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QUARTERLY JOURNAL

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EDITED BY

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VOLUME 51.—New SzErizs.
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CONTENTS.

CONTENTS OF No. 201, N.S., FEBRUARY, 1907.

MEMOIRS: PAGE
On the Parietal Sense-organs and Associated Structures inthe New =~
Zealand Lamprey (Geotria australis). By Artur Denby,
D.Se., F.L.S., F.Z.8., Professor of Zoology in King’s College
(University of London). (With Plates 1 and 2) ‘ iL
Studies in Spicule Formation. V.—The Scleroblastic Develp:
ment of the Spicules in Ophiuroidea and Echinoidea, and in the
Genera Antedon and Synapta. By W. Wooprianp, Demonstrator
of Zoology, King’s College, London. (With Plates 3 and 4) . 31
Studies in Spicule Formation. VI.—The Scleroblastic Develop-
ment of the Spicules in some Mollusca and in one Genus of
Colonial Ascidians. By W. Wooptanp, Demonstrator of
Zoology, King’s College, London. (With Plate 5) . . 45
A Preliminary Consideration as to the possible Factors concerned
in the Production of the various Forms of Spicules. By W.
Woop.anD, Demonstrator of Zoology, King’s College, London . 55
On Neurosporidium cephalodisci, n.g., n. sp., a Sporo-
zoon from the Nervous System of Cephalodiscus nigrescens,
By W. G. Riprewoop, D.Sc., Lecturer in Biology at St.
Mary’s Medical School, University of London; and H. B.
Fan Tuam, B.Sc., A.R.C.S., University College, London; Demon-
strator in Biology at St. Mary’s Medical School. (With Plates
6 and 7) : 2 el
Gametogenesis and Me rtilieation in ‘oN emilee Peete By L.
Doncaster, M.A., late Mackinnon Student of the Royal
Society; Lecturer in Zoology in the University of Birming-
ham. (With Plate 8) ; 5 akon
The Molluscan Radula: its Chemical Gonmtesitied and some Points
in its Development. By Ienrna B. J. Sotzas. (With Plate 9) 115
Observations on Tooth-Development in Ornithorhynchus. By
J. T. Witson, Professor of Anatomy, University of Sydney,
N.S.W., and J. P. Hitt, Jodrell Professor of Zoology, Univer-
sity College, London. (With Plates 10—12) : Se

iv CONTENTS.

CONTENTS OF No. 202, N.S., MAY, 1907.

MEMOIRS :

The Origin and Nature of the Green Cells of Convoluta ros-
coffensis. By Freprrick Kersiz, M.A., Se.D., University
College, Reading, and F. W. Gampie, D.Sc., Manchester
University. (With Plates 13 and 14)

On the Development of the Plumes in Buds of Gaphalodiaeee
By W. G. Riprwoop, |).Sc., Lecturer on Biology at St. Mary’s
Medical School, University of London. (With 11 Text-figures)

On the Structure of Anigma enigmatica, Chemnitz; a Con-
tribution to our Knowledge of the Anomiacea. By GixBert C.
Bourne, M.A., D.Sc., F.L.S., Fellow of Merton College ;
Linacre Professor of Comparative Anatomy in the University of
Oxford. (With Plates 15—17, and 2 Text-figures)

On the Chromatin Masses of Piroplasma bigeminum
(Babesia bovis), the Parasite of Texas Cattle-Fever. By
H. B. Fantuam, B.Se.Lond., A.R.C.S., University College,
London, and St. Mary’s Hospital Medical School. (With Plate
18, and 44 Text-figures) ;

The Skin, Hair, and Reproductive Coen of Wee S Con-
tributions to our Knowledge of the Anatomy of Notoryctes
typhlops, Stirling —Parts IV and V. By Groreina SWEET,
D.Se., Melbourne University. (With Plates 19 and 20, anda
Text-figure) .

Parorchis acanthus, the Type a a new Gene of Trematones
By Witt1am Nicotr, M.A., B.Se., Gatty Marine Laboratory,
St. Andrews. (With Plate 21) . ‘ ;

CONTENTS OF No. 203, N.S., AUGUST, 1907.

MEMOIRS:

The Chetognatha, or Primitive Mollusca. With a Bibliography.
By R. T. Ginruer, F.L.S., F.R.G.S., Fellow of Magdalen
College, Oxford. (With 10 Text-figures)

The Structure, Development, and Bionomics of the Rona Ay,
Musca domestica, Linn.—Part I. The Anatomy of the
Ily. By C. Gorpvon Hewitt, M.Se., Lecturer in Economic
Zoology, University of Manchester. (With Plates 22—26)

Trichomastix serpentis, usp. By C. Cxirrorp DoBE LL,
B.A., Scholar of Trinity College, Cambridge. (With Plate 27,
and 2 Text-figures) ‘ . . ‘

PAGE

167

221

253

297

325

345

357

449

CONTENTS.

Notes on Common Species of Trochus. By H. J. FLeure and
Mortet M. Gerrines, University College, ie (With
Plate 28) :

Note on the Formation of the Skeleton in the Metre yoraniey
By Marta M. Oettviz Gorpon, D.Se.(London), Ph.D.(Munich),
F.L.S. : : :

Studies in Spicule Mormacon! VII.—The Scleroblastic Develop-
ment of the Plate-and-Anchor Spicules of Synapta, and of the
Wheel Spicules of the Auricularia Larva. By W. Woop.anp,
The Zoological Laboratory, King’s College, London. (With
Plates 29 and 30, and 6 Text-figures) :

CONTENTS OF No. 204, N.S., NOVEMBER, 1907.

MEMOIRS:

The Development of the Head-muscles in Gallus domesticus,
and the Morphology of the Head-muscles in the Sauropsida.
By F. H. Epcewortu, M.B., D.Sc., Professor of Medicine,
University College, Bristol. (With 39 Text-figures) .

The Development of Ophiothrix fragilis. By E. W. Mac-
Brive, M.A., D.Se., F.R.S., Professor of Zoology in McGill
University, Montreal. (With Plates 31—36, and 4 Text-figures)

On the Segmentation of the Head of Diplopoda. By Marcaret
Rosinson, University College, London. (With Plate 37, and
6 Text-figures) .

The Fixation of the Gaps Rava of Seculini carcini
(Thompson) upon its Host, Carcinus menas. By Grorrrey
Smitn, M.A., New College, Oxford. (With 6 Text-figures)

Physiological Degeneration in Opalina. By C.Cutrrorp DoBELt,
B.A., Scholar of Trinity College, Cambridge. (With Plate 38,
and 9 Text-figures) : :

Some Facts in the Later Develanment of the Brae, Rana tempo-
raria, Part I.—The Segments of the Oveipital Region of the
Skull. By Acwes I. M. Extiot, B.Sc., Associate of Newnham
College, Cambridge. (With Plates 39 and 40)

TITLE, INDEX, AND CoNTENTs.

von. 51, PART 4.—NEW SERIES. b

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ee

New Series, No. 201 (Vol. 51, Part 1). Price 10s. net.

Subscription per volume (of 4 parts) 40s. net.

FEBRUARY, 1907.
THE

QUARTERLY JOURNAL

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EDITED BY

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HONORARY FELLOW OF EXETER COLLEGE, OXFORD}; CORRESPONDENT OF THE INSTITUTE OF FRANCE
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WITH THE CO-OPERATION OF

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SYDNEY J. HICKSON, M.A., F.RS.,

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T LONDON:
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CONTENTS OF No. 201.—New Series.

MEMOIRS:

PAGE
On the Parietal Sense-organs and Associated Structures in the New

Zealand Lamprey (Geotria australis). By Arruurn Denby,
D.Se., F.L.S., F.Z.8., Professor of Zoology in King’s College (Uni-
versity of London). (With Plates land 2) . . : i

Studies in Spicule Formation. V.—The Scleroblastic Development
of the Spicules in Ophiuroidea and Kchinoidea, and in the Genera
Antedon and Synapta. By W. Woopzianp, Demonstrator of
Zoology, King’s College, London. (With Plates 3 and 4) - foam

Studies in Spicule Formation. WI.—The Scleroblastic Development
of the Spicules in some Mollusca and in one Genus of Colonial
Ascidians. By W. Woopianp, Demonstrator of Zoology, King’s
College, London. (With Plate 5) . : ; Rk

A Preliminary Consideration as to the possible Factors concerned in
the Production of the various Forms of Spicules. By W. Woop-
LAND, Demonstrator of Zoology, King’s College, London . > 68

On Neurosporidium cephalodisci, n.g., n.sp., a Sporozodn
from the Nervous System of Cephalodiscus nigrescens. By
W. G. Ripewoop, D.Sc., Lecturer in Biology at St. Mary’s
Medical Schoo], University of London; and H. B. Faytuam,
B.Sc., A.R.C.S., University College, London; Demonstrator in
Biology at St. Mary’s Medical School. (With Plates6and7) . 81

Gametogenesis and Fertilisation in Nematus ribesii. By L. Don-
casteR, M.A., late Mackinnon Student of the Royal Society ;
Lecturer in Zoology in the University of Birmingham. (With
Plate 8) , ‘ ; d é - OE

The Molluscan Radula: its Chemical Composition, and some Points
in its Development. By Icerna B. J. Sotuas. (With Plate9) . 115

Observations on Tooth-Development in Ornithorhynchus. By J. T.
Wixson, Professor of Anatomy, University of Sydney, N.S.W., and
J. L. Hint, Jodrell Professor of Zoology, University College,
London. (With Plates 10—12) . ; ? - San

PARIETAL SENSE-ORGANS OF GEOTRIA. 1

On the Parietal Sense-organs and Associated
Structures in the New Zealand Lamprey
(Geotria australis).

By
Arthur Dendy, D.Sc., F.L.S., F.Z.S8.,
Professor of Zoology in King’s College (University of London).

With Plates 1 and 2.

(a) Inrropucrory Remarks.

Some years ago, whilst residing in New Zealand, I had the
good fortune to obtain a plentiful supply of living specimens
of the New Zealand freshwater Lamprey, Geotria australis.
These specimens were all in what is known as the “Velasia”’
stage of their development, none of them being sexually
mature, and all of them without the characteristic gular pouch
of the adult.!. They were, however, of considerable size,
averaging nearly a couple of feet in length, and the organs
which form the subject of the present memoir were probably
already fully developed. For the purposes of this investiga-
tion a considerable number of specimens were hardened and
preserved in a perfectly fresh condition by means of various
re-agents, of which absolute alcohol, Zenker’s fluid, and
Flemming’s solution yielded the most satisfactory results. In
some cases the head was simply cut off and hardened in toto,
while in others it was partially dissected in the fresh state
before being placed in the hardening re-agent.

Hight series of sections, longitudinal and transverse, were

1 For further particulars as to these specimens and the species to which
they belong vide Dendy and Olliver (1).

VoL. O1, parr 1,—NEW SERIES. 1

2 ARTHUR DENDY.

cut by the paraffin method, some through the entire head,
and some through the brain, after removal from the cranium.
Most of the material was stained in bulk with Ehrlich’s
hematoxylin, and some of the sections were counter-stained
on the slide by means of acid fuchsin or eosin.

Sections of material fixed in absolute alcohol were found to
be particularly valuable for demonstrating the arrangement
of the pigment in the “ pineal eye,” this pigment, as is well
known, being soluble in acids, and, therefore, often entirely
absent from material treated with strongly acid hardening
re-agents, such as Flemming’s solution or Zenker’s fluid.

I first observed the very well-developed “pineal eye” of
the New Zealand Lamprey in a Geotria Ammoccete which
had been preserved in chrom-osmic solution by my late col-
league Professor T. J. Parker, and given to me for investigation
by his successor at the Otago University Museum, Professor
W. B. Benham. The present investigation, however, was
largely stimulated by the remarkable results obtained by
Studnicka in lis researches on the minute histology of the
parietal sense-organs of the Huropean Lampreys (P etro-
myzon), and I am glad to be able to a large extent to
confirm these results, and perhaps even to still further extend
our knowledge of these remarkable structures. My work
has been greatly facilitated by the recent publication of
Studnicka’s admirable monograph on “Die Parietalorgane”
in Oppel’s ‘Lehrbuch der Vergleichenden mikroskopischen
Anatomie der Wirbeltiere’ (2), which renders detailed dis-
cussion of the writings of earlier investigators superfluous.

(sp) ToroarapHicAL ANATOMY OF THE FORE-BRAIN AND ITS
DERIVATIVES.

In its general characters the brain of Geotria in the
“Velasia” stage agrees closely with that of the adult Petro-
myzon. Hxternally perhaps the most striking difference
consists in the very distinct lobulation of the surface of the
large olfactory lobes (fig. 1, O.Z.), while internally the division

PARTETAL SENSE-ORGANS OF GEOTRIA. 3

of the cavity of the saccus vasculosus into right and left
halves by a well-developed longitudinal septum (fig. 2, Sept.)
deserves mention.

The thalamencephalon has, in its anterior part, a thin
membranous roof which rises upwards in a prominent dome.
This dome lies immediately behind and between the olfactory
lobes, and the thin roof bends down in front to form the
lamina terminalis (fig. 2, Z.7.). On this thin dome-
shaped roof of the third ventricle lie the organs with which we
are more immediately concerned, the pineal or parietal sense-
organs. There are in the Lampreys, as is well known, two of
these sense-organs, and in the genus Petromyzon one lies
beneath the other, the upper one being by very much the
better developed of the two, and being commonly spoken of
as the “pineal eye.” In the terminology of Studnicka the
upper one is described as the “pineal organ,” and the lower
one as the “‘parapineal organ.” According to the view adopted
by myself (8), and long since maintained by Gaskell (4), these
two sense-organs are really members of a pair which have
become displaced, the upper and better developed repre-
senting the right “ parietal eye,’’? and the lower the left one.
This view is supported in a very interesting manner by the
arrangement of the two organs in Geotria. It will be seen
from figs. 1 and 2 that the larger and better developed of the
two does not lie above but behind the smaller and less well-
developed organ, so that both are distinctly visible when the
brain is viewed from above. Moreover, I find in all cases
where the organs have been carefully examined in situ,
that the anterior one lies a little to the left side of the pos-
terior, the latter being approximately median in position.
This clear indication of the paired origin of the parietal
sense-organs affords a close parallel to the condition described
by myself in embryos of Sphenodon (3). In both cases there
is an anterior parietal organ lying immediately in front of and
a little to the left of a posterior one, but in S phenodon,
curiously enough, it is the left (anterior) organ which becomes
well developed as the apparently unpaired “pineal eye” of

4 ARTHUR DENDY.

the adult, while in Geotria and Petromyzon it is the right
(posterior) member of the pair which becomes dominant.

In Geotria the posterior (right) parietal sense-organ is
seen to be connected with an opaque-looking band of tissue
(fig. 1, P.S.), which runs backwards to the posterior com-
missure. ‘This is the pineal stalk, including the pineal nerve
(cf. fig. 6), and representing the hinder part of the original
outgrowth of the brain, whose anterior part forms the “ pineal
eye.” Owing, doubtless, to the enormous development of the
right habenular ganglion (fig. 1, G.H.R.), the pineal stalk is
pushed somewhat to the left side. Posteriorly, the pineal
nerve is connected, as will be shown later on, both with the
right habenular ganglion and with the posterior commissure.
The left habenular ganglion is, as in Petromyzon, very
much smaller than the right, and is divided into anterior
and posterior portions. ‘Ihe posterior portion (fig. 2, G.H.L.)
lies in immediate contact with the right habenular ganglion,
with which it is connected by a transverse band of fibres,
the commissura habenularis superior of Studnicka’s
terminology. The anterior portion (fig. 2, G.H.A.) lies imme-
diately beneath the left (anterior) parietal sense organ (para-
pineal organ), and is connected with the posterior portion by
means of a stout band of nerve-cells and fibres underlying
the pineal stalk and constituting the tractus habenularis
of Studnicka (fig. 2, 7.H.). (In fig. 2 both right and left
ganglia habenule are shown for diagrammatic purposes,
but it would not really be possible to see both in a strictly
median sagittal section such as is supposed to be represented.)

Almost immediately behind the right habenular ganglion,
but separated from it by a well-marked recess (the recessus
infrapinealis), hes the posterior commissure (figs. 1 and
2, C.P.). In a longitudinal section of the brain (fig. 2) the
posterior commissure is cut transversely, and appears as a
somewhat oval body projecting downwards and backwards
into the brain-cavity at the posterior dorsal limit of the third
ventricle. On the antero-ventral face of the posterior com-
missure lies a conspicuous longitudinal groove (fig. 2, Hp.G.)

PARIETAL SENSE-ORGANS OF GEOTRIA. 5)

lined by an epithelium composed of very much elongated
columnar cells. This groove is evidently formed by union
of the pair of grooves which I described in the Ammoccete
in 1902 (5); it has since been termed by Sargent (6) the
“ependymal groove.” In the “ Velasia,” though the two
grooves are closely approximated, they still show clear indi-
cations of their double origin (fig. 3, Hp. G.). The ependymal
groove is continued forwards into the recessus infra-
pinealis, and thence for a short distance beneath and to
the left of the right habenular ganglion, gradually losing
the special character of its epithelium.

According to Sargent (6) the epithelium of the ependymal
groove serves for the support and attachment of the anterior
branches of Reissner’s fibre on their way from the optic reflex
cells to the brain cavity, in which the main fibre lies freely.
From my own observations on Geotria I have come to the
conclusion that the anterior constituent fibrils of Reissner’s
fibre (fig. 2, R.F.) do leave the brain substance in the
ependymal groove as described by Sargent, but this discovery
by no means disproves the view which I previously (5) put
forward with regard to the function of the ependymal grooves
in the Ammoccete. Reissner’s fibre itself is very conspicuous
in Geotria,as shown in fig. 2, R.#., but further discussion of
this part of the subject may be conveniently left until later on.

(c) Tue PrnzaL Orean (Ricut Parrerat Eyer).

General form and structure.—The structure of this
organ (fig. 7) is in its general features very similar to that
described by Studnicka for the European lampreys. It con-
sists of a hollow vesicle, about half a millimetre in maximum
diameter, and having a very characteristic shape, not unlike
that of a pear, with the pineal stalk representing the stalk of
the pear. The upper surface of the optic vesicle, turned
towards the light, is perfectly circular in outline and very
much flattened like a button (fig. 4), while the lower surface
is strongly convex, especially posteriorly, where the wall of

6 ARTHUR DENDY.

the vesicle gradually passes into the pineal stalk. ‘The upper

and outer wall of the vesicle is formed by the unpigmented,

transparent “pellucida,” while the lower and inner wall is
formed by the “retina,” under which term we may include
both the retinal epithelium and the layer of ganglion cells
and nerve fibres which underlies it (fig. 7). ‘The retinal epi-
thelium has a characteristic opaque white appearance owing
to the abundant granules of white pigment imbedded in the
pigment cells, and this, seen through the transparent pellucida,
gives the whole organ its characteristic chalky appearance
even when seen with the naked eye or under a simple lens.
The line of junction of the pellucida with the retina, all round
the circular margin of the upper surface of the organ, is very
sharply defined ; the wall of the optic vesicle is here thinner
than in any other part, and the edge of the pigmented retinal
epithelium appears from above as a distinct, opaque, white
margin to the pellucida (fig. 4).

The thickest part of the wall of the optic vesicle lies pos-
teriorly just where it joins the stalk. The whole organ is
doubtless, as in Petromyzon, developed from the enlarged
distal extremity of a hollow pineal outgrowth, the proximal
portion (stalk) of which becomes solid and gives rise to the
pineal nerve. The original cavity of this outgrowth persists
distally as the cavity of the optic vesicle, and a smaller
portion of it persists in the thickness of the wall of the optic
vesicle, just in front of the point of entrance of the pineal
nerve, and forms the “atrium” of Studnicka. In Petro-
myzon, according to this author, the atrium communicates
freely with the main cavity of the optic vesicle, but yet shows
a tendency by enlargement to form an independent cavity.
In Geotria I have not been able to detect any communica-
tion between the atrium and the main cavity of the optic
vesicle; they appear to be completely separated from one
another. ‘The atrium (fig. 7 At.) usually appears both in
longitudinal and transverse sections as a small oval or
almost circular cavity lined by columnar cells. In one series
of longitudinal section there are indications of one or two

PARIETAL SENSE-ORGANS OF GEOTRIA. 7

subsidiary atrial cavities lying behind the principal one, and
doubtless representing a further remnant of the original
lumen of the pineal outgrowth; another series of sections
shows that this appearance may be due to curvature of
the atrium, whereby its lumen may be seen twice in the same
section.

The proper cavity of the optic vesicle is well developed
and usually of the shape shown in longitudinal section in
fio. 7, with a funnel-shaped depression in the middle of the
lower surface, probably indicating the original connection
with the atrium. ‘The funnel-shaped depression may, how-
ever, be almost, if not quite, unrecognisable (fig. 2). The
peculiar network of protoplasmic strands which occupies the
cavity of the optic vesicle will be described later on.

The whole organ is enclosed externally in a thin and ill-
defined layer of fibrous connective tissue, which may be
regarded, for the most part at any rate, as an extension of the
pia mater.

Histology of the pellucida (fig. 7, Pell.).—The |
outer surface of the pellucida is smooth and even, but its
inner surface is produced into large, irregular villi or pro-|
cesses which project into the cavity of the optic vesicle and |
are connected by thin strands of tissue with the retina
(fig. 7, P.St.). The pellucida is composed, for the most part |
at any rate, of a single layer of columnar cells, which are |
enormously elongated to form the projecting villi. These
cells contain conspicuous oval nuclei (fig. 7, N.C.C.) situate |
near their inner ends. Between the villi the inner surface of |
the pellucida, though uneven, is smooth, but at the inner ends |
of the villi the cells appear drawn out into threads, which go
to form the strands of tissue connecting the pellucida with the
retina. The columnar cells themselves appear to contain but
little cytoplasm, which is only shghtly granular and stains
lightly with Ehrlich’s hematoxylin and with acid fuchsin. |
Their outlines are well defined, but with a characteristic wavy
appearance, which I attribute to shrinkage. Amongst these \
columnar cells, in the outer part of the pellucida, one finds a —

8 ARTHUR DENDY.

number of almost spherical nuclei of doubtful significance,
but resembling the nuclei of the ganglion cells of the retina.
At various levels in the pellucida one also finds a small
number of very darkly staining, small, irregular nuclei, having
a shrivelled appearance, and closely resembling the “ con-
nective tissue”? nuclei found in the interior of the optic
vesicle and in the nervous layer of the retina.

Histology of the retina—The retina, as already
indicated, may be divided into two perfectly distinct layers,
the epithelial layer, composed of pigment cells and sense cells,
and the layer of ganglion cells and nerve fibres which lies
behind it, and which we may call, in short, the nervous layer.
Both these layers increase greatly in thickness as they recede
from the pellucida, and around the atrium the nervous layer
becomes so strongly developed as to form a veritable ganglion
(fig: 7).

The epithelial layer of the retina (figs. 5 and 7) is composed
of the same two kinds of elements as have been recognised by
Studnicka in Petromyzon—viz. sensory cells and pigment
cells. The former (fig. 5, R.S.C.) are greatly elongated,
slender rods whose inner ends project into the cavity of the
optic vesicle and terminate in irregularly rounded, swollen
knobs, while their outer ends branch into fibrils which lose
themselves.in the fibrillar network of the nervous layer. These
rods have large oval nuclei (fig. 5, N.S.C.) situated towards
their inner ends, and causing a fusiform swelling in the rod
itself. The end-knobs of the rod (fig. 5, S.C.K.), and the rods
‘themselves (apart from the nucleus), are only lightly stained
with Ehrlich’s hematoxylin, but take up acid fuchsin with
great avidity, whereby they are rendered very conspicuous.
Studnicka describes the end-knobs in Petromyzon as being
differentiated into inner and outer portions, but I have not
succeeded in detecting any such differentiation in the case of
Geotria, the knobs appearing to be practically homogeneous.
The adhering ends of the protoplasmic strands (fig. 5, P.St.)
which connect the sense-cells with the pellucida, may, however,
spread out on the knobs, and thus give rise to the appearance

PARIETAL SENSE-ORGANS OF GHOTRIA. 9

of an outer layer which stains less deeply with acid fuchsin.
Possibly this is the explanation of the appearance described
by Studnicka. In other respects the sensory cells appear to
be quite identical with those of Petromyzon.

The intervals between the sensory cells are filled by the
pigment cells, which Studnicka has, no doubt correctly,
identified with the ependymal cells of the general internal
lining of the brain cavities. The pigment cells (figs. 5, 7) are
vesicle, and taper gradually outwards till their slender,
thread-like outer extremities, which may apparently branch,
are lost in the fibrillar network of the nervous layer, along
with the outer ends of the sensory cells.

