipolar ganglion
cells were most frequently met with in the ganglia, bipolar
in the nerves.

The multipolar ganglion cells (fig. 58) have a reticulated
or spongy protoplasma, which stains dark blue with the
hematoxylin. From the processes of the cells the neuro-
fibrillee can often be traced for some distance. The nuclei
measure 8 to 9u long by 45,4 to 8u broad; they are
vesicular, and containa large nucleolus (up to 2 in diameter)
and one or two chromatin bodies. ‘The nucleus itself is
much paler than the surrounding plasma; the nucleolus
stains rather lighter than the chromatin.

In the nerves arising from the scolex gangha large bipolar
ganglion cells are frequent; fig. 39 represents a portion of
such a cell, one end not being in the plane of the section.
The nucleus resembles that of a multipolar ganglion cell; the
cell-plasma, however, contains tigroid bodies. From each
end of the cell neurofibrillee can be traced for a considerable
distance. ‘I'he long axis of the bipolar ganglion cells always
lies in the direction of the nerve.

In the suckers one also finds large multipolar ganglion
cells (fig. 40) ; they usually lie rather nearer to the delimiting
membrane than to the cuticula; their processes usually unite
with those of neighbouring nerve-cells, or they are connected
with each other by their neurofibrille. A connection between
the sucker ganglion cells and the ‘ Sommer-Landois”’ cells
of the subcuticular muscles of the suckers can frequently be
made out.

The nuclei of the sucker ganglion cells measure from 64
to 7 in diameter; they are usually globular and contain a
large nucleolus, measuring up to 24, and one or two chro-
matin bodies. Small glia cells are frequent along the surface
of the cell processes or the neurofibrille.

I am not aware that anyone has yet pointed out the presence
of ganglion cells in the suckers; I have already mentioned

368 LEWIS HENRY GOUGH.

them as occurring in Stilesia centripunctata (Rivolta)
and Anoplocephala magna (Abilgaard), (Gough, 1909).
I have recenti; also observed similar cells in Tenia, only
in Tenia they are smaller than in Anoplocephala or
Avitellina, agreeing in this respect with the other scolex
ganglion cells. Recently, similar cells have been observed
by Spitlich (1909), in the bothridia of Tetrabothrium
laccocephalus and IT’. macrocephalus; he describes
them as being large cells with granular plasma, which stains
deeply with hematoxylin, and with a large nucleus in which
several large chromatin masses are visible. ‘These cells have
branched processes and form a reticulation between the
muscle-fibres by the anastomosing of the processes. ‘These
branched cells are more or less restricted to the middle of
the bothridia, keeping distance from either surface. Spiitlich
thought that they might be glands, but states that his material
was not sufficiently well preserved to allow definite con-
clusions.

It is strange that nobody has yet looked for ganglion cells
in the suckers, as they could be expected to exist in order to
control the working of those complicated muscular organs.

Tae Genrran Oraans. Figs. 1-4 and 41-65; Text-fig. 2.

When the genital organs are in full activity, the recep-
taculum seminis is filled with spermatozoa and the odcytes
are passing from the ovarium into the uterus; the genital
organs are disposed as shown on Text-fig. 2 or fig. 1. The
position of the cirrus pouch to the vagina is extremely
variable, as shown on fig. 43; it can lie anterior or posterior,
dorsal or ventral to the vagina. The figure shows a sagittal
section through about nine sections, passing through four
cirri and vagine ; it will be seen that the utmost possible
irregularity has been realised.

The vagina leads into the spermiduct, which shortly after
having crossed the ventral canal widens and forms the pear-

shaped receptaculum seminis (‘l'ext-fig. 2) ; from the wider

TAPE-WORMS OF THE SUB-FAMILY AVITELLININA. 369

interior end of the receptaculum seminis arises the canalis
seminalis (Befruchtungskanal—fertilisation duct), which, how-
ever, soon branches, sending one arm, the oviduct, to the
ovarium, the other arm, the uterine duct, to the uterus.
Fertilisation of the eggs probably takes place at the point of
junction of the three ducts, as one cannot observe spermatozoa
penetrating into either the oviduct or the uterine duct. The
uterus originates as a simple, hollow, transverse tube, but
fills with cells soon after the arrival of the ova, cells derived
from the uterine walls completely surrounding and embedding
the eggs.

At the same time the male sexual organs consist of a cirrus,
which opens straight into the vagina; the vas deferens forms
several twists and turns within the cirrus-pouch (fig. 4).
Just before entering the cirrus pouch the vas deferens is
considerably swollen with spermatozoa, forming a kind of
vesicula seminalis. ‘I'he vas deferens runs straight across the
dorsal side of the segment, being dorsal to all the other
genital organs, to the nerve and to the excretory canals.
The testicles le near the transverse axis, in four groups of
from three to seven testicles, one group being lateral and one
median to each ventral canal.

The changes that follow as the segments proceed from the
anterior end of the strobila to the posterior end are, first the
disappearance of the ovary and oviduct, then of the canalis
seminalis, then the testicles disappear. ‘The uterine duct,
the receptaculum seminis and the cirrus pouch with the
vagina remain long after their function is past. When the
ovary is disappearing the paruterine organ begins to
develop; finally all the eggs are enclosed in the paruterine
organ (fig. 3).

In their first anlage the cirrus-pouch and the vagina
appear to have a common origin in a clamp of dark-staining
nuclei which collect near one of the lateral margins. Later
on this clump splits into two masses, which are surrounded
by myoblasts (fig. 44). In the middle of each of these
masses a central core of cells differentiates, those which are

370 LEWIS HENRY GOUGH.

to form the vagina advancing in their development perhaps
somewhat more rapidly than those from which the cirrus
arises. ‘I'he central core is at first solid, at this stage it cuts
itself off from the surrounding cells by the formation of a
basal membrane. ‘lhe cavity arises by the cells separating
in the middle of the epithelial cord. At this stage we find
the vagina lined with an epithelium; later on the cells of
the epithelium atrophy and their nuclei disappear. The
fully developed vagina (fig. 45) is lined with fine ciha, all
pointing away from the pore; it is surrounded by a sheath
of large cells, with round, dark-staining nuclei. The
plasma of these cells does not stain; their membranes are,
however, very distinct. In shape, the cells are prismatic,
with all the sides delimited by planes, the ends of the cells
bordering on the parenchym generally forming pyramids.
‘hese cells surround the vagina in a single layer; their
function may be glandular (7), and they certainly help to give
ereater rigidity to the vagina.

The female sexual canals, spermiduct, oviduct, uterine
duct, canalis seminalis and the receptaculum seminis all arise
as solid cords of epithelial cells, which, after having produced
a basal membrane, become hollow. As is the case with the
vagina, the epithelium atrophies, and finally disappears.
Oviduct, canalis seminalis and uterine duct are, when com-
pletely developed, lined with cilia. The receptaculum
seminis is not a simple dilation of the spermiduct due to the
action of the contents, but arises out of a clump of cells which
already show the final shape of the organ.

The ovarium forms at a very early stage a clump of darker
staining’ cells near the middle of the medullary layer. It is
remarkable, when fully developed, as compared with the
ovaries of other cestodes, on account of its compactness, and
also by reason of the fewness of the odcytes produced. The
ovary is bean- or kidney-shaped, the oviduct inserting in a
depression of its dorsal side ; it consists of a number of lobes,
separated from each other by septa (fig. 41); the lobes are all
enclosed by a common outer membrane. The single lobes do

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 371

not contain very many eggs; an ovarium produces at the most
fifty to one hundred eggs, a lobe probably not more than one
dozen. Besides the eggs, each ovarium contains a number
of smaller cells, probably abortive odcytes, which lie scattered
between the eggs and along the walls of the lobes. Fig. 42
represents a single such cell lying between three odcytes,
which have not been completely drawn. For these cells I
propose the name ovarial nutritive cells, on account of their
probable function. ‘They are much smaller than the odcytes

and are fairly rich in plasma. In shape they accommodate

3

themselves to the space at their disposal between the eggs.
They are very extensively in contact with the eges, sending
out plasmatic processes along and over the surface of the
surrounding odcytes. Their nuclei are oblong, rather pale,
especially as compared to those of the odcytes, and contain a
varying number of small round chromatin bodies. The
diameter of a nucleus averages 17 4 by 9 wu.

There seems to me to be no possible doubt about the
function of these cells. They act vicariously for the missing
yolk-cells, and supply the odcytes with nutriment by means
of the processes touching and covering their surface. <Ac-
cording to the accepted theory, the yolk-gland (vitelline
gland) is only a modified ovarium, and the yolk-cells modified
odcytes. Should this theory be correct, it would render the
mutation of odcytes into nutritive cells within the ovarium
easy to imagine, the chief difference from the state of things
existing elsewhere being, that here both nutritive cell and
odcyte arise in the one ovary, elsewhere the odcytes arise in
the ovary and the nutritive cells (yolk-cells) in a modified
ovary, the yolk-gland. A very important difference, however,
still remains. The yolk-cell of other cestodes becomes attached
to the oocyte, and remains closely united to it for a long
period after having handed over to the odcyte its supply of
yolk, being enclosed in the shell with the odcyte. In
Avitellina and Stilesia the nutritive cell is only tem-
porarily connected with the odcyte, it does not leave the
ovarium when the oocyte does, and finally one nutritive cell

372 LEWIS HENRY GOUGH.

has relations to several odcytes at the same time. For all
that, ovarial nutritive cell and yolk cell have in common, that
both are modified odcytes. The fact that in Avitellina
and Stilesia the ovarium has in some degree the double
function of ovary and vitelline gland, might be taken as
proving the two genera as being primitive.

The oocytes appear to mature in the ovarium, the reduction
of the chromatin taking place before the eggs leave the
ovarium. In this respect they differ from the odcytes of
Tenia serrata, studied by Janicki (1907), which mature in
the uterus after the penetration of the spermatozoon into the
egg-cell. The process of ripening is complicated by the
passing of chromatin out of the nucleus into the plasma.
Spatlich (1909) observed somewhat similar phenomena in
Tetrabothrium before the mitoses take place. For the
study of these changes it is necessary to use material that
has been stained with iron-hzematoxylin, and to compare
with sections stained by other means, as very much depends
on the technique of the specimen. Figs. 57-65 show in a
series of eggs the most important changes that take place
before maturation and immediately after fertilisation. Fig.
57 is the youngest stage illustrated; it represents an odcyte
that has already reached its full size. Its plasma shows no
modifications whatever, being perfectly homogeneous. A
nucleolus is not visible; the chromatin forms a large round
mass. Fig. 58 shows a somewhat older odcyte ; a nucleolus
has appeared, and in the plasma two centrosomes can be
observed. ‘lhe centrosomes in the cestode odcyte are quite
large objects, as already observed by other authors (e. ¢.
Janicki, 1907). When they first appear they have a light
centre surrounded by a dark ring. On the same cell one sees
that the chromatin is lying quite on the nuclear membrane,
causing it to press out somewhat. Fig. 59 is also of about the
same stage; in the cell shown a portion of the nucleus
appeared to be cutting itself off from the main portion. I
cannot say whether this is a regular occurrence or no, as I
have only observed it in a single cell. The next changes are

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 373

shown on fig. 60; here we find that the size of the chromatin
body in the nucleus is reduced, and that now there have
appeared two dark-staining bodies in the plasma, lying quite
close to the nucleus and just opposite the chromatin body.
In iron-hematoxylin these bodies stain to the same intense
black as the chromatin ; with Delafield’s hematoxylin they
also stain blue, but not quite as deep as the chromatin. I
consider them to be portions of the chromatin which have
been ejected from the nucleus ; in the course of the further
development of the egg they behave as the yolk-nucleus
observed by Janicki does. They are certainly not the same
as the yolk-nucleus observed by Spitlich in Tetrabothrium,
I have not seen any structure to compare with Spatlich’s
yolk-nucleus. There are usually only two of these problematic
bodies present ; occasionally, however, as shown by fig. 62, a
larger number can appear. Whilst they were at first situated
close to the nucleus, in older ovaria we find that they have
moved further away from it, until they have gone as far as
possible away from the odcyte nucleus. Fig. 61 shows the
emigrant chromatin bodies moving away from the nucleus,
and shows that they have also separated from each other at
the same time. A further change in the nucleus can be noted,
the linin threads are becoming distinct. Figs. 63-65 show
that a mitosis is now becoming imminent; the chromatin mass
has entirely disappeared, and the chromatin has rearranged
itself on the linin threads; the nucleolus, at first large, is
reducing its size. But it is also remarkable that not only has
the chromatin in the nucleus changed its arrangement, but
also the chromatin that had wandered into the plasma. In
the place where the emigrant chromatin bodies had been one
now finds a mass of fine granules, and finally the only remain-
ing trace is a darker staining of the plasma in the vicinity of
the place they had occupied. It is worth noting that a pair
of centrosomes are often seen close to the emigrant chro-
matin before it dissolves.

The mitoses that follow have been observed, but in speci-
mens stained with Ehrlich’s hematoxylin considerable luck

374 LEWIS HENRY GOUGH.

is necessary to obtain material where mitoses are taking
place, as they pass very quickly. Usually only two or three
segments at the most show mitoses; those before and after
not containing any; the difficulty of obtaining sections through
the exact portion of the strobila is consequently easy to
appreciate. There appear to be four chromosomes, the exact
number being hard to count, as the karyokinetic figure is
very small. I have not been able to make out the fate of
the pole bodies, but when the eggs pass into the oviduct
they are no longer to be found. The matured odcytes are
not enclosed in a membrane of any kind; they arrive naked
in the uterus, not having a membrane of their own, and
not receiving a shell, as there is no shell-gland. The
fertilised odcytes are at first not enclosed im shells or by
membranes. Fig. 49 shows four odcytes in the uterus in
various stages of development ; in odcytes a and b one
sees the spermatozoa as short rod-shaped bodies, stained
black by the hematoxylin, lying in a dark area, stained
bright red by eosin. Odcyte ¢ shows the sperma nucleus
and the egg nucleus fusing in the same manner as described
by v. Janicki (1907) for the eggs of Twenia serrata. The
plasma of the eggs in these first stages after fertilisation 1s not
homogeneous, but contains larger and smaller masses of
differently staining substance.

The later fate of the eggs has not been followed up; at
first, however, the multiplication of the nuclei is not followed
by division of the plasma, so that up to four nuclei can be
seen in an undivided mass of plasma. Later on one observes
quite regularly that the cell division gives rise to a few
macromeres, and to a greater number of micromeres, the
macromeres probably giving rise to the ege envelopes, the
micromeres to the embryo, as demonstrated by Janicki
(1907) for Tzwnia serrata. The exact number of macro-
meres is difficult to ascertain accurately: one is always larger
than the others ; two can usually be recognised, but I am
not certain whether there is a third or no.

Soon after the eggs arrive in the uterus they become

TAPE-WORMS OF THE SUB-FAMILY AVITELLININ’E. 375
surrounded by smaller cells, derived from the walls of the
uterus. These cells, which have already been observed by
Fuhrmann (1909) in Stilesia sjéstedti, Fuhrmann, can
be termed uterine nutritive cells; their function is without
doubt nutritive, as already suggested by Fuhrmann. The
uterine nutritive cells (fig. 49) soon fill all the space between
the eggs. ‘They are rich in plasma at first ; later on their
plasma decreases, and finally they atrophy and disappear
entirely at or before the stave when the egg envelopes have
developed.

Fuhrmann (1909) supposed the uterus of Stilesia to have
no cavity originally. This is not correct for Avitellina;
the uterus is a simple hollow tube at the time of the arrival
of the eges, the uterine nutritive cells appearing shortly after-
wards,

The absence of a vitelline gland, as can be seen, has had
such an influence on the cestode as to have caused cells of
two separate and distinct origins to arise in two different
organs to replace to some extent the nutritive function of the
missing organ, that is, if the vitelline gland has been lost in
the history of the genera. If, on the other hand, the lack of
a vitelline gland is a primitive character, the acquisition of
uterine nutritive cells must still be a recent adaptation, as one
would otherwise expect to meet snch cells in other cestodes.

The uterine wall cells, after having given rise to the uterine
nutritive cells, next supply the origin of the egg-pouches or
paruterine organ.' Inthe first stages of the development of
this organ we find parallel layers of plasma containing nuclei
splitting off the terminal wall of the uterus (fig. 51). The

' Tt is a question which I cannot attempt to decide, whether the par-
uterine organ of Stilesia and Avitellina is homologous to the par-
uterine organ of other cestodes, as where it has been observed previously
it has generally been held to arise outside the uterus. I am retaining
the name as being convenient and as referring to a more or less well-
known structure, but without prejudice as to its origin in other species.
The paruterine organs of various cestodes may quite possibly be of diffe-
rent origin, and may only be convergent structures, as Fuhrmann has
shown that they can arise independently in various unrelated genera.

VOL. 96, PART 2.—NEW SERIES. 26

35376 LEWIS HENRY GOUGH.

nuclei are oblone

oD?

resemble to some extent those of the uterine nutritive cells.

with two or three chromatin bodies, and

The presence of the developing paruterine organ causes the
wall of the uterus to bulge outwards at a very early stage.
The portion thus pressed outwards is at first hemispherical,
opening directly into the main body of the uterus, this portion
might be termed the paruterine pouch (fig. 53). The next
changes take place very quickly, and we find the mouth of the
pouch contracting, the contraction being effected by muscles
running around this portion of the uterus. As the contraction
proceeds the pouch becomes more nearly globular, until it is
finally almost spherical, remaining connected with the
remainder of the uterus by a very narrow passage. ‘These
changes of their receptacle are not without their effect on the
lamellee, whose shape has to accommodate itself to the changes
in form of the pouch; as they remain arranged parallel to the
walls of the pouch they, too, become spherical; but the
lamelle are also growing quickly, and some of them force
their way into the uterus as concentric hemispheres, whilst
others form concentric hemispheres within the pouch. The
lamellee projecting into the uterus appear to grow very
quickly, pressing the uterus and its eggs further and further
away from the pouch; the uterus, which was at first merely
a transverse tube, becomes globular through the invasion of
the lamelle, which have become almost spherical. Finally, as
the lamelle are still growing and as they have no more room
to spread outwards, the only outlet remaining for them is by
doubling back. Depressions appear on the surface of the
lamelle, deepen, pass through the neck imto the pouch,
varrying the eges, which are already enclosed in their two

oo”?

envelopes and contain embryos, with them into the paruterine
pouch. Finally, the lamella appear to complete the process
of retroversion and return entirely through the neck of the
pouch: none are left outside the pouch at all events. The
growing back always takes place at several points at the same
time, thus giving rise to more than one pocket; six or seven
are usually formed (figs. 55-56, pp.).

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 377

The lamellze are at first composed of a mass of plasma
belonging to several nuclei and are fairly thick ; with further
development the thickness of the lamellae becomes less and
less, and they finally resemble thin fibrillae on section. There
can, however, be no doubt that the structure is lamellar and
not fibrillar, as in whatever direction a section may pass
through a paruterine organ, one always sees a concentric
arrangement of ‘ fibres”? running within the plane of the
section, but never by any chance sections through fibrille.
As the lamelle grow older the nuclei atrophy and finally
disappear.

Directly anterior to the uteri lie pads of fibrous tissue
(fig. 52), whose probable function is to give support to the
paruterine organ, and perhaps also to act as cushions between
the uteri and paruterine organs of adjacent segments. ‘l‘hese
pads take their origin from myoblasts, which are very frequent
close in front of the uteri when the eggs are begining to enter
that organ. ‘The fibrous tissue, when fully developed, contains
scattered nuclei, and its fibrillar structure is fatrly apparent.
It stains fairly vividly with eosin (fig. 48).

The cirrus and cirrus pouch arise out of a common anlage
with the vagina, as already explained above. The dense
mass of dark-staining nuclei, which gives rise to both, first
splits into two masses; these become surrounded by myoblasts
(fig. 44) ; then, in the middle of one of the two masses the
vagina begins to develop, the cirrus and part of the vas
deferens in the other. The epithelial cord arising in the
middle of the cirrus pouch anlage is at a very early stage
already convoluted, probably giving rise to the twisted portion
of the vas deferens enclosed in the cirrus pouch

The vas deferens is, like the female ducts, formed of a solid
cord of epithelial cells surrounded by a basal membrane
(fig. 46); the hollow arises later. As in the female ducts, the
epithelium finally atrophies entirely. Its lumen is then
clothed with cilie.

The cirrus is straight, 344 long by 3, in diameter ; it is
devoid of hooks, bristles or cilia of any kind; it opens

ORS

378 LEWIS HENRY GOUGH.

directly into the vagina. I have never observed an intro-
mission of the cirrus into the vagina. The cirrus muscles
consist almost entirely of a circular muscle ; the longitudinal
muscle usually observed between the circular muscle and the
cirrus appears to be wanting or extremely weak. Cirrus and
vas deferens within the cirrus pouch are suspended by loose
parenchym cells, which stretch themselves from the walls of
the pouch, passing around and gripping the vas deferens.
They act as a kind of mesentery in function (fig. 50).

The muscles of the cirrus pouch are very feeble. The
whole cirrus apparatus gives one the impression that the
cestode has lost the habit of cross-fertilisation, and that self-
fertilisation has become the rule.

ConcLusions.
(1) There is no subcuticula in the suckers.
(2) The cuticula can arise independently of the subcuticula.
(3) The subcuticular cells stand in direct connection with
the dorsoventral and transverse muscles and form part of them.

(4) The parenchyma muscles are produced by three kinds
of myoblasts: (a) Bipolar myoblasts with terminal fibrillee ;
(b) bipolar myoblasts with lateral fibrilla; (c) elongate
bipolar myoblasts lying axially in the tubiform muscles of the
longitudinal muscle.

(5) The dorsal canals are surrounded by nephrocytes, which
are homologous with the parent cells of the flame-cells.

(6) There are ganglion cells in the suckers, connected by
neurofibrille with the “Sommer-Landois” cells of the
subcuticular muscles of the sucker.

(7) In addition to odcytes, nutritive cells are produced in
the ovaries. ,

(8) The odcytes mature before leaving the ovarium.

(9) The odcytes arrive fertilised in the uterus.

(10) The odcytes in the uterus are surrounded by nutritive
cells of uterine origin.

(11) The paruterine organ is contained in a pouch of the
uterus, and arises within the uterus; its structure is lamellar.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 379

LITERATURE.

Balss.—“ Uber die Entwicklung der Geschlechtsgiinge bei Cestoden,”
‘ Zeit. wiss. Zool.,’ vol. xci, 1908.

Bartels.—“ Cysticercus fasciolaris, Anatomie; Beitrige zur Ent-
wicklung und Umwandlung in Tenia crassicollis.” ‘ Zool.
Jahrb. Anat.,’ vol. xvi, 1892.

Blochmann.—* Uber freie Nervenendungen und Sinneszellen bei Band-
wirmern,” ‘ Biol. Centralbl.,’ vol. xv, 1895.

—— ‘ Die Epithelfrage bei den Cestoden und Trematoden, Hamburg,
1896.

Braun.—* Cestodes,’ Bronn’s ‘ Klassen und Ordnungen des Thier-
reichs, Leipzig, 1894-1900.

Bugge.— Zur Kenntniss des Excretions-gefiss-systems der Cestoden
und Trematoden,” ‘ Zool. Jahrb. Anat.,’ vol. xii, 1898.

Cohn.— Untersuchungen iiber das Centrale Nervensystem der
Cestoden,” ‘ Zool. Jahrb. Anat.,’ vol. xii, 1898.

——— “Zur Anatomie der Amphilina foliacea (Rud.),” * Zeit. wiss.
Zool., vol. lxxvi, 1904.

Fuhrmann.—* On the Anatomy of Prosthecotyle torulosa and
heteroclyta,” ‘P. R. 8. Edinb.,’ vol. xxii.

——— “Die Systematik der Cyclophyllidea,” ‘Zool. Anz.,’ vol. xxxii,
1908.

——— “ Cestodes,” ‘ Wissenschaftliche Ergebnisse der Schwedischen
Expedition nach dem Kilimandjaro, dem Meru und den umge-
benden Masaisteppen Deutsch Ost Afrikas,’ Stockholm, 1909.

Gliser.—* Die Entwicklungsgeschichte des Cysticercus longicollis
Rud.,” ‘ Zeit. wiss. Zool.,’ vol. xcii, 1909.

Gough.—* Notes on some South African Parasites,” ‘S. A. A. A. 8.,’
Grahamstown, 1908.

“The Anatomy of Stilesia centripunctata (Rivolta),”
‘The Transvaal Veterinary Bacteriological Laboratories,’
Pretoria, 1909. 3

Hamann.—* Tenia lineata Goeze, eine Teenie mit flichenstiindigen
Geschlechts 6ffnungen,”’ * Zeit. wiss. Zool.,’ vol. lvi, 1893.

Hein.—* Beitriige zur Kenntniss von Amphilina foliacea,” ‘ Zeit.
wiss. Zool.,’ vol. Ixxvi, 1904.

—_—— “Zur Epithelfrage der Trematoden,” ‘ Zeit. wiss. Zool., vol.
Ixxvii, 1904.

380 LEWIS HENRY GOUGH.

y. Janicki.—* Uber die Embryonalentwicklung von Tenia serrata
Goeze,” ‘ Zeit. wiss. Zool.,’ vol. Ixxxvii, 1907.
Krimer.— Beitriige zur Anatomie und Histologie der Cestoden der
Siisswasser fische,”’ ‘ Zeit. wiss. Zool.,’ vol. iii, 1892.
Khlodkowsky.—* Contributions 4 la Connaisance des Ténias des
Ruminants,” ‘Arch. parasit.,’ vol. vi, 1902.
Lang.—* Das Nervensystem der Cestoden im Allgemeinen und dasjenige
der Tetrarhynchen im Besonderen,” * Mittheilungen aus Neapel,’
vol. ii, 1881.
Leuckart.— Die menschlichen Parasiten und die von ihnen herrith-
renden Krankheiten,’ vol. i, 1879-1886.
y. Linstow.— Compendium der Helminthologie Hannover,’ 1889.
Liihe.—* Die Anordnung der Muskulatur bei Dibothrien,” ‘ Centralbl.
Bact.,’ vol. xxii.
“Beitriige zur Kenntniss des Rostellums und der Scolex-
muskulatur der Tzenien,” ‘ Zool. Anz.,’ vol. xvii, 1893.
“Zur Morphologie des Tznienscolex,” ‘ Inaugural Dissertation,’
Konigsberg, 1894.
—— “Zur Kenntniss der Muskulatur des Tzenienkorpers,” ‘ Zool.
Anz., vol. xix, 1895.
“Das Nervensystem von Ligula,” ‘ Zool. Anz.,’ vol. xix, 1895.
Lunewitz.—* Tenia ovilla Rivolta, ihr anatomischer Bau und die
Entwicklung ihrer Geschlechtsorgane,” ‘Archiv f. wiss. und
prakt. Thierheilkunde,’ vol. xxi, 1895.
Minckert.—“ Mitteilungen zur Histologie der Cestoden—(1) Uber Epi-
thelverhiltnisse und Struktur der Koérpercuticula,” ‘ Zool. Anz.,’
vol, xxix, 1905.
Mingazzini—* Sullesistenza di una secrezione emissa dalla superficie
del corpo dei Cestodi adulti, Atti Acad. Lincei.,” ‘Cl. Mat. Nat.,’
vol. x, 1907.
Neumann.— Traité des Maladies parasitaires non-microbiennes des
Animaux domestiques, Ist edition 1886, 2nd edition 1895.

“ Observations sur les Ténias du Mouton,” ‘C. R. Soc. Biol.,’
Toulouse, 1891.

Nitsche.—* Untersuchungen iiber den Bau der Tzenien,” ‘ Zeit. wiss.
Zool., vol. xxiii, 1873.
Perroncito— I parassiti dell’Uomo e degli Animali utili,” Milano, 1882.

‘Trattato teorico-pratico sulle malattie piu comuni degli
Animali domestici dal punto di vista agricolo, commerciale et
igienico, Torino, 1886.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 381

Perroncito.— Trattato teorico-pratico,’ 2nd edition, 1902.

Pinter.—* Untersuchungen iiber den Bau des Bandwurmkérpers mit
besonderer Beriicksichtigung der Tetrabothrien und Tetra-
rhynchen,” ‘ Arb. Zool. Inst. Wien.,’ vol. iii, 1881.

—— “Studien iiber Tetrarhynchen nebst Beobachtumgen an anderen
Bandwiirmern ” (iii Mitteilung), ‘Sitzber. k. Akad. Wiss. Wien.
Math. Nat. KL,’ Bd. exii, 1905.

Railliet.—* Elements de Zoologie Médicale et Agricole, Paris; 1st

edition 1886, 2nd edition 1895.

“Sur quelques parasites du Dromadaire,” *‘C. R. Soc. Biol.,’

1896.

Ransom.—* The Tznioid Cestodes of North American Birds,” ‘Smith-
sonian Institution Bull., lxix, 1909.

Rivolta.— Sopra aleune specie di Twenia della Pecora, Pisa, 1874.

Rossler.—* Uber den feineren Bau der Cysticerken,” ‘ Zool. Jahrb.
Anat.,’ vol. xvi, 1902.

Salensky—“ Uber den Bau und die Entwicklungsgeschichte von
Amphilina,” ‘ Zeit. wiss. Zool.,’ vol. xxiv, 1874.

Schiefferdecker.—* Beitrage zur Kenntniss des feineren Baues der
Tenien,” ‘ Jena Zeit. Naturw.,’ vol. viii, 1874.

Schneider.— Lehrbuch der vergleichenden Histologie der Tiere, Jena,
1902.

Spatlich—* Untersuchungen iiber Tetrabothrien, Ein Beitrag zur
Kenntniss des Cestodenkérpers,” ‘ Zool. Jahrb. Anat.,’ vol. xxviii,
1909.

Stiles.—* Bemerkunyen iiber Parasiten: (17), Uber die topographische
Anatomie des Gefiiss-systems der Familie Twniade,” * Centrbl.
Bact.,’ vol. xiii, 1895.

— “A Revision of the Adult Cestodes of Cattle, Sheep, and Allied
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No. 4, Washington, 1895.

Tempére.—* Parasites internes de homme et des animaux domestiques,”
‘ Micrographe préparateur, vol. xiv, 1906.

Vaullegeard.—* Métamorphoses et Migrations du Tetrarhynchus
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Will.—* Anatomie von Caryophylleus mutabilis, Rud., ein Beitrag

zur Kenntniss der Cestoden,” ‘ Zeit. wiss. Zool.,’ vol. lvi, 1893.

Wolffhiigel —“Stilesia hepatica, nov. spec., ein Bandwurm aus den

Gallengingen von Schafen und Ziegen Ost Afrikas,” ‘ Berliner
Tieriirzl. Wochenschr.,’ No. 43, 1903,

382 LEWIS HENRY GOUGH.

Zernecke.—* Untersuchungen iber den feineren Bau der Cestoden,”
‘Zool. Jahrb. Anat.,’ vol. xxvi, 1908.

Zschokke.— Recherches sur la structure anatomique et histologique
des Cestodes,’ Geneve, 1890.

EXPLANATION OF PLATES 12-14,

Illustrating Mr. Lewis Henry Gough’s ‘ Monograph of the
Tape-worms of the Sub-family Avitellinine, beme a
Revision of the Genus Stilesia, and an Account of the
Histology of Avitellina centripunctata (Riv.).”

PLATE 12.
Avitellina centripunctata (Rivolta).
Fig. 1.—Portion of three segments at male sexual ripeness. x 99.
Fig. 2—Oncosphere. x 588.
Fig. 5—Four segments with fully developed paruterine organs. x 99.
Fig. 4.—Cirrus-pouch and vagina. xX 380.
. 5.—Left half of the excretory canals of scolex, lateral view.
Fig. 6.—Right half of the excretory canals, lateral view.
Fig. 7.—Scolex.
Stilesia vittata, Railliet.
Fig. 8.—Pore-side of a segment at male sexual ripeness. x 99.

Fig. 9—Oncosphere. x 588.

Stilesia globipunctata (Rivolta).

Fig. 10.—Pore-side of a segment at male sexual ripeness. x 99.
Fig. 11.—Oncosphere. x 588.
r. 12.—Scolex.

Stilesia hepatica, Wolffhiigel.
Fig. 15.—Pore-side of a segment at male sexual ripeness. x 99.
Fig. 14.—Segment with developing paruterine organs. x 99.
Fig. 15.—Oncosphere. x 588.

Fig. 16,—Cirrus pouch and vagina. x 380,

TAPE-WORMS OF THE SUB-FAMILY AVITELLININE. 385

PLATE 13.

Avitellina centripunctata (Rivolta).

Fig. 17.—Cuticula of the suckers. x 784.

Fig. 18.—Cuticula of the proglottids, silver nitrate fixation. x 784.

Fig. 19.—Subcuticula and cuticula. x 784.

Fig. 2).—Subcuticula near the lateral margin. »x 784.

Fig. 21.—Cuticula and subcuticula in an old segment. x 392

Figs. 22, 25.—Dorso-ventral muscles. x 784.

Figs. 24, 25.—Transverse muscles. x 784.

Figs. 26, 27.—Longitudinal muscles. x 784.

Fig. 28.—‘ Sommer-Landois” cell, from the scolex. x 784.

Fig. 29.—** Sommer-Landois” cells of the “ subeuticular’’ muscles of
the suckers. x 784.

Fig. 50.—Ventral canal, 25 cm. from the scolex, showing epithelial
cells sinking into the parenchyma. x 784.

Fig. 51.—Dorsal canal, 50 em. from the scolex, showing nephrocytes.
x 784.

Fig. 52.—Epithelial cell of the ventral canal sinking into the paren-
chyma to become a parent of flame-cells. x 754.

Fig. 55.—Ventral canal, 1 cm. from the scolex, with developing flame-
cells and a group of young flame-cells. x 784.

Fig. 34.—Flame-cell. x 784.

Fig. 35.—Group of flame-cells, reconstructed from three consecutive
sections. xX 392.

Tenia serrata, Rud.
Fig. 56.—Developing flame-cells. x 784.

Avitellina centripunctata (Rivolta).

Fig. 37.—Flame-cell. x 2552.

Fig. 38.—Multipolar ganglion-cells from the central ganglion of the
scolex. x 784.

Fig. 39.—Bipolar ganglion-cell from one of the nerves in the scolex,
showing tigroid bodies. x 784.

Fig. 40.—Maultipolar ganglion-cell from the suckers. x 784.

Fig. 41.—Young ovarium. x 588.

Fig. 42.—Nutritive cell lying between three oocytes in a young
ovarium. x 1146.

384. LEWIS HENRY GOUGH.

PLATE 14.

Avitellina centripunctata (Rivolta).

Fig. 45.—Sagittal section through nine segments, showing cirrus
pouches and vagine of four segments. x 88.

Fig. 44.—Anlage of vagina and cirrus. X 784.

Fig. 45.—Transverse section of vagina. xX 784.

Fig. 46.—Anlage of vas deferens. x 1176.

Fig. 47.— Developing oviduct. x 1176.

Fig. 48.—Fibrous tissue. x 784. ;

Fig. 49.— Portion of uterus, with odcytes surrounded by nutritive
cells. x 1176.

Fig. 50.—Transverse section through a cirrus-pouch, showing the
vas deferens suspended by ‘loose parenchym cells.” x 754.

Fig. 51.—Anlage of paruterine organ on the uterus-wall; three
oocytes are represented surrounded by nutritive cells. x 784.

Fig. 52.—Developing paruterine organ. x 190.

Fig. 53.—Developing paruterine organ ; the “ pockets” (pp.) are just
forming. xX 190.

Fig. 54.—Paruterine organ: a somewhat older stage than fig. 53.
x 190.

Fig. 55.—Paruterine organ with eggs entering the pockets. x 190.

Fig. 56.—Fully developed paruterine organ; all the eggs have
entered the pockets of the pouch.

Figs. 57-65.—Oodcytes in various stages.

List oF ABBREVIATIONS USED.
|The abbreviations used are the same throughout the paper. |

a. Sucker. at. Cloaca. b. im. Basal membrane. b. p. Basal plate.
e. Cirrus. c¢.c¢. Connecting canal. ch. Chromatin. c. 1. Comidial
layer. c.p. Cirrus pouch. c.s. Canalis seminalis. cu. Cuticula.
d. Dividing nucleus. d.c. Dorsal canal. e. Egg. ep. Epithelium.
ep.c. Epithelial cell. f. Fibrilla. jf. c. Flame-cell. jf. ¢.d. Developing
flame-cell. ff. Flame. /. p. Fibrous pad. ju. Funnel. g. Gland-cells
surrounding the vagina. gl. Glia-cells. h. 1. Homogeneous layer. 7. d.
Interuterine duct. k. Capillary. k.c. Capillary cell. /. Lumen.
1.7. Inner layer. 7.0. Outer layer. m. Muscle. mb. Myoblast. m/f.
Muscle-fibrilla. mn. Nerve. wn. c. Nutritive cell. xf. Neurofibrilla.

TAPE-WORMS OF THE SUB-FAMILY AVITELLININ®. 385

nl. Nucleolus. iu. Nucleus. 0. Ovarium. od. Oviduct. p. Par-
uterine organ. par. * Parenchyma” cell. suspensory cell in the cirrus
pouch. pow. Paruterine pouch. +. Nephrocyte. +. f. Radial fibres.
vr. s. Receptaculum seminis. s. Spermatozoon. — s. ¢. Subcuticular cell.
s. 1. “Sommer-Landois ” cell. s. m. Subcuticular muscle. f¢. Testicle.
t.b. Tigroid bodies. ¢.c. Transverse canal. uv. Uterus. w.d. Uterine
duct. v. Vagina. v:c. Ventral canal. v.d. Vas deferens. v.s.
Vesicula seminalis. a. Corpuscle. y. Centrosome. z. Fusing sperma
and egg nucleus

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BRIEF NOTES ON SPIROCHETA ANODONTA KEYSSELITZ. 387

Brief Notes on the Structure and Development
of Spirocheta anodonte Keysselitz.

By
W. Cecil Bosanquet, M.D.

With Plate 15.

Mucw uncertainty still prevails as to the structure and
biological relationships of the spirochetes. Earlier observers
—not altogether influenced perhaps by a desire to prove
their affinities with the hemoflagellates—equipped them with
an undulating membrane and a complicated nuclear apparatus,
the existence of which is very problematical ; and much con-
troversy has existed as to their mode of division, longitudinal
or transverse. ‘The very claim of the organisms found in
Mollusca, as well as that of the smaller spirochetes, to be
called by this name, depending as it must on their relation-
ship to Sp. plicatilis Ehrenberg, the type-species, is not

very firmly established, since the structure of the latter—a
system of large waves, each broken by smaller undulations
superposed on them, with a central axial rod and a series of
dots, regarded as nuclei—is markedly different from that of
the later additions to the genus. It is desirable, therefore,
to record any observations which may tend to throw lght
on the morphology and life-history of these organisms.

My own cbservations have been carried out on Sp.
anodonte, a species easily obtained, and one which may be
regarded as fairly typical of the spirochetes which are
parasitic in Mollusca.

With regard to the so-called undulating membrane of

VOL. 56, PART 2,—NEW SERIES. 27

388 W. CECIL BOSANQUET.

Sp. anodonte I am inclined to agree with Schellack, who
holds that the appearance which has been thus interpreted
is an artifact, due to splitting of periplast, smce I found
that the better my preparations were fixed, the fewer were
the examples of this condition. It was found almost invari-
ably in films dried in the air (fig. 1), but very exceptionally
in those fixed in osmic vapour. It seems possible that the
periplast, if it is so to be called, adheres to the slide in the
position in which death of the spirochzte occurs, and that
subsequently the protoplasm of the organism shrinks and
thus straightens its outline a little, leaving a line of periplast
following a different line from that finally adopted. At
other times the periplast seems to shrink most and to form a
band uniting the cells of the spirochete (fig. 2).

I have seen a considerable number of specimens which
exhibit a dark line running along one side (figs. 5, 4). This
probably corresponds with the “ crest ” described by Gross.
Judging from the illustrations of spirochetes in section
given by him and previously by Fantham, it would seem that
the organism possesses a sheath which is loose enough to
form a fold along one side in certain conditions. ‘I'he
sheath or periplast stains more darkly than the body-sub-
stance of the spirocheete, and hence the double layer appears
as a dark line. Splitting of the sheath into fibrille seems
sometimes to occur, as in fig. 6.

On the other hand, I find it difficult to agree with Schellack
that two separate species of spirochete are present together
in Anodonta, one having blunt (figs. 1, 5, 4) and the other
sharp extremities (figs. 7, 8, 9). The amount of difference
seen is scarcely sufficient to suggest a specific differentiation.
Further, the great variation in the length and thickness of
the individuals in each class thus formed is such as to nega-
tive the possibility of distinguishing species by length, as he
further suggests. It is noteworthy that Schellack distinguishes
two species of spirochzete in more than one kind of mollusc,
and that Gross also describes two, very similarly differentiated,
in Pecten jacobeus. This rather strongly suggests that

BRIEF NOTES ON SPIROCH®TA ANODONTZ KEYSSELITZ. 389

the same spirochete may present different shapes and sizes.
I have never seen specimens of Sp. anodonte with trans-
verse lines near the middle of the length of the organism,
pointing to transverse division by means of a septum here
formed, as is depicted by Schellack.

As to the actual method of division, I had the opportunity
of observing an instance of what appeared to be longitudinal
fission. This was in a preparation containing living spiro-
chetes in a drop of fluid from the mantle-cavity of the
mussel. My attention was attracted to an individual which
remained fixed at one point, near to the edge of tle prepara-
tion, and wriggled feebly, instead of dashing about lke the
others. I then noticed that this spirochete seemed to
possess two “tails,” if this expression is allowable (fig. 11a) ;
and on watching it I noticed that a line, dark at one moment,
refringent at another, as the creature moved, was gradually
extending along the body of the spirochete from the point
of bifurcation towards the opposite extremity. Fission pro-
ceeded along this line, but not quite regularly as in tearing
a strip of calico, since at one moment there was visible a sort

‘

of loop in the ‘‘ anterior” part of the organism—using this
term for the portion which remained single longest—before
the process of division had extended quite to this point from
“behind ” (figs. 11b, c). When the longitudinal fissure had
extended nearly to the “anterior” end, the organism sud-
denly jerked itself away out of the field of the microscope-
and I was unable to see the final separation of the daughter,
individuals ; but as I was unable, on searching through the
preparation, to find the dividing spirochzte again, I could
only conclude that the separation took place almost imme-
diately, the two resulting spirochetes being then indistin-
guishable from the other individuals present.

A possible source of error in this observation hes in the
slenderness of the organisms and in the difficulty of accurately
observing them during movement. It is scarcely possible to
exclude the explanation that an organism may become folded
upon itself as a preliminary to undergoing transverse fission

390 W. CECIL BOSANQUET.

(figs. 12, 13). This alternative possibility was strongly
brought to my mind by a preparation in which many living
spirochetes appeared to have loops at one extremity, while
they revolved rapidly on a longitudinal axis. As their move-
ments slackened, these organisms appeared to have a shape
which might be compared with that of a hair-pin having its
“leas” twisted together. On fixing and staining this pre-
paration, specimens were found both of the condition just
described (fig. 14), and also of what seemed to be a succeed-
ing stage, in which division had taken place at the bend of
the loop (fig. 15). I could find no instance of a spirochaete
simply looped at one extremity, as might have been expected
to occur if longitudinal division took place, starting at a
point a little distant from the extremity of the spirochete.
I was at first tempted to regard the pairs of closely apposed
organisms here seen as being instances of conjugation, but
the explanation just given seems more probable in view of
the looped forms seen alive in the preparation. Division by
“incurvation ” is stated by Gross to occur in the spirochete
present in Pecten jacobeus (called by him Cristispira
pectinis), and he believes it to be the characteristic of all
spirochetes. Nuttall, Fantham and Porter state that in the
small spirochetes both transverse and longitudinal division
occurs. If this be so, it would be interesting to ascertain
whether any difference in environment is responsible for
their adoption of each method respectively, or whether the
two processes occur at different points in the life-cycle.
With regard to the internal structure of Sp. anodonte,
I have always found that specimens taken from the crystal-
line style of the mussel stain homogeneously like Bacteria
(fig. 7). Those, however, which are present in the stomach
of the mollusc, especially after the style has dissolved, show
beading of the substance of the organism, as is depicted by
Keyssehtz in his original memoir (figs. 16, 17, 18). Since
the style is apparently the most favourable medium for the
life of the spirochaete, I am inclined to look upon the beaded
forms as either involutionary or developmental. The fol-

BRIEF NOTES ON SPIROCHHTA ANODONT® KEYSSELITZ. 391

lowing observation supports the view that the change is, in
some cases at least, one of development.

A piece of crystalline style was dissolved in tap-water and
sealed up with vaseline for forty-eight hours under a cover-
slip on a slide. At the end of this period the cover was
removed, and the preparation was fixed in osmic vapour and
stained with thionine. The spirochxtes, which had originally
been very numerous, were found to have almost entirely dis-
appeared, while their place was taken by elongated, variously
curved bodies, closely corresponding with them in length and
thickness, containing irregular masses of staiming substance
(figs. 19 and 20). Some definite spirochaetes present showed
beading of protoplasm and separation into segments (segmen-
tation, plasmolysis?), and it was possible to trace a fairly
definite series of gradations between such spirochetes, as
seen in this and other preparations, and strings of bead-like
or coccoid bodies, which seemed to constitute the final stage
of development (figs. 16, 18, 21, 22). It seems then that
Sp. anodonte goes throngh a stage in development in
which it breaks up into coccoid bodies, just as, according to
Leishman, Sp. duttoni breaks up into similar bodies inside
the body of the tick. If this be so we have, on the one
hand interesting evidence of the generic unity of the small
and large spirochetes, and on the other, of the affinity of
the whole group with the Bacteria rather than with the
Protozoa.

The portion of style used for the above observation was
taken from a mussel which had been kept at a temperature
just above freezing-point. The spirochaetes seemed specially
numerous and lively at this temperature, and dividing forms
were numerous. I also found some organisms in the water
in which the mussels were kept. Most of these were stiff
and motionless, but one or two individuals were actively
moving when first seen. The addition of water to a piece of
the style seems to have the effect of rendering a large number
of the organisms motionless and rigid (fig. 23), and it is
tempting to speculate as to whether the difference between

392 W. CECIT. BOSANQUET.

what has been called a ‘'reponema, in which the curls are
nore or less rigid, and a Spirochete, in which there is
more flexibility, may not be dependent to some extent on the
medium in which the organism is examined. Accompanying
the spirochetes in the water were a considerable number of
spirillar organisms, exactly resembling them in microscopical
structure (figs. 25, 24a, b). It seems possible that the segment-
ing spirochetes (fig. 19) may divide first into short spirilla in
some instances before undergoing development into the coccoid
stage. At alow temperature the style of the mussel is more
slowly dissolved than at the temperature of the room. Ihave
found that, as a rule, this body entirely disappears in about a
week or ten days after the mussel is taken up from its native
place. One mussel, which was kept for three weeks in an
aquarium, where presumably food was plentiful, formed a
new style, so far as could be judged, since all the other
mussels of the same batch had lost theirs long before. This
re-formed style was quite free of spirochetes.

My best thanks are due to Prof. Minchin for allowing me
to use his laboratory, and for his advice and encouragement ;
also to Miss Rhodes, who has drawn the illustrations to this
paper.

LISTER INSTITUTE,

November 4th, 1910.

REFERENCES.

Fantham.—‘ Parasitology,’ 1910, iii, p. 392.

Gross.— Mitth. aus der Zoologischen Station zu Napel, 1910, Bd. xx,
Heft 1.

Keysselitz— Arb. a. d. kaiserlichem Gesundheitsamte,’ 1906, xxiii, p.
566.

Leishmnan.— Trans. Soc. Trop. Medicine and Hygiene, January, 1910,
| LY ACE

Nuttall, Fantham, and Porter.— Parasitology, 1908, i, No. 4.

Schellack.— Arb. a. d. kaiserlichem Gesundheitsamte, 1909, xxx, p. 379.

BRIEF NOTES ON SPIROCHETA ANODONT KEYSSELITZ. 393

EXPLANATION OF PLATE 15,

Illustrating Dr. W. Cecil Bosanquet’s paper on “ Brief Notes
on the Structure and Development of Spirocheta
anodontz Keysselitz.”

(All the figures, except Figs. 1 and 11, are from specimens fixed in
osmic vapour and stained with thionin, and all are drawn at a uniform
magnification of 2000. |

Fig. 1—Spirocheta anodontex, showing so-called undulating mem-
brane. From a film dried in the air and stained with iron-hematoxylin.
.2.—Sp.anodontx#x—long thin specimen from crystalline style.

oO

5
Fig. 3—Same. Long thick specimen.

a

5

vy. 4.—Same. Short thick specimen.
Fig.5.—Same. Short thin specimen. '

Fig. 6.—Same, showing splitting of (¥) sheath.

Fig. 7.—Same. Slender specimen showing uniformity of structure
and absence of * undulating membrane,” like a bacterium.

Fig. 8—Same. Short specimen with pointed ends.

Fig. 9.—Same. Longer specimen with pointed ends and somewhat
stiff curls (not well shown in the figure).

Fig. 10—Same. Short thin specimen with apparent undulating
membrane.

Fig. ll.—a, b, ¢, stages in apparent longitudinal division of Sp.
anodonte (diagrammatic).

Figs. 12, 15.—Stages in “ incurvation” (?) of Sp. anodontex.

Figs. 14, 15.—Complete incurvation (¥) and subsequent transverse
division.

Figs. 16, 17, 18.—Sp. anodontze, from the alimentary canal of the
mussel, showing plasmolysis (¥).

Figs. 19, 20, 21, 22.—Apparent development of spirochetes into
coccoid bodies. From a preparation of crystalline style dissolved in
water and kept for forty-eight hours.

Fig. 25.—Spirochete from water in which mussels had been kept, for
comparison of structure with figs. 24 a, b, showing spirilla from same

water.

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CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA.

395

Contributions to the Cytology of the Bacteria.

By

Cc. Clifford Dobell.

Fellow of Trinity College, Cambridge; Lecturer at the Imperial

College of Science and Technology, London.

With Plates 16-19, and 1 Text-figure.

Con'vrENTS.

INTRODUCTION
Historic
MATERIAL AND Merxops :
DESCRIPTIONS OF THE FORMS Tie ESTIGATED
1. Coccus Forms
(A) Micrococci .
(B) Sarcina
. Bacillar Forms
(A) Bacilli of flexilis tye
(B) Bacilli of spirogyra type
(c) Bacillus saccobranchi n. sp.
3. Spirillar Forms
(A) Spirilla with Chr aoa eacleus
(B) Spirilla with Filamentar Nucleus
(c) Spirilla with Spherical Nucleus
4, “ Fusiform Bacteria ” :
(A) From Lacerta mur Aly
(B) From Frogs and Toads.
(c) From Triton vulgaris .
(Dp) From Stylopyga orientalis . ;
5. On some Nucleated Bacterium-like Organisms .
SUMMARY OF RESULTS
GENERAL DiscusSsION
Metachromatic Granules
Morphological Evidence that Bacteria are pNuolete Cells

vou. 56, PART 3.—NEW SERIES. 28

Lo

PAGE
396
399
412
418
4.19
419
4.24.
426
426
A354.
44.1
44.6
4.4.6
448
451
452
452
453
454.
455
455
461
462
463
4.66

396 C. CLIFFORD DOBELL.

PAGE

Do Bacillar Forms with a Vesicular Nucleus exist ? . A479

Variability of the Nucleus at Different Periods in the

Life-cycle . : : ‘ : . 48
Pleomorphism : 3 : . A484

Do Enueleate aioe existe: ; ; . AB85

* Fusiform Bacteria” . 3 : . . 486
Affinities of Bacteria . : : : . 487
CONCLUSIONS : . 488
APPENDIX: ON THE Apnea Meroe OF BCT . 489
LITERATURE ; é : : ; . 492
EXPLANATION OF PLATES : : : - 499

“Teh hoffe zuversichtlich, dass wir nicht mehr allzu weit von dem
Augenblicke entfernt sind, wann es klar werden wird, dass die verschie-
denartigsten Angaben, insofern dieselben einer ernsten und gewissen-
haften wissenschaftlichen Arbeit entspringen, alle in reinen Hinklang
gebracht werden, so dass ein neues schénes Gebiude, das der Bacterien-
eytologie, in der allerfeinsten der Wissenschaften hoch emporragen
wird.’”’—Mencl (1910).

INTRODUCTION.

Iv is a remarkable fact that modern cytology, which has
recently made such rapid strides as the result of the enthu-
siastic investigations of a vast army of workers, has almost
lost sight of the Bacteria. Cytologists and protistologists
alike have been content, for the most part, with assuming that
the Bacteria are a group of simple organisms, possessing but
little structural differentiation, and have then left them alone.
Yet no biologist would deny, I think, that it is of the utmost
importance that we should possess exact detailed knowledge
of the structure and life-history of this immense group of
living beings. More than one of the current conceptions in
biology must undergo profound modification when we have
precise information regarding the Bacteria.

If anyone endeavours, at the present moment, to ascertain
from the vast bacteriological literature, which has been
pouring out for many years past, the present state of know-
ledge regarding the structure of Bacteria, he will find that the
whole matter is in a state of utter chaos. He will find that

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 397

the most divergent views are held regarding the various
structures present in the bacterial cell. He will find, for ex-
ample, regarding that most important of all cell-structures—
the nucleus, that all views regarding its existence are held—
from that which tells him that there is no nucleus of any sort,
to that which tells him that the whole cell is to be regarded
as a free nucieus.

Now the reason for this divergence of opinion is not far to
seek. For many years the Bacteria have been entrusted to
the bacteriologists, and only an occasional botanist or zoologist
has ventured to poach on their preserves. Yet to the bacterio-
logists, the Bacteria are but a means to an end—they study
them in order to cure a cold or make a cheese. Modern
bacteriological methods are excellent and adequate when
applied to medical diagnosis or industrial needs, but they are
inadequate when applied to astudy of the Bacteria themselves. !
It is for this reason that professed bacteriologists possess such
remarkably diverse opinions regarding the normal structure
of Bacteria, and it is for this reason also that what little is
definitely known of their cytology is due largely to the labours
of a few zoologists and botanists. The bacteriologists are, of
course, not to blame for this. Their aims are wholly different
from those of the protistologist or cytologist. It is from
these that-our knowledge of the structure of Bacteria must
come.

The great majority of Bacteria which have been described
have taught us nothing concerning the internal structure of
the bacterial cell. Nearly all the pathogenic forms are of
exceedingly small size; and in addition to this great dis-
advantage they have mainly been studied after fixing and
staining in the usual bacteriological manner, which renders
them worthless for cytological purposes. It is desirable, in
the first place, to study the largest and most easily investigated

1 The truth of this can easily be seen by anyone who will consult the
vast number of text-books on bacteriology which are in current use.
In the majority of these, the cytology of Bacteria is not noticed at all.
or else dismissed with a few inaccurate remarks made at random.

398 CG. CLIFFORD DOBELL.

forms, and to examine them after treatment by suitable cyto-
logical methods.

The foregoing considerations have led me to a study of the
cytology of the Bacteria. During the last four years I have
devoted a considerable amount of time and labour in an
endeavour to arrive at positive conclusions regarding the
structure of the bacterial cell. It has been my object to discover
large Bacteria which can be investigated cytologically with
comparative ease—both whilst living and after suitable fixation
and staining. ‘The present paper represents the greater part
of the results of my work, which—though still in progress—
has led me to conclusions which are sufficiently definite to
appear to me worth publication. I do not claim that the
problem of the cytology of the Bacteria has been solved. My
results are here given merely as a contribution towards a
solution of the problem: I know only too well how incom-
plete and imperfect they are.

My main object has been to discover whether the Bacteria
are nucleate or enucleate cells. It is useless to speculate upon
the “simplicity,” “ primitiveness,” ‘ lack of differentiation,”
etc., which this important group is supposed to display, when
such a simple point as this remains in doubt. I have en-
deavoured to find out whether a nucleus is present, and—if
present—what form or forms it may assume. As staining
reactions and micro-chemical tests appear to me to have been
a signal failure in this direction, I have attacked the problem
from another point of view—the morphological. I hoped—
and I confess I am not altogether disappointed—that a study
of the morphological elements present in the cell, and their
behaviour during the various phases of the life-cycle, would
throw considerable light upon the matter. Such results as I
have obtained are, at least, very definite. They are, moreover,
supported by the less important—as I believe—results derived
from staining reactions.

As there is already a very extensive and confusing litera-
ture dealing with the structure of Bacteria, I have thought it
advisable to give a brief historic review of the more important

CONTRIBUTIONS 'O THE CYTOLOGY OF THE BACTERIA. 399

work which has been done previously on the subject. I shall
then give my own observations—recording them quite in-
dependently of the work of others—and reserve a full discus-
sion of the whole matter to the final section of the paper.

My work was begun in the Zoological Laboratory in
Cambridge. Afterwards I continued it whilst working in the
Zoological Institute in Munich, and at the Zoological Station
in Naples.' Subsequently I was able to add to my results
whilst visiting Ceylon in 1909, during my tenure of the
Balfour Studentship of Cambridge University. I have com-
pleted my work up to its present state at the Imperial College
of Science and ‘’echnology, London. I desire here to record
my indebtedness to all those who have—in one way or another
—assisted in the furtherance of my work in the various places
mentioned,

Historic.

In the pages which now follow, I have attempted to give a
brief historic account of the most important work which has
been contributed towards a solution of the problem of the
nucleus in Bacteria. It is obviously impossible—in a paper of
the present scope—to enter encyclopzedically into all the
work which has been done in this connection.

In dealing with the cytology of Bacteria, it is of the very
greatest importance to consider the technique by means of
which the various workers have reached their results. I
shall therefore make a special point of noting in each case—
wherever possible—the methods of fixation, staining, etc.,
which have been used. When this is done, it becomes
apparent that a large part of what has been written upon the
bacterial nucleus is practically worthless—owing to the in-
adequacy of the technique employed.

The older observers were mostly content to regard the
Bacteria as enucleate—Monera, as Haeckel termed such

1 Whilst occupying the British Association Table in 1908, under a
grant from the Goldsmiths’ Company.

400 . CG. CLIFFORD DOBELL.

supposed forms.! Early workers (e.g. Cohn) noticed, indeed,
granular bodies in many Bacteria, but they were unable to
reach any definite conclusions regarding their significance.

If we turn to older books on bacteriology, we find it
usually stated that no nucleus is to be found in_ these
organisms. De Bary (1884) says: “ Nuclei have not yet
been observed in Bacteria” (p. 492). Similarly, Zopf (1885)
states: “ Until now, nuclei have been looked for in vain in
bacterial cells” (p. 14). Hiippe (1886), whilst pointing out
that no nucleus had ever been shown to exist in Bacteria,
suggested that the whole bacterial cell might be the homo-
logue of the nucleus of other forms. ‘This view has found
many subsequent adherents.

One of the very first to investigate the structure of Bacteria
was Kunstler (1887). He described in Spirillum tenue—
after fixation with osmic acid, and staining with “noir

? or hematoxylin—an alveolar structure ef the proto-

Collin
plasm, with numerous granules. In the later publications of
Kunstler and his colleagues, descriptions which seem essen-
tially similar are given of a number of different Bacteria.
The descriptions are usnally so incomplete, however, the
figures usually so diagrammatic, and the technique employed
usually so imperfectly indicated, that I find great difficulty in
interpreting his results. (See Kunstler et Busquet [1897,
1898], Kunstler [1900], Kunstler et Gineste [1906, 19064],
etc.) As a rule, Kunstler appears to think that there is, in
most Bacteria, no structure comparable with a nucleus.

Schottelius (1888) claimed to have found nuclei in various
Bacteria (B. anthracis, cocci, etc.). These nuclei are said
to be in the form of a short rod (bacilli) or spherule (cocci),
and to divide in the process of cell-division. .They are said
to be visible in the living cells, but more distinct in dry films
stained with gentian violet. ‘The method of fixation is not
given.

' Tt is perhaps worthy of note that, so late as 1894, it was still
dogmatically stated by Haeckel that Bacteria contain no nucleus
(‘Systematische Phylogenie der Protisten und Pflanzen’).

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 401

Babes (1889) found stainable granules—whose presence he
had recorded at an earlier date—in various bacterial cells.
Later (Babes, 1895), he named them “inetachromatic eranules,”
but he was unable to determine their precise significance.

Ernst (1888) found similar granules in the cells of Bacillus
xerosis. ‘They were observed in dry, flame-fixed cells,
stained with methylene blue and Bismarck brown. He
beheved that they took part in spore-formation. Subsequently
(Ernst, 1889) he found similar granules—using similar methods
—in a number of other Bacteria. He proposed the name
*“sporogenic granules” for them, and regarded them as
probably of a nuclear nature. Still later (Ernst, 1902), he
described “chromatophil” granules—of uncertain significance
—in many Bacteria (B. megatherium, water Bacteria, etc.).
These granules were coloured by intra-vitam staining with
methylene blue and neutral red.

The carefully conducted and classic work of Biitschli (1890,
1892, 1896, 1902) can here be considered in its main outlines
only. After studying the Cyanophycee, Butschli turned his

! Tn these he believes

attention to the large sulphur Bacteria.
that the protoplasm, which has an alveolar or honeycomb
structure, 1s differentiated into a peripheral layer and a denser
‘central body.’? In the meshes of the latter, granules which
stain red with hematoxylin (‘1red granules’’) are present.
He regards the
the homologue of the nucleus of other ceils, and the peripheral

“central body” with its “red granules” as

layer as the homologue of the cytoplasm. In the smaller
Bacteria which he investigated, he found that the peripheral
layer was relatively greatly reduced in size, or altogether
absent—the greater part, or the whole of the cell being there-
fore constituted by the ‘‘central body.’ He was therefore
led to regard the whole cell as homologous with a nucleus.
The observations were made not only upon living cells, but
also upon cells fixed, stained and variously treated by a
number of different reagents.

1 The earlier work of Winogradsky (1888) and others, upon this
group, did little to elucidate the structure of the cells.

402 C. CLIFFORD DOBELL.

Wahrlich (1890, 1891) studying a number of different
forms (B. subtilis, B. megatherium, etc.), arrived at con-
clusions essentially the same as those of Biitschh. He
believed, from their chemical and staining reactions, that
Bacteria contain chromatin. Young cells are homogeneous,
chromatic ; older cells show a reticulum of linin in which
granules of chromatin are suspended. The chromatin granules
fuse to form spores. He concludes that Bacteria are there-
fore really nuclei. All his work appears to be based upon a
study of dried cover-slip preparations.

Zettnow (1891), using Léffler’s flagellar stain—which has
agreed with

little value from a cytological point of view
Biitschli’s conclusions regarding small Bacteria. Later
(Zettnow, 1897) he extended his observations to large
S pirilla, using chiefly intra-vitam staining with methylene
blue, and drawing the same conclusions as before. Stall later
(Zettnow, 1899), he examined a number of Bacteria stained
by Romanowski’s method, but after lame-fixation. His
conclusions regarding structure were essentially the same
once more—that Bacteria consist entirely, or in some cases
chiefly, of nuclear substance.

Protopopoff (1891) found granules which stain with fuchsin
in a Bacillus from a cow’s tongue, and in Actinomyces.
He interpreted them as being of a nuclear nature, though on
very slender evidence. ‘The method of fixation is not stated.

Wager (1891) described a nucleus, containing two deeply
staining rods and surrounded bya very thin membrane, in a
Bacillus from the scum on water containing decaying S piro-
gyra. The division of the nucleus is briefly described. The
method of fixation is not given, but it is stated that cover-glass
preparations were stained with fuchsin. Wager (1895) again
described structures which he believed to be nuclei in various
other Bacteria, but gave only a very fragmentary account both
of the structures themselves and of the technique employed.

Frenzel (1891, 1892) gives a description of several species
of Bacteria—chiefly from a study of living cells—and draws
analogy between spores and nuclei.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 403

In 1892 Sjébring described large vesicular nuclei, which
divide by mitosis, in B. anthracis, hay Bacteria, the Vibrio
of fowl-cholera and several micrococci. Fixation is stated to
have been effected with nitric acid (alone, or with alcohol)
without previous drying. The stains used were carbol methy-
lene blue or carbol magenta.

T'rambusti and Galeotti (1892) investigated a large Bacillus
from water. The preparations were either dried, or fixed
with HNO,, and stained with safranin. ‘The organisms stain
at first uniformly, but later show a differentiation into darkly
staining longitudinally placed rods, and granules. Sub-
sequently young cells appear to be formed endogenously.
The authors compare the structural changes with mitosis,
though the reason for this is far from obvious.

Mitrophanow (1893) studied the structure of various sulphur
Bacteria (Beggiatoa, Chromatium, Ophidomonas, etc.),
also of Cladothrix, Spirilla, Bacilli, etc. He employed
intra-vitam staining with methylene blue, and also examined
organisms after fixation with various reagents and treatment
with various stains. He believed that a nucleus was present
in all the forms examined. Various modifications were des-
cribed and figured. He did not agree with Biitschli’s inter-
pretation of the structures present in the bacterial cell. He
believed “que toutes les bactéries que nous étudions ne
peuvent étre aucunement considérées comme des organismes
sans noyau ; de méme on ne peut pas leur attribuer exclusive-
inent une nature de noyau. Elles apparaissent commes des
cellules dans divers stades de complication, laquelle est ex-
primée par la séparation plus on moins compléte du noyau.”

Podwyssozki (1893) gives an account of the structure of the
cholera Vibrio, as seen in dried preparations stained with
Ziehl-Neelsen and in cells treated simply with fuchsin. He
finds a nucleus-like oval mass of “chromatin” in the cell, and
other bodies of different (undetermined) nature. In place of the
oval mass of chromatin, two or more masses may sometimes
be seen—appearances which he regards as due to degeneration.

Schewiakoff (1893) finds a structure like that described by

404. C. CLIFFORD DOBELL.

Biitschh in sulphur Bacteria, in a large freshwater organism
which he names Achromatium oxaliferum. This organism
resembles the sulphur Bacteria in general form, but contains
calcium oxalate—probably in combination with a carbohydrate
—instead of sulphur. There is a “central body” present,
containing colourable granules which undergo division.

Ilkewicz (1894), studying B. anthracis after flame-fixa-
tion and a complicated staining process, found darkly staining
bodies present, which he believed to be spore-rudiments. He
suggests that it is these structures which Sj6bring mistook
for nuclei.

A. Fischer (1894) explains the protoplasmic differentiation
described by Biitschli as due to plasmolysis. In this, as in
subsequent memoirs (Fischer, 1897, 1899, 1903), he maintains
that a “central body” does not exist: that the granules are
probably reserve material, and neither nuclei nor chromatin :
and that no nucleus has been demonstrated in Bacteria. The
cell is not the equivalent of a nucleus. His conclusions are
based upon elaborate studies of fixation and staining methods.
It is hardly necessary to enter here into the polemics which
have taken place between Fischer and Biitschli.

Migula (1894), after a study of Bacillus oxalaticus,
reaches the conclusion that no ‘ central body” is present in
this form. Colourable granules—insoluble in pepsin—are
present, but no definite interpretation of them is given. Ina
subsequent work (Migula, 1897), after reviewing the literature
he concludes: ‘‘ Ueber die Bedeutung der Kérnchen in der
Bakterienzelle lassen sich nur sehr subjektive Vermutungen
hegen ; ich méchte sie als die ersten Anfainge einer Zellkern-
bildung betrachten.” More recently (Migula 1904), he ex-
presses the opinion that the existence of a nucleus is still an
open question.

That nuclear structures occur in many Bacteria is believed
by Léwit (1896). His conclusions are based, however, upon
dried preparations stained with Loffler’s flagellar stain.

A. Meyer (1897), using various methods, found granules
which he interpreted as nuclei in B. asterosporus and B.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 4005

tumescens. In a later paper (Meyer, 1899) he extended
these observations to a number of other Bacteria. He
employed various methods—chiefly fixation with formol and
staining with fuchsin. The granules, which are nuclei, may
be from one to six in number in each cell. In 1904 he gave
a detailed account of the chemical and staiming reactions of
“volutin” granules in Bacteria and other organisms. More
recently (Meyer, 1908) he affirms that his “ nuclei” are not
volutin, but condemns the nuclear structures described by the
majority of other workers.

Wagner (1898) discovered a nucleus in the form of a
granule, dividing with a dumbbell figure—one in every cell
—in B. coli and B. typhosus. His preparations were

“dried in the usual way ”

and stained by a very elaborate
method.

“Chromatin” bodies were found in various forms of
Bacteria by Ziemann (1898). He made dry films, fixed in
the flame or in alcohol, and stained by Romanowski’s
method.

Macallum (1899) investigated three species of Beggiatoa,
after various methods of treatment. He finds no such
differentiation as described by Biitschli. Compounds of
masked iron and organic phosphorus are uniformly diffused
through all the protoplasm, and these compounds also occur
in certain granules which stain with hematoxylin. ‘ There
is no specialised chromatin-holding structure in the shape of
a nucleus of any kind.”

Rowland (1899) records the results of staining various
Bacteria—chiefly with roseine, without fixation. Deeply
stainable granules were found, though no very definite inter-
pretation was given to them. He appears to think that they
may be partly nuclear and partly excretory.

Under the name Bacterium gammari, a large nucleate
organism—inhabiting the body cavity and hemolymph of
Gammarus zschokkei (from Garschina Lake, Switzerland)
—was described by Vejdovsky (1900). ‘The organisms were
treated by various cytological methods. Each cell has a

406 C. CLIFFORD DOBELL.

distinct nucleus lying towards the centre. Later (Vejdovsky,
1904), he describes stages in the mitotic division of this
nucleus, and records similar nuclei in certain filamentous
Bacteria inhabiting the gut of Bryodrilus ehlersi.

Marpmann (1900) suggests—amonegst other things—that
enucleate Bacteria may exist. His observations are very
fragmentary, and all made upon flame-fixed organisms.

Feinberg (1900) describes ‘‘ nuclei” of various forms in
various species of Bacteria (B. coli, B. anthracis, Micro-
cocci, etc.). The observations were made upon organisms
stained by Romanowski’s method. The method of fixation
is not given; presumably the preparations were dried and
flame-fixed. (See here also Zettnow, 1900.)

Marx and Woithe (1900) arrive at the conclusion that the
Babes-Ernst granules afford an index of virulence

greater
numbers indicating a greater degree of pathogenicity. They
further state that the organisms containing these granules
are the “Trager und Hrhalter der Art.” They also make the
statement that ‘the Babes-Hrnst granules are products of
maximal condensation and typical localisation of the euchro-
matic substance of the bacterial cell.’ The illuminating
nature of such a statement is obvious. Regarding the relation
between the granules and virulence, the statement of Marx
and Woithe has been controverted by Ascoli (1901), Gauss
(1902), Schumburg (1902), Krompecher (1901), Ficker (1903),
Guilliermond (1906), and others.

Krompecher (1901), working on various organisms, draws
a distinction between “ metachromatic granules” and ‘“ Babes-
Ernst granules,” on the grounds of staining reactions. He
leaves the significance of the granules in doubt. (See here
also Mithlschlegel [1900], Marx [1902], etc.)

Hinze (1901) found scattered granules, which he believed
to be chromatin, in the cells of Beggiatoa. Later (Hinze,
1903), he described similar bodies in another large sulphur
bacterium—Thiophysa volutans. Various methods of
fixation (Flemming, etc.) and staining (Heidenhain, etc.)
were employed. The granules are said to divide by a process

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 407

of simple constriction. An ordinary form of nucleus was not
found. :

Nakanishi (1901) describes nuclei in a large number of
Bacteria (Cocci, Bacilli, Spirilla) stained with methylene
blue, either intra-vitam, or after fixation with formol vapour.
He finds minute spherical nuclei in Coccei; nuclei in the form
of a granule, rodlet, or filament in Bacilli; and granular or
filamentar nuclei in Spirilla. He also finds nuclei in
spores. He gives an excellent account of his technique, good
figures, and strong evidence for the conclusion that the
structures he observed are really nuclei. His interpretations
have been unfavourably criticised by Ascoli (1901 4), Ficker
(1905), Preisz (1904), Meyer (1908), and others.

Schaudinn (1902) inaugurated a new erain bacteriology by
studying cytologically the whole life-cycle of the gigantic
Bacillus bwttschlii in the gut of the common cockroach.
He described a nucleus in the form of scattered granules of a
chromatic substance (chromidia) throughout the greater part
of the life-cycle. During spore-formation the granules
arrange themselves in a spiral and finally become aggregated
into dense masses in the fully formed spore. A process
interpreted as a modified sexual act (autogamy) was dis-
covered. In the following year (Schaudinn, 1903) he
described analogous conditions in Bacillus sporonema, a
small marine organism.

Meyer’s pupil Grimme (1902) has given a lengthy and
elaborate account of the chemical and staining reactions
of many different kinds of granules which occur in many
different Bacteria. After a discussion of the various kinds of
granules which he studied—especially the ‘‘ metachromatic
granules ” (“ Volutanskugeln ”’)—he finally decides in favour
of the nuclear views of Meyer. The “nuclear” granules of
most other observers are probably not nuclei. (In connection
with these granules see also Guilliiermond [ 1906, 1910, ete. ],
Meyer [1904], Eisenberg [1910], etc.).

Under the name Spirillum colossus, Errera (1902)
describes an enormous spirillar form. Darkly staining masses

408 CG. CLIFFORD DOBELL.

of variable form are seen in dried and stained preparations.
Their interpretation is not indicated. (This organism is
certainly worthy of a careful cytological study.)
Federowitsch (1902) studied B. megatherium, B. pyo-
cyaneus, and other Bacteria. He found stainable granules,
which play a part in spore-formation, in the cells. But he

“no nucleus like that of higher cells” is

believes that
present. The method of fixation is not given; Weigert’s
stain was employed.

Ruzicka (1905) finds granules present in many Bacteria
after fixation with HeCl, and staining with methylene blue.
A definite interpretation is not given to the granules. In
later papers (Ruzicka, 1908, 1909, etc.) he advocates the
view that the bacterial cell represents a naked nucleus.

Ficker (1905) discusses the problem of the nucleus in
Bacteria. He expresses the opinion that it is premature to
draw any conclusions with regard to either granules or nuclei.

Mencl (1904), using careful cytological methods, finds
typical nuclei in Bacilli inhabiting the gut of the cockroach.
He also finds nuclei in B. megatherium. In 1905 he
describes nuclei of many different forms in filamentous water
Bacteria (Cladothrix, ete.), after staining intra-vitam with
polychrome methylene blue. Later (Mencl, 1907) he gives :
minute description of Bact. gammari, describing the various
appearances seen in resting and mitotically dividing nuclei.
He also published in the same year (Mencl, 19074) a more
detailed account of the symbiotic Bacteria of the cockroach.
Quite recently he has given a description of the nuclei in
Sarcinaand Micrococei as revealed by staining with
polychrome methylene blue intra-vitam and subsequently
clearing in glycerine. Mencl’s results have been adversely
criticised by Guilliermond (1907, 1908, 1910) and Meyer
(1908). ‘The latter states that Mencl’s nuclei are really volutin
granules; the former believes they are the septa formed in
the cells during cell-division. Menel (1909) has replied to
Guilliermond’s criticisms and maintains the correctness of his
own interpretations.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 409

Dietrich (1904), after reviewing the literature on the subject,
says: “ Wir wollen nur noch als Hauptergebnis betonen,
dass alle Versuche, Kerne in Bakterien zu finden, als
gescheitert zu betrachten sind.”

Preisz (1904) gives an elaborate account of the structure
of the anthrax Bacillus. He studied the organisms after
mixing them with alcoholic fuchsin, formol-fuchsin, or methy-
lene blue. He maintains that the nuclei described by
Schottelius, Nakanishi, etc., are really more deeply coloured
portions of the cytoplasm. ‘The real nuclei are in the form
of minute spherical corpuscles, one or more in each cell.
They undergo division. ‘hey are distinct from the meta-
chromatic granules of Babes and Ernst, and from the acid-
fast granules of Bunge. A nucleus enters into each spore.
He finds similar nuclei in B. coherens, B. tetani, and B.
asterosporus. His conclusions are therefore essentially
the same as those of Meyer. (Cf. here also Georgevitch
[1910].)