So far the pigment cells agree fairly well with those of
Petromyzon, as described by Studnicka (2), but there is
one very important difference. In Petromyzon it appears
that they terminate at their inner extremities in a smooth
surface, through which the knobbed ends of the sense-cells
project, while, according toc Studnicka’s latest account, they
themselves have no differentiated inner segments or knobs at
all. In Geotria, on the other hand, the inner end of each
pigment cell (fig. 5, [.S.P.C.) is very distinctly segmented off,
and separated from the outer and principal portion of the
cell (O.S.P.C.) by what looks like a limiting membrane (Z.M.).
In depigmented sections, as shown on the left-hand side of
fig. 5, this “limiting membrane” is very conspicuous, and
appears, at first sight, to form the inner surface of the retina ;
it has a characteristic dotted or beaded appearance. Careful
observation shows, however, that even in depigmented sections
the remains of the inner ends of the pigment cells, as well
as the projecting knobs of the sense cells, may be clearly
recognised as the inner side of the “limiting membrane,”
though not nearly so conspicuous as in sections in which the
pigment is preserved, as shown on the right-hand side of
fig. :

Thus in Geotria the pigment cells as well as the
sense-cells are provided with differentiated, knobbed inner

ns

10 ARTHUR DENDY.

extremities, but the knobs of the latter project further into
the cavity of the eye than those of the former. The knobs of
the pigment cells are much broader than the sense-cells at
the same level, so that they form almost a continuous layer
inside the limiting membrane, penetrated by the slender rods
of the sense cells on their way to the sense cell knobs (fig. 5,
S.C.K.) in the cavity of the optic vesicle.

The nuclei of the pigment cells (fig. 5, N.P.C.) are situated
towards the outer extremities of the outer segments, at about
the same level as the nuclei of the sensory cells, from which
they may be distinguished by their somewhat smaller size
and less dense-looking protoplasm. ‘The pigment granules
(composed of phosphate of lime ?) are minute spherical bodies
evenly distributed throughout the inner segment and the
greater part of the outer segment, but not, so far as my
observations show, occurring in the slender outermost portion
of the pigment cell beyond the nucleus.

Examination of Studniéka’s earlier figures (7, Pl]. III, figs.
6, 7,8) suggests that in Petromyzon also the pigment cells
may have differentiated knobbed inner extremities. The idea
that only one kind of cell is present in the retinal epithelium,
as shown in these figures, is doubtless, as Studniéka himself has
since pointed out, erroneous. According to his earlier obser-
vations, however, all the epithelial cells have knobbed (but
unpigmented) extremities, and it appears just possible that
he has abandoned too much of his previous results in making
the necessary correction. In spite of the precision of his
later account, it might be worth while to re-investigate this
point in the light of our knowledge of Geotria, in which
the segmentation of the pigment cells is so obvious as to
leave no room for doubt.

The nervous layer of the retina consists of ganglion cells,
nerve fibres, and connective-tissue cells. The ganglion cells
(figs. 5, 7, G.C.) are very conspict:ous on account of their
large spherical nuclei, surrounded by only a small quantity
of cytoplasm. ‘lhe cytoplasm is often scarcely recognisable,
while at other times it is more distinct and exhibits a multi-

PARIETAL SENSE-ORGANS OF GEOTRIA. 19

polar character. The nuclei contains a few well-defined,
darkly-staining chromatin granules. In the thinner parts of
the retina (fig. 5) the ganglion cells are comparatively few in
number, and occur chiefly towards the outside, just within
the connective-tissue capsule of the eye. In the neighbour-
hood of the atrium, however, they are accumulated in large
numbers, as already stated (fig. 7, G.C.).

The nerve fibres are extremely delicate and form a network
(together with connective-tissue fibres ?) in which the ganglion
cells are embedded (fig. 5, N.F'.N.). It is probable that there
is a special layer of nerve fibres between the ganglion cellls
and the connective-tissue sheath (fig. 7, C.7.S.), but I have
not found it possible to distinguish it clearly from the latter.

The connective-tissue cells of the retina are distinguished by
their elongated and very darkly-staining nuclei (fig. 5, C.7.N.’),
resembling those found in the connective-tissue sheath ; they
seem to indicate the presence of connective-tissue fibres,
running more or less vertically through the retina.

lregular masses of pigment granules, similar to those
found in the pigment cells of the epithelial layer, occasionally
occur in the nervous layer, but these can hardly be regarded
as essential constituents of this layer.

Histology of the wall of the atrium.—The atrium
is lined by a single layer of columnar ependymal cells, none
of which contain pigment, and I have not been able to demon-
strate the existence of sensory cells in this region,

Contents of the optic vesicle.—Much discussion has
taken place as to the nature of the irregular network which
so constantly appears in the interior of the pineal eye (fig. 7,
P.St.). The researches of Studniéka leave no room for doubt
that it is a normal constituent of the organ and not merely an
artifact, although probably it undergoes much alteration during
the processes of hardening and in the preparation of sections.
It is probably partly due to coagulation of the albuminous/
contents of the optic vesicle, but it is also undoubtedly in part
cellular in nature. As Studnicka has shown in Petromyzon,
the columnar cells of which the pellucida is composed are

12 ARTHUR DENDY.

connected with the sensory cells of the retina by delicate
strands of tissue which traverse the lumen of the optic vesicle.
This is very evident in the case of Geotria also, as indicated
on the left-hand side of fig. 7. This figure, however, repre-
sents a section of an eye which has been somewhat abnormally
distended in the processes of preparation, and in which, con-
sequently, most of the delicate connecting strands have been
ruptured; in other cases, where the pellucida has not become
artificially arched outwards, all the projections of its inner
surface are connected in this manner with the retina, and
the general direction of the connecting strands is vertical.
The strands themselves appear to be formed by outgrowth of
the inner ends of the long columnar cells of the pellucida,
which become attached to the knobs of the retinal sense cells.
In all cases which I have observed, however, they appear to
branch and form an irregular network (fig. 7), which may be
partially due to artificial entanglement. Entangled, as it
were, in the meshes of this network, one finds numerous small
nuclei, which often stain very darkly and exhibit a character-
istic shrivelled appearance as if undergoing degeneration.
Sometimes, also, one finds an irregular mass of almost homo-
geneous material with nuclei adhering to its surface—probably
identical with the “ syncytial mass” described and figured by
Studniéka in Petromyzon fluviatilis, but, in my opinion,
an artifact due to coagulation and entanglement. Such a
mass is shown in the middle of the optic vesicle in fig. 7.
Studniéka regards the “ plasmatischen Netze und Syncytien”
as representing the remains of a “corpus vitreum,” but this
appears to be a mere question of terminology, and it is
extremely doubtful whether it is desirable to apply the term
“corpus vitreum” to such very definite structures as the
protoplasmic strands which connect the pellucida with the
retina, although it is quite possible that these may be im-
bedded in a “ corpus vitreum” during life. It seems probable
that the function of these connecting threads may be to afford
support to the freely projecting knobs of the sense cells by
attaching them to the pellucida.

PARIWTAL SENSE-ORGANS OF GEOTRIA. 13

(p) Tue Pineat Nerve anp 117s CONNECTIONS.

It is well known that in Petromyzon the so-called
“pineal outgrowth” arises immediately in front of the pos-
terior commissure, and grows forward above the roof of the
fore-brain in the form of an elongated hollow sac, whose
distal extremity enlarges and becomes modified in structure
to form the pineal or parietal eye, while the proximal por-
tion, or ‘“stalk,’? becomes solid, and by histologicat differen-
tiation is, in part at any rate, converted into the pineal nerve.

In Geotria, as in Petromyzon, the original point of
connection of the pineal stalk with the brain is clearly indi-
cated by the depression between the posterior commissure and
the right habenular ganglion known as the recessus infra-
pinealis, as shown in figs. 2 (R.J.P.) and 6. At this spot
the epithelium of the ependymal groove, in sections, is usually
pulled out and separated from the rest of the ependymal epithe-
lium owing to the inevitable contraction in preparation, while
remaining closely adherent to the pineal stalk above it, to
which it is intimately attached by fibres which appear to belong
to the pineal nerve. This connection of the epithelium of the
ependymal groove with the pineal nerve has not, so far as I
am aware, been hitherto observed, and appears to me to be
a matter of considerable interest, though it must not be
forgotten that some at any rate of the connecting fibres may
be merely connective tissue.

The pineal stalk in Geotria is not, as a whole, very sharply
defined, but merges on either side in the mass of arachnoid
tissue which lies outside the brain. It thus appears much
more definite in longitudinal than in transverse sections,
forming a solid cord, apparently of loose connective tissue
(figs. 1, 6, P.S.), in which the pineal nerve itself is
imbedded. This nerve consists of a buudle of numerous
very slender, non-medullated fibres, containing elongated
nuclei, and indistinguishable from those of higher vertebrates,
as represented, for example, in fig. 138 of Schafer’s ‘ Essentials

14 ARTHUR DENDY.

of Histology’ (ed. vi). This bundle of fibres may easily be
traced from the nervous layer of the retina of the pineal eye,
in which it distinctly originates, to the surface of the brain
immediately behind the right habenular ganglion and above
the posterior commissure. The latter part of its course, as
seen ina series of longitudinal sections, is shown in fig. 6.
Shortly before reaching the brain it divides into several short
branches. One of these (P.N. 1) is directly connected with
the epithelium of the ependymal groove in the manner already
described. Another, or perhaps several small bunches of
fibres (P.N. 2), comes off more posteriorly, and its fibres
probably pass into the posterior commissure (C.P.), and
apparently through this to the inner surface of the ependymal
groove (Hp.G.). In all my sections, however, a small
shrinkage cavity (fig. 6, S.C.) is developed just above the
posterior commissure, and the fibres of this branch of the
pineal nerve are probably thereby ruptured, so that they
appear to terminate abruptly above the shrinkage cavity,
while from the lower surface of this cavity very delicate
(nerve ?) fibres run obliquely across the posterior commissure
to the inner surface of the ependymal groove. Another branch
(P.N. 8) of the pineal nerve comes off more anteriorly than
either of those yet mentioned, and, curving forwards between
the right habenular ganglion and the ependymal epithelium,
joins the Meynert’s bundle of the right side, and then, curving
upwards with the latter, forms a band of fibres which can
easily be traced into the middle of the habenular ganglion,
as shown in fig. 6.

It thus appears that the pineal nerve is connected (1) with
the epithelium of the ependymal groove (both directly and
possibly also by fibres which pass through the posterior com-
missure), (2) with the right habenular ganglion, and (3)
with the right bundle of Meynert. ‘The connection with the
habenular ganglion was long since maintained by Gaskell
(4), but has since been doubted by Studnicka (2), who
maintains that, whereas the “parapineal organ” is connected
with the left habenular ganglion and the superior (haben-

PARIETAL SENSE-ORGANS OF GEOTRIA. 15

ular) commissure, the pineal organ itself is connected with
the posterior commissure. Studni¢ka makes use of this
apparent discrepancy as an argument against the theory of
the paired origin of the parietal sense organs. We shall have
occasion to discuss this question somewhat more in detail at
a later stage.

According to the observations recorded above the con-
nection of the pineal nerve with the posterior commissure,
about the existence of which there can be very little doubt,
may be due simply to the fact that some of the nerve fibres
traverse this commissure in order to reach the epithelium of
the ependymal groove. Curiously enough, the existence of
this remarkable structure—the ependymal groove—appears
to have been hitherto ignored by those authors who have
investigated the pineal organs, and, conversely, those who
have dealt with the ependymal groove have entirely neglected
its relations to the pineal nerve.

In my memoir on the subject, published in 1902 (5), I
described a pair of these grooves in the Ammoceetes both of
Geotria and Petromyzon, and, believing that I had de-
tected cilia on the long columnar cells with which they are
lined, I termed them “ ciliated grooves,” and suggested that
they might serve to promote the circulation of the fluid in the
brain cavities, especially in relation to a highly vascular
vertical fold of the choroid plexus, which in the Ammoccete
hangs down, gill-like, into the brain cavity in the immediate
neighbourhood of the grooves in question. Sargent (6) in his
remarkable memoir on the optic reflex apparatus of verte-
brates, criticises this view, maintaining that what I had inter-
preted as cilia are really constituent fibrils of Reissner’s fibre,
and that the ependymal groove functions merely as an attach-
ment plate for these fibrils, which supports them as they leave
the brain on their way to join the main fibre lying freely in
the brain cavity. It does not seem to me that these two views
are incompatible with one another, and I find it difficult to
believe that such a remarkable and well-developed structure
as the ependymal groove should be required solely for the

16 ARTHUR DENDY.

function which Sargent assigns toit. The question of ciliation
must be left for future investigation to settle, but Sargent has
evidently misunderstood my observations on this subject, for
he makes me say that the cilia of the grooves are longer than
those of the ventricular walls generally, whereas I both de-
scribed and figured them as being much shorter. What I
described as cilia are, therefore, probably not the same struc-
tures as Sargent describes as constituents of Reissner’s fibre,
though I now believe myself that they may possibly indicate
merely a striated margin of the columnar epithelium. My
recent observations show, however, that in the Velasia stage
of Geotria it is possible to make out extremely fine threads
(fig. 6, RW”) projecting from the epithelium of the epen-
dymal groove, much longer than the supposed cilia, and these
are in all probability the nerve fibrils described by Sargent.
Sargent maintains, as already stated, that these fibrils are
connected with, and in fact go to make up, the fibre of
Reissner, and he regards the whole system as a short circuit
for optic reflexes. He has found Reissner’s fibre, with
similar relations, throughout the entire vertebrate phylum,
and brings forward experimental as well as_ histological
evidence in support of his views.

In Geotria Reissner’s fibre (fig. 2 and 6, R.F.) is con-
spicuously developed, and has in most respects the same
relations as described by Sargent in Petromyzon. It
appears to originate in the immediate neighbourhood of the
ependymal groove, beneath the posterior commissure, and is
made up of a number of branches (fig. 6, R.F.’, RF”) which
can be traced close up to the columnar epithelium of the
groove.! Though I have not been able actually to demon-
strate the connection between this epithelium and Reissner’s
fibre, I see no reason to doubt the correctness of Sargent’s
statement as to the existence of such a connection by means
of the delicate fibrils which emerge from the epithelium.

1 The two grooves in Geotria are so closely approximated as to form

practically a single groove (fig. 3, Zp.G@.), the form of which, however,
clearly indicates its double origin.

PARIETAL SENSE-ORGANS OF GEOTRIA. 17

These fibrils are so extremely slender that it is almost too
much to hope for to find unbroken continuity, especially
when we remember that the coagulation of the fluid in the
brain-cavity and the shrinkage in preparation must tend to
cause rupture. In fig. 6 a great tuft of extremely fine
branches (R.F.’) is shown coming off from Reissner’s fibre
beneath the hinder part of the posterior commissure, whilst
more anteriorly the main fibre divides into two approximately
equal branches (R.f.”), and at R.F.’” delicate fibrils are seen
emerging from the ependymal epithelium. hese appearances
strongly confirm the observations of Sargent as to the con-
nection of Reissner’s fibre with the ependymal groove. As
to the origin of the constituent fibrils from optic reflex cells
within the substance of the brain, however, I am not able to
make any definite statement. In fig. 3 I have shown the
existence of a group of large nerve-cells (N.C.) situated in
the anterior lateral part of the tectum opticum on either
side of the posterior commissure. ‘These obviously corre-
spond to two of the groups of optic reflex cells described
by Sargent in Petromyzon, and represented in his fig. 7,
but I have not seen any connection of these cells with the
ependymal groove, such as he figures. This, however, by
no means proves that no such connection exists, and it must
be remembered that my material was not specially prepared
for the purpose of tracing nerve fibres.

I have, however, already shown that fibres from the pineal
nerve are connected with the ependymal epithelium on its
inner aspect, while Sargent has shown that branches of
Reissner’s fibre are connected with the same epithelium on its
outer aspect. One is tempted to conclude, therefore, that the
pineal eye is connected with Reissner’s fibre through the pineal
nerve, and thus linked up with the optic reflex system. This
conclusion obviously involves one in what appears at first
sight to be a very serious difficulty. It must be remembered
that the pineal nerve is apparently a sensory nerve, while
Reissner’s fibre is a motor nerve, and a direct connection
between the two, without the intervention of ganglion cells in

voL. 01, pART ],—NEW SERIES 2

18 ARTHUR DENDY.

the central nervous system is, to say the least of it, extremely
improbable. This difficulty may be overcome, however, by
supposing that the “ reflex cells” required are situate in the
great ganglionic swelling which surrounds the atrium of the
pineal eye, and which, of course, is actually developed as an
outgrowth of the central nervous system.

I do not wish to press this suggestion too far, however, in
the present state of our knowledge, nor is it necessary to do
so in order to link up the pineal eye with the optic reflex
apparatus, for Sargent has shown that some of the con-
stituents of Reissner’s fibre issue from the base of the right
habenular ganglion. Now this ganglion is undoubtedly
connected with the pineal eye through the pineal nerve, as I
have already indicated, and it is extremely probable that we
have here reflex cells which transmit stimuli received through
the pineal nerve to Reissner’s fibre.

From the region of the posterior commissure it is quite
easy to trace Reissner’s fibre backwards through the iter and
the fourth ventricle, into the canalis centralis of the
spinal cord, as shown in fig. 2. It is worth noticing that, as
it passes beneath the cerebellum, it does not become imbedded
in the roof of the brain as takes place in adult Petromyzon,
but remains free throughout its course. This free condition
is also found in the young Petromyzon, so that it is not
unlikely that in Geotria also Reissner’s fibre may become
imbedded in the growing tissue of the cerebellum with
advancing age. I have not followed it backwards beyond
the commencement of the spinal cord.

(cr) THe ParapineAL Orcan (Lerr ParieraL Eye) anp Irs
RELATIONS TO THE Brain.

The parapineal organ, or left parietal eye (figs. 2,8, D.P.L.),
is, as already pointed out by Studnicka for Petromyzon,
essentially similar in structure to its larger and more perfectly
developed fellow of the primitive right side. Its position, in
front of and a little to the left of the “pineal organ,” has already

PARIETAL SENSE-ORGANS OF GEOTRIA. 19

been sufficiently described. Perhaps the most remarkable
difference which it exhibits as compared with its fellow con-
sists in the manner in which it is connected with the brain,
the organ itself lying immediately upon the anterior division
of the left habenular ganglion (figs. 2, 8, G.H.A.), while its
apparent nerve, the tractus habenularis of Studnicka
(figs. 2, 8, T.H.), is the long-drawn-out portion of the left
habenular ganglion which connects the anterior and posterior
portions of the latter. There is, therefore, strictly speaking,
no proper nerve to the left parietal eye, which remains seated
immediately upon the brain, though no doubt the tractus
habenularis functions as such.

The parapineal organ of Geotria is a hollow sac, much
smaller than the pineal organ and of different shape, flattened
dorso-ventrally and elongated transversely (figs. 1, 4, Z.P.H.).
It may be slightly, but distinctly, constricted in the middle
into right and left halves, or it may be more irregular in out-
line, as shown in fig. 4. The attachment to the anterior
division of the left habenular ganglion, though broad, does
not include by any means the whole of the ventral surface, so
that the parapineal organ is marked off from the ganglion by
a deep constriction, deeper in front and at the sides than it is
posteriorly. ‘he outer surface of the organ is covered with a
thin sheath of connective tissue (fig. 8, C.Z7.S.) continuous
with the pia mater of the brain.

The wall of the parapineal organ may be divided into
pellucida and retina exactly as in the case of the pineal eye
itself, but the distinction between the two is not nearly so well
marked. ‘The pellucida (fig. 8, Pell.) consists of a layer of
columnar cells, the inner surface of which is in places drawn
out into irregular processes projecting into the cavity of the
organ exactly as in the case of the pineal eye, only in a less
perfectly developed condition. Outside these columnar cells
are numerous spherical nuclei, probably indicating a layer of
ganglion cells similar to those found in the retina. In the
pineal organ such nuclei are almost absent from the pellucida,
and in Petromyzon Studnicka describes the pellucida of

20 ARTHUR DENDY.

the parapineal organ as consisting of only a single layer of
cells.

The pellucida passes quite gradually into the retina, which
consists of an epithelial layer of columnar cells facing the
cavity of the organ and backed by a nervous layer of ganglion
cells and nerve fibres. Thus the retina has a close general
similarity to that of the pineal organ, from which, however,
it differs strikingly in the entire absent of pigment. Accord-
ing to Studni¢ka the retinal epithelium of the parapineal
organ in Petromyzon consists of sensory cells and ordinary
ependymal cells, but I have not been able to distinguish
clearly between the two in Geotria. As in Petromyzon,
the characteristic knob-like projections of the retinal cells
into the cavity of the organ, which are so conspicuous in the
pineal eye, are not to be found in the parapineal.

In the interior of the parapineal organ we find, exactly as
in the pineal, a network of delicate threads connecting the
pellucida with the retina (fig. 8). Here again this network
appears to be formed by outgrowth of the columnar cells of
the pellucida, and contains small nuclei scattered in it.

The nervous layer of the retina must be considered in
connection with the underlying anterior division of the left
habenular ganglion (fig. 8, G.H.A.). This consists of a
central mass of finely granular or punctate matter devoid of
nuclei, but partially surrounded by nerve cells, as shown in
longitudinal section in fig. 8. In transverse sections (fig. 9)
the central mass is seen to extend laterally in a pair of hori-
zontal, wing-like projections, beneath which the nerve cells
are accumulated. From the upper surface of the central
mass stout bands or tracts of fibres are given off, which
curve upwards amongst the ganglion cells of the retina, and
sometimes appear to extend even into the pellucida. These
fibrous bands can, in part at any rate, be traced directly
backwards into the tractus habenularis, as shown in
fig. 8.

The anterior division of the left habenular ganglion passes
backwards quite gradually into the tractus habenularis.

PARIETAI SENSE-ORGANS OF GEOTRIA. Pail

The latter exhibits a very characteristic crescentic form in
transverse section, with the horns of the crescent, which are
directly continuous with the wing-like outgrowths of the
punctate substance in the anterior enlargement, turned up-
wards. The upper part of the crescent is composed chiefly
of longitudinal nerve fibres (compare fig. 8) cut across, while
the lower part is occupied by a somewhat thinner layer of
nerve cells covered by the ependymal epithelium.

(fF) AccEssoRY STRUCTURES OVERLYING THE PARIETAL SENSE
ORGANS.

The parietal sense organs lie in the cranial cavity imme-
diately beneath the connective tissue wall of the cranium,
between the nasal and occipital cartilages, as shown in fig. 2.
"he membranous wall of the cranium (fig. 2, C.7.C.), composed
of very dense fibrous connective tissue, thins out somewhat,
and is slightly arched upwards in this region, and the upper
surfaces of the sense organs are closely pressed against it.
Immediately above this there is a thick mass of very much
modified connective tissue forming the principal part of the
so-called cornea of Studnicka (fig. 2, C.7.P.). This mass of
connective tissue is a well-defined structure both in the
Lampreys and in Sphenodon, where it occupies the parietal
foramen, and it seems desirable to distinguish it by a special
name. I therefore propose to call it the “parietal plug.”
In Geotria it consists of a somewhat basin-shaped mass of
fibrous tissue, in which the fibres run almost vertically, but
converging somewhat below, where the plug is narrower than
it is above. The fibres are arranged in dense, multi-nucleate
bands, which branch and anastomose freely with one another
to form a network with lacunar meshes. Probably these
meshes are occupied in life by a gelatinous material, of which
traces are still recognisable. The upper ends of the fibrous
bands of which the plug is composed are closely attached to
the under surface of the corium or dermis. ‘This layer does
not appear to undergo any special modification as it passes

22 ARTHUR DENDY.

over the plug, except such as I have to mention shortly in
regard to the pigment. Above the corium comes the epi-
dermis, which again exhibits a perfectly normal structure.

When the dorsal surface of the head is examined carefully,
a small, light-coloured patch is visible a short distance behind
the nostril. ‘This patch is somewhat elongated and nearly
oval in outline (perhaps, rather, key-hole shaped), and con-
stitutes the well-known “ Scheitelfleck” of German authors.
It lies immediately above the parietal plug, and owes its pale
colour to the fact that the pigment, elsewhere so abundantly
developed in the integument, is here almost entirely absent.
Elsewhere we find the pigment cells arranged in two layers,
an outer and an inner. The outer one (fig. 2, Pig.') lies in
the corium at avery short distance beneath the epidermis,
and is but feebly developed, consisting of a sparse layer
of much-branched cells. This layer is still more feebly
developed in the region of the “ Scheitelfleck,” but not
entirely absent. The inner layer of pigment (fig. 2, Pig.*)
lies immediately beneath the corium, which it separates from
the underlying looser connective tissue. It is very much
denser than the outer layer, and is completely absent beneath
the “Scheitelfleck,” terminating abruptly on reaching the
upper margin of the parietal plug.