Rayman and Kruis (1904) describe typical nuclei—similar
to those found by Vejdovsky and Mencl—in a variety of
Bacteria (B. mycoides, B. tumescens, etc.). They are
found in young cells only. The method of treatment is
peculiar—fixation by desiccation (in a desiccator) and staining
with iron-hematoxylin and purpurin. Excellent photo-micro-
graphs are given. ‘The conclusions of these investigators are
challenged by Guilliermond (1908).

Swellengrebel (1906) records the results of a minute cyto-
logical and micro-chemical investigation of Bacillus maxi-
mus buccalis. He finds a nucleus present in the form of
a more or less complete spiral or zig-zag filament. In the
following year (Swellengrebel, 1907), he describes two large
spherical nuclei in Bacterium binucleatum—an organism
from the human mouth. He also describes spiral or zig-zag

nuclear filaments or rodlets in Spirillum giganteum
(Swellengrebel, 19074), and subsequently (19094) in certain
filamentous Bacteria (Spherotilus, Thiothrix), His
results have been questioned by Holling (1907), Zettnow

410 C. CLIFFORD DOBELL.

(1908), and Guilliermond (1908). he various objections raised
against his work have been answered by NSwellengrebel
(1908, 1909), who maintains the correctness of his conclu-
sions.

Guilliermond (1907) gives an excellent brief review of
previous results upon the cytology of Bacteria. In the follow-
ing year (Guilliermond, 1908) he describes the structure of a
number of Bacilli (B. radicosus, B. mycoides, B. mega-
therium, ete.). He believes that in all these the nucleus
is in the form of granules of chromatin (chromidia)—distinet
from the metachromatic granules—scattered through the
cytoplasm. ‘These granules become massed together to form
the spores. He criticises the results obtained by many other
investigators. Various cytological fixing and_ staining
methods were employed in his researches. In a more recent
paper, Guilliermond (1909) describes nuclei in the form of
spiral filaments—hke those found by myself—in two species
of Bacillus (from the gut of Hechinocardium) and a large
Spirillum.

In 1908 I gave the results of cytological researches which
IT had undertaken upon the structure and life-history of
several Bacteria. I described a new large disporic Bacillus
—B. flexilis, from the gut of frogs and toads—whose life-
history is essentially the same as that of B. biitsehlii
described by Schaudinn (1902). I also described another
organism—which I named Bacillus spirogyra—from the
same hosts, in which the nucleus is in the form of a spiral or
zig-zag filament. I described further in Spirillum mono-
a nucleus of the

spora—from the frog and toad also
chromidial form. ‘Che chromidia mass themselves together
in forming the spores. In 1909 I gave a more detailed
description of B. spirogyra. I discussed the nature of the
nuclear filament, and described the part it played in spore-
formation—a process which I described in detail. I descibed
in addition the structure and method of spore-formation in
B.lunula, which resembles in these respects B. spirogyra,
As a result of this work, I reached the conclusion that the

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 411

“autogamy ” of B. biitschlii (Schaudinn, 1902) and B.
flexilis was probably not a sexual process at all.

Amato (1908) describes results obtained by staining several
Bacteria (B. mycoides, Sp. volutans, ete.) intra-vitam
with Brillantcresylblau. He believes that in the spore, and
at the beginning of development, a relatively large spherical
nucleus is present, which breaks up subsequently into chro-
midia. The divergent views of different observers may have an
explanation in the fact that they observed similar organisms,
but at different stages in their development.

Dangeard (1909) records the results of a re-investigation
of Chromatium. By fixing with Flemming or Perenyi,
and staiming with various stains (especially Flemming’s
triple), he confirms the description of this organism given by
Biitschli. A “central body ” corresponding to a nucleus is
present. Additional evidence for regarding the ‘ central
body” as a nucleus is afforded by the fact that a rhizoplast
can sometimes be seen connecting the flagellum with this body.

Ambroz (1909) gives a lengthy description of Bacillus
nitri. Asa result, he reaches the same conclusion as Ruzi¢ke
—that Bacteria are nuclei. Fixation is said to have been
effected with a concentrated solution of “ HCl,,”! and staining
chiefly with Giemsa.

Under the name “ Hillhousia” mirabilis, West and
Grifiths (1909) describe a very large sulphur bacterium.
There is said to be a protoplasmic network present, containing
granules beheved not to be chromatin. ‘ Nothing of the
nature of a definite nucleus is present.” Details—especially
as regards the method of using formol as a fixative—are too
scanty for this conclusion to be accepted without further
evidence. No reference is made to the work of Biitschh,
Schewiakoft, Hinze and others, on similar forms.

Recently, an account of the structure of the long forms of
B. coli, B. typhosus, ete.—produced by growing these
organisms on culture media containing aniline dyes—has been

1 T presume this means HgCl,,— and not HCl, as given by Guillier-
mcnd in a review of this paper in ‘ Bull. Inst. Pasteur.’

VOL. 56, PART 3.—NEW SERIES. 29

412 C. CLIFFORD DOBELL.

eiven by Vay (1910). He finds large irregular masses of
darkly stained substance—which he calls chromatin—in these
organisms. He does not appear to be aware that the pro-
duction of these forms on coloured media had already been
described by Walker and Murray (1904).

Such, then, is a very condensed account of the chief work
which has hitherto been published concerning the problem of
the nucleus in Bacteria.

In all work in which inadequate technique has been em-
ployed—for example, in all studies in which only dried and
flame-fixed organisms have been examined—the conclusions
attained can have little value from a cytological point of view.
In many publications, moreover, the descriptions both of
results and of methods are so meagre as to render discussion
of them either unprofitable orimpossible. ‘Therefore I sha!l—
on either or both of these grounds—eliminate the following
works from any further discussion :

Kunstler and Busquet (1897, 1898), Kunstler (1900),
Kunstler and Gineste (1906, 19064), Schottelius (1888),
Zettnow (1891, 1899), Protopopoff (1891), Wager (1891,
1895), Sjobring (1892), Trambusti and Galeotti (1892),
Ilkewiez (1894), Loéwit (1896), Wagener (1898), Ziemann
(1898), Marpmann (1900), Feinberg (1900), Errera (1902),
Federowitsch (1902), West and Griffiths (1909), Vay (1910).

I think most cytologists will agree with me that no profitable
discussion of these papers is possible.

MarerRIAL AND MernHops.

As I have already indicated above, I have made a special
point of working upon the largest forms of Bacteria which I
have been able to find; but I have studied in addition a
number of small forms, when they have been suitable.

Small Bacteria are not only very difficult to investigate on
account of the limitations imposed by the microscope, but
they are also in many cases unsuitable in other ways for cyto-
logical study. They occur frequently in media which render

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 413

the making of good microscopical preparations exceedingly
difficult and laborious,.and they contain granules (reserve
material, etc.), which are relatively of sucha size as to obscure
much of the structure of the living substance itself. For the
latter reason, the sulphur Bacteria, in spite of their large size
in many cases, appear to me to be unfavourable objects for
study—as a starting-point, that is to say, on our way to a
comprehension of the organisation of Bacteria.

Another point that has seemed to me of some importance
is this. Much of the work which has been done upon the
structure of Bacteria has been based on a study of organisms
which have been kept in cultures for a greater or less period
of time. It seems to me highly probable that the discordant
results of different workers may in many cases be due to
cultural differences in the organisms studied. Different
culture media may be used in the cultivation of Bacteria :
but although “pure” cultures may be obtained in half a
dozen of these, it does not follow that all or any of the
colonies so obtained consist of normal individuals. Bacteria
are not found in nature as a rule in pure cultures, and this is
a point which should not be overlooked when considering
their normal structure. Culture methods are of the greatest
service in the separation of various microbes from one another,
but it does not at all follow that all pure cultures of a given
organism are identical, or that they contain individuals which
are in every way the same as those living in their natural en-
vironment. I have therefore not studied Bacteria grown in
artificial culture media, but have confined my attention for
the present to organisms in their natural habitat. The fact
that the Bacteria which I have investigated are not—for the
most part—previously described and named “ species” from
pure cultures, is therefore not an objection which can be urged
against my results, but a necessary consequence of the point
of view from which I have attacked the problem.

As a source of material, I have found the intestinal contents
of various animals most useful. ‘I'he contents of the large
intestine in many animals is swarming with Bacteria, frequently

414. C. CLIFFORD DOBELL.

of large size. The consistency of the intestinal contents,
moreover, is usually such as to render the making of micro-
scopic preparations (smears, etc.) comparatively easy. I
have found the contents of the large intestine of Amphibia
and Reptilia especially suitable; but insects, mammals and
other animals also contain a rich supply of suitable material
which is as yet almost untouched. Most of the organisms
which I am about to describe have been obtained from frogs,
toads and lizards.

I have found in all the animals which I have studied that
the Bacteria in the large intestine vary enormously—in different
individuals—both as regards the number of different forms,
and the number of microbes as a whole. In the frog, for
example, some individuals may contain very few Bacteria—
mostly of the same form—whilst others may contain countless
numbers of Bacteria of the most diverse forms. ‘This is, of
course, only what one would expect.

As the source of the material will be found under the
deseription of each organism, I shall here say nothing more
detailed regarding this, but will now devote a few words toa
description of the technique which L have employed in my
researches. JI have already (Dobell, 1908) given a brief
account of some of the methods which I have used.

I have tried most of the methods of fixation and staining
which are usually employed in cytological work. Itis usually
necessary to modify the ordinary procedure in one way or
another when dealing with Bacteria. In my experience,
the usual methods of fixation (e.g. corrosive sublimate,
Flemming’s solution, Hermann’s solution, osmic acid, formalin,
various picric acid and bichromate solutions, etc.) may all—
under suitable conditions, and with careful procedure—be
made to give excellent results. [Fixation is most easily and
effectively accomplished by making a wet film of the in-
testinal contents—or other medium in which the Bacteria
occur—on a coverslip, and then dropping it film-side down-
wards upon the fixing solution. Drying previous to fixation
is, of course, to be avoided. The usual bacteriological method

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 415

‘of making dry films and fixing them by passing them through
a flame is quite worthless from a cytological point of view,
owing to the plasmolysis and distortion which it brings about.

When the medium containing the Bacteria is too watery to
allow of fixable films beeing made, gelatine or albumen may
be added until a film of suitable consistency is obtained. If
the medium be too thick, one must of course be careful to use
isotonic salt solutions for its dilution.

Most of the ordinary cytological stains (e.g. Delafield’s
hematoxylin, carmine, safranin, etc.) I have found unsuitable
for Bacteria. They—lke most of the ordinary aniline deriva-
tives—are lable to stain the whole cell uniformly, without
differentiating the internal structures. This is largely due to
the marked affinity which the cell wall has for many stains,
causing it completely to obscure the finer structures present

in the protoplasm.

After trying a large number of combinations of fixatives
-and stains, I have latterly confined myself almost entirely to
two methods. Both of these have proved of the greatest
value. They are (1) fixation with osmic acid or formalin,
followed by staining with one of the modifications of Roman-
owski’s method, and (2) fixation with Schaudinn’s sublimate-
alcohol (2: 1) followed by staining with Heidenhain’s iron-alum
hematoxylin. ‘The latter method is now so well known (see,
for instance, Schaudinn, 1902) that I will not re-describe it.
Jt is of course a wet film method, and its only disadvantage is
that it is exceedingly difficult to use, owing to the difficulty of
‘obtaining exactly the right degree of differentiation. Indeed
with different degrees of differentiation quite different appear-
-ances may be produced in the same Bacteria, and it is there-
fore necessary to be very cautious in interpreting the results.
Nevertheless, I believe this method to be one of the most
valuable for the study of the structure of Bacteria.

With regard to the first method, I have found it so simple
-and easy to use that I can strongly recommend it to others.
My method of procedure is us follows. I take a drop of the
medium coutaining the Bacteria and place it in the centre of

416 C. CLIFFORD DOBELL.

a carefully cleaned glass slide (or coverslip) by means of a
platinum loop. I then place a drop of I per cent. osmic acid
or strong formol (40 per cent. formaldehyde, Schering) beside
the first drop, and then mix beth together and spread the
fluid in a thin and even film on the slide. I then allow the
film to dry, which usually takes a few minutes. No heating
should be used to accelerate the process. The slide or
coverslip with the dried film is then placed in absolute alcohol
for about ten to fifteen minutes. It is then removed, and the
film allowed to dry once more. I then stain the film with
Giemsa’s or Leishman’s stain in the usual way. After
staining I differentiate in 30 per cent. aleohol—wash in distilled
water—dry by blotting with a cigarette paper—and mount in
cedar wood oil or neutral Canada balsam. Chromatin
structures are coloured a bright red; the cytoplasm being
blue, lilac or pink, according to the degree of differentiation.
The structure of many Bacteria is revealed with remarkable
distinctness by this method—its chief disadvantage being that
the preparations sooner or later fade, and cannot as a rule be
satisfactorily re-stained.

The above method of fixation—which I term the drop
method—calls for some further comments. In the first place,
it might be urged that the drying which takes place would
be liable to injure the organisms, and give rise to misleading
appearances. ‘I'his is not so, however. If the Bacteria are
fixed with osmic acid or formol before drying is allowed
to take place they are not plasmolysed or injured in any
way. It is only when drying takes place before fixation
that such disastrous results ensue.

I have made many preparations by other methods as
controls. I have made wet films and fixed them by immersion
in 1 per cent. osmic acid or formol: I have also made wet films
and fixed them by exposure to osmic vapour : and I have then
stained these films by modifications of Romanowski’s method
and mounted them in balsam without allowing any drying to
take place at any stage in the proceedings (cf. Dobell, 1908).
The final results obtained in all these cases are almost in-

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 417

distinguishable from one another. The only real difference
observable is that the organisms which have been dried
appear slightly broader than those which have not—owing to
the slight flattening which drying brings about. ‘The internal
structures appear exactly alike. Controls with wet films fixed
with sublimate-alcohol and stained with Heidenhain’s iron-
hematoxylin give confirmatory results. I therefore think
that the drop method of fixation, when employed in the
manner described, gives reliable cytological results in the case
of Bacteria.! On account of the ease with which osmic acid
or formol may be employed in this manner, I have used them
more frequently than any other fixatives.

Another point which calls for comment concerns the use of
alcohol after fixation. When ‘ osmic acid” (more correctly,
osmium tetroxide, OsO,) is used—either in solution or in the
form of vapour—it is, of course, unnecessary to treat the pre-
parations subsequently with alcohol—so far as complete fixa-
tion is concerned. In practice, however, I find that films
fixed by osmic vapour or by the drop method adhere to the
slide or covershp better if they are hardened in absolute
alcohol tor a short time after fixation. When formol is
employed, however, it is absolutely necessary to employ
alcohol subsequently. As is well known, formaldehyde fixes
protoplasmic structures without precipitating them in an in-
soluble form. It is therefore necessary to place the fixed struc-
tures in strong alcohol before proceeding further—otherwise
fixation may be completely undone in subsequent treatment.”

1 T may add that beautiful preparations of small flagellates and
other Protista may also be obtained in this way.

> Cf. Gustav Mann (1902). This point seems worthy of attention.
I note that Swellengrebel (1906) fixes Bacteria by the drop method,
using formalin. But he does not appear to use alcohol subsequently,
so that many of the appearances which he describes may be due to
imperfect fixation. If it is desired to use formalin alone—without
using alcohol at all—and to use stains in watery solution, the fixation
may be preserved by adding a small percentage of formalin to all the
stains, etc., employed after the original fixation. If it is desired to
dilute the formalin used in fixation, this should be done with isotonic
salt solution—not with water.

418 C. CLIFFORD DOBELL.

Sometimes excellent results may be obtained by making
ordinary dry films, fixing with absolute alcohol, and staining
in the usual way with Giemsa or Leishman. This method is
not to be relied upon, however, and should never be employed
alone. Giemsa’s new wet method (vide Giemsa, 1909, 1910)
appears to give excellent results, but I have not used it myself
for Bacteria.

I have employed intra-vitam stains in many cases, but
with little suecess—so far as nuclear structures are concerned.
I have used neutral red, Brillantcresylblau, and methylene
blue. Many other workers appear to have been more
successful with these stains (e.g. Mencl, who has obtained
most striking results with polychrome methylene blue). In
my experience, only non-living structures in the cells (meta-
chromatic granules, etc.) can be stained during life. But
doubtless much depends upon the stain itself. Different

samples of methylene blue—for example—may give quite
iy I VS

different cytological results.

DESCRIPTIONS OF THE ForMS INVESTIGATED.

Having already given as briefly as possible the most im-
portant results which have been reached by previous work on
the cytology of Bacteria (see p. 399), I shall pass on to a detailed
description of my own observations. In this description I
shall make no attempt to compare or to correlate my own
results with those of others—my object being to give only the
facts which my own work has disclosed. A discussion of all
the results—obtained by other workers and by myself—will
be reserved for a subsequent section of the paper (see p.
462, et seq.).

In describing the various forms which I have investigated, I
have—for convenience—divided the organisms into five main
groups. These are the Cocci, Bacilli, Spirilla, “fusiform
Bacteria,’ and a group of other organisms which resemble—
but are not—non-motile rod Bacteria. I shall deal with each
of these groups separately, and in this order.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 419

1. Coccus Forms.

Cocci of various sizes are very common in the large in-
testines of many different animals. Unfortunately, however,
they are usually of very small size, and hence exceedingly
difficult to study accurately. I have examined many cocci
from the large intestines of frogs and toads (Rana tempo-
raria, R. esculenta, Bufo vulgaris), of newts (I'riton
vulgaris), of cockroaches (Periplaneta americana and
Stylopyga orientalis) and of several different snakes. All
these have proved to be of little value, because the organ-
isms were usually so small that I could not be certain of their
structure as seen under the microscope. The living orgauisms
were usually very refractile, and showed no internal structure
which could be definitely separated from appearances due to
optical phenomena. For instance ina small Micrococcus—
examined under a high power—a dark spot of varying size
can often be distinctly seen in the centre of the organism.!
This is not, I believe, a definite body—such as a nucleus—
lying in the cell, but is merely an appearance caused by optical
phenomena connected with the microscope.

(a) Micrococci.

Only two Micrococci of suitable size for investigation
have come under my notice, but they have both revealed a
structure which is quite unmistakable. Both forms were
found in the large intestines of lizards—Lacerta muralis
and Mabuia carinata.

Micrococci from Lacerta muralis.

The lizards were obtained in the neighbourhood of Naples.
[ found that nearly all of them harboured a large Micro-
coccus in greater or less numbers.

‘ These appearances probably led Schottelius (1888) to believe that
he could see a nucleus in living Bacteria.

420 C. CLIFFORD DOBELL.

The living Micrococei, examined in the contents of the
large intestine immediately after removal from the lizard,
showed no very definite structure. I have been unable to
convince myself of the presence of any internal structures
from an examination of living organisms alone.

With stained preparations, however, the case is very
different. I have obtained the best results after fixation with
1 per cent. osmic acid or formalin, and after staining with
Giemsa’s or Leishman’s stain in the manner already described
(see p. 415). The following descriptions apply to organisms
treated in this manner.

The Micrococci occur singly, in pairs, or in chains. They
are usually perfectly spherical, and have a diameter (in fixed
and stained specimens) varying from rather less than | jw, up
to 2. Allintermediate sizes may be found. It 1s possible,
of course, that the different sized forms are really different
species. ‘hey all occur together, and in company with many
other forms. But it 1s quite immaterial, for my purposes,
whether they are one species or one hundred, for they all show
a structure which is the same in each individual, and it is with
their structure that I am concerned.

Every individual, after fixing and staining (cf. Pl. 17,
fio. 45), shows a uniformly coloured cytoplasm, a well-marked
cell wall, and acentrally situated, darkly staining bedy. ‘This
central body is always present. It is roughly in the form of
a spherical granule, but may appear more or less square or
triangular in optical section. It always takes up the chromatin
stain strongly.

Among the ordinary “ resting ”

forms just described, a
number of dividing organisms can usually be found. The
details of the process of division can be followed in stained
specimens with great clearness, and present features of con-
siderable interest.

Division—which results in the formation of two equal

daughter-cells—takes place as follows (see figs. 46-49). In
the first place, the central body becomes elongated—the cell

itself also exhibiting a slightly rod-like form—and assumes a

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 421]

characteristic dumb-beil shape (fig. 46). The long axis of the
dumb-bell coincides frequently with the long axis of the cell,
but it is also often seen to be shghtly displaced from this axis
—occupying a somewhat oblique position in the cell. The
ends of the dumb-bell separate from one another, but remain
attached by the slender intermediate strand for some time.
When the central body has reached this stage, a constriction
appears in the middle of the cell in a plane at right angles to
the long axis of the dumb-bell figure. The cell now presents
the appearance shown in fig. 47 (PI. 17). A little later the ends
of the dumb-bell lose their connection with one another,
through the disappearance of the connecting strand. The
constriction of the cell wall is now more marked (fig. 48).
After the two new central bodies have been formed in this
way from the original body, the cytoplasmic constriction
becomes complete, and two daughter-cells are formed which
he at first in close contact with one another (fig. 49). In this
Diplococcus-condition the daughter-cells may remain; or
they may separate forming two free Micrococci; or they
may divide again, and so give rise to a chain of coccus forms.
Division always takes place in the manner just described—the
central body dividing with the formation of a characteristic
dumb-bell figure, and being followed by the fission of the
cytoplasm.

Now I think there can be little cause for complaint if I call
the central deeply staining body anucleus. ‘This body is a
constant morphological feature of every cell: it divides with
the formation of figures which are closely comparable with
those of a very simple amitosis—on a very small scale: and
is takes up the nuclear stain strongly. I shall discuss this
more tully in a later part of the paper, and will henceforward
call the central body the nucleus.

As I have pointed out above, the dividing nucleus not
uncommonly occupies a slightly oblique position in the cell.
It also shows occasionally another modification, which is of
the greatest interest—a modification which is characterised
by the dividing nucleus assuming the form of a zig-zag

A422 C. CLIFFORD DOBELL.

— Y

filament. ‘his condition may be more or less strongly
marked: it may take the form of a simple bend, or it may
take the form of a spiral filament consisting of one or more
turns (see fig. 52).

It might be urged that the bilobed cells which contain
a zig-zag, bent, or spiral filament are really different organisms
from those under consideration. The proof that this is not
the case lies in the fact that all stages can be found together
in the same chain of organisms (fig. 52). There can be very
little doubt that these chains are formed from the successive
divisions of an originally single Micrococcus. Inthe short
chain depicted in fig. 52, a pair of such forms is seen at the
lower end of the chain. Above these, four dividing cocci are
seen which show various modifications of the dividing
nucleus, from a slightly distorted dumb-bell figure to a
zig-zag or spiral filament.

I regard this configuration of the nucleus as of considerable
significance. he matter will be discussed at greater length
in a subsequent section of the paper (see p. 471).

Coceco-bacillar Forms from Lacerta muralis.

Now in addition to the coccus forms which I have just
described, there are many organisms which cannot be very
definitely classified with either coccus forms or bacillar forms,
but which occupy an intermediate position. These forms
(fig. 50) present the appearance of a shghtly elongated sphere,
or of a very short rod with rounded ends. ‘he shortest, most
spherical forms (fig. 50, upper right-hand individual) have a
nucleus which is in the form of a short and usually bent
rodlet. The longest forms (fig. 50, lower and upper left-
hand individuals) show a nucleus which is in the form of a
filament arranged in a more or less zig-zag or spiral manner.
That such organisms are in a “resting” (i.e. not dividing)
state, appears certain from the fact that no cytoplasmic
constriction can be seen (compare figs. 45-50 and 52).

As I have already noted, the ordinary Micrococcus

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 423:

forms show slight irregularities in the contour of the nucleus;
and it is, in fact, frequently impossible to decide whether an
individual should be describedas a Micrococcus or acocco-
bacillar form. All intermediate gradations occur, so that—
although an absolute proof is lacking—lI believe that all these
forms, from typical Micrococcus to typical Bacillus, are
really stages in the life-cycle of one and the same organism.!
For the present, however, I will confine myself to describing
the morphological features of these forms—merely pointing
out that, side by side in the same host, all forms occur from
typical, spherical cocci with a spherical nucleus, to typical
rod-shaped bacilh with a zig-zag or spiral nuclear filament.

Micrococci from Mabuia carinata.

These Micrococei were obtained from the large intestine
of the Brahminy lizard (Mabuia carinata), caught in
Ceylon (Colombo). They are of smaller size than those just
described, and I have examined, relatively, only a small
amount of material.

The organisms (PI. 16, figs. 42-44) have an average diameter
of about 1:5, or rather Jess. They are spherical, and show
a centrally placed nucleus just as in the case of the Micro-
cocci from Lacerta muralis (cf. fig. 44). The method
of division appears to be exactly the same, and I have there-
fore not figured it in detail. Allowing for the difference in
size, figures 46-49 would be equally good representations of
the dividing individuals of this form.

Coccus or cocco-bacillar forms in the gut of M. carinata
also show the zig-zag form of nucleus (fig. 45). I have not,
however, a complete set of stages between cocci and bacilli,
as in the case of the Bacteria from Lacerta muralis.

These Micrococci do not present any other features of
special interest. I have described them because they are the
only other cocci which have furnished me with unequivocal
evidence regarding their cytology.

1 For further consideration of this see p. 484.

424 C. CLIFFORD DOBELL.

(B) Sarcina.

After investigating the structure of the ordinary Micro-
coccus forms, I naturally became curious to see what sort of
structures could be found in the Sarcine. For some time
I endeavoured to ascertain the exact structure of a Sarcina
which is very common in the English frog and toad, but I
was unable to reach any definite conclusions owing to the very
small size of the individual cells. Other Sarcinee from other
animals proved equally difficult, but at last I discovered a
large and suitable form in the large intestine of a Ceylon toad.
This organism I will now describe.

Sarcine from Bufo melanostictus.

These Surcinz were obtained from a single toad which
was captured near Colombo. All the preparations were made
by fixing in | per cent. osmic acid and staining with Giemsa’s
stain. The following description therefore applies to organ-
isms treated in this manner.

Sarcina is, of course, simply a colony of cocci, arranged
typically in groups of eight individuals in three dimensions of
space. The groups originate by the successive “cleavages ”
—like a developing egg—of a single coccus cell.

The individual cocci which compose the cell-groups of the
Sarecina under consideration are of very large size. They
measure on the average a little over 2 w in diameter—some
cells attaining a diameter of 2°5 pu.

In the living organism, it can be seen that nearly every cell
contains one large refractile granule. ‘This is probably reserve
material of some sort. Sometimes this granule may be absent
and occasionally two such structures are to be seen. No other
internal structures can be made out with certainty in the fresh
state.

Upon staining the organisms, however, the structure of the
cell can be readily demonstrated (see figs. 24-29, Pl. 16). The
cytoplasm appears a uniform blue,’ and sometimes shows a

1 Or pink, if the blue is extracted with alcohol after staining.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 425

faint granular or alveolar structure. The refractile granules
remain unstained, or after prolonged staining may take on a
faint yellowish-pink tinge. In each cell a dark red granule—
corresponding with the nucleus described above in Micro-

coceci—can always be found. The position of this nucleus in
the cell varies. It does not always le in the centre, but is
usually near this point, and very often in contact with the
refractile granule (cf. figs. 24-29).

In resting cells, the nucleus las always this form ofa simple
granule, ‘I'his is seen in fig. 24, which shows a two-cell stage.
Division of the nucleus precedes the division of the cytoplasm,
and is effected in the same way as the nuclear division of the
Micrococeidescribedabove. The granule elongates slightly,
assumes a dumb-bell figure, and then separates into two
daughter-granules. Fig. 27 shows a three-cell stage, in which
the two daughter-cells on the left have completed division,
whilst the nucleus of the cell on the right is dividing. Fig.
26 shows a later stage. The two cells on the left contain
dividing nuclei, whilst the single cell on the right contains

two daughter-nuclei—cytoplasmic fission having

not yet
occurred. Fig. 28 shows a four-cell stage, each cell contain-
ing a resting nucleus. In fig. 25, one of the nuclei (upper
left-hand cell) has divided into two, and in fig. 29 three out of
the four cells show dividing nuclei. ‘The eight-cell stage
which results from the division of these four cells shows
exactly the same sort of nuclei.

Judging from the large number of cells which showed
dividing nuclei, I should think that cell division takes place
very slowly in this organism, but I made no observations on
this point on the living organisms.

It will be apparent, I think, to anyone who will compare
the figures of the Sarcina with those of the Microcoecci,
that the structure of the cell and its nucleus—both during
rest and during division—is essentially the same in both
forms.

I will now pass on to a description of the bacillar forms
which I have been able to investigate.

426 C. CLIFFORD DOBELL.

2. Bacittar Forms.

In two previous papers (Dobell, 1908, 1909), I have given a
description of the structure and method of spore-formation in
two large Bacteria which I obtained from the large intestines
of frogs and toads. These two forms I named Bacillus
flexilis and Bacillus spirogyra. The former is charac-
terised by having a nucleus in the form of chromidia scattered
through the cytoplasm: the latter by having a nucleus in the
form of a spiral or zig-zag filament. B. flexilis, moreover,
is a very large, flexible organism and forms two spores:
whereas B. spirogyra is considerably smaller, rigid, and
forms a single spore. As most of the Bacilli which I am
now about to describe are organised in a inanner similar to
that of these forms, I shall—for convenience—reter to them
frequently as Bacilli of the flexilis type or spirogyra
type; meaning thereby that the organisms under discussion
are structurally similar to one of these forms, though implying
nothing as regards difference or identity of species.

(a) Bacilli of the flexilis Type.

(1) Bacillus flexilis.—Although I have already given a
detailed account of this organism (Dobell, 1908), I shall here
add a few further observations on its structure, as 1t seems to
me of considerable importance that its cytology should be
made absolutely certain.

My original figures were drawn from preparations stained
by Giemsa’s method. The various modifications of this
method which I employed I have already given—as also
several other methods which gave me satisfactory results. I
would here emphasise the fact that all methods which give
reliable cytological results reveal exactly the same structure
in this organism. ‘I'hey show a number of deeply staining

granules scattered through the cytoplasm—an appearance
which I have interpreted as a nucleus in the form of
chromidia. Subsequent work on this and allied forms has

convinced me of the correctness of this interpretation.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 427

On account of its very large size, B. flexilis is particularly
well suited for observations upon its structure. I will now
describe the appearances which it presents when fixed by a
good wet method and stained by a good cytological stain.

I have given two figures of organisms so treated (Pl. 18,
figs. 119, 120). Fixation, sublimate-aleohol (Schaudinn) ;
stain, Heidenhain’s iron-hematoxylin. It may be noted here
that although this method gives good results on the whole, it
is very difficult to obtain uniformly sharp differentiation.
Different individuals behave differently towards the stain, so
that in the same preparation well-stained, over-stained and
under-stained organisms are often found side by side.

When examined under the highest magnification which I
have been able to use (Zeiss 2 mm. apochromatic oil-
immersion, compensating ocular 18) the following internal
structure can be made out. ‘The cytoplasm appears homo-
geneous and very finely granular (as it does in life) or else
shows a rather indistinct alveolar arrangement (cf. fig. 120).
The very well-marked cytoplasmic alveoli described by
Schaudinn (1902) in B. biitschliit are very much more
‘distinct than anything I have ever seen in B. flexilis. In
the latter the cytoplasm is, at most, slightly alveolar. <A
number of round black granules can be seen scattered
through each cell (figs. 119, 120). They are usually more
numerous towards the periphery than in the centre. ‘These
granules constitute—I believe—the nucleus, and are probably
composed largely of chromatin. Their behaviour during
spore-formation I have already described (Dobell, 1908). No
other structures are to be seen.

I believe that the figures (figs. 119, 120) give a faithful
picture of the structure of B. flexilis. J am convinced that
were other structures present—e. @. a vesicular nucleus, or a
nuclear filament—they would have been visible in some of my
preparations. I conclude, therefore, that the internal structure
of B. flexilis consists simply of a faintly alveolar cytoplasm
in which small granules of chromatin are imbedded.

Having said so much about B. flexilis itself, I will now

VOL. 56, PART 5.—NEW SERIES. 30

428 C. CLIFFORD DOBELL.

describe some very similar forms which I have been able to
examine.

(2) Bacilli of ‘the flexilis type from Lacerta
muralis.—I found these organisms in the large intestine of
Lacerta muralis captured in the neighbourhood of Naples.
Many lizards which I examined contained these organisms,
but I have no record of the exact percentage of animals
infected. Possibly it is the same organism which Prowazek!
observed in lizards in Rovigno. But froin his very brief
mention of it as ‘ein grosser mit zwei Polkérpern aus-
gestatteter Bazillus,” it is impossible to be certain.

These bacilli are of very large size, but they are usually
smaller than B. flexilis. The largest. individuals which I
have measured are about 30,” in length, with a breadth of
rather less than 2y. The ends are blunter than in B.
flexilis, but the organisms are flexible in the same way.
Figs. 83 and 84 (PI. 17) show two of these forms after fixation
with osmic vapour and staining with Giemsa. Figs. 133 and
134 (Pl. 18) show two other individuals—one of them (fig. 134)
undergoing division

after fixation with sublimate-aleohol and
staining with Heidenhain. It will be seen that all of them
show essentially the same structure

a structure, moreover,
identical with that of B. flexilis. There is a uniform or
slightly alveolar cytoplasm containing chromatin granules
scattered throngh it.

Division takes place in this organism by constriction—not
by septation

as in B. flexilis. I have not observed its
method of forming spores, but I believe that it is probably a
disporic form.

In addition to this organism of a typical flexilis form, I
found some smaller Bacteria, which show a similar kind of
organisation, in the large intestine of L. muralis. They
were of different sizes and forms, and may be different species
or stages in the life of the same species. It is impossible to
be certain from a comparison of the fixed and stained organ-

1 “Untersuchungen iiber einige parasitische Flagellaten,” * Arb.
kaiser!. Gesundheitsamte,’ xxi, 1904, p. 1.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 429

isms only. Some of these organisms are of large size, and it
is easy to determine their structure. ‘I'wo individuals of long,
slender form are shown in figs. 64 and 65 (Pl. 17). They
exhibit a pale, uniformly stained cytoplasm, with relatively
large chromatin granules distributed through it. The cyto-
plasm is generally free from chromatin granules at the extreme
ends of the organism.

This form divides like B. flexilis. It is slightly flexible,
and motile. I have not observed spore-formation. The
average length is about 11, the breadth a little less than
ypu:

I have seen a good many forms which are intermediate in
size between these forms and the large flexilis forms
described above in the same host. I think it possible that
there may be some genetic connection between them, though
it does not appear very probable.

(3) Bacilli of the flexilis type from Mabuia cari-
nata.—In the large intestine of a Ceylon lizard, Mabuia
carinata, I found an organism which is very similar to the
flexilis forms from European frogs, toads, and lizards. The
infected Mabuie were taken in Colombo.

This organism is shown in figs. 21-23, Pl. 16. It is motile,
flexible, disporic (see fig. 25), and divides by constriction. It
shows a nuclear apparatus exactly like that of B. flexilis.
I have not been able to obtain all the stages in spore-forma-
tion, so that I do not know whether it displays the same
remarkable phenomena during this process as are seen in B.
biitschlii and B. flexilis. But such stages as I have found
are very like those of B.flexilis. The average length of
the organism is about 17 ~—a good deal less than that of B.
flexilis or the flexilis forms from Lacerta muralis.

As far as my observations go, the ordinary forms of these
organisms are therefore closely similar. There are differences
which distinguish these three forms (i.e. those from the frog
and toad [ B. flexilis], from Lacerta, and from Mabuia)
from one another, but cytologically they are all three
essentially similar.

430 C. CLIFFORD DOBELL.

In the preparations of the contents of the large intestine
of Mabuia, I also found some small Bacteria which show a
similar structure. These forms (fig. 41, Pl. 16; fig. 76, Pl. 17)
do not exceed about 10m in length, and are very slender.
They ali show a nucleus consisting of scattered granules of
chromatin during the vegetative existence of the organism.

The Bacilh of the flexilis type from frogs, toads, Lacerta,
and Mabuia all appear to have but one form of nucleuas—
that of a diffuse system of chromidia. But in addition to
these forms, I have encountered two other organisms which
—whuilst apparently belonging to the same group—present
certain features which separate them from the others. These
two organisms were found in the common English newt and
the Naples hzard, but to my great regret my observations—
are very incomplete on both

recorded in the ensuing pages
of them,

(4) Bacilli of a modified flexilis form from Triton
vulgaris and Lacerta muralis.

(4) The form from Triton vulgaris.—lI recorded in a
previous paper (Dobell, 1908, p. 122) the fact that an organism
similar to B. flexilis occurs in the newt. Unfortunately it
is—as far as my observations go—exceedingly rare. 1 have
found it only once, inhabiting the large intestine of a T.
vulgaris captured in the vicinity of Cambridge. Subsequent
search for the organism in other newts has up to the present
been fruitless.

The organism is very long and slender. It is also the
most flexible of all the flexilis forms which I have encoun-
tered. Many individuals attain a length of 50 and more,
though the breadth is only about I wp.

The living organisms were actively motile. They showed a
number of granules of various sizes in the cytoplasm, but no
other clearly visible structures.

After fixation with formalin, and staining with Giemsa
(see p. 415), the organisms could be seen to have the following
structure (see figs. 79-82, Pl. 17). Most of the cells pre-
sented a nuclear apparatus like that of B. flexilis (fig. 79) ;

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 431

that is to say, they showed a number of red granules
scattered irregularly through the cytoplasm. The latter
showed no distinct structure as a rule.

Now, in addition to these forms of the characteristic
flexilis type, there were other forms which possessed quite
a different sort of nuclear arrangement (figs. 80, 82). They
showed a variable number of large, nucleus-like bodies
arranged in a single row along the whole length of the
organism. The cytoplasm of these forms was finely granular
(fig. 80), or sometimes distinctly alveolar (fig. 82). I observed
a number of forms which appeared to be intermediate in
structure between these forms and the ordinary chromidial
forms; but I cannot state with absolute certainty that these
different forms are not really different species. Though I[
believe that all the forms belong to one and the same species
—representing different phases in the life-history—the fact
that they are possibly different species living side by side
cannot be ignored.

This organism is—like the other flexilis forms—disporic.
It forms two terminal spores in each cell, but unfortunately
I did not succeed in observing all the stages of spore forma-
tion. I have seen the living, actively motile, spore-bearing
individuals, and also some isolated stages in the formation of
the spores. Fig. 81 shows one of these—with two large
chromatin spore-rudiments (as in B. flexilis) at the ends of
the cell. I have not found stages in which the chromatin is
arranged in a spiral filament—as in B. biitschlii and B.
flexilis during spore-formation. It is possible, therefore,
that spore-formation does not take place in the same manner
as in B. flexilis, and that the forms which show large
nuclei arranged in a row are really forms which are about to
sporulate. ‘This seems to me improbable, however, because
both long and short individuals (figs. 80, 82) may show this
arrangement of the chromatin, whilst it is usually in long
individuals only that spores are found.

As far as my observations go, therefore, it appears probable
that this particular organism, whilst presenting the flexible

432 C. CLIFFORD DOBELL.

form, disporic habit, and chromidial nucleus characteristic of
other flexilis forms, has also stages in its life-cycle during
which the chromatin is arranged in a series of large nucleus-
lke masses. The significance of this arrangement has not
been determined.

(s) The form from Lacerta muralis.—lI obtained this
organism in a single lizard which I caught near Naples
(Pozzuoli). As in the case of the preceding organism, I have
been able to make only very fragmentary observations upon
it. Unfortunately, I examined it only casually in the living
state—beheving it to be the ordinary flexilis form described
above (p.428). When alive it showed active movements and
was flexible—like the ordinary flexilis forms from the lizard.

To my great regret I made only a single preparation of this
organism—a dry smear on a coverslip, fixed in absolute
alcohol and stained by Giemsa’s method. All my observations,
therefore, are based on this scanty and unsatisfactorily treated
material.

It might be urged that the appearances presented by the
organisms in this preparation are due to imperfect fixation.
This may perhaps be true to some extent, but I do not think
it is altogether justifed. In the first place, other organisms
in the same preparation (e.g. some spirogyra forms and
other bacilli) appear quite normal. In the second place,
drying and fixing in this manner never produces similar
appearances in the ordinary flexilis forms.

Some of the organisms in this shde are shown in figs. 85-90
(Pl. 17). Many of them are of the form seen in fig. 89—that
is to say, they present the appearance of long, slender
Bacteria of the flexilis type. They possess a nucleus of the
chromidial type—many of the chromatin granules being, how-
ever, relatively very large. Large individuals reach rather
over 40 4 in length, though most of them are narrower than
the ordinary flexilis forms (cf. figs. 89 and 85).

IT have found in addition to these characteristic flexilis
forms a number of individuals which display quite a different
kind of nuclear arrangement. ‘he chromatin is in large,

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 433

more or less spherical, nucleus-like masses (figs. 85, 88, 90).
Some individuals possess three or four of these structures
(fig. 85), whilst others—usually much shorter—possess but
one (fig. 88). These “nuclei” bear a strong resemblance to
the nucleus-like spore-rudiments of B. flexilis and similar
forms, and it is possible that they are of a similar nature.
Spores, however, are not present in any of the organisms.

A third modification of the chromatin which I found is that
shown in figs. 86 and 87. It will be seen that the chromatic
elements are neither in the form of diffuse chromidia, nor in
the form of aggregated “ nuclei,” but in the form of a broken
spiral filament. In no single case did I observe a complete
filament like that of B. spirogyra. In the organism
depicted in fig. 86 the chromatin is disposed as follows: a
dumb-bell-shaped figure, two nucleus-like masses, and a short
spiral filament with enlarged ends. Fig. 87 shows a single,
nucleus-like mass, a short spiral filament, a small chromatin
rodlet, a small dumb-bell figure, and a second more drawn
out spiral filament.

After examining all these different forms of nuclear appa-
ratus, my impression is that they are different stages in the
life-history which are connected with one another in the
following way. The chromidial form (fig. 89) becomes con-
verted into the “nucleated” form (fig. 85) by the aggrega-
tion of chromatin at various centres ; this nucleated form then
gives rise to the form with the spiral filaments (fig. 87) owing
to the drawing out of the nuclei by a process similar to that
seen occasionally in the division of the nuclei in Micrococei
(compare fig. 52). This is very strongly suggested in the
organisms shown in figs. 86 and 87.

‘That this interpretation of the appearances is correct I can-
not, of course, be certain. But the appearances are so sug-
gestive that I cannot refrain from making the suggestion that
in this organism we have a clue to much that is obscure in
the morphology of the bacterial nucleus. It seems to me
highly probable that in this organism three different arrange-
ments of the chromatin—chromidia, spherical nucleus, and

454 C. CLIFFORD DOBELL.

spiral filament—succeed one another at different times in the
life-cycle. But I will reserve a discussion of this for another
part of the paper (see p. 481).

(8s) Bacilli of the spirogyra Type.

Under this heading I will describe those Bacilli which
possess a structure like that of Bacillus spirogyra. Ihave
encountered many Bacteria of this form, and believe the type
of organisation which they display to be a very common one.

(1) Bacillus spirogyra.—I have already given a full
account (Dobell, 1909) of the structure and life-history of
this Bacillus, which occurs in the large intestine of Rana
temporaria and Bufo vulgaris. My published figures
are all of organisms fixed with formalin and stained with
Giemsa. As this method involves drying before mounting
(see p. 415), I have thought it advisable to publish a few
more figures of the organism after treatment by a more
as I have pointed out on p. 416—

approved method, though
drying after fixation with osmic acid or formalin does not
appreciably affect the appearance of the cells as a rule.

Figs. 117 and 118 (Pl. 18) are accurate drawings of.
Bacillus spirogyra fixed with corrosive sublimate and
acetic acid and stained with Heidenhain’s iron-hematoxylin.
No drying was allowed to take place at any stage during the
making of the preparation—a moist film cover-glass prepara-
tion of the undiluted contents of the large intestine of a toad
(B. vulgaris). Itis very difficult to obtain good preparations
of this organism with iron-hematoxylin, on account of the
strong affinity of the pellicle for the stain. During differen-.
tiation the nuclear filament and the pellicle give up the stain
at almost the same rate, so that one is apt to over-differentiate.
But when differentiation has been stopped at exactly the.
right moment, very distinct figures of the cell-structure can
be obtained.

The appearances presented by the cells after this method.
of treatment are essentially the same as those obtained by.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 435-

osmic or formalin fixation and Giemsa staining. This can
easily be seen by anyone who will compare figs. 117 and 118
with the figures which I have already published (Dobell, 1909).
The cytoplasm has a uniform appearance—no granular or
alveolar structure being apparent. In each cell a darkly
staining filament (cf. fig. 117) can be seen. This filament
usually has a more or less strongly marked spiral or zig-zag
disposition, and extends throughout nearly the whole length
of the cell. Its behaviour durine cell-division and spore-
formation I have already described in detail (Dobell, 1909).

In my previous papers I described this filament as a
nucleus. As I now believe that this interpretation is com-
pletely justified from my subsequent observations,’ I shall
continue to refer to this structure by this name. I will say
no more about the nucleus of B. spirogyra here, as I have
already discussed it at some length in my previous papers,
but I will now pass to a description of some other Bacteria
which possess the same kind of structure.

(2) Bacilli of the spirogyra type from Lacerta
muralis.—In my description of the cocco-bacillar forms
which I found in the gut of Lacerta muralis, I pointed out
(p. 422) that many cocci and cocco-bacilli showed nuclei of a
more or less spiral form (cf. fig. 50, Pl. 17). Larger
Bacteria of a more definite bacillar form show an even
greater structural resemblance to B. spirogyra.

In the first place, there is a large Bacillus, which I found
very frequently in the lizards whose rectal contents I exainined,
which is almost identical—as regards structure—with the B.
spirogyra which I found in English frogs and toads. It is.
a large motile organism (figs. 55-60, Pl. 17), rod-shaped,
with rounded ends, and reaches a length of 9 w or slightly
over. Dividing forms frequently exceed 10 « in length.

The nucleus—as in B. spirogyra—is in the form of a fila-
ment of chromatin, arranged in a more or less spiral or zig-
zag manner. It may be almost linear (fig. 55), thrown into a
few coils (fig. 57), or much contorted (fig. 60). Sometimes.

1 See discussion on p. 466, et seq.

436 CG. CLIFFORD DOBELL.

it has a much shortened and thickened form (fig. 56). It
displays, iu fact, the same range of variations which I have
already described in B. spirogyra.

Division of the cell is preceded by division of the nucleus
into two portions (cf. figs. 59, 60), exactly as in B. spiro-
gyra. ‘The organism is, moveover, monosporic—forming a
single terminal spore in each cell (fig. 58). I have not
followed out the whole of the stages in spore formation, but
it appears closely similar to what 1 have already described in
detail in the form from the frog and toad. (See Dobell,
1909.)

These figures (figs. 55-60) are all from preparations treated
by the osmic-Giemsa method (p. 415). I have, however, ob-
tained confirmatory results by other methods. In figs. 91-95
(Pl. 18) organisms are shown which have been fixed
with sublimate-alcohol and stained with Heidenhain’s iron
hematoxylin. It will be seen that the structure of the cells
is essentially the same as in the osmic-Giemsa preparations.
This organism presents the same difficulties in staining by
iron-heematoxylin as B. spirogyra (see p. 454). The indivi-
duals depicted are therefore selected specimens in which
differentiation is particularly good.

As in the case of B. spirogyra, there can be no possible
doubt of the existence of the spiral filament which I regard
as the nucleus. Giemsa preparations reveal it always with
the greatest distinctness, and Heidenhain staining—when
successful—does the same. ‘Though my interpretation may
be wrong, the structures which I have described and figured
exist beyond all shadow of doubt.

I found a large number of organisms of the same type
which are intermediate in size between the forms just described
and the cocco-bacillar forms described on p. 422. In fact I
found such a gradually graded series of forms that it is difh-
cult to me to avoid the conclusion that the smallest cocci and
the largest bacilli are genetically connected. Direct proof of
this most important point is wanting. Nevertheless, whether
the different forms are different species or one and the same

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 457

does not in any way affect the fact that all the different forms
exist. I have represented some of the small bacillar forms in
fig. 51 (Pl. 17). They are so closely similar to the ordinary
spirogyra forms, that it will be unnecessary to enter into
a minute description of them. I would emphasise the fact,
however, that many organisms exist which are intermediate
im size and structure between these forms and the cocco-
bacillar forms (fig. 50) om the one hand, and the long spiro-
gyra forms (figs. 55-60) on the other. The importance of
this fact will be discussed later (see p. 484, et seq.).
Amongst the smaller bacilli inhabiting the large intestine of
L. muralis is a form which—tbough not of the characteristic
spirogyra type—may conveniently be described here. ‘The
organism (figs. 53, 54, Pl. 17) is a short rod, about 3 u in
length. Each cell contains a nucleus in the form of a short,
straight chromatin rod (fig. 53). This rod divides in the
process of cell division (fig. 54), behaving like the spiral
filament of ordinary spirogyra forms. This form is of
interest because it is but a little removed from many of the
cocco-bacillar forms. It may, indeed, be derived from a coccus-
form by simple elongation. It is—structurally—a drawn-
out coccus such as those in fig. 45, the nucleus assuming a

straight rod-like shape instead of the spiral or zig-zag of

many cocco-bacilli (cf. fig. 50). Some similar organisms
from another lizard are shown in fig. 109 (Pl. 18). The
drawings were made from a preparation fixed with sublimate
alcohol and stained with Heidenhain. Inside each cell a
short, darkly stained rod can be seen. I interpret this as
the nucleus, though it may possibly be a spore-rudiment. In
the case of the previously described individuals (figs. 53 and
54), however, such an explanation can hardly be advanced.
For the deeply staining rods are present in forms which are
undergoing division (fig. 54) and divide into two transversely
during this process.

Another form of Bacillus which I found is one of large
size (up to 8 w in length) which has its nucleus constantly in
the short, thick varicose condition which can sometimes be

438 C. CLIFFORD DOBELL.

seen in ordinary spirogyra forms (fig. 56). Some of these
organisms are shown in fig. 65, Pl. 17. The nucleus is con-
siderably shorter than the cell, and always has the thickened,
knotted appearance shown in the figures; otherwise it
resembles the nucleus of typical spirogyra forms.

(3) Bacilli of the spirogyra type from Mabuia
carinata.—In these hzards, captured in Colombo, I found
a large number of spirogyra forms in addition to the cocci
and flexilis forms already described. All the preparations
which I made of the contents of the large intestine were
fixed with osmie acid and stained with Giemsa’s or Leish-
man’s stain. The following descriptions are therefore based
upon material so treated.

The largest forms which I found (figs. 30-35, P]. 16) are of
considerable size. ‘They reach a length of 164. They show
the organisation characteristic of spirogyra type organisms
with great distinctness. Forms with a nearly straight
nuclear filament (fig. 35), forms with a simple spiral or
mg-zao (fie. 34) and forms with a very much twisted filament
(fig. 51) were found. In fact all the modifications which are
seen in B. spirogyra itself were encountered. A modifica-
tion which I have observed comparatively rarely, in B.
spirogyra was fairly common in this form. In this
modification the nuclear filament appears to be spht at
certain points, thus giving rise to open loops in these places
(cf. fig. 32).

Division takes place as in B. spirogyra (fig. 33). It will
be unnecessary therefore to describe it in detail. A number of
short forms (fig. 56), which measured about 7 in length,
were seen. They are apparently young forms resulting from
the transverse divisions of the lone individuals, but some of
them may possibly be another species.

This organism is—like B. spirogyra and the spirogyra
forms from Lacerta

monosporic. It forms a single terminal
spore in each cell (see fig. 39). Details of spore-formation
were not observed, but it appears to take place in a manner
similar to that of B. spirogyra.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 439

I found a few forms which appeared to belong to this
organism, but which presented unusual features. I have
figured two of these (figs. 37,58, Pl. 16). The organism repre-
sented in fig. 37 has finely granular, darkly coloured cytoplasm,
containing several large deeply stained chromatin masses.
The organism shown in fig. 38 contains what appears to be a
broken-up spiral filament together with some small chromatic
granules. Whether these forms are normal stages in the life-
cycle or degenerate forms, I am unable to decide. At first I
took them for degeneration forms, but in view of the nuclear
conditions which I have observed in B. saccobranchi (see
p. 441), it seems possible that they represent normal events
in the life-history. Their presence in my preparations seems
worthy of record.

Another form, which I believe to be certainly a degenerate
form, is that shown in fig. 40. I have seen and described
{Dobell, 1909, Pl. 13, fig. 19) similar degenerate forms in B.
spirogyra.

Many small Bacteria from the large intestine of Mabuia
carinata showed nuclei of the spirogyra type. Drawings
of some of these organisms will be found on Pl. 17. Fig. 69
shows a long slender form—I11y long, and less than Ip
broad. Through almost the entire length of the organism, a
delicate chromatin filament—irregularly coiled and zig-zagged
—can be seen to extend. In spite of its small size, the
filament can be seen with the greatest clearness. Figs. 70,
71 and 73 show similar, but smaller organisms. In each of
these the characteristic nuclear filament is plainly visible.
Figs. 72 and 74are smaller forms (length of fig. 72=2,) with
relatively short, thick and straight nuclear filaments. The
nucleus appears to be like that of the organism shown in fig.
56, but on a very small scale. Fig. 75 shows a dividing form
of the same kind of organism as fig. 72. With the exception
of certain forms from the blood of Saccobranchus (see
p. 441), these are the smallest Bacilli in which I have
succeeded in differentiating a nuclear filament with absolute
precision. All the figures are faithful copies of the organisms

44.0 C. CLIFFORD DOBELL.

which were in my preparations. Their structure was as.
distinct as that of much larger forms.

Two other organisms are shown in figs. 77 and 78. In the
form shown in fig. 77, there is apparently a large nuclear
mass occupying most of the middle region of the cell. I
found several organisms like this one. They were all equally
distinct, and appeared normal. But it is possible they are
really plasmolysed forms of one of the other varieties. I
cannot be absolutely certain that they are not, as I have not
sufficient material for comparison. Fig. 78 is drawn from an
organism which has its nucleus in the form of a shghtly bent
rod. I found a fair number of individuals of this form. It
appears to be a large form similar to the Lacerta organisms
shown in fig. 53. It is also somewhat similar to the forms

65.

shown in fig.

I believe that a number of very small Bacteria which occur
in frogs, toads, and lizards have the same structure as these
small Bacteria which I have just described. But I have, up
to the present, been unable to convince myself that this is so.
With very small Bacilli it is usually very difficult to be
absolutely certain of the exact structure. Only in very
favourable cases, when staining has been exactly right—as
in these minute forms from Mabuia—can satisfactory results
be attained. :

(4) Bacilli of the spirogyra type from Bufo
melanostictus.—lIn the large intestine of this toad—taken
near Colombo—I found several Bacilli which showed the
spirogyra type of nuclear structure. I have given figures
of two different forms which I encountered (see PI. 17, figs.
61, 62).

The larger forms (fig. 62) were usually of the shape of
a bent rod. ‘They attained a length of 10m. In these
organisms the nuclear filament was usually linear rather than
twisted—like the straight form of filament in B. spirogyra.
It may be described as a slender thread with here and there
large knot-like swellings on it (fig. 62). I did not see any

individuals with definite spiral or zig-zag filaments.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 441

The smaller forms measured about 2 4-275. in length.
They were of short, oblong form (fig. 61), and each contained
a very distinct spiral or S-shaped nuclear filament. I give
pictures of three individuals of this form in fig. 61, and will
not describe them in greater detail.

(c) Bacillus saccobranchi-_n. sp. and Associated
Forms.

IT shall now describe a new Bacillus which shows a form
of nuclear apparatus differmg in many ways from typical
flexilis or spirogyra organisms. As it 1s convenient to
have a name for this organism, I propose to call it Bacillus
saccobranchi.

(1) B. saccobranchi n. sp.—I obtained this Bacillus
in the following manner. Whilst I was working at the
Colombo Museum, I examined the blood of a number of fish
for trypanosomes. Among these I examined a number of
individuals of Saccobranchus fossilis caught in the
Colombo Lake. Some of them contained Tl’. saccobranchi
Cast. et Will. in their blood, but the majority showed no
blood-parasites of any sort.

One day two Saccobranchi were brought to me for
examination. As I was unable at the moment to examine
their blood microscopically, I placed them in water in a large
earthenware chatty. On the following day, when I was
about to make an examination of the blood of these two fish,
I found that one of them had died during the night. As it
appeared quite fresh, however, I decided to examine its
blood for Protozoa. The fresh blood, taken from the heart,
showed no Protozoa, but contained some very large and
actively motile bacilli, together with a number of smaller
Bacteria.t As the bacilli seemed to me particularly suitable
for study—both on account of their size and the medium in

' T never found Bacteria in the blood of other fish which I killed and
examined immediately after killing. It is possible that these Bacilli
caused the death of this particular fish, though there is no conclusive
evidence that this is so.

442 C. CLIFFORD DOBELL.

which they were livimg—I made some moist films in-the
following way. The blood was spread in a thin film upon a
slide and immediately—without drying—exposed to osmic
vapour for about 30 seconds. The slide was then transferred
immediately to absolute alcohol, where it was left to harden
for 15 minutes. After this treatment the film was stained in
the usual way in Giemsa’s stain—finally being allowed to
dry. I made several preparations in this way, and also by
the ordinary drying method with fixation in absolute alcohol.
In the preparations fixed with osmic vapour, both the blood-
corpuscles and Bacteria were beautifully preserved, and the
description of the organisms which follows is taken from
these preparations.

The largest individuals of this form are of considerable size
(figs. 4-6, Pl]. 16). They attain a length of as much as 16 p,
and a breadth of 24. The cytoplasm contains no inclusions
and appears usually homogeneous, though occasionally it has
a slightly alveolar structure. In all the Giemsa preparations
the cytoplasm is coloured a bright blue (see figs. 1-20)—as I
did not extract the colour with alcohol after staining.

In all the organisms the chromatin is stained red—in sharp
contrast with the cytoplasm (see figs. 1-20). It is distributed
through the cell in various ways, which can be classified
conveniently into three main types of structure, with inter-
mediate conditions.

The first type of nuclear structure is a typical spirogyra
form (figs. 1,2, 4,12). The nucleus is in the form of a spiral
or zig-zag filament, extending through the greater part of
the length of the cell. As in B. spirogyra, the filament
may be comparatively straight (fig. 1) or much contorted
(fig. 2). All intermediate conditions occur, During division
the filament behaves in the same way as in B. spirogyra:
that is to say, it divides transversely into two, a half of the
‘original filament remaining in each daughter-cell (see fig. 2).

The second type of nuclear structure is that shown in
fios. 3, 5-9. The organisms of this type show nuclei of very
irregular form. ‘They possess chromatin structures in the

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 443

form of short, irregular, broken, bent, branched, and twisted
filaments (cf. figs. 5, 6,etc.). These filaments are sometimes
connected with one another to form irregular networks (fig. 9).
In addition to the filamentar chromatin bodies, granules and
irregular masses of various shapes and sizes are frequently
present (figs. 3, 5, ete.). This irregular form of nucleus
appears to be derived from the spirogyra form through the
breaking up and rearrangement of the elements composing
the spiral filament. Figs. 6 and 8 show organisms which
suggest this very clearly. Indeed, I have seen so many
forms iutermediate between these forms and the typical
spirogyra forms that I have no doubt at all that they are
different nuclear stages which succeed one another at different
periods in the life-history.

The third type of nuclear structure is the chromidial type,
characteristic of the flexilis group of organisms (fig. 10).
In the organisms of this type—which were present in com-
paratively small numbers in my _ preparations—all the
chromatin is in the form of minute, diffusely arranged
granules (fig. 10). It can generally be seen that the
chromatin structures (filaments, networks, etc.) of the other
forms are built up of aggregated granules of chromatin.
The organisms with a chromidial type of nucleus appear to be
derived from the forms with irregular nuclei by the breaking
apart of these chromatin granules. In a form such as that
shown in fig. 5, the irregular chromatin filaments and masses
appear to be breaking up. Other organisms show later stages
in this process—intermediate between figs. 5 and 10. I
believe, therefore, that the chromidial type is derived from
the irregular type through the dispersal of the chromatin
granules.

As a result of examining a considerable number of indi-
viduals, therefore, I have come to the following conclusions :
B.saccobranchi may possess a nucleus of the characteristic
spirogyra type, or of the characteristic flexilis type. It
shows in addition a large number of forms intermediate
between these two types. The nucleus in the intermediate

VOL. 56, PART 3.—NEW SERIES. 31

444, C. CLIFFORD DOBELL.

forms may have the form of a broken filament, of an irregular
arrangement of branched filaments or chromatin masses, or
of an irregular network. So many different forms exist that
it is difficult to classify them accurately.

On account of the large number of intermediate forms, it
seems to me certain that all these different organisms are not
really different species. ‘They grade into one another almost
imperceptibly, so that [ regard the interpretation of them as
different species as excluded. Whether the spirogyra type
gives rise to the irregular type, and this in turn to the
flexilis type, or whether the process is in the reverse order,
cannot, of course, be stated with absolute certainty from an
examination of fixed and stained material alone. ‘lhe former
interpretation, however, appears to me the more probable.
It is really immaterial which of these interpretations is
correct. The point of interest is that all these different
nuclear modifications occur in the same organisms. | shall
discuss the matter more fully later (see p. 471).

This organism is, like typical spirogyra forms, mono-
sporic. The spore is formed towards—but not absolutely at
—one end of the cell (see fig. 14). I have not found all the
stages in spore-formation in my preparations, but such stages
as I have seen are similar to those of B. spirogyra. As in
this form also, the spore-forming individuals are short—
measuring on an average about 8 in length. <A certain
amount of chromatin, also, is left over in the formation of
the spore (fig. 14).

(2) Forms associated with B. saccobranchi.—I have
already noted above (p. 441) that I found a number of smaller
Bacteria in my preparations of the blood of Saccobranchus.
These forms I will now describe.

First of all, I should point out that although I found many
very small individuals, which were easily distinguished from
the large forms which I have called B. saccobranchi, yet
many of the smaller forms were really but little smaller than
this form. Indeed, I found so many organisms of all sizes
intermediate between the largest B. saccobranchi and the

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 445

most minute Bacilli, that I cannot avoid the impression that
all these different forms are really connected with one another.
This would be a point of great interest if it could be established,
but unfortunately no definite conclusion can be reached by
simply examining dead organisms.!

I found a number of individuals—of smaller size than the
large bacilli—which showed a very distinct nucleus of the
spirogyra type. Some of these are shown in figs. 11 and
15. They measure about 6 « in jength, and are consider-
ably narrower than the large organisms which I call B.
saccobranchi. ach individual will be seen to contain
a characteristic nuclear filament of the spirogyra type.

Some smaller forms, with a similar structure, are shown in
fig. 20. They measure about 3 w in length, and are much
narrower than the preceding forms: otherwise they are
closely similar. Nuclear filaments of various forms are
seen in each cell.

Some still smaller organisms—2 mx and less in length—are
depicted in fig. 19. In spite of their very minute size,
they show a nuclear filament with great distinctness. They
appear to possess a nucleus exactly like that of the spiro-
eyra forms of B. saccobranchi (fig. 2, etc.), but on a very
small scale. These are the smallest bacilli which I found in
the blood of Saccobranchus.

I would again emphasise the fact that the various indi-
viduals depicted in figs. 2, 11, 20, and 19 are organisms
selected on account of the difference in their sizes. Actually,
a number of intermediate-sized organisms of precisely similar
form were seen.

I have added some figures of other organisms which were
of interest. The Bacillus shown in fig. 15 shows a nucleus
of a form which I observed in a few individuals. It is in the

1 Tf the organisms are all of one species, it follows that the name
B. saccobranchi should be given to all. Asthe matter is so doubtful,
however, I have restricted this name to the large forms only, though I
do not wish to imply thereby that the large forms are specifically
distinct.

4.4.6 C. CLIFFORD DOBELL.

form of a short, irregular mass of chromatin, which appears
sometimes to be really a spiral filament contracted upon itself.
Fig. 17 shows a pair of Bacilli with rod-hke nuclei. Fig. 18
appears to be a similar kind of organism undergoing division.
Fig. 16 is an individual which is like a B. saccobranchus,
with a nucleus of the irregular type, on a smaller scale.
None of the smaller forms showed nuclei of a distinct
chromidial type.

3. SPIRILLAR Forms.

I have endeavoured to elucidate the structure of a number
of spirillar forms, but have succeeded in reaching definite
results in only four different forms. Yet these four different
forms have shown me three different types of nuclear apparatus,
and these I shall now describe.

(4) Spirilla with Nuclei of a Chromidial Type.

In a previous paper (Dobell, 1908) I described briefly the
structure and method of spore-formation which is found in
the Spirillum—which I named S$. monospora—which
occurs very commonly in the large intestine of frogs and
toads. I described this organism as containing a number of
scattered granules, stainable with chromatin stains. ‘hese
eranules | regarded as constituting a nucleus of the same
sort as that of B.flexilis and similar forms. They enter
into the formation of the spore, forming a nucleus-like spore
rudiment—as in B. flexilis, B. spirogyra, B, lunula,
and many other Bacteria.

I have made many attempts to obtain a more detailed
knowledge of S. monospora. My efforts, however, have
been unavailing. ‘lhe facts—as far as they go—which I
have already recorded are the only ones which I have so far
been able to discover. I therefore abandoned this form and
looked for other and more favourable objects for investiga-
tion.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 447

One of the first organisms to which I then turned my
attention was the large Spirillum which inhabits the hind
gut of the common cockroach, Stylopyga orientalis. I
hoped—from the large size of this organism, and from the
descriptions which have been given of it by others'—that I
should find some new and illuminating structures in this

Spirillum. But my hopes have not been altogether
fulfilled.

The only structures which I have been able to make out in
this form with absolute precision are essentially the same as
in the form from frogs and toads. The Spirillum of the
cockroach possesses an exceedingly thick cell wall, which has
a strong affinity for many stains, and which renders it very
difficult to study cytologically. I have obtained the most
satisfactory results with wet films fixed in sublimate-alcohol
and stained with iron-hematoxylin. But differentiation of
the contents of the cell is very difficult to obtain, and succeeds
only occasionally. It has usually been my experience that

1 According to Guilliermond (1907) and others, an organism contain-
ing a typical nucleus has been described by Kunstler and Gineste
((C. R. Assoc. Anatomistes,’ 1904), under the name Spirillum peri-
planeticum, from the intestine of Periplaneta orientalis.
Unfortunately I have not been able to see this paper. In two sub-
sequent notes, however (Kunstler and Gineste, 1906, 19064) these
authors describe and figure this organism, but do not mention or depict
a nucleus, and give the host as P. americana. In the first note
(1906) a reticular structure of the cytoplasm is figured and described.
In the second note (1906) ‘‘spherular elements” are described in the
cytoplasm, and the average dimensions are given as 8p X 3. It is
possible that this is the same organism which I call the “large Spiril-
lum from St. orientalis,” but I cannot be certain from the in-
complete notes of Kunstler and Gineste which are at my disposal.
Fantham (‘ Quart. Journ. Micros. Sci.,’ 1908) casually describes ** spiril-
lar forms occurring in the hind gut of the cockroach.” He observed
“a diffuse nucleus . . . consisting of a number of chromatin
masses seen to be connected by a lightly staining spiral in successfully
stained specimens. At other times the achromatic thread was not
evident.” Diagrammatic figures are given. They are not like anything
I have seen in Spirilla from S. orientalis. I do not attach any
importance to these fragmentary observations.

44.8 CG. CLIFFORD DOBELL.

when the colour has been sufficiently extracted from the cell
wall to enable one to see the internal structures, then the
cell contents have also been so much decolorised as to be
almost invisible. Now and then, however, clear pictures of
the protoplasmic structures can be obtained.

In well-stained individuals I have invariably found the
same structure present. ‘There is no vesicular nucleus visible
and no nuclear filament. The only structures which can be
seen are shown in figs. 110-112 (Pl. 18). The cytoplasm is
alveolar, the alveoli sometimes extending across the whole
width of the cell, sometimes being of smaller size. At various
points in the walls of the alveoli he deeply staining granules.
They are usually few in number, very irregularly arranged,
but generally in close proximity to the cell wall. Occasionally
two or more granules appear at first sight to be connected
with one another by fine intervening filaments; but it seems
to me that in all cases these filaments are really nothing more
than the alveolar walls. I have never found any structure at
all comparable with the nuclear filament of B. spirogyra
and similar forms.

My interpretation of the appearances observed in the large
Spirillum of the cockroach is therefore this: The structure
is essentially the same as that of B. flexilis and allied
organisms. The cytoplasm is alveolar and the nucleus is in
the form of chromidia—consisting of a few small, scattered
granules of chromatin lying in the walls of the alveoli.
Neither vesicular nucleus nor nuclear filament exists in the
forms which I have examined. ‘The structure is therefore
closely similar to that of S. monospora.

(Bs) Spirilla with Nuclei of Filamentar Form.

There is a large Spirillum! which lives in the gut of
Lacerta muralis. I have found it much easier to study
than the large forms from the frog and the cockroach, and

1 These are the organisms to which I referred in a previous paper
(Dobell, 1909, p. 583).

CONTRIBUTIONS T0 THE CYTOLOGY OF THE BACTERIA. 449

have found that it possesses a different kind of organisation.
It is—like the other forms—difficult to stain satisfactorily,
but I have obtained some beautiful results with iron-hema-
toxylin.. My figures (figs. 96-108, Pl. 18) are all drawn from
wet film preparations, fixed with sublimate alcohol and stained
with Heidenhain’s iron-hematoxylin. |My best preparations
are from the contents of the large intestine of lizards which
I obtained from a dealer in Munich; but similar—I beleve
the same—Spirilla also occur in Naples lizards, and have
furnished me with additional material.

The structure of these Spirilla is shown in fies. 96-108.
Long individuals attain a length of as much as 15 (fig. 102)
and more, whilst the shortest individuals measure about 4 pu
(figs. 105-105). All forms display a structure which is
essentially the same, and which differs markedly from that
of other forms which I have investigated.

In fixed and stained organisms! the cytoplasm has, as a
rule, a very distinct alveolar structure (cf. figs. 96, 100,
102, etc.). The alveoh may extend right across the cell,
being arranged as a single row of large chambers (fig. 96),
or they may be of variable smaller sizes (e. @. fig. 107). The
only other structure which is normally present in well-stained
individuals is a darkly staiming filament, like that of B.
spirogyra, which I interpret in this case also as the
nucleus (see figs. 96-108). Both the form and position of this
filament may vary. It is frequently in the form of a short
and nearly straight rod (figs. 96, 104, ete.), occupying a
central (figs. 104, 107) or terminal (fig. 96) position, or any
intermediate site inthe cell. It may be in the form of a more
or less twisted and varicose spiral or zig-zag (figs. 101, 102,
etc.), sometimes showing knob-lke thickenings at the ends
(figs. 100, 105), and sometimes running almost from one
extremity of the cell to the other (figs. 100, 101, etc.). Inspec-

1 As in the case of other Spirilla I have been unable to reach
any definite conclusions regarding structure from a study of the
living organisms. The living organisms, however, have been studied
in all cases.

450 C. CLIFFORD DOBELL.

tion of the figures on Pl. 18 will give a better idea of the
various modifications which occur in this filament than pages
of description.

The filament is a morphological element present in each
cell. It is a body which is independent of the walls of the
cytoplasmic alveoli: that is to say, it is not merely an acci-
dental appearance due to portions of the alveolar walls taking
up the stain more strongly than others. It can be seen in
many cases that the filament crosses the alveoli, and is a quite
independent structure (cf. figs. 96, 102, etc.). I believe there
can be no doubt whatsoever that it is a structure of precisely
the same sort as occurs in B. spirogyra and so many other
similar organisms.

Additional evidence in support of this view is derived from
a study of the behaviour of this filament during cell division.
Just as in the Bacilli of the spirogyra type, division of
the filament precedes division of the cell as a whole (see fig.
108). After the filament has divided into two approximately
equal parts, the cell divides across the middle (figs. 97, 98).
It will be noted that during division the filament must he in
a central position in the cell, and that immediately after
division it must occupy a more terminal position in the
danghter-cells. This no doubt to a large extent explains the
variable position of the filament, in different cells, alluded to
above. On purely morphological grounds, therefore, it seems
to me justifiable to regard the filament in these Spirilla as
a nucleus of the same sort as occurs in Bacilli of the
spirogyra type. I shall discuss these filaments and their
interpretation later (see p. 466): for the present [ wish
simply to establish the fact that a filament of this sort is
present in these Spirilla and in many Bacilli.

Occasionally darkly staining granules are present in the
cytoplasm of the Spirilla from the lizard. They are few in
number, and not a constant morphological feature of the
cell (cf. figs. 101, 102). I regard them as probably composed
of reserve material of some sort. Asarule these organisms
are free from granular inclusions.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 451

(c) Spirilla with Spherical Nuclei.

The fourth and last spirillar form concerning whose
structure I have reached definite conclusions is exceedingly
minute. It is an organism which occurs commonly in the
hind gut of the common cockroach (S. orientalis) in com-
pany with the larger form already described.

The organism in question has the characteristic form of a
small Spirillum or Vibrio (figs. 121-132, Pl. 18). The
smallest Vibrio forms have a length of rather less than 2n
(fig. 132) and upwards. In the largest Spirillum forms
the length rarely exceeds 4°5 m (fig. 130). In all cases the
organisms are very narrow—the largest having a breadth of
approximately 0°5 , the smallest, considerably less. Yet, im
spite of their very small size, these organisms possess an
internal structure which may be demonstrated with the
greatest clearness.

I have obtained very good results with wet films, fixed
with sublimate-alcohol and stained with Heidenhain. All
the figures (figs. 121-132) on Pl. 18 are from individuals
treated in this manner. Differentiation may be obtained
with comparative ease.

Every individual—when properly stained—shows a single,
darkly staining, spherical body situated somewhere within it
(cf. figs. 121-124, 130-132). . Lregard this body as a nucleus.
This body—or, as I shall call it, this nucleus, may he in the
centre of the cell (fig. 123, etc.) or at one extremity (fig. 121),
or in an intermediate position. It always has the form of a
minute round or oval granule, which appears—under the
highest magnification which has been available—quite homo-
geneous. It is a constant morphological feature of every cell.
No cytoplasmic structure can be made out with absolute
certainty, on account of the very small size of most of the
organisms. Faint indications of an alveolar structure seem
to me, however, to be sometimes present. In addition, it
may be noted that the ends of the cells frequently stain
deeply, so that in optical section an individual often appears

452 C. CLIFFORD DOBELL.

to have its ends covered with little dark caps—an appearance
seen in many stained Bacteria.

During the division of an organism into two, the nucleus
behaves in a characteristic manner. It les, before division,
towards the centre of the cell (fig. 180). Whilst in this
position, it divides into two daughter-nuclei, with the forma-
tion of a minute dumb-bell figure (figs. 125, 126). These
daughter-nuclei then separate to a variable distance from one
another (fig. 127) and cytoplasmic fission follows (figs. 128,
129). According to the degree of separation which has
occurred between the daughter-nuclei, the nuclei of the
daughter-cells may lie in a terminal (fig. 129) or central
(fig. 128) position.

The general appearance of the nucleus in this Spirillum
—both during rest, and during division—is therefore closely
similar to that of the nucleus which I have already described
in Micrococciand Sarcina.

4. “Fustrorm Bacteria.”

At various times, I have encountered a number of organisms
belonging to the group of so-called ‘ fusiform Bacteria.”
These organisms are very commonly found in the intestines
of animals. I have studied their structure in quite a number
of forms, and all appear to be organised in essentially the
same manner.

My own impression

after observing many different organ-
isms of this class—is that they are really not Bacteria at all,
but more probably belong to the Fungi. Perhaps they are
related to the yeasts. But as I have no conclusive evidence
—from their life-histories—of their real affinities, and as they
are usually regarded as Bacteria, I will describe here the
forms which I have investigated.

d

(4) “Fusiform Bacteria” from Lacerta muralis.

I found fusiform organisms fairly common in the large intes-
tines of the lizard which I captured near Naples. They were

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 4053

generally to be found in my preparations, but usually in small
numbers.

Good preparations can be obtained by the osmic acid or
formalin method with Giemsa staining (see p. 415), and Heid-
enhain’s iron-hematoxylin—when sharp differentiation has
been achieved—gives very good pictures of the structure of
these organisms.

In Pl. 18, figs. 115 and 114, will be seen some fusiform
organisms from a moist film preparation of the contents of
the large intestine of L. muralis, fixed with sublimate-
alcohol and stained with Heidenhain. It will be seen that
each cell contains a single, darkly staining, centrally situated,
spherical mass, which I believe to be the nucleus. I have
not obtained any satisfactory preparations which show a divi-
sion of this body in this particular form, but from analogy
with similar organisms, I have little doubt that it divides in
the course of cell division.

Small single individuals (fig. 114) have the characteristic
spindle shape, and measure 4y.—4°5 4 in length. The ends
are sharply pointed. No other structures besides the nucleus
were visible in the cells.

In this form—as in all the other fusiform organisms which
I have investigated—double individuals are very common
(fig. 115). They arise from dividing single individuals through
an incomplete separation of the daughter-cells—in the same
way that Diplococcus forms are produced by Micrococci.

Smaller forms than those just described also occur in the
lizard. They all show a similar structure. Fig. 68, Pl. 17
is of a small double form, stained with Giemsa’s stain. Hach
individual shows a distinct nucleus, as in the Heidenhain-
stained individuals. The length of this double organism was
approximately 54.

(zs) “Fusiform Bacteria” from Frogs and Toads.

The common English frog and toad (R. temporaria and
B. vulgaris) occasionally contain fusiform organisms in

454 (. CLIFFORD DOBELL.

their large intestines. They appear to be the same in both
hosts, but I have found them more often in the toad than in
the frog. Many of them, however, are of very small size, so
that they are easily overlooked unless one is specially on the
look-out for them.

As a rule these organisms occur in the double form (fig.
115)—single spindles being comparatively rare in any prepara-
tions. The ends of the double spindles are more rounded
than in the forms from the lizard. The nuclei are of the
same type. Fig. i15is an individual (double) from the large
intestine of Bufo vulgaris (moist film, sublimate-alcohol
and Heidenhain). Heidenhain and Giemsa reveal the same
structure. The length of the double individual depicted in
this figure was ca. 10 p.

(c) “Fusiform Bacteria” from Triton vulgaris.

The common newt contains several different forms of fusi-
form organism. ‘They are similar to those of the frog and lizard,
and usually occur in the double form. I have not made a
very careful study of these organisms, but they show certain
features of interest.

Hach individual (figs. 66, 67

- yy = a s -
nucleus. This may take the form of a simple, apparently

, Pl. 17), contains a single

solid granule of chromatin (cf. fig. 67, upper nucleus), or
occasionally it appears to be a vesicular structure with one
large karyosome (cf. fig. 67, lower nucleus). From the
small size of these nuclei it is often very difficult to be
quite certain of their exact structure. Another type of
nucleus, which I observed in some of the larger forms from
the newt, is that shown in fig. 66. This figure shows an
ordinary double form in which the upper individual is
dividing intotwo. It will be seen that all three nuclei which
are present are in the form of double granules, arranged
transversely across the cells. All these forms were seen in
Giemsa preparations, which were fixed with formalin, and
dried before mounting. It is therefore not impossible that

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 455

these double nuclei are artifacts produced by the breaking into
two of a single granule through drying. This seems to me
improbable, however.

(p) “Fusiform Bacteria” from Stylopyga orientalis.

The common cockroach harbours a fusiform organism
very like those which I have already described. ‘lhe forms
which I have encountered are usually of small size, and are
generally of the double spindle form.

Every cell contains a nucleus (see fig. 116). It is frequently
of a rather square shape, and sometimes I have found nuclei
which appear to be dividing (cf. lower nucleus in fig. 116).
All the dividing nuclei which I have seen are of this rather
curious form—that of two chromatin masses separated by a
variable distance. I have not been able to make out any
other details of the process of nuclear division, but I have not
made a very careful study of these organisms. My only
knowledge of them is derived from occasional individuals
which [ have found in preparations made for other organisms.

5. On somE NucLEATED BACTERIUM-LIKE ORGANISMS.

In this section I shall describe some curious organisms

which present—at certain stages in their lives—a strong
resemblance to Bacteria, but which are really to be placed
among the Fungi. For a considerable time I believed these
organisms to be Bacteria which possessed a typical vesicular
nucleus—like that of Bacterium gammari—and I believe
that other observers may have fallen into a similar error. I
think, therefore, that no excuse is needed for publishing my
results in some detail in the present paper.

The organisms under consideration have all been found in
the large intestines of animals. I have found them in several
insects, snakes, lizards, and Amphibia, and I have also en-
countered forms which I believe to be of a similar nature in the
rectal contents of mammals. However, I have made no careful

456 C. CLIFFORD DOBELL.

investigation of these. The forms which I have studied most
carefully are those occurring im frogs and toads (Rana
temporaria and Bufo vulgaris), in the common cockroach
(Stylopyga orientalis), andin Boaconstrictor. Itis
upon the organisms from the large intestine of this snake
that I have made the most complete series of observations,
and I will therefore record them in some detail at this point.

The living organisms, when removed from the host, and ex-
amined under an immersion, appear as large, bacterium-like,

TEXT-FIG:
_ iti wae So
Croce” Bg OE OD
— B
D
6 oo
G0. 8
~ =
[3 E
On Q
a (a
90 A p> ©
, 2 @
O6 .
. ov \ Oo
8 re) pg
OQ o Se

Bacterium-like organisms from large intestine of Boa con-
strictor. Living organisms. (Zeiss, 2°5 mm. apochromatic
water immersion X compens.-oc. 18.)

non-motile rods (see Text-fig.). They may occur singly, in
pairs, or in chains. The average length of the largest in-
dividuals is about 14 4. Smaller individuals are very common,
and many do not exceed 4. All intermediate sizes are to be
found.

The rods all have rounded ends, and many of the longer in-
dividuals show a slight curvature (cf. Text-fig. 5). The
internal structure in the living cells is very easily seen, though
the nucleus can be satisfactorily demonstrated in stained pre-
parations only. The cytoplasm is finely granular, and contains
as a rule a number of refractive bodies—probably reserve

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 457

material. In addition to these, pale vacuoles are usually to
be seen. ‘hey may be irregularly scattered through the cyto-
plasm (B, c) or arranged in a single line down the middle of
the organism (A). In the latter case the refractive bodies
frequently occur in pairs between the vacuoles—as shown in
fig. a. Intra-vitam staining’ brings out the refractive
bodies very sharply, but does not reveal the nucleus.
Multiplication can be easily observed under the microscope.
Tt is accomplished by the rods undergoing a transverse fission
in a manner which closely resembles that of many Bacteria.
If long cells be carefully scrutinised, some of them can be
found which show faint indications of a septum towards the
middle of the organism. ‘The septum makes its first appear-
ance as two faint transverse lines, extending towards one
another from opposite sides, in the centre of the cell. A little
later the lines appear to meet, so that a delicate septum
extends right across the middle of the cell (see Text-fig. ¢
—middle individual). Tle septum becomes thicker, and cuts
the parent cell into two equal daughter-cells. After separa-
tion—which now takes place—the contiguous ends of the
daughter-cells are square, but they rapidly assume a rounded
appearance (cf. text-fig. p). The whole process of division

as seen under the microscope—takes several hours, pro-
ceeding very slowly. I have not been able to follow the
division of the nucleus satisfactorily in the living organisms.

All the material upon which these observations are based
came from the iarge intestine of a single B. constrictor
which had been in captivity for some time. I have, therefore,
no data to indicate the frequency with which the parasite
occurs in this snake.

On examining the contents of the large intestine of the
snake, soon after death, I found a large number of organisms
present in the stages which I have just described. My first
conclusion—not unnaturally—was that I was dealing with a
large species of Bacterium. Had I not made further
observations upon the subsequent development of the organ-

} With neutral red, methylene blue or Brillantcresylblau.

458 C. CLIFFORD DOBELL.

isms, this conclusion might have appeared to some extent
justified. After fixing and staining some of the cells, a large
nucleus was seen to be present. It therefore appeared to me
at the time that I had discovered a new Bacterium which
possessed a well-marked nucleus, and hence belonged to the
eroup of organisms of which B. gammarzi is the type.

Stained examples of this organism from the boa are shown
in figs. 137, 138, 140, 141 and 144 (PI. 19) and in fig. 135
(Pl. 18). Owing to the watery nature of the rectal contents,
and to the large amount of grit present, it was found very
difficult to obtain good wet-film preparations. Most of my
stained preparations were therefore made by allowing some
of the fluid containing the organisms to dry upon a slide;!
then fixing the dried film in absolute alcohol; and finally
staining with Giemsa’s stain. As a cytological method this
is of course unsatisfactory ; but the results obtained were, in
the main, good enough for arriving at conclusions regarding
the general structure of the cells. In most cases the nucleus
had undergone a certain amount of fragmentation—owing to
drying—but it frequently showed its vesicular structure quite
clearly.

In fig. 187 a number of small individuals are depicted—
each showing a distinct nucleus. Fig. 1388 shows two larger
individuals, of the characteristic Bacterium form, with
rounded ends. Fig. 141 shows a similar organism, but with
ends of a squarer form. ‘The nucleus is in all cases unmistak-
able. In fig. 140 a chain consisting of four organisms of a
more or less bent form is seen. The nuclei are all somewhat
broken up through drying. Fig. 144 shows another chain
of four individuals, of smaller size, and each containing a vesi-
cular nucleus. Forms intermediate in size between these
small forms and the larger forms occur, so that there is no
reason for regarding them as different species. 1 propose to
vall all the individuals which have the rod-form characteristic

| At the time when these observations were made (1906) I had not

discovered the osmic acid drop method of fixation which has since
proved so useful.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 409

of Bacteria, the bacterioid forms—to distinguish them
from other forms.

The other forms which this organism is able to assume
appeared in the course of a few days in the contents of the
snake’s large intestine, which had been kept as a culture in
a glass vessel. They were not found inhabiting the snake.
Multiplication of the bacterioid forms contimued for several
days, after which the other forms made their appearance.
The ordinary bacterioid individuals (such as fig. 158, etc.)
were seen to become more rounded (fig. 142), finally assuming
the oval form characteristic of a yeast. In this yeast-like
condition the organisms continued to multiply—but by
budding, and not by transverse fission (see fig. 145). 1
propose to call these yeast-like forms the zymoid forms—
to distinguish them from the rod-like bacterioid forms.

The zymoid forms are exactly like any other ordinary yeast.
They possess an oval form, a vesicular nucleus, and repro-
duce by budding (cf. figs. 136 [Pl. 18] and 145 [PI. 19}).
They are, indeed, exactly like other yeasts with which [ am
familiar in the rectal contents of frogs, toads, lizards and
many other animals.

That the zymoid forms are directly derived from the bac-
and are not really independent organisms—I

terioid forms
can assert with absolute certainty. I have observed the
transformation in living organisms kept under observation for
several days. All intermediate forms, moreover, were found
in my fixed and stained preparations, and it was no un-
common thing to observe bacterioid and zymoid individuals
composing one and the same chain (fig. 151). Both bac-
terioid and zymoid forms existed side by side in my cultures
for many days, but finally the bacterioid forms were almost
completely supplanted by the zymoid forms.

Curious further changes were also observed. Many of the
bacterioid forms developed outgrowths, which sometimes
grew toa considerable length (see fig. 148). Many of the
zymoid forms also gave rise to outgrowths—in some cases of
very large size. These outgrowths began as short finger-like

VOL. 56, PART 3.—NEW SERIES. 32

460 C. CLIFFORD DOBELL.

processes (fig. 146), into which the nucleus sometimes
entered (fig. 149). In some cases, division of both nucleus
and cytoplasm occurred—the finger-like outgrowth being
separated off as a more or less bacterioid cell (fig. 150). At
other times, the outgrowths continued to grow in length
without cell division taking place. They often attained a
considerable length, and underwent branching (fig. 147)—
looking like the beginnings of a mycelium. Although I kept
the organisms under observation for many weeks, I never
found any other stages in development. Apparently, the
conditions under which the organisms were kept were such
as to inhibit further growth.

Now after observing the changes which my original
Bacterium-like organisms underwent, I came to the con-
clusion that I was really dealing with a fungus closely allied
to the yeasts. It seems to me more than probable that the
organisms are really fungi, a part only of whose life-cycle has
come under my notice. I believe the resemblance of the
original bacterioid forms to Bacteria is purely accidental, and
the organisms have nothing whatever to do with this group.

As I have already noted, forms similar to these from Boa
constrictor occur in the intestines of a variety of animals.
It is therefore necessary to be on one’s guard when investiga-
ting Bacteria derived from such sources. Unless observations
be made upon the development of the living organisms, one
may easily be led into error.

I must point out that the finer details of nuclear division
—of both bacterioid and zymoid forms—have not been
thoroughly investigated. ‘his is due to the fact that perfect
fixation was usually impossible. Division is, I believe,
amitotic: and this is certainly true of the form which occurs
in the frog—a form upon which I have made a number of
careful observations. As these, however, are still incomplete,
and indicate that this form is very closely similar to that
from the boa, I do not wish to enter into a fuller description
at present.

In conclusion, | would emphasise the fact that the foregoing

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 461

observations in no way invalidate the contentions of Vejdovsky
and Menclregarding Bacterium gammari. I see noreason
at present for doubting that this organism belongs to the
Bacteria. My own investigations have shown merely that
certain organisms, which appear to resemble B. gammari
at one stage in their lives, are really not Bacteria at all, but
belong to the Fungi.

Summary oF RESULTS.

I will now summarise the results which I have recorded in
some detail in the foregoing pages. In this section I shall
consider my own work only, without reference to the work of
others. A full discussion will be found in the next section of
the paper (p. 462 et seq.).

(1) All the Bacteria which I have been able to investigate
with precision contain a structure (or structures) which I
believe to be a nucleus. The reason for regarding these
structures as nuclei is two-fold—first, from purely morpho-
logical considerations ; secondly, from their staining reactions
(see discussion, p. 462).

(2) The Bacteria studied belong to four different groups—
namely, Cocei, Bacilli, Spirilla, and so-called “ fusiform
Bacteria.”

(3) The Coceus forms studied possess a single, centrally
placed, spherical nucleus in each cell. It divides by a
simple amitosis. This type of nuclear organisation has been
found in forms belonging to the genera Micrococcus and
Sarcina.

(4) Cocco-bacillar forms which have been investigated
show a nucleus in the form of a straight or bent rodlet, or of
a more or less spiral or zig-zag filament.

(5) Bacillar forms show several different types of nuclear
differentiation. ‘lhe nucleus may be in the form of chromidia
scattered through the cell (flexilis type, etc.) ; in the form
of a more cr less straight, spiral or zig-zag filament
(spirogyra type, etc.) ; or in the form of irregular strands

462 C. CLIFFORD DOBELL.

and networks (B. saccobranchi). There is evidence to
show that a nucleus in all these three forms may occur at
different times in the same organism (B. saccobranchi).
There is also evidence that spherical nuclei, filamentar nuclei,
and chromidial nuclei may occur in the same organism at
different stages in its life-history (Bacilli of modified
flexilis form from Triton and Lacerta).

(6) Spirillar forms which I have studied show three
different types of nucleus: the chromidial (Sp. monospora,
ete.) ; the filamentar (Spirillum from Lacerta) ; and the
spherical type (small Spirillum from Stylopyga), which
divides by amitosis, and resembles the nucleus of Coccus
forms.

(7
spherical, nucleus in each cell.

) “Fusiform Bacteria” possess a single, usually

(8) A number of large, parasitic, non-motile, rod-like
organisms, possessing a vesicular nucleus, which, appear at
first sight to be Bacteria, are really Fungi allied to the yeasts.

GENERAL DISCUSSION.

Now that I have briefly reviewed the more important
literature bearing upon the cytology of the Bacteria, and
have given my own observations in some detail, | am in a
position to discuss my results. My main object, as I have
already pointed out, has been to decide the question, whether
or not the Bacteria are nucleate cells. The chief part of this
discussion will therefore be directed towards answering this
question.

As I have already indicated, many of the observations
which have been made by others upon the cytology of the
3acteria, are based upon material which has been so imper-
fectly fixed and stained that it is useless to consider them.
Of the researches reviewed in the “‘ Historic ” section (p. 599),
therefore, only a part can be profitably considered here.
Furthermore, it is impossible to enter into a minute discus-

sion of many excellent contributions to the subject—extending,

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 463

as they do in the aggregate, over many hundreds of pages.
Consequently, I crave forgiveness for the many sins of
omission which must be apparent to anyone who reads the
ensuing remarks,

Metachromatic Granules.—Considerable confusion
exists in bacteriological literature regarding a number of
granular cell-inclusions which I shall call metachromatic
@ranules. Recent work has, however, done much to clear
up this confusion, and I believe that the interpretation of
these granules is now perfectly plain, and there is no cause
for any further misunderstanding regarding their nature and
significance. For an excellent summary of our present know-
ledge of these bodies, I would refer the reader to a recent
paper by Guilliermond (1910).

The first to observe these granules in Bacteria appears to
have been Babes. It was he, also, who subsequently named
them ‘“‘ metachromatische Kérperchen.” There seems to be
little doubt that the majority of colourable granules which
have been described in bacterial cells really belong to this
class of bodies. Different observers have given different
names to the granules, and this has been largely the cause of
the confusion which at present exists regarding them. It
appears to me certain that the ‘‘ metachromatic bodies” of
Babes, the ‘‘sporogenic granules”? of Ernst, the “red
grauules” (in part only) of Biitschh, the ‘chromatin
granules” (in part) of Wahrlich and many others and of
Meyer’s earlier papers, the “granules” of Fischer, the
“ Volutanskugeln” of Grimme, the “ volutine” granules of
Meyer, the ‘“toxigen granules” of von Behring, the
“ Babes-Ernst bodies” of many bacteriologists, and many
other kinds of granule described by many other workers—all
these are in reality the same, namely the bodies which I
shall call metachromatic granules. This name aptly
designates these bodies, and has been used throughout by
Guilliermond! in his important researches into their nature ;
and I hope—with him—that it will find universal acceptance

1 Guilliermond’s actual name is “ corpuscules métachromatiques.”

464. C. CLIFFORD DOBELL.

and so help to clear away the confusion which now surrounds
these bodies.

Metachromatic granules are found not only in many
Bacteria, but also in Fungi, Algw, Cyanophyceex, Protozoa,
and probably in many of the “higher” groups of animals and
plants. ‘Their presence in Bacteria can therefore not be used
as evidence of the affinities of this group.

Regarding the chemical and staining properties of these
granules, we now have a considerable mass of information—
chiefly from the work of Guilliermond, Grimme and A. Meyer.!
Their most characteristic property is that they stain red with
many blue or violet stains (e.g. methylene blue, hamatoxyhn,
etc.). After fixation they have a strong affinity for so-called
“nuclear” stains—which has given rise to their confusion
with chromatin.

Chemically considered, the metachromatic granules are
probably to be regarded as composed of nucleic acid combined
with an organic base (cf. Meyer, Guilliermond).

The biological significance of the metachromatic granules
appears to be definitely decided. They are non-living
(metaplasmic) reserve material, They are not living
morphological derivatives of either nucleus or cytoplasm, but
merely stored up food substance. The evidence for this
appears to me overwhelming. ‘'he most important fact has
been established, I believe, that they are not a constituent of
the living protoplasm: they are transient, non-living
elements of the cell. That they are in any way an index of
the virulence of the organisms containing them, as maintained
by Marx and Woithe (1900), is negatived by the work of
Ascoli (1901), Krompecher (1901), Gauss (1902), Schumburg
(1902), Ficker (1903), Guilhermond (1906) and others. ‘lhe
biological distribution of the granules throughout other
organisms also speaks strongly against such a view.

It might be urged, with some Justification, that the
“ chromidial nucleus ” described in Bacteria by Schaudinn,
Guilliermond and myself is really nothing more than a diffuse

1 See also Eisenberg (1910).

CONTRIBUTIONS TO THE CYLOLOGY OF THE BACTERIA. 465

system of metachromatic granules. Such a supposition has
already been considered and rejected by both Schaudinn and
Guilliermond. I have also had occasion already to speak
against this view, and I shall now enter into it more fully.
The Bacilli of the spitrogyra type which I have described,
also the Micrococci, and Spirilla with a filamentar or
spherical nucleus, are in the majority of cases entirely free
from granular inclusions in the cytoplasm. The nuclear struc-
tures which I have described are the only constant internal
structures present. It is therefore useless to argue about meta-
chromatic granules in these forms, unless it be assumed that
the nuclear filaments, etc., are metachromatic bodies—an
assumption for which there is not a shred of evidence, and
which is entirely opposed to the facts. It remains therefore to
consider the Bacilli and Spirilla (chiefly the organisms
of the flexilis type, and the Spirilla from the frog and
cockroach) in which I have described a chromidial nucleus.
In the first place, I must point out that the two methods of

staining—namely the Heidenhain and Romanowski methods
—which I have chiefly used are not sufficient to distinguish
between chromatic and metachromatic substances by means
of differential staining. Both chromatic and metachromatic
granules are stained black with Heidenhain and red with
Romanowski. Neither method, therefore, can be used as an
index of the chemical nature of the granules. In the second
place, I think it highly probable that metachromatic granules
do exist, side by side with the nuclear granules, in many
Bacteria with chromidial nuclei (cf. also Schaudinn [1903],
and Guilliermond [1908]). Bacillus flexilis itself, and
also other Bacilli of the same type, contain granular in-
clusions which may easily be stained intra-vitam with
neutral red, methylene blue and Brillantcresylblau. All
these granules have a faintly reddish tinge when so treated.
The same is true of Spirillum monospora. ‘These
colourable granules are few in number, however, as compared
with the number visible after Heidenhain or Romanowski
staining. I believe therefore that they are metachromatic

466. C. CLIFFORD DOBELL.

granules (reserve material) which are present in addition to
the granules constituting the nuclear apparatus.

That some of the “red granules” described by Biitschh,
and the ‘‘chromatin granules” of Wahrlich, A. Meyer and
others are also really metachromatic granules, I think ex-
tremely probable. Yet I believe that many of these granules
seen by these observers are of a nuclear nature—as in the
case of my own Bacteria. Guilliermond (1908), moreover,
found granules of both chromatic and metachromatic material
in a number of forms which he investigated.

Now the evidence for regarding the greater part of the
granules in my Bacteria as of a nuclear nature is not derived
chiefly from their staining reactions—which I regard as of
secondary importance—but is morphological. I shall
consider this in detail in the ensuing section.

Morphological Evidencethat Bacteria are Nucleate
Cells.—Before proceeding any further, it is necessary to
consider for a moment what is meant by the term nucleus.
Various more or less unsatisfactory definitions have been
given, and I do not propose to add to their number. ‘To
define any well-known thing—such as a nucleus—is merely
to confine one’s idea of the thing to certain arbitrarily chosen
properties which it possesses, and to lay oneself open to the
attacks of the verbal quibbler. It is absurd to define a
nucleus in terms of certain of its chemical characteristics
alone. Still more absurd is it to base a definition upon its

staining reactions; for—apart from the fact that it cannot,
in most cases, be definitely proved whether staming is a
chemical or physical phenomenon—it is well known to every
cytologist that different nuclei may display a very wide range
of difference in their staining capacities. And yet I think
every biologist knows what he means when he speaks about
a nucleus. He means a morphological element of the
living cell—a structure which could have been discovered
even if chemistry were completely unknown, and staining had

)

never been invented. ‘The concept ‘‘ nucleus ” ds fundament-

ally one of form—the idea, that is to say, belongs primarily

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 467

to the province of morphology, not of chemistry or physics.
It is necessary to bear this in mind when discussing it. Hence
whether a given body is a nucleus or not can only be decided
by studying its morphology and then comparing it with other
structures which we agree to call nuclei. Chemical properties
and staining reactions may aid us materially in reaching a
conclusion, but they cannot alone be used as criteria at
present.' If they could, then a pound of nuclear substance—
if it could be obtained—would be a nucleus.

One more point must be mentioned here. It has been
many times asserted that Bacteria consist entirely of nucleus,
or entirely of cytoplasm—because no cellular differentiation
like that of other organisms has been discovered. That
Bacteria are composed of cytoplasm is not frequently stated
in so many words, but it is often tacitly assumed when speak-
ing of these organisms as enucleate. But that Bacteria are
nuclei has been definitely stated by many workers—especially
in recent years by Ruzicka. Now, apart from any work
which may have led to such an interpretation, I should like
to point out that such statements are, a priori, nonsense.

By “nucleus ”

and “ cytoplasm” are meant definite morpho-
logical elements into which most—probably all—cells are
differentiated. There is good experimental evidence that
neither nucleus nor cytoplasm—specialised parts both of the
living protoplasm—is capable of living independently of the
other for any length of time. ‘To calla Bacillus a naked
nucleus is, therefore, a misapplication of a word in common
use. An organism may have a structure similar to that of
many nuclei, it may have similar chemical and staining

characters,” but to call it a nucleus in consequence is—far

‘In connection with the nucleus in Bacteria somewhat similar
views have already been expressed by Schaudinn (1903). It is curious
to note how many other writers are so profoundly impressed with the
importance of chromatin that they frequently use * chromatin” and
“nucleus” as though they were synonymous.

2 It should also be emphasised that the “special affinity for chromatin
stains,’ which is often attributed to Bacteria, is—as Fischer has
pointed out—a myth.

468 CG. CLIFFORD DOBELL.

from giving a satisfactory interpretation—simply to misuse
words. ‘Chat Bacteria are composed of a substance similar to
cytoplasm may readily be granted; but to say that they
consist of cytoplasm is merely to use the word “ cytoplasm”
in a sense which is not generally accepted. Hence, if it were
d

without
a nucleus, it would be necessary to employ some other word

proved—which it is not—that Bacteria were “ cells’

than cytoplasm to designate their contents—for instance
Van Beneden’s term “ plasson,” or some such word. At
present, however, there is no necessity to follow such a course.
If one chose arbitrarily to call nucleus cytoplasm, and cyto-
plasm nucleus, one could easily make the astounding gene-
ralisation that cytoplasm was really not cytoplasm, but
nucleus. Such, it seems to me, is the method of reasoning
which is occasionally applied in considering the structure otf
Bacteria.

In addition to the foregoing considerations, I should like
to emphasise another point. It is sometimes stated that the
Bacteria show a peculiar kind of protoplasmic organisation
in which nucleus and cytoplasm are not yet differen-
tiated from one another—that Bacteria show, in fact, a
primitive type of structure. Now-it has never been proved

and indeed the evidence is against it—that Bacteria
possess such a structure. It is obvious, therefore, that
the assumption of a condition supposed to be primitive
cannot be used as an argument in favour of the primitiveness
of the group—as is sometimes done.

Having said so much with regard to the nucleus in general,
T will pass on to an application of my reasoning to the
experimental results.

I shall begin with a consideration of the Coccus forms of
Bacteria. I have shown that certain Micrococcei and
Sarcinz contain, in each cell, a single, centrally placed
spherule. his body is a morphological feature common to
every cell. When the cell divides, the spherule also divides
—its division preceding that of the cell as a whole, and being
characterised by the formation of a dumb-bell-shaped figure

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 469

during the process. There is therefore every reason to believe
that the centrally placed body is a living constituent of the
cell. It cannot be maintained that it is a non-living structure
—for instance, a fat globule or metachromatic granule.
Now on purely morphological grounds, on analogy with what
is known of other cells, I think I am justified in calling this
centrally placed body in cocci a nucleus. It corresponds
as closely as could be desired with the structures which we
are accustomed to call nuclei in other cells. If it is not a
nucleus, then what is it? There is, I believe, only one
possible answer to such a question—that it may bea structure,
absent from other cells, which looks exactly like, and
behaves exactly like, a nucleus, but is really not a nucleus.
I think, therefore, that on morphological grounds it is com-
pletely justifiable to regard this body as a nucleus. More-
over, such a conclusion is considerably supported by the tact
that the structure is stained red by Romanowski’s method—
the colour which is assumed by structures which are uni-
versally admitted to be nuclei.

The observations which I have made do not stand alone.
They are supported by the quite independent observations of
Nakanishi (1901) and Menel (1910)!—both experienced
workers who employed reliable cytological technique. ‘The
organisms studied by Nakanishi, Mencl and myself, though
all Coccus forms of Bacteria, are all different organisms, and
the cytological methods used were different in each case.
Both Nakanishi and Mencl, moreover, draw the same conclu-
sion as I do—though not altogether from the same premisses.
They both believe that the structures which they discovered
are nuclei,

The contention of Meyer (1908), that the nuclei described
by Nakanishi are really vacuoles, is hardly worth discussing ;

1 J should like to point out—though of course I do not claim priority
in the discovery of nuclei in Cocci—that my observations were in no
way influenced by the work of Nakanishi or Mencl. My own observa-
tions were made before I had seen Nakanishi’s work, and two years
before the publication of Mencl’s paper.

4.70 C. CLIFFORD DOBELL.

for the same idea occurred to Nakanishi himself, and he
brought forward good experimental evidence to show that
this was not the case.

Asa result of my researches I regard it therefore as certain
that the Coceus forms of Bacteria contain a nucleus
of the form which I have described in the earlier part of
this paper.

And now let us consider the other Bacteria. I have
pointed out already that I have investigated a large number
of coceo-bacillar organisms which present every degree of form
between typical Cocci on the one hand, and typical Bacilli
on the other. With change inthe external shape, the nucleus
shows a corresponding modification. It becomes elongated
with the elongation of the cell, and hence assumes the form
of a filament. In round coccus forms, the nucleus is round.
In shehtly elongated cocci, the nucleus is in the form of a
short rodlet, which may be curved or shghtly bent. In still
more elongated cocco-bacillar organisms, the nucleus may
have the form of a zig-zag@ or spiral filament. ‘hese forms
merge gradually into the forms of the characteristic spiro -
gyra type.

I have not proved that the Coeci, spirogyra Bacilli and
intermediate cocco-bacillar forms, which I have found living
together, are genetically connected. A: proof of this is
immaterial for the present purposes. It suffices to know that
ail these forms occur. | Morphologically considered, therefore,
the spiral or zig-zag filament present in Bacilli of the
spirogyra type is the equivalent of the spherical body which
lies in the centre of the Coceus cells. Consequently, if it is
agreed that the latter is a nucleus, it follows that the spiral
filament of Bacilli of the spirogyra type is also a
nucleus. ‘his is a conclusion which is supported by the
behaviour of the filament during cell-division and spore-
formation, which I have described in detail in B. spirogyra.
My study of this form indicates beyond a doubt that the
filament is aliving element of the cell, and not a metaplasmic
structure.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 471

A further point in support of the morphological equivalence
of the spherical nuclei of Cocci and the filamentar nuclei of
certain Bacilli is furnished by the observations recorded on
p-421. Ihave shown that the nucleus of certain Micrococei,
when the cell is elongated during the process of cell-division,
may be drawn out into a zig-zag or spiral filament. We see
here directly, I believe, the way in which the filamentar
nucleus of some Bacilli has been derived from the spherical
nucleus of Micrococcus forms.

Again, staining reactions—so far as they go—support the
interpretation of the filament in Bacilli of the spirogyra
type as a nucleus.

At this point Bacillus saccobranchi must be considered.
I have shown that this organism possesses at one stage in its
life-history a nucleus of the characteristic spirogyra type
—that is to say, a spiral or zig-zag filament which is the
morphological equivalent of the nucleus of Coccus forms.
Now this structure undergoes a remarkable transformation
during the deveiopment of the organism. It becomes con-
verted into the form which I have called the “irregular
form ’’—assuming an appearance of an irregularly branching
filament or network. This structure in turn breaks up to
form a series of granules scattered diffusely through the
whole cell—the ‘chromidial form.’”! It follows, therefore,
with absolute certainty, that if the spiral filament is a
nucleus—as I have already shown is almost certainly the
case—then the chromidial structures are also the
morphological equivalent of a nucleus. ‘They are
developmental stages of the very same living constituent of
the cell which is represented at other times by a spiral
filament or irregularly branched filament or network. In
Bacillus saccobranch1, therefore, there is every reason to
believe that a nucleus in the form of scattered granules, or
chromidia, exists at certain stages in the life-cycle.

1 T have pointed out (p. 444) that itis possible that the changes in the
nuclear structures may take place in the reverse order to that given
above. It is immaterial tomy argument in which direction the sequence
of developmental changes takes place.

472 C. CLIFFORD DOBELL.

Again in this organism, staining results confirm the morpho-
ogical interpretation.

Arguing now on analogy, it becomes highly probable that
the scattered granules of Bacilli of the flexilis type—the

lo

are of the same nature as the

chromidia, in other words
eranules of Bacillus saccobranchi. They are the only
morphological elements distinguishable in the cells, and that

they are living structures—not reserve material
ine quite certain from the part which they play during spore-
formation. When it is further found that, in the course of
spore-formation, the granules arrange themselves in the form

appears to

of a spiral or zig-zag filament!—like that of Bacilli of the
spirogyra type—then the nuclear interpretation of the
granules is not merely strengthened, but becomes almost a
certainty. It appears to me that there is only one logical con-
that the chromidiaof

clusion to be drawn from these facts
Bacilli of the flexilis type represent the nucleus,
being the equivalent (morphologically) of the spherical
nucleus of Cocci and of the spiral filament of other Bacilli.
When we find that many smaller Bacilli show a structure
which is essentially the same as that of the large Bacilli of
the flexilis type, it 1s only natural to suppose that we see
here, also, structures which are capable of a similar inter-
pretation. ‘lhe assumption is justified that the chromidia
of small Bacilli constitute their nuclear apparatus.
In all these cases, moreover, staining reactions—so far as |
have tried them—support the morphological interpretation.
If we now consider the structures which are present in the
sacilli of a modified flexilis form (from the newt and
lizard—see p. 430) it becomes apparent that these structures
also represent phases of the nuclear apparatus. The actual
facts here are not so well established as in the forms which I
have hitherto considered, but it is at least exceedingly
probable that in these the nucleus exists, at some stages in
the life-history, in the form of a few large globular masses.
! Discovered by Schaudinn (1902) in Bacillus bitsehlii, and
confirmed by me (1908) in the case of B. flexilis.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 473

The aggregate of these masses in each cell is the morpho-
logical equivalent of the chromidia or the spiral filament.

Thus, we see here another modification of the nucleus
which may exist in Bacteria.

I will now consider the spirillar forms which I have in-
vestigated. I have found that three different types of
structure exist in these organisms. In one of these there is
a minute spherical body present in each cell : it divides with
a dumb-bell-shaped figure, its division preceding that of the
cell (small Spirillum from Stylopyga). It is aliving
element—a morphological feature of each cell. In the
second type, there is a filament of a zig-zag or spiral form,
which also divides into two during cell-division (Spirillum
from Lacerta muralis). Thirdly and lastly, there is a
type of Spirillum whose characteristic morphological feature
is a system of granules scattered through the cell (Sp.
monospora, large Spirillum from Stylopyga),. Froma
consideration of these spirillar forms alone we could, with
considerable justification, reach the conclusion that these
three different types of structure represent three different
modifications of the nuclear apparatus—upon morphological
grounds. When the analogy of these structures with the
nuclei of Coeciand Bacilli is considered, however, it appears
to me that only one logical deduction can be drawn, namely,
that the single spherule, the spiral filament, and
the chromidia of Spirilla are nuclei.

Staiming, again, gives results consistent with this inter-
pretation.

I believe my nuclear interpretation of the various
structures discussed above is the only logical
interpretation which can be given to the facts
known to us at present. And of the accuracy of the
facts which I have recorded, I have not the shghtest doubt.!

' Owing to the fugitive nature of the staining methods which I have
frequently employed, it is now impossible to demonstrate many of my

preparations satisfactorily. I have therefore at various times demon-
strated my preparations to competent observers, in order that they

A474, C. GLIFFORD DOBELL.

It remains now to consider how far these facts coincide
with those recorded by others.

First of all, I would point out that my results are in agree-
ment with those of Schaudinn (1902, 1903) and Guilliermond
(1908)—both of whom made accurate cytological investiga-
tions of different organisms. Both these observers, however,
examined Bacteria which possess a nucleus of the chromidial
form: it is with my chromidial forms, therefore, that their
results must be compared. Both Schaudinn and Guilliermond
—though on different grounds—arrived at an interpretation
similar to my own.

Quite recently, Guiliermond (1909) has recorded the exis-
tence of two species of Bacillus and a Spirillum which
possess nuclear filaments like those which I have described in
these forms. His observations appear to have been made
quite independently of mine, and may therefore be taken as
confirmatory.

I find it dificult to decide how far the results of Swellen-
erebel (1906, 1907, 19074, 1909, 19094) coincide with mine.
He finds in Bacilli and Spirilla remarkable filamentar
structures, usually in the form of an irregular or broken
spiral. On account of the micro-chemical and staining
reactions of these structures, he is led to interpret them as
nuclei. They are not exactly hike the filamentar structures
which occur in Baecilli of the spirogyra type. In many
cases they resemble certain of the nuclear modifications of B.
saccobranchi. It seems to me possible that in some cases
also the appearances are the result of imperfect fixation'—the
original spiral filament having been broken up in this process.

Sometimes, also, the filaments may be really chromatin

could confirm my statements as to the existence of the structures which
IT have described—if their existence were called in question. Among
those to whom I have shown one or other of my preparations may be
mentioned Sir Ray Lankester, Prof. Adam Sedgwick, Prof. J. B.
Farmer, and Prof. F. Vejdovsky—all of whom have agreed with me
as to the appearances presented.

1 See footnote on p. 417.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 475

granules connected by deeply stained cytoplasm—as main-
tained by Guilliermond (1908). As I have not myself madea
study of the forms which Swellengrebel describes, and as his
work has evidently been conducted with considerable care
and thoroughness, I hesitate to make any more definite
criticism of it at present.

The earlier observations of Biitschli (1890, 1896), Wahrlich
(1890), Zettnow (1897), and others are in agreement with
mine! if it be assumed—as appears highly probable—that
they investigated only those forms of Bacteria which possess
a chromidial nucleus. With Biitschli’s interpretations, how-
ever, I cannot agree.

Nuclei in the form of a few small granules in each cell,
described by Meyer (1897, 1899), and Preisz (1904), are
probably of the same nature as chromidial nuclei, and
the nuclei which occur in the Bacilli of modified flexilis
type.

The facts and their interpretations, given by Nakanishi
(1901) are—in many cases—closely parallel to my own.
Nakanishi found filamentar nuclei in Bacilli (e.g. B.
anthracis), and in Spirilla spherical and filamentar nuclei,
which are very like the structures which I have myself ob-
served in similar forms. After ably discussing his observa-
tions, Nakanishi arrived at an interpretation which agrees
with mine.

How far the observations of Amato (1908) can be brought
into line with my own I do not know. It is possible that
the “nuclear” structures which he describes are really meta-
chromatic granules—as suggested by Guilliermond (1910).

A point of considerable importance is to be found in the
work of Schewiakoff (1895). In Achromatium, he found
a number of minute chromatin granules scattered through the
cytoplasm—in other words, he found a nucleus of the
chromidiai type. He observed that these granules undergo
division—which is a further important piece of evidence

1 So far as the actual morphology of some of the smaller Bacteria
is concerned.

VOL. 96, PART 3.—NEW SERIES. 33

476 C. CLIFFORD DOBELL.

that they are living structures.! In Bacilli of the flexilis
type the chromidia are too small for their division to be
observed with accuracy, but I think they probably behave in
much the same way as the larger chromidia of Achro-
matium.

The remarkable work of Mencl (1905) upon filamentous
water Bacteria (Cladothrix, ete.) contains many observa-
tions which are in complete accord with mine. In the forms
he found nuclei of a

investigated—which are pleomorphic
spherical, filamentar, and chromidial form, with numerous
intermediate forms. He was able to observe the division of
these nuclei in the living cells—thus proving that they were
really living structures, and not metachromatic or other non-
living granules. He believes that the different nuclear forms
occur, at different stages in the life-history, in the same
organism. His results are therefore closely similar to mine.
The nuclear interpretation of the chromidial structures
present in Bacteria—as upheld by Schaudinn, Guillermond
and myself—has been controverted by Ruzicka (1909) on the
grounds that the whole bacterial cell is itself the equivalent
of a nucleus. Apart from the a priori absurdity of this
I must em-

view—which I have already pointed out above
phasise the fact that the observations recorded in the present
paper completely condemn such an interpretation. On the
other hand, I believe the chromidial view is completely
vindicated. ‘lhe statement made by Ambroz, who follows
Ruzicka, that the chromidial view has been “ reduced ad
absurdum”’ by the latter, is therefore entirely erroneous.
The observations of Mitrophanow (1895)? seem to me to be
capable of being brought into line with my own, when
allowance is made for the difference in technique. I find it
not always easy to comprehend Mitrophanow’s meaning ; his
methods of fixation and staining also seem to leave much to

1 Hinze (1903) made similar observations in the case of Thiophysa.
2 This paper is an abstract only of a larger work in Russian. It is
therefore possible that Mitrophanow’s observations and views are more
clearly given in the original—which is unfortunately inaccessible to me.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 477

be desired. Nevertheless, he appears to have found organ-
isms possessing nuclei in the form of chromidia, spiral fila-
ments and spherical masses of chromatin. He also dis-
tinguishes between nuclei and “ granulations,” and points
out the structural variability which the nucleus displays. On
the whole, his observations—so far as I understand them—
appear to be in agreement with mine.

Kunstler’s (1887) observations upon the structure of

Spirilla agree closely with what I have myself described in
Spirilla with a chromidial type of nucleus. Also the
chromatin structures described in the cholera Vibrio by
Podwyssozki (1893) bear a strong resemblance in many cases
to the nuclei which | have shown to occur in the small
Spirillum from the gut of Stylopyga orientalis.
I believe the “chromatin” granules described in sulphur
Bacteria by Hinze (1901, 1903) and Dangeard (1909) are—
like Biitschli’s findings in similar forms—to be interpreted as
nuclei in a chromidial condition. ‘The same interpretation
will apply to the granules of B. oxalaticus, described by
Migula (1894) ; and also to the iron- and phosphorus-containing
granules found in Beggiatoa by Macallum (1899).!

Rowland’s (1899) results can easily be explained if it be
supposed that the organisms which he studied possessed
nuclei in the form of chromidia in addition to metachromatic
bodies.

I think I may fairly claim, from what I have already
pointed out in the preceding pages, that not only do my own
observations furnish most conclusive evidence with regard to
the nucleus in Bacteria, but that in almost every case in
which careful investigation has been made by others, the
results are not inconsistent with mine. In many cases they
are, indeed, completely confirmatory. When good technique
has been employed, and careful observations have been made,

! Certain points in connection with fixation are, moreover, not quite
clear to me in the work of this author. It may also be pointed out that
Maeallum failed to find a nucleus in the yeasts—in which a typical
vesicular nucleus certainly occurs.

478 C. CLIFFORD DOBELL.

I do not believe a single fact of any importance has been
found which speaks against my results. In matters of inter-
pretation, of course there is considerable difference of opinion
already existing; but I am convinced that no interpretation,
other than that which I have given, can be found which will
fit all the facts known to us at present. How far such a con-
viction is justified further work alone can show.

So far I have considered only the Bacteria themselves, and
I believe the evidence which I have given from this group
alone is sufficient to establish the fact that Bacteria are
nucleate cells. Considerable additional evidence may, how-
ever, be adduced from analogy with two other groups of
organisms—the Protozoa and the Cyanophycee. In the
Protozoa, a chromidial form of nucleus occurs in many
different organisms, as a transient stage in the life-cycle. It
may also occur as the normal vegetative condition. It is un-
necessary to enter into this subject in detail here. The reader
will find a condensed account of chromidia in a paper which
I have previously published (see Dobell, 19098). A nucleus
in the form of irregular strands, networks, granules, etc.,

scattered through the cytoplasm, also occurs in Protozoa
especially in the Infusoria (cf. Dobell, 1909a).

In the Cyanophycez, analogous nuclear conditions probably
obtain. It is impossible in the present paper to enter into
a discussion of the vexed question of the nucleus in this group,
but I should like to call attention to two recent contributions
to the subject which have been made as a result of careful
cytological work. I refer to the work of Gardner (1906) and
Guilliermond (19074) Gardner describes and figures nuclei
in the form of networks, granules, and irregularly branched
filaments. Guilliermond describes similar structures, and also
nuclei in Nostoe which resemble those of Microcoecei, and
nuclear filaments, like those of Bacillus spirogyra, in

tivularia. If analogies were wanting for the structures
which I believe to be nuclei in Bacteria, they could be found
therefore without any great difficulty in the nuclei of other
organisms.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 479

Do Bacillar Forms with a Vesicular Nucleus
exist?—I have already had occasion to note that Bacilli
with a typical vesicular nucleus have never come under my
observation. All the organisms which I found to be con-
stituted in this manner have proved to be Fungi. Others,
however, have described very definite instances in which
vesicular nuclei occur, and the matter is of such moment that
a brief discussion is here necessary.

In the accounts of the older observers, the observations
are so incomplete, and the technique employed was_ so
impertect, that a discussion seems useless. his is not the
case with some more recent work, however. I refer to the
pubheations of the Bohemian investigators, Vejdoysky,
Mencl, and Rayman and Kruis.

There seems no doubt at all, from the very careful work
of Vejdovsky (1900, 1904) and Mencl (1907), that the organ-
ism which the former has named Bacterium gammari
really possesses a vesicular nucleus, which divides mitotically.!
The only point which requires to be settled is whether the
organism really belongs to the Bacteria or not. Considerable
discussion has already taken place regarding this. Some
observers (e. g. Guilliermond, 1907, 1908, 1910) are inclined
to regard it as a yeast-like fungus—not a Bacterium at all.
The resemblance between certain yeast forms and _ this
organism is certainly very striking (compare, for example,
the figures of Mencl [1907]—figs. 4, 7, 10, ete. [pl. x]—
with Wager’s [1898] figures—figs. 45, 46, 47 [pl]. xxx]—of
Saccharomyces pastorianus). After my own experi-
ences with Bacterium-like yeasts (see p. 455), I hesitate to
express an opinion with regard to B. gammari. It is
most important that further observations should be made
upon this most interesting organism ; and it is to be hoped that

1 My friend Prof. Vejdovsky has very kindly given me a preparation
of this organism, so that I have been able to examine it myself. To
my mind there can be no doubt as to the accuracy of the accounts
which have been given of it.

480 C. CLIFFORD DOBELL.

before long someone to whom fresh material is accessible
will reinvestigate the matter thoroughly.!

tegarding Vejdovsky’s filamentar forms from Bryodrilus,
my opinion is that they are really Fungi, similar to those
which I have myself described. Guilliermond (1907, 1908)
expresses a similar opinion—‘nous sommes a peu pres
certains, aprés V’examen attentif de ses (1.e. Vejdovsky’s)
préparations, qu’elle correspond a une moisissure. Nous
Wavons trouvé en tous cas, dans cette espéce aucun des
charactéres des Bactéries” (1908, p. 37).

I think there can be no doubt that the Bacterium-like
organisms, which I have already described (p. 455), are really
Fungi, allied to the Saccharomycetes. ‘lhe evidence for this
is chiefly derived from two features of their hfe history—(1)
the assumption of a characteristic yeast form, which repro-
duces by budding, (2) the formation of mycelium-hke out-
growths.” Similar outgrowths have been observed in yeasts
by other workers (cf. Janssens and Mertens, 1903). ‘To this
same group of organisms belong—I believe—two other forms
which have recently been described, namely, Kermincola,
a parasite of the body cavity of Coccid insects (Sule,
1906), and Bacillopsis stylopyge, from the cockroach
(Petschenko, 1908). Both these forms appear to me to be
indubitably Fungi, and not Bacteria (cf. also Vejdovsky,
1906). The fact that my organisms, Kermincola and

1 It is to be gathered from the discussion which has taken place regard-
ing B. gammari (Guilliermond, 1907, 1910; Menel, 1909) that Schau-
dinn—who saw Vejdovsky’s preparations at the Zoological Congress in
Berne—at first expressed the opinion that the organism was a yeast.
Later, however, he accepted Vejdovsky’s interpretation of it as a
Bacterium—an opinion shared also by Schewiakoff.

2 The formation of outgrowths is of course occasionally observable
in true Bacteria (Bacilli, Bacteria, Spirilla). It is usually
observed only in involution forms. Meyer (1901) interprets the out-
erowths as a reminiscence of mycelium formation in the ancestors of
Bacteria—believing them to be of fungal origin. For my own part, I
do not believe that the Bacteria have anything whatever to do with the

Fungi, and do not regard this as a correct interpretation of the pheno-
menon.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 481

Bacillopsis, are all Fungi, indicates of course nothing
regarding the existence or non-existence of true Bacteria with
a typical vesicular nucleus.

The vesicular nuclei described in Bacteria by Mencel (1904,
1905, 1907) and Rayman and Kruis (1904) are, according to
Guilliermond (1907, 1908, 1910), capable of a very different

‘

interpretation. According to him, the ‘ nuclei” are really
nothing more than various stages in the formation of trans-
verse septa in dividing cells. ‘This interpretation is vigorously
attacked by Mencl (1909), who maintains that vesicular
nuclei are actually present, and can be readily distinguished
from the transverse septa. Mencl’s figures certainly seem
clear enough—as do the photographs of Rayman and Kruis.
And J find it difficult to believe that so accurate and ex-
perienced an observer as Mencl could make such a mistake.
Swellengrebel’s (1907) results on B. binucleatum are also
favourable to his interpretations. Yet a certain amount of
imcertainty exists at present regarding these forms.

Finally I must say that it seems to me probable that

Bacteria do exist which possess—at any rate during part of

nuclei of the vesicular form characteristic of

their life-cycle
the cells of “higher” animals and plants. It is certainly not
legitimate to argue that because Bacteria have not been
previously found which contain a vesicular nucleus, therefore
that any form in which a vesicular nucleus can be demonstrated
—e.g. Bact. gammari—does not belong to the Bacteria,
but to the Fungi or some other group. This is simply begging
the question. There is absolutely no reason, either from my
own observations or from those of other workers, why typical
-vesicular nuclei should not occur in some Bacteria. The
evidence, in fact, is in favour of the view that such nuclei do
exist in certain Bacteria at certain stages in their lives.
Variability of the Nucleus at different Periodsin
the Life-cycle.—lIt will already be apparent to anyone who
has read the preceding part of this paper, that the nucleus of
any given bacterium is not necessarily constant in its form at

4.82 C. CLIFFORD DOBELL.

all stages in the life history. This point seems to me worth
special attention.

In the case of Bacillus saccobranchi, I have pointed
out that the nucleus may be in the form of a spiral filament,
or in the form of chromidia, or in forms intermediate between
these and characterised by having an appearance of irregular
strands, granules or networks of chromatin. There can be
no doubt that, in this Bacillus at least, the nucleus has a
variable structure. There is, however, no evidence to show
what relations these various nuclear modifications bear to the
life-cycle as a whole. All that can be said at present 1s that
these different nuclear forms exist.

When we turn to the Bacilliof the flexilis type, however,
we have exact knowledge of the relations between the nuclear
modifications and the phases in the life-cycle. From
Schaudinn’s (1902) study of B. biitschlii and my own re-
searches on B. flexilis and allied forms it can be definitely
stated that the chromidial stage represents the normal
vegetative condition of the nucleus, existing throughout the
vreater part of life. A nucleus in the form of a spiral
filament occurs as a transient stage connected with, and
immediately preceding, spore-formation. In the spore itself
a third nuclear modification is seen. ‘The chromatin is in the
form of a densely aggregated mass, which constitutes the
chief part of the living substance of the spore. From this
aggregated mass the chromidial condition is again assumed
in the process of germination from the spore.

In the Bacilli which I have termed those of a ‘ modified
flexilis form,” these three nuclear conditions are encountered
in a modified form, but their relation to the phases of the
life-cycle has not been determined.

In Spirillum monospora (Dobell, 1908), Bacillus
sporonema (Schaudinn, 1903) and many other Bacilli
(Guilliermond, 1908) only two modifications of the nucleus
have been established. During the vegetative condition the
nucleus is in the form of chromidia. It then assumes the
form of an aggregated mass, which enters into the formation

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 483

of the spore. These two different nuclear conditions therefore
coincide very definitely with two different phases of the life-
cycle.

In Bacillus spirogyra and allied organisms—as I have
shown (1909)—two nuclear conditions are also found.! Jn
the ordinary vegetative part of the life-history the nucleus
is in the form of a filament. A part of this gives rise to a
large, aggregated spherical mass of chromatin which enters
into the spore. Here, again, the nuclear changes are corre-
lated with definite stages in the life-history. I have not
studied the young Bacilli which emerge from the spores in
any organism of the spirogyra type. I cannot therefore
state with certainty that the observed nuclear changes are the
only ones which exist. On analogy with B. saccobranchi,
it is quite possible that a chromidial condition of the nucleus
occurs in Bacteria of this sort.

I have shown that three different nuclear conditions exist
in three different species of Spirilla which I have studied.
If one can argue on analogy in this case, it appears not
improbable that these nuclear conditions are temporary, and
that other phases in the nuclear structure exist in these
organisms also. It is quite possible, for example, that the
nuclear filament in the Spirillum from the intestine of
as

Lacerta muralis may at other stages in the life-cycle
in Bacillus saccobranchi—become modified into the
chromidial form of nucleus which exists in such an organism
as Sp. monospora.

My own belief is that the nucleus in Bacteria may
display not one, but many forms during the whole
life-cycle. Many of the nuclear structures which have
been shown to exist in these organisms should, I think, be
regarded as temporary stages rather than as permanent
conditions. The different results which have been reached

1 It may be emphasised also that the spiral filament itself in Bacteria
of this type shows a wide latitude of variation in form. Whether these
variations are correlated with special stages in the life-cycle is as yet
unknown.

484. C. CLIFFORD DOBELL.

by different workers when working, apparently, upon the
same species, may to some extent find an explanation in this
circumstance.

IT would call attention to the fact that Mencl'—whose
studies have been carried on with quite different Bacteria
from those which I have investigated—has arrived at a
similar conclusion. Many times Mencl has emphasised this
point

a point which is, I believe, of fundamental importance
for reaching a correct interpretation of the Bacteria. I am
rejoiced that in this we are both agreed.

Pleomorphism.—Though I have no conclusive evidence
to add to what has already been contributed to the hypothesis
of the pleomorphism of Bacteria, nevertheless, | must point
out that many of the facts recorded in the earlier part of this
paper are consistent with such a view.

Whilst investigating the Micrococcus, Cocco-bacillus
and Bacillus forms which I found in the gut of the lizard, I
was often impressed by the apparent genetic relations existing
between them. '’he same was the case with many of the
different bacillar forms which I found in the blood of Sacco-
branchus. I have already pointed this out in previous
pages, and although a direct proof of such genetic continuity
is wanting,
such an interpretation. This appears to me, in fact, the most

my observations are completely in accord with

probable hypothesis at present: otherwise it would be neces-
sary to assume the existence of an almost inconceivably large
uumber of species to account for the number of intermediate
forms which occur.

For my own part, I believe—although this is a view which
is not held by the majority of ‘ bacteriologists”’—that the
greater number of Bacteria are pleomorphic. ‘hat pleo-
morphism does exist in many Bacteria, | think there can be
no longer any doubt. Since the early work of Ray Lankester,
Cienkowski, Zopf, Metchnikoff and others, an immense mass
of evidence has been brought forward in favour of such a
view. It is outside the limits of the present paper to enter

1 See especially his studies on water Bacteria (Mencl, 1905).

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 485

into a discussion of this matter, but I should like to call
attention to the exhaustive—but almost completely ignored
—work of Billet (1890), and the remarkable researches of
Mencl (1905) in this connection. Here will be found an
immense collection of facts bearing upon the matter.

It appears to me probable that—just as in the case of
their nuclei—the majority of Bacteria may possess a wide
range of variation in their outward form at different stages in
their life-histories. The matter can be decided, however, by
further research only: but it offers a vast field for future
investigation—investigation which is not only of a most
fascinating nature, but of which the results also will be of
the greatest biological interest.

Do Enucleate Bacteria Exist?—I wish to say a few
words here about the belef which is often held, that the
Bacteria are a group of organisms which possess no structure
homologous with the nucleus present in the cells of other
protists, animals or plants.

From a survey of the work which has been done upon the
cytology of the Bacteria, I think it may be stated with
absolute certainty that not a single bacterial species
has been proved to be devoid of a nucleus. I do
not say that a nucleus has been proved to be present in every
bacterial species: but I do maintain that a nucleus has
been demonstrated in a large number of species
of Bacteria. The probability is, therefore, that all Bac-
teria are nucleate cells. That enucleate Bacteria may exist,
is, of course, a possibility which cannot be denied; but at
present there is absolutely not a vestige of evidence
im favour Or Smueh a View :

I should like also to draw attention to a sort of state-
ment about Bacteria which may be very frequently encountered
in biological writings. The following quotation will serve as
an instance of the sort of thing I mean: ‘It may be pointed
out that it is in these low forms of life that we must look for
a key to the secret of the origin of the cell nucleus, as well
as for data to determine the morphological character of the

486 (. CLIFFORD DOBELL.

primal life organism” (Macallum, 1899, p. 439). This is
one case in which this idea is definitely stated, but dozens of
other passages in the works of other writers can easily be
found in which a similar view is either formulated or tacitly
assumed.

In statements of this sort two assumptions are made:
first, that Bacteria are more simply organised than other
living beings; secondly, that the more simply organised
beings are phylogenetically the more primitive. There is no
real justification for either of these assumptions. By calling

Bacteria “low forms of life,”

it is easy enough to arrive at
the conclusion that they occupy a position near the bottom
of the phylogenetic tree. But this is nothing more than a
petitio principii—a using of the conclusion at which it is
desired to arrive as evidence for that conclusion. It is, of
course, open to anybody to make the assumption that the
Bacteria are like the most primitive forms of life; but the
fact should not be lost sight of that this is at present an
assumption, and nothing more.

“Fusiform Bacteria.’—All the so-called ‘fusiform
Bacteria?’ which I have examined possess a distinct nucleus,
usually in the form of a spherical mass of chromatin—one in
each cell. This nucleus divides previous to the division of
the cytoplasm.

Nuclei, which divide by amitosis, were originally described
in the fusiform organism (“Bacillus fusiformis”) which
occurs in the human mouth, by Miihlens and Hartmann
(1906). This—so far as I am aware—was the first record of
nuclei in these organisms. A detailed description of the
nucleus was not given, and no figures were published.

Quite recently, Hoelling (1910) has given a detailed
account of a fusiform organism—which he names F'usi-
formis termitidis!—which occurs in the gut of termites
(locality and species not stated). He also describes and

1 Presumably a mistake for termitis. Hoelling proposes for all

the fusiform organisms the generic name Fusiformis in place of the
obviously inapplicable name Bacillus.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 487

figures the fusiform organism from the human mouth, a
form from fresh water, and a form from the cecum of a
mouse. In all these, he finds nuclei which are essentially the
same as those which I have found in the various forms
described in the preceding pages.

Hoelling describes the formation of long, multinuclear
filaments by these organisms. He regards this as a degenera-
tion phenomenon. The occurrence of these filamentar (un-
segmented) forms lends, I think, some support to the view,
which I have already expressed (p. 452), that the “fusiform
Bacteria” are really Fungi.' At present there is no con-
clusive proof that this is so; but it should be noted also
that there is no proof that these protists are Bacteria.

Whatever be the systematic position of the ‘fusiform
Bacteria,’ I think there can be no longer any doubt that
they possess a characteristic nucleus, in the form usually of a
minute sphere or granule—one in each cell—which divides
by a simple process of amitosis.

Affinities of the Bacteria.

This is not the place to
discuss the affinities of the Bacteria in detail. Yet I believe
we have now arrived at the beginnings of a correct inter-
pretation of the structure and life-history of this group, so
that a discussion of their affinities would be more profitable
now than it would have been a few years ago.

Three chief views regarding the affinities of the Bacteria
have been advanced: namely, that they are allied to the
Fungi, to the Cyanophycee, or to the flagellate Protozoa. I
have previously expressed the opinion that the Bacteria do
not show affinities with the Fungi. ‘he cytological studies
recorded in this paper confirm this view completely. I
beheve there is not a particle of evidence to support the
hypothesis that the Bacteria and Fungi are connected. ‘The

* IT would call attention to the resemblance which these organisms
bear to a fungus described by Sule (1910) from the body-cavity of
Chermes strobilobius. This fungus—probably a yeast—which
Sule calls Schizosaccharomyces chermetis strobilobii, has a
“caraway-seed shape,” and the figures of it (fig. xv) certainly show a
strong similarity to many “ fusiform Bacteria” which I have observed,

488 C. CLIFFORD DOBELL.

name ‘ Schizomycetes’’—or “ Spaltpilze ”’—is a complete
misnomer. Similarly, with regard to the Protozoa, I see no
real evidence at all which indicates that affinities exist between
this group and the Bacteria. ‘There is no real similarity
between them.

There is, perhaps, rather more evidence of the affinities of
the Bacteria with the Cyanophycese. Nuclear resemblances
between the two groups certainly do exist, but on the other
hand there are many important differences. The evidence
is certainly very far from conclusive.!

I believe that at present there is no clear evidence of the
affinity of the Bacteria with any other group of organisms.
For the present they must be regarded as a group of Protista
which stands quite apart.

I believe, further, that amongst the Bacteria a number of
forms are included which do not really belong—that the
group Bacteria, as at present constituted, comprises a very
heterogeneous assemblage of forms.

Similar views to these have already been expressed by
Menel (1907) and Guilliermond (1907), when considering the
facts which were then known. I have myself also expressed
the same views on a previous occasion, and I believe that
they are now completely justified.

CONCLUSIONS.

I think, from the facts which have been given and analysed
in the foregoing pages, the following chief conclusions are
justified :

All Bacteria which have been adequately investi-
gated are—like all other Protista—nucleate cells.

' T should like to point out here that the cytology of the Cyano-
phycee and sulphur Bacteria does not furnish us with anything more
than analogical evidence regarding the structure of the smaller
Bacteria (i.e. Bacilli, Spirilla, ete.). I believe many sulphur
Bacteria are probably only distantly related to the majority of the
smaller forms, and there is no clear evidence that the Cyanophycex
have anything to do with them.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 489

The form of the nucleus is variable, not only in
different Bacteria, but also at different periods in
the life-cycle of the same species.

The nucleus may be in the form of a discrete
system of granules (chromidia); in the form of a
filament of variable configuration; in the form of
one or more relatively large aggregated masses of
nuclear substance; in the form of asystem of irre-
gularly branched or bent short strands, rods, or
networks; and probably also in the vesicular form
characteristic of the nuclei of many animals, plants,
and protists.

There is no evidence that enucleate Bacteria
exist.

Finally, in addition to these purely morphological con-
clusions concerning the nucleus, I think another conclusion
is rendered highly probable :

The Bacteria are in no way a group of simple
organisms, but rather a group displaying a high
degree of morphological differentiation coupled in
many cases with a life-cycle of considerable com-
plexity.

APPENDIX.

On the Alleged Autogamy of Bacteria.—In two
earlier papers I have discussed the so-called ‘‘ autogamy ” of
the disporic Bacteria in some detail. The actual facts
regarding this process were recorded by Schaudinn (1902,
1903), and myself (1908). In a second paper (1909) I
brought forward strong evidence to show that the so-called
“autogamy ” of Bacteria is not a sexual process at all, but has
a much simpler explanation. It seems necessary, however,
to refer to this matter once more, owing to the recent
appearance of a very misleading article by Dr. Ruzicka.!

After mentioning Schaudinn’s observations, the author

1 WV. Ruzicka, ‘‘ Ueber die experimentelle Autogamie der Bakterien,”
‘ Arch. Entw.-Mech.,’ Bd. xxx, Festschrift f. W. Roux, Teil. 1, p. 443,
1910.

490 C. CLIFFORD DOBELL.

proceeds (p. 443)—“‘ Kine Bestiitigung dieser Befunde ist bis
jetzt nur von Dobell' eingelanfen, und zwar insofern, als er
bei Bac. flexilis zum Teil ahnliche Bilder vorgefunden hat.
Er bestreitet indes die Deutungen Schaudinns, weil er die von
diesem Forscher geschilderten und seine Deutung eigentlich
bedingenden Plasmastrémungen nicht beobachten konnte.”
And further (p. 445)—“ Vielleicht ist der negative Befund
Dobells damit zu erkliren, dass er ohne vitale Farbung
untersucht hat.”

Now if Dr. Ruzicka had taken the trouble to read my first
paper, he would have found that my results were essentially
the same as Schaudinn’s; that I accepted then Schaudinn’s
interpretation that the phenomenon was probably a sexual
one; and that I did employ intra-vitam staiming methods,
and was unable to convince myself that streaming of the
eranules occurred in the living organisms on account of
their motility.? It is in my second paper (1909)—which Dr.
Ruzicka completely ignores—that I have given what is, I
believe, a definite proof that no sexual process occurs during
spore-formation in the disporic Bacteria. ‘There is very
strong evidence that the “sexual” phenomena are due
simply to a suppressed cell-division. I should like to point
out that Dr. Ruziéka’s own observations, recorded in this
paper, support my view. The “sexual act” which he invoked
by growing his Bacteria upon abnormal and innutritious
media may be quite simply explained by the fact—which he
himself records—that the organisms divided imperfectly and
then proceeded to form spores without developing typical
colonies. Dr. Riziéka’s incomplete observations and figures
of the formation of disporic individuals add nothing to the
facts observed and recorded by Schaudinn and myself.
Disporic, or coupled monosporic, individuals have already
been observed in many different Bacteria by many workers.

1 Here follows a reference to my 1908 paper.

2 But I have never used this as an argument against the sexual inter-
pretation of the phenomenon. That some of the granules do pass to
the ends of the cells I have, I think, helped to prove.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 491

As Dr. Ruzicka has added no new facts regarding the
method of spore-formation in these alleged autogamic forms,
it is only his interpretation of the phenomena that I can
dispute. But as I have already given my arguments against
the view which he adopts, I can suffice with referring him to
my secoud (1909) paper.

One or two other points appear worthy of mention. Dr.
Razicka says (p. 443)—“ Die Bakterien, bei welchen man
bislang geschlechtliche Vorgiinge festgestellt hat, waren als
zutalige Gaste oder Parasiten andrer Organismen vorge-
funden worden, ohne weiter und reingeziichtet worden zu
sein. Das hatte Skeptikern als Punktum fixum dienen
k6nnen, um ihre Zweifel an der Reihenfolge der Phasen des
besprochenen Vorganges und an seiner Zugehorigkeit zu den
sexuellen EKrschemungen weiter zu spinnen.” Now it may
be noted, in the first place, that B. sporonema is a free-
living form; and secondly, that phenomena continuously
observed in organisms in their natural environment are of
more, or at least equal, importance to those observed under
abnormal conditions, in which many of the factors are
unknown.

Dr. Ruzicka concludes his paper by stating (p. 458) that

the facts of the alleged “

autogamic ’ process are in accord
with his interpretation of Bacteria as nuclei. It seems
scarcely necessary to point out that such an opinion could be
arrived at only by a complete confusion of ideas coupled with
a misuse of words.

It seems to me unnecessary to discuss the speculative part

of Dr. Ruziéka’s paper, since it is based—I believe
per,

upon his
misinterpretation of the facts. Until it can be proven that
sexual phenomena occur, it is useless to construct further
speculations upon the mere assumption. And at present |
believe all the evidence speaks very definitely against the
view that a sexual process occurs at any stage in the life-
history of Bacteria.

IMPERIAL COLLEGE OF SCIENCE AND TECHNOLOGY,

Lonpon. October 5th, 1910.
VOL. 56, PART 3.—NEW SERIES. 34

492 C. CLIFFORD DOBELL.

LITERATURE.
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(1895).—** Beobachtungen ther die metachromatischen Kor-
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Biitschli, O. (1890) —‘ Ueber den Bau der Bacterien und verwandter
Organismen,’ Leipzig (Winter).
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das Protoplasma,’ Leipzig.
(1896).—* Weitere Ausftthrungen tiber den Bau der Cyano-
phyceen und Bacterien, Leipzig (Engelmann).
(1902).—** Bemerkungen ther Cyanophyceen und Bacteriaceen,”
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Journ. Micr. Sci.,’ vol. 52, p. 121.
—— (1909).—* On the so-called ‘ Sexual’ Method of Spore-formation
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——— (1909a).—* Some Observations on the Infusoria parasitic in
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CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 493

Dobell, C. C. (19098).—** Chromidia and the Binuclearity Hypotheses : a
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bildung,” ‘ Zeitschr. Hyg.,’ Bd. iv, p. 25.
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(1902).—** Ueber den Bau der Bakterien (Ergebnisse vitaler
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Fedorowitsch, A. (1902).—‘* Ueber die Kéornigkeit der Bakterien,”
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Feinberg (1900).—** Ueber den Bau der Bakterien,” ‘CB. Bakt., Abt.
i, Bd. xxvii, p. 417.

Ficker, M. (1905).—** Zur Frage der Kornchen und Kerne der Bakte-
rien,” ‘ Arch. Hyg.,’ Bd. xlvi, p. 171.

Fischer, A. (1894).—‘* Untersuchungen tiber Bakterien,” ‘ Jahrb. wiss.

Bot.,’ Bd. xxvii, p. 1.

——— (1897).— ‘ Untersuchungen iiber den Bau der Cyanophyceen
und Bakterien,’ Jena (Gustav Fischer).

——— (1899).—‘ Fixirung, Farbung und Bau des Protoplasmas,’ Jena
(Gustav Fischer).

—— (1903).— Vorlesungen iiber Bakterien,’ 2 Aufl., Jena.

Frenzel, J. (1891).—* Der Zellkern und die Bakterienspore,” ‘ Biol.
Centralbl.,’ Bd. xi, p. 757.

-~——— (1892).—‘* Ueber den Bau und die Sporenbildung griimer Kaul-
quappenbacillen,” ‘ Zeitschr. Hyg., Bd. xi, p. 207.

Gardner, N. L. (1906).—** Cytological Studies in Cyanophycez,” ‘ Univ.
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Gauss, C. J. (1902).—** Babes-Ernstsche Kérperchen und Virulenz bei
Bakterien,” ‘CB. Bakt., Abt. i, Bd. xxxi, p. 92.

Georgevitch, P. (1910)—‘ Bacillus thermophilus Jivoini nov.
spec. und Bacillus thermophilus Losanitchi, nov. spec.,”
‘CB. Bakt.,’ Abt. ii, Bd. xxvii, p. 150.

Giemsa, G. (1909, 1910) —* Farbung von Feuchtpraparaten und Schnitten
mit der Azureosinmethode,” usw, ‘ Deutsch. med. Wochenschr.,’

49 4. C. CLIFFORD DOBELL.

No. 40, 1909; ibid., No. 12, 1910; ‘ CB. Bakt.,’ Abt. i, Bd. liv, 1910,
p. 489.

Grimme, A. (1902).—‘* Die wichtigsten Methoden der Bakterienfarbung
und ihre Wirkung auf die Membran, den Protoplasten und die
Hinschliisse der Bakterienzelle,”’ ‘CB. Bakt., Abt. i, Bd. xxxii,
p. L

Guilliermond, A. (1906)—* Les corpuscules métachromatiques ou grains
de volutine,” ‘ Bull. Inst. Pasteur,’ t. iv, p. 145.

(1907) —‘“* La cytologie des Bactéries,’ ‘ Bull. Inst. Pasteur,’
t. v, p. 273.

—— (19074).—* Contribution a Vétude cytologique des Cyano-
phycées,” ‘ Rev. gen. Bot.,’ t. xviii, p. 392.

(1908).—* Contribution a l'étude cytologique des Bacilles endo-
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—— (1909).—** Observations sur la cytologie dun Bacille,” ‘ CR. Soe.
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(1910).—** A propos de la structure des Bacilles endosporés.
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(1903) —*Thiophysa volutans, ein neues Schwefelbak-
terium,” ‘ Ber. deutsch. bot. Ges.,’ Bd. xxi, p. 309.

Hoelling, A. (1907)—‘Spirillum giganteum und Spirochete
balbianii,” ‘CB. Bakt.,’ Abt. i, Bd. xliv, p. 665.

(1910).—** Die Kernverhaltnisse von Fu'siformis termitidis,
‘Arch. Protistenk., Bd. xix, p. 239.

Hueppe, F. (1886).—* Die Formen der Bakterien und ihre Beziehungen
zu Gattungen und Arten,’ Wiesbaden.

Ilkewiez, W. (1894).—‘** Ueber die Kerne der Milzbrandsporen,” ‘CB.
Bakt.,’ Bd. xv, p. 261.

Janssens, F’. A.,and Mertens, A. (1903).—* Etude microchimique et cyto-
logique dune Torula rose,” ‘ La Cellule,’ t. xx, p. 353.

Krompecher, E. (1901)—‘* Untersuchungen tiber das Vorkommen meta-
chromatischer Koérnchen bei sporentragenden Bakterien und
Beitrige zur Kenntnis der Babes-Ernst’schen Korperchen,” ‘ CB.
Bakt.,’ Abt. i, Bd. xxx, p. 385.

Kunstler, J. (1887).—‘* Contribution a la technique des Bactériacées,”
‘CR. Acad. Sci. Paris,’ t. cv, p. 684.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 495

Kunstler, J. (1900). Remarques sur certains points del’histoire de la vie
des organismes inférieurs,” ‘CR. Acad. Sci. Paris,’ t. exxx, p. 1416.
and Busquet, P. (1897)—*Sur la valeur nucléaire du corps
central des Bactériacées,” ‘CR. Acad. Sci. Paris, t. exxv. p.
1112.

(1898).— Observations sur la structure des Bactériacces
et des organismes voisins, Bordeaux (Imp. du Midi).

—— and Gineste, C. (1906)—‘Spirillum periplaneticum, nov.
spec.,’ ‘CR. Soe. Biol.,’ t. Iviii (2), p. 135.

(19064).—* Structure fibrillaire chez les Bactériacées,”
‘CR. Acad. Sci. Paris,’ t. exliii, p. 84.

Loewit. M. (1896).—*Zur Morphologie der Bakterien,” ‘CB. Bakt.,’
Abt. 1, Bd. xix, p: 673.

Macallum, A. B. (1899).—**On the Cytology of Non-nucleated
Organisms,” ‘ Trans. Canadian Inst., Toronto,’ vol. vi, p. 439.

Mann, G. (1902).— Physiological Histology, Methods and Theory,’
Oxford (Clarendon Press).

Marpmann, G. (1900).—‘* Ueber kernlose Bakterien,” ‘CB. Bakt.,’ Abt.
u, Bd. vi, p. 675.

Marx, H. (1902).—* Einige Bemerkungen zu Krompecher’s Arbeit ther
metachromatische Kérnchen und Babes-Ernstsche Korperchen,”
CB, Bake Abt. 1, Bd. xxxi, p. 107,

——— and Woithe, F. (1900)—** Morphologische Untersuchungen zur
Biologie der Bakterien,” ‘CB. Bakt.,’ Abt. i, Bd. xxviii, p. 1.

Mencl, E. (1904).—** Einige Beobachtungen tber die Strucktur und
Sporenbildung bei symbiotischen Bakterien,” ‘CB. Bakt.,’ Abt.
ii, Bd. xii, p. 559.

(1905).—** Cytologisches iiber die Bakterien der Prager Wasser-
leitung,” ‘ CB. Bakt.,’ Abt. 11, Bd. xv, p. 544.
— (1907).—* Nachtrage zu den Strukturverhaltnissen von Bac-
terium gammari Vejd.,” ‘ Arch. Protistenk.,’ Bd. viii, p. 259.

— (19074).—* Eine Bemerkung zur Organisation der Periplaneta-
Symbionten,” ‘ Arch. Protistenk., Bd. x, p. 188.

——— (1909).—‘** Die Bakterienkerne und die ‘cloisons transversales ”
Guilliermond’s,” ‘ Arch. Protistenk.,’ Bd. xvi, p. 62.

-_—— (1910).—‘ Ueber den Kern und seine Teilung bei Sarcinen und
Micrococcus ochraceus (butyricus),” ‘Arch. Protistenk.,
Bdraxip: L272

y/ Meyer, A. (1897).—* Studien tiber die Morphologie und Entwicke-

lungsgeschichte der Bakterien, ausgeftthrt an Astasia astero-
spora A. M. and Bacillus tumescens Zopf.,’ ‘ Flora, Bd.
Ixxxiv (Hrganzungsband), p. 185.