From the foregoing account it will be evident that the
light-transmitting tissues which overlie the parietal organs
have essentially the same structure and arrangement as in
Petromyzon, but, if we may judge from Studni¢ka’s figures,
the parietal plug is better defined in Geotria.

(a) GENERAL CONSIDERATIONS.

The function of the parietal sense organs.—In
considering the question of function, one must distinguish
sharply between the right and the left parietal sense organs.
The former is a well-developed “ pineal eye,” containing the
essential structural elements which one is accustomed to
associate with a light-perceiving organ, and, in common with

PARIETAL SENSE-ORGANS OF GEOTRIA. a3

Studnicka, I find it impossible to believe that, in the Lampreys,
it is not at the present day functional. It exhibits, in my
opinion, no sign of degeneration; sense cells, pigment cells, and
ganglion cells are all present in a high degree of perfection,
and the retina is connected with the brain by a well-developed
nerve. The enormous development of the right habenular
ganglion and of the right bundle of Meynert, with which the
pineal nerve is connected, also clearly indicate functional
activity.on the part of the pineal eye. Perhaps the most
striking evidence in favour of this view, however, is afforded
by the modification of the overlying tissues to form a light-
transmitting apparatus. It is one of the fundamental axioms
of biology that disuse leads to degeneration, and we may
safely assume that a high degree of structural differentiation
implies a corresponding degree of functional activity.
Everything points to the fact that the function of the pineal
organ is that of light perception, and therefore we are justified
in speaking of it as an “eye.” Its structure, however,
especially in the Lampreys, differs in important particulars
from that of any other eye known to us. In the Lampreys,
at any rate, it is not, as Studnicka has already pointed out,
a cameral eye, and we cannot suppose it to be capable of
forming animage. There is nothing which we are justified
in regarding as a lens, and the peculiar nature and arrangement
of the “ white”’ pigment is calculated to reflect the rays of light
in every direction, and thereby prevent the formation of an
image, even if the necessary dioptric apparatus were present.
On the other hand, it may well be that the brilliant white
pigment, by reflecting the light rays upon the knobs of the
sense cells, may thereby serve to intensify the light stimulus
and render the whole organ extremely sensitive to the
variations in the intensity of the illumination to which it
is exposed.! Such sensitiveness might be of great value
in giving timely warning of the approach of enemies from
1 This view of the function of the knobs of the sense cells is totally

opposed to that of Studni¢ka (12), who regards them as so many independent
lenses, each serving to focus the light upon its own particular sense cell.

24 ARTHUR DENDY.

above, before they come within range of the paired eyes, and
this I consider in all probability to be the function of this
organ. If the connection of the pineal eye with Reissner’s
fibre, which I have suggested above, really exists, we may
further conclude that the efficiency of the organ is greatly
increased by the “short circuiting” of the optic reflexes in
the same manner as has been described by Sargent in the
case of the ordinary paired eyes.

As regards the parapineal organ, or left parietal eye, it is
more difficult to express an opinion. Here we have, in the
absence of pigment and of the projecting knobs of the sense
cells, evidence either that the organ has never attained so
high a degree of organisation as its fellow, or that it has
suffered degeneration, and similar evidence is afforded by the
much smaller size of the left habenular ganglion and the left
bundle of Meynert. The fact that it usually les concealed
beneath the pineal eye also points to loss of function as a
light-perceiving organ ; and it is interesting to note that in
this respect the genus Geotria, in which both organs are
exposed to the light, one in front of the other, appears to be
in a less degenerate condition than Petromyzon. The
retention of the well-developed connection of the parapineal
organ with the left habenular ganglion, however, seems to
indicate that it is still in some degree functional. It is difficult
to understand why one member of the original pair should
tend to degenerate any more than the other, but the degene-
ration itself may be connected with a possible greater effici-
ency of a strictly median organ in appreciating what is taking
place immediately above the animal.

The paired origin of the parietal sense organs.—
The idea of a median, unpaired, Cyclopean eye on the top of
the head of the primitive Vertebrate ancestors has so struck
the popular imagination and become so firmly rooted even
in scientific literature that it is extremely difficult to gain
general acceptance for the modification of this somewhat
crude notion necessitated by modern research. Yet the
necessity for such modification confronts us at almost every

PARIETAL SENSE-ORGANS OF GEOTRIA. 25

point of view, morphological, embryological, and even palzon-
tological. In this connection we cannot content ourselves
with the consideration of any one Vertebrate group, but must
seek for evidence from as wide a field as possible. I
discussed the problem at some length in my memoir (8)
“On the Development of the Parietal Eye and Adjacent
Organs in Sphenodon (Hatteria),” published in this
‘Journal’ in 1899, with special reference to the Tuatara. Since
that date the embryological investigations of Cameron have
afforded striking confirmation of the views then adopted, and
my researches on the New Zealand Lamprey, described in
the present memoir, strongly confirm me in the opinion that
the so-called pineal and parapineal organs represent the
right and left members of a primitive pair.

The evidence derived from the study of Geotria may be
summarised as follows :

(1) The parapineal organ, in its position to the left of the
pineal, still shows evidence of its primitive paired character.

(2) The structure of the pineal and parapineal organs is
essentially identical, although the former is much more
highly developed than the latter.

(3) The connection of each of the two sense organs with
the corresponding member of the habenular ganglion pair
need no longer be questioned.

(4) The marked asymmetry in point of size of the two
habenular ganglia, and of the two bundles of Meynert,
corresponds exactly to the unequal development of the two
parietal sense organs with which they are connected, and
leaves no doubt as to the paired character of the whole system.

The embryological investigations of Cameron (8, 9) confirm
strongly the general results obtained by Hill, Locy, and
myself. In Amphibia and in the chick Cameron shows that
the ‘“‘ epiphysis” is in origin a bilateral structure, just as
Hill had shown for Teleosteans, Locy for Elasmobranchs, and
the present writer for Sphenodon. Insome cases, including
man, however, Cameron (10) maintains that there is a decussa-
tion of the nerve fibres at the base of the epiphysis, each lateral

26 ARTHUR DENDY.

half of the “ pineal body ” being supplied by fibres which come
from the habenular ganglion of the opposite side. This
may be true in certain types, but as Cameron himself
recognises, there is no evidence for any such decussation
in the Lampreys, where each of the parietal sense organs is
undoubtedly innervated from the habenular ganglion of its
own side.

By no means the least interesting evidence in favour
of the paired origin of the parietal sense organs is that
afforded by the study of fossil fishes, the history of which
affords a curious illustration of the influence of what we
may call the ‘‘Cyclopean theory” of the pineal eye. The
following quotation from Bashford Dean’s work (11) on
‘Fishes, Living and Fossil’ will serve to make this clear,
and at the same time to indicate the character of the
paleontological evidence in question: “The evidence, how-
ever, that the median opening in the head shields of ancient
fishes actually enclosed a pineal eye is now felt by the
present writer to be more than questionable. ‘lhe remarkable
pineal funnel of the Devonian Dinichthys (fig. 134) is evi-
dently to be compared with the median foramen of Ctenodus
and Paledaphus (= ‘Sirenoids,’ p. 122); but this can no
longer be looked upon as having possessed an optic function,
and thus practically renders worthless all the evidence of a
median eye presented by fossil fishes. It certainly appeared
that in the characters of the pineal foramen of Dinichthys
there existed strong grounds for believing that a median
visual organ was present.... But the function of this
pineal foramen, unfortunately for speculation, could not have
been optical. It occurs in a fish (Titanichthys) closely
related to Dinichthys, and, as the writer has recently found,
is of a distinctly paired character, its visceral and
outer openings bearing grooves and ridges which demon-
strate that the pineal structures must not only have been
paired, but must have entered the opening in a way which
precludes the admission of the epiphysis.... It must,
for the present, be concluded, accordingly, that the pineal

PARIETAL SENSE-ORGANS OF GEOTRIA. rate

structures of the true fishes do not tend to confirm the theory
that the epiphysis of the ancestral vertebrates was connected
with a median unpaired eye.”

If we once recognise the paired origin of the parietal
sense organs, the fact that a paired pineal foramen occurs in
the ancient Titanichthys need cause us no surprise.

REFERENCE List or LITERATURE.

(For further references vide Studnicka, 2.)

9

1. Denny and Ottiver.—‘‘ On the New Zealand Lamprey,” ‘ Transactions
New Zealand Institute,’ vol. xxxiv, 1902.
2. Srupnicka.— Die Parietalorgane,” in Oppel’s ‘ Lehrbuch der vergleich-

enden mikroskopischen Anatomie der Wirbelthiere,’ part v, Jena, 1905.

3. Denpy.— On the Development of the Parietal Eye and Adjacent Organs
in Sphenodon (Hatteria),”’ ‘Quart. Journ. Micr. Sci.,’ vol. xlii, n.s.,
p. 11), 1899.

4. GaskeLt.—‘‘On the Origin of Vertebrates from a Crustacean-like
Ancestor,” ‘ Quart. Journ. Micr. Sci.,’ vol. xxxi, n.s., p. 379, 1890.

5. Denpy.—“ On a Pair of Ciliated Grooves in the Brain of the Ammoccete,
apparently serving to promote the Circulation of the Fluid in the Brain-
cavity,” ‘ Proc. Roy. Soc. Lond.,’ vol. lxix, 1902.

6. Sarcent.—“ The Optic Reflex Apparatus of Vertebrates for Short-
circuit Transmission of Motor Reflexes through Reissner’s Fibre; its
Morphology, Ontogeny, Phylogeny, and Function.” Part I—‘ The
Fish-like Vertebrates,” ‘ Bulletin Mus. Comp. Zool. Harvard College,’
vol. xlv, No. 3, 1904.

7. Stupnicka.—“ Sur les Organes pariétaux de Petromyzon planeri,”
‘ Sitzungsber. der Kg. Ges. d. Wissensch. in Prag,’ 18938.

8. Camrron.—* On the Origin of the Pineal Body as an Amesial Structure,
deduced from the Study of its Development in Amphibia,” ‘ Proc.
Roy. Soc. Edin.,’ vol. xxiv, part 6, 1902-3.

9. CamEroyn.—“ On the Origin of the Epiphysis Cerebri as a Bilateral
Structure in the Chick,” ‘Proc. Roy. Soc. Edin.,’ vol. xxv, part 2,
1903-4,

10. Cameron.—“On the Presence and Significance of the Superior Com-
missure throughout the Vertebrata,” ‘Journal of Anatomy and
Physiology,’ vol. xxxviii, 1904.

11. Dean.—“ Fishes, Living and Fossil,” ‘Columbia University Biological
Series,’ New York, 1895.

28 ARTHUR DENDY.

12. Srupnicka.—“ Ueber den feineren Bau der Parietalorgane von Petro-
myzon marinus, L,” ‘Sitzungsber. der Konigl. bdlmischen Gesell-
schaft d. Wissenschaften,’ Prag, 1899.

EXPLANATION OF PLATES 1 & 2,

Illustrating Professor Arthur Dendy’s paper “ On the Parietal
Sense-organs and Associated Structures in the New
Zealand Lamprey (Geotria australis).

EXPLANATION OF LETTERING.

At. Atrium of pineal organ. C. Cerebellum. @C.H. Cerebral hemisphere.
C.H.S. Commissura habenularis (=C. superior). C.M/. Corpus mammillare
= lobus infundibuli). @.P. Posterior commissure. C.7. Connective tissue.
C.7.C. Connective tissue wall of cranium. (C.7.N. Nuclei of connective
tissue cells. €.7.N’. The same in nervous layer of retina of pineal organ.
C.7.P. Parietal plug of modified connective tissue overlying the parietal
sense organs. C.7'.S.Connective-tissue sheath. Hp.#. Ependymal epithelium.
Ep.G. Ependymal groove. pid. Epidermis. G.C. Ganglion cells. G.H.A.
Anterior division of left. habenular ganglion. G.H.Z. Posterior division of left
habenular ganglion. G.H.R. Right habenular ganglion. nf. Infundibulum.
I.S.P.C. Inner segments of pigment cells. Z.M. “Limiting membrane.”
L.M.B. Left bundle of Meynert. Z.P.#. Parapineal organ (= left parietal
eye). 2.7. Lamina terminalis. Med. Medulla oblongata. Muse. Muscle.
Na.C. Nasal cartilage. N.C. Nerve cells. W.C.C. Nuclei of columnar cells
of pellucida. W.F.N. Network of nerve fibres, ete. Not. Notochord.
N.P.C. Nuclei of pigment cells. W.S.C. Nuclei of sense cells. Oc.C.
Occipital cartilage. O.Ch. Optic chiasma. O.Z, Olfactory lobe. O.N.
Olfactory nerve. 0O.S.P.C. Outer segments of pigment cells. Par.C.
Parachordal cartilage. P.B. Pituitary body. Pell. Pellucida. Pig'., Pig.
Outer and inner pigment layers of integument. P/.Ch, Choroid plexuses.
P.M. Pia mater. P.N. Pineal nerve. P.N%. Branches of pineal nerve.
P.S. Pineal stalk. P.S¢. Protoplasmic strands in interior of pineal organ.
Ret. Retina. &.F. Reissner’s fibre. &.F'~’, Constituent branches of
Reissner’s fibre. 2.I.P. Recessus infrapinealis. 2.M.B. Right bundle of
Meynert. &.P. Recessus pre-opticus (= R. chiasmaticus). R.P.Z#. Pineal
organ (= right parietal eye). &.P.O. Recessus post-opticus. £&.8.C.
Retinal sense-cells. S.C. Shrinkage cavity above posterior commissure.
S.C.K. Terminal knobs of sensory cells of retina. Sept. Longitudinal septum

PARIETAL SENSE-ORGANS OF GEOTRIA. 29

dividing the saccus vasculosus into right and left halves. Sp.C. Spinal cord.
7.H. Tractus habenularis. ZAa/. Thalamencephalon. 1.0. Tectum opticum.
V3. Third ventricle. V4. Fourth ventricle. X. Point where the bundles of
Meynert reach the base of the brain.

(All the figures refer to Geotria australis [Velasia].)

PLATE 1.
Fie. 1.—Brain; dorsal view after removal of the choroid plexuses of the
mid- and hind-brain. x 10.
Fic. 2.—Sagittal section through the brain, with the surrounding cranium
and overlying structures (slightly diagrammatic, and with the arachnoid
tissue omitted).

Fic. 3.— Transverse section of the brain in the region of the posterior
commissure, showing the ependymal groove, bundles of Meynert, ete. Drawn
under Zeiss A, oc. 2.

PLATE 2.

Fic. 4.—The pineal and parapineal organs (right and left parietal eyes), as
seen from above under a dissecting lens.

Fic. 5.—Diagram showing the structure of the retina of the pineal organ
(right parietal eye).

Fic. 6.—Longitudinal vertical section through the recessus infrapinealis,
showing the relation of the pineal nerve to the posterior commissure, the right
habenular ganglion, the right bundle of Meynert, the ependymal groove, and
Reissner’s fibre (slightly diagrammatic).

Fic. 7.—Sagittal section of the pineal organ (right parietal eye), drawn
under Zeiss D, ocular 2 (slightly diagrammatic).

Fie. 8.—Longitudinal vertical section through the parapineal organ (left
parietal eye) and the anterior portion of the left habenular ganglion. Drawn
under Zeiss D, oc. 2 (slightly diagrammatic).

Fic. 9.—Transverse section through the hinder part of the auterior portion
of the left babenular ganglion, immediately behind the parapineal organ.
Drawn under Zeiss D, oc. 2.

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STUDIES IN SPICULE FORMATION. 31

Studies in Spicule Formation.

V.—tThe Scleroblastic Development of the Spicules in Ophiu-
roidea and Echinoidea, and in the Genera Antedon and
Synapta.

By

W. Woodland,
Demonstrator of Zoology, King’s College, London.

With Plates 3 and 4.

INTRODUCTORY.

Tue greater part of the material utilised in this inquiry
was obtained from the Marine Biological Laboratory at
Plymouth, and consisted of two common examples of the
Ophiuroidea, viz. the small Amphiura elegans (Amphi-
uride) and the much larger Ophiothrix fragilis (Ophio-
thricide), and one of the Echinoidea—Echinus esculentus.
Recently-metamorphosed specimens of this latter were mostly
obtained from the artificially-reared plutei referred to in
Study III (13), and were about a score in number; however,
one or two slightly larger specimens were also secured from
dredgings. Most of my results have been obtained from
observations on the viviparous Amphiura elegans. In
this Ophiurid the bursz, as is well known, function as brood-
chambers, and, in consequence, young animals are very easy
to obtain in every stage of development. The methods of
preparation adopted were as follows:—A hundred or more

32 W. WOODLAND.

living Amphiuras being obtained, the disc of each animal
was cracked, so as to permit the free access of the fixing,
staining, and preserving reagents to the young animals
contained within the burs, and these were then fixed with
1 per cent. osmic acid, stained with picro-carmine, and pre-
served in 90 per cent. alcohol in the manner already described
in previous studies. Some of my best slides, however, have
been obtained by merely fixing the live Amphiuras in absolute
alcohol, subsequently staining for a fortnight or more in a
saturated solution of safranin in absolute alcohol and washing
out in absolute alcohol for a month or so after (if the alcohol
be warmed less time will suffice), In many cases I also
employed lichtgriin as a plasma stain, but, if employed, the
solution in absolute alcohol must either be very weak, or, if
a saturated solution, the Amphiuras must only be immersed
in it for a minute or so, otherwise the tissues become opaque.
Lichtgriin, when successfully employed, undoubtedly gives
the best results; it is, however, quite possible to work without
it, though in this case it is more difficult to be quite certain
on occasion as to whether a particular cell belongs to a
particular spicule or not. When the Amphiuras have been
stained the discs are opened and the young ones extracted ;
these are then transferred to xylol, and finally mounted in
balsam. In general only the youngest Amphiuras (about 0°5
mm. in diameter; see fig. 1) yield satisfactory results, though
now and again it is possible to observe young spicules in the
arms of the older Amphiuras. It is surprising how very few
really satisfactory young Amphiuras are obtainable from
numerous parents: from at least five or six hundred adults
I have only managed to secure about a score of young ones
showing the origin of spicules in an unmistakable manner.

The above-described methods of preparation were also
employed in the examination of the specimens of Ophiothrix
and Hchinus.

My Antedon material consisted of very young specimens
which, after having their discs opened, were prepared by the
osmic and picro-carmine method. The imperforate thin plate

STUDIES IN SPICULE FORMATION. 33
spicules (which do not seem to have been previously described)
are lodged in the hypostroma of the integument, and in con-
sequence the portions of integument to be mounted must,
when freed from the viscera and musculature, be placed inside
upwards on the slide.

My Synapta material, consisting of 8. hispida and 8.
digitata, was obtained from Naples, and I believe was
simply fixed and preserved in alcohol. I stained portions of
the body-wall by the safranin and lichtgriin method and
obtained good results.

In many cases, especially of the older spicules of Amphiura,
it is difficult to decide upon the exact number of cells in con-
nection with a spicule, and such must be simply passed over.
The figures provided in the accompanying plate were all
drawn from spicules about which there existed no doubt
whatever as to the number of scleroblasts attached, and
these were only obtainable by careful and_ persistent
searching.

THe ORIGIN OF THE SPICULES IN AMPHIURA ELEGANS.

In Cucumariide (18) the plate-spicule originates as an
elongated granule (thick-set needle) with its length disposed
at right angles to the line joining the masses of the two
scleroblasts usually concerned in its deposition; in some
cases, however, four scleroblasts are concerned in the deposi-
tion, two being situated on each side of the needle. In
Amphiura elegans, on the other hand, the plate-spicule
originates in the same manner as the small superficial spicules
of the Cucumariidee do, viz. as an approximately spherical
granule contained within a single cell (fig. 2). This differ-
ence of origin between the holothurian and the ophiuroid
large plate-spicules can be correlated with other more general
differences affecting the manner of lime secretion as it occurs
in the two groups, and these I shall shortly indicate. For
the present it suffices to point out that the needle and the
granule correspond in form in both cases with the general

vou. 51, part 1.—NEW SERIES. 3

34 W. WOODLAND.

space disposition of the scleroblasts concerned in their indivi-
dual formation: the elongated needle is deposited by a bi-
nucleated mass of scleroplasm with maximum and minimum
diameters in the plane of the body-wall, and the spherical
granule by a scleroblast whose corresponding diameters
are all approximately equal. The granule in A. elegans
next assumes a flat three-cornered shape whilst still con-
tained by the single scleroblast, and the three corners
of the triangle thus formed then elongate to form a young
triradiate spicule (fig. 3). Shortly after this stage is reached
the nucleus of the scleroblast divides and two scleroblasts in
consequence appear in connection with the young triradiate
(fig. 4). Approaching nuclear division in the scleroblast is
always denoted by large size and faint coloration by picro-
carmine (fig. 5a, e.g.) ; as is well known picro-carmine is a
stain which never renders the details of karyokinesis visible.
The three arms of the spicule next show signs of bifurcation
at the extremities (figs. 5, 6), and, indeed, the whole of the
further development of the spicule (when this is not situated
at the extreme edges or ends of the arms, in which position
certain parts elongate greatly in a distal direction) consists,
as in Cucumariide, of a series of bifurcations resulting in a
more or less circular perforated plate (fig. 7), but it is notice-
able that the attached scleroblasts differ from those of Cucu-
mariide in their much greater number. The fact that
abnormally-large nuclei are so often met with in these
scleroblasts renders it exceedingly probable that they are all
(thirty or forty in the case of the larger plates) derived from
the original mother-scleroblast, although it is evidently im-
possible to make a decisive statement to that effect. This
repeated division of the mother-scleroblast might, indeed, be
attributed to the necessity for the proximity of nuclear sub-
stance to the constantly-increasing area of deposition (the
well-known experiments of Verworn on Polystomella proving
enucleated portions of protoplasm to be incapable of secreting
a shell pointing the argument) were it not that no such sub-
division occurs in the case of other spicules of equal size

STUDIES IN SPICULE FORMATION. 39

(Study IV). Why in Ophiuroids (and all other echinoderms
save holothurians) there should thus exist this definite relation
between the number of attached scleroblasts and the size of
the spicule, and not in holothurians I am quite unable to
say. (As a remarkable illustration of this fact compare the
stool spicules of Thyone [Study IV, fig. 55] with the young
spines of Ophiothrix [fig. 15]).

Not all of the spicules are derived from an original tri-
radiate structure; occasionally the spherical granule elon-
gates to form a rod which bifurcates at its extremities, as in
Cucumaride ; occasionally also the young spicule assumes a
more or less irregular form, and in Ophiothrix I have ob-
served four- and six-rayed young spicules (figs. 10 b and c),
but all these are very rare. The triradiate form of the
young spicule is by far the most usual. It must also be
mentioned that the largest of the perforated plates figured is
small when compared with most of the spicules present in
the young embryos of fig. 1, and of course the plates of the
adult Amphiura are still larger by comparison.

The scleroblasts in Amphiura are spherical cells which
assume a subspherical form when attached to the spicule;
the nucleus is relatively large, and if stained with picro-
carmine shows a distinct nucleolus. The cytoplasm is
faintly granular. Nuclei vary in size to some extent even in
the same individual, and, as already stated, become very
large previous to division. In specimens fixed with absolute
alcohol the nuclei appear somewhat contracted.

THE SPICULES IN OPHIOTHRIX FRAGILIS AND HCHINUS
ESCULENTUS.

My material for ascertaining the mode of spicule forma-
tion in these two genera being very limited I have merely
attempted to confirm the supposition that the process is here
identical with that in Amphiura elegans, and this I have
had no difficulty in doing. My Ophiothrix material con-
sisted of several young specimens with discs about 1 mm. in

36 W. WOODLAND.

diameter. Most of the plates in the young Ophiothrix are
extremely thick, and with comparatively few and small per-
forations (fig. 11) ; however, the ordinary thinner plates are
also present. The spicule originates in the same manner as
that already described (figs. 8—10). The young forms
represented by figs. 10 b and ¢ are, as already mentioned,
unusual. Fig. 10¢ indeed reminds one as regards shape of
a young stage in the formation of an auricularian wheel.
Fig. 13 represents a young spine which develops in much the
same way as the stool of Thyone, save of course that the
basal plate originates as a granule in one cell, and that the
number of scleroblasts concerned in its deposition is much
greater. It is quite possible that not all the cells repre-
sented in this figure are scleroblasts, some possibly belonging
to the surrounding membrane, and it is clearly impossible to
distinguish between them; it is quite safe to say, however,
that most of them are scleroblasts.