496 CG. CLIFFORD DOBELL.

Meyer, A. (1899).—* Ueber Geisseln, Reservestotte, Kerne und Sporen-
bildung der Bakterien,” ‘ Flora,’ Bd. Ixxxvi, p. 428.

— (1901).—‘ Ueber die Verzweigung der Bakterien,”’ ‘CB. Bakt.,’
Abt. i, Bd. xxx, p. 49.

——— (1904).—* Orientirende Untersuchungen iiber Verbreitung,
Morphologie und Chemie des Volutins,” ‘ Bot. Ztg.,’ Jahrg. Lx,
pestis:

— (1908).—* Der Zellkern der Bakterien,” ‘ Flora, Bd. xeviii, p. 335).

Migula, W. (1894).—** Ueber den Zellinhalt von Bacillus oxalaticus
Zopf.,” ‘ Arb. bakteriol. Inst. Karlsruhe,’ Bd. 7.

—— (1897, 1900).—‘ System der Bakterien,’ Jena (Mischer).

(1904).— Der Bau der Bakterienzelle,” Lafar’s ‘Handb. techn.

Mykol.,’ Bd. i, p. 48.

Mitrophanow, P. (1893).—* Etudes sur lorganisation des Bactéries,”
‘Monthly internat. Journ. Anat. Physiol.,’ vol. x, p. 475.

Miihlens, P., and Hartmann, M. (1905).—‘** Ueber Bacillus fusiformis
und Spirocheta dentium,”’ ‘ Zeitschr. Hyg.,’ Bd. lv, p. 81.

Mihlschlegel, A. (1900).—Ueber die Bildung und den Bau der Bakte-
riensporen,’ ‘CB. Bakt.,’ Abt. ii, Bd. vi, p. 65.

Nakanishi, K. (1901).—‘* Ueber den Bau der Bakterien,” ‘CB. Bakt.,

Petschenko, B. (1908).—‘* Sur la structure et le cycle évolutif de Bacil-
lopsis stylopyge nov. gen. et nov. spec.,” ‘ Bull. internat.
Acad. Sci. Cracovie,’ No. 4, p. 359.

Podwyssozki, W. W. (1895).—*‘ Zur Morphologie der Choleravibrionen,”
‘CB. allg. Pathol.,’ Bd. iv, p. 673.

Preisz, H. (1904).—* Studien tther Morphologie und Biologie des Milz-
brandbacillus,” ‘CB. Bakt., Abt. i, Bd. xxxv, p. 280.

Protopopoff (1891).—* Sur la question de la structure des Bactéries,”
‘Ann. Inst. Pasteur,’ t. v, p. 332.

Rayman, B., and Kruis, K. (1904).—* Des noyaux des Bactéries,” * Bull.
internat. Acad. Sci. Bohéme.’

Rowland, §S. (1899).—** Observations upon the structure of Bacteria,”
‘Trans. Jenner Inst. Prev. Med. London,’ Series ii, p. 143.
Ruzicka, V. (1903).—* Ueber die biologische Bedeutung der fiirbbaren

Kornchen des Bakterieninhaltes,” ‘ Arch. Hyg.,’ Bd. xlvi, p. 337.

(1908).—* Sporenbildung und andere biologische Vorginge bei
dem Bact. anthracis,” ‘ Arch. Hyg., Bd. lxiv, p. 219.

(1909).—* Die Cytologie der sporenbildenden Bakterien und ihr
Verhaltnis zur Chromidiallehre,’” ‘CB. Bakt.,’ Abt. ii, Bd. xxiii,
p. 289.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 497

VA Schaudinn, F. (1902).—* Beitrage zur Kenntnis der Bakterien und
verwandter Organismen : I. Bacillus butschlii, n. sp.,” ‘Arch.
Protistenk.,’ Bd. i, p. 306.
(1903). “II. Bacillus sporonema, n. sp.,” * Arch. Protistenk.,’
Bd. ii, p. 421.
Schewiakoff, W. (1893).—‘* Ueber einen neuen bakterienahnlichen
Organismus des Siisswassers,” * Verh. nat.-med. Ver. Heidelberg,’
N.F. 5, p. 44.

Schottelius, M. (1888).—** Beobachtung kernartiger Koérper im Innern
von Spaltpilzen,” * CB. Bakt.,’ Bd. iv, p. 705.

Schumburg, (1902).—‘* Die Beziehungen der Babes-Ernstschen Koérn-
chen zu der Virulenz der Bakterien,’ ‘CB. Bakt., Abt. i, Bd.
XXxl, p. 694.

Y/Y Sjobring, N. (1892).—** Ueber Kerne und Theilungen bei den Bakterien,”

‘CB. Bakt., Bd. xi, p. 65.

Sule, K. (1906)—* Kermincola kermesina n. g.n. sp. und physo-
kermina n. sp., neue Mikroendosymbiotiker der Cocciden,” ‘SB.
kel. bbhm. Ges. Wiss. Prag.’

(1910).—** Pseudovitellus und ahnliche Gewebe der Homopteren
sind Wobnstatten symbiotischer Saccharomyceten,’ “SB. kgl.
bohm. Ges. Wiss. Prag.’

Swellengrebel, N. H. (1906)—* Zur Kenntnis der Zytologie von
Bacillus maximus bucealis (Miller),” ‘CB. Bakt., Abt. 11,
Bd. xvi, p. 617.

(1907).—* Zur Kenntnis der Zytologie der Bakterien. I.
Bacterium binucleatum,”’ ‘CB. Bakt.,” Abt. ii, Bd.’ xix,
p. 193.

(19074).—* Sur la cytologie comparée des Spirochétes et de
Spirilles,” ‘ Ann. Inst. Pasteur,’ t. xxi, p. 448.

(1908).—** Erwiderung auf die Arbeit des Herrn Dr. Holling :
Spirillum giganteum und Spirocheta balbianii,” ‘CB.
Bakt.,’ Abt. ii, Bd. xlvi, p. 1.

(1909).—** Neuere Untersuchungen ber die vergleichende
Cytologie der Spirillen und Spirochaten,” ‘CB. Bakt., Abt. I,
Bd. xlix, p: 529.

(19094).—* Untersuchungen iiber die Zytologie einiger Faden-
bakterien,” ‘ Arch. Hyg.,’ Bd. lxx, p. 380.

Trambusti, A., and Galeotti, G. (1892).—** Neuer Beitrag zum Studium

der inneren Struktur der Bakterien,” ‘CB. Bakt., Bd. xi, p. 717.

Vay, F. (1910).—* Studien tber die Strukturverhaltnisse von Bakterien

mit Hilfe von farbehaltigen Nahrbéden.” ‘CB. Bakt.” Abt. i,
Rd. lv, p. 193.

498 C. CLIFFORD DOBELL.

Vejdovsky, F. (1900).—** Bemerkungen wher den Bau und Entwicklung
der Bakterien,’ ‘CB. Bakt.,’ Abt. 11, Bd. vi, p. 577.

(1904).—* Ueber den Kern der Bakterien und seine Teilung,”
‘CB. Bakt.,’ Abt. ii, Bd. xi, p. 481.

(1906). —** Bemerkungen zum Aufsatze des Herrn Dr. K. Sule
ber Kermincola kermesina, etc.,” ‘SB. kgl. bohm. Ges.
Wiss. Prag.’

Wager. H. (1891).—‘*On a Nuclear Structure in the Becteria,” ‘Ann.
Bot.,’ vol. v, p. 513.

(1895).—* Preliminary Note upon the Structure of Bacterial
Cells,” ‘ Ann. Bot.,’ vol. ix, p. 659.

(1898).—* The Nucleus of the Yeast-Plant,’ ‘Ann. Bot.,’ vol.
xii, p. 499.

Wagner, A. (1898).—‘“Coli- und Typhusbakterien sind einkernige
Zellen. Ein Beitrag zur Histologie der Bakterien,” ‘CB. Bakt.,’
Abt. i, Bd. xxiii, p. 435.

Wahrlich, W. (1890, 1891).—* Bakteriologische Studien, I and II”
(Russian), in ‘Seripta Botanica, St. Petersburg. See Review
in ‘ CB. Bakt.,’ Bd. xi, 1892, p. 49.

Walker, E. W. A., and Murray, W. (1904).—‘* The Effect of Certain
Dyes upon the Cultural Characters of the Bacillus typhosus
and some other Micro-organisms,” ‘ Brit. Med. Journ.,’ vol. ii,
p. 16.

West. G.S., and Griffiths, B. M. (1909).—* Hillhousia mirabilis, a
Giant Sulphur Bacterium,” ‘Proc. Roy. Soc. London,’ B. vol.
Ixxxi, p. 398.

Winogradsky, S. (1888).—‘ Beitrage zur Morphologie und Physiologie
der Bacterien. Heft i, zur Morphologie und Physiologie der
Schwefelbacterien,’ Leipzig (Felix).

Zettnow, E. (1891).—‘* Ueber den Bau der Bacterien,’ ‘CB. Bakt.,’

Bd. x, p. 690.

(1897).—** Ueber den Bau der Grossen Spirillen,” ‘ Zeitschr.

Hyg., Bd. xxiv, p. 72.

(1899).—** Romanowski’s Farbung bei Bakterien,” ‘ Zeitschr.

Hye; Bdixxx, p. 1.

(1900) —* Romanowski’s Farbung bei Bakterien,” ‘CB. Bakt.,

Abt. i, Bd. xxvii, p. 803.

(1908).—* Ueber Swellengrebels Chromatinbinder in Spirillum

volutans,” ‘CB. Bakt.,’ Abt. i, Bd. xlvi, p. 193.

Ziemann, H. (1898).—‘* Kine Methode der Doppelfarbung bei Flagel-
laten, Pilzen, Spirillen und Bakterien, sowie bei einigen Amében,”
‘CB. Bakt.,’ Abt. i, Bd. xxiv, p. 945.

Zopt. W. (1885).— Die Spaltpilze,’ Breslau.

GONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 499

EXPLANATION OF PLATES 16-19,

Ilustrating Mr. C. Clifford Dobell’s ‘‘ Contributions to the
Cytology of the Bacteria.”

[All the figures are drawn from fixed and stained organisms under
a Zeiss 2 mm. apochromatic oil-immersion (aperture 1-40) with the
aid of compensating oculars 6, 8, 12, and 18. The magnification of all
figures is the same, and is approximately 2000 diameters. The figures
are in no way diagrammatic. They are accurate representations of the
actual appearances observed. |

PLATE 16.
Figs. 1-20 are from wet film preparations of the blood of Sacco-
branchus fossilis, fixed with osmic vapour followed by absolute
alcohol, and stained with Giemsa’s stain.

Figs. 1-10, 12 and 14.—Bacillus saccobranchi n. sp.
Fig. 1—Short Bacillus, with nucleus in the form of a slightly bent
and varicose filament.
Fig. 2.—Two Bacilli with nuclei in the form of twisted zig-zag or
spiral filaments.

Fig. 3.—Bacillus with nucleus in the form of fragments of a zig-zag
filament.

Fig. 4.—Long Bacillus containing a long varicose zig-zag or spiral
nuclear filament. (Nucleus of spirogyra type.)

Fig. 5—Large Bacillus in which the nucleus is in the form of
granules and irregular short, curved, bent, and branched filaments.
(Irregular type of nucleus.)

Fig. 6.—Large Bacillus with nucleus partly in the form of an irregular
zig-zag or spiral filament and partly in the form of irregular branched
masses—connected with one another.

Fig. 7.—Two short Bacilli with irregular nuclei.

Fig. 8.—Large, slightly curved Bacillus, with nucleus in the form of
a broken varicose zig-zag or spiral filament.

Fig. 9.—Bacillus with nucleus of irregular type. A part of the
nucleus shows a very distinct reticular arrangement.

Fig. 10.—Bacillus with nucleus of chromidial type.

Fig. 12.—Bacillus with nucleus in the form of a thick varicose
filament.

500 C. CLIFFORD DOBELL.

Fig. 14.—Bacillus containing a large and almost fully formed spore.
Residual chromatin is seen lying in the cytoplasm outside the spore.

Figs. 11, 18, 15-20.—Smaller Bacteria, found in company with
B. saccobranchi.

Fig. 11.—Chain of three individuals with nuclei of spirogyra type.

Fig. 13.—Short, thick Bacillus with nucleus in the form of short, thick,
irregular vodlet, pointed at one end.

Fig. 15.—Bacillus with nucleus in the form of a varicose spiral or
zig-zag filament.

Fig. 16.—Bacillus with nucleus in irregular masses.

Fig. 17.—Two Bacilli with nuclei in the form of short, irregular
rodlets.

Fig. 18.—A similar organism, with nucleus undergoing division.

Fig. 19.—Three very small Bacilli with nuclei of spirogyra type.

Fig. 20.—Group of five small Bacilliwith spirogyra type of nucleus.
Figs, 21-23.—Bacilli of flexilis type, from large intestine of Mabuia

carinata. (Osmic acid 1 per cent., drop method ; Leishman’s stain.)

Fig. 21.—Ordinary individual, with chromidial nucleus.

Fig. 22.—Similar individual. The chromidia are smaller and more
numerous than in the preceding.

Fig. 25.—Spore-bearing (disporic) individual. The spore-coats are
stained blue, and a certain amount of residual chromatin material is
seen in the cytoplosm.

Figs. 24-29.—Sarcina from large intestine of Bufo melanostictus.
(Osmic acid 1 per cent., drop method; Giemsa’s stain.)

Fig. 24.

in each cell. The upper cell contains a refractile granule (white).

Organism in two-cell stage. Small spherical nuclei (red)

Fig. 25.—Four-cell stage. In the upper left-hand cell the nucleus
has divided into two. The three other cells each contain a single
nucleus. In each cell a single refractile granule is present.

Fig. 26.—Three-cell stage. The nucleus in the right-hand cell has
divided into two, but fission of the cytoplasm has not yet occurred.
The upper and lower left-hand cells contain dividing nuclei, of
characteristic dumb-bell form. A single large refractile granule is
present in the right-hand cell; the left-hand cells each contain a single
and smaller refractile granule.

Fig. 27.—Three-cell stage. The left-hand cells each contain a single
nucleus and a single refractile granule. The right-hand cell shows a
nucleus undergoing division.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERTA. 501

Fig. 28.—Fonr-cell stage. Each cell contains a nucleus and a retrac-
tile granule.

Fig. 29.—Four-cell stage. The lower right-hand cell contains a
single resting nucleus. The three other cells contain dividing nuclei.
The upper left-hand cell contains two small refractile granules—the
three others one.

Figs. 30-40.— Large Bacilli of spirogyra type from large intestine of
Mabuia carinata. (lL per cent. osmic acid, drop method ;
Leishman’s stain.)

Fig. 30.—Long Bacillus, with nucleus in the form of a spiral or zig-
zag filament.

Fig. 31—A similar form to the preceding, but with a longer and
more twisted nucleus.

Fig. 32.—Similar form, showing two loops in the nuclear filament.

Fig. 33.—Similar organism just completing division into two.

Fig. 34.—Shorter individual, with typical spirogyra type of nucleus.

hy

ig. 35.—Similar form with nucleus in the form of a straighter,
varicose filament.

Fig. 36.—Short Bacillus, with nucleus clearly seen to be composed of
chromatin granules, aggregated to form a spiral or zig-zag filament.

Fig. 37.—Bacillus containing finely granular cytoplasm and six
large nuclear granules. Possibly a degenerate or developmental form
of the preceding organisms.

Fig. 38.—Bacillus with nucleus in the form of a broken spiral fila-
ment. Degenerate or developmental form ?

Fig. 39.—Spore-bearing individual of spirogyra type.

Fig. 40.— Degeneration form.

Fig. 41—Long, slender Bacillus from large intestine of Mabuia
carinata. Nucleus of chromidial type. (1 per cent. osmic acid, drop
method ; Leishman’s stain.)

Figs. 42-44.—Micrococci from large intestine of Mabuia carinata.
(1 per cent. osmic acid, drop method ; Leishman’s stain.)

Fig. 42—Diplococcus form—each cell with a single nucleus.

Fig. 43.—Coccus with nucleus in the form of a short zig-zag filament.

Fig. 44.—Typical Micrococcus, with single nuclear granule.

PLATE 17.
Figs. 45-60.—Various Bacteria from large intestine of Lacerta
muralis. (1 per cent. osmic acid, drop method ; Giemsa’s stain.)
Fig. 45.—Group of five Micrococci of different sizes. The nucleus
is very obvious in each cell.

502 C. CLIFFORD DOBELL.

Figs. 46-49.—Four successive stages in the division of a Micro-
coccus similar to those seen in the preceding figure. Note the
characteristic dumb-bell figure assumed by the nucleus during division.
(Compare with figs. 24-29, Plate 16.)

Fig. 50.—Three coccobacillar forms. The nucleus is in the form of
a filament, bent in a more or less spiral or zig-zag manner.

Fig. 51.—Group of short Bacilli, with nuclei of characteristic spiro-
gyra form.

Fig. 52.—Chain of Cocci in which division is taking place. Note the
zig-zag or spiral form assumed by some of the dumb-bell figures of the
dividing nuclei. (This figure is drawn on a very slightly larger scale
than the others.)

Fie. 53.—Small Bacillus with nucleus in the form of a short rod.

Fig. 54.—Similar organism to the preceding, in the act of dividing
into two. The nuclear rod is completely divided into two parts.

Figs. 55-60.—Large Bacilli of spirogyra type.

Figs. 55-57.—Three individuals, showing three different arrangements
of the nuclear filament.

Fig. 58.—Spore-bearing individual of same species. (A single ter-
minal spore is formed—as in B. spirogyra.)

Fig. 59.—Dividing individual. The two halves of the nuclear fila-
ment are still joined by a very slender chromatin thread.

Fig. 60.—Another dividing individual. The nucleus—which is very
much contorted (cf. fig. 59)—has already separated into two parts.

Fig. 61—Three short Bacilli, with nuclei of spirogyra type, from
large intestine of Bufo melanostictus. (1 per cent. osmic acid, drop
method; Giemsa’s stain.)

Fig. 62.—Long, curved Bacillus, with irregular varicose nuclear
filament. Large intestine of Bufo melanostictus. (1 per cent.
osmic acid, drop method; Giemsa’s stain.)

Fig. 65.—Group of Bacilli from large intestine of Lacerta muralis.
The nucleus is in the form of an irregular knotted rodlet. The
lowest organism is undergoing division—the nucleus being already
divided into two. (1 per cent. osmic acid, drop method; Giemsa’s
stain.)

Figs. 64, 65.—Slender Bacilli from large intestine of Lacerta
muralis. Nucleus in the form of chromidia. (1 per cent. osmic acid,
drop method ; Giemsa’s stain.)

Figs. 66, 67.— Fusiform Bacteria’? from large intestine of Triton
vulgaris. The upper individual of the pair shown in fig. 66 is
dividing. Note the nuclei—in the form of double granules. Fig. 67 is

CONTRIBUTIONS T0 THE CYTOLOGY OF THE BACTERIA. 308

a double form, with one nucleus (upper) appearing as a solid mass of
chromatin, the other (lower) as a vesicular structure with a large karyo-
some. (40 per cent. formol, drop method, absolute alcohol; Giemsa’s
stain.)

Fig. 68.—‘“ Fusiform Bacterium” (double form) from large intestine
of Lacerta muralis. Each individual possesses a small spherical
nucleus. (Dry film, absolute alcohol; Giemsa’s stain.)

Figs. 69-78.—Bacilli from large intestine of Mabuia carinata.
(Osmie acid 1 per cent., drop method; Leishman’s stain).

Fig. 69.—Long slender Bacillus with nucleus of spirogyra type.

Figs. 70-75.—Smaller Bacilli of spirogyratype. Diverse forms and
S1ZeS.

Fig. 74.—Very small Bacillus with thick nuclear filament of spiro-
gyra type.

Fig. 75—A Bacillus, similar to that shown in fig. 72, undergoing
fission.

Fig 76.—Slender Bacillus with nucleus of chromidial type.

Fig. 77.—Slender Bacillus with large central nuclear mass (possibly a
plasmolysed form ”).

Fig. 78.—Bacillus with nucleus in the form of a short, irregular, and
slightly bent rod-like filament.

Figs. 79-82.—Bacilli of modified flexilis form from large intestine of
Triton vulgaris. (40 per cent. formol, absolute alcohol; Giemsa’s
stain.)

Fig. 79.—Bacillus of flexilis form, with chromidial nucleus.

Fig. 80 —Individual with finely granular, darkly staining cytoplasm,
and large nucleus-like masses of chromatin, eight in number.

Fig. 81.—Long, sporulating individual, bearing a large chromatin
spore-rudiment at each end. (The organism is normally disporic, like
B. flexilis.)

Fig. 82.—Long individual similar to that shown in fig. 80. The
cytoplasm is alveolar, and the chromatin in the form of large nucleus-
like masses.

Figs. 85, 84.—Long and short individuals respectively of Bacillus of
flexilis type from large intestine of Lacerta muralis. Nuclei of
chromidial form. (Osmic vapour [wet film], absolute alcohol; Giemsa’s
stain.)

DOA C. CLIFFORD DOBELL.

Figs. 85-90.—Bacilli of modified flexilis form from large intestine of

b=) 5

Lacerta muralis. (Dry film, absolute alcohol; Giemsa’s stain.)

Fig. 85.—Long individual, containing three large nucleus-like masses
of chromatin.

Fig. 86.—Short individual, with curious arrangement of the chromatin,

Fig. 87.—Large individual, somewhat similar to the preceding.

Fig. 88.—Short individual with a single, centrally placed, nucleus-like
body.

Fig. 89.—Long, sinuous individual, with chromidial nucleus of
characteristic flexilis type. Many of the chromidia are conspicuous
by their large size.

Fig. 90.—Long, straight Bacillus, with chromatin mainly in two large
masses. Possibly a plasmolysed or degenerate form.

PLATE 18.

(All the figures, unless otherwise stated, are drawn from wet film
preparations fixed with Schaudinn’s sublimate-alcohol, and stained with
Heidenhain’s iron-hematoxylin. |

Figs. 91-95.—Bacilli of spirogyra form from large intestine of
Lacerta muralis. Various forms of nuclear filament are shown.
The organism depicted in fig. 92 is dividing.

Figs. 96-108.—Large Spirilla from large intestine of Lacerta
muralis.

Fig. 96.—Short individual, showing large cytoplasmic alveoli and
nucleus in the form of a short rod-like filament at one end of the cell.

Figs. 97, 98.—Dividing forms. Note nuclear filaments.

Fig. 99.—Short individual, with nucleus in the form of a short and
somewhat zig-zag or spiral filament.

Fig. 100.—Short individual with long, varicose nuclear filament.

Fig. 101—Longer individual, with long spiral or zig-zag nuclear
filament.

Fig. 102.—Very long individual, with long nuclear filament similar
to that of the preceding.

Figs. 103-105.—Shortest individuals (Vibrio form) with nuclear
filaments.

Fig. 106.—A form similar to fig. 99, but with a longer nuclear filament.

Fig. 107.—Longer organism, with short, centrally placed nuclear
filament.

CONTRIBUTIONS TO THE CYTOLOGY OF THE BACTERIA. 505

Fig. 108.—Long individual, in which the nuclear filament has divided
into two preparatory to cell division.

Fig. 109.—Group of five small Bacilli with darkly staining nucleus-
like bodies—similar to those shown in fig. 53 (Pl. 17). (These * nuclei”
are possibly spore-rudiments.) From large intestine of Lacerta
muratlis.

Figs. 110-112.—Large Spirilla from the hind eut of Stylopyga
orientalis. The cytoplasm has an alveolar structure, and the
nucleus is of the chromidial type.

Figs. 115 and 114.—* Fusiform Bacteria” from the large intestine of
Lacerta muralis. Each cell shows a single spherical nucleus.

Fig. 115.—* Fusiform Bacterium,” of double form, from large intestine
of Bufo vulgaris. (Fixation: corrosive sublimate and acetic acid.)

Fig. 116.—* Fusiform Bacterium,” of double form, from large intestine
of Stylopyga orientalis. The lower nucleus in dividing.

Figs. 117 and 118—Bacillus spirogyra from large intestine of
Bufo vulgaris. Notethenuclear filaments. (Fixation: corrosive sub-
limate and acetic acid.)

Figs. 119 and 120.—Bacillus flexilis from large intestine of Bufo
vulgaris. Note the alveolar structure of the cytoplasm (rather in-
distinct) and the nucleus in the form of chromidia. The organism

shown in fig. 120 is undergoing division.

Figs. 121-132.—Small Spirilla from the hind eut of Stylopyga
orientalis.

Fig. 121.—Small Vibrio form with terminal nucleus.

Figs. 122-124.—Small individuals with centrally situate nuclei.

Figs. 125 and 126.—Longer individuals with dividing nuclei. Note
the characteristic dumb-bell figure which the nucleus assumes. (Compare
with Micrococci and Sarcina.)

Fig. 127.—Individual in which nucleus has divided into two, though
fission of the cytoplasm has not yet occurred.

Figs. 128 and 129.—Dividing organisms.

Fig. 150.—Long individual with centrally placed, undivided nucleus.

Fi

Fig. 152.—Smallest Vibrio form. Central nucleus in the form of a

* 131.—Small Vibrio form. Central nucleus.

(9 [)

minute chromatin granule.

Figs. 153 and 154.—Bacilli of flexilis type from large intestine of
Lacerta muralis. Chromidial nuclei. Fig. 154 shows a dividing
individual. Same forms as those shown in figs. 83, 84 (Plate 17).

506 C. CLIFFORD DOBELL.

Figs. 135 and 156.—Bacterium-like organism from large intestine
of Boa constrictor. (Wet film, absolute alcohol; Delafield’s
hematoxylin.)

Fig. 135.—Bacterioid forms—a chain of four.
Fig. 156.—Zymoid forms. A free single individual and another, which
has formed a bud.

PATE 19)

(All figures are of the nucleated Bacterium-like organism (or its
developmental forms) found in the large intestine of Boa constrictor.
(Dry film preparations: fixed absolute alcohol, stained Giemsa.) |

Figs. 157-141, 144.—Bacterioid forms.

Fig. 142.—Form intermediate between bacterioid and zymoid form.

Figs. 145 and 145.—Zymoid forms.

Figs. 146, 147, 149, 150—Zymoid forms, producing outgrowths. In
fig. 150 the outgrowth has divided off as a more or less bacterioid cell.

Fig. 148.—Four bacterioid forms in a chain—the two middle indi-
viduals producing outgrowths.

Fig. 151.— A chain composed of both zymoid and bacterioid indi-
viduals.

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ON CRISTISPIRA VENERIS NOV. SPEC.

507

On Cristispira veneris nov. spec., and the
Affinities and Classification of Spirochets.

By

Cc. Clifford Dobell,

Fellow of Trinity College, Cambridge; Lecturer at the Imperial

College of Science and Technology, London.

With Plate 20 and 2 Text-figures.

CoNnTENTs.

Introduction
Material and Methods ,
Occurrence of Micro-organisms in the Crystalline Styles
Lamellibranchs
Cristispira veneris n. sp.
(1) Structure
(2) Division . : ‘ :
The Morphology, Affinities, and Classification of Spirochiets
Literature References
Description of Plate

INTRODUCTION.

of

PAGE

Derine the last few years many memoirs have made their

appearance in connection with the remarkable group of

Protista which may be conveniently collected under the

common name “ Spirochets.’ Of these organisms the most

divergent descriptions have been given, and consequently

the most divergent views have been held regarding their

affinities with other organisms. Many workers consider that

VOL. 56, PART 3.—NEW SERIES. 35

508 ©. CLIFFORD DOBELL.

the Spirochets are allied to the flagellate Protozoa ; many
consider that their proper systematic position is among the
Bacteria. As I have devoted a considerable amount of study

to both these groups—as regards their cytology and life-
histories—I have naturally been anxious to extend my studies
to the Spirochets. The present paper represents a part of
the researches which I have made upon these organisms,
with the conclusions derived from them. I may state at the
outset that my own observations have led me to believe that
the Spirochets are really neither Protozoa nor Bacteria, but
for the present—must be held to

a group of Protista which
stand apart.

In the present paper I shall describe some researches
which I made upon the Spirochets of Molluses—to one species
of which I have devoted special attention. With the
exception of the work of Schellack (1909) and Gross (1910),
almost all the observations which have been made upon these
forms are, I believe, marred by incorrect interpretation.
My own observations and interpretations—made quite inde-
pendently, and upon different material—correspond in many
ways with those of Schellack and Gross.

I shall adopt the generic name Cristispira Gross to
denote the flexible, spiral organisms which occur in the
crystalline styles of so many Lamellibranchs. It is obvious
that the name ‘‘Spirocheta,’’ which has now for some
time been applied to them, is no longer applicable (see p. 534).

MATERIAL AND MeErtTHops.

The organism with which the present paper is chiefly
concerned is a large species of Cristispira which inhabits
the crystalline style of Venus (Meretrix) casta Chem.
As no Cristispira has previously been recorded from this
mollusc, | propose to name the new organism Cristispira
veneris 0. sp.

The discovery of this organism is due to Dr. Arthur
Willey, F'.R.S., who called my attention to it when I was

ON CRISTISPIRA VENERIS NOV. SPEC. 509

visiting Ceylon in 1909, during my tenure of the Balfour
Studentship of Cambridge University (cf. Dobell, 1910).
As the organism is of large size, I took the opportunity of
investigating its structure and lte-history as far as possible.

All the specimens of Venus casta which I examined were
taken from Tamblegam Lake,! in the Eastern Province of
Ceylon. These molluses—together with others—were collected
for me by Dr. Willey, at Niroddumunai, and sent thence to
me in Trincomalee—about eight miles distant. With Dr.
Willey’s assistance I also examined some of the molluses at
Niroddumunai soon after they had been captured. I take
this opportunity of again thanking Dr. Willey for his kind
collaboration.

A crystalline style was present in 50 per cent. of the
specimens of Venus casta which I examined. In every
instance in which a style was present it was found to be
infected with Cristispirze. Examination of the contents
of the cesophagus and stomach of individuals possessing no
crystalline style was in every case negative; but only a few
of these individuals were carefully examined. The Cristi-
spire were usually present in large numbers, and were always
very actively motile when first removed from their host.

After studying the living organisms, I made a number of
permanent preparations in order to investigate finer details
of structure. The method employed was the same as that
which [have frequently used with success in studying similar
forms, Bacteria and blood-inhabiting Protozoa. I made moist
films of the substance of the crystalline style, fixed them by
exposure to osmic vapour followed by absolute alcohol, and
then stained them by Giemsa’s or Leishman’s modification of
Romanowski’s stain. The films were then washed in water
and allowed to dry, or previously differentiated in weak
alcohol. They were examined under an immersion in cedar-
wood oil. This method gives, I believe, very accurate results
when properly employed. The fixation with osmic vapour

1 Tamblegam Lake is a salt-water lake, connected with Koddiyar
Bay. All my observations were made in September, 1909.

510 C. CLIFFORD DOBELL.

must be done with care; if this is the case, harmful effects
do not result from subsequent drying after staining. Minchin!
has found a similar method suitable for studying the structure
of trypanosomes. I have also obtained excellent preparations
of these and hemogregarines, etc., in this way.

I also made dry film preparations, fixed in absolute alcohol
and stained by Giemsa’s method in the usual way. This
method gives rise to most misleading appearances in the
organisms, but these are of considerable interest for com-
parison with those in other preparations which have been
properly fixed.

The appearances observable in different preparations will
be considered later, but it may be noted here that they vary
according to the length of time during which the osmic
vapour is allowed to act, and according to the degree to
which the stain is extracted with alcohol after staining.

During my stay in Trincomalee, I was unable to use other
methods of fixation and staining. But from my previous
experience of the method I employed, I believe that my pre-
parations are trustworthy, and give relable information
regarding the structure of the organisms. Comparison of
my results with those of others has served to strengthen this
opinion.

Tn all cases the films were made from the crystalline style
immediately after removal. Cristispire usually undergo
degenerative changes soon after they have been removed
from their host, and therefore exhibit a structure which is
very different from that of normal mdividuals. It is true
that they will often live in carefully made preparations for
several hours. But their motility, as a rule, diminishes
rapidly, and their internal and external structure becomes
modified by degenerative changes.

1B. A. Minchin, “The Structure of Trypanosoma lewisi in
Relation to Microscopical Technique,” ‘Quart. Journ. Mier. Sci.,’ vod.
53, 1909, p. 755.

ON CRISTISPIRA VENERIS NOV. SPEC. Pile

OccuRRENCE or Micro-OrGANISMS IN THE CRYSTALLINE S'TYLES
OF JiAMELLIBRANCHS.

In addition to making an examination of the crystalline
style of Venus casta, I searched for Cristispir in the
styles of eight other species of Lamellibranch. All these
were also obtained from Tamblegam Lake. I found Cris-
tispire present in the style of only a single species—
Soletellina acuminata Desh. Only three individuals out
of eleven examined harboured the parasites, though a crys-
talline style was present in every individual. In one style,
all the Cristispire were dead and degenerating when I
found them.

These Cristispire in the style of Soletellina acumi-
nata had been previously discovered by Dr. Willey (cf.
Dobell, 1910). On account of the small amount of material
which I obtained, I was unable to make any extensive
observations upon these organisms. The Cristispire of
this species are small, and resemble C. interrogationis
Gross. I found similar forms—possibly identical with these
—sometimes inhabiting the style of Venus casta, in com-
pany with the large C. veneris. As they were found in
relatively small numbers I have not been able to make a
careful study of them. I believe, however, that there can be
little doubt that they belong to a separate species, and are
not developmental forms of Cristispira veneris. ‘The
occurrence of more than one species of Cristispira in the
same style has already been described by Schellack (1909) in
several Lamellibranchs (Ostrea, Tapes, etc.), and by Gross
(1910) in Pecten.

In some of the other molluses which I examined, I found
that the crystalline style was infected with Bacteria. These
were not present simply as a few organisms—derived from
the gut contents—on the surface of the style, but permeated
the whole of its substance. In fact, the whole style appeared
to be a pure culture of the particular organism which was
inhabiting it. As far as I am aware, this has not been

O12 CG. CLIFFORD DOBELL.

observed previously in the styles of other Lamellibranchs,
and I wiiltherefore devote a few words to a description of my
observations.

Out of nine individuals of Circe gibbia Lam, which I
examined, five possessed a crystalline style, and three of these
were heavily infected with Bacteria—two being uninfected.
The Bacteria all appeared to be of the same species. They
were nov-motile Vibrio-like organisms of small size, and
many dividing forms were present.

A single individual of Cyrena impressa Desh. which
I examined contained a style heavily infected with a
Bacillus.

Seven individuals belonging to the species Psammotea
variegata Wood were found to possess crystalline styles.
Five of these contained large numbers of a Bacillus.

Texamined ten specimens of Arca (Scapharca) rhombea
Born, and found a style present in six of these. Four out of
these six styles were filled with curious branching filaments,
whose nature was not determined. I found the same sort of
filaments in the style of one specimen of Soletellina
acuminata. In the living state, the filaments look lke
fungal growths, and after staining by Giemsa’s method they
are seen to contain a large number of deeply staining gran-
a heavy thunder-

ules. Owing to an unfortunate accident
storm which overtook me when I was returning to Trinco-
malee with my preparations—my slides of these organisms
were much damaged, so that I can give no further particulars
regarding these peculiar growths.

The window-pane oyster (Placuna placenta), of which
I examined a few specimens, was always found to possess a
very long and well-developed style. No parasites were found
inhabiting it. Dr. Willey has also examined a number of
styles of this mollusc, and always with the same negative
results.

I give the results of my examination of the eight species of
Lamellibranch referred to in the following table :

ON CRISTISPIRA VENERIS NOV. SPEC. 513

Nore tl ou |
Molluse. individuals hich. 4 Observations.
examined, Sbyle hoes
present.
|
Arca (Scapharca) 10 6 4 styles were infected
rhombea Born. | with filaments; 2 un- |
infected.

Circe gibbia Lam. y) 5 5 styles were infected
with Bacteria; 2 un-
infected.

Cyrena impressa 1 I Style infected with

Desh. Bacteria
Placuna pla- 3 3 All styles uninfected.
centa L.
Psammotea vari- ff 7 5 styles infected with
egata Wood Bacteria; 2 uninfected.
| Solen (Ensis) | 1 0 —
regularis Dunk. |
|Soletellina acu- 11 11 2 styles infected with a
minata Desh. small Cristispira;
| 1 with dead Cristi-
| spira; 1 with fila-
| ments.
| Venus (Dosinia) 2, 1 Style uninfected.

cretacea Reeve
|
}

I take this opportunity of thanking the Rev. A. H. Cooke
for very kindly identifying these Lamellibranchs for me.

A good deal has already been written about the function of
the crystalline style of the Lamellibranchiata. It has been
suggested that it is a body of a secretory or excretory nature,
that it is a reserve supply of food material, and that it is a
mechanical device for catching and conglomerating food
particles. Mitra! has shown that it contains a proteid sub-
stance—which he showed to be a globulin—and that an
amylolytic ferment is present in it. He therefore regards it
as a body which is primarily connected with the digestion of

1 Mitra, “ The Crystalline Style of Lamellibranchia,” ‘ Quart. Journ.
Mier. Sci.,’ vol. 44, 1901.

514 C. CLIFFORD DOBELL.

food. Hornell,! from his own observations on the style of the
oyster, regards the style as a food-catching apparatus, as was
maintained earlier by Barrois. Pelseneer® states that “the
product of its solution forms a sort of cement which encrusts
any hard substances that may have been ingested and thus
protects the delicate walls of the intestine from injury.”

This is not the place to discuss these and other views which
have been put forward regarding the functions of the crystalline
style. But as this is of some importance in connection with
the organisms which inhabit it, the structure itself cannot be
ignored. It appears to me most probable—from the observa-
tions recorded by others—that the crystalline style serves
both to catch food particles and prepare them mechanically
for digestion and also to assist in the digestion of the amyloid
constituents of these particles.

In some Lamellibranchs—e. g. in Pecten (Gross, 1910)—
the Cristispire are found in the stomach and intestine, and
only rarely in the crystalline style. It therefore seems to me
probable that Cristispira is really a gut parasite, which
often happens to find the substance of the crystalline style a
suitable culture medium. ‘lhe same is also suggested by the
occurrence of Bacteria in the stvle. ‘The latter contains some
12 per cent. of globulin, with about 1 per cent. of salts and
88 per cent. water.2 It might therefore well serve as‘a
culture medium for many micro-organisms which reach it
accidentally. I do not think any deeper significance need
be attached to the association of Protista with the crystalline
style.

CRISTISPIRA VENERIS, N. SP.
I will now record my observations upon the structure

1 Hornell, ** Report on the Operations on the Pearl Banks during the
Fishery of 1905,” * Ceylon Marine Biological Reports,’ Part II, June,
1906.

2 Pelseneer, ‘Mollusca,’ in Lankester’s ‘Treatise on Zoology,’
London, 1906.

3 Mitra, loc. cit.

ON CRISTISPIRA VENERIS NOV. SPEC. 515

and mode of division of Cristispira veneris—the large
“molluse spirochet” which I found inhabiting the crystal-
line style of Venus (Meretrix) casta Chem. in Tamblegam
Lake. I shall here give my own observations only—reserving
an analysis of my own results and these of other workers for
the next section (p. 527).

(1) Structure.

Cristispira veneris is one of the largest members of the
genus, resembling C. balbianii Certes and C. pectinis
Gross. The average length is 50-60 mw, the average breadth—
in fixed and stained specimens—about 1°5 wu. A certain amount
of variation in the breadth of different individuals is observ-
able in fixed and stained organisms—tke narrowest being
shghtly over 1 uw, the broadest approximately 1:9 uw. Dried
films stained with Giemsa not uncommonly possess a width of
almost 2 4. The longest undivided individual which I have
measured was 74 u in length.

Living individuals appear to be of approximately the same
width, though it is almost impossible to make accurate
measurements of them on account of their great motility.
The differences in width observable in stained individuals are
due, I believe, to the greater or less degree of flattening which
takes place in the organisms in making the preparations. It
ean be seen in the living organisms that they are cylindrical—
that is to say, they are circular and not band-like in optical
transverse section. In the process of making films, the
cylindrical shape is modified by flattening to a band-like
shape, thus making the individuals appear broader. ‘Thus, if
the diameter of the cylinder constituting the organism were
1 pw, the circumference would be 7 uw. If complete flattening
of the cylinder occurred, the breadth of the organism would

29

appear to be $ x > or approximately 1°6 4. According to

the amount of flattening which occurred, different individuals

516 CG. CLIFFORD DOBELL.

might therefore display any breadth between 1 wand 1°6 p.
If the breadth of C. veneris is therefore a little more than
1 w»—that is, about 1:2 4, subject of course to slight individual
variation—then the different breadths observed in stained
specimens are easily accounted for by the different degrees of
flattening which different individuals have undergone in the
process of making the preparations. I believe, therefore, that
the body of C. veneris is cylindrical, and has an actual
uniform diameter of approximately 1:2 jm in the living
organism.

I have already described a similar apparent variation in
breadth—due, I believe, to the same causes—in the case of
Bacteria (see Dobell, 19104). The apparent variability in
the breadth of different individuals of Cristispira is a point
of some importance when considered in relation to the method
of division (see p. 526).

As in other members of the genus, the body of C. veneris
possesses a spiral, corkscrew-like shape. ‘lhe number of
complete turns in a full-grown individual is approximately
four. The number is greater than this in dividing indi-
viduals (five or six), and less in newly divided individuals
(two or three).

In the living organisms, I have not been able to distinguish
any structure in the protoplasm of the cell, which appears
homogeneous under the highest magnification which I was
able to employ (Leitz 34, in. oil-immersion x ocular 5, using
direct sunlight for illumination). A few small refractile
granular inclusions were usually to be seen in the proto-
plasm.

The ends of the organism are bluntly pointed (see fig. 1,
Pl. 20), being less rounded than the ends of C. balbianii
and less pointed than those of C. anodontw. The body
usually tapers very slightly towards the two ends. The
structures called “polar caps,’ described in C. balbianii,
C. pectinis, etc., are not observable in C. veneris. They
appear to be confined to the species which possess rounded

ends.

ON CRISTISPIRA VENERIS NOY. SPEC. 517

Neither in living nor in fixed and stained specimens can
any structures comparable with flagella be seen.

Like other Cristispire, C. veneris has a flexible body.
It may be noted, however, that in living and actively moving
individuals the body is kept relatively rigid—tlexibility being
chiefly observed in slowly moving (? abnormal) individuals,
and indicated by the irregular spiral conformation often
observable in fixed and stained organisms. I believe that
bending movements occur very seldom in normal active indi-
viduals. The ordinary movements of C. veneris are similar
to those of C. balbianii, which have already been described
by Perrin (1906).

The two most important characteristics of the Cristis pire
are the crista and the structure of the protoplasm. I will
now describe these in detail in C. veneris.

«and w=

The Crista.—This structure, formerly called the
lating membrane ” on account of its supposed homology with
the undulating membrane of trypanosomes, has hitherto been
correctly interpreted—I believe—by Gross alone. ‘The name
crista, or crest, which he has proposed for it, appears to me a
convenient and suitable one. I shall therefore adopt it.

A crista is present in every individual which I have
examined in the living condition or in properly fixed and
stained preparations. In dried Giemsa preparations, it may
be torn and distorted and sometimes appear completely
lacking, but this is due to the drying which has taken place
before fixation, and is therefore not a normal condition. In
all cases in which proper fixation with osmic vapour has been
effected, the crista is present and presents the same charac-
teristic appearance.

The crista is in the form of a narrow band, radially situate
on the surface of the organism, and spirally disposed (see fig.
2). It does not as a rule reach the extreme ends of the
organism, and appears to me to be a simple prolongation of
the membrane which clothes the body. At the ends it merges
gradually into this, and no structures comparable with basal
granules or blepharoplasts are present. It is homogeneous

518 C. CLIFFORD DOBELL.

throughout, and shows no fibrillar structure in living or
properly fixed specimens. It is stained a pink or violet colour
by Giemsa’s method, in marked contrast with the general
blue colour of the body (see fig. 2, etc.). There is no
thickened, chromatic edge to the crista. In fact, it does not
in any way resemble the undulating membrane of a trypano-
some.

In macerated individuals the crista may present a very
different appearance (fig. 3). It becomes greatly enlarged
and distorted, and shows a very definite fibrillar structure.
This is an artifact, and though it may indicate that the crista
is really composed of fibrils arranged longitudinally, it
must not be forgotten that in normal individuals it appears
absolutely homogeneous. This fibrillar appearance has often
been described as the normal structure of the “ undulating
membrane” of Cristispira—which it certainly is not.

The crista of C. veneris 1s therefore a delicate, uniform,
band-like appendage, wound spirally round the body, and
extending almost to the ends. It is always present, and has
no resemblance to the undulating membrane of a trypano-
some. It serves, apparently, as a rigid lateral fin-lke exten-
sion of the body, in the performance of the screw-like
movements of the organism. Some further account of some
of the previous interpretations of this structure will be found
on p. 528.

Structure of the Protoplasm.—As I have already
noted, the protoplasm of the living organisms appears homo-
geneous. In stained specimens, however, it has a distinct and
highly characteristic structure. ‘This structure has been
observed by Schellack and Gross, though the interpretations
of these two observers differ.

If a Cristispira be fixed by exposure to osmic vapour for
about thirty seconds, then transferred immediately (without
any drying being allowed to take place) to absolute alcohol
for ten minutes, then stamed by Giemsa’s method, and
examined in the manner already described (p. 509), it is seen
to possess a structure like that of the mdividual shown in fig.

ON CRISTISPIRA VENERIS NOV. SPEC. 519

1, Pl. 20. This organism is from a moist film preparation of
a crystalline style which was so treated. The whole organism
(fig. 1) shows a protoplasmic structure consisting of a single
row of chambers or alveoh. The walls of these chambers are
stained a deep blue, their contents a uniform pale blue. The
relative dimensions of these chambers are not always constant;
they may vary not only in different organisms, but at different
points in the same organism—being sometimes square, some-
times oblong (cf. figs. 7, 8, etc.). The alveolar walls
separating adjacent chambers from one another appear as
transverse septa in optical section (see figs. 1, 2, etc.). At
the point where the transverse septum joins the wall of the
cell a dark purple granule can be seen. ‘lhe whole organism
thus appears to contain a series of paired purple granules,
united by blue transverse lines—representing the alveolar
walls (fig. 1). This appearance is always presented by indi-
viduals treated in the manner described. If the exposure to
osmic vapour has been limited to about thirty seconds, and no
overstaining has taken place, then the appearances are con-
stantly encountered. ‘The difference in size observable in the
purple granules should be noted

also the fact that they
always lie at the edges of the organism, and never centrally
digs: 15-7).

Ti the osmic vapour be allowed to act for a longer period
of time—i. e. for several minutes—then the organisms present
a different appearance after Giemsa staining. The granules
appear much smaller, and are stained a deep blue (figs. 2, 8).
The chambers are easily visible, but the granules have
dwindled to tiny dark blue points. In some cases they
cannot be distinguished with precision at all levels in the
body (cf. fig. 6).

Organisms which have been dried previous to fixation, fixed
in absolute alcohol, and then stained by Giemsa’s method in
the usual way, often present appearances which are quite
different from those seen in osmic-fixed organisms. ‘hey
show, in fact, all the remarkable “ chromatin” configurations
which have been described by Perrin and others. The

520 C. CLIFFORD DOBELL.

chambers are often indistinctly seen, or absent. Vacuoles are
not infrequently present. Red “chromatin” structures of
varying form are seen in different individuals and at different
points in the same individual. Fig. 10 shows some of the
“nuclear ”’ structures observable in dried organisms. It is
drawn from a part of a Cristispira which was dried before
fixation, fixed in absolute alcohol, and stained by Giemsa’s
method. It will be seen that the “ chromatin” is in the form

of spiral or zig-zag filaments, rods, granules, “ tetrads,” ete.

‘ ?

These arrangements of the “chromatin” are found side by
side in the same organism at the same time.

The appearances which are observable in organisms which
have undergone plasmoptysis are instructive. Such an indi-
vidual is shown in fig. 3. The whole organism is filled with
red granules, of variable size and irregular distribution. At
the points where the cell membrane has burst, the protoplasm
has flowed out, and it can be seen that it consists of two
different substances—a bluish or lilac coloured substance and
a denser dark-red substance.

How are all these different appearances to be interpreted ?
I believe the correct interpretation is as follows: The
structure of Cristispira may be compared with that of a
bamboo stem. ‘The whole body is in the form of a hollow
cylinder, divided into a single series of chambers by means of
a series of transverse disc-like partitions like the nodes of a
bamboo rod. The cytoplasm forming the walls of the cylinder
and the disc-like partitions is dense and deeply stainable ; the
cytoplasm which fills the chambers is less dense and less
deeply stainable. Text-fig. 1 illustrates diagrammatically the
structure of a portion of a Cristispira which is supposed to
have been split longitudinally, so as to divide the body into
two equal parts. When viewed from inside, an appearance
such as is shown in 'l'ext-fig. 1 would be seen.

The tube forming the body is divided into cylindrical
chambers by transverse disc-like partitions—only half of each
dise and chamber being seen, of course, when the other halt
is split off. In a Cristispira all the solid structures dia-

ON CRISTISPIRA VENERIS NOV. SPEC. yA

grammatically represented in Text-fig. 1 are composed of the
denser part of the cytoplasm, the chambers—represented
empty in the diagram—being filled with the less dense cyto-
plasmic matter.

Now I believe that the only. other morphological con-

TrxtT-FIG. 1.

Explanation in text.

stituents of the cell are a number of small granules, which
are arranged round the circumference of the disc-like parti-
tions—in the dense cytoplasm which lines the cell. The
position of these granules

which I suppose to form a ring
when a partition is seen in a transverse section of the whole

TrxtT-Fic. 2.

A. B.
Explanation in text.

cell—is shown in the diagram (text-fig. 1, Gr.). A transverse
section of the cell, passing through a partition, would present
an appearance similar to that shown in Text-fig. 24. In this
diagram it will be seen that the granules are arranged in the
form of a ring round the circumference of the disc. A

522 C. CLIFFORD DOBELL.

transverse section of a cell, passing through the middle of a
chamber, would present an appearance lke that shown
diagrammatically in Text-fig. 28. The appearance is that of
a tube—the wall composed of dense cytoplasm, the inside
filled with less dense and more lightly staining cytoplasm.

Now I think that those individuals which have been
exposed to osmic vapour for several minutes and then stained
with Giemsa’s stain, present appearances which must be
interpreted as representing a structure such as I have just
described. The chambered structure of the cytoplasm, with
the ring of small granules round the circumference of each
partition, is quite clearly seen in these individuals. In optical
section, of course, only a single pair of granules is seen—
lying at the point where the partition joins the cell wall. The
granules are of very small size, and are therefore visible
under the highest powers only, and after correct differentia-
tion. It is difficult to be absolutely certain that a ring of
granules is present round each partition, but I believe that
this can often be demonstrated. As the bodies of Cristi-
spire treated in this way remain cylindrical—or undergo
only a very slight flattening—in the process of fixing and
staining, it is necessary to suppose such an arrangement of
the granules to account for their constant appearance at the
edges of the organism—at the points where the septa and
cell-walls unite (cf. figs. 2, 8, etc.).

As I have pointed out, the organisms which have been fixed
by exposure for a shorter time to osmic vapour show pairs of
much larger purple granules situated at the points where the
partitions join the sides of the cell. A ring of granules is
not present in these forms. I believe the correct interpreta-
tion of such organisms (figs. 1, 7) is as follows: In the course
of making the preparation the organisms have become
flattened, as a result of drying following upon inadequate
fixation, Exposure to osmic vapour for about half a minute
is not sufficient to fix the organisms properly. As they dry
on the slide the granules run together into small masses at
the edges of the organism, and so give rise to the appear-

ON CRISTISPIRA VENERIS NOV. SPEU. 523

ances which [ have described. It is easy to understand why
the granules—in reality masses of granules—appear to be of
different sizes in such organisms (fig. 1), and why they
always appear at the edges of the organism, which we know
to be really cylindrical when alive.

When no fixation previous to drying occurs, the cell under-
goes plasmolysis and complete flattening on the slide. The
small granules run together in various ways, giving rise to
the various “ nuclear” figures which have been described (ct.
fig. 10). It is easy to understand how the flowing of the
granules through the walls of the chambers, and their massing
together in various ways, can give rise to the appearance of
transverse bars, spirals, tetrads, etc.,of ‘‘chromatin.” It will
hardly be necessary to describe in detail the several ways in
which such appearances may be caused.

The staining reactions of the substance of which thie
granules are composed require a brief consideration. I have
already noted that the granules staina deep blue after a long
exposure to osmic vapour, purple after a brief exposure, red
when osmic fixation is omitted, and only absolute alcohol is
employed after previous drying. I believe these differences
are directly due to the action of the osmic vapour—pro-
longed action of which so changes the granules that they are
unable to take up the red-staining element in the Romanowski
stain. I have observed this action of osmic acid in the case
ot Bacteria and many Protozoa, and I believe it must have
been noticed by many other workers who employ Romanowski
staining after osmic fixation. A short exposure to osmic
vapour permits the granules to stain red—as they do when
not acted upon by it. A longer exposure permits them to
stain red to a less extent, and gives rise to a purple colora-
tion. Still longer action of the osmic vapour renders the
granules incapable of taking up the red element in the stain,
and they therefore appear blue—the blue element alone being
capable of staining.

There can be no doubt, I believe, that the granules are
composed of a substance which is different from that of the

VOL. 56, PART 3.—NEW SERIES. 36

524 ©. CLIFFORD DOBELL.

cytoplasm. Itis a substance, moreover, which may be stained
red with Giemsa’s stain (ef. figs. 3, 10). From this it may
perhaps be inferred that the granules are composed of a
chromatin substance, and are therefore of a nuclear nature.
This consideration, however, does not really justify the con-
clusion that the granules constitute the nuclear apparatus.
Further evidence of the behaviour of the granules during
other phases of the life-history is required before their true
significance can be settled. Yet for the present, I regard
the nuclear hypothesis as the most probable, and believe that
the granules represent a chromidial nucleus somewhat similar
to that which occurs in many Bacteria (see Dobell, 1910a),
and some Protozoa.

To summarise my interpretation of the protoplasmic
structure of Cristispira: The whole body is composed of
a single series of cylindrical chambers or alveoli, separated
from one another by disc-like partitions. These structures
are composed of a denser cytoplasm constituting their walls,
and a less dense cytoplasm which fills the chambers. Very
small granules—probably constituting, as a whole, a nucleus
of a chromidial form—are arranged round the circumference
of each disc-like partition. Various appearances—such as
a series of pairs of large granules, tetrads, transverse bars,
spiral filaments, etc., of chromatin-—which are often encoun-
tered, and have been frequently described by others, are
artifacts.

One more point in the protoplasmic structure of Cristi-
spira veneris requires consideration. It often happens
that here and there, in the body of an individual, certain
chambers appear more darkly stained than the remainder.
This appearance is well seen at the point marked a in the
individual depicted in fig. 6. At other times the partitions
between the chambers appear thickened (see fig. 8, b, etc.),
and appearances which are intermediate between a darker
chamber and a thickened septum are also to be seen (see
fig. 9, where this is shown in two places). Similar appearances
have been figured by Gross and others.

ON CRISTISPIRA VENERIS NOV. SPEC. 525

The explanation of these appearances is, I believe, quite
simple. As will be shown in the next section of this paper
(vide infra), the method of multiplication is by transverse
fission. The daughter-individuals which arise from the
transverse division of a long individual are therefore short—
being only half the length of the original organism. Before
they undergo a subsequent division they must grow in length,
and must therefore form new chambers. I believe that these
new chambers are formed at various points in the body, and
arise by the gradual thickening of a partition and its subse-
quent hollowing out. ‘Thickened partitions therefore corre-
spond to the points where new chambers are beginning to be
formed—more darkly stained chambers are newly formed
chambers. Successive stages in the formation of chambers
in this way are shown in fig. 8 (where a thickened septum
is seen at b), fig. 9 (which shows the hollowing of the septa at
two points), and fig. 6—where a darkly staining (newly
formed) chamber is seen at a.

(2) Division.

Although I have not been able to observe every stage in
division in the living organism, I have encountered a number
of dividing forms in my stained preparations which leave no
room for doubt as to the essential features of the process.
Division is transverse, and is effected in the manner described
by Gross in the case of C. pectinis. I have never seen
any indications of a longitudinal division, and all the observa-
tions which I have made speak strongly against the view that
such a method of multiplication occurs in these organisms.

The long individuals which are about to divide into two
transversely are in the form of spirals consisting of five or
six complete turns. Before dividing, they bend themselves
double—the two halves becoming intertwisted (see fig. 4). ’
This phenomenon has been described in C. pectinis by
Gross, who calls it “ incurvation.” The transverse fission of
the organism begins when it is in this condition. It occurs

026 C. CLIFFORD DOBELL.

in the middle of the incurved individual, at a point where a
transverse partition separates two adjacent protoplasmic
chambers from one another (cf. fig. 4). The partially
divided organism then untwists itself—passing out of the
condition of incurvation to the original form of a simple
spiral (fig. 6). In this condition fission is completed, and the
two daughter-individuals separate from one another. The
latter are, of course, short individuals in the form of spirals
consisting of two or three turns.

In the division of the body the crista is also involved. It
divides with the rest of the body, in the manner shown in
fie. 7. This figure shows the middle region of a dividing
Cristispira which is just straightening itself after being in
the state of incurvation.

The whole process of division is extremely simple, and
apart from the incurvation—the process of divi-

resembles
sion which can be seen in many Spirilla and other
Bacteria.

I think there can be no doubt at all that the imcurved
individuals are not really stages in a longitudinal division—
as they seem frequently to have been interpreted by other
workers. The crista does not split longitudinally. I have
never seen partially longitudinally split individuals; the
transverse division of the looped end of the incurved organism
is often very easily seen; the number of turns in the spiral
in a newly divided individual is half that of the undivided
individual ; and finally, the width of all individuals—when
allowance is made for the differences due to technique (see
p. 515)—is fairly constant. These facts indicate most clearly
that division is transverse and not longitudinal, as Schellack
and Gross have maintained in the case of other species of
Cristispira. I believe, with these two observers, that all
cases of longitudinal division which have been described in
Cristispira are due to misinterpretation of the observed
appearances.

Formation of gametes, conjugation and encystation I have
never encountered. These phenomena—first described by

ON CRISTISPIRA VENERIS NOV. SPEC. 527

Perrin

have been said to occur by several observers, but
their statements are based, I believe, upon a wrong inter-
pretation of the facts. This has already been pointed out by
Schellack and others, so I will therefore omit further dis-
cussion of the matter here.

THe MorpHonocy, AFFINITIES AND CLASSIFICATION OF
SPIROCHRTS.

In the following pages I shall discuss the most important
features in the morphology and life-history of the Cristi-
spire, or, as they are commonly called, ‘mollnse Spirochets.”
A discussion of these features is necessary in order to arrive
at conclusions regarding the affinities of this remarkable
group of organisms, and of Spirocheets in general.

Two excellent contributions to this subject have recently
been made—that of Schellack (1909) and that of Gross (1910).
Both these workers employed good cytological methods, and
made careful detailed observations on the forms which they
investigated. As they have both discussed the earlier work
at some length, and entered fully into the literature on the
subject, I will confine myself chiefly to pointing out wherein
my results agree with or differ from those of these two
workers.

The Cell Membrane.—The body of a Cristispira 1s
bounded by a cuticle-like covering, which I shall call the cell
membrane. This membrane is usually termed the “ peri-
plast ”

a name originally applied to it by Perrin, who
believed the organisms to be Trypanosomes. The use of this
special word for the cuticular covering in these two groups of
organisms—Spirochets and ‘rypanosomes—appears to have
led many people to believe that the cell membranes are so
similar to one another, and different from other cell mem-
branes, as to indicate affinities between the two groups. ‘The
only real similarity between the cell membrane of a Cristi-
spira and that of a Trypanosoma is that the same word is
used for both. Both are, of course, modified forms of mem-

528 C. CLIFFORD DOBELL.

brane which bound the protoplasm of the body; but such
inembranes are found in the majority of Protista, only they
are not usually called “ periplasts.”’ I shall therefore avoid
using this term, as I believe it leads to a confusion of ideas ;
and I shall speak of the cuticular covering of a Cristispira
as the “cell membrane,” or simply as “ the membrane.”

A membrane certainly exists in Cristispira. Unless this
were present, it is difficult to see how the contours of the
body are preserved. The appearance of burst individuals
also indicates that a membrane of some sort is present (see
fig. 3). Moreover, the presence of a membrane is clearly
demonstrated when the organisms undergo plasmolysis. This
has been clearly shown by Swellengrebel (1909) in C. bal-
bianil.

It has frequently been stated that the “ periplast”’ of
Cristispira possesses a fibrillar structure, which can be
seen when the organisms are macerated. I have seen many
individuals of C. veneris which show the appearances which
have been thus interpreted, and I believe the fibrils are
derived in all cases from the crista (see fig. 3). The cell-
membrane itself possesses no structure. Schellack (1909)
states that “bei den grossen Spirocheeten! ist ein fibrillarer

fo

Periplast sicher nachgewiesen; er kann kiinstlich aufgefasert
werden.” I believe this is incorrect. My own view is the
same as that expressed by Gross— Der Periplast existirt gar
nicht. Die Cristispiren haben einfach eine ziemlich starke,
aber farberisch nicht differenzirbare Zellmembran.” As
Gross has discussed the matter fully I will say nothing
further about it—merely pointing out that my interpretation
agrees with his.

The Crista.—Schellack (1909) interprets the crista as
an artifact—‘als ein durch kiinstliche Veranderung des
Periplasts hervorgerufenes Gebilde.” I believe this inter-
pretation to be quite incorrect. The crista is easily visible in
slowly moving, living organisms, and is constantly present in
properly fixed specimens. It is homogeneous and possesses

1 T.e. Cristispire.

ON CRISTISPIRA VENERIS NOV. SPEC. 529

no chromatic border. It is totally different from the undulating
membrane of a 'l'rypanosome, to which most previous workers
have likened it. My interpretation of this characteristic
structure is the same as that of Gross (1910). “Die Crista
ist ein Organell sui generis.”

A deeply staining (“chromatic ”) edge to the crista and a
fibrillar structure can only be seen in macerated organisms,
or organisms which have been imperfectly fixed. Such strue-
tures must therefore be regarded as artifacts. The normal
evista of C. veneris stains pink or violet with Giemsa’s
stain, but this does not necessarily indicate that it contains
chromatin.

Flagella.
Protozoa or Bacteria, are not present in Cristispire. ‘The

Flagelia or cilia, such as occur in flagellate

matter has been fully discussed by Schellack (1909) and Gross
(1910), who have both come to this same conclusion. Further
discussion will therefore be superfluous.

Protoplasmic Structure.—Vhe chambered structrue of
the protoplasm, which I have described in C. veneris, has
already been clearly recognised in other Cristispire by
Schellack and Gross. Iam convinced, with these two observers,
that the various nuclear figures (spiral filaments, transverse
rodlets, tetrads, etc.) described by Perrin and others are really
artifacts. Moreover, Perrin’s account (1906) of the relations
existing between the various nuclear figures and the longi-
tudinal division of the organism must be discarded. For the
nuclear figures are artifacts, and longitudinal division does
not occur.

‘The interpretations of the appearances observed by Schellack
and Gross differ from that which I have given in preceding
pages. It will therefore be necessary to discuss their views
briefly.

Schellack’s (1909) interpretation of the protoplasmic
structure of Cristispira is somewhat similar to mine.
His deseription of the structure of the chambers is in close
agreement with my own description. In one point, however,
Schellack’s interpretation differs from mine. He believes

550 C. CLIFFORD DOBELL.

that chromatin granules are scattered through all the walls
of the chambers, whereas I believe that—in C. veneris—the
granules are confined to the circumference of each transverse
dise-like partition. Schellack thus regards a Cristispira as
containing a nucleus of a kind of chromidial form.!

Gross’s (1910) interpretation is peculiar. Although he
appears to have observed the same structures as Schellack
and myself, he comes to the conclusion that the protoplasm
is really structureless, and there is no nucleus of any sort
present. The chambers are artifacts, because they can be
seen neither in the living organisms nor in organisms fixed
with Flemming’s fluid and stained with iron-hematoxyhln.
Gross always found the chambered structure present after
fixation with corrosive sublimate, but he attributes this
structure to the action of the fixative.

I beheve that another explanation is correct. I believe
that the invisibility of the chambered structure after fixation
with Flemming’s fluid is the direct result of the action of the
fixative. It is often exceedingly difficult to obtain good
differentiation of the internal structure of Bacteria after they
have been fixed with Flemming’s fluid, and I believe that this
is due to the action of the fluid upon the cell-membrane and
the protoplasm. Hvery cytologist must have experienced, at
some time or other, a difficulty in staining cells after fixation
in Flemmine’s fluid. At all events my own experience leads
me to believe that this must be so. I would also point out
that, in the case of C. veneris, not only does a prolonged
action of osmic vapour—in the course of fixation—cause a
change in the staining reactions of the granules, but it also gives

! This statement requires some qualification. For although Schellack
describes the chromatin as being in the form of granules (‘Die
Kammerwinde scheinen aus einer festeren Substanz zu bestehen und
es sind ihnen Koérnchen aufgelagert,’ p. 400), he seems inclined in
another place to regard the nucleus as being constituted by the whole
of the substance of the chamber walls. He says: “ Die Gesamtheit der
Waben in einer normalen Spirochete bildet einen ziemlich fest in sich

haltbaren, kompakten Stab, den sogenannten Kernstab. Die Periplast-
hiille liegt ihm direkt auf,” ete. (p. 401).

ON CRISTISPIRA VENERIS NOV. SPEC. Hei

rise to a less precise staining of the cell as a whole. Cristi-
spire which have been subjected to osmic vapour for many
minutes tend to take up a more diffuse blue stain, and show
the chambered structure less distinctly in consequence. But
although this is the case, the chambers can always be seen.
They never disappear completely, though they do become
fainter after more prolonged fixation. hat the chambered
structure cannot be seen in the living organism I do not
regard as any proof of its non-existence. For the width
of the cell is small (less than 2 py): the cell-membrane is fairly
thick and possesses a considerable degree of refractivity :
and the difference in refractivity between the protoplasm
forming the walls of the chambers and that which fills them
is probably not very great in the living organism. ‘The
chambered structure appears with such constancy in organ-
isms fixed with osmic acid or corrosive sublimate that it will
require a good deal more evidence than that furnished by
Gross to prove that it does not exist.

Swellengrebel’s (1907) original account of C. balbianii
differs in some ways from his later description (1909), in
which he records appearances which are consistent with my
interpretations. ‘The transverse bars of chromatin which he
describes are, I believe, similar to the transverse bars which
I have frequently seen, and are produced in precisely the
same way—by imperfect fixation. Swellengrebel states that
he fixed the organisms in formaldehyde (1907, p. 19), but he
appears to have overlooked the fact that fixation in the way
he describes is inadequate unless employed in conjunction
with after-treatment with alcohol—a point which I have
already had occasion to point out elsewhere (Dobell, 1910a).

It is apparent from the foregoing, therefore, that whereas
Schellack appears to regard the body of a Cristispira as
being chiefly composed of a nuclear structure, Gross regards
it as enucleate, and I regard the nucleus as being in all
probability represented by chromidial structures arranged in
the manner described in previous pages (see p. 521).

Plasmolysis.—Swellengrebel (1909) has proved that

532 C. CLIFFORD DOBELL.

Cristispire are plasmolysable. The phenomenon is so often
seen in organisms which have been dried, or are drying, in a
drop of sea-water, that it is almost inconceivable that anyone
should have stated that the organisms are implasmolysable.
I think there can be no doubt whatever that plasmolysis may
be caused in these organisms, and that it is similar to that
which may be seen in many Bacteria.

Division.—My own conclusions regarding division are
completely in accord with those of Schellack and Gross.
Division is transverse, and not longitudinal. The errors of
interpretation which have led many workers to believe that
longitudinal division occurs have been fully discussed and
elucidated by Gross. Further discussion of the matter there-
fore appears to me unnecessary.

Polarity.—A point of considerable importance, but one
which has received hardly any attention from those who have
discussed the affimties of the Cristispire and similar
organisms, les in connection with what I may term the
“olarity”’ of the cell. All flagellate Protozoa possess an
antero-posterior differentiation—that is to say, they show by
their movements that one end of the body is the front end,
the other the hind end. It is therefore correct to speak of
their movements as backward or forward movements. ‘The
front end is usually the end which bears the flagellum. Now
in the Bacteria no such differentiation can be observed.
Spirilla and Bacilli cannot correctly be said to move backwards
or forwards, because neither end is definitely differentiated
as anterior or posterior. In other words, either end is a
facultative anterior or posterior end.

In this respect Cristispira and the other so-called Spiro-
cheets are similar to the Bacteria, and stand in sharp contrast
with the flagellate Protozoa.

The point is not one to be ignored. For it is evident that
a differentiation of this sort must involve the organisation of
the whole organism, and must therefore be of profound
significance.

Flexibility.—It has more than once been urged that

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ON CRISTISPIRA VENERIS NOV. SPEC.

Cristispira and its allies, being flexible and not rigid
organisms, show affinities with the Protozoa and not with the
Bacteria in consequence. I do not know who is responsible
for the original statement that all Bacteria are rigid organisms,
but it is certain that such a statement cannot be accepted.
Many Bacteria of large size are flexible to a considerable
extent. [have shown this to be the case in Bacillus flexilis
(Dobell, 1908) and a number of allied forms (Dobell, 1910).
It is therefore manifest that flexibility cannot be used as a
criterion for judging whether the Spirochets are to be ranked
among Protozoa or Bacteria.

ee

Conjugation.—The organisms described as “ gametes”
by Perrin and others, and the stages said by them to repre-
sent conjugation stages, are all quite arbitrarily so designated.
I believe there is absolutely not a vestige of evidence that
conjugation occurs in these organisms. Neither Swellen-
grebel, nor Schellack, nor Gross, nor myself could find any
indication of sexual phenomena in this group. Both Schellack
and Gross have discussed the matter more fully, and I am in
complete agreement with their conclusions.
Eneystment.—Whether Cristispire encyst or not is a
point which is still undetermined. I believe the “cysts”
described by Perrin and others are really to be regarded as
the results of degeneration or plasmoptysis. Schellack and
Gross both appear to be of the same opinion. At all events,
it may be said with justice, I believe, that no clear case of
encystinent has yet been described in Cristispire.

Affinities and Classification.

Having now briefly noted the more important features in
the structure and life-cycle of the Cristispire, it is possible
to discuss the affinities and classification of these most
remarkable organisms.

At the present moment it is customary to assemble under
the common name “ Spirochets” three different groups of
unicellular organisms. These are (1) the Cristispire,

D034 C. CLIFFORD DOBELL.

parasitic in Lamellibranchs, (2) the much smaller parasitic
organisins like “S pirocheta” pallida, “8S.” buccalis,
the organisms of relapsing fevers, etc., (3) the free-living
forms Spirocheta plicatilis and its allies.

Now the name Spirocheta! was introduced by Hhrenberg
in 1835 for the free-living organism 8. plicatilis. It must
therefore be applied to this and similar organisms. ‘The
structure of S. plicatilis has been described by Schaudinn
(1905, 1907). According to him there is an undulating
membrane and a nucleus in the form of a longitudinal
filament surrounded by chromidia—these two elements corre-
sponding respectively to the kinetic and trophic nuclei of a
trypanosome. Reproduction occurs by multiple transverse
fission.

(Quite recently these organisms have been more carefully
studied by Ziilzer (1910), whose observations differ greatly
from those of Schaudinn. She interprets the axial filament
as an elastic body—not part of the nucleus. The latter 1s
represented by large, regularly arranged chromatin granules.
There is no undulating membrane. If this description is
correct,” it is obvious that 8S. plicatilis is a very different
organism from Cristispira. Anyone who has observed
living specimens of S. plicatilis would, I should think, be
impressed by their dissimilarity to Cristispire—both as
regards movements and general appearance. ‘l'his, at all
events, is my own impression. The bodies of both are flexible
and spirally wound, but beyond this there is no great resem-
blance. The differences are at least sufficiently great to
justify the bestowal of different generic names upon the two
organisms. As Gross has introduced the name Cristispira
for the molluse Spirochets it should henceforth be adopted.

The smaller parasitic Spirochzets—such as the syphilis

' The correct spelling of this name is Spirocheta, and not Spiro-
chete, as adopted by Doflein (1909) and numerous other writers.

> T have every reason to believe it is, as I had an opportunity of con-
versing with Frl. Dr. Ziilzer and seeing some of her preparations at the
International Zoological Congress in Graz this year (1910).

ON CRISTISPIRA VENERIS NOV. SPEC. 535

organism, the organisms of relapsing fevers, etc.—differ not
a little from Cristispira and Spirocheta. In the forms
which I have been able to study myself,! I have never been
able to make out any definite structure—chiefly on account of
their very small size. I believe that no protoplasmic structure
similar to that of either S. plicatilis or Cristispira is
visible. I also regard it as exceedingly doubtful thata crista
is present. ‘The method of division is, I believe, in all
probability always transverse. Although the facies of these
organisms is very similar to that of Cristispire, I think it
is advisable to keep the two groups of organisms in separate
genera for the present.

Regarding the generic name which must be applied to these
organisms, it is obvious that as neither Spirocheta nor
Cristispira can be used, some other name must be selected.
The name Spironema, proposed by Vuillemin (1905) for the
syphilis organism, is pre-occupied—having been used by Kiebs
for a flagellate. Schaudinn (1905a) therefore proposed the

name T'reponema—a name which must stand, according to
the rules of nomenclature. If it be allowed that the small
parasitic Spirochets are similar to the syphilis organism, it
therefore follows that they must all be placed in the genus
Treponema. It appears to me advisable to adopt this
system.

For the three groups of organisms which are included in
there are therefore three

the common name “ Spirocheets ”’

veneric names already in existence. On the assumption, then,
that these three groups are sufficiently akin to one another to
justify their being collected into a common class—an assump-
tion which appears to me to be justified in our present state of
knowledge—I propose to classify the Spirochzets as follows :

1 These are especially forms from the gut of the frog and toad
(Dobell, 1908), from termites (Dobell, 1910), and “8.” bueccalis and
“$.° dentium (unpublished observations).

2 T see no valid reason for drawing a generic distinction between
Treponema pallidum and such forms as “Spirocheta” recur-
rentis, “S.” duttoni, “S.” dentium, etc.

Or
(J)
Oo

©. CLIFFORD DOBELL.

Spirochetoidea.

Genus 1.—Spirocheta Ehrenberg. Free-living forms,
freshwater or marine. Examples: 8S. plicatilis
Ehrenberg, S. gigantea Warming.

Genus 2.—Treponema Schaudinn. Parasitic in animals
(Vertebrates and Invertebrates). Examples: T.
pallidum Schaudinn, T. recurrentis Lebert,
T. dentium Koch, T. gallinarum Blanchard;
etc., etc.

Genus 3.—Cristispira Gross. Parasitic in Lamelh-
branchia. Examples: C. balbianii Certes, C.
anodontez Keysselitz, C. pectinis Gross, C.
veneris, etc., etc.

The exact classificatory value to be attached to the group
S pirochwtoidea cannot at present be accurately determined.
The name stands for a group of Protista which, like several
other groups (e.g. Bacteria, Mycetozoa, Myxobacteria),
cannot at present be regarded as a “ class,” “order,” or any
other sort of subdivision of another group, but must be
regarded as an independent group of unicellular organisms
which show very little affinity to any other group.

This last statement requires some further qualification.
Many workers regard the Spirochzts as showing affinities to
other Protista. It has been suggested that there are resem-
blances between them and the flagellate Protozoa, the Bacteria,
and the Cyanophycee.

Schaudinn was the first to express the opinion that the
Spirochets are allied to the Trypanosomes, and hence to the
flagellate Protozoa. Krzysztalowicz and Siedlecki (1908) go
so far as to place them in a group Spirilloflagellata
among the Mastigophora. Doflein (1909) places them in a
between the Bacteria and the

erou_p—Proflagellata
Mastigophora. Now I think that I am completely justified—
from what I have already pointed out in the preceding part
in stating that there is not one character of

of this paper

ON CRISTISPIRA VENERIS NOV. SPEC. 537

importance which is common to Spirochets and Flagellates—
save that both are unicellular. It is, to me, most remarkable
that anyone can see any real resemblance between a Spiro-
chet and a Trypanosome. ‘lhe nuclear and cytoplasmic
structures are wholly different: a 'Trypanosome has a flagellum,
a Spirochet has none:! the crista is not lke an undulating
membrane: the cell-membranes are not similar: and moreover,
the method of division is quite different in the two groups of
organisms. As regards conjugation, nothing has been proved
either in Trypanosomes or Spirocheets, so that its occurrence
or non-occurrence can furnish no grounds for discussion of
affinities between the two groups. The flexibility of Spiro-
chets also, as I have pointed out, affords no criterion for
determining their protozoal or bacterial affinities.

Many workers regard the Spirochets as Bacteria. Novy
and Knapp (1906) place them in the genus Spirillum.
Swellengrebel (1907) places the Spirochats and Spinilla in
the same family (Spirillaceze) among the Bacteria. Schellack
(1909) suggests that the Spirochets are related to the
Cyanophycee by way of Spirulina and similar forms. Gross
(1910) finally places Spirocheta with the Cyanophycee,
and Cristispiraand Treponema with the Bacteria. Ziilzer
(1910), however, who has made a special study of S. plica-
tilis and the spiral forms of Cyanophycee (Spirulina,
Arthrospira), has shown that there is no real similarity
between these organisms. Affinities between Spirocheets and
Cyanophycez appear therefore not to exist. |

Now beyond a certain superficial similarity of form between
certain Spirochets and Spirilla, there is really no reason for
regarding Spirocheets as Bacteria. The points of similarity
are chiefly these—both possess the same sort of cell polarity
(see p. 532), both divide transversely, both are plasmolysable.

1 The “ flagella’ of various species of Treponema are probably—as
has often been pointed out already—merely the drawn-out ends of
organisms which have just resulted from the transverse division of a
longer organism. They have nothing to do with the flagella of Protozoa
or Bacteria.

538 C. CLIFFORD DOBELL.

But the same might be said of many other Protista. Two
most important characters of the Bacteria—the formation of
are not encountered
in the Spirochets. The structure of the cell, especially as

endospores and the possession of flagella

regards the nucleus, in Cristispira and Spirocheta is
quite different from that of Spirilla. With regard to the
latter, I would refer the reader to my work on the cytology
of the Bacteria (Dobell, 19104). There is, in fact, no real
reason for regarding Spirochets as Bacteria.

There seems to be a curious tendency on the part of many
workers to reason thus: Spirochets are not Protozoa, there-
fore they are Bacteria; or conversely, they are not Bacteria,
therefore they are Protozoa. ‘The premisses are both correct,
I believe, but the deductions are both wrong. Spirochets
are neither Protozoa nor Bacteria; they are a group of Protista
which stands alone. ‘They certainly have a few characters in
common with Bacteria, but the differences greatly outweigh
these.

In conclusion, I will summarise the results to which my
work has led me. They are as follows :

The organisms commonly called Spirochets may be con-
veniently collected into a single group, for which I propose
the name Spirochetoidea. This group comprises three
different sets of forms, which may be correspondingly classified
in three different genera — Spirocheta, Treponema,
Cristispira. These three groups of organisms, whilst
showing certain resemblances to one another, possess no
definite relations with Protozoa,. Bacteria, or Cyanophycex.
The Spirochetoidea should therefore be regarded—for the
present—as a group of Protista which stands apart.

IMPERIAL COLLEGE OF SCIENCE AND
TECHNOLOGY, LONDON.
November, 1910.

ON CRISTISPIRA VENERIS NOV. SPEC.

LIrERATURE REFERENCES.

The following list contains only those memoirs to which reference is
made in the text. Fuller bibliographies of works dealing with the
subject will be found in the papers of Schellack (1909) and Gross (1910)
cited below.

Dobell. C. C. (1908).—* Notes on some Parasitic Protists,” * Quart.
Journ. Mier. Sci.,’ vol. 52, p. 121.

(1910).—** On some Parasitic Protozoa from Ceylon,” *Spolia
Zeylanica, vol. vii, p. 65.

(19104).—* Contributions to the Cytology of the Bacteria,”
‘Quart. Journ. Micr. Sci.,’ in the press.

Doflein, F. (1909) —‘ Lehrbuch der Protozoenkunde,’ Jena (Fischer).
Gross, J. (1910).—* Cristispira nov. gen. Ein Beitrag zur Spiro-
chatenfrage,”’ ‘ Mittheil. zool. Stat. Neapel,’ Bd. xx, p. 41.
Krzysztalowicz, F., and Siedlecki, M. (1908).—* Etude expérimentale de

la Syphilis; morphologie de Spirocheta pallida,” * Bull.
Acad. Sci. Cracovie,’ p. 173.
Novy, F. G., and Knapp, R. E. (1906).—‘‘ Studies. in Spirillum
obermeieri and Related Organisms,” ‘ Journ. Infect. Dis.,’ vol.
iy p. 201.

Perrin, W. S. (1906).—** Researches upon the Life-history of Try pano-
soma balbianii (Certes),” ‘Arch. Protistenk., Bd. vii, p. 151.

Schaudinn, F. (1905).—* Zur Kenntnis der Spirochete pallida.”
‘ Deutsch. med. Wochenschr.,’ p. 1665.

(19054).—Correspondence in ‘ Deutsch. med. Wochenschr.,’ p.
1728.

(1907).—* Zur Kenntnis der Spirocheta pallida und anderer
Spirocheten”’ (Aus dem Nachlass Schaudinn’s herausgegeben von
Hartmann u. Prowazek), ‘Arb. kaiserl. Gesundheitsamte, Bd.

xVI5 P: Lle
Schellack, C. (1909)—* Studien zur Morphologie und Systematik der
Spirocheten aus Muscheln,” ‘Arb. kaiser]. Gesundheitsamte,’
Bd. xxx, p. 379.
Swellengrebel, N. H. (1907).—* Sur la Cytologie comparée des Spiro-
chétes et des Spirilles,” ‘ Ann. Inst. Pasteur,’ t. 21, p. 448.
(1909).—* Neuere Untersuchungen iiber die vergleichende
Cytologie der Spirillen und Spirochiten,” ‘C. B. Bakt., Abt. I,
Orig., Bd. xlix, p. 529.
YOL. 56, PART 3.—NEW SERIES. By

540 C. CLIFFORD DOBELL.

Vuillemin, P. (1905).—‘‘ Sur la dénomination de Vagent présumé de la
syphilis,” ‘C. R. Acad. Sci.,’ vol. exl, p. 1567.

Ziilzer, M. (1910).—** Ueber Spirochexta plicatilis und Spirulina,”
“Zool. Anz:., Bd. xxxv, p. 795.

EXPLANATION OF PLATE 20,

Illustrating Mr. C. Clifford Dobell’s paper “ On Cristispira
veneris nov. spec., and the Affinities and Classification
of Spirochets.”

[All figures are drawn from stained preparations of Cristispira
veneris n. sp.,from the crystalline style of Venus (Meretrix) casta
Chem., taken in Tamblegam Lake, E. Province. Ceylon. The drawings
were made under a Zeiss 2 mm. apochromatic oil-immersion, with com-
pensating oculars 6, 12, and 18. The magnification of the figures is
approximately 2000 diameters. |

Fig. 1.—An average-sized individual, in optical section. The general
form of the body is well seen. Note also the chambered structure of
the cytoplasm and the arrangement of the purple-staimed granules.
(Osmic vapour 350 secs. absolute alcohol; Giemsa’s stain.)

Fig. 2.—A somewhat extended individual, showing the disposition of
the crista and the structure of the protoplasm. The body is seen in
optical section, but the crista is shown as it appears when focussed
carefully at different levels. (Osmic vapour {several minutes |; absolute
alcohol, Giemsa.)

Fig. 3.—An individual which has been macerated in a drop of sea-
water, allowed to dry, then fixed in absolute alcohol and stained by
Giemsa’s method. The organism has undergone plasmoptysis, and
the crista shows a fibrillar structure.

Fig. 4.—A dividing organism in the stage of incurvation. (Osmic
vapour, absolute alcohol, Giemsa.)

Fig. 5.—Part of an almost completely divided organism in incurvation
stage. The upper end corresponds with the upper end of fig. 4, being
the point at which fission ocenrs. (Slightly more highly magnified
than the other figures.) (Osmic vapour, absolute alcohol, Giemsa.)

Fig. 6.—An individual which is almost completely divided into two.
Stage following incurvation. At a, a darkly stained chamber. (Osmic
vapour [several minutes], absolute alcoho!, Giemsa.)

ON CRISTISPIRA VENERIS NOV. SPEC. 541

Fig. 7.—Middle region of an individual which is opening out after
incurvation. Division of body and crista is seen. (Osmic vapour
30 sees., absolute alcohol, Giemsa.)

Fig. 8.—Part of the body of an organism which has been fixed by
exposure for several minutes to osmic vapour, then treated with absolute
alcohol, stained with Giemsa, and differentiated in alcohol. At a the
dark blue granules are distinctly seen ; at bis seen a thickened partition.

Fig. 9.—Part of another organism, treated like the preceding, but
more deeply stained. The granules are not sharply differentiated from
the walls of the chambers. At two points new chambers are being
formed.

Fig. 10.—Part of a dried organism fixed in absolute alcohol and
stained with Giemsa. “ Nuclear” structures in the form of a spiral or
zig-zag filament (a), a transverse bar (b), granules (c), a tetrad (d), ete.,
are seen.

Iuant. fourn Mier Sci. Vo, 56. NS LLCO

C.C.D.del. Huth, Lith’ London
CRISTISPIRA VENERIS.

»

TROCHOPHORE OF HYDROIDES UNCINATUS (£UPOMATUS). 543

On the Development and Structure of the Tro-
chophore of Hydroides uncinatus (Eupo-
matus).

By
Cresswell Shearer, VI.A.,
Trinity College, Cambridge.

With Plates 21-25 and 29 Text-figures.

CONTENTS.

PAGE
1. Introduction . . 5643
2. Review of Literature. . 558
3. Material and Methods . ; . 563
4. Segmentation and Gastrulation . _ 566
5. The Eetomesoblast . 568
6. The Calomesoblast : : : . ole
7. The Early Trochophoral Stages . ows
8. Summary : . 584.

1. [wrropucrion.

WuiLr working at Naples some years ago, I was led to
investigate the early development of the Annelid Eupomatus
with a view to determining the origin of the mesoblast bands
and their relation to the head-kidneys. ‘his species is
common at Naples and breeds throughout the year. ‘The
biastule and gastrule are very hardy, and development is
normal under the adverse conditions of heat and impure sea-
water incidental to their study under laboratory conditions.
Fertilisation takes place quickly when the ripe generative

O44 CRESSWELL SHEARER.

products are brought together, and material can be easily
obtained of any stage. The trochophores can be readily
reared to the adult worm in small jars of sea-water to which
sufficient food is added from time to time, in the form of
cultures of the common Diatom Nitzschia closterium.
On this they rapidly grow, and soon attach themselves to
the sides of the culture jars and form their tubes.

The minuteness of the egg is a serious disadvantage, how-
ever, in following the changes that lead up to the establish-
ment of the trochophore. The fully formed larva barely
measures 654 in diameter, and the pre-trochophoral stages
are very sinall, and the cells of the blastule and gastrule are
unusually minute. In following the origin and growth of
the head-kidneys one is forced to depend almost wholly on
sections, and sectioning larve of this size is a_ tedious
proceeding.

in the Serpulid Pomatoceros I soon found a more
suitable object in which to trace the development of the
head-kidneys. The egg is larger and more deeply pigmented.

In the arms of the “ cross-cells ”

this pigment quickly
becomes segregated on development, where it affords a ready
means of orientation. For these reasons I early abandoned
the study of Hupomatus for that of Pomatoceros, on
which I hope shortly to complete my “Studies on the
Development of Larval Nephridia,” by publishing a
full account of the origin and growth of these organs in
this animal.

The present notes dealing with Kupomatus, although
incomplete, | have thought worthy of publication, as they
deal with the formation of the trochophore and the appear-
ance of the coelomesoblast. They derive some importance
from the fact that on this Annelid, Hatschek (17) conducted
his classical investigations on the development of the meso-
blast bands—investigations which have played so prominent
a part in all our speculations concerning the mesoderm, Any
revision, therefore, of the subject on the same material as
that studied by him is not without interest.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 545

In the following account I have incorporated some drawings
and notes of Hydroides pectinata, kindly placed at my
disposal by Prof. E. B. Wilson, which I believe were made by
him some years back.

One object I have kept in view has been that of following
the changes leading from the gastrula to the formation of
the trochophore. In the numerous careful accounts of the
development of Aunelids that have been published few
attempts exist to connect the cell regions of the early stages
with the organs of the trochophore. Many of the early
embryologists, as Kowalevsky (28), Agassiz (1), Hatschek (18)
and Salensky (30) seem to start their studies only with the
young larva, when the rudiments of the larval organs have
already appeared. On the other hand, many of the more
recent investigators, commencing with the unsegmented egg,
frequently fail to carry their studies far enough when they
stop short at the end of gastrulation, and before the definitive
organs of the larva have appeared.

Some considerable confusion has arisen through taking
the conditions found in relation with the mesoderm at rela-
tively late stages, and considering these same relations to
hold in the early phases. This is seen in the work of
Hatschek (18) and Fraipont (12) on Polygordius; and has
resulted in some error with regard to the head-kidney rudi-
ments, larval and ecelomesoblast.

In all Annelids with a free-swimming larva such as that of
Jupomatus there is always a considerable interval between
the end of gastrulation and the assumption of the full tro-
chophoral condition. ‘This period, for the sake of convenience,
IT shall refer to as that of the pre-trochophoral stage. It is
the period of which we know the least in the development of
Annelids.

The excellent papers of Woltereck (52) on Polygordius
and Torrey (41) on Thalassema have done much to advance
our knowledge. ‘I'he early cell-regions have here been traced
clearly to their ultimate fate in the organs of the trochophore.
Woltereck has shown that the head-kidneys arise early and

546 CRESSWELL SHEARER.
Trext-FIes. 1-6.

Podarke

Planocera

Text-figs. 1-5.—Karly segmentation stages of Planocera (Surface).
Text-figs. 4-6.—Podarke (Treadwell). HE. Ist endoderm cell
budded off from 4d,. Hn.{Entomeres. 1.m.1. Left portion of the
ectomesoblast. /.m.m. Median portion of the same. “.m.i. Right
portion of the same. M.E. Ceelomesoblast. Stim. Stomodeum.
X,,5. Anal cell.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 547

before the mesoblast bands. They are already functional
before the bands have -appeared, for the pole-cells so con-
spicuous in Hatschek’s figures have no existence at this
stage.

It is true that Meyer (27), from the study of late stages,
came to the conclusion that larval mesoblast was a structure
apart from the ccelomesoblast, but I doubt if the evidence
derived from the study of these late stages in Polygordius
alone is sufficiently convincing.

TART MTG. OVA.

Podarke

Section through a late gastrula stage of Podarke (Treadwell).
a. Archenteron. «.r. Apical rosette. EH. First endoderm
cell budded off from 4d,. lm. Larval or ectomesoblast.
ME. Ccelomesoblast.

Within the last twenty-five years a large literature has
grown up with regard to the question of the mesoderm, and
embryologists have held many opinions regarding its origin
and significance. ‘These conflicting views are roughly
reducible, however, to two groups, each of which has been
advocated with more or less success. ‘lo the first belong
those who consider the mesenchyme (larval mesoblast,
ectomesoblast) and mesothelium (definite mesoderm or
ccelemesoblast) as one and the same structure; to the second

belong those who consider them as two separate structures.

548 CRESSWELL SHEARER.

The first consider they have a common, while the second
claim they have a separate origin.

Hatschek, as the result of his studies on Polygordius (18),
Echiurus (16), Eupomatus, and Teredo (1% and 15),
many years ago pointed out the difference between the
irregular scattered cells of the mesenchyme and the definite
cells of the mesoblast bands. He claimed, however, to have
observed the origin of the mesenchyme cells from the meso-
blast bands. In his opinion mesenchyme and mesothelium
arise from a common foundation. This was followed by
Wilson’s (48 and 47) work on Hydroides, Polygordius
and Lumbricus, where he found a complete gradation from
the stellate cells of the mesenchyme scattered through the
blastoccel to the round fixed cells of the anterior ends of the
germ bands. Many other observers have pointed out more
or less the same thing, as, for instance, Roule (29) in Huchy-
treoides, Fraipont (12) in Polygordius, Biirger (5) in
Nephelis, Hirudo and Aulastoma, Hacker (18) in
Polynoe. The common nature of both mesenchyme and
mesoderm at one time gained wide acceptance through its
adoption and elaboration by the brothers Hertwig (19) in
their well-known ‘ Ccelomtheorie.’

On the other hand, the majority of those embryologists
who have recently investigated the development of Annelids
and Molluses hold that these structures are both ontologically
and phylogenetically distinct ; that the mesenchyme has an
origin apart from the ccelomesoblast, that it arises in a
pecuhar fashion from the ectoderm; hence they have sought
to denote this in the name they have applied to it, i.e. that
of ectomesoblast. ‘The ccelomesoblast, on the contrary, is
usually segregated in a single large cell seen in the ventral
lip of the blastophore.

Kieinenbergh (21) was perhaps the first to lead the way
towards this conception of the nature of mesoderm and
mesenchyme, in his paper on the development of Lopado-
rhynchus, where he pointed out that the mesoderm arises
as a membrane between the two primary layers, and, as he

TROCHOPHORE OF HYDROIDES UNCINATUS (HUPOMATUS). 549

thought, direct from the ventral side of the ectoderm. This
was followed by the work of Whitman (45) on Clepsine,
Bergh (8) on Lumbricus, Vejdovsky (44) on Oligocheets.
Schimkewitsch (32) in Dinophilus described a separate
origin of the mesenchyme in the anterior end of the larva
from the definite mesoderm of the posterior region. Finally
the separate nature of mesenchyme and ccelomesoblast has
been most ably championed in the very extensive researches
ot Meyer (27) on the mesoderm of Annelids.

In the work of the cell-lineage investigators, however, the
distinction between larval and ccelomesoblast has been most
definitely brought to ight. Inall Annelids, Lamellibranchs,
and Gasteropods studied by them, with one exception, the
ceelomesoblast invariably arises from a large cell in the
ventral side of the blastophore (4d). The one exception is
the Annelid Capitella, where, according to Hisig (11), it
arises from the third and fourth quadrants of the third
quartette. Herethe cell 4d contains a little larval mesoblast,
but the main portion contains ectoderm. In Molluscs, accord-
ing to Conklin (7), 47, while containing the ccelomesoblast, is
more than half endoderm. Inthe Annelid Podarke, accord-
ing to ‘Treadwell (42), 4d divides and then sinks in, and
takes up its position in the endoderm of the archenteron
(Text-figs. 4,5, 6). Here at a later stage it gives rise to the
coelomesoblast.

At the time 4d is being invaginated, or even before,
irregular ectoderm cells are given off into the interior of the
blastoccel; these are the larval mesoblast cells. They
migrate inwards and scatter throughout the cavity. ‘heir
origin has been determined in a large number of forms,
first by Lillie (25) in Unio, and then by Conklin (7) in
Crepiduia, 'lreadwell (42) in Podarke (Text-fig. 5, 1. m.r.,
l.m.l., l.m.m.), Wierzejski (46) in Physa (Text-fig. 11, /.m.r.,
l.m.l.), Torrey (41) in Thalassema (Text-figs. 8 and 9, l.m.r.,
l.m.l.,l.m.m.). The mode of origin of the ectomesoblast, there-
fore, is distinctly in opposition to that of the ccelomesoblast.
In Unio it arises asymmetrically, and only afterwards takes

590 CRESSWELL SHEARER.

up a bilateral position. In Thalassema it arises from the
first and third quartettes. In some thirty Annelids it can be
said definitely that the ccelomesoblast arises from the posterior
cell of the fourth quartette, while the larval mesoblast arises
from the firstand third. This title of “larval mesoblast” does
not mean necessarily that it is confined alone to the organisa-
tion of the larva, for the greater part of it enters into the
structure of the adult. The same has been shown to be the
case in a number of other Annelids, as in Polygordius,
Podarke, and Thalassema.

Meyer long ago put forward the theory that the mesen-
chyme of higher forms corresponds with the mesoderm of the
lower ; that the larval mesoblast of Annelids and Molluscs is
to be homologised with the adult mesoderm of Platodes.
Wilson (50) has shown in Leptoplana that the mesoderm
in this Polyeclad arises from the second and third quartettes,
while in Annelids the larval mesoblast, as I have mentioned
ubove, takes its origin from the same quartettes. He has
established that here the large cell 4d is almost entirely
entoblastic. The early development of the Polyclad Plano-
cera has been studied by Surface (40): ‘ At the forty-four
cell stage the posterior cell of the fourth quartette (4d) buds
a single large cell into the interior of the embryo; both of
these subsequently divide bilaterally (lext-fig. 3). Of these
four cells the two upper and inner (Text-fig. 3, 2d) give rise
to a portion of the mesoderm, and possibly a small part of
the endoderm (‘T'ext-figs. 1-3, 4d). The lower pair lying on
the surface of the embryo give rise practically to all the
endodermal part of the alimentary canal.” ‘Thus the history
of this cell (4d) in this Polyclad shows a remarkable resem-
blance to its homologue in Molluscs and Annelids. ‘A
portion of the mesoderm, chiefly that part lying round the
pharynx, is derived from cells of the second quartette, and
thus corresponds with the secondary mesoblast or larval
mesenchyme of Annelids and Molluscs (Text-fig. 1, 2b). In
the spiral cleavage the segregation of the ectoblast in three
quartettes, the formation of a large portion of the mesoderm

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 551]

from 4d, the formation of the apical cells, and in many other
details, the development corresponds to that of Annelids and
higher forms.”’

In Platodes the mesoderm has a radial origin, and this is
also the case in a number of Annelids with regard to the
larval mesoblast. JI have also mentioned in a number of
Annelids and Molluses that a small portion of 4d is ento-
blastic. The condition in Polyclads, where the greater part
of 4d is entoblastic, is suggestive of a more primitive condi-
tion than that found in Annelids. If the germ-cells in the
Polyclad arise from the 4d portion of the mesoderm, then
the homology of this cell with the cell 4d of Annelids would
be complete. ‘The history of the posterior cell of the fourth
quartette in Polyclads, Annelids, Lamellibranchs, and Gastro-
pods has a remarkable resemblance in all these forms, and the
relation it shows with the endoderm of the gut points clearly,
as Wilson (50) has said, to the way in which teloblasts have
arisen by progressive specialisation from a purely endodermic
origin of the ccelomesoblast as retained in an unaltered con-
dition in the Echinodermata to-day. As he says, it is difficult
to explain these facts otherwise than on the grounds “ that
cell outlines represent definite boundaries of differentiation
areas in the developing embryo.”

Child (6), on the contrary, claims that no importance can
be attached to resemblances of this nature, and that in the
case of the cell 4d they are purely ccenogenetic, and have to
do with the formation of the larval body from a growing
region at its posterior extremity, and the resulting segrega-
tion of material at this point. J think this cannot be said of
all cases where there is a similar segregation of the ccelo-
mesoblast. The growth of the adult from the Glochidium
larva is different in many respects from the growth of the
adult worm from the trochophore, yet in both we ect a marked
segregation of the ccelomesoblast.

With the high degree of specialisation shown by eges that
give rise to a free-existing larva, the ccoelomesoblast, which
primitively arose as diverticula from the gut, became restricted

5H CRESSWELL SHEARER.

Trext-FIGs. 7-12.

Text-figs. 7-9.—Gastrulation stages of Thalassema (Torrey).
Text-figs. 10-12—Physa (Wierzejski). M.D. Coelomesoblast.
L.m.l. Left portion of theectomesoblast. U.m.m. Median portion
of same. é,m.7. Right portion of same.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 553

in the course of time to certain cells in the wall of the archen-
teron, and as development became progressively specialised
their origin became localised in the posterior cell of the
fourth quartette.

It is from the wider and more definite facts of comparative
anatomy rather than from those drawn from development
alone that the real value of Meyer’s theory hes. It is evident
that the germ-cells—the foundation of the later ccoelomesoblast
—are much older in the phyletic sense, as Kleinenbergh (21)
and Meyer (27) long ago pointed out, and as Eisig (11) has
recently stated, than the two primary germ layers, and that
they were differentiated long before the ectoderm and endo-
derm had been evolved as separate structures, as is the case
to-day in Volvox. Meyer’s theory has been recently con-
sidered by Lang (24) and Hisig (11) so exhaustively that it is
unnecessary for me to go into it here. No theory certainly
accounts for so many facts or has been so widely supported
by evidence, both anatomical and embryological.

Thus the separation of the mesoderm into two definite
portions is a characteristic feature of the development of
Polyclads, Annelids, and Molluscs. It remains to be men-
tioned that in a few Molluscs a larval or ecto-mesoblast has
not been observed or is apparently wanting. This would
seem to be definitely the case in Aplysia, the late stages of
which have recently been studied by Carr Saunders and Miss
Poole (81). In Umbrella, Heymons (20) has been unable
to find this structure, but he suggests that possibly in stages
later than those he studied ectoderm cells may migrate into
the interior of the larva and form mesoderm. In Neritina
Blochmann (4) also fails to figure it; but both Heymons and
Blochmann’s work was done at a time when the importance
of the larval mesoderm was hardly recognised, and ultimately
it may prove to be present in these forms. Its absence in
Aplysia, however, seems to be clearly established. It is
hard to understand why this should be the case, as the

majority of Molluscs possess a larval mesoblast, and one is
present in Fiona.

04 CRESSWELL SHEARER.

Korschelt (22) has called attention to the relation of the
ecto- and ccelomesoblast in Physa. If we take a section of
such a stage of Physa as is shown in Text-fig. 11, he points
out that the ecto- and ccelomesoblast between them form a
complete ring round the blastopore. He thinks this condition
points to the conclusion that in Annelids and Molluscs ecto-
and coelomesoblast were originally one structure, which has
been divided and specialised as the result of larval develop-
ment. In Phoronis and the other great group of animals
of the Deuterostomia type this has not taken place.
Phoronisis undoubtedly closely related to the Annelids in the
Actinotrocha stage, with its solenocyte-bearing nephridia and
ciliated rings, but shows no segregation of the coelomesoblast
into pole-cells.

From the work of De Selys Longchamps (84) we know that
the mesoderm consists of a large number of irregular cells
scattered throughout the blastoccel. I have shown (86), and
it has also been clearly demonstrated by the work of other
investigators, that these cells arise in the region of the
blastopore, or from the line along which the blastopore has
already closed. The cells resemble the larval mesenchyme
of Annelids more than the cells of the Annelid ccelomeso-
blast.

In Brachiopods the mesoderm is also of the irregular
variety, and arises from the caelom, which is here a direct
outgrowth from the anterior end of the primitive archenteron,
as Conklin (7) has recenly described in Terebratulina.
No division into ecto- and ccelomesoblast can be distin-
guished, and it is purely ccelomesoblastic.

There would thus seem to be a sharp division between
Phoronis and Brachiopods on the one hand, and Annelids
and Molluses on the other. In one we get a sharp division
of the mesoderm into two portions, while in the other there 1s
no such division. Korschelt (22) thinks that without a more
definite knowledge of how the ccelomesoblast arose in the
hypothetical Annelids, we cannot reconcile these two types
of mesoderm formation.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 555

It appears to me, however, that in Phoronis, or at least
in the early stages of the Actinotrocha larva, we have exactly
the same thing as in Annelids.

I have said that in Phoronis the mesoderm arises in the
region of the biastopore as a number of irregular cells, which
are budded off into the blastoccel. These scatter throughout
the cavity, where they give rise to the mesodermic structures.
According to De Selys Longchamps (84), some of these cells
in the trank region collect to form a rather imperfect ccelomic
sac about the rectum or posterior portion of the stomach. I
was of opinion, however (86), that the cells that gave rise to
this sac had their origin in the gut wall, but of this I was by
no means certain. In any case, in Phoronis we have the
inesoderm showing a specialisation into a coelomic portion,
forming the primitive coelomic sac, and the irregular meso-
dermal cells scattered throughout the blastoccel. Whether
we regard the ccelomic portion as arising from the gut wall
or not, if seems to me we have here the two forms of meso-
derm as in Annelids, and that Phoronis is intermediate
between Annelids and animals in which the mesoderm is
entirely coelomic. Korschelt (22) sums up the mesoderm
formation under five heads, which are worth reviewing in
this connection.

Ist. Mesoderm band formation from teloblasts or pole-cells,
as in Annelids and Molluscs.

2nd. Secondary mesoderm band formation, a modification
of the above process, and re-multiphed in Arthropods and
Cephalopods.

5rd. Formation of mesoderm from gut pouches.

4th. Formation of mesoderm from solid out-growths of the
eut.

Sth. The mesenchyme cells alone give rise to the coelom
and all the mesodermic structures.

To the trochophore originally described by Hatschek (17)
in Eupomatus undue importance has perhaps been attached,
tor such a trochophore is possessed by only a limited number
of Annelids, and is almost exclusively confined to the group

»

VOL. 56, PART 3.—NEW SERIES. 35

296 CRESSWELL SHEARER.

of the Serpulids. Our text-books frequently cite it as a
typical trochophore, although most Annelids possess a
trochophore quite different. The trochophore characteristic
of the majority of Polycheets is one such as that of Sabella
or Nereis, and not that of the Serpulids. ‘This possesses no
head-kidney, and the mesoderm bands develop under con-
ditions that modify their growth as compared with those of
Kupomatus. The blastoccelic cavity in these is always
greatly reduced or entirely obliterated, and gastrulation is
usually epibohe ; while in the Serpulid larvee there is always
a large blastoceelic cavity, and gastrulation is by invagina-
tion. The egg in the majority of the Serpulids, again, is
small and contains very little yolk, although forms like
Spirorbis and Sabella contain a considerable quantity.
It is hard to make any fast distinctions, however, for larvee
occur in the same family, and even in the same genus, which
differ entirely in this respect. ‘The principal cause of this
ereat diversity of form is due in most cases to the modification
undergone by their locomotor organs, as the result of their
adoption of different life-habits. Frequently closely related
larval forms differ greatly in this respect. If they live a
free-swimming pelagic existence, or the contrary, their loco-
motor organs are correspondingly developed or reduced.
Terebella conchilega, leading a pelagic life, possesses
strongly developed ciliated rings, and is a powerful swimmer,
while Terebella meckelii, for the most part spending its
larval existence in the jelly-like mass in which the eggs are
deposited, is uniformly ciliated, and lacks these structures.
Thus the tuberculous Polychets can be divided roughly
into two classes on the basis of their possession or non-
possession of a trochophoral stage. The first of these,
including Eupomatus, Pomatoceros, and Psygmo-
branchus, possess typical free-swimming larvee with well-
developed prototroch and ciliated rings; while a second
group, 1ncluding some of the Terebellids, Aricia and
Arenicola, do not possess a free-swimming stage, are often
uniformly ciliated, and are poor swimmers. In addition, we

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 597
Text-Fies. 13-18.

Euvnomatus

Sections of segmentation and gastrulation stages of Eupomatus.
bl. Blastocel. end. Endoderm. ME. Celomesoblast. lint.
Left portion of ectomesoblast. lmr. Right portion of same.

998 CRESSWELL SHEARER.

find a very large number of errant forms, which have under-
gone so much modification that in many cases it is difficult
to say to which group they belong. In the first class of the
pelagic type we have the larve of Nereis, Phyllodoce,
and Aphrodite, while in the second we have forms like
Diopatra, Ophryotrocha, and many of the Eunicid larve.
Probably the most difficult to class of all are these last, on
account of their great variation (Hicker, 13).

Without some knowledge, therefore, of the mode of hfe of
the primitive ancestors of the Polychets, and the conditions
under which they existed, it is difficuit to decide which of
these various larval types is the most primitive. It is pro-
bable that the free-swimming type has been recently evolved,
and is a more highly modified one than the uniformly ciliated
type, that the trochal has been derived from the atrochal
form. And this is borne out by the fact that in its most
perfect form, as in Eupomatus, it is found in so relatively
few Annelids.

2. Review or LITERATURE.

The early development of the Serpulid Annelids has been
studied by a number of investigators. The earliest account
is that of Stossich (89) in 1878, who described in some detail
the development of Serpula uncinatus (Hupomatus) and
S. glomerata. Itis clear from Iis figures that many of
his larves were abnormal. IJ have obtained many similar
larvee during the hot months in Naples, when the temperature
of the Laboratory sea-water was unusually high. ‘Through
the study of these larve Stossich came to many erroneous
conclusions.

Salensky (30), in 1885, studied the development of Psy gm o-
branchus and Terebella. In these forms the presence ot
a considerable quantity of yolk and the absence of a true
trochophoral stage considerably modify the course of deve-
lopment. He arrived at no certain conclusions regarding
the origin of the mesoderm, although he observed the teleo-
blast cells of the mesoderm bands.

TROCHOPHORE OF HYDROLDES UNCLNATUS (EUPOMATUS). 559

Conn (9) pointed out that in Serpula the egg-chorion is
never thrown off, but remains as the cuticle of the larva.
The gastrula has three noticeable features. The blastopore
is not round but slit-like, and arranged round its margin is a
cireular band of locomotor cilia. Right opposite the blasto-
pore is the apical thickening, bearing a tuft of hair-lke cilia.
The growth of the gastrula is not accompanied by elongation
in the main axis, but obliquely to this in such a way as to
pass through one end of the slit-like blastopore. One end of
the blastopore is thus carried backwards away from the
other, which remains more or less fixed. The blastopore
becomes an elongated slit, the lips of which meet in the
middle and close, forming the rudiment of the future gut.
For a short time the digestive tract remains attached to the
ectoderm throughout the length of the blastopore, but after
a little it only retains this connection at either end. With
further growth the embryo is converted into the trochophore.
The digestive tract becomes hollow and acquires two openings
to the exterior at the two points of its previous connection
with the ectoderm. That near the ciliated band becomes the
mouth, while the other becomes the anus.

“Just before the formation of the anus a number of ecto-
dermal cells near the region of the future anus become
separated from the rest of the digestive tract and form a mass
of cells lying outside the alimentary canal in the body-cavity.
These cells form the mesoderm. Some of these cells increase
in size and form stellate mesenchyme cells, and finally a few
of them stretch across the body-cavity near the anus, forming
a membrane which separates a small portion of the body-
cavity from the rest, forming the anal vesicle. | Occasionally
another partition grows across it, separating it into two
smaller divisions.’ Certain other mesoderm cells form the
true mesoderm. ‘They multiply quite rapidly, and soon give
rise to the mesoderm bands. One of the eye-spots develops
much before the other” (p. 671).

Vou Drasche (10), in 1884, gave an account of the develop-
ment of Pomatoceros, but the early stages and the forma-

560 CRESSWELL SHEARER.

tion of the trochophore were very briefly studied. He did
not observe the origin of the mesoderm cells.

Hatschek (17), in 1885, studied the development of
Kupomatus at Trieste. He supplemented these observa-
tions by the examination of a small trochophore found in the
Pantano, at Faro, Sicily. The identity of this larva he did
not definitely estabiish. The eggs studied at Trieste were
fertilised by the addition of ripe sperm, and were studied in
the living state. Segmentation is equal, and of the spiral
type characteristic of many Polychets. In the resulting
blastula the cells from which the germ layers form are already
differentiated. The greater part of the lower hemisphere of
the blastula produces endoderm. — 'I'wo cells here larger than
the rest give rise to the primitive mesoderm cells, or
teloblasts. The region where they lie corresponds to the anal
end of the larva. At this time the pre-oral baud of cilia
makes its appearance as an equatorial circle of cilia. Shortly
afterwards the apical cilia appear. The endodermic part of
the blastula invaginates about nine hours after fertilisation.
The two mesoderm cells at the same time move to the interior
of the segmentation cavity and detach themselves from their
connection with the other cells. The invaginated portion of
the endoderm forming the gut then bends towards the anal
side of the larva, and fuses with a slight depression of the
ectoderm and produces the anus and proctodeum. At the
same time the blastopore has become narrowed toaslit, which
gradually closes from behind forwards. At the place where
the last trace of the blastopore remains the ectoderm invagi-
nates and forms the cesophagus. At the same time the two
primitive mesoderm cells divide, giving rise to the mesoderm
bands, while other cells near the pole-cells of the bands give
rise to the head-kidneys ; these increase greatly in length and
become hollow. ‘The head-kidney then extends from the pole-
cells in the region of the anus to the wall of the cesophagus,
to which they are attached by a thin protoplasmic strand,
while another runs up in the apical region. They open,
according to Hatschek, on the exterior on either side of the

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 561

anus. ‘Ihe eye-spot is located in a cell in the apical region.
There is a peri-anal circle of cilia.

H. B. Wilson (48) brietiy studied (1890) the segmentation
of the ege of a species of Hydroides found at Naples. The
order and direction of the early cleavage planes coincide
very closely with those of Hupomatus, and segmentation is
of the equal spiral type. The spiral symmetry would seem to
be retained until a late stage. He did not definitely observe
the cell 4d or follow its history. In his early paper (48) on
the origin of the mesoblast bands of Annelids he was of
opinion that the bands gave origin to the mesenchyme cells.
He did not observe the pole-cells of the bands as described
by Hatschek. He pointed out that the head-kidney probably
opened into the proctodseum.

The later development of Psygmobranchus has been
studied by Meyer (27), who made some important observa-
tions on the mesoderm. He pointed out that in the young
trochophore it can be divided under three headings: First,
the mesoderm bands, which are closely applied to the ventral
surtace of the endoderm; secondly, a collection of irregular
cells attached to both ecto- and endoderm, which we can cal!
the embryonic mesenchyme; thirdly, a row of functional
primary larval muscles. ‘he mesoderm bands appear as a
paired plate of cells converging on one another posteriorly,
each ending in a pole-cell—the so-called teloblasts. The
plates extend forward into the oral region. ‘The cells of the
mesoderm bands can be clearly distinguished from the irre-
gular cells of the mesenchyme by their polygonal outlines
and their dark-staining nuclei. The larval mesenchyme cells,
on the contrary, are irregular in outline, and their nuclei
stain less deeply than do those of the bands. ‘lhe mesen-
chyme does not form a compact structure, but is somewhat
irregularly arranged into masses on the inner wall of the
ectoderm or the wall of the gut. It is divided into a median
and a lateral portion, which is again divided into a trunk
aud head portion.

The lateral trunk mesenchyme lies on either side of the

562 CRESSWELL SHEARER.

inner surface of the body-wall, and commences behind the
teloblast cells of the mesoderm bands, and runs forward in
the region of the oral ciliated ring. In the pre-oral region
one finds a number of these mesenchyme cells under the
body-wall, where they form a portion of the head division of
the mesenchyme in relation with the prototroch and apical
O regan .

The median trunk mesoderm begins behind in front of
the anal vesicle, and is continued forward in the median line
under the gut into the region of the stomatodeeum between
the mesoderm bands. The functional Jarval muscles of the
mesenchyme type consist of a ventral and dorsal longi-
tudinal set and the pre-oral circular muscles of the proto-
troch. With the growth of the larva the greater part of the
larval mesenchyme is converted into the definite musculature
of the adult. The mesoderm bands in no instance give rise
to mesenchyme cells, and the two can be sharply distin-
gushed throughout the course of the larval development.

The development of Spirorbis borealis has been briefly
described by Schively (33). There is a very small blastoccel,
and the blastopore is a median ventral slit. It closes from
the posterior end forwards until nothing remains but a small
aperture at the anterior end, which becomes the future mouth.
The endoderm on invagination forms the archenteron. The
mesoderm can be traced to the left posterior macromere,
which sinks into the segmentation-cavity, giving rise by
a bilateral division to the primitive mesoderm cells. No
mention is made of the larval mesoblast.

The early development of Serpula infundibulum has
been studied by Souler (38) in 1902. The main outcome of
his work has been to confirm very closely Hatschek’s results
for Eupomatus. The mesoderm cells are recognisable as
two large cells in the endoderm at the time of invagination.
They arise at the point of union of ecto- and endoderm, and
pass into the segmentation-cavity, where they give rise to
the mesoderm bands. heir relation to the irregular cells of
the mesenchyme was not determined.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 563

Apart from the Serpulids, the development of Thalas-
sema, Podarke, and Polygordius closely resembles that
of Kupomatus in its essential features. The cleavage in
these is of the equal spiral type of that of Eupomatus, and,
in fact, the early cleavage of Podarke, Thalassema, and
Kupomatus are almost similar cell for cell until the time of
gastrulation. In each the gastrula is formed by invagina-
tion, and a well marked blastoccelic cavity is present. In the
later stages of gastrulation Thalassema and Poly gordius
more closely approach KEupomatus than does Podarke.
This is possibly due to the fact that the trochophore ot
Podarke is somewhat modified, apparently not having any
head-kidney. Polygordius, with its large blastopore,
represents possibly a more primitive condition than do
the others. Thalassema in the pre-trochophoral stage
approaches nearer Eupomatus than do the others, for in
Polygordius the head-kidneys form some time before the
mesoderm bands. The details of the resemblance between
these four will be considered further on.

3. MarertaL AND MeruHops.

When the sexual products are ripe in Eupomatus it is an
easy matter to distinguish the sexes from the colour of the
body. The female is bright yellow, while the male is white.
In Naples they grow in dense colonies attached to stones,
the sexes being evenly proportioned, although the males and
females show a slight tendency to occur together in separate
spots inthecolony. Their tubes stand upright, being attached
by one end.

In effecting fertilisation under artificial means, it is
unnecessary to wait until the eggs are deposited as in many
Annelids, as Nereis, Podarke, Phyllodoce. ‘The ripe
eges cut from the body-cavity fertilise as readily as those
laid in the normal manner.

The egg of the Neapolitan Eupomatus seems to be more
opaque than that studied by Hatschek at Trieste, for I have

564. CRESSWELL SHEARER.

been unable to follow the fate of the invaginated cells during
gastrulation in surface views of the living egg as he was
able to do. In the following work I have relied entirely on
the evidence of sections. By means of the combined celloidin-
paraffin method of embedding, one is able to obtain aood
sections of small gastrule such as these. For fixing I have
found sublimate acetic and Flemming solution give satis-

TExt-FieG. 19.

Section of early trochophore of Eupomatus. cw. m. Colomeso-
blast. mese. Mesenchyme. mm. Mouth. sfin. Stomodeum.

factory results. From these sections I have followed the
formation of the gastrula cell by cell. No mistake can be
made, therefore, in the position of these cells, as is frequently
done in the study of surface views alone, and one does not
vet flattening and distortion from the pressure of the cover-
alass, as is invariably the case in the study of living prepara-
tions. The fertilised egg measures about 55 4 in diameter.
‘he eggs laid under normal conditions are almost spherical,

TROCHOPHORE OF HYDROIDES UNCINATUS (EBUPOMATUS). 565

but those obtained from the body-cavity are always flattened
and lenticular in shape. On being placed in sea-water, after
a short time they fill out and become spherical and regular
in outline. They are covered by a thin membrane which
remains attached throughout segmentation and gastrulation,

Trxt-Frie. 20.

Section through a trochophore of Hupomatus three days old.
an.v. Anal vesicle. ce.m. Colomesoblast. hk. Head-kidney.
stme. Stomach,

becoming the cuticle of the trochophore. This in the living
egg is smooth and transparent, but shrinks and becomes con-
siderably wrinkled under the reaction of reagents, especially
sublimate. This renders the study of fixed material difficult,
as the cuticle has a strong affinity for stains, obscuring the
underlying cells and adding to the uncertainty of orientation,

566 CRESSWELL SHEARER.

This cuticle has been noticed by Stossich (39), Conn (9), and
Hatschek (17) ; the first of these investigators observed that
it became the cuticle of the larva. A similar though some-
what thinner membrane surrounds the eggs of Podarke.
In Serpulaitis even thicker than in Eupomatus, where
at the animal pole it leaves quite a space surrounding the
polar bodies. A smaller space is found in Hupomatus, in
which two dark polar bodies are seen. There is no micropyle,
and the sperm seems to be able to penetrate the membrane
at any point.

It must be remembered that the type of cleavage of such
widely separated forms as Hydroides, Thalassema,
Podarke, and Lepidonotus resemble one another on
account of their possession of a trochophore. ‘They all
possess a free-swimming stage of considerable duration, and
as the initial size of the blastomeres stands in direct relation
to the size of the part to which they give rise, as pointed out
by Lillie (25), the resulting cleavage conforms to the same

type.

4. SEGMENTATION AND GASTRULATION.

Segmentation begins about one to two hours after the sperm
have been added to the eggs. The rate of development
naturally varies greatly, being increased with any rise and
decreased with any fall in the temperature. With the hot
weather in Naples during the summer months, development
quickly becomes abnormal unless precautions are taken to
keep the water cool in the culture dishes. Segmentation 1s
rapid and regular once it has set in, and results in a blastula
containing a segmentation-cavity of variable dimensions. It
is of the equal type, and resembles very closely that of the
Anunelid Podarke, which is remarkable for the fact that
the spiral symmetry is retained almost complete up till an
unusually late stage. The first cleavage furrow cuts through
the egg, sinking in more rapidly at the upper than at the lower
pole, and produces the two-cell stage. The first cleavage is

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 567

exactly equal; the two nuclei of the cells are opposite one
another, and show no.tendency to rotate as Conklin (7) has
described in Crepidula. The subsequent divisions follow
in rapid succession. With the third cleavage four slightly
smaller upper cells are separated by a dexiotropic division
from the lower macromeres. In the fourth cleavage the
micromeres of the second group are of the same size, and
are very slightly smaller than the macromeres.

Invagination produces a typical gastrula. Gastrulation
usually commences about seven or eight hours after fertilisa-
tion, and consists of a sinking in of the ventral ectoblastic
plate, all the entomeres of which are alike during the early
stage of the process. Gastrulation is of the modified embolic
type, with considerable preparatory flattening of the ventral
plate. The cells about to sink in elongate, and their nuclei
take up a position at their inner swollen ends. While this
flattening is taking place the apical portion of the gastrula
is rounding out, the apical tuft of cilia commences to
appear, and the endoderm cells sink in till they come in con-
tact with the inner wail of the ectoderm, in the region of the
“rosette cells.” At first there is a complete obliteration of
the segmentation - cavity, the endoderm folding up close
against the ectoderm; but in the immediate filling out of
the gastrula, which takes place almost simultaneously, the
ectoderm is again drawn away and the segmentation-cavity
reappears (Text-fig. 18). At this stage a number of viscid
protoplasmic threads are seen connecting the two layers, and
one blastomere with another. They have been observed in
Podarke by Treadwell (48),in Serpula by Soulier (38), and
in Thalassema by Torrey (41) ; I have already drawn atten-
tion (85) to them in Kupomatus, and pointed out that they
are probably similar to the filose strands first described by
Andrews (2), and considered by him as cell connections.
Prof. Loeb has suggested to me, however, that they are
rather more in the nature of the fine cytoplasmic strands so
frequently seen in membrane formation during fertilisation
than definite cell communications.

568 CRESSWELL SHEARER.

The blastopore at first hes exactly in the middle of the
ventral plate, and is marked out behind by two large cells,
which, as in Nereis, probably belong to the X group (fig. 11).
When fully formed it is an elongated slit, somewhat enlarged
at its anterior end. ‘This end never completely closes, but
after the formation of the stomach becomes the future mouth.
The posterior portion closes completely, the anus breaking

TExt-Fies. 21 AND 22.

Sections through early trochophores of Kupomatus. g. Gut.
es. (isophagus. m. Mouth. hk. Head kidney. ce@.m. Ceelo-
mesoblast. stme. Stomach.

through almost immediately at the point where the last portion
of this part of the blastopore disappears. ‘Thus the closure of
the blastopore in Eupomatus is essentially the same as in
Polygordius, although the different steps in the process are
not so evident. Inthe majority of Annelids the blastopore
usually closes completely, as in Capitella.

>. Tae EcromMrsoBLasr.

Towards the end of gastrulation some irregular cells are

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 569

seen in the segmentation-cavity. Their origin I have not
succeeded in observing. They are shown in Text-fig. 18,
Imr. and Iml. I believe they arise from cells of the third
quartette, but as I have not followed the cell-lineage care-
fully, Iam by no means certain of their exact origin. They
sink into the cleavage-cavity during gastrulation, and take
up a bilateral position on either side of the blastopore, as
shown in Text-fig. 18. They immediately divide, giving rise
to some irregular small cells that apply themselves closely to
the wall of the stomodzum, and later form larval muscles.
One large cell on either side gives rise to a string of cells,
which enter into close relation with the ecelomesoblast. From
their mode of origin and their subsequent behaviour I think
there can be no doubt that they represent the larval or
ectomesoblast of Podarke and Thalassema. In addition
to these cells, some mesenchyme cells are also constantly seen
in slightly later stages (figs. 9 and 10) in the apical region
under the “cross-cells.”. Whether they arise by migration
of some of the cells from the stomodeum, or by the sinking
in of ectoderm ceils in the apical region, which last I think
is more likely, I have not determined. As in Podarke and
Thalassema and molluscs, therefore, the larval mesoblast
can be divided into the median, the portion under the apical
organ, and the right and left portion on either side of the
blastopore. These cells (Jr. and Jml. of Text-fig. 18)
would correspond with the right and left parts of the ecto-
mesoblast of Podarke and Thalassema.

It is worth repeating the description of these structures in
these forms. In Thalassema, Torrey (41) states, “The
most important source of functional mesenchyme, in Thalas-
sema, are the three cells*from the third quartette, namely,

=

$05 644, 2 95, aud and 3a,,,. ‘The first two sink into
the cleavage-cavity, just before gastrulation, and lie at first
close to the ccelomesoblast cells. They soon migrate laterally,
and bud off simultaneously small cells towards the mesoblast
cells, dividing like teloblasts, but in the reverse of the

ordinary direction. So close is the connection of these cells

570 CRESSWELL SHEARER.

with the ccelomesoblast (see Text-fig. 9, Zml.) that one would
certainly be led to think that they formed part of these bands,
unless their cytogeny had been carefully followed” (p. 223).
‘hey have been described as follows in Podarke by 'l'read-
well (43). They arise as in Thalassema from the 3d, 3e,
and Ja, and sink into the segmentation-cavity, where they
arrange themselves symmetrically, forming bands of three or

TEXT-FIG. 23.

Oblique corneal section through early trochophore of Eupomatus.
Lettering as in fig. 22.
more cells. “ Since the posterior end of each band lies very
close to the definitive mesoblast, the effect is that of a well-
developed mesoblast band, lying in the usual position in the
segmentation-cavity ” (p. 427).

The median portion of the ectomesoblast in NHupomatus
retains its position untransformed into larval musculature
until a very late stage, when the trochophore becomes seg-
mented. Itis shown under the apical organ of the early
trochophore in figs. 9,10, 16 (mesc.). In the fully formed

trochophore it is shown in figs. 2, 3, and 6 (mesc.).

WROCHOPHORE OF HYDROUDES UNCINATUS (EUPOMATUS). 571

I will now describe in detail the changes undergone by
these cells. Ina late gastrula stage such as that shown in
this text-figure (Text-fig. 18) these cells have already divided ;
the division usually is an unequal one, in which one of the
daughter-cells is much smaller and moreirregular in shape than
the other. They seldom divide simultaneously on both sides,
but the right. usually precedes the left. If we refer to fig. 9,
we see the larger of these cells attached to the ventral wall
of the oesophagus (lk.). The smaller seems to give rise to
some of the mesenchyme cells that are attached to the wall
of the cesophagus. These are very irregular in shape and
size. At this early stage they are only seen with difficulty,
as they are few in number, and are closely pressed against the
surface of the cesophagus. Although the stage represented
in fig. 9 has already assumed the shape of the early trocho-
phore, it is but shghtly older than the late gastrula stage
represented in Text-fig. 18.

‘he head-kidney strand is derived from the division of the
large cell (hk.) seen in fig. 9. This divides once, and then by a
second division of one of the daughter-cells a band of three
cells is formed (Text-fig. 23). The nuclei of these cells so
arrange themselves that two remain in the end of the strand
attached to the cesophagus, while one moves to the distal
end, which abuts against the anal endof the gut. This stage
is represented in fig. 12. In fig. 10 the head-kidney cell has
divided, forming two daughter-cells, one of which is apphed
close to the wall of the cesophagus, while the other rests
against the inner lower surface of the larval hemisphere.
In fig. 12 the two distal nuclei of this band have moved
apart, one resting against the anal end of the gut, while the
other remains close to the cesophagus.

In Text-figs. 21 and 22 the strand of cells forming the
head-kidney is shown in sections. In Text-fig. 21 (coe. m.) is
shown part of the nucleus of the ccelomesoblast cell. This
figure is aluiost in the median plane, while the plane of section
of Text-fig. 22 is quite oblique, showing only a portion of the
stomach and gut-wall. The cell boundaries disappear, so

VOL. 56, PART 3.—NEW SERIES. 39

572 CRESSWELL SHEARER.

that the head-kidney strand consists of a thin thread of
cytoplasm, at either end of which are the nuclei. A fine
lumen begins to appear in the middle about the second day ;
this increases in size and works its way towards either end,
and by the middle or end of the third day the organ becomes
functional as the head-kidney, having acquired an opening

into the proctodzum.

6. THe CaLoMESOBLAst.

Towards the end of gastrulation, and after the period when
the ectomesoblast has already appeared in the blastoccel, two
large cells are seen side by side in the ventral lp of the
blastopore. In surface views they seem to lie more in the
ventral ectodermic plate than in the endoderm. From
sections, however (Text-fig. 17, me.), they are seen to be part
of the endoderm at its point of junction with the ventral
plate. They are not free in the segmentation-cavity, and
during the course of invagination they come to lie in the wall
of the primitive archenteron. They finally rest in the anal
end of this structure, where, at much later stages, by a series
of rapid divisions, they give rise to a number of cells which
push out into the blastoccel and form the mesoderm bands.
They are, therefore, the coelomesoblast cells.

In the stage represented in section in Text-fig. 17 they are
usually seen in the ventral hp of the blastopore undergoing
division. The fate of the smaller of the resulting daughter-
cells I have been unable to determine, but I believe they
represent the small cells forming part of the wall of the
archenteron in Podarke. ‘The larger of the two cells
becomes the ccelomesoblast. As development advances they
are carried back in the wall of the archenteron, and do not
lie free in the blastoccel till a later stage. In late stages
they are seen in the anal end of the archenteron as in Text-
fies. 19-25 (cw. m.); here they always project slightly from
the gut-wall.

After their division, as shown in Text-fig. 17, the various

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 573

steps by which the larger of the two cells is shifted back
into the anal region are somewhat difficult to follow. Some-
times they do not appear to differ greatly from the surround-

TExT-FIG. 24.

Diagrammatic figure of an early trochophore of HKupomatus
before the formation of the mesoblast bands, and showing the
opening of the head-kidney into the proctodewm. ce@.m. Ccelo-
mesoblast. an. Anus. an.v. Anal vesicle. mesc. Mesen-
chyme or ectomesoblast. hk. Head kidney. m. Mouth. stme.
Stomach.

ing cells, but they can usually be distinguished by their
greater affinity for stains; and in late stages they can
always be recognised by the way they are wedged into the

O74. CRESSWELL SHEARER.

gut wall above the anal vesicle. Close examination of the
sections shows them first as two cells in the ventral wall of
the stomach, and then the gut. The change that has to do
mostly with bringing this about is the great increase in the
dorsal surface of the gastrula and the consequent narrowing
of the blastoporal surface, changing the large ventral to a
small ventro-lateral surface. At a time when the anal open-
ing of the gut has not been established they occupy about the
mid-region of the archenteron. At the period when the anus
breaks through they have already moved into the anal end.

The blastoccel during this time is still small, and has not
undergone the great increase it shows shortly after this
period, as only a trace of it can be seen between the gut and
the ectoderm. This adds somewhat to the difficulty of deter-
mining how the various steps in the process take piace. The
primitive trochophore about this time begins to assume its
typical shape ; up to this the round shape of the gastrula has
been retained. During early gastrulation before the division
of the ccelomesoblast cell, as shown in Text-fig. 17, I have
been quite unable to distinguish it from any of the other endo-
derm cells. No conspicuous cell is seen forcing its way into
the segmentation-cavity as shown by Hatschek (17) and Souler
(38), and I believe that both these investigators have been
mistaken in their identification of the ccoelomesoblast cell.
The cell shown in Hatschek’s figs. 25-56, and in Soulier’s
figs. 25-27 and 33 and 34, and identified by them as the
ccelomesoblast, are really the right and left portions of the
ectomesoblast. At a later stage they give rise to the head-
kidneys. The real coelomesoblast at this period still les in
the gut-wall, and not free in the blastoccel.

In the late gastrula stages the right and left portions of
the larval mesoblast appear as shown in Text-fig. 18. In all
respects these cells answer to the mesoderm cells of Hatschek’s
figs. 25-37, fio. 9 of this paper corresponding ue Hatschek’s
fic. 33. By a comparison of figs. 9, 10, 12, 13, 15, 16, the
various changes will be seen by which these cells are trans-
formed almost entirely into the head-kidneys. In fig. 16 the

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 575

mesoblast bands have not appeared. In the young trocho-
phore shown in fig. 1. they are just appearing as they grow
out from the gut-wall. As these cells just mentioned are
converted into the head-kidneys before the mesoblast bands
have appeared, it is fair to assume that they do not represent
the ccelomesoblast cells as Hatschek and Soulier claim. It
must also be recalled that both these investigators have not
followed the cell lineage, and therefore they have no definite
grounds of cytological importance on which to substantiate
their claim as to the nature of these cells.

In the early stages of invagination it is certain that the
coelomesoblast cells cannot be distinguished, as these investi-
gators state, by their conspicuous size and the manner in
which they force their way into the segmentation-cavity. In
fact, I have been only able to distinguish them satisfactorily
in early stages by following their development backwards
from a stage when these are clearly recognisable in the anal
end of the gut to a stage towards the end of gastrulation ;
prior to this I cannot see that they differ from any of the
other endoderm cells.

In fact, the condition in Hupomatus is simply a more
marked type of that found in Podarke. In this Annelid,
according to ‘Treadwell (48), at the sixty-four-cell stage the
fourth group of micromeres have just formed. ‘They are all
alike, but shortly one of them divides bilaterally, thus
aiding substantially im the establishment of the bilateral
symmetry. ‘Then each buds off a small cell ventrally ; these
small cells form a part of the wall of the archenteron.
During the course of gastrulation the mesoblast cells lie in
the wall of the archenteron, with which they are carried
inwards, finally coming to he in the anal region. They
protrude considerably, and in sections that pass (Text-fig. 6a)
a little to one side of the sagittal plane they seem to lie
actually free in the segmentation-cavity.

The condition in Podarke and HKupomatus, again, is
only a more marked state of that found in Crepidula,
where the greater portion of the primary mesoblast cell

576 CRESSWELL SHEARER.

contains endoderm, remaining a mesendoblastic cell for eight
divisions before the mesoblastic is separated from the endo-
dermic portion. he endodermic part enters into the wall of
the archenteron. In Eupomatus and Podarke the greater
part of the mesoblast cell (4d) is mesoblastic, while in Crep1-
dula only a small part of it is mesoblastic. In Amphitrite,
Mead (26) represents the mesoderm cell similarly giving off
a small cell. The spindle of this division, as in Hupomatus,
lies in the short diameter of the cell, which at this moment is
compressed between the ventral wall of the ectoderm and the
main mass of the invaginated endoderm. ‘The axis of the
spindle is in the direction of greatest pressure.

In Thalassema the mesoblast cells, which at first are
pressed together under the ventral lip of the blastopore,
separate and move apart towards tbe sides, lying well up
towards the prototroch (Text-fig. 9, me.). As they move
apart they divide rapidly, each giving rise to a group of five
or more cells, which form the mesoblast bands as in Annelids.
They are quite free in the blastoccel, and enter into close
relation with the right and left portions of the larval mesoblast,
from which they can be distinguished, as in Hupomatus,
by their different stainime reaction. ‘hus Thalassema
represeuts a condition midway between that of Hupomatus
and Physa and other Molluscs, where the mesoderm cell
lies free in the blastoccel from the time of invagination.

In Polygordius I have shown (87) that the head-
kidneys form early and before the mesoderm bands have
appeared; that the rudiments of these organs are first
recognisable as two cells in the ventral plate of the ecto-
derm. They grow out into the blastoccel, and by division
give rise to a string of cells, as in HKupomatus, that run up
to the cesophagus. They fuse together and become one strand
of cytoplasm, with three or more nuclei. ‘This then hollows
out, develops a flagellum, and becomes functional as a head-
kidney, at an age when the mesoderm bands are represented
by a few cells on either side of the anal opening.

I have advanced reasons for believing that the head-kidney

TROCHOPHORE OF HYDROIDES UNCINATUS (BUPOMATUS). 577

strands in Polygordius are in many ways comparable to the
lateral portions of the larval or ectomesoblast of Thalassema
and Annelids. The condition in Polygordius, where the
ectomesoblast arises and becomes functional so much earlier
than the ccelomesoblast, shows that the head-kidney strands
do not form from the bands, and this point is borne out by
the cell-lineage as worked out by Woltereck (52), In
Kupomatus the formation of the ccelomesoblast follows so

TEXT-FIGS. 25 AND 26.

i es. mesc.
\ s —

toy , O =
| \ Q) sof s: en eae Tese.

i fey roc tkle RE Wy” a * go f z
’ piace eer a /_ 4 is
Caen: S48 >---C0e Mm: y Pa vi}
3
|
|

Sections through the anal ends of early larve of Eupomatus.
an. Anus. ai.v. Anal vesicle. c@.m. Cclomesoblast. hk.
Head kidney. ect. Ectoderm. g. Gut. os. Cisophagus.
closely on that of the ectomesoblast that this difference is not
so marked.

To sum up: the ccelomesoblast in Hupomatus is not
recognisable until a relatively late stage in gastrulation,
and the cells described by Hatschek and Soulier as the
mesoderm cells are probably portions of the larval or ecto-
mesoblast. At the time the ectomesoblast is represented by
two cells on each side of the mouth, the ccelomesoblast is
represented by a cell in either side of the gut-wall above

578 CRESSWELL SHEARER.

the proctodeum. Only in the trochophoral stage does the
ccelomesoblast divide, giving rise to the mesoblast bands,
which gradually grow up the head-kidney ducts to the region
of the cesophagus. There is relatively a considerable period
during the trochophoral stage, when the larva is without
mesoderm bands, and the rudiments of the bands are repre-
sented by a single cell on either side of the gut-wall in the
anal region.

7. Tue Earty TrocHopHoraL StraGes.

In part the early trochophoral stages have been considered
in the foregoing section. Before the completion of gastrula-
tion the larva begins to assume the shape of the trochophore.
Figs. 9, 10, 12, 18, 15 and 16 show the shape of the early
larvee ; of these probably fig. 12 is the most typical. In these
figures the upper and lower larval hemisphere is dome-shaped
and rounded, as compared with the pointed and more conical
appearance of the mature larve shown in figs. I, 2, and 3.
The apical cilia, cilia of the mouth, prototroch and paratroch,
are, for the sake of simplicity, not shown in these figures,
which are drawn from fixed material, and are therefore more
granular looking than the living larve. ‘These stages are
derived from the gastrula about the twentieth to the thirtieth
hour of development. At this time there is a great thinning
out of the tissues, and the larva rapidly increases in size. In
the region of the prototroch a very active proliferation of the
cells is taking place, by which the gastrula is lengthened out
into the conical dome-shaped larva. The primitive archen-
teron becomes sharply divided into the cylindrical esophagus,
cubical stomach, and narrow gut. ‘The cells of its walls are
seen dividing rapidly. ‘The inner surface of the cesophagus
secretes a cuticle, as in Thalassema (Text-fig. 19). The
archenteron is lined throughout with strong cilia. Those of
the cesophagus are remarkably long and powerful. The inner
wall of the stomach is covered uniformly with fine cia, which
keep the food contents in constant motion. The cilia of the

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 579

gut are somewhat longer and more powerful than those of the
stomach. Immediately above the proctodeeum the lumen of
the gut is narrowed down by a projecting ridge. This is
well shown in the trochophore of Hydroides pectinata
(fig. 18). Below this constriction the gut opens into the
proctodzeum, which, like the stomodzum, also secretes a fine
cuticle. At first the cells of the archenteron are uniformly
cubical in appearance, but those of the cesophagus and the gut
soon thin out, while those of the stomach alone retain their
primitive appearance.

The larva at this time has the shape represented in figs.
9, 10, and 12. The anal vesicle begins to appear as a small
vacuole in one of the ectoderm cells of the anal region. ‘This
at first connects with the exterior by a small duct, but this
soon closes, and the vesicle increases rapidly in size. The
cytoplasm of the cell stretches so that a thin envelope alone
is left which surrounds the vesicle. It then becomes con-
stricted into two portions, as shown in fig. 14. The original
nucleus of the cell is seen projecting into the blastoccel from
the upper wall of the vesicle.

On either side of the gut, just above the anal vesicle, a
large conspicuous nucleus is seen embedded in the wall.
This is the nucleus of the ccelomesoblast cell. In the stages
represented in figs. 9 and 10 it is not so prominent as in the
later stages shown in figs. 12, 15, 15, and 16. As develop-
ment proceeds it is pushed out more and more into the
blastoceel. In Text-fig. 19 it appears to be free in the
blastoccel, but examination of the subsequent sections of
this series clearly shows it to lie in the gut-wall. As I have
mentioned, it is of somewhat different staining reaction to
the surrounding cells, and this contrast is shown somewhat
in this text-figure, which is from a camera drawing of an
actual section. ‘The section passes a little to one side of the
median line, and is slightly oblique, as the mouth and
cesophagus are cut in the median plane, while the section
passes through the lateral wall of the stomach and the gut.
In Text-fig. 20 is shown a section of an older stage in which

580 CRESSWELL SHEARER.

the head-kidney has formed, and the mesoderm cell is seen
wedged in between the anal vesicle and the head-kidney.
The growth of the bands from these cells is not that of a
true teloblastic one; when the ccelomesoblast cells start to
divide they do so quite irregularly. The bands at first consist

TEXxT-FIG. 27.

ws

> oe Ne ef arN,.

Section through trochophore of Eupomatus older than those of
the foregoing figures. an.v. Anal vesicle. ca.m. Coelomeso-
blast. g. Gut. hk. Headkidney. mesc. Mesenchyme or ecto-
mesoblast. stis. Stomach.

of groups of three or four cells; they divide in all directions,
so that after the first division it is not possible to speak of a
pole-cell, the divisions always being equal. Hatschek’s rather
elaborate account of the origin of the bands by teloblastic
growth conveys quite an erroneous impression of the process.
The ccelomesoblast cell first divides into two equal cells, and

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 581

these, again, in turn divide equally. One cell remains attached
to the gut-wall, as shown in a late stage in fig. 17 (pm.),
but this cell does not divide in this stage, and the growing
point of the bands is not here, but towards the ends nearest
the ceosphagus. The position of this cell alone gives it the
appearance of being a _ pole-cell.

According to Wilson (48) there is a complete absence of
teloblastic growth in the species of Hydroides studied by
him, for he states: ‘I have carefully studied the develop-
ment of Hydroides dianthus (a form nearly allied to
Kupomatus) by following the cleavage of the living ovum,
by examination of stained and cleared embryos, and actual
sections. ‘he cleavage is in every detail identical with that
of Kupomatus, the gastrulation takes place in essentially
the same manner, and the trochophore is of quite the same
type. Yet I have been unable to identify the teloblasts at
any period. ‘They are certainly not present at a stage when
the mesoblast bands consist of not more than four or five
cells each. At this period each band ends posteriorly in a
group of about three cells, two of which are not perceptibly
larger than the others, are jomed by a narrow bridge of
protoplasm stretching across in the angle between the procto-
dzeum and the wall of the anal vesicle” (p. 215).

In Thalassema, Torrey (41) has not been able to find a
teloblastic growth of the bands. “Itis a fact,” he says, ‘‘as
far as 1 know, without exception, that in all forms where
there is a trochophore stage of long duration (as in the case
of all Annelids with equal cleavage), the two ccelomesoblast
cells do not, in the earlier stages at least, bud like teloblasts”
(p. 222).

As the bands grow out from the gut-wall in Eupomatus,
they keep quite apart from the mesenchyme cells of the
blastoccel, nor have I been able in any of the stages I have
studied to observe the origin of these cells from the ends
of the bands. ‘his is a very debated point in Annelid
embryology. Are not the numerous mesenchyme cells of the
blastoccel in part derived from the ends of the bands? So

or
ice)
i)

CRESSWELL SHEARER.

TExT-FIG, 28.

Head-kidneys and mesoblast-bands in a late larva of Hupomatus.
ce.m. Ccelomesoblast. hk. Head kidney. os. Césophagus.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS), 583

recent an investigator as Treadwell (48) is of opinion that they
have such anorigin. He, however, did not trace the bands in
Podarke beyond a stage when they were represented by a
few cells, so he obtained no definite information on this point.

In Kupomatus itis clear that the anterior ends of the
bands never give off cells into the blastoccel as Hatschek
has described. They can be plainly observed throughout the
course of their growth; they are always a compact mass of
cells, clearly distinct from the larval mesenchyme. The larval
mesenchyme cells enter into close relation with the cells of
the bands, as may be seen in Text-figs. 25 and 26, and in part
overgrow them, but even in the living condition they can
usually be distinguished. In sections in which the fixation
has been rapid they can readily be separated on account of

their different staining properties—a point that has been exten-
sively used by Meyer in his numerous studies on this question.

In Eupomatus a large part of the larval musculature has
already been laid down before the formation of the bands has
taken place, the greater part of this musculature persisting
and ultimately forming a very considerable portion of the
adult body.

Meyer (27) has criticised Hatschek’s statement regarding
the origin of the mesenchyme in Kupomatus, and has
expressed himself as being very sceptical as to whether cells
arise from the anterior ends of the bands. He is of opinion
that, with more modern technique than that employed by
Hatschek, whose observations were restricted to living
material and optical sections, the facts of the case will prove
different. He points out that while Hatschek describes the
ccelomesoblast pole-cells as giving off cells into the blastoccel
before they form the bands, he neither figures nor appears to
have seen the division of these cells. Torrey (41), in speaking
of the resemblance of the ectomesoblast in Podarke and
Thalassema, says, “ The striking similarity in the origin of
the ectomesoblast in these two forms justifies us, I believe,
in supposing that we may have the same condition of affairs
in Eupomatus where the cleavage is also equal ” (p. 226).

584. CRESSWELL SHEARER.

From 'Text-figs. 27, 28, which represent sections through
the growing bands and head-kidneys of the four-day trocho-
phore, it will be seen that there are numerous mesenchyme
cells about the head-kidneys which could hardly have arisen
from the ccelomesoblast cells (c@.m.), which, moreover,
show no evidence of having recently divided. I have
examined a large number of such sections without observing
in a single instance the division of these cells to form mesen-
chyme.

Treadwell (48) holds the view that this separation of the
mesoderm in Annelids into apparently distinct portions is
only a mechanical result of development, but the varied con-
ditions under which a larval mesenchyme is present in Annelids
seems to me to be against this view. ‘Treadwell (48) has
pointed out that we are forced to believe in two non-homolo-
gous sets of larval mesenchyme, the one arising from the
ectoderm as in Thalassema and Podarke, and the other
from the anterior ends of the germ-bands, as in Nereis and
Lumbricus. These two sets do not, as arule, exist together.
“On the other hand,” he says, ‘no one has proved, as far as
I know, that no ‘mesenchyme’ arises from the germ-bands in
cases where a larval mesenchyme exists.” I have attempted
to show that in Kupomatus, where a larval mesenchyme
exists, no evidences of its origin from the bands can be
observed, and the main result of my work has been to empha-
sise the distinction between ecto- and ccelomesoblast. I have
already considered in the “Introduction” whether we are
justified in laying any stress on this point. In Annelids we
are at least certain that this separation seems general and
definite.

SuMMARY.

Segmentation results in a round blastula with a very
reduced blastocele. Invagination produces at first an almost
spherical gastrula. But this soon begins to assume the conical
shape of the early trochophore. The blastopore, which is

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 585

small, closes from behind forwards, the anterior portion
remaining as the mouth, while the posterior closes completely,
the anus breaking through immediately at this point. The
blastopore, which was originally ventral, becomes shifted to
a ventro-lateral position. At a time when gastrulation is
about half completed, some cells appear on either side of the
endoderm and take up a bilateral position. They probably
correspond to the lateral portions of the larval or ectomesoblast
of Thalassema. ‘They subsequently form the head-kidneys
in Kupomatus. At the same time two conspicuous cells are
usually distinguishable in the ventral lip of the blastopore.
These are the ccelomesoblast cells. In the further progress of
invagination, they are carried inwards in the wall of the arch-
enteron, finally coming to lie in the anal end of the gut. Here
at a considerably later stage they give rise to the mesoderm
bands. There is a short stage in the early trochophore when
the head-kidneys are already functional while the mesoderm
bands are alone represented by these two cells in the gut-
wall. With the formation of the bands the organisation of
the trochophore is completed. The bands during their
growth are never seen to bud off cells into the blastoccel.
They remain from the first a compact mass of cells clearly
distinguishable from the irregular cells of the ectomesoblast
and the head-kidneys. The head-kidneys open into the
proctodeum. They are formed from the ectomesoblast.

LITERATURE.

1. Agassiz, A.—‘* On the Young Stages of a Few Annelids,” ‘ Annals
Lyceum Nat. Hist. New York,’ vol. viii, 1867.

2. Andrews, E. A.—‘‘ Filose Activity in Metazoan Eggs,” ‘ Zool.
Bullet.,’ vol. ii, 1898, p. 1.

8. Bergh, R. 8.—‘ Zur Entwicklung und Differenzirung des Keims-
triefens von Lumbricus,” ‘Zeit. f. wiss. Zool.,’ vol. 1, 1890,
p. 469.

4. Blochmann, F.—* Ueber die Entwicklung der Neritina fluvia-
tilis,” ‘Zeit. f. wiss. Zool.,’ vol. xxxviii, 1883, p. 392.

586 CRESSWELE SHEARER.

5.

10.

iT

12.

13.

14.

15.

16.

U7 fe

18.

22.

23.

Burger, O.—* Beitrage zur Entwicklungsgeschichte der Hirundi-
nean,”’ ‘ Zool. Jahrb., vol. iv, 1891, p. 677.

Child, C. M.—** The Early Development of Arenicola and Stern-
apsis,” ‘Arch. f. Entwickl.,’ vol. ix, 1900, p. 587.

Conklin, EK. G.—* The Embryology of Cre pidula,” ‘Journ. Morph.,’
vol. xiii, 1897, p. 1.

“The Embryology of a Brachiopod: Terebratulina sep-
tentrionalis,” ‘Proc. Acad. Nat. Sci. Phil.’ 1902, p. 41.

Conn, H. W.—** Development of Serpula,” * Zool. Anzeig.,’ vol. vil,
1884, p. 669.

Drasche, C. von.— Beitrige zur Entwicklung der Polychzten.”
I. ‘ Entwicklung von Pomatoceros triqueter, Wien, 1884,
IL. ‘Entwicklung von Sabellaria spinulosa, Hermione hys-
trix, und einer Phyllodocide, Wien, 1885.

HKisig, H.—‘ Zur Entwicklung der Capitelliden,’ *‘Mitth. a. d.
Zool. Stat. Neapel,’ vol. xiii, 1899, p. 1.

Fraipont, J.—** Le genre Polygordius,” ‘Fauna u. Flora Golf v.
Neapel,’ Mon. 14, 1884.

Hacker, V.—‘‘ Die spiitere Entwicklung der Polynoé-Larve,”
‘Zool. Jahrb., vol. xviii, 1895, p. 245.