In Echinus esculentus (and miliaris) also the deve-
lopment of the spicule follows on the same lines (figs. 14—18).

Tue PLATE-SPICULES IN ANTEDON BIFIDA.

The majority of the spicules situated in the region of the
disc of Antedon lie towards the inner side of the soft mtegu-
ment, and are of two kinds—the imperforate thin “ glas-
plattchen ” of comparatively wide diameter and concentric-
ally marked (see accompanying text-figure), and the ordinary
perforated plates, which, however, often assume a very irre-
gular shape, and occasionally become mere branching struc-
tures. All transitions (fig. 21) are to be found between the
“ olasplattchen,”’ which are very unechinoderm in appear-
ance, and the ordinary perforated plates; the former, how-
ever, are much the more common, and exist in great numbers.
Since the development of the perforate plates and branching
spicules is quite normal, i.e. the same as that already
described for Amphiura, Ophiothrix, and Kchinus, I shall
merely describe the simple development of the imperforate

STUDIES IN SPICULE FORMATION. 37

spicules. ‘These plates each originate as a spherical granule
contained within a single cell (fig. 19), and this granule
gradually becomes larger (fig. 20), and early assumes the
plate-like form (fig. 22). There is thus no rod or triradiate
stage—so common in the development of echinoderm spicules
—in the growth of these plates, and the plate is never of the
perforate type. The nucleus of the original mother-scleroblast
divides at an early stage of growth of the spicule and the
number of nuclei present at different stages of growth (a

Full-sized imperforate plates of Antedon bifida x cir. 270
diameters. These plates are very thin, easily fractured, show con-
centric lines (presumably of growth), and are to be found in large
numbers in the hypostroma of the integument.

score or more on fully-developed plates) is strictly propor-
tional to the size attained by the spicule. These large thin
squamose spicules of Antedon do not seem to be generally
known, although they exist in great numbers. They are
very easily decalcified in virtue of their extreme thinness,
and, as I have already mentioned, are very unlike echinoderm
spicules (so much so that I at first mistook them for artefacts).
As Dr. Bather has very kindly pointed out to me, similar,
but much thicker, imperforate plates (with no concentric
markings) have been described, among others, by Ludwig
in Holothurians (4), Ophiuroids (5) (also Mortenson [8]), and
Asteroids (Astropectinide) (6).

38 W. WOODLAND.

I may also mention that the dark granules, so conspicuous
in the scleroblasts of many Cucumariide, are also present in
the scleroblasts of Antedon; indeed, they are usually dark
without any additional staining with lichtgriin.

Ture SMALL Puate-SpPICULES OF SYNAPTA HISPIDA AND
S. DIGITATA.

In addition to the plate-and-anchor spicules of S.
inhaerens I have examined the similar (though easily
distinguishable) spicules of S. hispida and 8S. digitata,
and, as might have been expected, I find that the disposition
of the scleroplasm concerned in their formation is essentially
the same as that which I described in Study IV. Through
lack of material I have not as yet been able to ascertain the
disposition of the scleroplasm in the early stages of develop-
ment, though I hope to do so shortly.

Besides these conspicuous plate-and-anchor spicules there
exist in S. hispida and 8. digitata (though not in S.
inhaerens) small elongate plate-spicules, containing in the
former species a single perforation in their centre, and the
development of these perforate plates of S. hispida is
remarkable. I may mention that these small plates are
particularly numerous in the region of the muscle-bands,
though they are also to be found in the intervening spaces.

The plate in both species arises quite normally as a more
or less spherical granule in the centre of a single scleroblast
(fig. 23), and, as in Antedon, this gradually becomes elon-
gated (fig. 24) and plate-like, but, differing from Antedon,
the nucleus of the scleroblast remains single throughout the
entire development. In §S. digitata these plate-spicules
cease growth at the stage depicted in fig. 30, which, it will
be noticed, is identical with the stage of development repre-
sented by fig. 24 of the plate-spicule of 8. hispida. In this
latter species the plate elongates considerably, and expands
laterally to some extent except in the vicinity of the nucleus,

STUDIES IN SPICULE FORMATION. 39

the result being that the nucleus becomes lodged in a depres-
sion to one side of the plate (fig. 25). The further develop-
ment of the spicule consists of the enclosure of the nucleus
by the extension of the calcite, as shown in figs. 26—28.
Two arms of calcareous matter extend round the nucleus on
either side, meet, and finally fuse, and the adult plate, in
consequence, contains a central perforation, in which the
nucleus is imprisoned (fig. 28). Occasionally, when the per-
foration is larger than usual, a secondary ingrowth of calcite
occurs (fig. 28b); occasionally also the two arms of calcite
first overlap each other instead of fusing immediately (figs.
27a and 27b), and sometimes, but rarely, secondary pairs of
arms are formed, which tend to emulate the first pair in their
direction of growth (fig. 28a). This last feature (as also that
represented by fig. 29) proves, however much it may appear
to the contrary, that the presence of the nucleus is not the
only stimulus giving rise to the peculiar mode of extension of
the calcite just described. In fact, here, as in the cases
described in the foregoing parts of this paper and elsewhere,
the nucleus, with its associated mass of cytoplasm, probably
has very little to do with the direction of growth of the
calcite—with the form of the spicule—and must largely be
discounted as a factor in the production of spicular forms.

THEORETICAL CONSIDERATIONS AND Previous Work.

From the results described above, and from the figures of
young spicules in the various classes of echinoderms provided
by Agassiz (1), Ludwig (7), Seeliger (9), Fewkes (2, 3),
Théel (11, 12), and many others, we may assume what has,
indeed, been already implied, viz. that in Ophiuroidea,
Asteroidea, Echinoidea, and Crinoidea, the typical mode of
scleroblastic development of the spicules is that described
above for Amphiura elegans, i.e. the spicule originates
as a triradiate structure contained within a single cell. From
the figures of these and other authors, on the other hand, we
may also assume that the typical mode of development of

40 W. WOODLAND.

the plates of Holothuroidea is that described by me for the
Cucumariide (Study IV), viz. the origin of the elongated
calcareous needle between two or four cells, its growth
to form a rod, the bifurcation of the extremities of this
rod, and so on. Up to the present I only know of one
exception to this rule, Semon (10) describing and figuring
most distinctly the triradiate mode of origin of certain
spicules in the holothurian Chiridota venusta. But this,
and possibly a few other exceptions, do not invalidate the
general rule, and, as before mentioned, this difference of
origin between most echinoderm spicules and the spicules of
holothurians can be correlated with a general difference
which exists between the modes of skeleton formation in the
two groups, 1.e. this rule can be justified by a reason for its
existence. The quantity of lime respectively secreted by
most echinoderms and by holothurians differs greatly—in the
former group the stroma is packed with a calcareous stereom,
whereas in most individuals of the latter the skeleton is only
represented by isolated spicules—and correlated with this
difference is (a) the fact that in the former group every
scleroblast gives rise to a spicule, whereas in the latter at
least two scleroblasts have to co-operate for the same
purpose, and (b) the equally cogent fact that in most echino-
derms scleroblasts multiply very rapidly (shown by the
number of scleroblasts per spicule), whereas in holothurians
they multiply very slowly. In other words, the difference in
the origin of the spicule in the two groups is correlated with
the amount of the skeleton present—with the skeleton-pro-
ducing capacity.

Previous work on the subject of the present paper, so far
as I have been able to discover, has been very small in
amount. In fact, the only paper that I know of describing
the origin of the spherical granule and the young triradiate in
a single scleroblast is that of Semon (10) on the holothurian
Chiridota just referred to. Semon also figures very dis-
tinctly the young triradiate with two scleroblasts (similar to
fig. 4). At the same time Semon represents the triangle-

STUDIES IN SPICULE FORMATION. 41

shaped spicule as a tetrahedron, and, curiously enough,
represents this tetrahedron as forming the distinct centre
or basis of the older spicules, secondary calcareous matter, so
to speak, prolonging the solid angles of the tetrahedron. As
I have stated in Study ITJ, I believe this tetrahedron struc-
ture to be quite imaginary, and I certainly cannot credit
without more evidence its persistence as the-visible basis of
older spicules.

Fewkes (2), describing the metamorphosis of Echin-
arachnius parma, says that “ the first limestone formation
which was observed is a trifid spicule in the wall of the body
of the growing sea-urchin. In its very first. form this trifid
spicule is spherical in contour. Later it assumes a trifid
shape, and seems to be enclosed in a transparent sac, the
outer wall of which is believed to be formed of epiblast, the
calcareous body being formed possibly in mesoblast’’!
Fewkes also adds that he does not know whether these trifid
bodies develop into the plates or not.

Théel, in his papers on HEchinus miliaris (12) and
Echinocyamus pusillus (11), quite correctly describes
the development of the spicules, and also states his opinion
that “ they first originate from cells which have wandered in
between the tissues,” but he gives no details of the sclero-
blastic development.

Ludwig, Fewkes, and others provide numerous figures
showing the young triradiate spicules and older stages, but
from not employing suitable staining reagents they entirely
overlooked the scleroblasts in connection with the young
spicules.

42, W. WOODLAND.

LITERATURE.
1. AGassiz, A.—‘ Bull. Mus. Comp. Zool. Harvard,’ vol. x, 1882.
2. Fewxes, J. W.—Op. cit., vol. xii, 1885.
3. Op. cit., vols, xiii (1886) and xvii (1890).
4, Lupwic, H.—‘ Holothurien der Hamburger Magalhaensischen Sammel-
reise,’ Lief. iii, 1898.
5. ‘Zool. Jabrb.,’ Suppl. 4; ‘Fauna Chilensis,’ Heft. iii, 1898.
6. ——— ‘Fauna Stat. Neapel,’ xxiv, 1897.
ie ‘Zeitschr. f. wiss. Zooi.,’ vols. xxxiv (1880) and xxxvi (1882).
8. Mortenson, T.—‘ Medd. Gronland,’ vol. xxix, 1903.
9. SreticER, O.—‘ Zool. Jahrb., Abth. f. Morph.,’ vol. vi, 18938.

10. Semon, R.—‘ Mittheil. Zool. Stat. Neapel,’ vol. vii, 1887.
11. Tute1t, Hs.—‘ Nova Acta R. Soc. Sci. Upsala,’ ser. iii, 1892.
12. ‘ Bib. Svenska Akad. Handl.,’ vol. xxviii, ser. 4, 1902.

18. Wooptanp, W.—“<Studies in Spicule Formation” (I—IV), ‘Quart.
Journ. Mier, Sci.,’ vol. 49, 1905—1906.

EXPLANATION OF PLATES 3 anp 4,

Illustrating Mr. W. Woodland’s “ Studies in Spicule
Formation” (V).

Fig. 1 drawn natural size; Figs. 2, 3, 8, 9, 14, 15, and 23 x 1280 dia-
meters; all other figures x 640 diameters. All figures (save Fig. 1) drawn
with the camera lucida.

PLATE 3.

Development of the plate spicules of Amphiura elegans (Figs. 1—7),
Ophiothrix fragilis (Figs. 8—13), and Echinus esculentus (Figs.
14—18).

Fie. 1.—Young Amphiura in which the spicules were observed. (Several
specimens of Amphiura were considerably smaller than those here depicted.)

STUDIES IN SPICULE FORMATION. 43

Fie. 2.—The origin of the plate-spicule in Aimphiura elegans as a
spherical granule contained within a single scleroblast.

Fic. 3.—The triangular and subsequently triradiate forms assumed by the
initial granule within the scleroblast.

Fie. 4.—Young (usually) trifid spicules with two scleroblasts attached
(derived from division of the mother scleroblast).

Fie. 5.—Older stage of the triradiate. Ina one nucleus is dividing; in 4
one nucleus has divided, three scleroblasts being present.

Fie. 6.—The bifurcation of the arms of the triradiate. The number of
scleroblasts has increased, and nuclei are seen to be about to divide.

Fic. 7.—Older spicules with numerous scleroblasts.

Figs. 8—12 represent stages in the formation of the usually thick plates
(with small perforations) of Ophiothrix fragilis. In Fig. 10, 4 and ¢
represent young stages of unusual shape. Fig. 12 is a young spicule of the
thin plate variety.

Tic. 13 represents one of the lateral trifid spines of O. fragilis. The
numerous scleroblasts in connection with the spicule is striking when com-
pared with the two scleroblasts associated with the somewhat similar “stool”
spicule of Thyone (Study IV, pl. 34).

Fics. 14—18 represent young stages in the formation of the plates in
Echinus esculentus similar to those above.

PLATE 4.

Development of the imperforate plate-spicules of Antedon bifida (Figs,
19—22), and of the plate-spicules of Synapta hispida (Figs. 23—29), and
S. digitata (Fig. 30).

Fics. 19—22.—The development of the imperforate plate-spicule of
Antedon bifida, Fig. 21 represents spicules which show some affinity in
their form to the ordinary perforate plate-spicules of echinoderms. ‘The size
attained by the adult thin perforate plate may be realised by comparing the
present figures of young plates (Xx 640) with the text-figure showing adult
plates (x 270).

Fics. 23—80 represent the development of the curious plate-spicules o
Synapta hispida and S. digitata, which are very small in comparison
with the plate-and-anchor spicules. Fig. 24, representing a young stage in
the development of the plates of S. hispida, shows forms very similar to
those of Fig. 30, which represents adult spicules of S. digitata. Figs.
25—28 illustrate the enclosure of the nucleus within the central perforation.

Fig. 29 shows plates in which the central aperture is considerably larger than
the average,

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STUDIES IN SPICULE FORMATION. 45

Studies in Spicule Formation.

VI.—The Scleroblastic Development of the Spicules in some
Mollusca and in one Genus of Colonial Ascidians.

By

W. Woodland,
Demonstrator of Zoology, King’s College, London.

With Plate 5.

SPICULES IN NupiprancH Mo.uuusca.

The nudibranchs examined, all obtained from Plymouth,
consisted of three genera—Goniodoris castanea, Archi-
doris tuberculata, and Lamellidoris bilamellata.
Most of my specimens were simply fixed and preserved in
absolute alcohol, and were, in consequence, much contracted ;
others were fixed with 1 per cent. osmic acid, opened to give
admission to the stain, and stained as usual with picro-carmine
(three hours), and I received them in this condition. In both
cases I divided the animals horizontally into halves (with
scissors), and in each instance scraped away with a scalpel
the viscera and most of the musculature of the body-wall,
until the portion of integument left looked to some extent
translucent (whether unstained or stained). I usually found
that the integument of the foot showed the spicules better
than that of the dorsum. ‘The unstamed portions of mtegu-
ment (alcohol specimens) | stained in a saturated solution of

46 W. WOODLAND.

safranin (nigrosin is also good) in absolute alcohol for a fort-
night, washed thoroughly in warm absolute for a day or so,
stained for a few minutes in a saturated solution of lichtgriin
in absolute alcohol, again washed well in absolute, cleared in
xylol and finally mounted in balsam. In most cases the picro-
carmine specimens became too dark if also stained with
lichtgriin.

The spicules of these three genera are all somewhat irre-
gular monaxons with a concentric structure when viewed in
section, and containing a larger or smaller amount of organic
matter, but they differ slightly both in size and shape accord-
ing to the genus. In Archidoris the spicules are much more
slender than in the other two genera, and do not attain such
a great length; moreover, they do not possess a sudden
thickening situated midway in the length of the spicule like
those of the other two genera, and the nucleus of the sclero-
blast is much smaller both relatively to the size of the spicule
and absolutely, is spherical instead of oval and possesses a
distinct nucleolus. The spicules of both Goniodoris and
Lamellidoris are, as just mentioned, thickened at the middle
and in the latter genus this midway thickening (which is not
so prominent as in Goniodoris) often possesses one or two
large spikes. The spicules of Lamellidoris attain a much
greater size than those of Goniodoris, and are much smoother
in general outline. Corroded spicules exhibit straight-sided
regular outlines at the exposed edges, whence we may suppose
that nudibranch spicules, like most other calcareous spicules,
are essentially aggregates of calcite crystals.

The nearest approach to the spherical concretion stage of
the spicule which I have observed is that represented in fig.
1. Without doubt the spicule originates, as in every other
case of simple spicules, in this form, but unfortunately my
specimens are not young enough to show this, although the
stages figured afford sufficient proof that this is the case.
The granule becomes a rod and the rod assumes the form of
the adult monaxon, growing over its entire surface (but, of
course, chiefly at the extremities) by the deposition of cal-

STUDIES. IN SPICULE FORMATION. AT

careous matter derived from the scleroblast cytoplasm which
entirely surrounds the spicule. The median portion of the
adult spicule is formed first (evident when this is thickened),
the tapering extremities growing out from this. The sclero-
blast, i.e. single nucleus, never divides, so that the spicules
are purely unicellular products, and in most cases the body
of the scleroblast (the small mass of protoplasm immediately
surrounding the nucleus) is constantly situated midway in the
length of the spicule, i. e. in the vicinity of the thickening in
those spicules possessing this feature.

Spicules in Aplacophorous Mollusca.

My material consisted of specimens of Proneomenia
aglaopheniw and Dondersia banyulensis, specially
prepared at Plymouth by the osmic acid and picro-carmine
method which gave excellent results.

So much has been previously written and so many good
figures provided in connection with the development of these
characteristic calcareous spicules of the Aplacophora that
my sole excuse for re-considering the subject is the uncertainty
which still prevails as to whether these spicules are unicellu-
lar (Thiele [8], Wiren [9]) or multicellular (Heuscher [8],
Hubrecht [4], Kowalevsky and Marion [5], Pruvot [7]) in
growth. There is also a misapprehension to correct, which
is that the spicules embedded in the cuticle of Proneomenia
are “in relation internally with epithelial papillae” (Sedgwick’s
‘Text-book of Zoology,’ p. 353, likewise Pelseneer’s ‘Mollusca’
in Lankester’s ‘ Treatise on Zoology ’).

To state the results of my direct observations and inquiries
as briefly as possible, I may say that it is now quite certain
that the spicules of the Aplacophora, like those of the Poly-
placophora (Pelseneer), all arise individually in a single cell
of the hypodermis (figs. 7—13). It is also certain that the
spicules in Proneomenia (and other Aplacophora which pos-
sess an integument of similar type) do not in the majority of
cases bear any relation to the hypodermal papille, and that

48 W. WOODLAND.

when they do the relation is purely an accidental one. This
occasional accidental relation apparently originates thus : the
young spicules arise each in a single cell situated in the
hypodermis, and in the majority of cases the portion of the
hypodermis containing this spiculiferous cell remains in its
ordinary position, but if, as sometimes happens (say in 20 per

(-\

iG,
S M
ae omaaaaanas

D A

Semi-diagrammatic drawing of the hypodermis and cuticle of Pro-
neomenia aglaophenie, illustrating the “‘carrying up” of the
spiculiferous cells and spicules by the hypodermal papille. The
figure (X 400 diam.) is composed of drawings of the actual objects
brought together into one field. In A the spicule is in its normal
position ; in B the scleroblast is being detached from the hypodermis ;
in C the scleroblast has lost its spicule, this lying free as at K; in
D a scleroblast with a well-grown spicule has been caught up in the
young papillary elevation; in E a fairly young spicule has been
carried up some distance bya papilla; in F the spicule has similarly
been carried up, but is older; G and H represent young papille;
J a full grown papilla, with its pigmented swollen extremity lying
just below the outer limit of the cuticle. It must be understood
that normally the papilla and spicules are quite distinct, not being
in any way associated. L represents débris on the exterior of the
thick cuticle; M is the hypodermis.

cent. of cases), it becomes raised up into a papilla, the spiculi-
ferous cell is inevitably carried up with it, and then the
spicule superficially appears to be a product of the papilla
(see the accompanying text-figure). The majority of the
spicules are quite separate from the papillae, but in those
cases in which they are associated the purely accidental nature
of this association is proved by the various positions which
the spicule assume relative to the papilla, these illustrating
the various stages of ‘‘ carrying up ” referred to above.

STUDIES IN SPICULE FORMATION. 49

The spicules of Proneomenia, from their first appearance
each as a small needle contained within a single cell (figs. 7,
8), grow in a more or less vertical direction, and soon burst
through the cell-membrane (fig. 9). From this stage onward
the further growth of the spicule is confined to the basal
extremity (so resembling the growth of a hair), which alone
is enveloped by the cell-substance (fig. 10). An axial cavity
appears in the spicule substance before the spicule is half
grown, but closes up proximally before the full size is
attained. Many of the scleroblasts attached to the larger
spicules become more or less withdrawn from the hypodermis
(text-figure, B, c), and subsequently also lose connection with
the spicules themselves, which, perhaps owing to contrac-
tions of the integument, often come to he, when full grown,
near the outer limit of the cuticle (kK in text-figure).

In Dondersia the monaxon spicule, instead of being ver-
tical in position as in Proneomenia, is disposed more or less
horizontally from the first (figs. 11, 12) in correspondence
with the thin cuticle (thin as compared with that of Pro-
neomenia, being little more than the thickness of the hypo-
dermis). ‘These spicules also when adult become separated
from the hypodermis, and lose their scleroblasts.

I may add that the figures and statements of Heuscher
and Hubrecht, affirming the multicellular papillary origin of
the spicules of Proneomenia, are misleading, though super-
ficially they appear to be correct. They have solely resulted
from insufficient attention being paid to detail, and, bearing
in mind the facts described above, it will easily be seen how
the mistake has arisen.

It may also be mentioned that the hypodermis contains
many gland-cells with mucilaginous contents, which have
been, on at least one occasion, figured as young spicules!
Needless to say the two are readily distinguishable.

With reference to the mode of growth of these spicules of
the Aplacophora, I may here point out that the production
of a straight “finished”? mineral structure by terminal
accretion due solely to the activity of a single scleroblats

vot. 51, part 1.—NEW SERIES. 4,

50 W. WOODLAND.

under undisturbed conditions is here clearly demonstrated ;
there is in these aplacophore spicules no question whatever
as to whether or no the symmetrical form is due to ecrystal-
lisation or the like. And, bearing this example in mind, it
further cannot be denied that the straightest rays of the
triradiates of clathrinid Calcarea in all probability owe their
symmetry to the same cause. Crystalline matter is deposited
by the terminal cell in a similar manner in both cases, and,
so long as the conditions remain undisturbed, the growth of
the spicule must continue ina straight line. “ Bio-crystal-
lisation ” and the rest are here at least superfluous. On the
other hand, introduce disturbed conditions and, as might be
expected, the more irregular spicules of Leucosoleniide and
Sycons are the result. Further, granting placid conditions,
bring three cells into such close apposition that their inner
surfaces become adpressed into the outline of a triradiate
and let each of these cells divide centripetally, the distal cell
in each case producing, in relation with the fixed proximal
cell, a straight monaxon,' and personally I can see no reason
why cumbrous hypotheses should be invented in order to
explain why the three contained angles of such a triradiate
spicule (found in most calcarea) should in almost all cases be
equal. Surely the equiangularity is the direct result of the
apposition of the three scleroblasts, as I have previously
contended [10].

No such explanations, however, apply to the young tri-
radiate ‘ stars’? of most echinoderms described in the last
Study, which, however, differ, like most other spicules, from
the calcareous spicules of sponges and Aplacophora in that
they are entirely invested by the formative protoplasm.

Tur SPICULES IN THE ASCIDIAN GENUS LEPTOCLINUM.

I have examined three species of Leptoclinum—L. com-
mune, L. maculosum, and L. sp.— all obtained from
1 This monaxon is, under these conditions, pointed at both ends. In

Aplacophora each spicule, corresponding to its one-celled basal origin, is
truncated at its proximal end.

STUDIES IN SPICULE FORMATION. 51

Naples and preserved in alcohol. In all cases I embedded
portions of the colony in paraffin wax and cut thin free-
hand sections with a razor; I then stained these sections in
the manner described below. ‘The spicules are identical in
the three species. Staining methods which do not differ-
entiate the nuclei very prominently do not give good results
for ascertaining the number of scleroblasts in connection
with the adult stellate spicules, chiefly because the mass of
the spicule more or less effectually hides all objects situated
underneath, and the conical protuberances largely obscure
objects situated to the side. The use of picro-carmine would
doubtless give better results, but I was unable to employ this
stain. The only effectual method which I employed was the
ordinary borax-carmine method, the differentiation with acid-
alcohol dissolving the spicules sufficiently to render the
scleroplasm apparent. This method merely revealed one
scleroblast (nucleus) in connection with each spicule, the
layer of scleroplasm closely investing the entire spi-
cule and following allits outlines, and the large nucleus
being situated in a mound of protoplasm at the periphery
(figs. 17, 18). The stellate spicule originates in a scleroblast
as a spherical granule (fig. 14; these early stages are quite
visible in the non-decalcified safranin and lichtgriin-stained
preparations) which later acquires spines on its surface as
it increases in size (fig. 16), and which spines ultimately
become the conical processes of the adult spicule.