* Pelagische Polychetenlarven,”’ * Zeit. f. wiss. Zool., vol. 1xii,
1897, p. 74.
Hatschek, B.—** Entwicklunesgeschichte von Teredo,” * Arbt.
Zool. Inst. Wien,’ vol. iii, 1881, p. 1.
“Uber Entwicklungsgeschichte von Echiurus,” * Arbt.
Zool. Inst. Wien,’ vol. iii, 1881, p. 45.
—* Entwicklung der Trochopora von Eupomatus wunci-
natus,” ‘ Arbt. Zool. Inst. Wien,’ vol. vi, 1885, p. 121.
“Studien iiber Entwicklungsgeschichte der Anneliden,”
‘Arbt. Zool. Inst. Wien,’ vol. vi, 1885, p. 199.

. Hertwig, O., and Hertwig, R.—‘ Die Celomtheorie,’ Jena, 1881.

. Heymons, R.—“ Zur Entwicklungsgeschichte von Umbrella

mediterranea,” ‘Zeit. f. wiss. Zool.,’ vol. lvi, 1893, p. 245.

. Kleinenbergh, N.—‘t Die Entstehung des Annelids aus der Larve

von Lopadorhynchus,’’* Zeit. f. wiss. Zool.,’ vol. xliv, 1886, p. 1.
Korschelt, E.,and Heider, K.—‘* Lehrbuch der verglichenden Ent-
wicklungsgeschichte der Wirbellosen Thiere,” ‘ Allgemeiner
Theil., Vierte Lieferung, 1910.
Kowalevsky, A.—‘* Embryologische Studien an Wirmer und
Arthropoden,”’ ‘Mem. Acad. St. Petersbourgh,’ ser. 7, vol. xvi, 1871.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 587

24. Lang, A.—‘ Beitrage zu einer Trochocéltheorie,” Jena, * Zeit. f.

25.

26.

Zit

28.

29.

30.

31.

Natwiss.,’ vol. xxxvili, 1903, p. 1.

Lillie, F. R.—** The Embryology of the Unionide,” ‘ Jown. Morph.,’
VOlex, [895, p.l:

Mead, A. D.—** Embryology of Marine Annelids,” ‘ Journ. Morph..,’
vol. xiii, 1897, p. 227.

Meyer, E.—* Die Korperbau der Anneliden,” * Mitth. a. d. Zool.
Stat. Neapel,’ vol. vii, 1887,-p. 592; vol. viii, 1887, p. 462; vol. xiv,
1901, p. 262.

Montgomery, T. H.—** The Modes of Development of the Mesoderm
and Mesenchyme,” ‘ Journ. Morph.,’ vol. xii, 1896, p. 355.

Roule, L.—* Etudes sur le développement des Annélides,” ‘Ann.
Sci. Nat.,’ vol. vii, 1889, p. 105.

Salensky, W.—‘ Etudes sur le développement des Annélides.” 1”
Pte. “Psygmobranchus.” 2” Pte. “Nereis.” ‘Arch. d.
Biol.,’ vol. iii, 1882, p. 345. “ Pileolaria, Aricia, Terebella,”
‘Arch. d. Biol.,’ vol. iv, 1883, p. 143. “Branchiobdella,” ‘ Arch.
d. Biol.,’ vol. vii, 1887, pp. 1 and 589. ‘ Ueber den Bau der
Echiuruslarve,” ‘Mem. Acad. St. Petersbourgh’, vol. xvi, 1905,
p. 1:

Saunders, A. M. Carr, and Margaret Poole.—‘* The Development of
Aplysia punctata,” ‘Quart. Journ. Micr. Sci.,’ vol. 55, 1910
p. 497.

32. Schimkewitsch, W.—* Zur Kenntniss des Baues und der Entwick-
lung des Dinophilus,” ‘ Zeit. f. wiss. Zool.,’ vol. lix, 1895, p. 46.

38. Schively, M. A.—‘ The Anatomy and Development of Spirorbis
borealis,” ‘ Proc. Acad. Nat. Sci: Phil.,’ 1897, p. 153.

34. Selys-Longchamps, M. de.—** Recherches sur les developpement des
Phoronis,” ‘ Arch. d. Biol.,’ vol. xviii, 1902, p. 495.

85. Shearer, C.—‘* On the Existence of Cell-communications between
Blastomeres,” ‘Proc. Roy. Soc. London,’ Ser. B., vol. Ixxvii,
1906, p. 498.

36. “ Studies on the Development of Larval Nephridia.” Part I,
“Phoronis,” ‘Mitth. a. d. Zool. Stat. Neapel,’ vol. xvii, 1906,
p. 487.

37. * Studies on the Development of Larval Nephridia.” Part IJ,
“Polygordius,” ‘Phil. Trans. Roy. Soc. London,’ Ser. B,
vol. exeix, 1907, p. 199:

38. Soulier, A.—* Les Premiers stades Embryologiques dela Serpule,”

‘Trav. d. Inst. d. Zool. d. Univer. d. Montpellier,’ Mem. ix, 1902,
pel:

VOL. 06, PART 3.—NEW SERIES. 40

Ot
CO
@'<)

CRESSWELL SHEARER.

89. Stossich, M.—‘ Beitrage zur Entwicklungsgeschichte d. Cheto-
poden,” ‘Sitzb. d. Acad. d. Wiss.,’ vol. Ixxvii, 1878.

40. Surface, F. M.—* The Early Development of a Polyclad, Planocera
inquilina,” ‘ Proc. Acad. Nat. Sci. Phil.,’ 1907, p. 514.

Al. Torrey, J. C.—“‘The Early Development of Thalassema,” ‘ Ann.
N.Y. Acad. Sci.,’ vol. xiv, 1903, p. 165.

42. Treadwell, A. L.—* Equal and Unequal Cleavage in Annelids,”
‘ Biol. Lect. Woods Holl.,’ 1899, p. 93.

43. ——— “The Cytogeny of Podarke obscura,” ‘Journ. Morph..,’
Vol. xvii, 19015 p: 399:

44. Vejdovsky, F.—‘ Entwicklungsgeschichtliche Untersuchungen,”
Prag, 1890-91.

45. Whitman, C. O.—‘‘ Embryology of Clepsine,” ‘Quart. Journ.
Mier. Sci.,’ vol. xviii, 1878, p. 215.

46. Wierzejski, A——‘‘ Embryology von Physa frontinalis,” ‘ Zeit. f.
Wiss. Zool.,’ vol. Ixxxiii, 1905, p. 592.

47. Wilson, E. B.—‘*The Germ-bands of Lumbricus,” ‘ Journ.
Morph.,’ vol. i, 1887, p. 183.

48. ——— * The Origin of the Mesoblast-Bands in Annelids,” ‘ Journ.
Morph.,’ vol. iv, 1890, p. 205.

49. ———“ The Cell-Lineage of Nereis,” ‘Journ. Morph.,’ vol. vi,
1892, p. 561.

50. ——— “Cell-Lineage and Ancestral Reminiscence,’ ‘Ann. N.Y.

Acad. Sci.,’ vol. xi, 1898, p. 1.

51. Woltereck, R.—* Wurmkopf, Wurmrumpf, und Trochophora,”
‘Zool. Anzg.,’ vol. xxviii, 1904, p. 273.

52. ——— “ Beitrige zur praktische Analyse der Polygordius,
Entwicklung,” ‘ Arch. f. Entwickl.,’ vol. xviii, 1904, p. 377.

TROCHOPHORE OF HYDROIDES UNCINATUS (EUPOMATUS). 589

EXPLANATION OF PLATES 21-23.

Illustrating Mr. Cresswell Shearer’s paper “On the Develop-
ment and Structure of the Trochophore of Hydroides

uncinatus (Eupomatus).”

LETTERING.

es, Gsophagus. an. Anus. an. v. Anal vesicle. ap. s. Apical
muscle-strand. 6/. Blastopore. ce. m. Colomesoblast. co. Otocyst.
e. Hye spot. ect. Ectoderm. end. Endoderm. g.Gut. hk. Head-kidney.
m. Mouth. mesc. Mesenchyme or ectomesoblast. oc. Otocyst. seg. e.
Segmentation cavity or blastoceel. Stm. Stomach.

BASE eek

Fig. 1.—Fully-grown free-swimming trochophore of Eupomatus
three days old. The mesoderm bands are just commencing to appear.
The head-kidney is shown opening into the proctodzeum while the closed
end is attached to the esophagus. This and the subsequent figs. 3, 7 and
8 are drawn from living larve compressed slightly under a cover-
glass.

Fig. 2.—Trochophore of Hy droides norvegica.

Fig. 3.—Trochophore of Eupomatus four days old, showing the
otocyst and mesoderm bands well formed.

Fig. 4.—Head-kidney in a three-day old larva of Eupomatus.

Fig. 5.—Head-kidney in larva of Hydroides norvegica.

Fig. 6.—Trochophore of an unknown Annelid (probably Hydroides
pectinata) from an outline drawing by Professor E. B. Wilson,
showing the opening of the head-kidney into the proctodzeum.

Fig. 7.—Trochophore of Eupomatus three days old. Seen from the
ventral surface, showing the junction of the head-kidney on one side
with the gut.

Fig. 8.—Trochophore of Eupomatus three days old seen from the
oral side. The head-kidneys are shown on either side running down to
open into the proctodxeum.

PLATE 22.

Fig. 9.—Whole preparation of a larva of Eupomatus twenty-four
hours old. In this and in the subsequent figures of this plate the cilia

590 CRESSWELL SHEARER.

on the external surface are not shown, for the sake of clearness. The
head-kidney cell is seen on the ventral side of the cesophagus. In the
apical region some ectomesoblast cells are shown.

Fig. 10.—Slightly older stage than that of the last figure. The head-
kidney is represented by a string of three cells.

Fig. 11.—External view of a late gastrula of Eupomatus showing
the portion of the blastopore that remains as part of the mouth.

Fig. 12.—Still later stage than that shown in fig. 10. This stage is
about thirty-six hours old.

Fig. 15.—Still later stage than the last.
Fig. 14.—Anal end of a young trochophore of Eupomatus showing
the double formation of the anal vesicle.

Fig. 15—Early trochophore of Eupomatus older than that of
fig. 15.

5
Fig. 16.—Early trochophore of Eupomatus forty-eight hours old.

The ccelomesoblast cell is seen in the wall of the gut above the anal
aperture.

PLATE 23.

Fig. 17.—Hydroides norvegica. The trochophore in this figure
is represented as tilted up and seen from the oral surface. The ccelo-
mesoblast is seen arising from two cells in the gut-wall dorsal to the
anal vesicle.

Fig. 18.—Hy droides norvegica. The lower portion of the trocho-
phore is shown under high magnification and slightly compressed under

the cover-glass. The opening of the head-kidneys into the proctodeum
is shown, and the ccelomesoblast.

Quart. Journ. of Microsc. Science Vol. 56.N.S.Pl. 21.

"SE Gate

Ith Aust v. EA Funke Leipzig.

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Quart. Journ. of Microsc. Science Vol. 56.N.8.P1L. 23.

C.S.del. Lith Anst v- EA Funke, Leipzig.

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 591

Studies in the Experimental Analysis of Sex.

By

Geofirey Smith, .A.,
Fellow of New College, Oxford.

(From the Department of Pathology, University of Oxford.)

Part 5.—On the Effects of Testis-extract Injections upon
Fowls.

In the ‘ Proceedings of the Royal Society of Medicine,’
vol. i, “ Pathology,” p. 153, 1907-8, Dr. C. E. Walker
describes an experiment in which he injected two adult
hens with extract of cock’s testis for a certain period, with
the apparent result that the combs grew very rapidly to quite
twice their original area. On ceasing the injections they
shrank gradually until they nearly reassumed their original
size. A further experiment is alluded to in which several
young hens of two months’ age were treated in the same
manner, and it is stated that, though the results differed
somewhat from the first experiment, they were entirely
satisfactory, but no further details, as far as I am aware,
have been given.

On the strength of the above experiments Dr. Walker
concludes, firstly, that the hen bird possesses the potentiality
of developing the comb as in the male; secondly, that there
is present some internal secretion in the testis which, when
injected subcutaneously into the hen, calls forth the produc-
tion of this and other secondary sexual characters proper to
the male, e. g. the wattles and temperament.

592 GEOFFREY SMITH.

This conclusion has been accepted, as proved by Dr.
Walker’s experiments, by a number of authorities on sex.

Since this conclusion, that the injection of testis extract
into the female calls forth the production of certain secondary
sexual characters of the male, is one of considerable theoretical
importance, and since it rests at present entirely on the experi-
mental evidence furnished by Dr. Walker, it appeared to me
desirable to repeat the experiment on a rather larger scale,
and to control the experiment with measurements on normal
hens. It may be at once stated that the result of this inquiry
has convinced me that the comb of the adult hen is usually in
a state of fluctuating growth, varying between wide limits,
and that this fluctuation is entirely uninfluenced by the injec-
tion of extracts of the cock’s testis. Out of nine birds injected
only one showed fluctuations in growth which fell outside the
variability of the control birds, the other eight giving abso-
lutely negative results. In the course of the experiments I
also tested the influence of the injections upon the fertility of
the eggs and upon the properties of the blood-serum of the
injected birds.

The whole of the experiments have been done in the Depart-
ment of Pathology, Oxford, under the supervision of Prof.
Dreyer, to whom I tender my most hearty thanks for the help
he has given me.

1. MeruHops EMpLoyeD.

As I was desirous of repeating Dr. Walker’s experiments
in the same manner as he performed them, the extract was
made by crushing up the fresh testes of a cock with twice their
weight of sterile saline and straining the emulsion through
gauze. In this way everything except skin and connective
tissue passes into the extract, which forms a fine emulsion.
Dr. Walker injected the hens with °5 c.c. of such an emulsion
every day. I have used various methods, in certain cases
injecting the birds with greater amounts, up to as much as
10 c.c., at intervals of a few days, in other cases injecting

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 593

them every day with about 3 c.c. In all cases rather more
extract was administered in my experiments in a given time
than in Dr. Walker’s. Since Dr. Walker obtained pronounced
effects in three weeks, and very pronounced effects in less
than two months, I have not continued the injections for
more than a month except in a few cases. That this does not
vitiate my results is, however, most clearly shown by the fact
that in the two birds in which very marked variations in the
comb were observed, comparable to Dr. Walker’s, the full
increase took place three weeks after the first injection, the
injections being performed at intervals of two or three days.
The injections were made with aseptic precautions in the
pectoral muscles. In measuring the comb two methods have
been used: firstly, by tracing an cutline of the comb onto
cardboard, and secondly, by measuring the two greatest
dimensions of the comb directly with a pair of compasses.
Both methods were used with the four birdsin Experiment 1,
but the numbers given in referring to this experiment in the
schedule at the end were all taken from the tracings, as the
direct measurements did not form a complete series for all the
birds. As a consequence of this the numbers referring to
comb measurement in these birds do not vary so smoothly as
in the case of the later experiments. In Experiments 2 and
3, relating to fifteen birds, I relied entirely ou direct measure-
ments, which I consider liable to less experimental error.
Measurements were not made of the wattles, as being too
inconvenient. In calculating the percentage increase the
following method is used. The height of the comb multiplied
by the length, the same points being, of course, always taken,
is considered as giving roughly the area of the comb. ‘The
increase of area observed is calculated as a percentage on the
original area when the experiment began. Thus in Bird No. |
the original area was 50 x 25; the area at the end of the
injections was 72 x 37, which gives, as the percentage
increase—

(72 x 37) — (50 x 25) x 100

DU x 25

= 115; per centr

594, GEOFFREY SMITH.

Besides measuring the combs the weights of the birds in
orammes were taken at regular intervals, and as many observa-
tions as possible were made upon the fertility of the eggs by
incubation. In certain cases samples of blood were drawn
and their action upon suspensions of the testis extract was
observed.

29. Tue Errecr on THE GRowrH OF THE CoMB.

In Experiment No. 1 four birds were used, three of
them being white Leghorn hens of two years’ age and one a
buff Orpington of the same age. Two of the Leghorns were
injected, namely, Nos. 1 and 2 in the schedule. No. 1 was
injected with 35 c.c. extract in the course of twenty-four
days. During this period the comb increased 115 per cent.,
the largest increase observed in any of the experiments.
After the cessation of the injections the comb decreased a
little and showed subsequent fluctuations of no very decided
character, sometimes increasing considerably (see p. 11).

Bird No. 2 was injected with 102 ¢.c. ina period of seventy -
five days. The comb fluctuated slightly in size, the greatest
increase being 25 per cent.

In the two control birds, which were kept under the same
conditions, but were not injected, one showed an increase of
24 per cent., the other remained constant.

The result of this experiment is that in one case the increase
of comb in an injected bird was much greater than in the
controls ; the other injected bird, which was injected for a
longer period, showed about the same increase as one of the
control birds, and therefore gave a negative result. In the
case of the injected bird which showed a large increase in
the comb, there was no constant shrinking of the comb after
the injections ceased.

The fluctuations in weight of all the birds did not show any
correspondence with the fluctuations in the comb area.

In Experiment No. 2 six birds were used, all belonging
to the same breed, viz. the Indian Jungle fowl, a small breed

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 595

a little larger than the ordinary Bantam. The combs in all
these birds were similar single combs, but in Nos. 8, 9, and
10 the combs were larger, these birds being two years old
and having a strain of Silky in them. The other three birds,
Nos. 5, 6, and 7, were pure Jungle fowls of one year’s age.

No. 5 was injected with 117 c.c. extract in sixty-two days.
The increase of the comb was 76 per cent. After the cessa-
tion of the injections the comb fluctuated in size, but after
having decreased a little it increased again, and five months
after the injections had ceased it was rather larger in area
than ever before. There was therefore no tendency to
decrease after the cessation of the injections. Result doubtful,
perhaps positive.

No. 6 was kept as a control bird from February 22nd to
September 7th, during which period it showed an increase of
comb of 29 per cent. It was then injected with 53 c.c.
extract in twenty-one days, during which period the comb
remained quite constant, showing no increase. Result entirely
negative.

No. 7 was kept as a control from April 7th to June 14th,
during which period the comb increased 16 per cent. It was
then injected with 45 c.c. extract in fifteen days, during which
it showed an increase of 35 per cent. The comb decreased
again in August, but spontaneously increased 35 per cent. in
September without any injections being administered, this
increase being the same as that observed while the injections
were going on. Subsequently, in November, the comb again
increased, bringing up the percentage increase to 78. The
result of injection was therefore entirely negative.

Bird No. 8 was injected with 714 c.c. extract in fifty-two
days, during which period the comb increased 14 per cent.
Result negative.

Nos. 9 and 10 were kept as controls during the whole
period of the injections of the other birds, and they showed
percentage increases of 33 and 31,

The result of this experiment, then, is that in no case was
the percentage increase of the comb greater in the injected

596 GEOFFREY SMITH.

bird during injection than in the control birds. The comb of
the injected bird No. 5, which showed a large increase during
injection, not only did not constantly shrink after the imjec-
tions ceased, but actually, five months subsequently, attained
its maximum size. Again, in these birds there was no
correlation between growth of comb and general body-
weight.

In Experiment No. 3 nine young birds, three months
old, of the same parentage and brought up together, were
used. They belonged to the Indian Jungle fowl breed, and
all had similar combs. ‘Three birds, Nos. 11, 12, and 13,
were injected each with 59 c.c. extract in a period of twenty-
one days, during which period thei combs showed a per-
centage increase of 45, 62, and 30 respectively. The six
control birds showed the followime percentage increases
during the same period: 53, 60, 62, 38, 14, and 30.

In case it might be argued that the effects of the injection
might show themselves some time after the cessation of
injection, measurements of the comb were continued fora
month after the last injection. In that month the injected
birds gave percentage increases of 26, 20, and 28, while the
control birds in the same period gave 97, 9°8, 13, 35, 36,
and 36.

The result of this experiment, therefore, conclusively showed
that in young birds of three months old the injection of
39 c.c. of testis extract in a period of twenty-one days had
absolutely no effect on the growth of the comb.

Summarising the results of the three experiments it will
be seen that out of nine injected birds, eight gave
absolutely negative results when compared with
the controls. It cannot be objected to these negative
results that the hens were not injected with enough extract or
for a sufficiently long period, since all of them received as
much, or in most cases more, and for an equal or longer
period than the bird No. 1, which might be claimed as
showing positive results. ‘This bird showed an increase
greater than in any of the controls (115 per cent.), but con-

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 597

sidering the wide limits of variation in uninjected birds, from
0 to 78 per cent., it is certain that no significance can be
attached to this single case. Neither did the behaviour of
this bird, subsequent to the cessation of the injections, lend
any support to the idea that the injections were the cause of
the increase. In Dr. Walker’s two birds, after the injections
ceased, the combs steadily shrank back nearly to their
original dimensions. In my bird the comb shrank a little,
but then afterwards increased again. Doubtless a larger
series of measurements during the spring months on white
Leghorn fowls would reveal as wide a range of fluctuation in
untreated as in this injected bird. JI have now under
observation four Leghorn hens, whose combs have decreased
to less than half their area during moulting, and doubtless
they will again increase in the spring.! ‘lhe measurements
both on injected and control birds establish the fact that in
adult as well as in young hens the comb is in a state of
fluctuating growth, the fluctuations being often marked
within a few days. If we attempt to correlate the variations
in the comb with the variations of body-weight as given in
the fourth column of the schedule, it will be seen that a
simultaneous increase in the comb and in the body-weight is
only to be observed in the young hens in Experiment 3,
where such a correspondence would be naturally expected.
It appears to me that an increase of comb is to be observed
just before the hens begin laying.” Thus a reference to the
fifth column in the schedule will show that the correspond-
ence is marked, especially in Bird No. 7. It will be seen that
the increase of comb is not confined to any particular period
of the year, but may take place in autumn as well as in

spring.

) This supposition has been confirmed, a normal Leghorn hen giving
a percentage increase of 130 in twenty days. This is the greatest
increase observed in any bird, normal or injected.

> In the next study evidence will be produced proving that the
sudden increase of the comb is strictly correlated with egg-laying.

598 GEOFFREY SMITH.

3. THe Errecr on THE Bopy-wericHt, GENERAL HEALTH, ETC.

The series of weights of injected and non-injected hens
shows that the injections do not have any constant effect on
the weight of the body, even in the young animals used in
Experiment 3. With regard to effect on general health it is
true that one of the injected birds (No. 1) became unwell
after the injections, appearing anzmic and with reduced
temperature, and another (No. 8) died soon after the injec-
tions ceased, but seven of the injected birds showed no
symptoms ot any discomfort, laid well, and maintained their
weight. Itis therefore very probable that the two ill-haps
were either purely coincidents or else due to accidental
infection, and not to any poisonous action of the extract
injected. Dr. Walker gives as the result of his injections
that the hens became quarrelsome and attacked cocks that
were put in with them. It may be mentioned that no such
characteristics were developed by any out of my nine
injected birds.

4. Tue Errect ON THE FERTILITY OF THE Eacs.

Since the extract contains a large quantity of ripe and
partially ripe spermatozoa, it was interesting to inquire if the
injected hens might be rendered immune against the cock’s
spermatozoa, and if the presence of an immune substance
in the body-fluids might render fertilisation impossible
or abnormal. In order to test this, eggs laid by injected
hens during the period of their injection were incubated to
the third or fourth day together with eggs from control
birds and the result noted. As will be seen from the schedule,
four of the injected birds laid eges during the full period of

injection, and these eggs were in all cases fertile and

o
normal in development. From some of these eggs healthy
young were actually raised which showed no abnormalities.
We may conclude, therefore, that the injection of the extract

has no influence either on ego-laying or on the fertilisation

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 599

or development of the egg. It is a remarkable fact that one
of the control birds (No. 3) laid a very large proportion
of eggs which developed abnormally, abnormalities of a
greater or less degree being observed in more than 50 per
cent. of the eges. Some of these abnormally developing
egos were simply abnormal in having the chick not sufficiently
forward at the fourth day of incubation with the area vascu-
losa rather bloodless, but others showed actual structural
abnormalities, in two cases double-headed or double-bodied
monsters being produced, in others the back being twisted
into a peculiar shape. I have recorded this fact, as it demon-
strates very clearly that the production of these abnormalities
must have been a fixed character in this hen and not an
accidental occurrence.

5. Tue Errect on tHE Boop.

The normal serum of the fowl has a very powerful agglu-
tinating effect upon the live spermatozoa of the cock. If a
small quantity of the spermatozoa from the vas deferens is
mixed with a smal! quantity of normal blood-serum, it is
observed that in a few moments the spermatozoa, instead of
being dispersed through the fluid, are agglutinated in clumps
and stringy masses. In the space of a quarter of an hour
most of the spermatozoa will be found to be motionless, and on
transferring them to saline they do not recover their mobility
but are shown to be irrecoverably damaged. If we perform
this experiment with a suspension of the testis extract used
in our experiments, the same agelutinating effect is observed
in test-tube experiments, but the agglutination does not pro-
ceed so rapidly even when the tubes are incubated at 37° C.,
because the suspension does not consist only of spermatozoa
but very largely of fatty materials and cellulardébris. The
agelutinating effect of the serum obtained from an injected
hen (No. 2) was tested twice against a suspension of sperma-
tozoa and of testis extract and the result compared with
samples of normal serum. No increased agglutinating power

600 GEOFFREY SMITH.

was observed in the serum drawn from the injected bird, nor
did this serum stop the motion of spermatozoa any quicker
than in the case of normal birds. The various dilutions of
the serums obtained from injected and non-injected birds
also gave similar results.

It was therefore found in the limited experiments per-
formed that no immunising process could be detected as
the result of the injections, and this negative effect is in
accordance with the observations on health, comb growth,
and fertility, as affected by the injections.

SUMMARY OF RESULTS.

(1) The injection of testis extract into hens was found to
have an entirely negative effect on the increase of the comb
in eight out of nine adult and young hens when compared
with the fluctuations in growth observed in control birds.
In one case the injected hen showed an increase of comb
shehtly greater than any observed in the control birds,! but
the comb in this bird did not show the constant shrinkage,
after cessation of injections, observed in the cases cited by
Dr. C. E. Walker, and regarded by him as an essential
feature of the experiment.

(2) The injections had no observable effect on the health,
body-weight, fertility, blood properties, or any other features,
although very large quantities were administered over periods
varying from fifteen to seventy-five days.

(3) The result of the experiments is to show that, although
Dr. Walker’s observations were doubtless correct, his con-
clusion that the increase of the comb was due to the testis
extract injected was erroneous. ‘There is, therefore, no
evidence that the testis contains an internal secretion which,
when injected into the female, can call forth the production
of any of the male secondary sexual characters.

1 Since the MS. was in proof, a control bird of two and half years age
has given a percentage increase in comb of 130 in a period of twenty
days, thus exceeding that of any of the injected birds (see note on p. 7).

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 601

Exprriment No. 1.—No. of Bird, 1; White Leghorn,
"2 years old.

Comb :
Date. Treatment. eee ee Remarks.
millimetres.
1910
Feb. 1 Injected 2 c.c. extract) 50 x 25 —
ne 3 2 DU xX 25 -— =
7 4, 00 x 25 ~- -
10 Attar 58 x 29 1520
12 ol a¢ X 28 1420
15 on a9 x 30 380 :
17 = 39 X 30 1510 ~
19 Injected 5 ¢.c. extract 67 x 3 1520
22 3 5 67 x 3 1650 ~~
24, 5 OXON 1670 ~~ Increase of 113 %.
28 — ier eM 1500 From this date to
‘March 7th the bird
was very unwell,
too weak to perch,
and anemic in
March 4 — ~- 320 appearance.
7 — 67 x 34. 1320 Recovering.
14 — 67 x 34 1580 Recovered.
18 ~- 67 x 34 | 1470 —
22 ~- 68 x 34 1510 =
24 = —— = Fertile normal egg.
25 —- 70 x 36) 1550 Increase of 10 %
since March 7th.
55 sel!) — 70 x 36 1420 —
April 2 = — = Fertile normal egg.
es 3 == — =
4. as a i
6 = =: =
7 — 70 x 34 | 1320 —
ie -— 69 x 34 —- -=
14 -— 67 x 34 ~=1080 =
| 19 — 67 x 34 | 1150 —
22 + 67 x 34) 1200 =
{oe BE -- 66 x 33 | 1320 —-
‘May 3 — 64 x 32 | 1410 -—
ie 14 -—— of <X 30} 13850 —
eee 2 a | 62 x 30 | 1240 -—
June 1 — 61 x 28 1270 —-
14 = 63 x 29 | 1290 = |

No.
Date.

1910
Feb. 1
fe 3
7

D4,
March 3
pee iy,

eee

oe
ot 14
16
18

‘June 1

GEOFFREY

SMITH.

of Bird, 2; White Leghorn,

Treatment.

Injected 2 c.c. extract

»

Oreo C3 RL

os

Injected 5 c.c. extract
5
Injected 5 ¢.c. extract
SY 38 5
OP. 5
D455 %
10
10

10

Injected 10 ¢.c. extract

(

Jomb

measure-

m

95

95
95
97
97
98

LOl

98
98
99
99
98
98
98
98

ent in

millimetres.

Pe Ra I CG OK KO |
one
a

Weight in
grammes.

2070
1900
1900
1850
1970
1900
1820
2050
2020
1870
1860
1700
1680
1629

1650
1620
1570

1790

1670

1560
1290
1520
1300

2 years old.

Remarks.

Fertile normal egg.

Blood drawn for
agglutination ex-

periment.

Fertile normal egg.

Normal fertile egg.
Increase of 23%. |
Blood drawn for

agglutination ex- |
periment.

Normal fertile egg.

ss

Normal fertile egg.

Normal fertile egg.

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 603

No. of Bird, 3; White Leghorn, 2 years old;
Control.

| Comb
Date. Treatment. measure- | Weight in Remarks.
ment 1 grammes.
millimetres,
1910
Feb. 22 - 85 x46 PAOLO) |
pi 5 we : 87 x50 1990 |
March 1 - 87 x52 1920 |
15 -- 92° <5 1940 |
Is a 90 x54 | 1900 | |
22, 90 x53 1900
25 91°5 x 54. 1920 | Several fertile
30 87°5x53°5| 1790 || eggs, many ab-
April 4 -- 87° x<oe 1720 |f normal in de-
» 14 89 x52 1670 | | velopment.
Is — = |
OD, — 87 X02 1750 |
Ob 89 x52 1790 |
May 3 - 89 x51 | 1600 |
a: - 87 ~x5l 1560
i ae == 85 x50 1520 |)
June 14 ~- 86, x53 1720 Increase of 24%.

No. of Bird, 4; Buff Orpington, 2 years old;
Control.

Comb ee :
| Date. Treatment. Sean ve es Remarks.
| millimetres.
| 1910
Feb. 23 — DOS Lo 3270)
March 2 — yay Beas, 3250 ||
15 — ye ee 3300
18 —- Ha! S20 3200
22 — as Ie Oa) 3160 | Numerous fertile
25 — 53 x 25 3270 | eggs, normal
7, 00 — 53 x 25 3070 | development.
April 7 53x25 2920
e 14 = Ia) 5-6 5s 2830s!
» 20 Say SpA) 2840
May 3 i OD. 2820" 4
June 24. —- Soop 2850 | No variations in
3} comb.

VOL. 56, PART 3.—NEW SERIES, Al

604. GEOFFREY SMITH.

EXPERIME nt No. 2.—N O. Obes birds, 2 5 Jungle Fowl i
5) : to) 5)
year oO Na

| Comb measure- Weight |

Date. | Treatment | ment in in Remarks.
millimetres erammes
1910 ae Sib eke 75 ae
Feb. 19 Injected 2 ¢.c. extract) 23-5 «10°25 720
22 - 31 : 93°5 10°25) 720 —
24. et » Nes bx lO bel) 740 =
~ 20 rs by 24 SOD 750 —
March 3 | 3 a ee is 25 «10°25! 780
Dl ss 45 53 26 «105 760 ~~
8 : 3S 3 ; 20. <ul R00 —
gy UE os Dir » (275 X115| 800
14, = BY ate % 980 <i 820
7a * BS) | oe 3 Sepa Sen) 800 —
DO nS ee. Zo. Sale 800 =
A a5 = 29 x<le 760 —
.. 28 Injected 10 ¢.c. extract = — |Normal fertile egg.
30 is IO » 129 x12 720 %
April 6 i LO tye At | Beery 740 | —-
i ote 7 — —_ = Normal female ego,
WP ee ets — 30 xls 690 |
ae LO “= = | — {Normal fonilees ro",
14|Injected 10 c¢.c. extracti30 «12 | 740
1G) es Oi EY Sek 74) |Normal foilee eo,
LOY ees 1) ee 7 e0 SebRee Zl) |
a I By ae 3 (BO 15 770 “Increase of 76%.
3) 320: -— SLES < iBone cs0ng| =
May 3 — Is25<lis 810 | a
By alos) -— 131-25 x18 720 —
ly — Bila salts 650 =
edi) — 29) S<lZ) 1) 1630
yy ols = AS Sey} 620 —
June 6 = 30 «12:5 670 | _-
be aba a 30) << 1250\ 670" |
Hie a0 — 30 x12 670 —
eel —- I3 x13 680 |
July 1 — 30°5 x 12°75) 690 =)
33 7 = 3 x 12:25) 700 | se
3 9| -- 3 Seip eK) | =
| 95 14. — 3 x< 1 | 720 | ar
Pee —- 38. x145 | 740 | Largest dimen-
| sions of comb
| | reached.
Aug. 16 -- 30 x125 | 770 =
Sept. 9 — 20) Baio 700 -~
poke iD — tei baal 720 —
» 14 as 29 x12 770 —
16 — 30 «°x<1255 | 800 =
19 — BO” <2 720
23 | — 29) ssed2 790 Egg » laid.
28 — ag) ec Tatas 820 =
30 | ae Se wcale 840 Kee laid.
Oct. 38 = B20 LS: 840 | =

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 605

No. of Bird, 6; cunene Fowl, 1 year old.

|
Comb | Weight |
Date. Treatment. poirihe: a in Remarks.
millimetres. Srammes
fo tO
Keb. 22 — 22—-x7 670 —
March 1 == 22 Xho 660 —
8 | = eT 720 ==:
paallicll -—- 23.5 ator Pio =
CT 22 — 23°5 x 7°25) 690 _-
25 — 235 x75 720 = |
30 == 23°5 x75 700 — |
7 il 6) — 129-5* 57-95) 700: 15
14. 3 23 x75 | 640
19 | — 23°5 x8 64.0
99 C)Q.r S) Pan
36 eG etc 2 a Several normal
_ | avo XO eae fertile eggs
May 3) — 23:5) <8 720 55”

me) al) — 2A XS 670

Li) = 2b) Xs 670

COA — 24:5 x75 | 600

a = 24-5 7°25) 650 =
June 7. — 24-25 X7 660 — |
| 14. | = 2A a7 600 —
| 20) = 24 x7 630 =
Wa = (24 = DA med 650 —

July 1 — 245 X7 690 —
. i — i245 7-25) C70" | — |
I a 95 ~<7-25| 690 | = |

14| — 245 x7 | 660 —

» 22 — 945 x7 630 --
Aug. 16| _— 24 x7 700 = |
Sept. 7| = 24° x7 690 | Greatest increase, |
| : | | 29 %.
ease LO. enna 4¢.c. extract/24 «7 690 ~=
ese See ee van a0 =
14 A, 24 x7 670 —

16) Joe 4 x7 670 =
\ircor eds] 4, = — —
ee eli wth PLES. 7 670 | —-
lly Aum 24 KT 64.0 -—
| 23 | A, DA) XT 650 —
24. | Ay — — =
2.63) Ac DAS AT 670 | -—
ee Aa DAs EX 0 6100 | =
» oO 4, 23°75 X65 | 670 --
Oct. 1 Ane — 670 | —-
| ; 3 —_ 24 x65) 690 |Noincreaseas result
| of injection, slight
| decrease.

606

No. ot

Bid;

GEOFFREY SMITH.

Jungle

\Comb eseacel Weight

Fowl,

Date. | Treatment. ment in in
| | millimetres. grammes
1910

April 7 - 26°25 x 9:25) 3840
| ee ae! 27 x 9°75) 840
| 19 wy Seil0 820
22 27 x 10 850

= AS) 96°5 « 10°25) 870
May 3| 27 x105 | 820
- 10) 26°5 x10-25| 820
17| 27 10-25) 710

24; 26°75 x10 670
eee: 265 x10°25) 670
June 7. 26 = S<0 730
14 Ye SII) 770

15 Injected 3 ¢.c. extract -— 740

16. 3 Wee Se) 770

17 | 3 27°25 x 10 720

18 | 3 28 =x ll 750

19 | 3} 28) Salil 74.0)

» 20} pase ok - 23°51 xs 74.0
All| on 2810 <1 720

22 | Se 2Sreclilise 720

23 33 98°25 «12 740

24. 3 28°5 x12 710

2 3 2875) <2 700

27 3 28 <2 700

28 3 28:5. 12 720

29 3 DO eel? 710

ena ()}| Bk Ks 2979) x<ali2ie 750
July 1} - 29°75 x 12 720
eB 7 | 29°25 x 11 670

9 | 29 Sil: 690

14. Msi SEMPRE) 660

- 22, — 2S Ore 650
|Aug. 16 27°5 x10 750
Sept. 7 2 x11 700
= 10) = 285 10°75) 750
; 14. egoeciles 790
lees 16 - BON ><il2 S00
| 19| = 2025 exc lilcomile vo)
21) - 30°5 « 12:25) 850

93 | at 29°5 11:5 | 820

26 | 29°5° Selisd 820
28) = 29°5 «11:5 820
tap cal} a= 29°55 x11 | 840
|Oct. 3 | —- 129-5 11:25) 840
a 18 | — 31°75 x12 890
ay as — 34 x13 900
Nov. 1 Hy die 850
8 _ 3l5 x13 800

|

|
|
|

1 year old.

Remarks.

Several normal
| fertile eggs.

Increase 16 °/,.

=

Eee laid, normal.

boo)
Hox

a
ss

x laid, normal.

| Ege laid, normal. |

Increase of 36°/,. |

Egg laid.

Increase of

Egg laid.

Eggs laid.

5

Tiereuse. of thea
since August 16th.

35°/ 0. |
since August 16th.

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX.

No. of Bird, 8; Jungle Fowl, 2 years old.

a
55°

607

Comb measure- Weight
Date. | Treatment. | ment in in Remarks.
| millimetres. grammes.
STO" | :
March 1 Injected 33 ¢.c. extract 26°5 x12 800 —
Heese bls a; Dass 27 x12 790 =
14” fart _ pA press Al [4 740 —-
1 a Bo ey wre ay 730 ==
Steel ss ot) se) (20ior ce 730 --
20 8 26:5 «12 790 =
oe 30) 8 27°35 X12°25 740 Normal fertile ege.
April 6] _,, Sn Be arid eocal 770 Normal fertile e:
os 8 — PAF (PAST. A Wed 750 —
» 14 Injected 5 c.c. extracti27 «1225 790 -
ko dls, As Sle ak 5 Dio Xo S20 —-
S| A eae) ae a Zee els 820 Increase of 14%. |
26 -— 27° x12°75 800 a

30) =

Found dead on
nest. Peritoneal

infection

| cavity full of clear yellow pus. Slight

ot tuberculosis in liver,
| _ hardly sufficient as cause of death.

No. of Bird, 9; Jungle Fowl, 2 vears old.

Comb measure-

Weight
in

grammes. |

Remarks.

900
900
870
870
890
920)
920
950
950
970
950
| 990
| 940
| 970
990
990
1000
950

|

fertile eggs.

ne normal
|
5

Increase of 33%.
Suffering from
hard tumour-like

Killed, and on dissection oviduct

Date. | Treatment. r ment in
millimetres.
1910 | en Pe oe eer
March 1 _ SOs LD
cs 11 a 386 x14
sollte} = BOO. KIA
, 22 = 134°5 X13°5 |
2k | = 35 x14
» 20 = 30° X13°5
April 7 — 39 = X135
pee 14 = By ole |
ve al) — SOM liosno|
en 22 = 36 x4
» 26] — 36 x14
May 5 — 37°15 x14
10 — BED SCID
> ily, — 38 x lb
; 24. — 39 =x 15:25)
7 31 — a9) X16
June : | — 39 «6x16
swelling in abdomen.
of large-yolked eggs.

found occluded with large masses of yolk; ovary also full

|

608

GEOFFREY SMITH.

No. of Bard, 10; Jungle Fowl, 2%years old:

Date.

1910
Feb. 23
March 3

ail)
April 25
June 2
» 24
Sept. 26

Treatment.

EXPERIMENT No.

Comb
measure-
ment in
millimetres.

Weight.

Remarks.

20:5 9¢12275)
20°5 x 12°75}
PAD Salas
Zon DGD
22 ale
20 x13
BOOS

Comb measure-

Date. Treatment. | meut in
millimetres.
1910
Sept. 7 — 20° «6:5
9 Injected 3 c.c. extracti20 x 6°75
2 a 3 20:20 6770
to eee eh a 3 20 x 675
eee lly a) A: 2 PAD) = Se (OED
ee eal?) 3 —
ee ee} = By" kn 208 <a 725
21 | 3 Bs 20;253< 7-25
23 3 PADRES See FAS
24. 3 5 =
26 3 se DAY xe ie:
28 ‘3 a 21:°25x 8
= ail) BS A 21-255< 18
Jet. 5 _ =
is : = Wle7ox 9
18 — 23°25 x 10
Ae OE — 23°75 x 10
Nov. 1 _— 245 x10
8 — 24°75 x 10

Weight

grammes.

in

Increase of 31

Numerous fertile
normal eggs laid.

O04

7O-

Remarks.

Increase of 45 °/,.

Increase of 26 °/,

|
|
|
|
|
|
|
|

3.—No. of Bird, 11; Jungle Fowl, 3

months old.

since October 3rd.

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 609

No. of Bird, 12; Jungle Fowl, 3 months old.

Comb

Weight
| Date. Treatment. 2baNef ASME in Remarks.
ment in grrammes.
millimetres.
1910
Sept. 7 = 18 «x5 350 ==
. 9 Injected 3 c.c. extract)18'5—x5°25 350 ~-
1 : ay 19 «5:75 420 —
14. Oy 19 x6 410 —
16 By. <2 19 x6 450 =
17 Be — ~- —
19) But 195 x6 ATO —
21 | ae 195 x6 500 ee
23 3: 19°75 X65 500 =
24. 3: — = a3
26 3% 20) <615 00 —=
28 3 Hon Sez 500 —
esi 3 205X725 520 oe
Oca Wy Sea = 520 —
3 | — 21, x75 | 520 | Increase of 62 °/..
18 -— ZO xo 64.0 oo i
oe 25: os ye CTA) 620 —
Nov. 1 — 93°25 x8 600 =
8 — 23°75 x8 600 Increase of 20 °/,

|
|

since October 3rd.

No. of Bird,-13; Jungle Fowl, 3 months old.

|

| Comb Weight |
Date. Treatment. sen T DSSS in Remarks.
ment in grammes.
millimetres. ~
1910 :
Sept. 7 — 165 x475 270 —
eve: 9 Injected 3 c.c. extract 16 «475 270 —
Ve pee ety ome » |1625x*475! 340 —
ee 3 a Gaeta 340) -—
LG 3 16°75 x5 380 —
1% : Sy ae. | = == =
19 ; 3 5 |G 7b5co 400 —
ae Bh 3 (Be 44.0 =
2: 3) ic 16°75 x5 440) | =
of ; aye 4 re — —_— =
26 By * 17°25 x 5 430 —
28 35 17-25 x 5°25) 420 =
Gal) By. bp WED) oro 44.) —-
Oct. 1 5 ate a el —- 44.0) —
s 3 = 18 x55 450 | Increase of 30 °/..
els — 19 <6 550 —
ieee) — IIS) SE AU =
Nov. 1 — 10 5eexowe) 620) | —=
2 8 — 195 x65 600 : Increase of 28 °/,

' since October 3ru.

610

GEOFFREY SMITH.

No. of Bird, 14; Jungle Fowl, 3 months old;

Control.

I ‘omb measure-| Weight
Date. Treatment. | ment in in Remarks.
| millimetres. |grammes.|
1910 ' lowe
Sept. 7| ~- plist se 410 —
= 9 — W185 x 6 420 =
12 = 19 x 6 500 =
14. —~ 18-75 x 6 490 =
= PG == 19 x 651] 500 = |
~ - 1) == 19:25 x 6°75] 530 = |
21 — 195 <7 570 a) |
23 ~- 19°75 x 7 570 | —
26 -- PAV Sve) 550) |} ==
28 —- 20a x *7 5AO =
ay GN) — 21 SEED 5d0 =
Oct. 3 — 21:25x 8 540 | Inerease of 53°/.. |
18 ~- 2ae. = SiD 600 =
a es = 255 «10 630 | =
Nov. Il -- Pye SEIN) (635K) 4) =
8 — 280 2 650 | Increase of 97°/.
since October 5rd.

Date.

1910
Sept. 7
x 9

12
14
16
19
2|
23
243
28
30

»

»
18

25

Noy eaml

of Bird, 15; Jungie Fowl, 3 months old;

Treatment.

Control.

Comb
measure-
ment in

millimetres.

SSS Sa5=

J “I “I au ~J “I Cap Sa

Weight |
in
crammes. |

300
310
320
320
350
370
350
370
380
400
400
ALO
480
500
500
570

Remarks.

Increase of 60°/..
Increase of 9°8°/,
since October 3rd.

STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 611

No. of Bird, 16; Jungle Fowl, 3 months old;
Control.

| |
| | Comb Weight |
| Date. Treatment. pect ae in | Remarks.
millimetres. ae ae
1910 | |
Sept. 7 = 155 x3 250 —
3 = 15°75 x3°5 | 260
12 — 15°75 x3°5 | 300 -~
14 — 1G x35 | 300: | —
16) = 15°75 x35 | 320° | —
19 = 115°5 x38°75| 320 aaa
21 —- 155 x4 350 |
23 16 x4 B00 |
28 16:25 x 4°25) 370 | —
io _ 165 x425) 37 =
Oct. 3 16°75 x45 | 390 |. Increase of 62°/,.
pols 175 xo 490
lee eee 17°75 x 4°75) 800
Nov. l 18 4°75) 500 ~
has 8 = 18 %«4°75) 500 | Increase of 13°/,
_ since October 5rd.
|

No. of Bird, 17; Jungle Fowl, 3 months old;

Control.
l |
Comb =e |
Date. | Treatment. peeves relay | Remarks. |
| millimetres. acer al
| 1910 | waa |
Sept. 7| — Ge. Se4 1) “250 | —
a 9 = 165 x425) 270 —
12 | = 116-25 x 4°25} 310
14.) = 16 x45 | 320
16! — 165 x4°75| 340 =
19 ae 16 x4°75) 380 —
Dall _ 165 x4°75| 370 —
23 = 16°75 x 4°75) 380 —
26 = L255 380 —
28 | =— ye S<e53 390 -—
2 0) ae 7 Sash 4.00 _-
Oct; 3] = Wieioo<o, || ALO Increase of 38°/..
a Del ed = 19 ~x575! 500 —
ae) == 19:25 x5°75) 510 —
Nov. 1 == 20' 5:25) 500 ==
8 — 20 x6 500 | Increase of 35°/,
since October 3rd.

VOL. 56, PART 8.—NEW SERIES. A2

612

No. of Bird, 18; Jungle Fowl, 3

GEOFFREY SMITH.

months old;

Control.
ate reatment. : in eCmarks,
eM, eramnmes.
1910 |
Sept. 7 = 17) x45 | 300 —
by 9 — 7s 3<4c5) «350 =
: 12 -— 175 x45 | 370 =
ee ela: Se e425 e370 —
als — 18 »~x425) 390 =
ey 9} —- 175 x4:25} 420 —
fee ee — Vib <4 430 | —
eee ee -— 17-75 x45 | 450 =
AS —- 17°75 x45 | 460 =
ASI — 18 ~x<4°75} 470 —
ym asl) —— 18°25 x 4°75) 470 =
Oct. 3 a 185 x4°75| 470 | Increase of 14°/,.
5 dS! — 19 »x5:75) 580 —
sy PAD -- 20) =x<bsvo| 680 =
Nov 1 — 20" x6 570 =
> 8 | — 20 x6 570 | Increase of 36°/,
| since October 3rd.
No. of Bird, 19; Jungle Fowl], 3 months old;
Control.
Comb ‘
| Date | Treatment. ett ee Remarks.
| | millimetres. |ST@™mmes.
| 1910 | |
Sept. 7 | = IN a5) 280 | --
he 9 | oo 17°25 x5 280 =
2 12) _- iS es<on al) O20 —
14 | — 117°25 x 5°25} 320
1G) - 17°25 x5 340 =
i 19! —- Iga Seiytay |) S570) =
a» all — 7d <0) |) 380 =
yD — 18 x5 390 | =
by XB 18 5°75} 400 | —
28 — Se eSd6 400 | —
0) — ise (63 410 | ==
Oct. 3 - 185 x6 410 | Increase of 30 °/,.
At 18 — 195 x6:25|) 510 | —
eg 435 — NPE Score] 0) | =
Nov. 1 = 20 6575) 000) | =
;5 8 — 21 725) 550 | Increase of 36 °/,
since October 3rd.

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 613

Cortical Cell Lamination of the Hemispheres of
Papio Hamadryas.

By

E. H. J. Schuster, M.A... D.Se.,
Fellow of New College, Oxford.

With Plates 24-50.

PREFACE.

THe localisation of function in the cerebral cortex is a
problem on the importance of which it is hardly necessary
to dwell. Its practical value to the surgeon and its theoretical
interest to the physiologist and psychologist are obvious and
have been sufficiently recognised. It is a problem which
concerns many of the biological sciences, since it can be
approached in many different ways. The physiologist has,
by the electric stimulation of different parts of the surface of
the brain in various animals, obtained responses from different
parts of the body, and has thus been enabled to say what areas
control the movements of the various groups of muscles.
Occasional opportunities are afforded to the surgeon for
making similar experiments on the human subject. The
ablation of different parts of the cerebral cortex in the living
animal is another physiological method from which valuable
conclusions have been drawn. Observations on the alterations
in function associated with local cortical lesions due to
accident or to disease form a parallel method of research
in the domains of pathology.

The contribution called for from the anatomist and histo-

voL. 56, PART 4.—NEW SERIES. 43

614. KE. H. J. SCHUSTER.

logist is the mapping out of the cortex into areas differing
from one another in structure. In doing this he may take
into account the form and arrangement either of the nerve-
cells or of the nerve-fibres, or he may divide the surface into
regions distinguished by the period at which they arrive at
maturity. In an exhaustive treatment of the subject evidence
obtained in all these ways must be considered and co-ordinated
—a difficult task, since the interpretation of the observations
is beset with pitfalls, and in consequence a number of apparent
inconsistencies have to be explained away or harmonised.

On some points already a considerable degree of certainty
has been arrived at, namely the extent of the area concerned
directly with the movement of the voluntary muscles, and of
that region in which the visual impulses set up in the retina
reach the cortex. With regard to these points the evidence
from all sources is in agreement, and the definite function is
in each case correlated with a perfectly definite structure,
which, particularly in the case of the visual area, sharply
delimits it. A perfectly definite structure also marks the
olfactory cortex, while to many other structurally distinct
areas a special function may be assigned with some degree
of confidence.

That a better appreciation of a physiological and anatomical
problem may be obtained by extending it into the realms of
comparative physiology and comparative anatomy is generally
accepted. Both the points of agreement and the points of
divergence in structure between animals with different habits
living in different environments may elucidate the relation
between structure and function. The present paper aims at
making a small but definite contribution to the subject, in
that it consists of a description of a special aspect of the
histology of the cortex in an animal not hitherto treated in
this way. The histological study of the cortex may be
pursued in at least three different ways. Firstly, the nerve-
fibres may be stained by some method like that of Weigert ;
secondly, the nerve-cells may be stained either by the Golgi
or Cajal process. These are uncertain in action and only

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 615

select a few cells for staining, but show them complete with
dendrites and axon, and may enable one to trace for a con-
siderable distance the path of the latter, in some cases even
as far as its terminal arborisation. ‘I'hese methods are of
great importance, and throw light onthe relations of one cell
to another within a particular cortical area, and thus on the
individual functions of the different types of cell. They are,
however, unsuitable for the survey of the arrangement of the
cells in their several layers, which is necessary in order to
divide the cortex into regions according to the type of cell
lamination present in each. For this purpose it is necessary
to employ a method of a third type, such as that of Nissl, in
which every cell is rendered visibie by the stain. Nissl’s
method shows the general shape of each cell and its principal
dendrites, also the structure of the nucleus and the arrange-
ment of the extra-nuclear chromatin; if does not show the
axon nor the nerve-fibrils runuing through the body of the
cell.

It is a variation of Nissl’s methylene-blue which has been
used for the purposes of the present paper, namely Unna’s
polychrome methylene-blue, as the particular point dealt with
is the arrangement of the layers. Much work of the kind
has been done on the human brain, without, however, exhaust-
ing the subject, but the other Primates have been somewhat
neglected. Of the few papers dealing with them the earhest
is that of Schlapp (‘ Archiv f. Psychiat.,? Bd. xxx), “ Der
Lebenbau der Gross linn rinde des Affen Macacus cyno-
mologus.’’ Schlapp does not go into any great detail:
three types only of cortex are described and somewhat
roughly figured, their extent being shown by a sketch of the
lateral surface of the hemisphere.

Campbell (‘ Histological Studies on the Localisation —of
Cerebral Function,’ Cambridge, 1905) describes the brains
of the chimpanzee and orang-outang. ‘This work, which fills
a volume of considerable size, deals principally with the
human brain, and treats the subject with praiseworthy
thoroughness. Not only is the arrangement of the cells

616 E. HH. J. “SCHUSTER.

described, but that of the fibres also. The human brain 1s
compared with that of the two apes, and the clinical, patho-
logical, and experimental evidence is fully discussed in its
relation with the histological results.

An excellent series of papers on the cell lamination have
been published by Brodmann in the ‘Journal f. Psychologie
und Neurology, 1903 and onward, and summarised in his
book, ‘ Vergleichende Lokalisations lehre der Grosshimrinde,’
Leipsig, 1909. The human brain is described in great detail,
a very large number of separate areas (more than fifty) bemg
distinguished histologically. Similar detailed descriptions of
the brains of Cercopithecus and of the lemurs are given, while
the marmoset (Hapale) is dealt with more shortly. The homo-
logies between different regions in the human and _ other
Primate brains are dealt with as far as possible.

Mott and Keliey publish a “Complete Survey of the Cell
Lamination of the Cerebral Cortex of the Lemur” (‘ Proceed-
ines of the Royal Society,’ B., vol. Ixxx, 1908), in which the
histological results are compared with the results of stimula-
tion experiments performed on the lemur’s brain by Prof.
Halliburton, and a paper of a similar nature, but dealing with
the brain of the marmoset, has been produced by Mott,
Halliburton, and the present author (Mott, Schuster, and
Halliburton, ‘‘ Cortical Lamination and Localisation in the
Brain of the Marmoset,” ‘ Proceedings of the Royal Society,’
By vol, ixxxu; 1910):

MaTERIAL AND MEtHops.

The material used in the present paper consisted of the
brain of a specimen of Papio hamadryas which had died
in the gardens of the Zoological Society.

“ach hemisphere was cut into a number of blocks (seventy
or more), arranged in such a way as best to secure that as
much as possible of the cortex was cut in planes at meht
angles to its surface, since in oblique sections the nature of
the cell lamination is almost impossible to determine. The

CORTICAL CELL LAMINATION. OF PAPIO HAMADRYAS. 617

outlines of the blocks were carefully plotted in drawings of
the brain. From each block sections 10 thick were taken
and stained with polychrome methylene-blue. As no suatis-
factory sections of the insula were obtained, the cortex in
this part of the brain has not been described.

In the investigation of these sections the admirable works
of Brodmann! on Cercopithecus and Campbell? on the chim-
panzee and orang-outang were of great assistance to me, and as
far as possible my observations have been brought into line
with theirs.

My observations differ from Brodmann’s in that I have not
described so many types of cortex. This is due to a difference
in judgment and not to antagonism with regard to matters of
fact. Since in the area covered by each main type local
differences are present, and since the main types in many
cases shade into one another, it would be possible to describe
many more varieties of structure than are mentioned by
Brodmann. One must arbitrarily fix a limit to the number
of types distinguished, and I have fixed mine at a smaller
number than he.

For illustrating the structures described in the text, outline
drawings of the cells were made under the Abbé Zeichen-
upparat, and these were afterwards blacked in with ink so
that the cells are represented in silhouette.

It was thought advisable to prefix a short description of
the fissures to the principal part of the paper.

FissuRES IN THE Brain or ParPio HAMADRYAS.

The Sylvian fissure ( F'S.) and sulcus centralis (c.) call for
no special comment. Anterior to the latter the following
fissures are present :

' K. Brodmann, “ Beitrage zur histologischen Lokalisation der
Grossheikrinde Dritte Mitheilung; Die Rindenfelder der Niederen
Affen,” * Journ. fiir psych. u. Neurol.,’ Bd. iv, Heft 5/6, 1905.

7 A. W. Campbell, ‘ Histological Studies in the Localisation of

Cerebral Function,’ Cambridge, 1905.

618 i. He. a SCHUSDER.

Suleus arcuatus (arc.).—From the most posterior portion
of the backwardly directed convexity of this fissure a straight
branch runs upwards and backwards. The upper limb in the
right hemisphere is united at its extremity with one of the
sulci of the frontalis superior series. ‘The lower limb m both
hemispheres runs straight downwards and forwards, and ends
about a centimetre above the anterior end of the Sylvian
fissure.

Suleus inferior transversus (v.) runs obliquely
upwards and forwards between the lower ends of the sulcus
centralis and sulcus arcuatus; in the right hemisphere it 1s
connected with the Sylvian fissure.

Suleus rectus (rect.) runs horizontally forwards from a
position about 5 mim. in front of the sulcus arcuatus, to end
about the same distance from the frontal pole. Between its
posterior end and the lower portion of the sulcus arcuatus
lies the sulcus diagonalis (d.), a shallow oblique fissure
better marked in the right hemisphere than in the left.

The sulcus precentralis superior and sulcus
tronbalis superior™( ss. 2,02, 3,-4) are together repre-
sented by a series of shallow fissures, of which the posterior
(fs) is the best developed. ‘This hes m a more or less
sagittal direction, while the other members of the series are
more transversely placed.

On the orbital surface is found a sulcus orbitalis (orb.)
lying near the mesial border; this is triradiate in the right
hemisphere, but a simple, more or less sagittal, fissure in
the left. On the lateral side of this is the obliquely placed
suleus fronto-orbitalis (fo.), the anterior outer end of
which just reaches the lateral surface of the hemisphere.
This fissure is quite well defined, though not more than three
or four millimetres deep. Its shape is complicated in the left
hemisphere by the presence of a shallow forwardly directed
bifurcated branch.

The suleus temporalis superior (Zs.) is a long, well-
developed fissure extending both in front of and behind the
Sylvian fissure. Its posterior end is bifurcated in the right

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 619

hemisphere but not in the left. Below it, in the temporal
lobe, lie one or two shallow grooves (Z.4, 9, ete.), which may
be taken to represent the sulcus temporalis medius.

The sulcus post-centralis superior (pes.) 1s a shallow
groove lying almost horizontally opposite the upturned
posterior end of the sulcus cinguh.

The sulcus intra-parietalis (¢p.) starts anteriorly in
the space which is bounded above and in front by the sulcus
centralis, and below and behind by the Sylvian fissure. I[t
runs at first upwards and backwards, then turning backwards,
disappears underneath the occipital operculum. Here it
bifurcates. Its outer branch runs outwards on the anterior
wall of the sulcus lunatus, and then divides into two rami,
of which one runs to the bottom of that fissure while the
other runs upwards. The inner branch, by far the larger of
the two, is called the ramus parieto-occipitalis (rpo.) of
the sulcus intra-parietalis. It cuts very deeply through the
supero-mesial border of the hemisphere, then runs straight
downwards to end in a bifurcation, the branches of which he
more or less parallel to the sulcus calcarinus.

Owing to the extent of the occipital operculum, this ramus
parieto-occipitalis appears to be a mesial continuation of
the sulcus lunatus; in reality the two fissures are partially
separated by a submerged gyrus lying near the supero-mesial
border.

The fissura parieto-occipitalis (fpo.) is a triradiate
fissure, the two posterior rays of which arch round thie
anterior limb of the terminal bifurcation of the parieto-
occipital branch of the sulcus intra-parietalhs. ‘These two
rays are better developed in the left hemisphere than in the
right, and there the lower one forms a superficial connection
with the posterior limb of rpo. The anterior ray is poorly
developed.

The sulcus subparietalis (sp.) (posterior hmbic sulcus)
hes between the fissura parieto-occipitalis (fpo.) and the
sulcus cinguli. The main portion of this fissure runs upwards
and shghtly backwards; its lower end is divided into two

620 t. H. J. SCHUSTER.

branches, which are better marked in the left hemisphere
than in the right.

The suleus cinguli (Sc.) (intercalary or calloso-marginal
fissure) is quite typical in its arrangement. At its anterior
end lie two shallow sulci rostrales (ro.), with the upper of
which it is, in the right hemisphere, in connection.

The sulcus collateralis (Col.) is in the right hemi-
sphere divided into three segments ; the most anterior of these
(col.,) lies obliquely between the sulcus rhinalis and the
anterior end of the sulcus temporalis superior. The middle
segment (col.,) is the longest; it runs sagitally backward.
Between its posterior portion and the sulcus calearinus lies
the third segment (col.,), the main portion of which runs
parallel to that fissure, to end posteriorly in a transverse
member. In the left hemisphere only two segments are
present, the anterior (col.,) corresponding exactly to the
anterior segment in the right hemisphere, while the posterior
segment of the left hemisphere corresponds to the two
posterior segments of the right; at its hinder end it runs
upwards and ends very close to the sulcus calcarinus.

In the left hemisphere a shallow T-shaped sulcus (7.) is
present lying between the posterior part of the sulcus colla-
teralis and the sulcus calcarinus.

The sulcus rhinalis (rh.) is quite typical.

The suleus lunatus (lun.) (‘ Affenspalte’”’) is well
developed in both hemispheres ; its posterior lip is produced
forwards to form an operculum. Curving round its lower
end is the deep sulcus occipitalis inferior.

The suleus occipitalis inferior (Oi.), the upper lip of
which is produced downwards to form a well-marked oper-
culum. Behind this fissure, curving round the tentorio-lateral
margin, is another suleus, which is independent in the right
hemisphere, but connected with it in the left.

The sulcus occipitalis lateralis (Ol.) lies horizontally
between the sulcus lunatus and the occipital pole. It is a
very deep fissure, which cuts obliquely downwards into the
hemisphere so that its lower lip forms an operculum. — Its

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 621

anterior end is bifurcated in the right hemisphere. One or
two shallow grooves lie between this and the sulcus occipi-
talis inferior.

The sulcus calecarinus (cal.) at its anterior end joins
the fissura hippocampi; it runs a sigmoid course backwards,
to end in a long bifurcation near the occipital pole. ‘The
upper branch is by far the longest, and its upper end just
reaches the lateral surface of the hemisphere. In the nght
hemisphere it gives off a short downward branch.

Frontrat Lose.

Precentral (Motor) Type (fig. 1).

This type of cortex is characterised by its great depth,
which reaches to over 2°7 mm., the presence of the Betz cells,
and the absence of a distinct layer of
drawing (fig. 1) is taken from the lateral surface of the
hemisphere a short way in front of the upper end of the
sulcus centralis. Here the breadth of the cortex is about
2°7 mm., but only the upper 2°4 mm. are drawn. The Betz
cells occupy a broad zoue, otherwise rather poor in cells,
which stretches from 1°3-1°9 mm. from the surface. ‘hey
appear to be larger here than in other parts of the pre-
central area. The cell bodies of the largest are about 70
in length by 50 » in breadth, but the massive processes given
off from the apex and base render accurate measurements
impossible, since it is difficult to say where the cell-body
ends and the bases of the apical process or other dendrites
begin. ‘These cells are slightly smaller on the mesial surface,
and gradually diminish in size as one passes downwards over
the lateral surface towards the Sylvian fissure. About half-
way down they measure only 50 x 30 u.

The other cell layers are as follows:

“oranules.’”’ The

622 i. H. J. SCHUSTER.

(1) The lamina zonalis is about 1 mm. broad.

(1) Lamina granularis externa reaches to a depth of about
‘3mm. Its constituent cells are small pyramids and granules ;
these vary in size in different regions, and appear to be rather
larger on the mesial than on the lateral surface. The largest
of the small pyramids are about 15 x 12 pu.

(ii) Lamina pyramidalis is about 1 mm. broad, extending
from a depth of -3 to a depth of 1°3mm.; as is usual, the
cells increase in size from above downwards. ‘The largest
cells in the area figured are about 35 in length x 20 win
breadth ; but this particular strip of cortex, though exception-
ally rich in large Betz cells, is rather poorer than others in
large pyramids of layer iii.

(iv) Lamina granularis interna is represented by a few
scattered granules in the lower part of layer iii and upper
part of layer v.

(v) Lamina ganglionaris. The Betz cells have already been
described; the other cells contained in the layer resemble
either those of layer iii or of layer vi.

(vi) Lamina multiformis is rather poor in cells. ‘These are
arranged in regular radial rows, and many of them have
strong, downwardly directed processes.

Anterior Precentral Type (fig. 2).
J

The whole depth of the cortex is about 2°2 mm. ; it is built
up as follows :

(i) Lamina zonalis, breadth just over ‘1 mm.

(i) Lamina granularis externa, breadth about °2 mm.
This is composed of small pyramids and granule cells, the
former measuring 10-15 » in length by 9-12 w in breadth,
and the latter being some 9-12 w in diameter.

(iii) (iv) and (v) Lamina pyramidalis, lamina granularis
interna, lamina ganglionaris are not very clearly separated ; a

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 623

narrow strip, such as is figured, hardly shows any lamination,
but if a wide area of cortex is examined under the low power
of the microscope ove can distinguish (1) an upper zone
(lia) consisting mostly of medium-sized pyramids, which
stretches downwards to a depth of about -8 mm. (2) A
tendency to form a definite layer of large pyramids (1115) at
a depth of ‘9-1-2 mm. from the surface. The size of these
cells may be 30-50 x in length by 20-25 in breadth. (3) A
stratum, rather poor in cells, extending from 1:2-1'4 mm.
(4) Another, but less well-defined, layer of large pyramidal
cells (v) at 1'-4-1°6 mm. These celis are in some places rather
larger than those at ib, in other places about the same size
or rather smaller ; they are in all places much less numerous.
A few granule cells and small triangular and irregular-shaped
cells are present between, and interspersed with, both the
upper and lower layers of large pyramids.

(vi) Lamina multiformis occupies the rest of the cortex ; it
contains cells of varied shapes, the most prevalent being
roughly triangular cells with long processes extending both
upwards and downwards.

Extent and Boundaries.—The precentral cortex on the
mesial surface is bounded below by the sulcus cinguli; on the
lateral surface it extends posteriorly to the bottom of the sulcus
centralis; below it falls considerably short of the Sylvian fissure.
Along its free anterior and lower border it passes gradually
into the anterior precentral type.

The anterior precentral type is also bounded below on the
mesial surface by the sulcus cinguli; along the supero-mesial
border it extends to more than half the distance between the
upper end of the sulcus centralis and the frontal pole. Its
upper anterior extension forms a broad band on the lateral
surface, the posterior part of which is bounded below by the
horizontal limb of the sulcus arcuatus. The vertical portion of
that fissure serves as the anterior boundary of the narrow strip
of this cortex, which lies in front of the lower portion of the
precentral area. Approaching the Sylvian fissure the anterior
precentral type alters considerably in character; the large

624 H. Ha. J. SCHUSTER.

pyramids, both supra- and sub-granular, become smaller and
more densely packed together, and the lamina granularis
interna is more easily recognisable. In this condition the
cortex is prolonged forward beyond the lower end of the
sulcus arcuatus, where it passes gradually, on the one hand,
into the frontal, and, on the other, into the posterior orbital
type. In structure tlis lower forward extension of the
anterior precentral bears a marked resemblance to the
anterior part of the posterior orbital cortex. In position it
has some points in common with Brodmann’s type 10, which
it resembles in having a layer of granules distinguishable but
not well developed.

Posterior orbital type (fig. 5).

The position of the particular strip of cortex shown in
fig. 5 was near to the posterior border of the orbital surface
just lateral to the suleus orbitalis. The cortex is here
1'8 mm. in depth. The lamina zonalis extends to a depth of
‘25 mm., and is succeeded by a very poorly developed lamina
eranularis externa; this in turn gives place to the lamina
pyramidalis. The lower and larger pyramids of the latter
form together with the cells of the lamina ganglionaris a zone
rich in cells of moderate size, which extends from a depth of
‘7 mm. to a depth of 1°3 mm. A faint indication of the lamina
granularis interna lying at a distance of about °‘9 mm. from
the surface suggests that only the upper border of this zone
should be reckoned to the lamina pyramidalis, and that the
greater part of it is derived from the lamina ganglionaris.
The lower half millimetre of the cortex is occupied by the
lamina multiformis, the greater proportion of the cells of
which are of a broad pyramidal form with basal processes
extending horizontally. These cells tend to be arranged in
horizontal rows, separated by tangentially running bundles

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 625

of fibres, a type of arrangement which, though obvious even
in a narrow strip lke that figured, becomes very conspicuous
when a considerable width of cortex is examined under a low
power.

The posterior orbital type is of practically identical structure
with Brodmann’s type 11, but it shows some differences in its
extent and boundaries.

According to Brodmann, this type covers the greater part
of the orbital surface in the hemisphere of Cercopithecus,
being absent only from its anterior portion and the gyrus
rectus. In Papio a type of cell-lamination exactly answering
to the description given above is found only in the posterior
quarter of this part of the hemisphere, and there it extends
across the gyrus rectus, and is in continuity with the anterior
limbic type over the orbito-mesial border. In front it extends
further forward along the lateral than alone the mesial
border, but 1t gradually changes its character; the internal
layer of granules becomes more marked, and the tangential
arrangement of the lamina tmulitiformis less marked, till
finally, at about the position indicated in the figure, it is
definitely replaced by frontal or prefrontal cortex, though
where the exact boundary may be it is impossible to say
owing to the gradual nature of the transition.

The intermediate precentral area of Campbell is of approxi-
mately the same structure and extent both in man and in the
higher apes (orang and chimpanzee). In_ structure it
resembles the precentral (motor) cortex in almost all points,
except that no giant Betz cells are present in the internal
layer of large pyramidal cells. The elements of this layer
are smaller and less numerous than those of the outer layer
of large pyramids. Between the two layers is a stratum
poor in cells, which occupies the position of the layer of
stellate (granule) cells present in most other regions. ‘his
type of cortex extends from the mesial surface, over the
supero-mesial border, then downwards and outwards over the
lateral surface, and ends by occupying a considerable area of
the orbital face of the hemisphere. On the mesial surface it

626 i. H. J. SCHUSTER.

is bounded behind by the precentral area, below by the
calloso-marginal fissure, and extends forward for a cousider-
able distance. Its anterior boundary crosses the supero-
mesial border about 2 em. in front of the precentral area in
the chimpanzee, and somewhat further forward in the orang.
On the lateral surface it forms a zone lying in front of the
precentral area, which is broad above, but becomes narrower
lower down, being bounded in front for a short distance by
the sulcus arcuatus.

As it nears the Sylvian fissure it extends forwards over the
lower portion of the suleus arcuatus and occupies a more or
less rectangular area bounded above by the sulcus rectus and
below by the superior limiting sulcus of the island of Reil—
the part of the hemisphere which, according to Campbell,
corresponds to the pars basilaris of the human brain. In
front it invests the upper end of the suleus fronto-orbitalis,
“then turning abruptly downwards, and still following the
fronto-orbital sulcus, it coats the convolution forming its
anterior wall and is finally arrested well down on the inferior
(orbital) surface by the sulcus orbitalis.”

In Brodmann’s description of the brain of Cercopithecus
the area which corresponds to Campbell’s intermediate pre-
central is occupied by three types of cortex. ‘ype 6, which
agrees in structure with the intermediate precentral, has
approximately the same extent on the mesial surface and on
the upper and middle portions of the lateral surface, but it
never extends far beyond the sulcus arcuatus, which lies along
its whole length just behind the anterior boundary. The
region roughly corresponding to the pars basilaris is occupied
by type 10, which in the orbital surface gives place to
type 11.

Type 11 resembles type 6 in the absence of a layer of
granules but differs from it in the following points: (1) The
external and internal layers of large pyramids (Brodmann’s
layers 11 6 and v) are fused to form a clearly delimited band,
the cortex is narrower, and to judge from the photographs
the cells are considerably smaller.

CORTICAL CELL. LAMINATION OF PAPIO HAMADRYAS. 627

In Type 10 the granule layer is present but poorly deve-
loped. Types 10 and 11 agree in a point which Brodmann
regards as important, namely, that owing to the tangential
arrangement of the fibres in the lower part of the cortex the
cells of the innermost layer tend to lie in tangential rows
and not in radiating rows more or less vertical to the
surface.

The Frontal and Prefrontal Types (figs. 3 and 4).

OO! ONOGZOTOVSV SS
° 9 oO

The drawing of the frontal cortex (fig. 3) is taken from the
lateral surface of the hemisphere on the upper side of the
sulcus rectus, about two thirds of its length from its posterior
end. Here the depth of the cortex is approximately 2 mm.
The lamina zonalis (i) occupies the upper ‘2 mm. of this.
The lamina granularis externa (1) extends to a depth of
rather over *5 mm., while the lamina pyramidalis (iii) forms a
zone of which the lower border lies ‘9 mm. from the surface.

There is nothing remarkable about any of these layers; as
is usually the case the pyramids of layer iii increases in size
from above downwards, but the large cells near the lower
border do not form at all a well-marked layer. The lamina
granularis interna (iv) extends from a depth of 9 to a depth
of 1-2 mm. or lower, but it is overlapped by the lamina
ganglionaris (v), the large cells of which spread upwards
among the granules and downward to a depth of about
14mm. The rest of the cortex is occupied by the lamina
multiformis (vi), which fades away into the white matter.
The cells of this layer are more concentrated near its upper
border, thus forming a distinct band between which and (v)
is a slightly clearer stratum. This type of cortex corresponds
exactly with Brodmann’s type 9 or Campbell’s “ frontal.”

The prefrontal type is illustrated in fig. 4—a drawing of a
strip of cortex taken from the gyrus rectus about half-way
along it.

628 E. H. J. SCHUSTER.

The prefrontal type differs from the frontal in its compara-
tive poverty in cells and in the smaller size of the cells of
layers 111) and v, particularly the former. Its depth is also
less. Measurements of the cells in the two layers mentioned
were made near the posterior boundary of the frontal region
and in the prefrontal. It was found that in the former
position the largest cells at 1115 were about 40 in length x
304 in breadth, while in the latter 50,4 x 23 represents
approximately their dimensions. ‘lhe general average is,
however, in each case much less. The corresponding figures
for layer v are : in posterior part of the frontal area about the
same as for layer ili b, while in the prefrontal area, 35 x
27 « roughly indicates their size. In fig. 3, which is taken
fairly far forward in the frontal region, it will be noticed that
the subgranular pyramids are rather larger than those
situated above the granules, and that the cells of each layer
are slightly larger than the corresponding cells in fig. 4.

The extent of the lamina multiformis and the shape of its
cells depend to a great extent to the prevalent direction of
the fibres of the underlying white matter; where this is
tangential the layer is shallow and its cells tend to be drawn
out tangentially ; where the fibres radiate upwards towards
the surface the cells are elongated along an axis vertical to
the surface, and the layer tends to be deep. This effect of
the direction of the fibres is not always confined to the lamina
multiformis, but the layers lying above it may all be involved.
[t is not contined to one part of the hemisphere, but may be
noted in all. It is mentioned here because, in the part of the
prefrontal region figured, the fibres are arranged tangentially,
while in fig. 3 they are radial in direction. It seemed desir-
able to explain that this difference is not an essential difference
between the two types. In the region of the frontal pole the
direction of the fibres of the prefrontal cortex is radial, ani
the result is a marked difference of appearance in the shape
and arrangement of the cells, particularly of the lower layers.
The condition described by Brodmann as type 12 may thus
be distinguished: “slender pyramids,” “layer vi much

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 629

extended and passing gradually into the white matter through
isolated, scattered spindle-cells.”

Extent of Frontal and Prefrontal Types.—The
extent and boundaries of the frontal and prefrontal types are
shown in the figures, and hardly call for verbal description.
The limits are in most places vague and indefinite, but an
exception to this statement must be made in the case of the
sulcus arcuatus, which forms the posterior, and, to a certain
extent the upper boundary of the frontal area on the lateral
surface. On the anterior wall of that fissure the cortex is
intermediate in character between the frontal and anterior
precentral types.

The granule layer is slightly less accentuated than in the
former, while a few very well developed pyramidal cells, lying
some above it and some below it, are distinctly suggestive of
the latter type. This intermediate strip corresponds to
Brodmann’s type 8. A difference will be noted in the region
between Papio and Cercopithecus, namely, that in the latter
the anterior precentral cortex extends in front of the sulcus
arcuatus and the intermediate type 8 lies well in the surface ;
in the former the anterior precentral type stops at the bottom
of the fissure.

The whole of the frontal lobe in the chimpanzee and the
orang lying in front of the intermediate precentral and
limbic areas is covered by the frontal and prefrontal types
of cortex. ‘These agree in the possession of a distinct layer
of granules, which is broad and well-defined, but not so
densely crowded with cells as the corresponding stratum of
the parietal or temporal areas. Above and below there is in
each case a layer of large pyramids. ‘The chief point of
difference lies in the smaller size, slenderer forms, and
reduced numbers of the constituent elements of these two
layers in the prefrontal area. ‘here is no well-marked line
of demarcation, but the large pyramids, which are best
developed near the posterior border of the frontal area,
evadually undergo reduction in size and number.

Brodmann’s description of the corresponding region in the

VOL. 56, PART 4.—NEW SERIES. 44

630 E. H. J. SCHUSTER:

hemisphere of Cercopithecus agrees in the main very well
with Campbell’s account summarised above. ‘he slight
differences to be noted may be due to the gradual nature of
the transition between one type of cortex and its neighbours.
Thus Brodmann recognises four different types against
Campbell’s two, namely, Nos. 8, 9, 12, and 25. Of these,
No. 9 clearly corresponds with Campbell’s frontal type, and
No. 12 with the prefrontal, though the extent of the area
occupied by No. 12 is relatively considerably less.

No. 8 lies immediately in front of the sulcus arcuatus ; it
occupies a narrow strip of the lateral surface of the hemi-
sphere, bounded behind by type No. 6 and in front by type
No. 9. In structure it resembles the posterior portion of
Campbell’s frontal area, being intermediate between the two
types, which it separates. No. 25 lies completely on the
mesial surface, where it occupies a comma-shaped area between
No. 9 (frontal area) and No. 24 (the anterior limbic area).
Its structure is intermediate between these.

ParreraL OccrprraL AND TEMPORAL LOBES.

Post-central Type (fig. 6).

The post-central type of cortex is illustrated in fig. 6,
which is taken from the crown of the post-central gyrus a
little way below the anterior end of the sulcus post-centralis
superior (pes.). The cortex is here 1°7 mm. in depth, of which
the lamina zonalis (i) occupies the upper*limm. ‘The Jamina
granularis externa (i1), composed as usual of small pyramids
and granules, is fairly well developed, but not so closely
crowded with cells as to be sharply marked off from the under-
lying lamina pyramidalis; it reaches to a depth of about
25mm. The lamina pyramidalis (iii) is very distinctly sub-
divided into an upper stratum (iii @) containing medium-sized

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 6381

pyramids in no wise remarkable, and a lower stratum (iiid),
which is very conspicuous, and constitutes the most charac-
teristic feature of the post-central cortex. i115 extends from
a depth of 5 mm. to a depth of *85 mm.,and its principal
constituents are large cells closely crowded together, and
lying about three deep. Their shape and approximate size
may be gathered from the drawing; they are elongated pear-
shaped or pyramidal cells drawn out above into a broad
process which takes the stain rather faintly. It is difficult
to give a numerical indication of their size, but this may
be as much as 60 or 70 x30. The lamina granularis
interna is a dense layer of granules intermixed with small
pyramids ; its lower limit is*l mm. below that of i116, but
above it is extended by columns of small cells which lie
between the large elements of that lamina.

Closely underlying iv is the lamina ganglionaris v. This
consists of a number of triangular quadrilateral and other
elements of comparatively small size, among which are
scattered occasional pyramids of notably superior size; these
celis, which lie near the lower border of the layer, at a depth
of 1:2 mm., are inferior in size to the supra-granular pyramids,
aud differ from them also in shape; their cell-bodies are
more slender, and their apical processes narrower and more
darkly staining. ‘The rest of the cortex is occupied by the
lamina multiformis (vi), which is separated from v by a zone
comparatively poor in cells. It cannot be subdivided into
a lamina triangularis and a lamina fusiformis.

The cortex described above corresponds to Brodmann’s
types Nos. land 2. As it dips over in front into the sulcus
centralis it alters somewhat in character; the whole cortex
becomes narrower ; the large cells at i115 become reduced in
size and number; the layer of granules (iv) grows less
distinct, and the lamina ganglionaris suffers the same changes
as 1116.

According to Brodmann, the large cells at 116 and v, which
form the most characteristic feature of the post-central
cortex, are larger and more numerous near the posterior lip

632 E. H. J..SCHUSTER.

of the gyrus centralis posterior than at the summit of the
eyrus. He thus distinguishes two types—No. 1 clothing the
summit of the gyrus, and No. 2 its posterior lip. I do not
find that this is the case in Papio. Here the post-central
type is at its best in the middle portion of the gyrus, becomes
less well-developed near the posterior border, and as it passes
downwards into the intra-parietal fissure it loses its distinctive
features as it changes into the neighbouring type. A gradual
reduction of the size and numbers of these large cells, par-
ticularly those of the lamina ganglionaris, can also be
observed as one passes downwards over the lateral surface ;
they are largest and numerous in the upper half of the gyrus,
and become very much less conspicuous near the lower end
of the sulcus centralis. On the mesial surface the large cells
of the lamina ganglhionaris are particularly well represented,
while those of the lamina pyramidalis inferior are neither so
large nor so numerous.

Extent of the Post-central Type.—The post-central
cortex, modified locally as described above, occupies on the
mesial surface the posterior portion of the para-central lobule.
On the lateral surface it extends anteriorly to the bottom of
the sulcus centralis, and occupies the greater part of the
gyrus centralis posterior. Above, where the sulcus intra-
parietalis bends backwards, it passes into the superior parietal
type, while below it extends in front of the lower end of the
sulcus centralis, and becomes continuous anteriorly with the
lower portion of the anterior precentral, while posteriorly it
changes into the inferior parietal cortex.

Superior Parietal Type (fig. 7).

The superior parietal type is illustrated in fig. 7. The
general plan of its layers and their relative extent is very
much the same as that of the post-central type. ‘The most

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 688

striking difference is to be found in the lamina pyramidalis
inferior (1b). Here, though numerous large pyramids are
present forming a fairly definite layer, they are notably inferior
in size to those of the post-central type, while in number also
they are conspicuously less. ‘l'hey are confined to a narrow
zone lying immediately above the granules, while in the post-
central cortex they form a broad stratum which occupies
more than half of the whole Jamina pyramuidalis,

Extent and Boundaries.—the superior parietal cortex
occupies on the lateral surface the greater part of the area
bounded laterally by the sulcus intra-parietalis, mesially by
the border of the hemisphere, posteriorly by the upper part
of the sulcus lunatus, and anteriorly by the post-central
cortex. It extends also on to the mesial surface, where it
lies between the sulcus cinguli and the ascending limb of the
sulcus subparietalis. Below it passes into the posterior
limbic type, and behind into the inferior parietal.

Inferior Parietal Type (fig. 8).

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The inferior parietal cortex is illustrated in fig. 8, which
is taken from the lateral surface of the hemisphere just
posterior to the sulcus intra-parietalis at a level a little below
the posterior end of the sulcus temporalis superior. The
cortex is here 1:9 mm. deep. The lamina granularis in-
terna (iv) is very well marked ; it 1s about *2 mm. in breadth,
its upper margin lying at a depth of °8 mm. Above it lesa
zone of large pyramids (i 6) of approximately the same
breadth. ‘The cells contained in it are smaller than their
homologues in the superior parietal cortex. In shape they
are rather siender : their length may be as much as 40 p, but
their breadth is never much over 20 u.
Below the granules is a fairly well-developed lamina
ganglionaris (v). ‘lhe majority of the cells in this layer are

634 E. H. J. SCHUSTER.

much smaller than those at iii b, but scattered among them
are a few of outstanding size, yet not so large as the corres-
ponding cells of the post-central or superior parietal regions.
The lamina multiformis may be divided into an upper lamina
triangularis and a lower lamina fusiformis. ‘The cells of the
former are large and numerous with broad triangular or
quadrilateral forms predominating, while those of the latter
are smaller and slenderer, and many of them approach the
fusiform in shape. On the mesial surface the breadth of the
cortex is about + mm. less. In this reduction, which is
associated with the more tangential arrangement of the fibres
in the underlying white matter, the Jamina multiformis is
alone much affected. Not only is this layer reduced in
breadth, but its constituent cells are reduced in size and
numbers.

This type of cortex bears a considerable resemblance to
the temporal and some to the frontal cortex. It differs from
the latter, however, in many important points. It is of about
the same breadth but much richer in cells. The lamina
zonalis is narrower, the lamina granularis externa more
clearly defined. The large pyramids at ii b are much more
numerous and form a more distinct layer. ‘The lamina
granularis interna is also much richer in cells, and more
obviously separated from the adjacent strata. The lamina
ganghonaris is not so well developed, but the lamina imulti-
formis is broader and richer in cells, and does not show the
gradual transition to the white matter which Brodmann
observes is a characteristic of the frontal lobe.

Hxtent and Boundaries.—The inferior parietal cortex
hes behind and below the superior parietal both on the lateral
and on the mesial surface. On the mesial surface it covers a
broad area bounded in front by the sulcus subparietalis and
behind by the ramus parieto-occipitalis of the intra-parietal
fissure. Below it passes gradually into the posterior limbic and
occipital types. Above it passes over the supero-mesial border
and forms a narrow strip between the superior parietal area and
the upper end of the sulcus lunatus, then crosses the sulcus

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 635

intra-parietalis, and invests the triangular area lying between
that fissure and the upper end of the sulcus temporalis
superior; below this it extends forwards and downwards on
the upper side of the Sylvian fissure; it gradually becomes
narrower, and finally dies away a little in front of the lower
end of the sulcus centralis.

The inferior parietal cortex corresponds in position and
structure to Brodmann’s type 7. hat part of it which lies
in the mesial surface and on the lateral surface above the
intra-parietal corresponds with Campbell’s parietal area, but
is notably less in extent than in the higher apes or in ian.
The remaining area is according to Campbell clothed with
temporal cortex, but there is so great a similarity between
the two types that there is no great antagonism in the two
descriptions.

The calcarine type of cortex is illustrated in fig. 9, a draw-
ing taken from the wall of the lower branch of the posterior
bifurcation of the calcarine fissure. Here the cortex is just
over 1*5 mm. in breadth.

The lamina zonalis occupies rather more than the upper
‘1 mm. of this, and the lamina granularis externa forms a
narrow border of small pyramids and granules below it. The
lamina pyramidalis, consisting of small- and medium-sized
pyramids, extends to a depth of ‘4mm. It is densely crowded
with cells, which follow the usual arrangement in being larger
near the lower border. The largest of them do not appear to
exceed a length of 25 uw. What is considered by Brodmann to
be homologous with the lamina granularis interna occupies
about half the total breadth of the cortex, extending, as it
does, from a depth of -4 to a depth of 1:15 mm. It cau be
divided into three strata: (1) An upper layer of granules

636 i, H. J. SCHUSTER.

interspersed with a few small pyramidal cells; this extends
from ‘4-05 mm. (2) A middle layer, 25 mim. wide, corre-
sponding to Campbell’s layer of large stellate cells. This is
rather poorer in cells than the rest of the cortex, being the
region occupied by the stria of Gennari. A considerable
number of cells are, however, present, and these seem to be
concentrated rather nearer to the upper than to the lower
border. ‘The most conspicuous are the large stellate cells
themselves, the length and breadth of which may be about
254. The majority of these seem to be in the form of rather
broad pyramids, sending out an apical process and horizontal
basal processes. Scattered among them are granules and
smaller pyramids. (3) The lower layer of granules extends
from a depth of -8 to a depth of 1:15 mm. This can be clearly
subdivided again into an upper layer of large pyramidal and
irregular-shaped cells and a lower layer consisting almost
entirely of small granules. The lamina ganglionaris is com-
paratively poor in cells, and the cells themselves are small.
The solitary cells of Meynert, which are reckoned to belong
to this layer, are actually sunk within the upper part of the
underlying lamina multiformis; they are not very numerous,
and only one of them is shown in the drawing; in size they
may be about 30 « in length by about the same breadth.
The lamina multiformis is very narrow, but richer in cells
than the layer above it; the cells themselves are small.

Kxtent and Boundaries.—The calcarine cortex is on
the mesial surface largely confined to the calcarine fissure ; at
the posterior bifurcation of the latter it emerges on to the
surface and covers the whole area lying behind it. Spreading
on to the lateral face of the hemisphere it occupies the greater
part of the large area, bounded in front by the sulcus lunatus,
to the hp of which it almost extends. It covers the walls of
the lateral occipital fissure. Near the occipital pole it does
not extend much below that suleus, but in front it spreads
downwards much farther, yet always falls far short of the
sulcus occipitalis inferior.

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 637

Occipital Type (fig. 10).

The occipital type is illustrated in fig. 10, which is drawn
from the lateral surface of the hemisphere between the sulcus
lunatus and the upturned end of the sulcus temporalis superior.
The cortex is here 1-6 mm. deep, of which the lamina zonalis
occupies rather over ‘1mm. ‘lhe lamina granularis externa
is not clearly separated from the subjacent lamina pyramidahs.
The latter extends downwards to a depth of ‘75 mm., and its
lower half is characterised by the presence of a number of
elongated pyramids, which reach a length of 50 4 and more.
The lamina granularis interna is narrow but very well marked ;
the granules are arranged closely together, and very few cells
of other forms are present among them except im so far as
they themselves extend upwards among the bases of the large
pyramids. The lower border of this layer lies at a depth of
‘9mm. The lamina ganglionaris is composed of rather small
pyramids ; it is poor in cells and appears as a clear band
under the low power cf the microscope. It is the conspicuous
presence of a layer of large pyramids above the granules,
coupled with their conspicuous absence below, which gives
the distinctive character to this type of cortex. The lamina
multiformis is well developed and about ‘4 nm. broad. Along
its upper border the elements are larger and more triangular,
while near its lower edge they are smaller and more fusiform.

The occipital type, which corresponds to Campbell’s visuo-
psychic and Brodmann’s types 18 and 19, is not of uniform
structure over the whole area which it invests. Immediately
adjacent to the calcarine area, which it completely surrounds,
it is much narrower than in the position selected for illus-
tration, fewer cells are present, and the lamina ganglionaris
is relatively clearer. As it approaches the temporal .or
inferior parietal areas which form the greater part of its
anterior boundary it assumes more the character of those

638 E. H. J. SCHUSTER.

types. It is this intermediate structure which forms Brod-
inann’s type 19.

ixtent and Boundaries.—The occipital type completely
surrounds the calcarine, the boundary between the one type
and the other being quite abrupt and definite. Very different
is the transition between the occipital and the inferior parietal
temporal and posterior limbic types, into which it passes
anteriorly. This is so gradual that it is almost impossible to
say where the oue ends and the other begins, and the boun-
dary is in consequence very difficult to describe. Under
these circumstances I do not attempt a verbal description,
but merely refer to the figures 4, B, ©, D.

Temporal Type (fig. 11).

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The essential features of the cortex covering the greater
part of the temporal lobe are shown in fig. 11, which is taken
from the middle temporal gyrus in its posterior half. Its
depth is here about 2 mm.

The lamina zonalis extends to a depth of -2 mm. The
lamina granularis externa is closely packed with cells and
fairly well separated from the underlying lamina pyramudalis.
The latter extends to a depth of rather more than half the
whole thickness of the cortex. ‘lhe large pyramids (ub),
which reach a length of 30 jc or more, form a broad zone near
the inner border of the layer.

The lamina granularis interna is exceptionally well
developed, and stretches from 1'l mm. to 13 mm. from the
surface.

Beneath this layer the development of the cortex is poor in
comparison with the parts above, but the lamina ganglonaris
is fairly well developed, though its cells are shghtly smailer
than those at ib. A slightly clearer space separates it from
the lamina multiformis.

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 6389

The description given above applies in its details only to
the middle temporal gyrus. The cortex of the superior
temporal gyrus differs in many respects. It is broader and
poorer in cells. ‘he lamina granularis interna is not so well
developed. ‘he lamina ganglionaris is, on the whole, not so
clear, but contains a few large cells scattered through it.

A change in structure is also apparent as one moves round
the temporal lobe in the other direction. As one approaches
the hippocampal gyrus the cortex becomes narrower, and the
layer of granules less well developed.

The extent of the olfactory cortex is shown in fig. c, but it
has not been thought necessary to give a special description
or drawing of its structure.

Anterior Limbic Cortex (fig. 12).

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‘he anterior limbic type is illustrated in fig. 12, which is
taken from the mesial surface of the hemisphere just above
the anterior portion of the corpus callosum. The cortex here
reaches a depth of 1:9 mm. Its chief characteristic is the
absence of any very definite system of stratification. ‘lhe
lamina granularis externa is very poorly developed ; it passes
gradually into the lamina zonalis above it and is hardly
possible to separate from the lamina pyramidalis below. The
latter is somewhat sparsely populated with small and medium-
sized pyramids until a depth of °6 or*7 mm. is reached ; below
this, extending for about halt a millimetre, is a zone of cells
considerably larger in size and more closely arranged, which
probably represents the lower part of the lamina pyramidalis
(iii b) and the lamina ganglionaris (v). ‘here is no trace ot
the lamina granularis interna. ‘The lamina multiformis is
also poor in cells, of which the upper ones are of fair size and
triangular form, while the lower ones are smaller and more

spindle-shaped.

640 a. H. J. SCHUSTER:

Posterior Limbic Cortex (fig. 13).

‘The posterior limbic cortex is illustrated in fig. 18, whichis
taken from the mesial surface of the hemisphere just above
the posterior end of the corpus callosum. It differs from the
anterior limbic type in (1) a greater richness of cells, (2) the
better development of the lamina granularis externa, (3)
the presence of a lamina granularis interna. ‘he latter,
though clearly defined, is not so well developed as in the
inferior parietal or occipital types, which it resembles in some
respects. ‘lhe lamina ganghonaris follows closely below the
internal layer of granules. It is well developed, the cells on
the whole being larger and more numerous than in the strip
actually drawn.

Extent and Boundaries.—The anterior limbic cortex
lies between the suleus cinguli and the corpus callosum ; it 1s
continued forward as a broad band round the anterior end of
the latter. ‘This anterior portion is separated from the frontal
cortex by an area intermediate in character. Posteriorly it
extends a little way behind the region of the mesial surface,
in which the anterior precentral changes into the precentra!|
type. At this point, by the gradual acquisition of an internal
layer of granules, it becomes transformed into the posterior
limbic type. The latter extends round the posterior end of
the corpus callosum, changing gradually above and behind
into the types of cortex on which it abuts.

The posterior limbic coxtex corresponds to Brodmann’s
type 23, the anterior to his type 24, while the area inter-
mediate in character between the limbic and the frontal is
equivalent to his type 25.

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 641

EXPLANATION OF PLATES 24-30,

Illustrating Mr. EH. H. J. Schuster’s paper, ‘ Cortical Cell
Lamination of the Hemispheres of Papio Hamadryas.”

PLATE 24.

The two hemispheres seen from different points of view to show
fissures and distribution of various types of cortex.

Fig. A.—Dorsal view of both hemispheres.

Fig. 8.—Ventral view of both hemispheres.

Fig. c.—Mesial view of left hemisphere.

Fig. p.—Lateral view of left hemisphere.

Fig. —E.—Mesial view of right hemisphere.

Fig. r.—Lateral view of right hemisphere.

The left hemisphere in figs. A, B, Cc, D is shaded to show the super-
ficial extent of the different types of cortex described. The lettering
on the right hemisphere in figs. A, B, E, and F refers to the fissures.
while the numbers show approximately the position from which the
strips of cortex drawn in figs. 1-15 are taken. These drawings were,
many of them, taken from the left hemisphere, but their position has
for the sake of simplicity been transferred to the right hemisphere in
the diagrams.

FS. Sylvian fissure. c¢. Suleus centralis. are. Sulcus arcuatus.
it. Sulcus inferior transversus. rect. Sulcus rectus. fs. 1, 2,3, 4. Sulcus
precentralis superior and sulcus frontalis superior. orb. Sulcus
orbitalis. jo. Sulcus fronto-orbitalis. T's. Sulcus temporalis superior.
Tm. Sulcus temporalis medius. pes. Sulcus post-centralis superior.
ip. Sulcus intra-parietalis. rpo. Ramus parieto-occipitalis of sulcus
intra-parietalis. jfpo. Fissura parieto-occipitalis. sp. Sulcus subparie-
talis. Sc. Suleus cinguli. 70. Sulcus rostralis. Col. Sulcus collateralis.
rh. Suleus rhinalis. lun. Sulcus lunatus. o7. Suleus occipitalis inferior.
ol. Suleus oecipitalis lateralis. Cal. Sulcus calearinus.

Magnification in figures 1-15 = 130.

EXPLANATION OF SHADING IN FicuREs A, B, C, D, Puate 24.

Precentral cortex (Pl. 25, fig. 1).

Anterior precentral cortex (Pl. 25, fig. 2).

Frontal cortex (Pl. 26, fig. 3).

Prefrontal cortex (Pl. 26, fig. 4).

Posterior orbital cortex (Pl. 27, fig. 5).

Post-central cortex (P1. 27, fig. 6).

Superior parietal cortex (Pl. 28, fig. 7).

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Occipital cortex (Pl. 29, fig. 10).

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Temporal cortex (Pl. 30, fig. 11).

Anterior limbie cortex (Pl. 30, fig. 12).

Posterior limbie cortex (Pl. 29, fig. 15).

Olfactory cortex (Pl. 24, fig. E, rh, and fig. C).

CORTICAL CELL LAMINATION OF PAPIO HAMADRYAS. 643

PLATE 25.

The shading on figs. A, B, C, D in Pl. 24, as shown in the diagram on
the opposite page, corresponds in position to the sections of cerebral
cortex shown in the several figures in Pls. 25 to 30.

Fig. 1.—Precentral motor cortex.
Fig. 2.— Anterior precentral cortex.
PLATE 26.
Fig. 3.—Frontal cortex.
Fig. 4.—Prefrontal cortex.
PLATE 27,
Fig. 5.—Posterior orbital cortex.
Fig. 6.—Posterior central cortex.
PLATE 28.
g. 7.—Superior parietal cortex.
Fig. 8.—Inferior parietal cortex.
PLATE 29.
Fig. 9.—Calearine (visual) cortex.
Fig. 10.—Occipital cortex.
Fig. 15.—Posterior limbic cortex.
PLATE 30.
Fig. 11.—Temporal cortex.

Fig. 12.—Anterior limbic cortex.

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LIFE-HISTORY OF LEPTOMONAS MUSCA DOMESTICR. 645

On Some Stages in the Life-History of Lepto-
monas musce domestics, with some re-
marks on the Relationships of the Flagellate
Parasites of Insects.

By

di. Ss. Dunkerly.

With Plate 31.

In an endeavour to examine the biflagellate character of
Herpetomonas as described by Prowazek (20), I have dis-
sected and examined a number of house-flies. At first
Musca domestica was investigated, as I had supposed that
this was the animal indicated by the word ‘ Stubenfliege.”
In this country, however, M. domestica does not seem to
be commonly infected, as I was unable to find the parasite in
it, and Hewitt (6), who examined a good number of these flies,
was similarly unsuccessful. In the smaller house-fly, Homa-
lomyia canicularis, flagellate parasites were found to be
present, but Hewitt had confined himself to M. domestica.
Still, the infections were very rare. I examined these flies
taken in three distinct localities: Chelsea and Wandsworth
in London, and Benfleet in Hssex. Parasites were found in
flies from each place, but always in a low percentage, about
4. per cent., of the flies examined. Other species of flies! have
been examined, but not in large numbers, so that it is not
surprising that no Herpetomonads have been met with in
them as yet.

‘I take this opportunity of expressing my thanks to Mr. Austen,
who kindly assisted me in the identification of these flies.

NEW SERIES. 45

VOL. 06, PART 4.

646 J. SS) DUNKERIEY.

I will first describe the forms met with by me in Homa-
lomyia canicularis, and afterwards discuss their signi-
ficance. (1) In the midgut (ventriculus) of two flies were
seen some large typical Herpetomonas forms (Pl. 51, fig. 1).
This form had a body 25 n to 30 long, and the flagellum was
30 wlong. Its movements were characteristic, the body being
clumsily swung from side to side by the lashing of the long
thick flagellum. With Giemsa’s stain the double character
of the flagellum described by Prowazek is evident in most
cases. If, as Patton (17) states, this is merely a stage in
division, then it is an unusual type of division, since the
kinetonucleus is not even transversely elongated when the
flagellum has divided along its whole length, this being very
unlike the state of affairs found by me in dividing forms
(Pl. 3], fig. 9). My material for the study of these forms
has been very scanty, and I can only say that they are very
different, both in appearance and size, from the other flagel-
lates met with in the fly.

(2) A commoner form (PI. 31, figs. 2-14) was found in
the intestine, and once in the Malpighian tubules. ‘These
infections were always heavy ones, the parasites occurring in
dense clusters, either on the intestinal wall or free in the
lumen of the intestine. On slides the clusters were seen to
be formed by the typical rosettes, or more correctly, agglo-
merations, with the flagella pointing to the centre, as described
by Woodcock (26) for cultural forms of trypanosomes. ‘I'he
body of this form was 15 to 18, long, and its movement
was rapid and graceful, the anterior part of the body often
undulating. A large number of dividing forms were usually
present (Pl. 31, figs. 6-10). In one case, in which the
forms were particularly elongated (Pl. 31, fig. 14), cysts
were also found, and doubtful intermediate stumpy forms.
The characters to which I wish to draw particular attention
are the varying position of the kinetonucleus and the presence
of an undulating membrane (PI. 31, figs. 3, 4, and 11-14).
(The forms shown in figs. 3, 4, and 11 are from the same fly).
All intermediate stages between the short form, with its

LIFE-HISTORY OF LEPTOMONAS MUSCH DOMESTICM. 647

anterior end truncated, and the elongated one with anterior
extremity drawn out into a membrane attached to the
flagellum, are found (Pl. 31, figs. 3, 4, and 11). A form
resembling that in fig. 11 was found by Chatton and
Alilaire (2) in Drosophila confusa, and described under
the name Trypanosoma drosophila, although the authors
recognised the possibility of its being a stage in the life-
history of a Leptomonas found by them in the same fly. Werner
(24) also described the same form from “Stubenfliegen,”
and named it Crithidia muscw domestice to distinguish
it from the biflagellate Herpetomonas musce domestice
of Prowazek. Miss Mackinnon (14) also, whose paper ap-
peared while this work was in progress, in describing what she
regards as a Herpetomonas from Homalomyia corvina (?)
pointed out the similarity between some of the forms found
by her and the Crithidia of Werner, which would not be
surprising if both are stages in the life-history of organisms
belonging to the same genus, possibly to the same species.
The forms shown in Pl. 31, figs. 12, 13, and 14 possess,
undoubtedly, an undulating membrane, though the flagellum
is not produced beyond it, and these resemble in a striking
manner some stages of Trypanosoma cazalboui in cul-
tures, described by Roubaud (22, pl. viii, figs. 2 and 6), thus
indicating a close relationship between the parasite of a non-
blood-sucking fly like Homalomyia and the trypanosomes of
vertebrates. Patton (18, and 17, p. 142, note), in objecting
to Prowazek’s account of Herpetomonas (which, however,
has been supported by other observers, Lingard and Jen-
nings [12], Roubaud [22]) decided that all uni-flagellate
parasites of insects with the kinetonucleus anterior to the
trophonucleus and without undulating membrane are to be
called Herpetomonas, and that those having the kinetonucleus
posterior to the trophonucleus, and possessing an undulating
membrane, should receive the generic name of Crithidia.
Litthe (18) and Hartmann and Jollos (5) have pointed out that
Patton’s failure to see the characters observed by Prowazek
and others does not prove their non-existence ; and as to his
ADS

648 J. S. DUNKERLY.

use of the name Crithidia, this is certainly a misuse of Leger’s

ce en

term, which Jie applied (8) to a short rounded form,
form de grain d’orge légérement aplati et tronqué a ’extrémité
antérieure . . . , and usually without an undulating mem-
brane. However, it seems from the evidence of the forms
found in Homalomyia that the same organism may be without
an undulating membrane at one stage of its life-history, while
possessing one at another stage. I shall return later in
this paper to this question of nomenclature.

(3) In the rectum, near the rectal glands, were found
masses of small oval bodies (PI. 31, fig. 15) attached to the
rectal epithelium. On examining these in water I was able
to observe the mass apparently swell, as though the walls of
the oval bodies were gelatinous, and after a short time some
of the bodies were seen to become actively motile, with a
small. anterior flagellum (PI. 51, figs. 16 and 17). The flies
containing these cysts had no other flagellate stages in them,
but came from the same locality as those that had. Similar
cysts have been described by Minchin (15) for T. grayi, by
Prowazek (20), Rosenbusch (21), and Mackinnon (14) for
Herpetomonas, the latter having observed them giving rise
to flagellates. The cysts stained with Giemsa (PI. 31, fig. 15)
show a faint trophonucleus and a distinct kinetonucleus, with
a large number of scattered granules stained a deep purple
colour, and have a definite wall surrounded by a remark-
able substance which stains deeply, and may be gelatinous in
nature (vide supra). But iron-hematoxylin shows little of
these peculiar effects (Pl. 51, fig. 17a). The commencement
of development of the flagellum is indicated bya clear area in
Giemsa preparations (Pl. 31, fig. 158), the borders of which
appear to stain with iron-hematoxylin, showing a triangular
area with the kinetonucleus as base (PI. 31, fig. 17a and p),
and the same appearance has been seen in non-flagellate
forms of ‘I’. lewisi by Prof. Minchin, who kindly showed
me his original drawings.

In the life-cycle of Leptomonas, as far as I have investi-
gated it, we find the following forms: (1) A typical Lepto-

LIFE-HISTORY OF LEPTOMONAS MUSCH DOMESTIC. 649

monas (fig. 2), which actively divides in the intestine or in
the Malpighian tubules of the fly (figs. 6-10), producing
(2) very active, slender forms, often with undulating mem-
brane (figs. 11-14). These probably encyst while attached in
large numbers to the rectal wall, and the cysts (figs. 15 and
17a) may be passed out with the feces to give rise to flagellate
forms in another fly, as described by Patton (19) for the
Herpetomonas (? Leptomonas) of Musca nebulo, the Madras
bazaar fly. Bat whether the large Herpetomonas form (fig. 1)
should have a place in this life-history I am at present unable to
decide. Almost certainly the above is but a part of the whole
life-cycle, and the low percentage of infections have prevented
the completion of it up to the present. It might be thought
improbable on & priori grounds that flies in England and in
India should be infected by the same pair of parasites, yet in
smears of house-flies’ guts which Dr. Row brought from India
and kindly left at the Lister Institute, there are large
Herpetomonads and small Leptomonads just as in H. cani-
cularis in England. If these should prove to be different
forms of the same organism, and at the same time have a
try panosome-stage in their life-history, considerable changes
in our nomenclature of flagellate parasites will be necessitated.

As to Prowazek’s description of elaborate autogamy and
hereditary infection in Herpetomonas, one is tempted to
interpret some of his figures (which hardly bear out his
account), as being those of a Sporozoan infection, and [|
hope to publish shortly an account of a Microsporidian which
I have found in Homalomyia.

‘The nomenclature of these forms, interesting on account of
their probable relationship with the trypanosomes, is in a very
confused state, and it is with a view to the clearing up of at
least one part of the vexed question that I wish to re-state the
following facts in their history.

Saville Kent in 1881 (23) established the genera Lepto-
monas and Herpetomonas for uniflagellate parasites found in
a Nematode, Trilobus; and in Musca domestica respec-
tively. The only points of distinction mentioned by him

650 J. S. DUNKERLY.

)
long, and formed rosettes, while Herpetomonas was zz to
44, In. long, and had, at any rate, not been seen in rosettes
or agglomerations. In 1902 Leger (9) found flagellate
parasites in Homalomyia and other Diptera, and named an
elongated form Herpetomonas (sp. var.), while a short
> he called
Crithidia (sp. var.). Later (10, a and Bs), he described
H.subulata from Tabanus as possessing an undulating
membrane, still retaining the name Crithidia for short
pyriform forms. Prowazek (20) in 1904 had investigated
the parasite of the house-fly, and described it as possessing

rounded form, “en form de grain dorge,’

two flagella united by a membrane and arising from an
anterior double basal-granule or diplosome. Novy, MacNeal,
and Torrey, in 1907 (16) followed Leger’s nomenclature for
types found in mosquitoes, their Herpetomonas in cultures
showing an undulating membrane. ‘They described a diplo-
some, not where Prowazek had placed it, but at the posterior
end of the body, and bearing, as they themselves point out,
a considerable resemblance to a Diplococcus, which was
generally adherent to the body of Herpetomonas in the
cultures. Lingard and Jennings (12) in 1906 found in a
Muscid fly forms showing the typical diplosome described by
Prowazek, but most of their figures are not clear, and they
claim to have seen the actual folding of the flagellate to
form the biflagellate condition according to the Prowazek-
Schaudinn theory respecting the origin of the double
flagellum.

The history of Herpetomonas up to this point has been
related in greater detail by Woodcock (25). His conclusions
are—(1) That some of these parasites of mosquitoes are
probably connected with 'rypanosomes of vertebrates ; (2)
some of the typical Herpetomonads found may be simply and
primarily parasites of the insects; (3) that forms adapted
for life in sanguivorous insects, by which are meant
“ Crithidia”’ forms with an undulating membrane, following
Patton’s nomenclature, may be unrelated to any trypanosome

LIFE-HISTORY OF LEPTOMONAS MUSCA DOMESTICM. 651

of a vertebrate. But no forms were then known with an
undulating membrane in a truly non-sanguivorous insect.
In 1908, however, Chatton and Alilaire (2) described flagel-
lates found in Drosophila confusa—a Leptomounas (as
distinct from Prowazek’s Herpetomonas) anda Try pano-
soma without a clear undulating membrane, but with the
blepharoplast at the posterior end of the body. They named
these forms L. drosophile and I’. drosophile, but at the
same time put forward the suggestion that they are really
two stages of the same life-cycle. Werner (24) in 1909, and
Rosenbusch (21) in 1910, have stated that there are two
distinct parasites of the house-fly, a Herpetomonas of Prowazek
and a Crithidia with posterior kinetonucleus, of which Rosen-
busch describes the encystation. Rouband, in an interesting
article in 1909 (22), has used an old generic term, “ Lepto-
monas,” for the uniflagellate parasite of the fly Pyecnogonum,
excluding Herpetomonas of Prowazek, which he also found
in the same fly. He regards, then, Herpetomonas of Prowazek
as biflagellate, and Leptomonas as uniflagellate, with kineto-
nucleus usually anterior, but with a so-called trypanosome
stage in its life-history. The evidence of Rosenbusch (21),
Chatton and Alilaire (2), and Mackinnon (14), and that given
by my figures, all goes to show that a form resembling
Leptomonas of Saville Kent is found in non-sanguivorous
Hies (in three cases, house-flies), developing in the course of
its life-history a form resembling a cultural trypanosome, and
having an encysted stage. The fact that many observers
have seen a large form (shown in PI. 31, fig. 1), which differs
very much in appearance from Leptomonas, renders it possible
that the other observers who fail to see the two flagella are
dealing with a different organism.

This much, however, seems certain: (1) That Leger’s
original pear-shaped Crithidia is only a stage of the Lepto-
monas life-history; also (2) that the “ Crithidia” of later
authors— Patton (18), Woodcock (25)—found in blood-sucking
flies, or in cultures, are in some cases developmental stages of
a Trypanosoma, The evidence of the forms found by me

652 J. S. DUNKERLY.

(Pl. 31, figs. 11-14) in the house-fly, Homalomyia canicu-
laris, shows that Rosenbusch’s Crithidia musecw domes-
tice, and therefore probably Trypanosoma drosophile
of Chatton and Alilaire, are merely forms assumed by a
Leptomonas.

Should Leptomonas or Herpetomonas be the name given
to these parasites of the Insecta? The Leptomonas of
Saville Kent was described as being of a size comparable
with that of the small Leptomonas, of, e. ¢., Homalomyia,
whereas Herpetomonas was evidently a huge form. Again,
Leptomonas was said to form rosettes. A diagnosis based on
morphological grounds is of more value than one depending
upon habitat. At present, therefore, Leptomonas would
appear to be a correct name for the uniflagellate parasites
found in the gut of non-sanguivorous insects, including house-
flies, Pyecnogonum (22), Bombyx (11), and in some plants (7),
while Herpetomonas may be retained as a provisional name
for a large form with peculiar flagellar apparatus and a com-
plicated life-history, as described by Prowazek. Should the
latter prove to be but a stage in the Leptomonas’ life-history,
then Herpetomonas should be merged in Leptomonas, since
the latter would then have been the first which was accurately
described. Crithidia cannot be applied as a generic name to
any form, as 1b has simply been the name given to two stages
in the hfe-history of Leptomonas, or in other cases to what
are probably stages of Trypanosoma. ‘That Leptomonas had
priority over Crithidia was pointed out by Hartmann and
Jollos (5), but it was not clear then that ‘ Crithidia ” was a
form in the Leptomonas’ life-history.

A paper by Flu on parasites of the house-fly, Musca
domestica, appeared (‘ Centralblatt f. Bakt., etc.,’ Bd. lvii,
1911, p. 522) after this paper had been sent to press, and is
in the main confirmatory of the chief points emphasised above.

PROTOZOOLOGICAL LABORATORY,
ListER INSTITUTE,
LONDON.

LIFE-HISTORY OF LEPTOMONAS MUSCH DOMESTIC. 653

REFERENCES.

1. Chagas. — “* Nova tripanozomiaze humana,” ‘Mem. do Inst.
Oswaldo Cruz,’ t. 2. f. 2, p. 159 (Abs. in ‘Bull. Inst. Pust.,’
vill, p. 373).

2. Chatton et Alilaire.—* Coexistence d'un Leptomonas (Herpeto-
monas) et dun Trypanosoma chez un Muscide non vulnerant,
Drosophila confusa, Staeger,” ‘C. R. Soe. de Biol.,’ t. 64, 1908,
p. 1004.

3. Doflein.—‘ Lehrb. d. Prot., 2 Aufl., 1909.

4. Donovan.— Kala Azar in Madras’ (read before Bombay Med.
Congr., February 24th, 1909.)

5. Hartmann and Jollos——‘ Die Flagellatenordnung Binucleata,”
‘Asch: f. Prot, Bd. xix, 1910) Heft, 1.

6. Hewitt.—** The Bionomies, Allies, Parasites, and the Relations of
M. domestica to Human Disease,” ‘ Quart. Journ. Micr. Sci.,’
54, 1909, p. 347.

7. Lafont.—‘ Sur la presence d'un Leptomonas dans trois Euphor-
biacées,’ ‘ Ann. de l’Inst. Past.,’ xxiv, 1910, p. 205.

8. Léeger.—‘“Sur un flagellé parasite de lAnopheles maculi-
pennis,” ‘C. R. Soc. de Biol.,’ liv, 1902, p. 354.

9. “Sur quelques Cercomonadines nouvelles ou peu connues
parasites de intestine des Insectes,” * Arch. f. Prot.,’ Bd. ii, 1902,
p. 180.

10a. ——— “Sur un nouveau Flagellé parasite des Tabanides,” ‘C. R.
Soc. de Biol.,’ t. 57, p. 613.

108. ——— “Sur les affinités de (Herpetomonas subulata, et la
phylogenie des Trypanosomes,” ‘C R. Soc. de Biol, t. 57, p.
615;

11. Levaditi.—* Sur un nouveau Fiagellé parasite du Bombyx mori,”
‘C. R. Acad. des Sciences,’ exli, 1905, p. 631.

12. Lingard and Jennings.—‘Some Flagellate Forms found in

13

14

15

Diptera,’ Adlard and Son, London, 1906.
. Lihe.—*“ Die im Blute schmarotzenden Protozoen und ihre nachsten
Verwandten,” Mense’s ‘ Handbuch der Tropenkrakh., 1906.

. Mackinnon. —‘* Herpetomonads from the Alimentary Tract of
certain Dung-flies,” ‘ Parasitology,” iii, September, 1910, p. 255.
. Minchin.—* Investigations on the Development of Trypanosomes

in Tsetse-flies, etc.,” ‘Quart. Journ. Micr. Sci.,’ 52, p. 159.

654 J. S. DUNKERLY.

16. Novy, MacNeal and Torrey.—‘ The Trypanosomes of Mosquitoes
and other Insects,” ‘Journ. Infect. Diseases,’ iv, No. 2, p. 223.

17. Patton.—* The Life cycle of a Species of Crithidia parasitic in
Gerris fossarum,” ‘ Arch. f. Prot.,’ xii, 1908, p. 131.

18. ——— “A Critical Review of our Present Knowledge of the
Hemoflagellates and Allied Forms,” ‘Parasitology, ii, May,
1909, p. 91.

19. ———— * Experimental Infection of the Madras Bazaar fly, Musca
nebulo, with Herpetomonas muscew domestic,” * Bull.
Soc. Patholog. Exot.,’ iii, p. 264.

20. Prowazek.—*‘ Die Entwickelung von Herpetomonas,”’ ‘Arb. a. d.
Kais. Ges.,’ xx, 1904, p. 440.

21. Rosenbusch.—* Kine neue Encystierung bei Crithidia musce
domesticex,” ‘Centr. f. Bakt.,’ Bd. lv, 1910, p. 387.

22. Roubaud.—* Les Trypanosomes pathogenes et la Glossina
palpalis,” * La Maladie du Sommeil au Congo Frangais,’ 1909.

23. Saville Kent.—‘ Manual of the Infusoria,’ 1880-81.

24. Werner.—* Uber eine eingeisselige Flagellatenform im Darm der
Stubenfliege,” ‘Arch. f. Prot.,’ xiii, 1909.

25. Woodcock.—* The Hwmoflagellates and Allied Forms,” in Lan-
kester’s ‘ Treatise on Zoology,’ vol. i, fase. i, 1909, p. 193.

26. ——— “Studies on Avian Hemoprotozoa,” ‘Quart. Journ. Mier.
Sci.,’ 55, 1910, p. 641.

EXPLANATION OF SPLATEH 31;

Illustrating Mr. J. S. Dunkerly’s paper “ On Some Stages in
the Life-history of Leptomonas musce domestica,
with Some Remarks on the Relationships of the Flagel-
late Parasites of Insects.’

| All figures are outlined with the aid of Zeiss-Abbé drawing apparatus,
and are drawn at a magnification of 2400. ]

Fig. 1.—Large Herpetomonas from stomach of Homalomyia
canicularis. Osmic vapour, Giemsa.

Fig. 2.—Leptomonas from intestine of H. canicularis, showing
distinct blepharoplast. Flemm.-Fe. ham.

Figs. 3 and 4.—Leptomonas from intestine of H. ca nicularis,
showing varying positions of the kinetonucleus. Schaud-Fe. hem.

LIFE-HISTORY OF LEPITOMONAS MUSCH DOMESTICH. 655

Figs. 5-10.—Leptomonas from intestine of H. ¢ suoublen is; various
stages in division. Flemm.-Fe. hem.

Figs. 11-14.—Leptomonas from intestine of H. canicularis;
trypaniform individuals, Schaud.-Fe. hem.

Fig. 15.—Cysts of Leptomonas musce domestice from rectum
of H. canicularis, showing scattered nuclear material. Osmic vapour,
Giemsa.

Fig. 16.—Small flagellate forms a few minutes after leaving cyst.
Osmic vapour, Giemsa.

Fig. 17.—Small flagellate forms a few minutes after leaving cyst
showing development of the flagellum. Flemm.-Fe. hem.

Quart. fournMicr: Se. Vol. ENS L.31

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LEPTOMONAS MUSCAE DOMESTICAE.

ON MERLIA NORMANI. 657

On Merlia normani,a Sponge with a Siliceous
and Calcareous Skeleton.

By
R. Kirkpatrick.

With Plates 32-38 and 5 Text-figures.

A coop deal of the work in connection with the following
investigation was done at the Lister Institute, in the labora-
tory of Professor E. A. Minchin, and I take this opportunity
of thanking him for the continual help and instruction which
he very kindly gave me in methods of technique.

Also I would express my sincere thanks to Senhor A. C.
Noronha, who accompanied me to Porto Santo to help with
dredging for Merlia.

Most of the drawings have been done by Mr. P. Highley,
who has put them on stone. The drawings of young stages
of Merlia on Pl. 38 and figs. 1-4 on Pl. 56 were done by
Miss Rhodes.

In 1908 Canon Norman, who had been working out the
Polyzoa of Madeira, sent to the Natural History Museum four
small, dried, incrusting calcareous organisms resembling
Polyzoa. The crusts were about a square centimetre in area,
anda millimetre or less in thickness. They had been detached
from a small mass of calcareous conglomerate hooked up by a
fisherman from sixty fathoms off Porto Santo island, about
twenty miles N.E. of Madeira.

The specimens were covered with a thin yellow pellicle
stretching across a white calcareous network, with very
minute polygonal meshes, and with small rough tubercles
rising from the nodes and pushing up the pellicle. A vertical

VOL. 56, PART 4.—NEW SERIES. 46

658 R. KIRKPATRICK.

section showed a series of vertical tubes divided up by
horizontal partitions or tabule. The vertical walls were
imperforate, but marked with longitudinal vertical sutures ex-
tending from the upper surface to the base, one suture being
between any two tubercles. The horizontal tabule usually
had a central hole or slit, but were sometimes imperforate. In
the uppermost spaces of this honeycomb-like framework were
bundles of slender pin-shaped spicules. In the small frag-
ment that could be spared for the making of preparations |
found a ‘“‘tuning-fork ” spicule, seemingly imbedded in one
of the tabule (PI. 38, fig. 6).

Apparently the pin-shaped spicules were not present in the
particle of Merlia used for decalcification, and I wrongly
concluded that these spicules were calcareous and that they
had been dissolved in the acid.

I named the incrusting organism Merlia normani, and
regarded it as a Pharetronid sponge.’ (1) Even if I

1 Tf it had been lawful to base any opinion at ail on the investigation
of such scanty material, then the conclusion arrived at was, I think, a
legitimate one. * Tuning-fork” spicules with thick, parallel, wide-apart
prongs have been found only in Pharetronid sponges. Finding this
rare and peculiar form of spicule seemingly imbedded in one of the
tabule of a mysterious calcareous skeleton unlike that of any known
recent organism, it seemed justifiable to conclude that the spicule
belonged to the framework, and that therefore the latter had been
made by a calcareous sponge. The upper surface of the skeleton of
Merlia shows, too, certain resemblances to that of the Pharetronid
sponges, Porosphera and Plectroninia. Further, a Pharetronid
—Murrayona phanolepis, Kirkp. (‘ Proc. Roy. Soe.,’ 1910)—has
now been found, in which solid skeleton fibres are devoid of an axial
core of spicules, and it was on these characters that I founded the sub-
family Merlinew. The spicule, which sent me on the wrong track, was
a genuine “tuning-fork” and not a simulacrum made by some boring
fungus or Alga, for when I crushed under the cover-slip the fragment
of sponge containing the spicule, the latter floated out solid and free
into the balsam. At present only three Pharetronid Lithonine sponges
are known with a similar kind of tuning-fork, and these have been
recorded from the Indian Ocean and Pacific. Off Porto Santo Island,
in submarine holes or caves, possibly almost inaccessible to dredges,
there must be a Pharetronid sponge. Unfortunately I failed to secure
examples, in spite of twelve days’ dredging.

ON MERLIA NORMANI. 659

had known that the pin-shaped spicules were siliceous—as,
indeed, they were—I would have considered them as part
of a siliceous sponge growing overa Pharetronid. In January,
1909, mainly with the object of procuring living specimens
of Merlia, I spent a winter holiday at Madeira and Porto
Santo. After dredging for nine days off the latter island I
found the sponge in sixty fathoms off a little rocky islet
called Cima, at the 8.E. corner of Porto Santo. The living
specimens were in the form of little bright vermilion crusts,
with a smooth surface. At first, when removed from
the water, nothing else was seen excepting the bright
smooth patch of colour, but soon the surface sank a little,
and the porcelain-white skeleton network with its nodal
tubercles became visible, thus enabling Merlia to be distin-
guished from certain other small red incrusting organisms
brought up in the dredge, viz. a red Ectyonine sponge, a
polyzoan, a compound ascidian and a coralline alga. It is
true these latter all had slightly different shades of red, but
Merlia itself varied slightly in this respect. <A crust of
Merlia broken in half showed the cavities of a calcareous
framework filled with orange-coloured jelly.

Specimens were fixed in ‘5 per cent. osmic acid in sea-
water, washed; stained immediately in Weigert’s picrocarmine,
and either graded into alcohol or put into glycerine. Others
were fixed in Flemming mixture, and others again in absolute
alcohol.

When I came to examine the first decalcified sections of the
fresh material I was expecting to find a Pharetronid sponge,
and great was my surprise on seeing at the surface a siliceous
sponge, and below the latter and in continuity with it a series
of separate but closely packed parallel moniliform cylinders
chiefly made up of large granular cells imbedded in a
tough transparent maltha. In specimens decalcified whole
the cylinders hang down from the thin surface lamina, the
various segments of each cylinder being united by narrow
central isthmuses of tissue which had passed through the
holes in the centre of the tabule. After a long search

660 Rk. KIRKPATRICK.

among many specimens I failed to tind any calcareous
spicules, ‘‘ tuning-fork ” or otherwise.

At first sight it seemed that Merlia must be some unknown
calcareous organism, viz. Foraminiferan, Pharetronid sponge,
Coral or Polyzoan, infested by a remarkable siliceous sponge.
Weltner, (2) who had seen some of the fresh specimens and
sections, published a short paper entitled “Ist Merlia
Normani Kirkp. ein Schwamm?” in which he stated his
belief that Merlia was an unknown calcareous organism, and
that the siliceous sponge associated with it was merely a
“raumparasit ” which would probably be discovered sheltering
itself in other suitable situations. Later, in a paper on
Astrosclera willeyana, Weltner (3) again expressed his
opinion that the siliceous sponge had simply incrusted and
grown into the calcareous organism. ‘There was, however, an
important objection to this theory. When shells on which
the Merlia was growing were ground down till the base ot the
Merlia appeared, the cavities or “ crypts”?! of the calcareous
framework were found crammed with the large granular
“crypt” cells even five stories down below the surface, and
frequently such crypts were roofed over by tabule in
which the central hole has become reduced to an almost
imperceptible slit from 1 to 3m in diameter. A propos
of the crypts and the cells inside them, to the question
“Whose grave is this?” the answer seemed to be, ‘’Tis
indeed thine, since thou liest in it.” I concluded that the
cells had been formed in situ and had not grown down from
the surface. I named the siliceous sponge Noronha
scalariformis, but failed to arrive at any definite opimon
coucerning the calcareous structure, pointing out, however,
its resemblance to certain Paleozoic fossils (Monticuli-
pora sensu lato, Rhaphidopora) and to Heliopora. (4)

Further examination showed that the masses of crypt-tissue
were in vital continuity with the sponge at the surface. In
some instances bundles of siliceous spicules were found
imbedded amongst them. The existence of a sponge with

‘ A name suggested by Prof. E. A. Minchin.

ON MERLIA NORMANI. 661

siliceous and calcareous skeleton had seemed to me as improb-
able as that of a centaur, for no distinction between siliceous
and calcareous spouges has been considered so profound as
that of the chemical constitution of the skeleton.

After examining about 1000 specimens, and tracing the
development of the soft tissues and skeleton at the growing
edges, I became convinced that Merlia was, beyond doubt,
a sponge with a siliceous and calcareous skeleton (5). The
grounds on which this conviction is based are so firm that the
final proof, such as would be afforded by seeing an embryo
of Merlia settle down and develop the siliceous and
calcareous elements, is, in my opinion, no longer necessary. I
am having, however, relays of specimens sent from Porto Santo
every fortnight, in the hope of discovering when Merlia
forms its reproductive cells and embryos, and of thereby
being enabled to work out the development.

DESCRIPTION OF MERLIA NORMANI.!

The great majority of the specimens, of which there are
about 1000, were obtained from sixty fathoms off Porto Santo
Island. All the specimens from this depth are in the form
of small incrustations, on an average about a square centi-
metre in area and a millimeter or less in thickness, The
crusts grow on shells, branches of corallines, Foraminifera,
worm-tubes, ete., are bright vermilion in colour, and have a
smooth surface. They conform closely to the surface of the
objects upon which they grow, creeping round the edges of
shells and encircling the branches of corallines (Pl. 382, figs.
1-3). They are flat and thin when spread over the smooth
inner surface of a shell, but in other situations they may
form slightly thicker convex bosses. Merlia always grows
on hard unyielding surfaces, and never on soft objects such
as sponges or Ascidians. The Merlia crust is always so
intimately united to the surface of foreign objects that 1t is
almost impossible to flake it off without removing some of the

1 A certain amount of repetition of details given in the introductory
account seems unavoidable.

662 R. KIRKPATRICK.

foundation on which it is growing. ‘This close union is one
of the characters that has made the problem of Merlia
difficult to solve, and has led to false inferences being drawn,
as I shall endeavour to explain later.

In addition to the above-mentioned specimens there are two
much larger ones requiring special notice. One of these, a fine
example forming a large patch over 30 square centimetres in
area on a small block of volcanic rock, was hooked up by a
fisherman from ninety fathoms off Porto Santo Island (Pl. 32,
fig. 4). The specimen was nearly dry when brought to me, but
it still retained its red colour. Lastly, in the Seminario
Museum at Madeira there is a large fragment of a dead
Dendrophyllia covered over a considerable area with a thin
crust of Merlia. ‘The coral was hooked up from ninety
fathoms off Cape Garajau, Madeira. PI. 32, figs. 5, 5a shows a
small piece of incrusted coral, which the authorities of the
Seminario Museum kindly allowed me to break off. The
Merlia incrusts both the sides and free end of the coral
branch. Evidently the sponge flourishes better in ninety
fathoms than in sixty, and it may be inferred that the habit
of Merlia is always incrusting.

When living specimens are removed from the sea the soft,
smooth surface sinks downa little, thus allowing the porcelain-
like network, with its minute circular or polygonal meshes
and little nodal tubercles, to be seen imbedded in the red
flesh. Usually no openings of any kind are to be seen, but
in the case of two specimens killed suddenly by dropping
alive into Flemming’s mixture, several very small circular or
oval holes, from 20 to 60 win diameter, were visible (Pl. 52,
figs. 7, 8). These two specimens were the best preserved of
all, for they were in the expanded condition, and the fixing
fluid had penetrated well into the interior.

The thin growing edge of the sponge extends well beyond
the edge of the calcareous skeleton in examples in which the
crust spreads freely over a surface, but often the growing or
creeping edge is dammed up, so to speak, and the sponge
and its calcareous skeleton increase in depth at such places.

ON MERLIA NORMANI. 663

A living sponge broken in half—1. e. in vertical section—
shows (PI. 32, fig. 10), beneath the soft surface-layer, a more
or less regular white calcareous honeycomb, with blocks of
reddish orange-coloured jelly filling in the spaces, the appear-
ance being that of pots of jelly superposed one on another in
from one to three or four or, rarely, five storeys from edge to
centre of the sponge. The blocks often form fairly regular
horizontal and vertical rows. Occasionally the uniformity of
a vertical row is interrupted by a block of double breadth,
and very frequently the regularity of the horizontal rows is
broken owing to the blocks of “ jelly ” being longer or shorter
than the average. Pl. 52, fig. 9, shows variation both in the
length and (once) in the breadth of the blocks of soft tissue
filling in the spaces or crypts in the calcareous skeleton. PI.
39, fig. 17, shows a section of the skeleton with nearly equal
and uniformly arranged compartments. In the youngest
specimens, which form little red spots only 2 or 3 mm. in dia-
meter, there are no crypts at all, the calcareous skeleton con-
sisting merely of slender bars of a wide-meshed polygonal
network. Also, in the case of specimens which can extend
freely over the flat or curved, smooth inner surfaces of old
bi-valve shells, there may be only a few crypts in perhaps
one or two storeys at the centre of the crust, the rest of the
skeleton being composed of deeper or shallower pits, with
the floor formed by the surface of the shell.

Not infrequently the sponge grows in depth rather than in
extent, and the crypts may then become four, or even five,
storeys deep, or rather, it should be said, high, for the growth
is from below upwards. In such specimens the storeys may
diminish gradually to four, three, two, one, and finally to
none at the growing edge.

On viewing by powerful reflected hght under a low magni-
fication a stained specimen in glycerine, it is possible to see,
below the transparent surface, circular masses of flagellated
chambers fillig in the uppermost spaces of the calcareous
framework, and joined to each other by radiating spokes,
also composed of masses of flagellated chambers, crossing

664. R. KIRKPATRICK.

over the upper edges of the calcareous walls and between
any two tubercles (Pl. 33, fig. 1). The circular masses show
a lighter central area, or occasionally two suchareas. Further,
from one to three nearly vertical bundles of fine needles are
present in or above each circular mass. Scattered mostly in
or upon the surface of the sponge are very minute, oval siliceous
rings. ‘The siliceous spicules are not visible in glycerine pre-
parations, but clearly so in balsam ones. Thin specimens
decalcified whole show very clearly the darker node-like
circular areas of masses of flagellated chambers joined to each
other by five to seven radiating spokes, the blank spaces
between the spokes having been occupied by the calcareous
tubercles. ‘I'he under-surface of fairly thick decalcified
specimens presents an extraordinary appearance, such as has
never been observed before in any sponge. Hanging down
from the thin surface-layer of the sponge are closely packed
but separate parallel moniliform cylinders. The segments of
each cylinder vary in breadth from 75 to 100 uw (or rarely
150 w), and in length from 75 to 2504. The segments are
joined to those above and below by narrow central strands,
which had passed through the holes or slits in the tabule.

Fig. 9 on Pl. 32 shows a vertical section of a decalcified
specimen. ‘Tle spaces between the cylinders extend from the
base of the sponge to the apex of the gaps left by the dis-
solved tubercles, not taking into account the fine organic
basis of the skeleton remaining after decalcification. The
upper part of the section, including the ectosome and part of
the choanosome, is extremely contracted, but below this the
flagellated chambers, bundles of spicules, and the cylindrical
masses of large granular cells are to be seen.

Fig. 5 on Pl. 35 shows a decalcified vertical section of one
of the two expanded specimens; in the part of the section
shown in the figure there happens to be only one mass of
crypt cells that had been covered bya tabula. All the other
basal extensions of soft tissue had filled in calcareous pits,
the floors of which had been formed by the surface of the shell
on which the sponge grew (see below, Section 3).

665

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666 R. KIRKPATRICK.

By macerating specimens in Kau de Javelle the calcareous
framework stands out clear (Pl. 32, fig. 6, and Text-fig. 3
see Section 2b).

*)

Having given a general account of the external appearance
and coarse anatomy of Merlia, I will now describe the
sponge more in detail under the following headings :

(1) The canal system.

The skeleton ; (a) siliceous, (b) calcareous.
The soft tissues and cells.
4) Young stages of Merlia.

© bo
5 i
ns

Theory of construction of the caleareous skeleton.
Systematic position of Merlia.

On some resemblances between the calcareous skeleton
ertain Paleozoic fossils.

Summary.

Q,

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ee

(1) Tae Canat System: a New Tpke.

It would have been very difficult to have learned the
structure of the canal system from contracted specimens.
Under such conditions no surface openings are to be seen,
and the upper part of the sponge is squeezed down—like a
closed concertina—into the open crypts.! Some examples of

this very contractile and sensitive sponge, dragged up with a
mass of shells and débris from sixty fathoms, were resuscitated
by trailing them in a bottle in the sea, and then suddenly
dropping them into Flemming. In these specimens not only
are some of the oscules and pores open, but the whole sponge
is expanded and shows wide ectosomal spaces. In contracted
specimens the sponge surface almost rests on the calcareous
tubercles, but in expanded examples the latter are a consider-
able distance below.

The larger openings or oscules measure about 60, in

* Open crypts—the uppermost spaces in the calcareous skeleton. In

the macerated skeleton these spaces are open and not roofed over by
tabule.

ON MERLIA NORMANI. 667

diameter, and the smaller ones or pores about 20-30 p. Both
kinds are oval or circular, and quite flush with the surface.

The pores may form a circle round an oscule (PI. 32, fig. 7),
but sometimes no regular arrangement is perceptible (fig. 8).
The regular plan of a central oscule and a ring of pores would
seem to conform to the shape of the upper crypts and tubercles,
but the reverse is probably the case, the crypts conforming to
the arrangement of the canal system, as I hope to make clear
later.

Both oscules and pores are provided with a very well-
developed sphincter apparatus, consisting of concentric and
radial contractile cells for respectively closing and opening
the orifices. Some of these cells are remarkable in shape,
viz. with long processes which curve round the orifices and
which may actually anastomose, thereby becoming porocytes.
These cells seem to indicate the mode in which porocytes may
have arisen, viz. by the fusion of processes curving round an
orifice, and not by the appearance of a hole in the solid body
of the cell (Pl. 57, fig. 5).

The incurrent canals pass down and bifurcate, passing right
and left (as seen in sections). Some of them present a
puzzling appearance, for they are surrounded by flagellated
chambers. Sometimes a string or tube of flagellated chambers
is seen traversing an open space. The lumen of the tube
is the incurrent canal and the open space is an excurrent
one, into which the apopyles on the outer surface of the tube
open.

Fig. 13, on Pl. 33, which depicts the growing edge of a very
young sponge, will show how this arrangement has probably
come about. In the youngest stage the canal structure is that
of a simple rhagon, i.e. of a gastral cavity with much folded
walls, the folds branching out to the periphery. ‘The spaces
between and outside the folds constitute the imcurrent, and
those in the lumen of the folds, channels or canals, the
excurrent system.

The incurrent canals pass between the little clusters of
flagellated chambers, which clusters at this stage have a

668 R. KIRKPATRICK,

narrow lumen (or excurrent canal). In the course of growth
the clusters expand at the same time that their lumen
increases, and they encroach on the incurrent spaces, which
may now consist of narrow channels or canals completely sur-
rounded by flagellated chambers.

A tube of flagellated chambers may be compared to a
hollow central column (say with a “pore” at the summit
opening on the roof) supporting a groyned vault, the concave
surfaces of which face the interior of the building which
leads to a door (oscule).

The excurrent spaces or channels in the upper part of the
sponge often form wide pouch-like out-foldings, but those in
the deeper parts which are crowded into the uppermost open
crypts of the hard skeleton are in the form of narrow
canals.

In a section the in- and excurrent canals are easily distin-
guished by the direction of the apopyles and the collar-cells.
A terminal excurrent canal or space opens into the floor of one
of the large ectosomal spaces, and the latter open to the
exterior by an oscule.

The flagellated chambers possess a remarkable structure
not known to occur elsewhere. ‘They are semi-oval or hemi-
spherical, and about 33 in diameter. They are usually
closely approximated to each other, being joined—or sepa-
rated—only by a few connective-tissue cells.

Stretched across the excurrent aspect of each chamber is a
membrane with a thin-edged circular apopyle in the centre.
Nearly surrounding eavh apopyle is a single contractile cell
which acts as a sphincter muscle. The apopyles vary in
diameter from a point up to nearly the width of the flagellated
chamber.

The prosopyles consist simply of spaces between the fused
rays of the stellate bases of the collar-cells (Pl. 35, fig. 6)
—a character recalling the membrana reticularis of Hexacti-
nellida. This stellate arrangement of the cell-plasma is
not easy to see, and I am thereby led to believe that this
mode of union may possibly occur among other tetraxonid

ON MERLIA NORMANI. 669

sponges, especially among those with a eurypylous type of
canal system.1 ;

The collar-cells vary greatly in appearance according to
their state of contraction. Figs. 8-12 on Pl. 33 are drawings
of collar-cells on one and the same slide. Some are relatively
short and thick, with the collar contracted down and joined to
the collars of neighbouring cells, and with a wide opening
with polygonal outline. Others are elongated and with
separate cylindrical collars. The collar-cells vary in height
from 454 to 8° uw. The usually spheroidal but sometimes
hemispherical body is about 3°35 4 in diameter. ‘lhe spherical
nucleus is at or near the base of the cell. The flagellum
passes down through the cell to the nucleus (PI. 33, fig. 12).
The name “ hymenopylous’’* is proposed for the new type of
canal system, which seems to be a modification of the eury-
pylous type.

(24) Tae Sintckous SKELETON.

The siliceous skeleton consists mainly of upright bundles of

' Fig. 6, Pl. 55, is, I find, slightly diagrammatic in places, but it is
sufficiently accurate in places to justify the statement that the collar-cells
are Joined to each other by stellate basal processes. Further investi-
gations of new, well-fixed material, prepared with a good plasma stain, are
desirable. It is not surprising that we have so little knowledge concerning
the inter-relations of collar-cells in siliceous sponges. The difference
between the tissues of healthy living sponges and dead ones fixed under
the best possible conditions must be very great, especially in the matter
of contraction. Minchin and Reid have shown (‘ Proc. Zool. Soc.,
London,’ 1908, p. 674, pl. xxxvii, fig. 24) that in Clathrina contorta
the bodies of the collar-cells are separated by delicate extensions of the
gelatinous ground-substance of the sponge. Muriel Robertson and
Minchin (‘ Quart. Journ. Micr. Sci.,,; November, 1910, p. 621) write that
probably the collar-cells are connected across this intervening substance
by protoplasmic fibrils, though they have not yet seen such connections.
Certainly the manner in which a contracted Ascon with its collar-cells
squeezed out among the porocytes expands again with all its collar-cells
“dressed” and in line would lead one to suspect that here also there
may be a kind of collar-cell membrane, as in Hexactinellids.

> tpny, évoc, membrane ; win, gate.

670 R. KIRKPATRICK.

slender tylostyles' with pointed ends outwards, along with a
few slender rhaphides, which form more or less vertical pillars
of support round the large ectosomal spaces and incurrent
canals. In contracted sponges one to four of these vertical
wisps are drawn down into the upper crypts, but in expanded
specimens the wisps are outside the crypts and form supporting
pulars to the ectosome and canals (PI. 32, fig. 10, and PI.
33, fig. 3). Sometimes a bundle of spicules lies transversely
on the floor of an upper open crypt. Only very rarely are
spicules of any kind found in the lower crypts, but neverthe-

TExT-niG. 2:

Sections of masses of crypt-tissue showing siliceous spicules.

a. An elongated calcocyte in neck of crypt. Soft crypt-tissue
mostly disintegrated owing to insufficient fixation. x 225,

less they do occur there. (The probable reason for this rarity
is explained in Section 5).

Numerous microscleres in the form of oval rings—for
which I propose the name “ clavidiscs ”’*—are scattered about
on the surface, and also, but much less abundantly, deeper
down.

‘In the report on the ‘“ Discovery ” Tetraxonida I have used the
term “tyle” in place of “tylostyle,” because it was short, and by way
of antithesis to “amphityle,” but I now return to the commonly used
designation “ tylostyle.”

* Clavis, key, referring to the key-hole notches; discus, quoit.

ON MERLIA NORMANI. 671

A second kind of microsclere, viz. a very minute, slender,
simple sigma is found in fair abundance in the choanosome,
especially in the immediate neighbourhood of the flagellated
chambers. At one time I thought these spicules were the
broken curved ends of rhaphides, but latterly I have seen the
little spicules in their scleroblasts.

Rhaphides and trichodragmata constitute a third and
fourth kind of microsclere.

The Spicules.—The slender tylostyles (Pl. 35, fig. 1)
which are commonly curved at the distal end, though some-
times nearly straight, are about 140 long, 18, thick, and
with oval heads 5 by 2°24 in length and breadth. The
rhaphides (PI. 35, fig. 2a) are about 804 long. They are found
separately or mixed in with tylostyles. Trichodragmata (PI.
39, fig. 2) occur, but are rather rare. In one specimen there
are toxa-like spicula with a central kink or bend (PI. 35, fig.
13), but this is an exceptional feature. The clavidises (PI.
39, figs. 3-9) are about 454 long, 50 u broad, and with the
rim, which is bevelled inwards to a thin edge, 3 broad. A

)

key-hole shaped sinus or notch is present on the inner margin
at each end of the long axis. ‘lhe axial canal is in the centre
of the thickness of the outer edge of the rim. Numerous
variations and sports occur, which are interesting because
they show the mode of origin of these spicules, viz. from
deeply curved rods which have bent round till the ends met
and joined. Sometimes the ends cross or do not meet at all,
or a transverse bar may cross from side to side (PI. 35, figs.
7, 8). Fig. 14 shows clavidises with a disc-like plate in place
of the key-hole sinus. Fig. 7 shows a sigma-shaped spicule
which is probably merely a deviation from the ring shape.
Again, the key-hole sinuses may be absent from one or both
ends. Lastly, the clavidise may sometimes be in the form,
not of a ring, but of a solid dise (not figured).

I had formerly supposed (6) that the clavidises were related
to chelate spicules of Desmacidonide, but I now consider
their affinities to be with the diancistra of Hamacantha
(see Section 6 on the affinities of Merlia). These spicules

672 R. KIRKPATRICK.

are mostly scattered at the surface, in which they lie hori-
zontally.

The oval rings found deeper down in the sponge usually
have thinner rims. In one instance six rings followed at
equal intervals on one side and five on the other side of the
mass of sponge filling an open crypt. Hence I called the
upper part of the sponge Noronha scalariformis.

The very fine primitive simple sigmata are commonly found
in the neighbourhood of the flagellated chambers.

There seems to be no transition between the sigmata and
the clavidiscs. At the same time the clavidiscs probably
developed from some such form. In one or two of the
myocytes acting as sphincters round the apopyles there seemed
to be an appearance of a slender curved axial rod of silex.
Possibly the slender sigmata may originally have come into
existence owing to the presence of sphincters, which surround
not only the pores and oscules, but also the apopyles of the
flagellated chambers.

To sum up, normally there are five kinds of spicules in
Merlia, viz. tylostyles, long rhaphides, trichodragmata, clavi-
dises, and slender sigmata. Rarely thicker sigmata and toxa
occur.

Pl. 35, figs. 11-15 show abnormal forms of spicules, all found
in one specimen.

(28) Tau CALCAREOUS SKELETON.

When a living sponge is taken from the water presently the
semi-transparent, red, fleshy surface sinks a little, and
the porcelain-white calcareous skeleton becomes visible.
Under a lens it is possible to see the very minute circular or
polygonal meshes of a fine network, and the still more minute
tubercles rising from the nodes. In dried specimens the
flesh forms merely a thin yellowish pellicle, covering the
surface, which has a uniformly granular aspect due to the
tubercles below, with here and there a polygonal pattern
where the soft tissues have sunk more deeply into the spaces
of the skeleton beneath.

ON MERLIA NORMANI. 673

For the investigation of the skeleton specimens were
macerated in Eau de Javelle, and vertical and horizontal
sections ground down, and some examples were incinerated.
To the naked eye the surface of a macerated-out skeleton or
of a dried specimen like that encrusting the Dendrophyllia
(PI. 32, fig. 5, 5a) has a very finely porous appearance, the
meshes being barely visible.

The meshes are about *18 to 22 mm. in total diameter, i.e.
four and a fraction to a millimetre, the actual spaces or holes

TEXT-FIG. 3.

Surface of calcareous skeleton. The large dark circle is the
mouth of a worm-tube. x about 40.

being about ‘12 to °15 mm. across, and the walls about ‘04 to
‘06 mm. thick. The number of tubercles round a mesh varies
from four to seven or eight, five or six being the average
number. Occasionally two meshes are combined into one
larger oval one, with ten to twelve tubercles.

The tubercles are about 75 high and 754 broad at the
base, and are covered with very minute sharp-pointed conules
about 10 high and 16 broad at the base, but varying both in
shape and size. The point of the conule is generally nipple-
shaped and may lean over a little to one side. Again the

vot. 56, PART 4,—NEW SERIES. 47

674 R. KIRKPATRICK.

conules may be rounded at the summit, or more elongated than
usual. <A little below the surface the uppermost tabul are
visible, each with a central, circular or oval hole or nearly
closed slit (Pl. 35, fig. 16). Sometimes the tabula is merely a
rim or ledge round the inner wall of the calcareous tube, and
the central hole is correspondingly large; or, again, the tabula
may be imperforate. The tabule show five or six sutural lines
radiating from the central hole or slit to the circumference.

On the surface of the specimen incrusting the Dendro-
phyllia there are numerous small circular holes due to worm-
tubes, and here and there among the ordinary meshes of the
Merlia are larger ones nearly *5 mm. in diameter, with a
shallow floor, on which there are radiating ridges and even a
central columella-like- knob, the whole somewhat resembling
avery small coral calycle. ‘The large meshes are here simply
due to the effort of the sponge to repair the lesion caused by
the presence of the worm-tubes, the openings of which are
found just beneath the floors of the supposed calycles.

A vertical section of the skeleton shows parallel vertical
tubes divided up by tabule (PI. 35, fig. 17). Often the tabule
form a series of parallel horizontal planes at regular intervals
of about ‘15 mm., but the regularity is frequently disturbed,
as will be obvious from fig. 9 on Pl. 32, showing the soft
tissues which filled the crypts.

The magnified surface view of the skeleton (Pl. 35, fig.
16) shows three flanges or wings radiating from the base of
each tubercle and meeting opposing flanges from neighbour-
ing tubercles. A sutural line marks the junction of the
opposing flanges, and the most prominent radial sutures on
the tabule are continuous with those between the flanges.
A vertical section shows the interflange sutures to be con-
tinued as vertical lines down the walls of the tubes from
surface to base.

The tabule are about ‘015 mm. thick. On both surfaces,
in addition to the radial sutures there are fine concentric
markings confined to those surfaces, the thickness of the
tabula being homogeneous. ‘he radial sutures typically

ON MERLIA NORMANI. 675

run from the central region to the vertical sutures between
the flanges and are continuous with them, though occasionally
this arrangement is a little obscured. Fig. 19, Pl. 35, shows

a thick ground-down section in balsam as viewed by powerful

TExtT-FIG. 4.

A. Diagram showing a structural unit of the calcareous skeleton.
Horizontal section at level of three tabule. a. Curved surface.
b. Suture of flange. c, c’, ec’. Three segments of tabule. d.
Margin of portion of central hole in tabula. e. Central vertical
axis below apex of a tubercle. 8B. Diagram showing five struc-
tural units joined to form one tube and tabula, and parts of
five other neighbouring tubes and tabule. Horizontal section
at level of tabule. Lettering as before. c. Three structural
units in elevation (diagram). Lettering asin a. f. Tubercle.

oblique reflected light. The walls of the tubes were here,
i.e. at the base of the sponge, very thick, but, both the hori-

676 R. KIRKPATRICK.

zontal tabular and vertical (or columnar) sutures were
beautifully lighted up.

The disposition of these vertical and horizontal sutures
clearly shows how the skeleton has been bmlt up. The
structural unit is a broadly based pillar with three vertical
longitudinal wings or flanges (which unite with the flanges of
three juxtaposed columns), and with three vertical longi-
tudinal curved surfaces or flutings which form a part or
segment of the walls of three tubes, and lastly with tiers
usually of three horizontal triangular tabular segments in one
plane, the number of tiers varying with the number of tabule.
(See diagrams 4, B, C in text.)

It was a surprise to find that there was no continuous
floor such as was depicted (im vertical section) in my original
figure.(7) ‘he seeming existence of a floor in certain places
is apparently due to the section cutting through the thick
outspread base of a vertical wall.

It is certain there is no continuous basal lamina at the
periphery of thin spreading crusts, but occasionally I
have seen what appear to be complete floors at the base of
some of the basal crypts. ‘he floor of Merlia is formed by
the shells, coralline alga, Foraminifera, etc., on which the
sponge is growing. ‘The vertical walls of the lowest crypts,
often thickened and spread out a little at their bases, are
intimately fused with the foreign foundation.