Previous literature dealing with the scleroblastic develop-
ment of Ascidian spicules is very small in amount (see
Herdman [2] for the literature relating to Ascidian spicules
up to 1885, since which year, I believe, no literature on the
present subject has appeared). Loewig and Kolliker [6] in
1846 provided very poor figures of the stellate “ cellules
incrustées” in Didemnum, and illustrated one of these
“cellules” partly decalcified, showing the cell-wall spherical
in outline. Giard [1] in 1872 figured stellate spicules of
Euccelium and Didemnum also situated each within a circle
which is supposed to represent the cell-wall; he figured as

52 W. WOODLAND.

well several small spicules situated within one cell. No
nuclei are shown in any of these figures. In the first place I
may say that I do not remember ever having observed more
than one spicule contained within a single cell; and secondly,
that the above authors are greatly mistaken in supposing the
outline of the scleroblast containing the adult spicule to be
spherical. A scleroblast containing a spicule only remains
spherical when the contained spicule is small compared with
the original size of the cell (microscleres of some siliceous
sponges and the young stages of larger spicules e.g.) ; when
as in Leptoclinum or Didemnum, the spicule is many times
larger than the original scleroblast, this latter necessarily
becomes distended and envelops the entire spicule with a thin
layer of scleroplasm, which of course assumes the shape of the
spicule. If the spicules are too hastily decalcified it is quite
conceivable that the evolution of gas, being coincident with
the disappearance of the mass of the spicule, inflates the
scleroblast, and thus causes it to artificially assume a spherical
shape; indeed, I have some evidence that this is liable to
occur. Decalcification by means of the acid-alcohol used in
the borax-carmine method is very slow, and the scleroplasm
retains its stellate form, though the spicule has largely or
wholly disappeared.

LITERATURE.

1. Grarp, A.—‘‘ Recherches sur les Ascidies Composées ou Synascidies,”
‘Archiv Zool. expér.,’ t. 1, 1872.

2. Herpman, W. A.—‘ The Presence of Calcareous Spicula in the Tuni-
cata,” ‘P. Geol. Soc. Liverpool,’ 1884-85.

8. Hruscner, J.—‘Zur Anatomie und Histologie der Proneomenia
sluiteri, Hubrecht,” ‘Jena Zeitschr.,’ Bd. xxvii, 1892.

4, Husrecut, A. A. W.—“Proneomenia sluiteri; with Remarks upon
the Anatomy and Histology of the Amphineura,” ‘Archiv ftir Zoologie,”
Suppl., Bd. i, 1881.

5. Kowatevsky, A. O., and Marion, A. F'.—‘‘ Contributions a |’Histoire
des Solenogastres ou Aplacophores,” ‘Annales du Musée d’Histoire
Naturelle de Marseille,’ ‘‘ Zoologie,” t. ili, 1887.

STUDIES IN SPICULE FORMATION. 53

6. Lorwic and Ké1i1r1KER.—“ De Ja composition et de la Structure des En-
veloppes des Tuniciers,” ‘Ann. Sci. Nat.,’ 3rd sér., t. v, 1846.
7. Pruvor.—‘ Sur lorganization de quelques Néoméniens des cétes de
France,” ‘Arch. Zool. Exp.’ (2), ix, 1891.
8. TuIELE, J.— Beitrage zur Vergleichenden Anatomie der Amphineuren,”
‘Zeit. f. wiss. Zoologie,’ Bd. lviii, 1894.
9. Wiren.—“ Studien tb. die Solenogastres,” I and II. ‘Svenska vet. Akad.
Handl.,’ xxiv, xxv, 1892-93.
10. Woopianp, W. N. F.—“ Studies in Spicule Formation. I. Sycon
Sponges,” ‘ Quart. Journ. Micr. Sci.,’ vol. 49, 1905.
** Studies in Spicule Formation,” V. ‘Quart. Journ. Mier. Sci.,’
vol. 51, 1907.

ile

EXPLANATION OF PLATE 5,

Illustrating Mr. W. Woodland’s “Studies in Spicule
Formation” (VI).

Figs. land 2, x 640; Figs. 3—6, x 192; Figs. 7—18, x 1280. All drawings
were made with the camera lucida.

Fic. 1.—Young stages in the development of the monaxon spicules of
Goniodoris castanea. The thick median portion is the first to be formed.

Fie. 2.—Apparently an abnormal spicule of G. castanea possessing two
thickened club-like extremities and two nuclei, and so somewhat resembling a
young alcyonarian spicule.

Fires. 3 and 4.—Older spicules of G. castanea, each entirely enveloped in
cell-substance and with one nucleus.

Fic. 5.—Small monaxon spicules of Archidoris tuberculata. These
spicules possess no central thickening and have small spherical nuclei.

Fic. 6.—Small (young) spicule of Lamellidoris bilamellata; large
type of spicule, and usually possessing a median spine.

Fics. 7 and 8.—Young spicules of Proneomenia aglaopheniz.

Fias. 9 and 10.—Older spicules of the same.

Fies. 11 and 12.—Young spicules of Dondersia banyulensis.

Fic. 13.—Older spicule of the same.

Fics. 14—18.—Stages in development of the stellate spicules of Lepto-
clinum commune,

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FORMS OF SPICULES. ay

A Preliminary Consideration as to the possible
Factors concerned in the Production of
the various Forms of Spicules.

By

W. Woodland,
Demonstrator of Zoology, King’s College, London.

So many misconceptions prevailing as to the essential
nature of spicules, and in consequence as to the causes which
can possibly give rise to the multitudinous and remarkable
forms which spicules assume, a brief consideration of the
subject seems to be called for. Such at least will serve to
remove sundry erroneous ideas which are continually being
put forward in explanation of these phenomena and will also
point out the direction in which further investigation is
necessary for their better comprehension.

SpicuLes DEFINED.

Recalling to mind among the more obvious features of
spicules that these bodies (a) always possess a form other than
the spherical (otherwise being termed concretions), (b) always
possess curved surfaces and never plane facets (so being dis-
tinguished from crystals whether these be simple or aggre-
gate), and (c) that, although always intra-cellular in origin
and usually so during their whole subsequent development,
yet certain spicules (calcareous spicules of sponges, plutei and
aplacophore Mollusca, and some siliceous sponge spicules)
are extra-cellularly produced during the later part of their

56 W. WOODLAND.

erowth,! we may define a spicule as a hard, crystalline or
colloidal deposit, of more or less extended and
often definite and complex form, always possessing
curved surfaces and never plane facets, formed
initially within a cell or a cell-fusion, and whose
subsequent growth, which may be intra- or extra-
cellular, is due either solely to the activity of the
mother-cell or cells and its or their division-pro-
ducts if formed, or also partly to the activity of
cells not derived from the original mother-cell or
cells. Spicules which originate as a single deposit in the
interior either of a single cell or of two or more cells which
are more or less fused (and by a “ cell”? we mean a nucleus
associated with a mass of protoplasm whose limits may or
may not be distinguishable from other similar cells, thus con-
sidering a syncytium as a collection of cells) and whose sub-
sequent growth is solely associated with this cell or these
cells or its or their division-products are termed simple
spicules (spicules of Spongilla and apparently most other
Monaxonida, many tetraxonid spicules, radiolarian spicules,
aleyonarian spicules, monaxons of calcareous sponges, many
holothurian spicules, etc.) ; simple spicules which arise in
juxtaposition, and which subsequently become fused so as to
form a complex whole of a higher order of individuality (e. g.
triradiates of calcareous sponges, the rosettes of the Esperia
larva and possibly the plate-and-anchor spicules of Synaptide)
give rise to what may be termed aggregate spicules; finally,
spicules whose later growth is in part effected by adventitious

1 By intra-cellular growth we mean that the deposit is enclosed on all sides
by the substance of the cell, as is the case in most spicules. In Calearea, how-
ever, the apical actinoblasts, e. g. of the calcareous triradiates, are cylindrical
structures enclosing the rays at their apical extremity and a certain portion
of their length, and it is therefore clear that the deposits which lengthen the
rays are not intra-cellularly produced as above defined, since they are not
entirely surrounded by the cell; and from this cylindrical disposition of the
scleroblast to the simple adhesion of the cell to the surface of the spicule
there is obviously every transition.

FORMS OF SPICULES. Me

cells, i.e. cells not derived from the original mother cell or
cells concerned in their production (spicules of the pluteus
larva, lithistid desmas, etc.) are termed secondary spicules.

Arr THE Forms or SpicuLtes INHERITED ?

In considering the possible factors giving rise to the various
forms which spicules assume, it will facilitate matters, more
especially as showing in what direction the factors are to be
found, if it can possibly be decided first of all whether or no
the forms of spicules are inheritable. But previously to dis-
cussing this particular subject we must have clear ideas as to
what exactly we mean when we decide that any particular
skeletal or other structure is inherited. Heredity must after
all be regarded as a complex of physical causes identical in
nature with those which produce purely inorganic phenomena,
and, mentally, at any rate, we must carefully distinguish these
physical causes constituting heredity from the physical
(ontogenetic) causes giving rise to non-inheritable organic
structures, though this is admittedly usually very difficult to
do in fact. No one will deny that the form of a bone, tooth,
nail, valve of a diatom or other similar non-living part of an
organism is inherited, but in asserting this we evidently must
mean that the form of the protoplasmic mould which produces
the bone, tooth, nail, or valve is the part inheritable, since
the non-living matter composing these structures being unable
to reproduce itself obviously cannot inherit properties. Hvi-
dently then to assert that the form of a siliceous or calcareous
spicule is inherited implies that the disposition of the sclero-
blasts associated with any given type of spicule is that which
is inherited, and that the spicule itself, like the bone or
diatom-valve, is simply deposited in a mould already formed
for it by the scleroblasts. That is, the disposition of the
scleroplasm on this hypothesis determines the form of the
spicule and not vice versa.

1 “T must rather maintain that the form of the sponge spicules is deter-
mined by the organic matrix in and from which they originate, and that the

58 W. WOODLAND.

Now the only direct way in which to prove or disprove
this supposition that spicular forms are inherited is to rear
spicule-bearing organisms in water deprived of the material
necessary for the formation of the skeleton. If we do this
we shall then, if the supposition be correct,' find that the
scleroblasts in an echinoderm, e. g. will form moulds of the
plate or more complicated spicules, differing only from those
formed under normal conditions in that they do not contain
spicules. Is there any evidence to prove or disprove this?
The only experiments conducted on the lines just described
that I know of are those which were performed by Maas
(13, 14) in 1904 on calcareous sponges, and by Herbst (9) in
1892 on echinoplutei. Unfortunately, these experiments
were but of little value so far as they relate to the present
problem since the spicules of Calcarea and plutei are not
wholly enclosed by cell-substance (the hypothetical moulds) as
most other spicules are, but simply increase in length and thick-
ness in the same way as a hair or horny fibre does (Study VI),
and the dispositions of the cells? associated with the formation
of these spicules (which dispositions were produced in these
experiments) are not such as to be solely related to the forms
of the spicule but are brought about by other causes (Studies
ITand III). Up to the present then we possess no experi-
mental evidence that the form of the spicule is determined
by a scleroblastic mould,® and the direct proof or disproof of

formative forces are in no essential way different from those which are
everywhere exhibited in the shaping of the living organism and its parts.”
(Schulze [28]; similarly Maas [15)).

1 Aud if the altered chemical conditions of existence have no pathological
effect on the development of the organism—which substantially appears to be
the case according to the experiments of Maas and Herbst, referred to below.

* Herbst says nothing about a triradiate mould being formed in one of the
constituent cells of each lateral cluster; but then, of course, he did not look
for one.

3 ven in calcareous sponges, in which a kind of ‘‘mould”’ is stated to
normally occur (Minchin [17], Woodland [Study I]), this in all probability is
but a preliminary deposit of horny matter, which later forms the spicule-
sheath (Minchin). The instances of cell “moulds” described by Chun

FORMS OF SPICULES. 59

this supposition that the forms of spicules are inherited is
still a desideratum.

Although, therefore, we cannot as yet state decisively that
the forms of spicules are not inherited, yet there exist certain
facts which appear to justify us in provisionally making this
statement.! In the first place the results of recent inquiries
in experimental embryology prove that for any given cell
(blastomere) of a growing organism to be able to produce a
living part of the adult organism adapted in form to the other
parts (an organ), this must be connected with the
other cells. The part which a given blastomere plays in
the formation of the adult organism is a function of its con-
stant position relative to the other blastomeres, i. e. a function
of its localised connection with the other blastomeres.
Detach a blastomere from its fellows, and it either gives rise
to a complete though diminutive organism or becomes a
wandering-cell or a germ-cell; in no case does it give rise to
a part of the adult organism adapted to the general economy
in structure and relative position, i.e. an organ.” And so far

(Auricularia wheels) and Théel (spicules of Elasipoda) were, without doubt,
merely cases of decalcification, as I have elsewhere pointed out (Study IV).

1 In what follows the general tone of the argument would imply that I
altogether reject as unwarranted the hypothesis of the inheritance of spicular
forms. This is not the case. I have taken up, somewhat vigorously perhaps,
the opposite position to that held by most zoologists who have worked at
spicules solely in order to see what can be made of that position, and, rather
to my surprise, I find that there is a good deal to be made of it. For
example, in connection with my main argument, based upon the results of
experimental embryology, if it should turn out that the forms of spicules are
inheritable, it seems to me that, in view of these additional facts, the problem
of heredity will be rendered more complex than it already is. I do not reject
as unwarranted the inheritance hypothesis in the case of spicules, for the
obvious reason that at present we are so ignorant as to what heredity can and
cannot do. Nor, without definite proof to the contrary, is it justifiable
to summarily reject the convictions of those who have worked for years on
siliceous sponges e.g. I think it right, however, that the objections to. the
inheritance hypothesis here elaborated should be published (see Note at end).

* Occasionally, as in Ctenophora, detached blastomeres, in virtue of once
having been joined to the other blastomeres in a definite position, produce

60 W. WOODLAND.

as it is legitimate to assert such a thing it is impossible that
it should; an ovum is a mechanism which evolves into an
organism—“ crystallises out” so to speak—and any portion
which separates off necessarily remains uninfluenced by the
“crystallising out”?! process. The whole fabric of
Weismannism is based upon this assumption, and whether
the superstructure be sound or not no one doubts but that the
assumption is valid. Moreover, all histological researches of
recent years confirm the supposition that, apart from the
various classes of wandering-cells, the whole organism is a
syncytium. Now the theory of the inheritance of spicule
forms asserts that most scleroblasts (not all, since many
spicules are not in any way adapted to the architecture of
the organism) are unique in this respect, being able, though
having lost connection with the rest of the organism, to give
rise to a complicated structure (an organ—the spicule-mould)
adapted in shape and function by inheritance to
that part of the organism in which it happens to
be situated. ‘Take, as a concrete example, one of the com-
plicated siliceous hexactines of hexactinellid sponges so
marvellously adapted to fit in, so to speak, with the architec-
ture of the sponge-wall which it inhabits. The hexactine,
like other spicules, has undoubtedly originated as a simple
deposit within one or more scleroblasts (see Ijima* [11]), and
separately that part of the organism which they would have done if unde-
tached. But this capacity would be of no use in the case of a wandering
cell which, as the term implies, is constantly changing its position, since the
organ produced would not be in relation with the rest of the organism—in

Ctenophora it would be futile e.g. for a detached blastomere to produce say
an eighth of an embryo in one of the tentacles (see Wilson’s “The Cell,”
chap. ix).

1 This likening of the developmental process to crystallisation is a truer
analogy than might be supposed. In the formation of complex aggregate
crystals (the familiar fronds on the window-pane, e. g.) the co-ordination of the
multitudinous simple crystals to form one pattern is only possible by mutual
contact between the constituent crystals; the least separation leads to the
production of another centre of crystallisation.

2 Tam at present working on hexactinellid spicules, and can amply con-
firm the statements of Ijima and Schulze (‘Hexactinellida,’ “ Expedition auf

FORMS OF SPICULES. 61

these scleroblasts are wandering-cells. Now the theory of
the inheritance of spicule forms asserts at the very least that
this scleroblast or scleroblast-fusion inherits the property of
becoming enormously distended in size, of so distending as
to assume externally the form of aspicule adapted in its main
features to its immediate environment, and of correspondingly
forming an internal mould necessarily identical in shape in
every detail with the spicule to be deposited, which shape, as
just implied, varies according to the position in the organism
which the wandering-scleroblast happens to occupy at the
time of deposition! And these wandering-cells termed
scleroblasts are supposed to inherit the property of undergoing
all these changes (in the main definitely related to the archi-
tecture of the rest of the organism and complex in the
extreme) without having any connection with the other com-
ponent cells of the body—an assumption which, if my above
remarks are true, is quite unwarranted, to say the least. ‘This
hypothesis of the inheritance of the forms of spicules, in fact,
either contradicts the ascertained truths of experimental
embryology or implies that scleroblasts are not wandering-
cells.

It is true that in the development of Balanoglossus,
annelids and other animals, masses of cells are stated to be
often completely detached from the rest of the embryo, and
that these sometimes undergo changes of form related to the
surrounding tissues whilst so detached, but it will invariably
be found that either these changes of shape are simple
mechanical adaptations to the architecture of the organism
(formation of ccelomic sacs, etc.), or are due to the fact that
these cells have quite recently been connected with the rest
of the organism, the changes of shape being adapted to the
general scheme of development because the mass of cells has

dem Dampfer ‘ Valdivia,’” Bd. iv, 1904) that these spicules are completely
invested by scleroplasm.

1 A scleroblast can be supposed, on the hypothesis, to inherit the property
of giving rise to spicules of different shapes, according to its localisation in
the organism, on the principle of the ‘‘ equipotentiality ’’ of blastomeres,

62 W. WOODLAND.

retained its relative position in the organism (for-
mation of evaginations of the separate enteron in Balano-
elossus to meet the stomodzal and proctodzal invaginations
of the epiblast, etc.). Including these apparently anomalous
cases, it is one of the most certain facts in Biology that if a
detached cell (or cells) is to produce an organ of the adult
organism, it must become connected with its fellows. And
as I have pointed out above, the hypothesis of the inherit-
ance of spicule forms denies this.

The above argument, however, strictly speaking, solely
applies to spicules adapted in form to the architecture of the
organism (most of the large complicated spicules), but many
spicules exist which exhibit no such relationship (micro-
scleres of siliceous sponges, stellate spicules of Ascidians,
many holothurian and alcyonarian spicules, etc.) But even
in this case the scleroblasts will be unique amongst
wandering-cells if they produce by heredity an internal
spicule-mould (often also, as just stated, undergoing disten-
sion and external change of form), 1.e.if they assume a
complicated structure.” Apart from this very hypothetical
case of the scleroblasts I know of no instance of wandering-
cells becoming complicated in form at all. Spermatozoa are
cells separated off from the rest of the organism, and which
become very complex in structure, but this complexity (defi-
nitely related to a function) arises in the cell before it is
detached, and is analogous to the production of the ciliated
cells of a Vorticella or of medusz from a hydrozoan colony ;

1 Prof. Dendy has kindly brought to my notice the following striking
illustration of my argument. Lefevre (“‘Jour. Morph.,” 1898), in a paper
on ‘ Budding in Perophora,” states that wandering cells (free blood-cells)
produce the heart, neural ganglion, gonads, and some other organs of the
buds, but in order to do this they must become closely attached to
the organism. As I have argued in the text, cells which remain free
either become wandering cells or, as shown, e.g. by the rediz and cercarise of
the common Distomum, produce miniature organisms.

? The mere idea of a wandering cell possessing the capacity to form an
internal mould of the complexity required for the production of a hexacti-
nellid floricome or onychaster, e.g, seems absurd,

FORMS OF SPICULES. 63

in other words, the complexity is a part of the continuous
development of the organism.

Only in the case of such organisms as Radiolaria, in which
the scleroplasm, i.e. the portion of the body-substance in
which the deposition of spicule-substance occurs, is con-
tinuous with the rest of the organism, do the above argu-
ments fail to apply. Granting this we may still ask what is
the evidence that in Radiolaria the forms of the spicules are
inherited? And the answer from most quarters will be: the
same class of evidence which leads us to state that the forms
and patterns of the siliceous valves of diatoms, e.g. are
inherited. The siliceous valves of diatoms and the siliceous
spicules of Radiolaria are both cytoplasmic secretions, and
surely what applies to one applies to the other. In replying
to this criticism, a distinction must first be drawn between
the general region of deposition of the silica and the par-
ticular distribution of the silica within that region. The
general shapes of the diatom valve and the radiolarian shell
(or region containing the spicules in those Radiolaria and
higher organisms which possess no shell) indicate the former
and are undoubtedly inherited—are determined, like the
form of bone, by the disposition of the scleroplasm, i.e. by a
mould. But, on the other hand, it is altogether another
question as to whether the forms of the individual spicules or
the patterns on the radiolarian shell or diatom valve are
inherited.1. Anyone who questions the inheritance of these
patterns is at once met with the answer that particular pat-
terns are constantly reproduced by particular organisms, and
that therefore they must be inherited (!)—an answer alto-
gether beside the mark. Given a colloid of the same compo-
sition under similar conditions, and if it produces a particular
pattern on one occasion for purely physical reasons (onto-
genetic causes) it will on another, just as calcite crystals are
always produced whenever the required conditions are con-
formed to. Previous to the experiments of Herbst, the arms

1 The patterns on dinoflagellate and desmid plates, coats of pollen-grains
and seeds, and scales of ganoid fishes may also perhaps be included here,

64 W. WOODLAND.

of plutei were supposed to be inherited for just this same
reason, viz. because echinoid larve always had them ; never-
theless, as Herbst has shown, they are not inherited, since
they merely result from the elongation of the spicular rods.
It is indeed more than possible that the patterns on diatom
valves, radiolarian shells, and the rest are purely physical
products—ontogenetically determined—and that physicists
will some day be able to reproduce these patterns under
artificial conditions as Rainey did in the case of certain
organic calcareous structures. The perforations in diatom
valves and radiolarian shells it is true undoubtedly serve a
physiological necessity (since were there no perforations, the
imprisoned organisms would die) and the organism is thus
evidently able to control to some extent the deposition of
silica, but physiological necessities ipso facto never yet pro-
duced ornamentations of no use or only incidentally of use to
the organism, and until the contrary is proved we are fully
justified in assuming for the reasons given and about to be
given that the forms of radiolarian and all other spicules are
not inherited.!

But there is a still more satisfactory answer with which to
reply to the criticism that radiolarian (and other) spicules,
being, like cuticular products, cytoplasmic secretions, there-
fore owe their forms to the same cause, and this is that, as
all observers have agreed, spicules as a class do differ in
several very important respects from every other class of
cell-deposits. One of these differences is that, excepting
crystals, spicules are the only deposits assuming definite

1 Or only inherited in the sense that their form is determined by the
colloidal nature (see below) and architecture of the organism, which pro-
perties of course are inherited. Assuming that the forms of spicules are
“inherited” in this sense is very different to assuming either (4) that every
individual scleroblast is guided by some unimaginable means to precisely that
position in the organism in which the spicule, which it is alone capable of
producing (by a power which no other class of cells is known to be capable of,
i.e. by forming an intracellular mould), is adapted to the economy of the
organism; or (B) that every individual scleroblast is capable (in addition to
possessing the unique power just mentioned) of producing by heredity

FORMS OF SPICULES. 65

(other than spherical or approximately spherical) forms
which arise in the interior of cells: all other definitely-
shaped deposits arise on the exterior of cells, and indi-
vidually owe their (inherited) form as a whole (though not
necessarily their patterns and the like) to that of the cyto-
plasmic surface (mould) which produces them. As before
stated, there is no evidence that the forms of spicules are
determined by internal moulds; on the contrary, evidence
exists which leads us to suppose that such moulds cannot
exist.