Under a fairly igh power the skeleton shows a finely
fibrillar structure (Pl. 55, figs. 16,17). When the skeleton is
viewed from the surface, the fibrilla are seen to radiate out
from below the base of each tubercle towards the longitudinal
sutures, where they meet but do not blend with the fibrille
of opposite flanges (Pl. 35, fig. 16). Superposed zones of
fan-like bands of striz radiate upwards and outwards from
the vertical axes of the columns of the skeleton and pass into
the tabule and walls of the tubes (Pl. 35, fig. 17).

An English colleague was engaged, at the time this was
being written, in the attempt to show that Merlia was a
Foraminiferan. He had only a dried skeleton, coming, I

ON MERLIA NORMANI. 677

believe, from the West Indies, and two of my specimens of
the real Merlia from Porto Santo, on which to base his
conclusions. If I had had such scanty material, I also might
easily have come to the same conclusion, because Merlia often
grows on a spreading crust of Polytrema or other Forami-
nifera, and the cancellated structure of the latter appears
to belong to the Merlia; but having had hundreds of
specimens to deal with, I have found Merlia growing on
shells, on gneiss, on spreading lamine of thin incrusting
Corallines, etc. Foraminifera with a superficial reticulate
pattern with nodal tubercles are not uncommon. Dr.
Harmer showed me one he had found among the ‘‘ Siboga”’
Polyzoa. Professor Stanley Gardiner collected a similar
species incrusting Lithothamnion from Providence Reef. I
myself dredged a Carpenteria-like species off Christmas
Island. In all these instances the Foraminiferal nature is
clearly shown by the finely perforated floor at the base of
each mesh. At one time I myself thought Merlia was a
Foraminiferan, not only on account of the calcareous skeleton,
but also because the masses of crypt-cells (in glycerine)
looked like lobose pseudopodia, the maltha in which they
were imbedded being invisible.

At the growing edges of the sponge and in whole young
specimens there is simply a delicate network of calcareous
bars. In the case of young specimens on thin bivalve shells
one can often see through the whole structure. The soft
sponge tissues cover the calcareous network, which has no
basal lamina, and there is no trace of any other organism what-
soever. Even this strong circumstantial evidence in favour of
the theory that Merlia is a sponge is unimportant in com-
parison with the positive evidence afforded by the soft tissues.

The growing edge of the skeleton shows the delicate poly-
gonal network, with half- or open polygons at the edges, and
here and there isolated small Jumps or even bars (PI. 38, fig. 5).
In the older parts the slender bars of the delicate meshwork
have increased in height and the meshes have become pits,
and still later tubes with one or more tabula.

678 R. KIRKPATRICK.

The growth of the skeleton is much influenced by the
conformation of the base. When growing on shells or worm
tubes with ridges, the sponge makes squarish oblong meshes
with parellel or concentric arrangement (Pl. 35, fig. 20).

This last figure of a specimen macerated in Hau de
Javelle shows well also the earliest beginnings of the
skeleton before bars or tubercles are formed. Here, in
places, little smears of calcareous scales are beautifully dis-
tinguished on the reddish surface of the worm-tube. ‘These
glistening bands have a crystalline appearance as if a brush
dipped in strong solution of sugar had been drawn over the
surface and allowed to crystallise. The individual scales
have a finely punctate structure, and each one shows a little
elevation which corresponds, I believe, to the nucleus of the
cell which, made the scale (Pl. 35, fig. 21). Viewed as
opaque objects, the fine points and the little nuclear hump in
each scale strongly reflect the light. I was unable to detach
the glistening scales from the worm-tube so as to examine
them by transmitted light under a high power.

The calcareous skeleton of Merlia is formed of calcite.
Dr. G. I’. Prior found that the specific gravity was 2°65.
That of pure calcite is 2°7, the difference being due to a certain
amount of organic matter remaining inthe Merlia even after
bemg finely ground and macerated. The specific gravity of
arragonite is much higher, being 2°9, and, moreover, Meigen’s
test gave no reaction.

(3) Tue Sorr Tissurs anp CELLS.

Merlia is semi-transparent, and its tissues are of a soft
texture, but, at the same time, rather tough. ‘The bright red
colour resides in the granules of certain cells—the amcebo-
cytes. The colouring matter is soluble in alcohol, in which
it forms an orange-yellow solution. The cells will be de-
scribed under the following headings: (A) Collencytes, and
ground substance or maltha; (B) gland-cells and cuticle ;

ON MERLIA NORMANI. 679

(c) canalar epithelium; (D) myocytes; (k) scleroblasts ; (r)
choanocytes; (G) amcebocytes; (H) tokocytes.

(a) Collencytes or Connective-Tissue Cells.

The connective-tissue cells or collencytes (Pl. 37, fig. 2)
are finely granular with an oval vesicular nucleus devoid of
a distinct nucleolus, and with branching processes which
anastomose with those of other collencytes to form a network.
The ground substance or maltha at first sight seems almost
homogeneous, but in good preparations under a high power
it shows a very finely fibrillar structure. When bundles of
fibrille are cut across they form finely granular areas in
section (Pl. 54, fig. 4).

(s) Gland-cells and cuticle.

The sponge surface is often coated over with a very thin
layer of structureless cuticle, apparently the product of
elongated granular cells vertically orientated just below the
surface (PI. 37, fig. 4). In these cells there is a minute dark
body like a nucleolus, but situated just outside the vesicular
nucleus.

(c) Canalar Epithelium.

A remarkable feature of great interest, and, I believe,
unique, is the absence of a surface layer of epithelium. In all
the best preserved material I found the cells at the surface
precisely the same as those in the body of the sponge, i.e.
they were branching collencytes well separated from each
other by the maltha in which they were embedded. Knowing
how difficult it often is to see the outlines of surface epithelial
cells, at first I concluded that the cuticle—usually, but not
always present—was an epithehal layer. Further, in one or
two very contracted specimens, which had not been properly
fixed, the collencytes had become so pressed down in the

680 R. KIRKPATRICK.

maltha as to resemble an epithelium when viewed from the
surface. In some very thick vertical sections, well preserved
and stained, it was possible to see a considerable area of the
surface as well as the depth of the sponge, and to note the
entire absence of any trace of an epithelium. Accordingly,
Merlia in its adult condition affords a confirmation of the
theory put forward by Haeckel and confirmed by the embryo-
logical researches of Maas, Yves Delage and Minchin, that
the sponge is a two-layered organism. Merlia has an “ exo-
derm” and an “endoderm,” but no mesoderm.

Although there is no epithelium on the surface of the
sponge the canals have an epithelial ning, which apparently
consists of cells of the same nature as the collencytes in the
maltha, for sometimes a cell on the surface of a canal has
branching processes extending back into the maltha.

(D) The Myocytes.

The myocytes form concentric or radial groups round the
incurrent and excurrent orifices. The myocytes have a
granular plasma, the granules being coarser than those of the
collencytes, and the nucleus is vesicular and without a nucle-
olus. They vary greatly in shape, but generally have branched
prolongations—indeed, they do not differ greatly from the
collencytes. Sometimes the branched prolongations surround
an orifice and fuse, so that the myocyte becomes a porocyte
(Pl. 37, fig. 3). Further, one or more small, curved, fusiform
myocytes surround the apopyle of each flagellated chamber.

(E) Scleroblasts.

The scleroblasts are very finely granular, and with a clear
spherical nucleus. PI. 36, fig. 2 shows these cells both in
longitudinal and transverse sections of spicule bundles. PI.
36, fig. 10 shows a scleroblast of one of the clavidiscs.

ON MERLIA NORMANI. 651

(rz) Choanocytes.

(See under ‘Canal System,” Section 1, and PI. 33, figs. 8-12.)

(a) Caleigenous Amobocytes or Calcocytes.

It is to these cells that the extraordinary character ot
Merlia is due, for they are caleigenous, and it is their
function to build the calcareous skeleton.

Quite apart from their lime-forming function, the arrange-
ment of these masses of cells in the form of moniliform
cylinders is a very remarkable feature.

Before I had understood the structure of Merlia I called
these cells crypt-cells, because many of them are found in
spaces of the calcareous skeleton covered over by tabule.
As I am now practically certain that these cells which fill up
the crypts also build those structures, I suggest the designa-
tion “calcocyte,” which is in keeping with the cytological
terms now in use. Since these lime-forming cells of a sili-
ceous sponge constitute a special and unique phenomena, |]
think they should be distinguished by a separate name from
the telmatoblasts, or better, telmatocytes, which form the
cement in Pharetron sponges. (8)

The lime-forming celis of Merlia are exceptionally de-
serving of the designation ‘amcebocyte,” for they exhibit
« remarkable variation in shape in accordance with their
position in the sponge and the kind of work they have to do.
Before settling down to build the skeleton and in places
where they are not compressed in an enclosed space they are
more or less elongated (PI. 38, fig. 1). When caught in the
isthmus between two crypts they may be vermiform (Pi2o6.
fig. 3, and Text-fig. 2). In the crypts, where the cells are
pressed against each other, they are massive and cylindrical
or pyriform (Pl. 36, figs. 1-4), excepting on the outer surface
of the crypt masses, in which situation they become flattened
(Pl. 34, fig. 6a, and Text-fig. 5). When forming the bars of the
young calcareous skeleton or the inner smooth walls of crypts

682 R. KIRKPATRICK.

they form large flat plates with the nuclear region projecting
out from the free surface (Pl. 36, figs. 6, 7, 8).

Whatever their disguise, they possess certain common
characters which enable them easily to be recognised. They
are crammed with large orange-coloured pigment-granules,
the colour being retained for a long time in specimens pre-
served in glycerine or formalin. Further, they have a large,
soft, spheroidal or oval, vesicular nucleus and a distinct
nucleolus. The granules, which stain easily and deeply, vary
apparently according to the state of the metabolism—that is
to say, in the early stages in the history of a calcocyte the
granules are often of unequal size, but when they are ina
position to lay down lime they become more or less uniform.

When the granules are pressed out of a cell and viewed
under a high power slightly out of focus they appear as
uniformly light discs, and gradually, as they are brought
into focus, a dark point appears in the centre surrounded by
a light circle; on focussing down further the dark point
enlarges till the circle becomes uniformly dark. ‘The appear-
ance of the dark point in the hght circle is of some importance,
because it is seen all over the surfaces of the calcareous
skeleton, where the ends of the fibrille give this appearance,
and even in the interior of the conules on the tubercles (PI.
35, fig. 22, and Pl. 36, fig. 9). The granules appear to be the
essential calcigenous elements.

Wherever the skeleton is being laid down these granular
calcocytes abound, and in decalcified specimens they are
found in the closest relation with the organic basis of the
dissolved skeleton.

Pl. 34, fig. 3, shows a decalcified tubercle and side of a
polygonal mesk with the calcocytes closely appled.

Fig. 1 on PI. 37, which represents the extreme growing
edge of the sponge, is especially interesting, for it shows
the calcocytes planning out the first foundations of a polygonal
mesh on the surface of the shell on which the Merlia is
growing. ‘lhe calcareous products of calcocytes in this
position are flakes or scales (see Section 28, and PI. 35, fig. 21).

Mo

ON MERLIA NORMANL. 68:

C

I believe the whole skeleton to be made of calcified calco-
cytes. Fig. 22 on Pl. 35 shows a camera lucida drawing by
Mr. Highley of a conule, which seems to me to be nothing
else than a petrified calcocyte. For the conule fits hke a cap
onthe rounded surface to which it is attached; also the edges
of its base are rounded and show a lobose process. Further,
there is an appearance of an oval nucleus in the interior, and
the fine structure shows the granules each with the dark
central point.

In the crypts and on the bars of the polygonal network the

TEXT=RIG! Oo.

Flattened calcocytes on the surface of a mass of crypt tissue.
x 750.
surface calcocytes are spread out in the form of plates (PI.
34, fig. 6, and Pl. 56, fig. 1, both in vertical section), and
the nucleus forms a little hump-like projection on the surface
facing the soft tissues.

The supposed epithelium on the surface of the masses of
cry pt-cells is composed simply of flattened-out calcocytes of the
same nature as the massive cells in the interior of the mass.
Fig. 1 on PI. 36 is drawn from sections made from material in
which the fixing fluid did not well penetrate. The large nuclei
of the plate-like calcocytes are shrunk and compressed, and cut
into vertical sections. Fig.6 on Pl. 34 shows much better the

684. R. KIRKPATRICK.

vertical sections of the caleocytes on the surface of a crypt;
here the nucleus is pressed outwards or away from the
skeletal face of the cell to form a little mound on the opposite
face. The most difficult part of this investigation was the
discovery of the precise nature of the surface of the masses of
crypt-tissue. This difficulty was partly due to the slowness
with which fixing reagents penetrated into the nearly inacces-
sible crypts, and partly, I think, to actual variation in the
condition of the surface-cells owing to functional causes.
Pl. 34, fig. 6, shows a well-fixed Flemming preparation, and
PI. 36, fig. 1, a badly fixed osmic-picro-carmine one. Some-
times flattened surface-caleocytes are very well shown; at
other times one can make out only a diffused layer of granules,
the boundaries of cells being invisible.

There is a possibility that the large inner cells may be
trophic stores, but in my opinion the surface crypt-cells are
amoebocytes of, the sane nature as the inner ones, and also I
think they are calcigenous, and not by secretion, but by
actual transformation.

Fig. 6 on Pl. 36 shows the caleocytes forming an epi-
thelium-like layer closely moulded to one of the calcareous
bars of a polygonal mesh at the growing edge of a crust of
Merlia.

The theory that the calcocytes become wholly transformed
into calcified lumps or scales is confronted with one difficulty,
viz. to account for the fan-like, fibrillar structure of the
skeleton. I can only suggest that possibly the fibrillation is
a secondary change. A decalcified section of a tubercle (PI.
34, fig. 3b) shows radiating, fibrillar structure apparently
corresponding with that of the hard skeleton.

A propos of the calcocytes, it only remains to say that the
evlindrical, moniliform masses of these cells are simply basal
prolongations of the sponge body in which they have accumu-
lated. The cells are imbedded in the finely fibrillar maltha,
just as are the rest of the tissues of the sponge. Occasionally
the maltha in the crypts contains collencytes, scleroblasts,
and spicules, and, very rarely, undeveloped wedge-shaped

ON MERLIA NORMANI. 685

collar-cells, such as occur in the youngest specimen. The
reasons for this sharp separation between hypersome and
hyposome now seem to me obvious. The calcocytes migrate
down and accumulate in the base of the sponge, leaving but
little or no room for the other elements, which can only
expand upwards. The continued growth of the soft tissues
thus separated into upper and lower, or superficial and basal,
within the confined space of calcareous pits or tubes which have
gradually been forming, leads to an hour-glass constriction
and forming of tabule, with still more complete separation of
the hyper- and hyposomal elements (see Section 5 below).

Often the calcocytes migrate to the periphery of a crypt,
leaving the central part of the maltha clear and free of cells,
and giving to the mass of cells the appearance of being
arranged as a columnar epithelium against the crypt-wall and
surrounding a cavity (PI. 54, fig. 4). Partly for this reason I
was led to think that Merlia might be a coral of some sort,
since both the hard and soft tissues ‘‘ conspired” to produce
this impression.

(nH) Tokocytes.

At one time I thought that the cylindrical masses of ecalco-
cytes in the crypts were gemmule cells, but unless they were
capable of dissolving the calcareous skeleton, in which most
of them were interred and cut off from the world, it was diffi-
cult to understand what could be the object of forming
gemmule cells in nearly closed crypts, roofed in by tabulee
with central holes or slits sometimes only from 1 to 3 in
diameter.

Just below the choanosome in one section are some granular
cells with the nucleus and nucleolus larger even than in the
calcocytes (Pl. 34, fig. 8). Such cells may be egg-cells. The
specimen from which the section was made was captured in
May.

(4) Youna Srace or Mertra.

The youngest example found was in the form of a little red

686 R. KIRKPATRICK.

spot, abont 2 mm. in diameter, on the inner surface of a shell.
The figure (Pl. 52, fig. 12), magnified twenty-five times
linear, shows the delicate polygonal network below the surface
of the sponge. There are no crypts at this stage, but simply
the slender strands or bars of the wide-meshed network,
with small tubercles at the nodes.

A. vertical decalcified section, highly magnified (Pl. 38,
figs. 1-3), shows a layer of cells all over the sponge at, or
rather just below, the surface. The cells have processes
which branch dendritically into innumerable slender fibrillz
or filaments. The fibrille passing to the surface form a thin,
surface feltwork, which shows as a thin line in the section.

Bundles of fibrilles pass down from the cells of the upper
surface, and up from the basal cells. Occasionally the opposing
filaments meet and almost form a central, horizontal lamina,
especially near the periphery of the sponge, where the upper
and lower surfaces are very close together. The waving
masses of branched fibrils almost resemble the flames of a
conflagration as conventionally depicted.

The calcocytes are elongated, and mostly congregated near
the base of the crust and orientated at right angles to the
base, like a field of stakes or hop-poles. Many of these cells
are embraced between bundles of fibrille: passing up from the
basal branched cells.

The flagellated chambers are not yet formed, but the collar-
cells are present as small wedge-shaped cells which here and
there show a tendency to be arranged in half circles (as seen
in section). Further, some wedged-shaped cells appear to be
prolonged at their acute angle into a filament which joins on
to a main filament, like the main stalk and secondary stalks
of a pinnatifid leaf. Apparently the main stem and lateral
stems represent the beginnings of the canal system. I have
not been able to determine the nature and relations of these
branched filaments; possibly they are the processes of con-
nective-tissue cells.

Scleroblasts and their spicules are abundantly present.

The little grooves or arches at the base of the section are

ON -MERLIA NORMANI. 687

the gaps left by the dissolved bars of the calcareous skeleton.
In this situation a few calcocytes are seen in the walls of the
arches.

(5) THrory oF CoNSTRUCTION OF THE CALCAREOUS SKELETON.

The skeleton is formed by the granular amcebocytes or
ealcocytes. The youngest stages of the skeleton (PI. 35, figs.
20, 21) consist simply of microscopic scales, flakes or lumps
laid down on the surface of the shell or other body which
Merlia incrusts.

Within the growing edge of a flat, thin, spread-out specimen
of the sponge the edge of the skeleton is visible in the form
of slender bars forming a polygonal network, the polygons
being incomplete at the extreme edge. In a still younger
stage, visible only under fairly high powers, the slender bars
are no more than smears or streaks of flakes, scales or lumps,
somewhat higher at points where the tubercles will be formed.
In course of growth the smears become ridges, and the poly-
gonal outlines grow into circular pits with a rim of tubercles,
and finally the pits become tubes with tabule varying in
number from one to five, but commonly with one or two.

I believe that this complicated structure can be accounted
for in the followmg way: Merlia is a thin incrusting
siliceous sponge which has acquired the character of forming a
calcareous skeleton. ‘This sponge, like many other Tetraxonid
sponges, 1s a modification of the simple ‘‘rhagon,” i.e. of a
thin-walled flattened sack with the choanosomal folds branch-
ing out all round. The mode of growth is that of dichotomous
branchlets extending out from centre to periphery and possibly
anastomosing with or in close juxtaposition to neighbouring
branches (see radiating spokes on Pl. 33, fig. 1). At some
epoch in its history—a point to be discussed later—the meta-
bolic cells, the amcebocytes, became fed up with carbonate of
lime,and either underwent calcification or re-secreted the lime.
These cells would necessarily occupy the spaces in the sponge
between the choanosomal branches, especially at points ot

688 R. KIRKPATRICK.

bifurcation. Accordingly heaps of amcebocytes would accu-
mulate at these points, and would extend along lines
at right angles to the choanosomal branches till they
met neighbouring ridges, in such a way as to enclose poly-
gonal or circular areas, over the edges of which the choano-
somal branches would extend (and between the tubercles or
points of greatest heaping). At last the shallow meshes
would become pits into which the heavy amcebocytes loaded
with calcigenous granules would migrate. The heavy masses
of loaded amoebocytes in the pits would tend to stay there,
but the choanosome and ectosome would continue their
growth in the direction of the surface. A tendency to a
cleavage into two zones, hypersome and hyposome, would
gradually become accentuated, especially in cases where the
sponge can increase considerably in depth.

The result of the pull between opposing forces would be
to cause in the little cylinders of soft tissue an hour-glass
constriction. The continual growth of cells and tissues
in a confined space would fill in the space round the hour-
glass neck so that the ‘glasses’? would become two closely
approximated cylinders, between which the amcebocytes,
or rather, caleocytes, would form a tabula leaving only
an isthmus of tissue. Often even this little isthmus is nipped
in and finally cut through by the total closure of the tabula, and
the calecocytes below become cut off and buried in their crypt.

Obviously with varying factors there will be varying
results. With uniform migration of calcocytes over a flat
surface there is a tendency to peripheral extension rather
than to growth in depth, and a uniform pull between hyper-
some and hyposome will lead to uniformity in size of crypts
and to deposition of tabulee in horizontal planes, and so on.

' The laying down of calcareous bars in the earliest stages before the
flagellated chambers are formed would seem at first to furnish an argu-
ment against the theory that the form of the calcareous skeleton has
been, so to speak, moulded by the choanosome. It must be remembered
that siliceous spicules are found, too, in the youngest stage, and these
must originally have been developed later than the flagellated chambers.
By “choanosome” is here meant chiefly the canal system and flagellated
chambers.

ON MERLIA NORMANI. 689

A simple model such as a wide india-rubber tube attached
at one end and stretched so as to form a “ node ”—which could
be tied with string—may serve to represent hyposome and
hypersome. If air be forced in at each end, the two sections
will become cylinders tending to approximate at the node.
The pumped-in air will represent the growing tissues and cell-
masses. Perhaps the rubber tube should be encompassed by
a hard cylinder corresponding to the calcareous pit or tube.

Where growing protoplasm surrounds itself with a nearly
closed calcareous cell or wall this opposition between the
pull of basal inertia and that of centrifugal growth force tends
to bring about the formation of a tabula or its equivalent.

In cases where the soft tissues in a tube are lifted bodily up
and secrete a new floor at their base the comparison with what
takes place in Merlia does not quite hold. The hour-glass-
shaped siphonoglyphe of Tubipora and the cystiphragms in
certain Monticuliporas appear to be due to special kinds of
pulls or strains on the epithelial and other soft tissues.

The varying style of construction of the calcareous skeleton
is due, I believe, to the fact that the calcocytes either settle
down and deposit heaps of lumps (the conules of the tubercles)
or—in the case of crypt-walls and tabulee—layers of flakes.
When crowded in the crypts the actively secreting calcocytes
are spread or squeezed out flat and make smooth walls, filling
in irregularities between the already formed conules. Some-
times from difficulties of terrain, such as the presence of
steep ridges or shells, the little builders form thick conical
main pillars conulated from base to summit and with scarcely
any flanges or tube-walls.!

1 The researches of Koch and Bourne seem to prove that the skeleton
of stony corals is formed as a secretion from calicoblasts. Heider and
Maria Ogilvie, on the other hand, have concluded that this skeleton is
the product of calcified cells. At present the former theory is in the
_ascendant. I have given evidence, though not so much as I could have
wished, to show that the almost coral-like skeleton of Merlia is built
up of calcified cells. The amcebocytes of a sponge are so very different
from the epithelial cells of a coral that it is doubtful whether the know-
ledge of the mode of skeleton formation in Merlia would throw light
on the question of stony coral formation.

VOL. 56, PART 4,—NEW SERIES. 48

690 R. KIRKPATRICK.

Porto Santo is a little island about six miles long from east
to west, with a small islet, Cima,! at its south-east corner,
and another, Ilheo de Baixo, at its south-west corner. Baixo,
which is a volcanic island, is extremely interesting geolo-
gically, for it has beautifully preserved coral reefs sand-
wiched between layers of basalt. The Madeirans use the
coral for lime, and the English name of the island, viz,
Lime Island, is of some significance from the point of view of
a theory Iam about to suggest in explanation of the peculiar
feature of Merlia, viz. that this little sponge may have
acquired its lime-forming character at the epoch when the
coral reefs of Baixo were being formed, that is to say, durmg
the Miocene period.’

There would probably have been a larger amount of carbo-
nate of lime in solution and of fine particles of that substance
in suspension in the sea-water in that area than at present.

Possibly the metabolic cells of Merlia became fed up (if a
vulgar colloquialism may be permitted in the classic pages of
this journal) with calcareous matter, and either re-secreted it,
or became wholly calcified into lumps orflakes.* Nature,always
selecting what is helpful for survival, ended in making use
of this property to elevate the delicate little crust—so liable
and to form shelter-pits

to be overlaid by other. organisms
for the soft tissues. Support and shelter are two very
important factors in sponge economy, as witness the boring
sponges, which have acquired the character of working their
way into shells and limestone. Merlia constructs its own
shelter and supports. Morphologically the calcareous skeleton
is wholly on the basal surface of the sponge, and, indeed.
outside of it.

1 Cima above, Baixo (cf. basis) low. The Portuguese refer to a loca-
tion to the east as cima and one to the west as baixo, because their own
mountains are east and their coasts west.

2 It would be interesting to discover Merlia incrusting the shells,
corals, etc., so abundant in these reefs. It seems to me very probable
that it will be found there.

% Compare Haeckel’s theory of the origin of calcareous and siliceous
sponges respectively on calcareous and siliceous oozes.

ON MERLIA NORMANI. 691

If it be asked why other siliceous sponges, especially those
living in coral-reet -areas, do not form calcareous skeletons,
the reply would be that it is too much to expect to be able
to explain why any apparently spontaneous variation takes
place in any living organism, because the ultimate mole-
cular and chemical factors elude the power of analysis. As
Weismann writes (12)—(but a propos of the trophic
effect of functional stimulus)—‘‘ We are here face to
face with the fundamental phenomenon of life, metabolism ;
and since we do not understand the cause of this, we are not
in a position to say why it varies in this way or that accord-
ing to the stimulus.” Possibly the orange-coloured proto-
plasmic granules of the calcocytes happened to acquire
under favourable conditions just that molecular constitution
that rendered carbonate of lime attractive, but apparently
under penalty of becoming petrified. Later the “ penalty ”
of the individual cells became transformed into an advantage
for the common good.

The theory that Merlia constructed its calcareous skeleton
in comparatively recent times is rendered plausible by the
fact that no fossils, even with a superficial resemblance to the
latter, occur till Paleozoic times. In any case the resem-
blance between Merlia and certain Paleozoic fossils may be
purely homceomorphous.

If, as I believe, Merlia is asiliceous sponge! which has taken
to forming a calcareous skeleton, then this sponge furnishes
a good example of the hereditary transmission of an acquired
character, and further, a striking instance of the tendency,
which Herbert Spencer (9) first called attention to, of one
part of an organism to increase in size and importance at the
expense of the rest.

Weismann gave the name “ intra-selection” to this ten-

1 Whether Merlia acquired its calcigenous properties in Silurian or
in Miocene times, it is difficult to regard it as any other than a siliceous
sponge related to Desmacellinw. No spongologist would believe

that Merlia got its tylostyles, sigmata, trichodragmata and occasional
toxa by a process of convergent evolution.

692 R. KIRKPATRICK.

dency. It is surprising to realise that the extensive crust of
Merliaon the Dendrophyllia is the product of a very thin
incrusting siliceous sponge, and, again, it is equally surprising
to observe the great bulk of the masses of calcocytes in
proportion to the rest of the tissues.

The English colleague already mentioned, who is, I believe,
publishing a report on the dried calcareous skeleton of an
organism which he thinks is Merlia, considers it to be a
Foraminiferan. I think the specimen comes from the West
Indies. Assuming the skeleton to be that of a Merlia, in
which case it will be part of a sponge, the existence of Merlia
at Porto Santo may have been due to embryos being carried
by the Gulf Stream. On the north shore of the island
Columbus collected Hntada beans, which led him to speculate
on the existence of land beyond the western horizon. The
currents which carried the Entada beans might also carry the
Merlia embryos which would settle on the first submarine
peaks they would come to after their long voyage.

The theory that Merlia is a local survival from the periods
when the coral reefs of Ilheo da Baixo were laid down seems
to me more probable, however, than that of the West indian
origin of the sponge.

(6) Systematic Position or Merit.

The calcareous skeleton may be a character of no great
importance from the systematic point of view.

The canal system—in spite of its novel or hymenopylous
character—and the siliceous skeleton undoubtedly resemble in
many respects the types commonly found in Monaxonellid

sponges.

The calcareous skeleton is, I believe, an accidental and
acquired character due to the activities of cells usually con-
cerned in the metabolism of food and not occupied with skeleto-
eenous functions, these latter belonging to a wholly different
ty pe of cells—the scleroblasts. Merlia,then,is a Monaxonellid

ON MERLIA NORMANI. 693

sponge, and further, there need be no hesitation in placing
it in the family Haploscleride. At one time I had regarded
the clavidises as modified chele, on account of the existence
of two minute knobs on one of the “teeth” of a developing
form, but it soon became evident that the affinities of these
spicules were with the sigmata. If the free ends of the
diancistra of Hamacantha were united it would become a
clavidisc. Merlia, with its tylote megascleres, its rhaphides,
trichodragmata, sigmata and toxa, is nearly related to the
Desmacelline, a sub-family into which Lundbeck (11) places
the three genera, Biemma, Desmacella, and Hamacantha.
The scattered vertical tufts visible through the surface of
Merlia in whole decalcified specimens somewhat resemble
similar tufts at the oscular and poral surfaces of Biemma
rosea Fristedt (11). Occasional horizontal bundles of
spicules lying on the tabule in Merlia might be compared
with the deeper-lying spicular network in B. rosea.

The presence, however, of clavidiscs and of a peculiar type
of canal system in Merlia necessitate the founding of a new
sub-family, viz., Merlin, which should be placed next to
Desmacelline.

It would be an interesting experiment to cultivate Merlia
in lime-free sea-water. If the sponge survived such depri-
yation the calcocytes would probably at first continue to heap
up or build a soft skeleton similar in shape to the hard one,
but later, being unburdened with lime, would resume their
original functions as ordinary amcebocytes.

(7) ON Some ReseMBLANCES BETWEEN THE CALCAREOUS SKELE-
TON OF Meriia AND CrrraIn PaLxozoic ORGANISMS.

The calcareous skeleton of Merlia undoubtedly presents
striking superficial resemblances to certain Paleozoic fossils,
such as certain Monticuliporas, and to Rhaphidopora
stromatoporoides from the Devonian of Gerolstein (PI. 38,
figs. 7, 8, 9). In each case there is a surface polygonal

694, R. KIRKPATRICK.

network with tubercles at the nodes, and tabular floors. In
Rhaphidop ora the tabule have a central hole and radial
(? sutural) lines. In a former paper on Merlia I pointed
out that these resemblances might be merely homceomor-
phous ; at the same time I hoped that Merlia might throw
light on the problem of the nature of Monticulipora and
kindred organisms that have been placed by various authors
in widely different groups; and I was rash enough to state
that there was now more evidence in favour of the theory
that some Monticulipores were sponges than in that of any
other theory concerning the nature of these fossils.

After re-examining the large British Museum collection of
fossils, and sections of Monticulipora and allied forms,
especially the American series presented by Prof. Ray
Bassler, I doubt whether Merlia is going to throw light on
the problems concerning the nature of these organisms. It
would, indeed, be an astounding paradox to state that some
of the Paleeozoic so-called Tabulate corals (Nicholson) or
Polyzoa (Bassler) might prove to be the product of siliceous
sponges, but the theory here put forward concerning the
nature of Merlia is just such a paradox. At any rate the
fact must be taken into account that it is possible for a
siliceous sponge to make a tubular tabulate calcareous skeleton
with certain resemblances to some of the Paleozoic M onti-
culiporas sensu lato. If Merlia had been found fossil,
Nicholson would certainly have classed it among the T'abulate
corals. Nickles and Bassler would, I think—but I write
subject to correction—have placed it among the Polyzoa,
possibly in a new sub-order, or in the Trepostomata, though
not in the family Monticuliporidee Nicholson (emend Ulrich)
(10), seeing that Merlia has no cystiphragms, mesopores, or
acanthopores. The zoologist who would propose to classify
a recent Merlia among the Foraminifera would naturally
place the fossil Merlia in that group. I myself at first
described the dried skeleton as that of a Pharetron sponge.
Certainly no paleontologist or zoologist would have regarded
such fossils or dried recent skeletons as products of siliceous

ON -MERLIA NORMANI. 695

sponges. Yet in this instance I may say, in view of my own
error, that truth is stranger than fiction.

It seems to me unlikely that Merlia could form anything
else than a thin crust, 1.e. that it, or forms allied to
it, could have formed a laminate or branched growth, for
instance, but sometimes the living sponge grows over a dead
crust of its own kind.

What has happened in one instance may have taken place
on other occasions. Ameoebocytes of ‘letraxonid sponges
may have become calcocytes under favourable conditions at
any epoch, and these calcocytes may have formed calcareous
skeletons, just as Merlia has done.

(8) Summary.

Merlia is a vermilion coloured incrusting Monaxonellid
sponge belonging to a new sub-family—Merlinze—of the
Haploscleridee. Large granular amcebocytes (caleocytes) have
constructed a basal calcareous skeleton formed of vertical tubes
divided up by horizontal tabule. The tubes are built up of
columns, each with three vertical wings which unite with
wings of neighbouring columns to form tubes. This mode
of construction was probably primarily determined by the
disposition of the branches of the choanosome which led to
the deposition of amcebocytes at the points of bifurcation of
the lines of flagellated chambers.

Apparently the caleocytes become wholly transformed into
lumps, conules or flakes.

The calcareous skeleton shows certain resemblances, espe-
cially at the surface, to certain Paleozoic fossils, classed
among “ Tabulate corals”? or Polyzoa.

There is no dermal epithelium, and the canal system is
hymenopylous.

The sponge has been found in 60-90 fathoms off Porto
Santo Island and Madeira.

A few more words remain to be said. It has been denied
that Merlia isa sponge. I can only say that my opinion

696 R. KIRKPATRICK.

whether right or wrong, is based on the prolonged investiga-
tion of abundance of good material, whereas other opinions
seem to me to be founded mainly on a priori considerations.

I have examined over 500 specimens of Merlia and have
always found the tissues of the sponge in most intimate
association! with a caleareous structure, which grows as the
sponge grows. Granular amoebocytes varying in shape
according to circumstances are found everywhere in contact
with the skeleton, and apparently in continuity with the
organic matrix of the same. Also there is a curious
simuarity in appearance between the granules of these super-
ficially placed cells and the surface view of the ends of the
fibrillee of the skeleton. The amcebocytes in the upper part
of the sponge have the same fundamental characters as those
in the interior of the crypts.

Amoebocytes are found deep down in crypts nearly closed
over by tabule, and it is incredible that these large masses
of cells could have worked their way down through the
alinost closed slits which are found in many tabule. Apart
from these mechanical difficulties, one cannot imagine, from
the point of view of common-sense, why the under surface of
a supposed parasitic sponge should send down cylindrical
moniliform masses of granular cells into the empty spaces
of a foreign organism, thereby carrying out a seemingly use-
less and exhausting procedure. In young sponges on delicate
shells, well stained and perfectly transparent, it can be
clearly seen that there is not the least trace of any other
organism than the sponge (on the shell). Weltner writes—
of the caleareous structure as that of an

and not unnaturally
uuknown organism in which a spouge has settled. If this be
so the said organism has preserved its incognito ina marvellous
manuer. ‘The theory that Merliais a sponge that has formed
both a siliceous and a calcareous skeleton seems to me the only
one possible, and further, the theory that it is a siliceous

' Of course, it is usual for a parasite to be closely associated with its
host, but I trust that this investigation will make it clear that Merlia
is not an instance of an association of this nature.

ON MERLIA NORMANI. 697

sponge that has taken to forming carbonate of lime one that
is extremely probable.

Personal Note.—The finding of Merlia was associated
with a curious incident which I trust may be of sufficient
interest to be worth mentioning. I went out dredging daily
at Porto Santo in a small whaling boat with Senhor Noronha
and a crew of seven men. As day after day passed without
result, I reluctantly decided to give up the search and return
to Madeira. On the ninth day of dredging I secured as
usual several large tubfuls of various specimens, but
apparently Merlia was not amongst them. One of the tubs
contained coarse sand, and I had told the men to throw it
overboard in order to refill with more likely material, such
as shells, pieces of rock, etc. I had arranged to leave the
island that evening, and shortly after landing from the
dredging I sent my luggage down to the beach. While the
men were carrying away my boxes | casually picked up a
small worm-tube out of the tub of coarse sand which had
been brought ashore after all. Great was my pleasure on
recognising in the fading light the long-sought-for Merlia.
I at once altered my plans and went next morning to the
spot whence the sand had been dredged, and secured a
number of living specimens of the little sponge.

LITERATURE.

er

. Kirkpatrick, R.—‘*On Two New Genera of Recent Pharetronid
Sponges,” ‘ Ann. Mag. Nat. Hist.,’ 1908 (8), ii, p. 503.

2. Weltner, W.—‘* Ist Merlia normani Kirkp. ein Schwamm?”
‘Archiv fiir Naturg., Jahrb. 75, Bd. i, Heft 1, 1909, p. 159.

“Ist Astrosclera willeyana Lister eine Spongie?”
‘Archiv fiir Naturg.,’ Jahrb. 76, Bd. 1, Heft 1, 1910, pp. 128-184.

4. Kirkpatrick, R.—“ Notes on Merlia normani Kirkp.,” * Ann.

Mag. Nat. Hist.,’ 1909 (8), iv, p. 42.

5. “Further Notes on Merlia normani Kirkp.,’‘ Ann. Mag.
Nat. Hist.,’ 1910 (8), v, pp. 288-291.
6. “Notes on Merlia normani Kirkp.,” ‘Ann. Mag. Nat.

Hist.,’ 1909 (8), iv, p. 47.

698 R. KIRKPATRICK.

7. Kirkpatrick R.— Ann. Mag. Nat. Hist.,’ 1908 (8), ii, p. 5038, pl. xv,
fig. 14.
3. ——_— “Ann? Mag: Nat. Hist:, 1908 (8), 11; p: 509:
9. Spencer, Herbert.—‘* Weismannism Once More,” ‘ Contemporary
Review,’ reprint, 1894, p. 7.
10. Nickles, J. M., and Bassler, Ray S.—‘A Synopsis of American
Fossil Bryozoa,’ 1900, p. 28.
11. Lundbeck, W.—‘ The Danish Ingolf-Exp. VI, i, Porifera (1),’ 1902,
DP: 62; DL xy, MoS. 74.6.
12. Weismann, A.—‘ The Evolution Theory,’ transl. J. A. and M. Thom-
son, 1904, vol. i, p. 244.

EXPLANATION OF PLATES 382-38,
Illustrating Mr. R. Kirkpatrick’s paper “On Merlia nor-
mani: a Sponge with a Siliceous and Calcareous
Skeleton.”’
PLATE 32.
Fig. 1—Merlia normani. Piece of shell incrusted with three
small specimens. Natural size.
Fig. 2.—Specimen on coralline. Natural size.
Fig. 5.

Fig. 4.—Specimen on voleanic block. Natural size.

2

Specimen on shell. x 3.

Fig. 5.—Specimen, dried and mostly denuded of soft tissues, incrusting
Dendrophyllia. Natural size.

Fig. 5a.—Part of same. The larger circular holes are due to presence
of worm-tubes. x 5. (See also Text-fig. 3, p. 373).

Fig. 6.—Macerated and incinerated specimen. xX 15.

Fig. 7.—Part of surface of sponge showing oscule surrounded by
a ring of pores. x 50.

Fig. 8.—Another part of surface showing irregular distribution of
pores. x 50.

Fig. 9.—Decalcified thick vertical section of sponge showing several
storeys of masses of crypt-cells or calcocytes ; sponge much contracted
at surface. x 50.

Fig. 10.—Vertical section of well-expanded specimen. a. Subdermal
spaces. 6, Flagellated chambers. c. Crypt-cells or caleocytes. d. Long

ON MERLIA NORMANIT. 699

siliceous spicules. e. Clavidiscs. f. Tubercles of calcareous skeleton.
g. Skeletal wall showing zones of fibrille fanning out. x 125.
Fig. 11.—Crypt-cells in glycerine. x 1500.

Fig. 12.—Youngest specimen (A) forming a minute red spot on a
shell. x 25.

PLATE 33.

Fig. 1—Upper surface of decalcified specimen showing “nodal”
masses, a, of flagellated chambers joined by radial bands, b, of flagel-
lated chambers. c. Spaces left by decalcified tubercles. x 100.

Fig. 2.—Under surface of another specimen (decalcified). a. Masses
of choanosome filling uppermost spaces of calcareous skeleton. b. Radial
“spokes” joining these masses. c. Spaces left by decalcified tubercles,
d. Crypt cells. x 120.

Fig. 3.—Decalcified vertical section of well-expanded specimen
(showing only one mass of crypt-cells). a. Subdermal space. b. Flagel-
lated chambers. c. Soft tissues (in uppermost crypts). d. Soft tissues,
mostly calcocytes, in a lower crypt. e. Bundles of siliceous spicules.
jf. Clavidises. g. Inhalant pore. x 140.

Fig. 4.—Flagellated chambers. a. Apopyle in sphinctrate membrane.
b. Sphincter * muscle ’’-cell. x 750.

Fig. 5.—Flagellated chambers. Lateral view. x 750.

Fig. 6.—Flagellated chamber, convex surface, showing prosopyles
between stellate bases of collar-cells. x 1600.

Fig. 7.—Section of a flagellated chamber. x 2425.

Figs. 8-11.—Collar-cells in different conditions of contraction, drawn
from the same section. x 1800.

Fig. 12.—Collar-cell showing flagellum passing down to nucleus. x
1800.

Fig. 15.—Growing edge of young specimen. a. Flagellated chambers.
b. Radiating tufts of spicules. c. Clavidise. x 175.

PLATE 34.

Fig. 1.—Section of a small decalcified specimen showing gradual
development of calcareous skeleton from edge to centre of the crust
(the skeleton being represented by spaces in figure). x 25.

Fig. 2.—Surface of a young specimen. a. Oscule. 6. Pores. x 425,

Fig. 3.—Decalcified tubercle and upper edge of wall of skeleton,
showing large granular cells (caleocytes) and radiating fibrillar structure
of organic matrix. a. Granular cells. b. Radiating fibrille in body of
tubercle. c. Flagellated chamber. x 375.

700 R. KIRKPATRICK.

Fig. 4.—Transverse section of mass of crypt-cells (caleocytes). a.
Fibrille of maltha. b. The same cut across. x 540. (N.B.—Calco-
cytes in crypts are, for convenience, termed crypt-cells.)

Fig. 5.—Portion of same. x 2000.

Fig. 6.—Edge of mass of crypt-cells, showing cut edges of surface
ones forming a flat epithelium (a), with the large oval nucleus forming
a hump on inner aspect facing cell-mass. x 1025.

Fig. 7.—A crypt-cell showing spherical nucleus and nucleolus. x
5000.

Fig. 8.—Granular cell in an uppermost or open crypt, with very
large nucleus and nucleolus, a. ? Tokocyte (egg-cell). x 3000.

PLATE 35.
Fig. 1—Tylostyles. x 525.
Fig. 2.—Trichodragma. x 750.
Fig. 2a—Rhaphides. x 750.
Fig. 5.—Clavidise, most frequent shape. x 750.
Fig. 4.—Broken clayvidise showing axial canal. x 1000.

or
o

Fig. 5.—Clavidise with narrower rim, deeply situated in the sponge.

Fig. 6.—Developmental form of clavidise, with ends joined by slender
filament. x 750.

Fig. 7.—Incomplete clavidise, like a sigma. x 750.
Fig. 8.—Clavidise with crossed ends. x 750.

Fig. 9.—Young twisted form of clavidise—abnormal. x 750.

Fig. 10.—Very slender, simple sigmata. x 750.

Figs. 11-15.—Abnormal spicules, all from one specimen. Fig. 11,
tylostyle with three knobs. Fig. 12, tylostyle with double curve. Fig.
13, toxa-shaped rhaphide. Fig. 14, clavidise with flat discs in place of
keyhole sinuses. Fig. 15, slender sigmata with knobs at each end.
All x 750.

Fig. 16.—Calcareous skeleton, surface view, showing surface tabula,
with central hole, or slit, which may be quite closed. The tabula
show concentric circles and radial sutures radiating from centre to the
inter-flange suture. x 130.

Fig. 17.—Vertical section of skeleton, showing several crypts, one
series being laid open and two others closed over (by vertical flanges or
wings). x 130.

Fig. 18.—Base of skeleton, showing some tabule nearly closed, which

ON MERLIA NORMANI. 701

roofed over crypts which had been full of calcocytes, later macerated
out. x 100.

Fig. 19.—Ground-down basal section with soft tissues remaining.
Thick walls of tubes as seen by strong, oblique reflected light, showing
well how vertical flanges or wings join to form walls of tubes. a.
Tabula. x 160.

Fig. 20.—Calcareous skeleton of Merlia on surface of carinate worm-
tube, macerated in Eau de Javelle, the youngest parts of skeleton being
lumps and seales. x 100.

Fig. 21.—Caleareous scales on surface of worm-tube (see fig. 20)
having appearance of calcified flattened cells, with a nuclear hump and
granular structure. xX 425.

Fig. 22—A conule at base of tubercle having the appearance of a
petrified amcebocyte, fitting cap-like on rounded surface below, with
rounded basal rim and lobate basal process, with oval nucleus and
granular structure. x 1500.

PLATE 36.

Fig. 1—Part of mass of erypt-cells. a. An isthmus of tissue. b.
Flattened crypt-cells in section, much contracted, showing sections of
nuclei. c. Connective tissue cell. x 1000.

Fig. 2.—Part of sponge tissue at bottom of an upper or open crypt.
a. Collar-cells. b. Longitudinal bundles of spicules with their sclero-
blasts. c. Transverse section of spicule bundle. x 1500.

Fig. 3.—A neck or isthmus joining upper part of sponge to a mass of
crypt-tissue. a. Large granular crypt-cells. b. Connective-tissue cells
(collencytes). x 1500.

Fig. 4.—Large granular crypt-cells. x 1500.

Fig. 5.—Three caleocytes on summit of a tubercle. Slightly separated
by pressing down cover-slip ; separation indicated by ¥ line. [Note.—
The view is in projection; the cells extend some distance vertically
down.| x 2200.

Fig. 6—Growing edge of skeleton. Calcareous bar covered with
layer of caleocytes. x 650.

Fig. 7.—One calcocyte from those depicted in fig. 6, showing
granular contents (each granule with dark central spot), large oval
nucleus, and the nucleolus. x 4000.

Fig, 8—Two flattened granular cells. x 2220.

Fig. 9.—Caleareous skeleton, showing granular appearance (due to
ends of fibrillee) at surface, and fibrillar structure in section. x 1800.

Fig. 10.—Clavidise with its scleroblast. a. Nucleus. x 1450.

702 R. KIRKPATRICK.

PLATE 37,

Fig. 1.—Growing edge of a specimen of Merlia (osmic-picro-carmine
preparation in glycerine) showing elongated calcocytes planning out
calcareous skeleton on smooth surface of shell. a. Calcocytes. b.
Flagellated chambers and excurrent canal. ec. Siliceous spicules. d.
Edge of sponge. e. Connective-tissue cells (collencytes). x 750.

Fig. 2.—Collencytes. x 2425.

Fig. 3.—Myocytes arranged radially and concentrically round a pore.
x 1450.

Fig. 4.—Gland-cells. x 2425.

PLATE 38.

Fig. 1.—Vertical section of very young specimen figured on PI. 32,
fig. 13, decalcified. a. Spaces left by dissolved skeleton. b. Klon-
gated caleocytes. ¢. Connective-tissue cells, with finely and abundantly
branched processes. d. Young wedge-shaped collar-cells. x 1000.

Fig. 2.—Vertical section of same showing a tylostyle and a bundle of
siliceous spicules cut transversely. x 1000.

Fig. 3.—Vertical section of same nearer to edge of sponge than in
fig. 1. x 540.

Fig. 4.—Part of a collencyte (mostly cut away) showing many tufts
of branching processes. x 1500.

Fig. 5.—Growing edge of a specimen. a. Edge of sponge. 6. Cal-
careous skeleton. x 120.

Fig. 6.—The tuning-fork spicule from the original dried specimen
of Merlia. x 1050.

Fig. 7.—Monticulipora (Heterotrypa) monilformis Nich.
(from Devonian of Ontario), surface, showing polygonal network and
tubercles. Copied from Nicholson’s * Paleeozoic Corals,’ ‘* Monticuli-
pora,’ 1871, pl.i, fig. la. x 18.

Fig. 8.—Rhaphidopora (Chetetes) stromatoporoides
Roemer. Devonian of Gerolstein. ‘Tangential section” (but ? sur-
R.K.). From Nicholson and Foord, ‘Ann. Mag. Nat. Hist.’

(5), xvii, pl. xvi, fig. 5, showing polygonal network, tubercles, and tabule,

face view.

some with central hole and apparently with radial sutural lines. x 20.

Fig. 9.—The same as fig. 8, deeper transverse section. xX 20.

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INDEX "EO VOL. 56;

NEW

Acanthohamingia Shipleyi: a
new Echiuroid, by Iwaji Ikeda,
135

Arnold and Barratt, on the blood of
Coleoptera, 149

Avitellina centripunctata, his-
tology of, by Gough, 317

Bacteria, cytology of, by Clifford
Dobell, 395

Barratt, see Arnold.

Blood of Coleoptera, by Barratt and
Arnold, 149

Bosanquet on Spirochexta ano-
donte, 387

Brain-hemispheres, structure of, in
Papio hamadryas, by Schuster,
613

Coleoptera, blood of, by Barratt and
Arnold, 149

Cranial muscles of Vertebrates, by
Edgeworth, 167

Cristispira veneris and classi-

fication of Spirochetes, by Clifford
Dobell, 507

Dasyurus viverrinus, embryo-
logy of, by Prof. J.P. Hill, D.Se.,1

Dobell on Cristispira veneris
nov. spec., and on the affinities
and classification of Spirochetes,
507

SERIES.

| Dobell on the cytology of Bacteria,

395

Dunkerly on the life-history of
Leptomonas musce domes-
tice and on the relations of the
flagellate parasites of insects, 645

_ Echiuroid, on a deep-sea, by Iwaji

Ikeda, 135

Edgeworth, F. H., on the morpho-
logy of the cranial muscles of some
Vertebrates, 167

Embryology of the Marsupialia, con-
tributions to, by Prof. J. P. Hill, 1

Eupomatus, development and struc-
ture of the trochophore of, by
Cresswell Shearer, 543

Flagellate parasites of insects, by
Dunkerly, 645

Gough on tapeworms of the family
Avitelline, 317

Hamingia, remarks on species of, by
Ikeda, 135

Hill, Prof. J. P., on the early de-
velopment of the Marsupialia, 1

Hydroides uncinatus, develop-
ment of the trochophore of, by
Cresswell Shearer, 543

704

Ikeda on a deep-sea Echiuroid,
Acanthohamingia Shipleyi,
n.g. and n.sp., 135

Kirkpatrick on Merlia Normani,
with a siliceous and
calcareous skeleton, 657

a sponge

Leptomonas musce domes-
tice, life-history of, by Dunkerly,
645

Marsupialia, early development of,
lone lergoyi, dio Jen la bubly 1Ossiers al

Merlia Normani, a sponge with a
siliceous and calcareous skeleton,
by Kirkpatrick, 657

Muscles, cranial, of Vertebrates, by
Edgeworth, 167

Papio hamadryas, cortical cell
lamination of the cerebral hemi-
spheres of, by Schuster, 613

INDEX.

Schuster on the cortical cell lamina-
tion of the cerebral hemispheres of
Papio hamadryas, 613

Sex, experimental analysis of, by
Geoffrey Smith, 591

Shearer on the development and
structure of the trochophore of
Hydroides uncinatus (Hupo-
matus ), 543

Smith, Geoffrey, on the experimental
analysis of sex, 591

Spirocheta anodonte, by
Bosanquet, 387

Spirochetes, affinities and classifica-
tion of, by Clifford Dobell, 507

Sponge (Merlia) with a siliceous
and calcareous skeleton, by Kirk-
patrick, 657

Stilesia, a genus of tapeworms, by
Gough, 317

Tapeworms of the genus Stilesia,
by Gough, 317

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CONTENTS OF No. 221.—New Series.

MEMOIRS:
PAGE

The Early Development of the Marsupialia, with Special Reference to
the Native Cat (Dasyurus viverrinus). (Contributions to the
Embryology of the Marsupialia, IV.) By J. P. Hin, D.8ce.,
Jodrell Professor of Zoology and Comparative Anatomy, University
of London, University BE, (With Plates 1-9 and 2 Text-
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Notes on a Deep-Sea chided! ice neonatal gia peapics (n.g
et n. sp.), with Remarks on the species Hamingia ijimai iikedat
By Iwast Ixepa. (With Plate 10) . . 185
A Study of the Blood of certain Coleoptera : Diapciaeds marginalis
and Hydrophiluspiceus. By J. O. Waxetin Barratt, M.D.,
D.Se., and Groree ARNOLD, M.Sce., from the Cancer Research Labo-
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MEMOIRS :

On the Morphology of the Cranial Muscles in Some Vertebrates. By
F. H. Epceworra, M.D., D.Sc., Professor of Medicine, University
of Bristol. (With 100 Text-figures)

A Monograph of the Tape-worms of the Sub-family Ayitelesace
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water as required. Half-tone reproduction is recommended for
uncoloured drawings of sections and of Protozoa.

Drawings for text-figures should nor be inserted in the MS.,
but sent in a separate envelope to the Editor.

Contributors to this Journal requiring ewtra copies of their
communications at their own expense can have them by applying
to the Printers,

Messrs. AbLARD & Son, 23, Bartholomew Close, E.C., on
the following terms:

For every four pages or less—

25 copies ; : 5/-
abc 5 . : 6/-
(Dae : 6/6
100% =. 7/-

Plates, 2/- per 26 if uncoloured ; if coloured, at the same rate for
every colour.
Prepayment by P.O. Order is requested.
ALL CoMMUNICATIONS FOR THE EDITORS TO BE ADDRESSED 'O THE CARE
or Messrs. J. & A. Courcuiit, 7 Great Mariporoues Srreet,

Lonpon, W.

THE MARINE BIOLOGICAL ASSOCIATION

OF THE

UNITED KINGDOM.
Patron—HIS MAJESTY THE KING.

President—Sir RAY LANKESTER, K.C.B., LL.D., F.R.S.
:0:

THE ASSOCIATION WAS FOUNDED “‘ TO ESTABLISH AND MAINTAIN LABORATORIES ON
THE COAST OF THE UNITED KINGDOM, WHERE ACCURATE RESEARCHES MAY BE CARRIED
ON, LEADING 10 THE IMPROVEMENT OF ZOOLOGICAL AND BOTANICAL SCIENCE, AND TO
AN INCREASE OF OUR KNOWLEDGE AS REGARDS THE FOOD, LIFE CONDITIONS, AND HABITS
OF BRI’TISH FOOD-FISHES AND MOLLUSCS.”’

The Laboratory at Plymouth

was opened in 1888. Since that time investigations, practical and scientific, have
been constantly pursued by naturalists appainted by the Association, as well as by
those from England and abroad who have carried on independent researches.

Naturalists desiring to work at the Laboratory

should communicate with the Director, who will supply all information as to
terms, etc.

Works published by the Association

include the following :—‘ A ‘Treatise on the Common Sole,’ J. ‘I’. Cunningham, M.A,,
4to, 25/-. ‘The Natural History of the Marketable Marine Fishes of the British
Islands,’ J. 1. Cunningham, M.A., 7/6 net (published for the Association by
Messrs. Macmillan & Co.).

The Journal of the Marine Biological Association
is issued half-yearly, price 3/6 each number.
In addition to these publications, the results of work done in the Laboratory
are recorded in the ‘Quarterly Journal of Microscopical Science,’ and in other
scientific journals, British and foreign.

Specimens of Marine Animals and Plants,

both living and preserved, according to the best methods, are supplied to the
principal British Laboratories and Museums. Detailed price lists will be forwarded
on application.

TERMS OF MEMBERSHIP.

ANNUAL MEMBERS : : - £1 1 Oper annun.
LIFE MEMBERS . ‘ : : A 15 15 0 Composition Fee.
FOUNDERS . : ; LOO ORO: 9 =

Governors (Life Membersof Council) 500 0 0

Members have the following rights and privileges:—They elect annually the
Officers and Council; they receive the Journal free by post; they are admitted to
view the Laboratory at any time, and may introduce friends with them; they have the
first claim to rent a table in the Laboratory for research, with use of tanks, boats, etc. ;
and have access to the Library at Plymouth. Special privileges ure granted to Governors,
Founders, and Life Members.

Persons desirous of becoming members, or of obtaining any information with
regard to the Association, should communicate with—

The DIRECTOR,
The Laboratory,
Plymouth.

Rae

~~

New Series, No. 223 (Vol. 56, Part 3), Price 10s, net.

Subscription per volume (of 4 parts) 40s. net.

APRIL; 1911.
THE

QUARTERLY JOURNAL

OF

MICROSCOPICAL SCIENCE.

EDITED BY

Siz RAY LANKESTER, K.C.B., M.A., D.Sc., LL.D., F.R.S.,

HONORARY FELLOW OF EXETER COLLEGE, OXFORD;
MEMBER OF THE INSTITUTE OF FRANCE (ASSOCIK ETRANGER DE L’ACADEMIE DES SCIENCES) ;
CORRESPONDENT OF THE IMPERIAL ACADEMY OF SCIENCES OF 8T. PETERSBURG, AND OF THE
ACADKMY OF SCIENCES OF PHILADELPHIA, AND-OF THE ROYAL ACADEMY OF SCIENCES
OF TURIN; FOREIGN MEMBER OF THE ROYAL SOCIETY OF SCIENCES OF
GOTTINGEN, AND OF THE KOYAL BOHEMIAN SOCIETY OF SCIENCES, AND
OF THE ACADEMY OF THE LINCEI OF ROME, AND OF THE AMERICAN
ACADEMY OF ARTS AND SCIENCES OF BOSTON; ASSOCIATE OF THE
ROYAL ACADEMY OF BELGIUM; HONORARY MEM2®R OF THE
NEW YORK ACADEMY OF SCIENCES, AND OF THR
CAMBRIDGE PHILOSOPHICAL SOCIETY, AND OF
THE ROYAL PHYSICAL SOCIETY OF EDIN-

BURGH, AND OF THE
BIOLOGICAL SOCIETY OF PARIS, AND OF THE CALIFORNIA ACADEMY OF SCIENCES OF SAN FRANCISCO, AND
OF THE ROYAL ZOOLOGICAL AND MALACOLOGICAL SOCIETY OF BELGIUM;

CORRESPONDING MEMBER OF THE SENKENBERG ACADEMY OF FRANKFURT-A-M;

FOREIGN ASSOCIATE OF THE NATIONAL ACADEMY OF SCIENCES, U.8., AND MEMBER OF THE
AMERICAN PHILOSOPHICAL SOCIETY 3
HONORARY FELLOW OF THE ROYAL SOCIETY OF EDINBURGH,

LATE DIRECTOR OF THE NATURAL HISTORY DEPARTMENTS OF THE BRITISH MUSEUM; LATE PRESIDENT OF THE
BRITISH ASSOCIATION FORK THE ADVANCEMENT OF SCIENCE; LATE FULLKERIAN PROFESSOR OF
PHYSIOLOGY IN THE ROYAL INSTITUTION OF GREAT BRITAIN,

LATE LINACRE PROFESSOR OF COMPARATIVE ANATOMY AND FELLOW OF MERTON COLLEGE, OXFORD;
EMERITUS PROFESSOR OF ZOOLOGY AND COMPARATIVE ANATOMY IN UNIVERSITY COLLEGE, UNIVERSITY OF LONDON.

WITH THE CO-OPERATION OF

ADAM SEDGWICK, M.A., F.RS.,

FELLOW OF TRINITY COLLEGE, CAMBRIDGE, AND PROFESSOR OF ZOOLOGY IN THE IMPERIAL COLLEGE OF
SCEENCE AND TECHNOLOGY, LONDON ;

SYDNEY J. HICKSON, M.A., F.RB.S.,

BEYER PROFESSOR OP ZOOLOGY IN THE UNIVERSITY OF MANCHESTER,

K. A. MINCHIN, M.A.,

PROFESSOR OF PROTOZOOLOGY IN THE UNIVERSITY OF LONDON;
AND

GILBERT C. BOURNE, M.A., D.Sc., F.R.S.,

LINACRE PROFERSSOR OF COMPARATIVE ANATOMY, AND FKILLOW OF MERTON COLLEGE, OXFORD.

WITH LITHOGRAPHIC PLATES AND TEXT-FIGURES.

LON DOWN:
J. & A. CHURCHILL, 7 GREAT MARLBOROUGH STREET.

OLE
LS CLE LE ALI DIL AP MT I Bs EE

Adlard and Son, Impr.,] [London and Dorking,

CONTENTS OF No. 223.—New Series.

MEMOIRS:

Contributions to the Cytology of the Bacteria. By C. CLirrorp
Doser, Fellow of Trinity College, Cambridge; Lecturer at the
Imperial College of Science and Technology, London. (With
Plates 16-19 and 1 Text-figure)

On Cristispira Veneris nov. spec., aad the Affinities aaa Glace:
fication of Spirochets. By C.Currrorp DosrEtt, Fellow of Trinity
College, Cambridge ; Lecturer at the Imperial College of Science
and Technology, London. (With Plate 20 and 2 Text-figures)

On the Development and Structure of the Trochophore of Hy droides
uncinatus (Eupomatus). By CrEesswELL SHEARER, M.A.,

Trinity College, Cambridge. (With Plates 21-23 and 29 Text-
figures) : ; :

Studies in the Bepeseeael deals of Sex. By Grorrrey Smirx,
M.A., Fellow of New College, Oxford

PAGE

395

507

With Ten Plates, Royal Ato, 5s.
CONTRIBUTIONS TO THE KNOWLEDGE OF RHABDOPLEURA
AND AMPHIOXUS.
By Sir RAY LANKESTER, K.C.B., M.A., LL.D., F.R.S.
London: J. & A. CHURCHILL, 7 Great Marlborough Street.

Quarterly Journal of Microscopical

Science.

The SUBSCRIPTION is £2 for the Volume of Four Numbers;
for this sum (prepaid) the JouRNAL is sent Post Free to any part
of the world.

BACK NUMBERS of the Journat, which remain in print, are
now sold at an uniform price of 10/- net.

The issue of Surptemenr Nomegrs being found inconvenient,
and there being often in the [Hditor’s hands an accumulation of
valuable material, it has been decided to publish this Journal at
such intervals as may seem desirable, rather than delay the appear-
ance of Memoirs for a regular quarterly publication.

The title remains unaltered, though more than Four Numbers
may be published in the course of a year.

Each Number is sold at 10/- net, and Four Numbers make
up a Volume.

of Microscopical Science receive fifty copies of their communica-
tion gratis.

All expenses of publication and illustration are paid by the
publishers.

Lithographic plates and text-figures are used in illustration.
Shaded drawings intended for photographic reproduction as half-
tone blocks should be executed in “ Process Black”’ diluted with
water as required. Half-tone reproduction is recommended for
uncoloured drawings of sections and of Protozoa.

Drawings for text-figures should nor be inserted in the MS.,
but sent in a separate envelope to the Editor.

Contributors to this Journal requiring ewtra copies of their
communications at their own expense can have them by applying
to the Printers,

Messrs. ApirarD & Son, 23, Bartholomew Close, H.C., on
the following terms:

For every four pages or less—

25 copies : ; 5/-
eRe 6/-
FGM Sh : 6/6

LOGS ~;, : 7/-
Plates, 2/- per 25 if uncoloured; if coloured, at the same rate for
every colour.
Prepayment by P.O. Order is requested.
ALL COMMUNICATIONS FOR THE EDITORS TO BE ADDRESSED TO THE CARE
or Messrs. J. & A. CHurcHILL, 7 Great MARLBOROUGH STREET,
Lonpon, W.

THE MARINE BIOLOGICAL ASSOCIATION

OF THE

UNITED KINGDOM.
Patron—HIS MAJESTY THE, Kine:

President—Sir RAY LANKESTER, K.C.B., LL.D., F.R.S.
LO)

‘THE ASSOCIATION WAS FOUNDED “ TO ESTABLISH AND MAINTAIN LABORATORIES ON
THE COAST OF THE UNITED KINGDOM, WHERE ACCURATE RESEARCHES MAY BE CARRIED
ON, LEADING TO THE IMPROVEMENT OF ZOOLOGICAL AND BOTANICAL SCIENCE, AND TO
AN INCREASE OF OUR KNOWLEDGE AS REGARDS THE FOOD, LIFE CONDITIONS, AND HABITS
OF BRITISH FOOD-FISHES AND MOLLUSCS.”

The Laboratory at Plymouth

was opened in 1888. Since that time investigations, practical and scientific, have
been constantly pursued by naturalists appointed by the Association, as well as by
those from England and abroad who have carried on independent researches.

Naturalists desiring to work at the Laboratory

should communicate with the Director, who will supply all information as to
terms, etc.

Works published by the Association

include the following :—‘ A ‘Treatise on the Common Sole,’ J. T. Cunningham, M.A.,
4to, 25/-. ‘The Natural History of the Marketable Marine Fishes of the British
Islands, J. I. Cunningham, M.A., 7/6 net (published for the Association by
Messrs. Macmillan & Co.).

The Journal of the Marine Biological Association
is issued half-yearly, price 3/6 each number.
In addition to these publications, the results of work done in the Laboratory
are recorded in the ‘Quarterly Journal of Microscopical Science,’ and in other
scientific journals, British and foreign.

Specimens of Marine Animals and Plants,

both living and preserved, according to the best methods, are supplied to the
principal British Laboratories and Museums. Detailed price lists will be forwarded
on application.

TERMS OF MEMBERSHIP.

ANNUAL MEMBERS . : : . £1 1 Oper annum.
LIFE MEMBERS . , : : . 15 15 0 Composition Fee.
FOUNDERS . ; LOO Opn

” ”

Governors (Life Members of Council) 500 O O

Members have the following rights and privileges:—They elect annually the
Officers and Council; they receive the Journal free by post; they are admitted to
view the Laboratory at any time, and may introduce friends with them; they have the
first claim to rent a table in the Laboratory for research, with use of tanks, boats, etc. ;
and have access to the Library at Plymouth. Special privileges ure granted to Governors,
Founders, and Life Members.

Persons desirous of becoming members, or of obtaining any information with
regard to the Association, should communicate with—

The DIRECTOR,
The Laboratory,
Plymouth.

New Series, No. 224 (Vol. 56, Part 4). Price 10s. net.

Subscription per volume (of 4 parts) 40s. net.

JUNE, 1911.
THE

QUARTERLY JOURNAL

OF

MICROSCOPICAL SCIENCE.

EDITED BY

Sir RAY LANKESTER, K.C.B., M.A., D.Sc., LL.D., F.B.S.,

HONOKARY FELLOW OF EXETER COLLEGE, OXFORD ;

MEMBER OF THE INSTITUTE OF FKANCE (associf ETRANGER DE L’ACADEMIE DES SCIENCES) ;
CORRESPONDENT OF THR IMPERIAL ACADEMY OF S8CIKNCES OF 8T. PETERSBURG, AND OF THE
ACADEMY OF SCIENCES OF PHILADELPHIA, AND OF THE ROYAL ACADEMY OF SCIENCES
O¥ TURIN} FORKIGN MEMBER OF TIE ROYAL SOCIETY OF SCIENCES OF
GOTTINGEN, AND OF THE ROYAL BOHEMIAN SOCIETY OF 8SCIENCES, AND
OF THE ACADEMY OF THE LINCEI OF ROME, AND OF THR AMERICAN
ACADEMY OF ARTS AND SCIENCES OF BOSTON} ASSOCIATE OF THB
ROYAL ACADEMY OF BELGIUM; HONORARY MEM2ER OF THE
NEW YORK ACADEMY OP SCIKNCES, AND OF THE
CAMBRIDGE PHILOSOPHICAL SOCIETY, AND OP
THE ROYAL PHYSICAL SOCIETY OF EDIN-

BURGH, AND OF THE
BIOLOGICAL SOCIETY OF PARIS, AND OF THK CALIFORNIA ACADEMY OF SCIENCES OF SAN FRANCISCO, AND
OF THE ROYAL ZOOLOGICAL AND MALACOLOGICAL SOCIETY OF BELGIUM;
CORRESPONDING MEMBER OF THE SENKENBERG ACADEMY OF FRANKFURT-A-M;3
FOREIGN ASSOCIATE OF THK NATIONAL ACADEMY OF SCIENCES, U.S.) AND MEMBER OF THE
AMERICAN PHILOSOPHICAL SOCIETY 3
HONORARY FELLOW OF THE ROYAL SOCIETY OF EDINBURGH;

LATE DIKECTOR OF THE NATURAL HISTORY DEPARTMENTS OF THE BRITISH MUSKUM; LATE PRESIDENT OF THE
BRITISH ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE; LATK FULLKEKIAN PROFESSOR OP
PHYSIOLOGY IN THE KOYAY INSTITUTION OF GREAT BRITAIN ;

LATER LINACRE PROFESSOR OF COMPARATIVE ANATOMY AND FELLOW OF MERTON COLLEGE, OXFORD
EMERITUS PROFESSOR OF ZOOLOGY AND COMPARATIVE ANATOMY IN UNIVERSITY COLLEGE, UNIVERSITY OF LONDON

WITH THE CO-OPERATION OF

‘ADAM SEDGWICK, M.A., F.RS.,

FELLOW OF TRINITY COLLEGE, CAMBRIDGE. AND PROFESSOR OF ZOOLOGY IN THE IMPERIAL COLLEGE OF
SCIENCE AND TECHNOLOGY, LONDON;

SYDNEY J. HICKSON, M.A., F.RB.S.,

BEYER PROFESSOK OP ZOOLOGY IN THE UNIVERSITY OF MANCHESTEE,

KE. A. MINCHIN, M.A., F.R.S.,

PROFESSOR OF PROTOZOOLOGY IN THE UNIVERSITY OF LONDON;
AND

GILBERT C. BOURNH, M.A., D.Sc., F.R.S.,

LINACRE PROFRSSOR OF COMPARATIVE ANATOMY, AND FKLLOW OF MERTON COLLEGE, OXFORD.

WITH LITHOGRAPHIC PLATES AND TEXT-FIGURES.

LON DON?
CHURCHILL, 7 GREAT MARLBOROUGH STREET.

1911.

Adlard and Son, Impr.,] ; oe ee ee PONUOI HH DOP RIE.

CONTENTS OF No, 224.—New Series.

MEMOIRS :

PAGE
Cortical Cell Lamination of the Hemispheres of Papio Hama-
dryas. By E. H. J. Scuustrrr, M.A., D.Se., Fellow of New
College, Oxford. (With Plates 24-30) . 613
On Some Stages in the Life-History of Teno, Muses
Domestice, with some Remarks on the Relationships of the
Flagellate Parasites of Insects. By J. 8. Dunxreriy. (With
Plate 31) . : - 645
On Merlia normani, a Sronee rae a Sica and Galearedue
Skeleton. By R. Kirkpatrick. (With Plates 32-38 andj5 Text-
figures) . ; : : : : . 657

With Ten Plates, Royal 4to, 5s.
CONTRIBUTIONS TO THE KNOWLEDGE OF RHABDOPLEURA
AND AMPHIOXUS.
By Stir RAY LANKESTER, K.C.B., M.A., LL.D., F.R.S.
London: J. . A. SDE oe 7 Great ee Street.

Quarterly Journal of Microscopical

Science.

The SUBSCRIPTION is £2 for the Volume of Four Numbers;
for this sum (prepaid) the JourNAL is sent Post Free to any part
of the world.

BACK NUMBERS of the Journat, which remain in print, are
now sold at an uniform price of 10/- net.

The issue of SuppLemenr Numpers being found inconvenient,
and there being often in the Hditor’s hands an accumulation of
valuable material, it has been decided to publish this Journal at
such intervals as may seem desirable, rather than delay the appear-
ance of Memoirs for a regular quarterly publication.

The title remains unaltered, though more than Four Numbers
may be published in the course of a year.

Kach Number is sold at 10/- net, and Four Numbers make
up a Volume.

London: J. & A. CHURCHILL, 7 Great Marlborough Street.
TO CORRESPONDENTS.

Authors of original papers published i in the Quarterly Journal
of Microscopical Science receive fifty copies of their communica-
tion gratis.

All expenses of publication and illustration are paid by the
publishers.

Lithographic plates and text-figures are used in illustration.
Shaded drawings intended for photographic reproduction as half-
tone blocks should be executed in ‘‘ Process Black” diluted with
water as required. Half-tone reproduction is recommended for
uncoloured drawings of sections and of Protozoa.

Drawings for text-figures should nor be inserted in the MS.,
but sent in a separate envelope to the Editor.

Contributors to this Journal requiring ewtra copies of their
communications at their own expense can have them by applying
to the Printers,

Messrs. ApitarD & Son, 23, Bartholomew Close, E.C., on
the following terms:

For every four pages or less—

25 copies : : : : 3/-
a eae : : : : 6/-
iS Serer : ; : : 6/6
100: #,, 2/-

eo? 2/- per 25 if uncoloured ; if coloured, at the same rate for
every colour.
Prepayment by P.O. Order is requested.
Att COMMUNICATIONS FOR THE EDITORS TO BE ADDRESSED TO THE CARE
or Messrs. J. & A. CuurcHiLt, 7 Great MarLBorouGas Street,
Lonpon, W.

THE MARINE BIOLOGIGAL ASSOCIATION

OF THE

UNITED KINGDOM.
Patron—HIS MAJESTY THE KING.

President—Sir RAY LANKESTER, K.C.B., LL.D., F.R.S.
=(0)=

‘THE ASSOCIATION WAS FOUNDED “ TO ESTABLISH AND MAINTAIN LABORATORIES ON
THE COAST OF THE UNITED KINGDOM, WHERE ACCURATE RESEARCHES MAY BE CARRIED
ON, LEADING TO THE IMPROVEMENT OF ZOOLOGICAL AND BOTANICAL SCIENCE, AND TO
AN INCREASE OF OUR KNOWLEDGE AS REGARDS THE FOOD, LIFE CONDITIONS, AND HABITS
OF BRITISH FOOD-FISHES AND MOLLUSCS.”

The Laboratory at Plymouth

was opened in 1888. Since that time investigations, practical and scientific, have
been constantly pursued by naturalists appointed by the Association, as well as by
those from England and abroad who have carried on independent researches.

Naturalists desiring to work at the Laboratory

should communicate with the Director, who will supply all information as to
terms, etc.

Works published by the Association

include the following :—‘ A Treatise on the Common Sole,’ J. ‘Il’. Cunningham, M.A.,
4to, 25/-. ‘The Natural History of the Marketable Marine Fishes of the British
Islands, J. IT. Cunningham, M.A., 7/6 net (published for the Association by
Messrs. Macmillan & Co.).

The Journal of the Marine Biological Association

is issued half-yearly, price 3/6 each number.

In addition to these publications, the results of work done in the Laboratory
are recorded in the ‘Quarterly Journal of Microscopical Science,’ and in- other
scientific journals, British and foreign.

Specimens of Marine Animals and Plants,
both living and preserved, according to the best methods, sre supplied to the

principal British Laboratories and Museums. Detailed price lists will be forwarded
on application.

TERMS OF MEMBERSHIP.

ANNUAL MEMBERS . ; : eel 1) 0 per'anhum,
Lire MemBERs . ; : . . 15 15 O Composition Fee.
FOUNDERS . - LOO Os 20 a

Governors (Life Members of Council) 500 O 0

Members have the following rights and privileges:—They elect annually the
Officers and Council; they receive the Journal free by post; they are admitted to
view the Laboratory at any time, and may introduce friends with them; they have the
first claim to rent a table in the Laboratory for research, with use of tanks, boats, etc. ;
and have access to the Library at Plymouth. Special privileges ure granted to Governors,
Founders, and Life Members.

Persons desirous of becoming members, or of obtaining any information with
regard to the Association, should communicate with—

The DIRECTOR,

The Laboratory,
Plymouth.

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