Another and indeed the principal feature distinguishing
spicules from other cell-deposits is their general nature
which, as all authorities agree, is closely allied to that of
crystals and similar bodies. The geometrical symmetry of
the forms of many spicules, and their physical nature, are
both characteristics pointing to the close affinity which exists
between spicules and crystals, or bodies allied to crystals,
such as those which I have below termed “‘ crystallomorphs.”
It has already been shown how the forms of spicules differ
from those of crystals, but it has not yet been shown how
very similar, both as regards condition of formation, physical
properties, and variety, symmetry and complexity of form,
spicules are to crystallomorphs. The one feature in which
spicules differ from crystal-like bodies is their, in many cases,
obvious adaptation of form to the architecture of the or-
ganism—the feature which of course has led to the supposi-
tion that their forms are inherited—but as I shall show later,
this feature can be otherwise accounted for. The assertion
that, despite the facts, spicules have no affinity with crystal-
line bodies, but are more closely allied to utter dissimilar
various forms of spicules, according to its position in the organism, whilst
quite uninfluenced by the rest of the organism (since it is uncon-
nected with the rest of the organism). To say that the scleroblast is influenced
by “heredity” at a distance is merely to assert what I assert, viz. that direct
ontogenetic causes determine the form of the spicule and not heredity. A
scleroblast in all probability no more produces a spicule-mould, in the sense
that a nephroblast produces a nephridium, than a cell of adipose tissue swells
out by heredity to produce a spherical oil-drop.

vou. 51, part 1.—NeEW SERIES. )

66 W. WOODLAND.

though often also symmetrical structures like bones, feathers,
and the like is the assertion necessarily implied in the suppo-
sition that the forms of spicules are inherited.

Another very important difference distinguishing spicules
from other cell-deposits relates to their disposition in the
organism. Apart from the fact already sufficiently insisted
upon that all hard deposits save spicules (not those of
Radiolaria) arise in cells connected with the rest of the
organism, there is the additional significant fact that all
(except concretions and crystals) these non-spicular deposits
(bones, teeth, nails, hairs, scales, feathers, etc.) are, on
account of the connection of the secreting cells with the rest
of the organism, usually laid down (obviously by inheritance)
only in those particular parts of the organism where they
are required, the appendicular skeleton, e. g. being formed
in the axes of limbs where the greatest stresses exist, dermal
bones protect particular viscera in particular places, nails
occur on the terminal phalanges where most contact occurs,
and so on. Spicules, on the other hand, are not limited in
their distribution in this manner, but tend to occur wherever
the purely physical conditions permit the wandering cells to
secrete them (calcareous spicules, e.g. cannot occur in the
vicinity of digestive or other organs where acid solutions
abound), and their local adaptations in form to the archi-
tecture of the organism are, there is good reason to believe,
determined by purely physical causes which influence the
scleroblasts during the development of the spicule.

In view, then, of the above arguments, which, collectively,
are, in my opinion, of considerable weight, and, in the
absence of direct proof to the contrary, we are, I think, for
the present justified in declining to entertain the hypothesis
of the inheritance of spicule forms. ‘he hypothesis of the
inheritance of spicule forms, it is true, does not necessarily
imply that such forms have arisen by the process known as
natural selection ; natural selection, as even the most ardent
selectionists admit, is obviously incompetent to account for
the complex symmetry of spicule forms. The hypothesis

FORMS OF SPICULES. 67

simply implies that the variations of spicule forms are non-
controlled, and that they persist without reference to the
economy of the organism. My objections to this hypothesis
are, in short, that the implied capacity of the protoplasm to
form the requisite spicule moulds is shown, in all probability,
to be non-existent by the facts both of experimental embryo-
logy and cell physiology, and that the resources of physical
science can provide examples of structures much more closely
allied to spicules than any class of bodies known to be
organically produced, i.e. by inheritance.

FACTORS POSSIBLY CONCERNED IN THE PRODUCTION OF
SpicuLe Forms.

Assuming the above conclusion to be valid, we are thus at
liberty to consider purely physical factors as being fully
competent to account for the varied forms which spicules
assume. Before enumerating possible factors it will be as
well to state definitely that we do not consider crystallisation,
in the strict sense of the term,! to be one of these. ‘To put
the argument in brief, we may reiterate that no crystal
possesses curved surfaces, and, since no spicule exists without
them, therefore no spicule is a crystal. But, apart from this,
we may point out that all spicules composed of crystalline
matter consist of calcite, and that the various crystalline
forms of calcite being strictly limited in number (none of
which bear the slightest resemblance to any form of cal-
careous spicule) and all modifications of one type, it is
evidently impossible to refer the multitudinous and widely-
different forms of calcareous spicules to any such factor as
the crystalline properties of their substance. In fact, the

1 It is important, in view of the loose application of the term, that I should
explain that by a crystal I mean solely a mass of matter which has assumed,
on soldification from a dissolved or fused condition, a form bounded by plane
surfaces referable to one of the six systems recognised by crystallographers.
Crystals, as thus defined, may be simple (most compact crystals) or aggregate
(snow-crystals, e. g.).

68 W. WOODLAND.

crystalline nature of calcite, as a factor in determining form,
seems to exhaust itself, in the case of calcareous spicules, in
producing the individual crystals, of which these spicules are
composed ; certainly there is no reason to suppose that it has
any influence on the form of the aggregate (see Maas [15]).
With regard to siliceous spicules, hydrated silica (opal), so
far as I know, has never been observed to assume a crys-
talline form, although some of the siliceous concretions
figured by Maas (16) are angular in outline.

Several authors have contended for the recognition of
certain calcareous spicules as crystals on the ground that,
like true calcite crystals, they behave optically (with polarised
light) as “crystal individuals” (Bidder [1] and others). I
have not here the space to discuss such a large subject, but I
may point out that the value of this supposed criterion of a
crystal is easily shown, in the case of spicules at least, to be
naught by the fact that, according to this criterion, the shape
of a simple spicule like one of those of Alcyonium is not due
to crystallisation (though undoubtedly a spicule individual),
whereas the shape of a compound quadriradiate spicule of
Calcarea is (though this spicule is composed of four spicule-
individuals secondarily joined together to form a system).

The above arguments, it must be admitted, are quite valid,
but they are only entirely valid provided that the term
crystal retains the definition I have above supplied. Many
facts, however, point to the conclusion that the form of a
crystal is as much a function of the medium in which the
crystal is deposited as of the properties of the crystal
substance itself, and since, as is well known, a given
crystalline substance will, in the presence of different media,
give rise to the most varied forms (these forming, however, a
continuous series on account of numerous transitional forms
—facets giving place to curved surfaces among other
changes), it becomes questionable as to whether we are
justified in restricting the term crystal to the old meaning.
Now the media most potent in these effects on crystalline and

FORMS OF SPICULES. 69

amorphous deposits are undoubtedly colloidal media, and,
since complex shapes produced by colloidal media are almost
always characterised by the possession of curved surfaces,
and thus, though connected by all transitional forms with
those of simple crystals, constitute a class of bodies possess-
ing common characters, I propose that, to distinguish them
from crystals as above defined, they shall be termed “ crys-
tallomorphs.”” Whether crystallomorphs are always modified
ageregate crystals (as in many cases they are) or sometimes
equivalent, as regards the order of crystal individuality, to a
simple crystal is not at all clear from published accounts on
the subject. If, then, we distinguish crystallomorphs from
crystals as just suggested, we are still justified in stating that
spicules are not crystals ; whether spicules are crystallomorphs
is a question I must discuss later.

Three factors in the production of spicule forms are con-
ceivable: (a) The gross mechanical factor, or the shaping of
a structure due either to actual contact with surrounding
objects (contact which, in this case, would affect the shape of
the spicule by influencing the scleroblasts depositing it and
not the spicule itself, which is a rigid structure) or to the
configuration of the secreting substance; (b) the influence at
a distance—actio in distans—of different parts of the organ-
ism on the scleroplasm; and (c) the factor which produces
crystallomorphs. These three factors I will discuss as briefly
as possible.

Factor (a) has been employed by several authors in the
interpretation of spicule forms with, however, but varying
success. ‘The well-known line-of-least resistance theory of

1 So far as I know, the influence of other crystalline substances in solution
on developing crystals is solely to give rise to very complex aggregate crystals
—not possessing curved surfaces (see Lehmann [12]). It is very probable,
however, that the presence of such crystalloid solutions greatly facilitates the
colloids in the production of complex crystallomorphs, probably being largely
instrumental in giving rise to variety of form.

2 I originally proposed (British Association, York, 1906) the term “col-
loidomorph,” but this is evidently defective. ‘‘Crystallomorph” is somewhat
awkward, but it seems to me preferable to Rainey’s ‘‘ coalescence body.”

70 W. WOODLAND.

Sollas and the alveolar theory of Dreyer are notable examples.
With reference to the latter, the inability of the theory to
account for the well-ascertained fact that all spicules arise
from approximately spherical concretions, and that com-
paratively very few ever assume the tetraxon form, releases
us from the necessity of considering it.1 The theory of
Sollas, on the other hand, logical enough in its premises, and
possibly providing an explanation of some forms of spicules,
most lamentably fails when confronted, e.g. with the com-
plicated siliceous spicules of many sponges and the calcareous
spicules of many holothurians. Sollas’s theory, however, if
properly understood and applied, accounts for a good many
of the facts. Consider, e.g. the significant fact that the
forms of spicules in general are constantly adapted as regards
their space dimensions to their position in the organism con-
taining them; flat spicules, e.g. are generally found in
situations limited by two more or less definite parallel
surfaces (triradiates of Calcarea, plate-spicules of holothurians,
etc.), and spicules of three dimensions are always found
either in situations far removed from limiting surfaces (most
alcyonarian spicules, asters of siliceous sponges, and some
colonial ascidians, etc.), or with their parts disposed in
relation to these (quadriradiates of Calcarea, the hexactine
macrosclere and its modifications in hexactinellids, etc.). More-
over, definiteness of form of the spicule generally, if not
always, exhibits some correspondence with the definiteness of
its immediate enviroment. Most symmetrical spicules occur
in regions of the organism which are symmetrically disposed
with regard to the spicule, and irregularly-formed spicules,
on the other hand, are generally found in situations charac-
terised by the absence of definite architecture. Such corre-
spondences, to be found both in spicules contained in different
organisms and in different parts of the same organism, must
be due to the effects of either factor (a) or factor (b) on the

1 Dreyer’s similar explanations referring the forms of radiolarian shells to

an alveolar conformation of the scleroplasm have no facts whatever to support
them. There is no evidence of this particular alveolar conformation,

FORMS OF SPICULES. 71

spicules, or, rather, on the scleroblasts which deposit them,
and this seems still more probable when we remember that
scleroblasts are constituted of a soft and highly complex
sensitive substance which must be readily influenced by
mechanical and other forces, which are in all cases transmitted
through the gelatinous matrix surrounding the spicules.
Further, in considering mechanical factors as applied to
biological phenomena, ‘‘mechanists”’ are too apt to forget
that the substance of organisms is after all living, in other
words, possesses among other features the capacity of “ spon-
taneously ” altering its configuration within certain limits.
For example it is quite possible that the spinous processes
and elongated and branching forms of many spicules are
attributable to the pseudopodial activity of the scleroplasm
resulting from physiological requirements, and it is certain,
as I have elsewhere stated, that the perforate character of
most echinoderm plate-spicules and of radiolarian shells is
due to the necessity for communication across the area occu-
pied by the spicule or skeleton, and is probably determined
ontogenetically in each case, though exactly how it is diffi-
cult to say. But at present I have not the space to do more
than suggest this form of activity of the protoplasm as a
possibly important factor in the production of spicule forms.
I may finally remark that even the crystal-like symmetry
of some (certainly not of most) spicules (in Calcarea and
aplacophore Mollusca) can be referred to purely mechanical
conditions,! as I have pointed outin Studies land VI. How-
ever, although I believe that many individual features of
spicules can be attributed to purely mechanical causes, yet it
is quite evident that factor (a) in all its aspects is but a sub-
1 The fact stated by Sollas [25] with reference to the spicules of calcareous
sponges (aggregates of calcite crystals), viz. that ‘‘the position of the rhom-
bohedra relative to the surface of the spicules is very similar to that which
may be observed in rhombohedra of calcite filling up a cavity within a lime-
stone rock, or inside the chamber of an ammonite,” is suggestive. Sollas
adds that “we must suppose that the deposition of calcite within the spicule-

sheath occurs according to just the same laws which are followed in the
purely mineral world.”

72 W. WOODLAND.

sidiary one when the more complicated forms of spicules are
concerned.

Concerning factor (b) this has been so little considered
that, though in all probability it is a very important one from
our present standpoint it is impossible to discuss it at any
length. It is probable that factor (b) (in conjunction with
factor [a]) has a lot to do with producing that adaptation of
the form of the spicule to the architecture of the organism
(occasionally it is the reverse) which is often so conspicuous.
Crystallomorphs plainly exhibit this feature—the presence of
an adjacent though separate object to one side, e.g. clearly
modifying the shape of the crystallomorph on that side.
Moreover, the organism seems to exert a decided influence
on the disposition of the spicules in such cases as in certain
Radiolaria, e.g. and therefore similar influences may be at
work in more complicated organisms. However, we possess
no data as yet in connection with this subject, and beyond
making the preceding suggestions it is impossible to say any-
thing about it.

Concerning factor (c) there exist, as already stated, a suffi-
cient number of facts which seem to me to indicate that we
must rely in the main on this factor (in conjunction with fac-
tors (a) and (b) for our future comprehension of spicule forms.
It is significant that, of colloidal media, albumen was found
by Ord and others to be the most effective in the production
of what he terms “ coalescence bodies,” and what I venture
to term crystallomorphs. ‘To gain an adequate idea as to the
nature of these bodies it is necessary to refer to the works of
Rainey (20, 21), Harting (8), Ord (18, 19), Vogelsang (26),
Slack (24), Lehmann (12), Bowman (2), and others on
the subject, but for present purposes a few statements
descriptive of the nature of some of the simpler crystallo-
morphs will suffice! Thus Ord, employing albumen and
other colloidal media, states that ‘‘triple phosphate being

It is perhaps a fact of some significance that the most definite and

complex forms of calcareous spicules are those containing least organic
(horny) substance in their composition.

FORMS OF SPICULES. 73

used, the stalactitic crystals were found turned into rounded
rods, bulging at many points into beads and variously bent,
twisted, and interwoven, so as to bear some resemblance to
the form in which mineral matter is deposited in the skeletons
of some of the Echinodermata. Other phosphates (phosphate
of calcium) were found in irregular, elongated, curved, and
branching masses.” Calcium carbonate is deposited in albu-
men in the form of small spheres covered with curved and
pointed spines ; calcium oxalate in gelatin forms mulberry
masses, spheres, dumb-bells, feathered octahedra with curved
pinne, etc., and uric acid is still more protean. The curious
ball- and cone-shaped deposits which corrosive sublimate
forms in balsam are known to everbody. And, as Bowman,
Vogelsang, and others have shown, some of the forms assumed
in colloidal media by common salt, santonin, salol, pyrogallol,
antipyrin and other substances are marvellous in their com-
plexity and beauty. And it must not be forgotten that all
these forms are determined by colloidal media alone. As
stated above, crystalloid media have an immense influence
in producing the most varied complex aggregate crystals,
and if, in addition to these, there are present, as is the
case in living organisms, colloidal media of highly complex
and variable constitution, the possibility of the develop-
ment of elaborate crystallomorph forms is obvious. It is
true that these crystallomorphs are all composed of so-
called crystalloid matter, whereas the greater number of
spicules are composed of colloid matter (mainly opal), and,
as I have before stated, I am not aware of any experimental
or other evidence that colloids are capable of assuming
crystallomorphic forms. However, it is well known that
typical colloids lke egg- and serum-albumen and certain
globulin proteids are easily capable of ordinary crystallisation
and in consequence we have some reason to suppose that
colloid crystallomorphs are at least possible. Applying our
present scanty knowledge of crystallomorphs to the subject
of spicules, the researches of Gautier (7) and others have
made known to us that the protoplasms, so to speak, even of

74. W. WOODLAND.

different species of animals in all cases appreciably differ in
chemical constitution from each other, and even supposing
that the substance of all calcareous spicules, e. g. is constant
in composition, the different colloidal natures of the various
organisms in which spicules are found are in all probability
amply sufficient, in conjunction with factors (a) and (}), and
the different crystalloid media present to account for the
various spicule forms encountered.?

As showing in some degree the complexity of this subject
of the causes determining the forms of spicules I may instance
the extraordinary nature of even the common process of
simple crystallisation, as ascertained by Frankenheim,
Vogelsang, and several other observers. Vogelsang observed
that the first visible stage in the formation of certain crystals
(sulphur crystallising from carbon bisulphide solution, e. g.)
was the appearance of liquid globules, which subsequently
aggregate to form small solid isotropic spheres which he
termed globulites, these again arranging themselves in de-
finite patterns successively coalesce to form still higher aggre-
gates (crystallites, margarites, etc.) until finally the
crystalloids (the “ integrant molecules” or crystal particles)
are produced.’ It would be interesting to know whether the

1 Also the well-known fact that different tissues and regions of the
substance of the same organism differ widely in chemical constitution must
be remembered, this possibly largely accounting for the different forms of
spicules found in different parts of the same animal (Chiridota and other
Holothurians, Hexactinellids, ete.) ; moreover, in some cases, as Maas (16)
has shown in Tethya, e.g. the scleroblasts differ among themselves as regards
total size, size of nucleus, character of cytoplasm, etc., and this consequently
introduces another cause for the diversity of form of spicules contained in the
same organism.

2 Arguments for the inheritance of spicule forms, based upon the numerous
transitional forms connecting together spicules of different types, are of little
value, since all crystallomorphs exhibit exactly the same phenomenon on a
very large scale, and, as is well known, all transitions exist even between true
crystals of very different shapes. Moreover, in the case of spicules, there
exist more causes to produce these transitional forms than in the case of
crystals or crystallomorphs.

% Dr. Rosenheim [22] has brought to my notice certain incidental

FORMS OF SPICULES. 7

alveolar structure of calcareous and siliceous spicules described
by Biitschli (3) has anything to do with the globulites of
crystal formation.

Whatever may be the value of the above suggestions, one
fact is certain, viz. that prolonged investigation (largely ex-
perimental) on the entire subject is absolutely essential before
we can hope to interpret in at all an exact manner the shapes
of spicules.

Note.

One important and, indeed, vital source of evidence
respecting the inheritance or otherwise of spicule forms,
which I have left almost unconsidered in the text, lies in the
answer we must give to the question as to whether modifica-
tions of spicule forms, evidently due to ontogenetic causes,
ever appear in the development of the spicule apart from
these causes. Obviously, if this can be shown to be a fact in
a single instance, then the inheritance of this particular
spicule form is proved, and that of others rendered probable,
though how the arguments I have advanced above in opposi-
tion to this are to be met I fail to see. Only one such
apparent example of the inheritance of spicule forms, which
might be cited in evidence against my position occurs to me
just before returning the corrected proofs of this essay, and
this example is taken from a paper I have myself published.
In the first of my Studies in Spicule Formation (Sycon
sponges) I have suggested that the elongation of the ‘ pos-
terior ” ray in certain Sycon triradiates is directly due to its
vertical basal position, the stresses incident upon the sponge
being largely borne by the longitudinal element of the
skeleton, especially at the sponge base, and the actinoblasts
of the basal ray being thus stimulated to secrete more actively.
Now it so happens that in Sycon ciliata and some other
sponges the superior size of one of the rays is apparent in the

observations of his own on the formation of choline crystals, in which
similar phenomena are described,

76 W. WOODLAND.

development of the triradiate even before the three minute
needles secreted by the sextet have united to form an aggre-
gate or compound spicule (Study I, fig. 32), and therefore
long before any stresses in the sponge-wall could possibly
have produced any such modification. Therefore, if this
larger ray of the young triradiate is the large “ posterior ”
ray of the adult spicule, it seems to me that the inheritance
of this particular form of spicule is proved. But the fact is
that this larger ray of the young triradiate does not, by any
means, always become the “posterior” ray of the adult
spicule, though it often does (probably for the reasons
assigned in Study I, pp. 263—265). Observations of the
Sycon oscular rim show that the young triradiates are dis-
posed very irregularly, and only exceptionally does the large
ray point exactly towards the base of the sponge. As I have
stated in Study I “the large ray is often found pointing as
much as 80° from the downward vertical line,” and more
recent observations prove that the large ray may not
uncommonly lie in exactly the opposite direction to that in
which the majority of the basal rays of the adult spicules lie ;
in short, the large ray of the young triradiates may point in
any direction, whereas that of the adult triradiates generally
points basally. These facts seem to me to show that the
large ray of the young Sycon triradiate is not the inherited
large “ posterior” ray of the adult spicule, since, were it so,
the cause which is capable of thus reproducing in the young
spicule a structural characteristic only directly producible
in the adult spicule should be equally capable of determining
the appropriate disposition of that structural characteristic.
As an alternative to the inheritance hypothesis I can only
suggest that the above feature of the Sycon triradiate is of
the same order of form-modifications as the clubbed extremi-
ties of the triradiates of Clathrina clathrus, the gastral
ray spikelets of C. cerebrum, etc.—modifications produced
ontogenetically without reference to the economy of the
organism, and possibly mechanical or crystallomorphiec in
nature. Assuming this to be the explanation of the large

FORMS OF SPICULES. “ih

ray of young Sycon triradiates it is noteworthy that this
feature has been made use of to some extent by the organism,
since if the triradiates are rotated sufficiently (Study I, pp.
263—265) the large basally-directed rays afford extra support
aud enable the sponge to assume a more erect posture. ‘Thus
the shapes of spicules are not only in part determined by the
form of the organism, but the contrary is also the case.

This large ray of the young Sycon triradiate is the only
instance apparently affording support to the theory of the
inheritance of spicule forms which I can at present call to
mind. But even if later evidence definitely proves the inherit-
ance of spicule forms to be a fact I shall not regret having
published a discussion which at the least illustrates the com-
plexity of the whole subject and suggests inquiry in more
than one direction.

LITERATURE.
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‘Proc. Roy. Soc. Lond.,’ vol. Ixiv, 1898.

2. Bowman, J. H.—“‘ A Study in Crystallisation,” ‘Journ, Soc. Chem.
Industry,’ vol. xxv (4), 1906.

8. Bitscui1, O.—‘ Mechanismus und Vitalismus,’ Leipzig, 1901.
4,

** Kinige Beobachtungen tiber Kiesel- und Kalknadeln von Spon-
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5. Darser, A.—‘ Microskopie der Harnsedimente,’ Wiesbaden, 1896.

5a. Dreyer, F.—“ Die Prinzipien der Geritistbildung bei Rhizopoden,
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6. Esner, V. von.— Ueber den feineren Bau der Skelettheile der Kalk-
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7. Gautier, A—‘ Du Mécanisme de la Variation des Etres vivants, etc.,”
in ‘Hommage a M. Chevreul a |’Occasion de son Centenaire,’ 1886.

7a. Hicker, V.—“ Ueber die biologische Bedeutung der feineren Struk-
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8. Hartinc.—‘*On the Artificial Production of some of the Principal

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24,

W. WOODLAND.

. Hersst, C.— Experimentelle Untersuchungen iiber den Einfluss der

Veranderten chemischen Zusommensetzung des Umgebenden mediums
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. Hesset.—‘ Einfluss des Organischen Kérpers auf den Unorganischen,’

Marburg, 1826.

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. Lenmany, O.—‘ Molekularphysik,’ 2 vols., Leipzig, 1888 and 1889.
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“Ueber der Wirkung der Kalkentziehung auf die Entwicklung
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“ Ueber die sogen Biokrystalle und die Skeletbildungen niederer
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- “Ueber Entstehung und Wachstum der Kieselgebilde bei Spon-
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‘The Influence of Colloids upon Crystalline Form and Cohesion,’
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Rainry, G.—‘On the Mode of Formation of Shells of Animals, of Bone,
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Some Further Experiments and Observations on the Mode of
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RosenuEerm, O.—“ New Tests for Choline in Physiological Fluids,”
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FORMS OF SPICULES. 79

25. Souuas, W. J.—“‘ On the Physical Characters of Calcareous and Siliceous
Sponge Spicules and other Structures,” ‘ Proc. Roy. Dub. Soc.,’ N.S.,
vol. iv, 1885.

26. Vocetsane, H.—‘ Die Krystalliten,’ Bonn, 1875.

27. Wooptanp, W.— Studies in Spicule Formation,’ I— VI, ‘ Quart. Journ.
Mier. Sci.,’ vols. 49—51, 1905—1907.

ioe)
—

NEUROSPORIDIUM.

On Neurosporidium cephalodisc¢i, n.g., n.sp.,
a Sporozoon from the Nervous System of
Cephalodiscus nigrescens.

By
W. G. Ridewood, D.Sc.,
Lecturer in Biology at St. Mary’s Medical School, University of London ;
AND

H. B. Fantham, B.S8c., A.R.C.S.,
University Coilege, London; Demonstrator in Biology at St. Mary’s
Medical School.

With Plates 6 and 7.

INTRODUCTION.

The organism described in this paper occurs in the
nervous layer of the ectoderm of Cephalodisecus (Idio-
thecia) nigrescens, Lank., a large and massive form of
Cephalodiscus dredged by the “ Discovery ” on January
13th, 1902, in 100 fathoms, off Coulmann Island, near
Victoria Land, in the Antarctic Ocean.

The specific name nigrescens was given to this Cephalo-
discus by Prof. Ray Lankester,! the polypides being to the
naked eye of a sooty black colour; a detailed description of
the polypides and the tubarium or “ house” of Cephalo-
discus nigrescens is given in the “ Reports of the ‘ Dis-
covery’ Expedition,” published by the British Museum.

The Sporozoon that occurs in Cephalodiscus nigres-

1 * Proc. Roy. Soc.,’ 1905, pp. 400—402.
voL. 51, PART 1.—NEW SERIES. 6

82 W. G. RIDEWOOD AND H. B. FANTHAM.

cens is found in relation with the nervous, deeper layer of
the ectoderm. It does not always occur actually within that
layer, since it may bulge outwards into the more superficial
cells of the ectoderm, or may project inwards into the under-
lying tissues, but the new organisms seem always to be
situated in the nerve tissue before they begin to enlarge and
sporulate. On account of this peculiarity we apply to the
organism the generic name Neurosporidium. Cephalo-
discus being the host in which the parasite has been
encountered, we base the specific name upon that. The
organism is thus denominated Neurosporidium ceplhalo-
disci.

No unicellular parasites of Cephalodiscus have up to
the present been described. In Cephalodiscus gilchristi
there is a Copepod Crustacean occurring parasitically in the
stomach of a large proportion of the polypides!; this parasite
is closely allied to the Copepods that occur in the gut of
Tunicates, and belong to the family Ascidicolide of
Giesbrecht.

Masterman? has drawn attention to “ cyst-like structures ”
occurring near the anus of Cephalodiscus dodecalophus,
which he is disposed to regard as larvee of Cephalodiscus,
but which from their position are more likely to be parasites.
They are of comparatively large size, and judging by the
large size of the cells as drawn in the figure, they are more
probably the young of some metazoon parasite than cysts of
Sporozoa.

M’Intosh* marks by the symbol bp. a series of three spheri-
cal bodies occurring in the deeper part of the nervous layer
of the ectoderm, which might at first glance be taken for
Sporozoa. Their position is one commonly occupied by the
parasite which forms the subject of the present paper, and
their size and general shape carry the resemblance with

1 Ridewood, W. G., “Cephalodiscus gilchristi,” ‘Marine Invest.
South Africa,’ iv, 1906, p. 181.

2 «Trans. Roy. Soc. Edinb.,’ 1898, p. 513, last paragraph ; also pl. 5, fig. 86.

3 « «Challenger ” Reports,’ part 62, vol. xx, 1887, pl. 6, fig. 3.

NEUROSPORIDIUM. 83

Neurosporidiuin still further; but it is to be noticed that
the central, uninucleate cells that one would take to be the
spores, are enclosed within a wall composed of a single layer
of regular cells, and not by a structureless cyst or capsule.
In the explanation of the figure, the parts marked bp. are said
to be “sections apparently of the shield pores in their pro-
eress outwards,” but itis highly improbable that the proboscis
canals could, in a section taken at right angles to the buccal
shield, be so cut as to present a circular outline, and even
then the presence of the third body is difficult to explain, for
the proboscis canals are short, and with firm walls of closely-
set epithelial cells, and could not, even in the most contorted
polypides, be cut across twice in the same section.

In view of the close relationship which is admitted to exist
between Balanoglossus and Cephalodiscus it is worthy
of mention in this connection that Spengel! has found in the
celom of a species of Balanoglossus (Ptychodera
minuta) masses of small, uniform, nucleated cells which he
is inclined to regard as of a parasitic nature. Caullery and
Mesnil* agree with Spengel that these bodies are parasitic
organisms, and they refer them provisionally to the new
order of the class Sporozoa to which they have given the
name Haplosporidia, the order in which we propose to place
the Neurosporidium of Cephalodiscus.

MarertaAL AND MeErdHops.

The specimens of Cephalodiscus nigrescens obtained
by the ‘‘ Discovery ” were fixed, some in a 5 per cent. solution
of formalin, some in Perenyi’s fluid, and some in picric acid
solution. Serial sections of the polypides were cut for the

1 «Fauna and Flora des Golfes von Neapel,’ Monogr. 18, 1893, pp. 661,
662; pl. 2, figs. 19, 20; pl. 3, figs. 50, 51; pl. 4, figs. 60, 61, 76, 79, 80;
pl. 5, fig. 105.

« Haplosporidies,” ‘Arch. de Zool. Exp. et Gén.,’ iv, 3, 1905, p. 164, and
pl. 18, fig. 125. E

84, W. G. RIDEWOOD AND H. B. FANTHAM.

purpose of investigating the anatomical] structure of this new
species of Cephalodiscus, and these sections, and a few
additional ones cut more recently, were utilised for the study
of the Sporozoén. The sections were 5y, 6m, and 75m in
thickness. The majority of them were stained with Ehrlich’s
hematoxylin and eosin, the others with hematoxylin and
orange G, or Mayer’s hemalum, or borax carmine. ‘The
structural details of the parasite were most satisfactorily
shown by the sections that were cut from material fixed in 5
per cent. formalin and stained with hematoxylin and orange
G, or hematoxylin and eosin. ‘The parasites were studied
with a Zeiss 3 mm. apochromatic homogeneous immersion
objective, used in combination with compensating oculars 4
and 8, and occasionally 12. In some of the stages they are
of large size (pl. 6, figs. 1 and 2, Nsp.), and can be readily
recognised with a 2 inch objective.

OccURRENCE OF THE PARASITE.

The infection appears always to commence in the nervous
tissue, and although the parasites when at their largest may
project beyond the nerve layer into other tissues (pl. 6,
fig. 1, Nsp.), they remain in relation with nerve tissue at one
part of their surface. The parts of the nervous system in
which the parasite has been found are the central nerve mass
(pl. 6, fig. 2, Nsp.), the nerve layer of the dorsal wall of
the buccal shield (practically a continuation of the central
tract), the nerve layer of the ventral wall of the shield
(pl. 6, fig. 1, Nsp.), and the lateral nerve tracts near the
collar pores. ‘he parasite has not been found in the nerve
tracts of the plumes, nor in those of the stolon.

The occurrence of Neurosporidium in the nerve tracts
of Cephalodiscus is not the first instance of a Sporozoén
infecting nerve tissues, although infection of such tissues is
not common. ‘Three instances are known of Neosporidia

NEUROSPORIDIUM. 85

infecting the nervous system of Teleostean fishes. Pfeiffer!
in 1892 discovered a Myxosporidian in the cranial and spinal
nerves of the Grayling (Thymallus vulgaris), the cysts
lying between the medullary sheath of the nerves and the
sheath of Schwann; Schuberg and Schréder? in 1905
described a similar, but not identical Myxosporidian, which
they named Myxobolus neurobius, in the nerves of the
Brook Trout (Salmo fario) ; and in 1898 Doflein® described
a Microsporidian, Glugea lophii (now known as Nosema
lophii), occurring in the ganglion cells of the central nervous
system of the Angler (Lophius piscatorius).

The proportion of infected specimens of C. nigrescens is
considerable. Out of a total of twenty polypides examined,
Neurosporidium was found to occur in eight.

Spherical or shghtly oval spores of Neurosporidium
appear to set up a loca] degeneration in the nervous, deeper
layer of the external epithelium of Cephalodiscus, and in
the oval spaces so formed the parasites grow and sporulate,
and become surrounded by an ill-defined, somewhat irre-
gularly alveolar capsule (pl. 7, figs. 9, 12, 15, cps.). This
capsule is not of the nature of an ordinary cyst, secreted by
the parasite peripherally, but would appear rather to be formed
by the surrounding tissue of the host, much in the same
way as is the enveloping membrane of the Sarcosporidia.

The capsule is not in all cases clearly evident. Some of
the parasites, appearing as groups of spherical bodies when
examined under a Zeiss A or D objective, nearly fill the
spaces in the host tissue in which they lie, while others are
more or less closely surrounded by a capsule (pl. 7, fig. 12,
cps.), which itself is separated from the wall of the host tissue
by a space. The space (fig. 12, cav.) is bounded externally
by a thin layer of the same material (fig. 12, cps!.) lining the

1 Pfeiffer, L., ‘Untersuchungen tiber den Krebs; die Zellerkrankungen
und die Geschwulstbildungen durch Sporozoen,’ 1893, p. 75.

2 Schuberg, A., and Schroder, O., ‘ Archiv fiir Protistenkunde,’ iv, 1, 1905,
pp. 50 et seq.

3 Doflein, F., ‘Zool. Jahrb.,’ Anat., xi, 1898, p. 332.

86 W. G. RIDEWOOD AND H. B. FANTHAM.

host tissue. The capsule and this latter layer are refringent,
and yellowish-brown in colour; they seem to be chitinous,
but special tests were not applied.

These chitinoid layers are not always present, and even
when they are to be recognised, they are so indefinite and
irregular that we prefer not to use the word cyst at all, but
to employ a more indefinite term, such as capsule. Probably
the layers in question are produced by the host tissue in an
attempt, unsuccessful as it appears, to isolate the parasite
from organic connection with itself, or else they consist of
broken-down nerve tissue which has taken a more or less
spherical shape in accommodating itself to the space between
the parasite and the still healthy part of the host tissue.

In some of the cavities a coagulum is to be noticed sur-
rounding the parasite (fig. 7, coag., also figs. 5 and 6). The
coagulum is probably directly connected in its origin with the
degeneration of the host cells, and the production of the
capsule.

SrrucruRE AND Lire-cycLE or NEUROSPORIDIUM.

The parasites in their smallest phase occur as little round
or oval cells (free spores or ameebulee), about 3 wu (2 to 4) in
diameter (see fig. 3), each with a single nucleus. These are
found lying in or among the nerve cells of the host, having
apparently entered this tissue by infiltration. It is not
possible, with the material at our disposal, to say definitely
if there is an intracellular phase of the parasite within a
nervous epithelial cell at the beginning of the life-history of
the parasite. Nuclear division takes place in the amebula,
and young trophozoites, with two or three nuclei in a some-
what irregular mass of protoplasm (fig. 3, 6, ¢, d) are of
common occurrence, often lying in a space among the
nervous tissue cells of the host. Figures of nuclear division
(fig. 8, c) are very rarely and imperfectly seen in our material.

The young trophozoites are usually about 5 u in diameter.
The protoplasm of the trophozoite at this stage is granular

NEUROSPORIDIUM. 87

and easily stained, while in the one-cell or oval corpuscle
stage the protoplasm is hyaline and only slightly granular,
and does not stain deeply. Several young trophozoites may
occur in one space (fig. 6).

Further nuclear division rapidly takes place, and multi-
nucleate trophozoites, spherical, ovoid, or cylindrical in shape,
are found with their many nuclei embedded in a more or less
rounded or slightly lobulated mass of deeply-staining granular
protoplasm (figs. 8 and 9). The multinucleate trophozoites
correspond with the “plasmodium” stage of the Haplosporidia
of Caullery and Mesnil,! though they are not markedly irregu-
lar or ameeboid in outline. These trophozoites are 30 to
40 uw long, and 25 4 to 354 broad.

A “plasmodium ” may be formed by the nuclear division
and growth of the oval corpuscle or amebula that constitutes
the earliest stage of the parasite. It may also be formed by
the fusion and growth of several young trophozoites (fig. 7),
as can be seen by careful examination of a series of sections
passing through a cavity containing several parasites at this
stage. Such a fusion or concrescence approximates very
closely to the formation of a true plasmodium.

In the multinucleate trophozoite phase the protoplasm of
the parasite is not surrounded by a cuticle, and Neuro-
sporidium hereby differs from such a type as Bertramia,
which it otherwise much resembles at this stage. The proto-
plasm is opaque, and crowded with closely distributed granules.
Around each nucleus, however, is usually an area of clear
protoplasm (fig. 8). Some of the nuclei are rather large, 1
to 1:5, or even 2 in diameter, but no definite nucleolus or
karyosome is visible ; the chromatin of these nuclei seems to
be evenly distributed. No refringent granules such as occur
in Bertramia asperospora? were noticed in the deeply-
staining protoplasm. It is to be noted, however, that while

1 Caullery, M., and Mesnil, F., ‘‘ Recherches sur les Haplosporidies,”
‘Arch. Zool. Exp. et Gén.,’ iv, 1905, pp. 101—181, three plates.

2 Minchin, E. A., ‘ Sporozoa,’”’ Lankester’s ‘Treatise on Zoology,’ part 1,
fase. 2, 1903, p. 310.

88 W. G. RIDEWOOD AND H. B. FANTHAM.

our Neurosporidium consists of preserved material only,
Bertramia was described from living specimens.!

In the larger and older multinucleate trophozoites the
nuclei and their surrounding zones of clear cytoplasm are
distinctly seen (fig. 9), marking the beginning of the segrega-
tion of the plasmodial mass into pansporoblasts.

The term pansporoblast was first employed by Gurley * in
connection with the Myxosporidia (in the wider sense, includ-
ing Microsporidia). A pansporoblast (‘sphére primitive’ of
Thélohan) may be described as a spore-mother-cell differenti-
ated within the substance of the trophozoite, destined to give
rise to sporoblasts, from which spores are developed. (The
term sporoblast is applied to cells which develop each into a
single spore, and the term pansporoblast is applied to a cell
which by division gives rise to sporoblasts.) In the Myxo-
sporidia the differentiation takes place by the concentration
of cytoplasm around one of the nuclei of the endoplasm, and
as a rule the protoplasm becomes more transparent. ‘The
pansporoblasts of a trophozoite may be many or few, or even
a single one. A greater or less amount of the protoplasm of
the trophozoite usually remains over, together with a number
of residuary nuclei of the endoplasm, although in some
Microsporidia, e.g., Thelohania and Pleistophora, the
whole trophozoite becomes converted into a single pansporo-
blast. In the typical Myxosporidia each pansporoblast gives
rise ultimately to two spores; in the Microsporidia, on the
other hand, each pansporoblast may give rise to four, eight,
or a larger number of spores.*

‘he term pansporoblast may be extended in its applica-
tion to the Haplosporidia, for although in Bertramia the

1 See also Warren, E., “On Bertramia kirkmani,” ‘Annals Natal Gov.
Mus.,’ vol. i, London, 1906, pp. 7—17.

2 Gurley, R. R., ‘On the Classification of the Myxosporidia,” ‘ Bull. U. 8.
Fish. Comm. Rept. for 1891,’ xi (1893), pp. 407—420: see his footnote on
p. 408.

3 Except perhaps in Nosema pulvis. See Perez, Ch., “ Microsporidies
parasites des crabes d’Arcachon,” ‘Soc. Sci. d’Arcachon, Stat. Biol.,’ ann.
viii, Paris, 1905, pp. 15—36.

NEUROSPORIDIUM. 89

trophozoite divides into a number of bodies, each of which
becomes a single spore, yet in the case of Haplospori-
dium scolopliand H. marchouxi! the bodies into which
the trophozoite divides undergo each a division into four
spores.” The ‘ bodies” in question are thus pansporoblasts
in the sense described above.

In Neurosporidium a definite segregation of the cyto-
plasm of the trophozoite around the nuclei marks the begin-
ning of the formation of pansporoblasts (figs. 7, 10, and 11).
The general shape of the trophozoite at this stage is ovoid
(fig. 11), or sometimes nearly sausage-like (fig. 10). ‘he pan-
sporoblasts are uninucleate cells with hyaline protoplasm,
lying embedded in the opaque, granular protoplasm of the
trophozoite. They are irregularly, but fairly closely packed
in this granular mass, sometimes shghtly more crowded
together at the periphery of the trophozoite than in the
centre (fig. 10). They are spherical or oval in shape, and 3p
or 4 in diameter.

The pansporoblasts in some trophozoites are very quickly
formed, the phase of nuclear division being rapidly passed
through, or even omitted, since this stage may be reached,
in true ‘ plasmodial” manner, by the fusion of several
young trophozoites, all lying within a common cavity in the
host-tissue, as already noted. By careful examination of
series of sections, what appears to be the fusion of such
masses of young trophozoites can be seen, and a stage in the
process is shown in fig. 7.

The cavity in the host-tissue enclosing the parasite in-
creases in size as the growth of the parasite proceeds ; in all
probability the parasite causes progressive degeneration of
the neighbouring host tissue.

Up to this point the development of Neurosporidium
has been simple, in many respects closely following that of a
typical Haplosporidian, such as Bertramia. The mode of

1 Caullery and Mesnil, loc. cit., p. 114 and p. 117.
2 In Haplosporidia there is no visible distinction between sporoblast and
spore; the former passes directly into the latter.

90 W. G. RIDEWOOD AND H. B. FANTHAM.

sporulation, however, is distinctive, and of considerable im-
portance in determining the systematic position of the para-
site.

All the pansporoblasts in a trophozoite of Neurospori-
dium are formed, by internal segmentation, at the same
time. They then commence to increase in size, growing at
the expense of the protoplasmic ground mass of the tropho-
zoite. Hach pansporoblast becomes less well-marked in out-
line ; its protoplasm is still rather homogeneous (non-granu-
lar), but deeply staining, and its centrally-placed nucleus
divides and discharges chromidia throughout the cytoplasm
of the pansporoblast. Hach pansporoblast thus becomes a
spore-morula, for the division of its nucleus into groups of
chromidia, or small portions of chromatin (daughter-nuclei),
results in the formation of many small unicellular sporoblasts,
distributed not only peripherally, but throughout the sub-
stance of the pansporoblast, as may be seen by careful
focussing.

The division of the nucleus of the pansporoblast is not of
the serial karyokinetic type, first into two, then into four, and
so on, but is a kind of simultaneous “ multiple fission ” of the
reproductive chromatin into groups of chromatin forming the
nuclei of the many sporoblasts, each of which soon becomes
a spore with little or no further differentiation. ‘The vegeta-
tive chromatin of the nucleus of the pansporoblast remains
in the centre of the spore-morula; it is a pale-staining body,
and cannot be recognised until the spores have scattered
(see fig. 14, 7.m.).

Each spore is about 1 or 2m in diameter, and stains
deeply ; its small nucleus is surrounded by hyaline proto-
plasm. From a full-grown trophozoite a very large number
of spores is formed, since the trophozoite gives rise to many
pansporoblasts, and each pansporoblast grows and divides
into many spores. A full-grown trophozoite containing
spore-morule in sporulation is more or less spherical, and
measures about 40 to 70m in diameter (figs. 12 and 13).
The spore-morule are spherical, and 10 u to 15, in diameter,

NEUROSPORIDIUM. 91

The uninucleate spores, which are small gymnospores or
amzbule, gradually pass out of the cavity around the parent
organism by more or less irregular apertures in the capsule,
and so invade the surrounding tissue of the host, and start
fresh infections in other parts of the nervous system of the
Cephalodiscus.

Kach spore on becoming free grows to about 2, 3, or 4 u
in diameter, and division of its nucleus begins, while its
cytoplasm becomes granular and a little more opaque. In
this way young trophozoites are formed, such as those de-
scribed in the first paragraph of this section.

All the cytoplasm of the spore-morula is not used up in the
formation of spores, and there is also nuclear material left
over. These residuary masses are clearly shown in our pre-
parations, and their fate may here be considered in some
detail. When the spores have left the parent spore-morula,
the latter has a fairly definite contour, rather more distinct
than during sporulation. The protoplasm is granular, and
there is usually a pale, centrally-placed nucleus. The proto-
plasm stains feebly, and the granules init are distinct. The
residual nucleus is pale in colour, and reddish or brownish-
red after staining with hematoxylin, and it thus stands in
marked contrast with the deeply purple nuclei of the spores in
the same preparation. In a few spore-morule examined little
or no residual nuclear matter was found, even after the
examination of all the sections into which they had been
cut.

One or two rather more deeply staining dots may be seen
in the pale residual nucleus in some, rather rare, cases
(fig. 15). The residual nuclear matter is, as already stated,
the remains of the vegetative chromatin of the spore-morula,
the reproductive or generative chromatin being distributed
in the nuclei of the spores. Deeply staining granules of
chromatin are seen in capsules at this stage, sometimes in
two or three groups (fig. 15, chr.), but usually aggregated
towards the centre of the mass (fig. 17, chr.). These are
groups of chromidia, or small masses of chromatin, of about

92 W. G. RIDEWOOD AND H. B. FANTHAM.

half the diameter of the nuclei of the spores. Some of them
are surrounded by a clear area of cytoplasm, and may form
spores, but many would appear to be residual, and not
destined to give rise to spores.

The actual transition between the stage of sporulation in
a spore-morula and that of residual matter is depicted in
fig. 14. The capsule, of which a small part only is shown, is
full of ripe spore morulz, and these are in process of divi-
sion into uninucleate spores; one of them, however, is
slightly in advance of the rest, and has already divided into
spores, which have scattered, leaving the remains of the spore-
morula, pale in colour, with granular protoplasm and remains
of nucleus. The outline of this relic (fig. 14, 7. sp. m.) in the
capsule is more clearly seen than that of its neighbours; its
protoplasm is of a light brownish tint, and its nuclear residue
of a brownish pink, after staining with hematoxylin, which
imparts a characteristic purple coloration to the neighbour-
ing sporulating masses, forming a clear and striking con-
trast.

The remains of spore-morule, after sporulation, undergo
degeneration in old capsules (fig. 16). Their outlines become
less distinct, and vacuoles appear in the now coalescing mass
of protoplasm (fig. 17). The residual nuclei become less
clear, but chromidia may still be seen near the centre of the
degenerating mass (fig. 17). Some of the spores, formed
from the spore-morulez before they degenerated, may for a
time be seen in the neighbouring tissue and in the cavity in
which the parent capsule lies (fig. 16).

While the above account of the life-history of Neuro-
sporidium suggests how the infection may spread from one
part of the host to another, by the amebule and young
trophozoites migrating along the course of the nerve tracts,
we have no evidence to offer which can explain the spread
of the parasite to new hosts. In Bertramia aspero-
spora, of Rotifers, the mode of spread has been watched by
Bertram ; the spores have no power of independent move-
ment, but are disseminated passively on the death and disin-

NEUROSPORIDIUM. 93

tegration of the body of the host.! Possibly the same may
happen in the present instance.

SYSTEMATIC POSITION oF NEUROSPORIDIUM.

In determining the systematic position of Neurospo-
ridium cephalodisci the following features in the life-
history, so far as we have been able to trace it, should be
kept in view:

1. From a capsule lying in a cavity in the nervous system
of Cephalodiscus gymnospores or amebule are liberated,
as rounded masses of clear, naked protoplasm, 2 to 4 jy in
diameter, each with a centrally placed nucleus.

2. The next stage is one in which a small multinucleate
trophozoite lies in a small cavity in the nervous system.
This stage is reached, either by the rapid growth and nuclear
division of a single ameebula, or by the coalescence of several
ameebule (plasmodium).

3. Nuclear division of the trophozoite continues, and the
size increases, until the body is about 30 to 50 mw in diameter,
and ovoid in shape. ‘The general protoplasm is granular and
opaque, but there is a clear zone of protoplasm around many
of the large nuclei.

4. The large ovoid trophozoite segments into pansporo-
blasts, each a single cell, 3 to 5 « in diameter, consisting of a
large nucleus surrounded by clear cytoplasm.

5. The nucleus of each pansporoblast divides into many
daughter-nuclei, and the pansporoblast enlarges and becomes
a spore-morula, 10 to 15 q« in diameter. Each daughter-
nucleus, with its small mass of clear cytoplasm, becomes a
sporoblast, and then a spore. The spores are generally dis-
tributed throughout the spore-morule.

6. The spores ultimately separate, and pass out into the
adjacent parts of the nervous system of the host. The remains
of the spore-morule, consisting of granular protoplasm, pale-
staining remains of nuclei, and some free chromidia, undergo
gradual degeneration.

1 Caullery and Mesnil, loc. cit., p. 136.

94. W. G. RIDEWOOD AND H. B. FANTHAM.

7. Around the parasite in its various stages (except the
amezebula stage) is an ill-defined capsule, partly alveolar, and
probably secreted by the cells of the host.

The systematic position of Neurosporidium may be
readily determined by reference to the characters summarised
above. The somewhat irregular form of the trophozoite and
its possession of several nuclei; the appearance of pansporo-
blasts, denoting the commencement of spore-formation, at an
early stage in the growth of the parasite; the division of the
pansporoblast to form many spores; and the intercellular
(histozoic) habitat of the parasite; all these are characteristic
of the Neosporidia.

Further, on account of the small, simple uninucleate spores,
without polar capsules, the increase in the number of nuclei
during the trophic stage, and the segmentation of the full-
grown trophozoite into ovoid pansporoblasts, we place the
parasite among the Haplosporidia.

The division of the pansporoblast into many spores is a
feature which does not occur in the life-history of the typical —
Haplosporidian. In Haplosporidium scolopli and H.
marchouxi the number of spores formed from each pan-
sporoblast is four; in Bertramia it is one. In Rhino-
sporidium kinealyi, however, a parasite from the nasal
mucous membrane of man, recently described by one of us
(Fantham)? in collaboration with Prof. Minchin, and also by
Beattie,” the portion of the life-history in question finds a
fairly close parallel.

In Rhinosporidium there is a multinucleate trophozoite
phase, followed by segmentation into many closely packed
pansporoblasts lying within a thin peripheral layer of un-

1 Minchin, E. A., and Fantham, H. B., “Rhinosporidium kinealyis
n.g., n.sp.: a Sporozodn from the Mucous Membrane of the Septum Nasi of
Man,” ‘Quart. Journ. Mier. Sci.,’ n. s., xlix, 3, 1905, pp. 521—532, two
plates.

2 Beattie, J. M., “Rhinosporidium kinealyi: a Sporozoon from the
Nasal Mucous Membrane,” ‘Journ. Path. and Bact.,’ xi, 3, 1906, pp. 270—
275, two plates.

NEUROSPORIDIUM. 95

differentiated protoplasm; each pansporoblast becomes a
spore-morula, dividing into about a dozen small, round, uni-
nucleate spores. In_ these respects Rhinosporidium
resembles Neurosporidium. In Rhinosporidium, how-
ever, there is a well-defined cyst secreted by the parasite,
and the outline of the spore-morula is rather more distinct,
there being a definite membrane round each, and the outhne
of each spore in a full-grown spore-morula is also well marked.
This sheht difference in the definiteness of contours is of
little importance as compared with the segmentation of pan-
sporoblasts into many spores, which is in marked contrast
with what occurs in the typical Haplosporidia.

Neurosporidium differs also from Rhinosporidium
in that the spore-formation from pansporoblasts is not pro-
gressive, as it is in the latter. In Rhinosporidium the
pansporoblasts, within a large cyst, are disposed in three
zones, merging the one into the other, namely, a peripheral
zone of uninucleate pansporoblasts just within a thin layer of
undifferentiated protoplasm, an intermediate zone of pan-
sporoblasts with two or three nuclei each, in which zone
spore-formation is proceeding, and a central mass in which
each pansporoblast, now a spore-morula, contains about a
dozen uninucleate spores. In other words, spore-formation
proceeds in a centrifugal direction within the cyst, the central
pansporoblasts completing their sporulation before the peri-
pheral ones begin to divide.

In Neurosporidium, on the other hand, there is an
interval between the simultaneous formation of the uninucleate
pansporoblasts and their segmentation, and the segmentation
of the spore-morule occurs simultaneously throughout the
same capsule.

As pointed out by Minchin and Fantham,! Rhinospori-
dium, in the successive formation of its pansporoblasts,
resembles Schewiakoff’s parasite of the Cyclopide,? to which

' Loc. cit., p. 529.
* Schewiakoff, W., ‘‘ Ueber einige ekto- und ento-parasitische Protozoen der
Cyclopiden,” ‘ Bull. Soc. Imp. Nat. Moscou,’ n. s., vii, 1893, pp. 1—29, pl. 1.

96 W. G. RIDEWOOD AND H. B. FANTHAM.

Caullery and Mesnil? have recently given the generic name
Scheviakovella.

The degree of importance to be attached to the mode of
sporulation, whether simultaneous or successive within the
same cyst, is for future investigations to decide; for the
present, our opinion is that the production of many spores by
each pansporoblast instead of a few spores or a single spore,
a feature possessed in common by Rhinosporidium and
Neurosporidium, is of more importance than the former
feature, in which the two genera differ.

After due consideration of the various points above set
forth, we definitely place Neurosporidium and Rhino-
sporidium in the order Haplosporidia, extending the order
to include these forms. Further, we divide the extended
order Haplosporidia into two sections ; (1) the Oligosporulea
(nom. nov.) for forms ike Haplosporidium, Bertramia
and Coelosporidium, in which each pansporoblast pro-
duces only a small number of spores or a single spore, and
(2), the Polysporulea (nom. nov.) for forms hke Rhino-
sporidium and Neurosporidium, in which the pansporo-
blast gives rise to many spores, either successively or almost
simultaneously.

Some protozoologists express doubt as to the homogeneity
of the Haplosporidia as a group, and not altogether without
reason. But Coelosporidium, Bertramia, and Haplo-
sporidium form a well-defined section, and we have shown
above the relation of Neurosporidium and Rhinospori-
dium to this section. The precise limits of the order Haplo-
sporidia are difficult to define because of the elementary
structure and simple developmental cycle of the organisms
which that order includes.

The Haplosporidia show points of resemblance with the
Mycetozoa and with the Rhizopoda. The possibility, how-
ever, must not be overlooked that while some of the forms
are truly primitive, others may be spuriously so, and may
owe their simplicity to degradation from a higher stock, a

1 Loe. cit., p. 156.

NEUROSPORIDIUM. 97

process which in so many groups of animals is known to
result from indulgence in a parasitic mode of life.
For the present the Neosporidia may be divided as follows :
1. Cnidosporidia (Doflein), with polar capsules.
Myxosporidia, e.g. Myxobolus, Spherospora.
Microsporidia, e.g. Glugea, Pleistophora.
Actinomyxidia, e.g, Hexactinomyxon.
? Sarcosporidia, e.g. Sarcocystis.
2. Haplosporidia (Caullery and Mesnil), simpler forms than
the above, without polar capsules.
Oligosporulea, e.g. Bertramia, Haplosporidium.
Polysporulea, e.g. Rhinosporidium, Neurospori-
dium.

SUMMARY.

The parasite begins its life-cycle as around or oval gymno-
spore or ameebula in the nervous system of the host (figs. 3
and 4).

The ameebule cause a degeneration of the nerve-tissue
immediately around them, and come to lie within cavities,
one amebula in a cavity, or several (fig. 5).

The amebula becomes a multinucleate trophozoite, either
by enlarging and undergoing nuclear division, or by coales-
cence with other amzbule or young trophozoites, or by a
combination of both processes (figs. 6, 7, 8, 9).

The capsule surrounding the parasite is ill-defined, and is
probably formed by the host (fig. 12, etc., cps.).

The trophozoite segments into uninucleate pansporoblasts
(figs. 10, 11), each of which enlarges and becomes a spore-
morula.

The spore-morula gives rise to many small spores (figs.
12, 15), and after the liberation of these, there remains a mass
of granular protoplasm, with residual nuclei (figs. 15, 16, 17).

The infection probably spreads through the nervous system
of the host by the migration of the amebulez and trophozoites.
The mode of cross-infection from host to host is not known.

It is proposed to divide the Haplosporidia into the Poly-

VoL. 51, part 1.—NEW SERIES. 7

98 W. G.. RIDEWOOD AND H. B. FANTHAM.

sporulea (pansporoblasts giving rise to a number of spores)
and the Oligosporulea (pansporoblasts giving rise to a few
spores or to a single spore each).

The systematic position of Neurosporidium is as follows:

Phylum.—Protozoa. |
Class.—Sporozoa.
Sub-class.—NosporipIiA.
Order.—Haplosporidia.
Section.—Polysporulea (nov. sec.).
Genus.—Neurosporidium (noy. gen.).
Species.—cephalodisci (nov. sp.).

EXPLANATION OF PLATES 6 AND 7,

Illustrating Dr. Ridewood and Mr. Fantham’s paper on
Neurosporidium cephalodisci, n.g., n. sp., a Sporo-
zoon from the Nervous System of Cephalodiscus
nigrescens.”

PLATE 6.

For the two photomicrographs from which figs. 1 and 2 were drawn we are
indebted to Dr. N. H. Alcock, Lecturer on Physiology in St. Mary’s Hospital
Medical School, and to him we hereby tender our best thanks. The photo-
graphs were taken by monochromatic light obtained by a Thorpe’s grating,
and Zeiss’s apochromatic objectives were used.

Fic. 1.—Section of a polypide of Cephalodiscus nigrescens taken
transversely to the length of the body, and passing behind the pedicle of the
buccal shield and behind the mouth. x 120. MNsp. Neurosporidium in
the thick ventral wall of the buccal shield. pA. Wall of pharynx. ¢.c. Trunk
celom. m. Muscles of the ventral body-wall.

Fic. 2.—Section of a polypide of Cephalodiscus nigrescens taken
transversely to the length of the body, and passing through the central nerve
mass and the notochord. x 100. Msp. Neurosporidium in the central
nerve mass. s. Septum between the right and left collar cavities. 20. Noto-
chord. p.c. Proboscis cavity, or cavity of the buccal shield. 4.8, Thick
ventral wall of the buccal shield. /p/. Base of the lophophore, cut obliquely.
a. Ectoderm around the anus, cut tangentially.

NEUROSPORIDIUM. 99

PLATE 7.

All the figures on Plate 7 were outlined with camera lucida (Abbé), using
apockromatic objective 3 mm. homogeneous immersion (Zeiss) and compen-
sating oculars 4 and 8.

All the figures of this plate, except fig. 16, are enlarged 1000 diameters.

SIGNIFICANCE OF THE LETTERING.

cav. The cavity in the host tissue in which the parasite lies; it often con-
tains a coagulum (coag.). chr. Chromidia or daughter-nuclei resulting from
the division of the nucleus of the spore-morula in the formation of spores,
principally those daughter-nuclei remaining over in old and degenerating cap-
sules. cps. Capsule, an ill-defined, sometimes alveolated membrane around
the parasite; it is not a true cyst, aud is apparently formed by the tissue of
the host. cys’. A similar layer lining the healthy tissue of the host. za.
Nucleus (figs. 4 to 9), or daughter-nucleus (figs. 12 to 14). p’spd/. Pan-
sporoblast. 7.2. Residual nucleus of the spore-morula. r.sp.m. Residual
cytoplasm of the spore-morula. vac. Vacuole.

Fic. 3.—A number of free spores. a. An amebula, with one nucleus.
6 and d. Later stage, young trophozoites with two and three nuclei respec-
tively. e¢ shows nuclear division, rather indistinctly, but as well as could be
expected from the material to hand.

Fic. 4.—Deeply staining spore (amebula) lying in a definite cavity in the
nervous tissue of the host.

Fig. 5.-—Several such spores in a cavity, surrounded by a thin envelope
(cps’.).

Fic. 6.—Several young trophozoites lying in a cavity.

Fic. 7.—Young trophozoites coalesced into a plasmodium, lying in a cavity,
with another young trophozoite, which examination of the series of sections
shows to be continuous with it. A coagulum is also seen, probably resulting
from the remains of the host cells. This specimen is in the stage of pansporo-
blast formation. Although smaller than those shown in figs. 8 and 9, it is in
a later stage of the life cycle.

Fic. 8.—Maultinucleate trophozoite or ‘ plasmodium.”

Fic. 9.—Multinucleate trophozoite, surrounded by a capsule, lying in a
cavity in the host tissue.

Fic. 10.—Elongate trophozoite, with oval pansporoblasts lying in deeply
staining granular protoplasm. ‘There is also a small trophozoite by the side,
lying in the same cavity.

Fie. 11.—Ovoid trophozoite, full of pansporoblasts.

Fie. 12.—Several spore-morule (full-grown, segmenting pansporoblasts),
which are dividing into spores. The parasite is seen surrounded ill-

100 WwW. G. RIDEWOOD AND H. B. FANTHAM.

defined capsule (cps.), somewhat alveolar in character, and apparently formed
by degenerating host tissue; it lies in a cavity in the host tissue, which
latter is lined by a membrane (eps’.).

Fie. 13.—Typical capsule, full of spore-morule, each dividing into many
spores.

Fic. 14.—Part of a capsule containing spore-morule, one of which, having
shed its spores, shows a residual granular mass of cytoplasm, and a pale, ill-
defined nucleus. The difference in the staining reaction of this remnant of the
spore-morula from that of its sporulating neighbours is very striking.

Fic. 15.—Capsule containing residua of spore-morule. Groups of nuclear
granules (chromidia), some of which may perhaps form spores, while others
will degenerate, are scattered about, chiefly in the centre. These chromatic
dots are smaller than the daughter-nuclei shown in fig. 13, each of which
latter becomes the nucleus of a spore. Some of these smaller nuclei (cir.)
are surrounded by a layer of clear, refractive protoplasm.

Iie. 16.—Further stage in degeneration of residua of spore-morule.
These consist, at this stage, chiefly of residual. granular protoplasm, which is
becoming vacuolated. At the periphery of the cavity, now ill-defined, in
which the large mass lies, are seen some of the spores and young trophozoites
formed from these spore-morule. »x 500.

Fie. 17.—Still further degeneration of residual matter of the spore-
morule, showing vacuolated protoplasm and remains of nuclei (7.7.) of some
of the spore-morule. Some chromidia are grouped in the centre, but these
were probably not destined to form spores.

GAMETOGENESIS AND FERTILISATION IN NEMATUS RIBESIT. 101

Gametogenesis and Fertilisation in Nematus
ribesii.

By
L. Doneaster, M.A.,

Late Mackinnon Student of the Royal Society; Lecturer in Zoology in
the University of Birmingham.

With Plate 8.

Iy a previous paper! I gave an account of the maturation
and behaviour of the polar nuclei in several species of
sawflies which develop parthenogenetically.

In all these species there were two maturation divisions,
giving rise to an egg nucleus and three polar nuclei, and in
some cases fusion took place between the second polar
nucleus and the inner half of the first. The ege nucleus
sank into the yolk and began to divide to form the embryo,
while the polar nuclei in all cases ultimately disintegrated.
Since whenever the chromosomes were clearly visible their
number appeared to be eight, both in the maturation mitoses
and in the later divisions in body-cells, it was concluded that
no reduction in the ordinary sense took place. But if
fertilisation ever takes place by conjugation of male and
female pronuclei, an obvious difficulty arises with regard to
the chromosome number in fertilised eggs, and since the
process of fertilisation had not been thoroughly examined at
the time when the paper referred to was written, it was
necessary to leave the question open in the hope of finding a
satisfactory answer later. This paper gives an account of

1 *Quart Journ. Micr. Sci.,’ vol. 49, 1906, p. 561.

102 L. DONCASTER.

the work done on the fertilised egg in Nematus ribesii
and on the gametogenesis in that and other species.

The methods used were generally the same as before, but
it was found that, in searching for male pronuclei in the eggs
of impregnated females, thionin or gentian violet were more
satisfactory stains than iron hematoxylin, since they stain
nucleus and cytoplasm but leave the yolk uncoloured. In
the work on spermatogenesis and the development of the
ovarian egg, osmic fixatives (e.g. Flemming’s fluid) were
largely used in addition to sublimate.

Tue Fertitisep Ecc In N. RIBESII.

In some animals, e.g. the bee, the fertilised egg is easily
distinguished from the virgin by the presence of sperm asters
in the yolk, but in the sawflies nothing of the kind can be
found, and over 200 eggs had to be cut and examined before
it became certain that conjugation of male and female
pronuclei takes place. Invery young eggs I had occasionally
found minute rod-like bodies in the peripheral protoplasm
near the anterior end, which are probably the heads of
spermatozoa, and in somewhat later eggs bodies which
appeared to be degenerating nuclei sometimes appear in a
similar position. In eggs laid by impregnated females there
are frequently in the yolk in front of the polar region more
or less numerous small radiating patches of protoplasm
which sometimes appear to contain indistinct nuclei, but
protoplasmic masses not certainly distinguishable from these
are found also in virgin eggs, although with less regularity.
In eggs which are probably fertilised there are also fre-
quently lines of protoplasm running inward from the edge
of the egg near the point where the spermatozoa had been
found. But in no case have I been able to recognise with
complete certainty the male pronucleus before the maturation
divisions of the egg are completed, and after that stage
nuclei found in the yolk may always be derived from the

GAMETOGENESIS AND FERTILISATION IN NEMATUS RIBESII. 103

egg-nucleus itself. It is never possible, therefore, to say
with certainty that a given egg is fertilised or not.

But after much time spent in vainly trying to follow the
entrance of the spermatozoon and its conversion into the
male pronucleus, | at last was able to observe the conjugation
of the sperm-nucleus with that of the egg, and so to prove
that true fertilisation does take place (fig. 1). It occurs
immediately after the maturation divisions; the three polar
nuclei lie near the edge of the egg (two of them in the same
section as the egg and sperm nuclei), and the fusion of the
two inner polar nuclei has not yet taken place. The male
and female pronuclei are in contact, the male being distinctly
smaller than the female, but in another egg in which the
same stage is seen the two are of about equal size. The
subsequent stages of the conjugation and division of the
zygote nucleus have not been observed, but the section
represented in fig. 1 leaves no reasonable doubt that normal
conjugation takes place. It therefore became necessary to
reconsider my previous conclusions with regard to the number
of chromosomes, since never more than eight have been
found in either fertilised or virgin eggs. I was thus led to
work out the spermatogenesis, and to the fresh work on the
maturation divisions to be described later.

In my previous paper I mentioned that the behaviour of
the polar nuclei appeared to be slightly different in fertilised
and in virgin eggs, and subsequent work has confirmed this.

In the virgin egg of N. ribesii the two inner polar nuclei
fuse and give rise to a group of chromosomes, which is
generally clearly double, with eight in each half. The two .
halves of the group do not lie far apart, and commonly
remain without much change for some time. But in the
majority of eggs from impregnated females the chromosome
groups derived from the two inner polar nuclei lie completely
and sometimes widely separated, as if the conjugation
between the nuclei had been much less complete than in
virgin eges (figs. 2, 3,and 4). Further, in virgin eggs the
polar chromosomes usually do not divide, at least for some

104 L. DONCASTER.

time, but in fertilised egos they frequently divide compara-
tively early, giving groups containing as many as sixteen
chromosomes rather irregularly arranged in the “polar
protoplasm.” That this difference in behaviour is really
connected with fertilisation is made probable by the fact
that it rarely, if ever, occurs in eggs which are certainly
virgin, but in the eggs laid by impregnated females it is
frequent. Further, in several eggs laid by impregnated
females the polar nuclei follow the typical virgin arrange-
ment, and in these the little rayed protoplasm masses in the
yolk, characteristic of fertilised eggs, are absent; but
other eggs laid by the same female have the fertilised type
of polar chromosomes, and in these the rayed protoplasm
patches are also present.

It appears, therefore, that the fertilisation of the egg
nucleus, or the presence of spermatozoa in the egg, in some
way influences the behaviour of the polar nuclei.

SPERMATOGENESIS.

When it had been shown that normal fertilization could
take place in N. ribesii, it became necessary to re-examine
the maturation divisions in order to make certain about the
chromosome number, which I asserted in the previous paper
to be eight both in the maturation and in the somatic
mitoses, and also apparently in fertilized eggs. The matura-
tion of the egg begins immediately after it is laid, so that it
is very difficult to get good preparations of the early stages,

-and I therefore decided to examine the matter first in the
development of the spermatozoa.

In very young male pup, shortly after the larval skin is
cast in the cocoon, the testes consist of compact groups of
cells at the sides of the alimentary canal. These cells
(spermatogonia) have relatively large nuclei containing a
conspicuous nucleolus (plasmosome) and eight or about eight
chromatin masses apparently attached to the nuclear mem-
brane (fig. 5). Division figures are scarce, but when found

GAMETOGENKESIS AND FERTILISATION IN NEMATUS RIBESIT. 105

they show clearly about eight rather large chromosomes
in the equatorial plate, which split so that eight travel
towards each centrosome (fig. 6a, b). Ata later stage the
testis becomes larger, and consists of lobes or compartments
in each of which all the cells are in about the same stage.
By the time the colours of the mature fly are beginning to
appear the testis contains nothing but spermatids and nearly
mature spermatozoa, but when the pupa is still white all
stages from spermatogonia to spermatids are found in dif-
ferent lobes, often in the same section.

In the nucleus before the first maturation divisions the
chromatin consists of a number of irregular masses (appa-
rently about eight, but they are always rather indistinct).
Shortly afterwards it becomes condensed into four more
concentrated masses, each of which frequently appears double
or quadruple (fig. 7a, b,c). A spindle is then formed, and
the four chromatic masses become tightly packed together in
the equatorial plate, which is much smaller than in the
spermatogonial divisions. There are conspicuous centro-
somes. ‘I'he chromosomes in the spindle are so tightly packed
together that it is difficult to be certain of their number, but
a comparison of many mitoses leaves little doubt that there
are four, each of which is bivalent (fig. 8a, b). ‘The mitosis
appears to be of the heterotype form, resembling the figures
found by Moore in the cockroach! except that the chromo-
somes are fewer and very much smaller (fig. 9a, b). They
are, however, appreciably larger than the chromosomes in the
maturation mitoses of the egg.

The second maturation division is easily distinguished from
the first by the fact that the spindle is of about half the
diameter; the chromosomes are usually even more tightly
packed, so as frequently to appear as a single body, but in
clearer cases there is little doubt that there are four (fig.
10 a, b, c). At the telophase a vesicular spermatid nucleus
is formed, with the chromatin arranged round the edge

1+ Quart. Journ, Micr. Sci.,’ vol. 48, 1905, pp. 489 and 571.

106 L. DONCASTER.

giving it a characteristic appearance (fig. 11). This becomes
converted into the head of the spermatozoon.

It must be concluded therefore that in the male the normal
somatic number of chromosomes is eight ; that four ‘‘ gemini”
appear in the prophase of the first maturation division, and
that finally four chromosomes are distributed by heterotype
and homotype divisions to each spermatid nucleus.

There is no trace of the ‘‘ polar body ” formation described
by Meves in the spermatogenesis of the bee.”

OoGENESIS.

In the larva before it casts its skin within the cocoon the
ovaries are much like the testes of the male, but larger, with
bigger nuclei. The ovary is enclosed in a cellular sheath,
and some ovarian cells are already larger than others ; these
will form the eggs, while the more numerous smaller cells
give rise to the nutritive and probably to the follicle-cells.
All the nuclei at this stage contain about eight chromatin
masses and one to three nucleoli (fig. 12). In the young
pupa the ege tubes are already differentiated, and in a
longitudinal section of a tube the changes in the nucleus can
easily be followed. At the apex of the tube the nuclei are
like those in the larval ovary; below this zone the chromatin
becomes distributed through the nucleus as fine dots, which
are often aggregated together in one part, as in a sort of
synapsis (fig. 13). The egg nucleus then enlarges consider-
ably, and the chromatin appears as an irregular thread; at
this stage two or three nucleoli are generally conspicuous
(fig. 14). After this stage yolk begins to be deposited, and
before the egg is ripe the nucleus, which has been very
large, dwindles so that in nearly ripe eggs I have been
totally unable to find it.

In the larval ovary mitoses may be found in the ovarian
cells and in the sheath; those actually in the ovary appear to
have eight chromosomes (fig. 15 a, b). But in the sheath in

> * Anat, Anzeiger,’ xxiv, 1903, p. 29,

GAMETOGENESIS AND FERTILISATION IN NEMATUS RIBESII. 107

all the mitoses observed the number is more than eight ;
usually it seems to be sixteen, but in some cases the figure
suggests more than sixteen very small chromosomes (fig.
16a—f). Wilson! has described spindles with double the
somatic number in the ovary-sheath of Hemiptera, and
regards them as abnormal, but the figures seen in N. ribesii
certainly suggest that the eight chromosomes in the primi-
tive germ-cells are compound, composed of a greater number
of smaller units, possibly more than sixteen.

In the pupal ovary the egg-cells are already definitely
formed, and do not divide further, but merely undergo the
usual growth with deposition of yolk. The follicle cells are
now quite small, and an occasional mitotic figure is visible ;
these are rarely clearly defined, but appear to have eight
chromosomes. When the egg has reached its full size the
follicle cells become degenerate, with obscure dark-staining
nuclei. Groups of similar degenerating cells are found here
and there in the larval ovary.

The fact that the chromosome number in the ovary, and
probably in the follicle cells, is smaller than that found in the
sheath is of considerable interest.

In addition to the case described by Wilson, and referred
to above, the same kind of thing has been observed by
Petrunkewitsch in the bee,” in which he found the unreduced
number to be sixteen in the egg, but sixty-four in the blasto-
derm, and it is still more conspicuous in Ascaris, which,
according to Boveri, has a large number of very small chromo-
somes in the somatic cells, but only four in all the cells on the
‘“‘ verm-track,” from the fertilised egg up to the maturation
divisions of the germ cells.3 These facts suggest that it may
happen not infrequently that the chromosomes in cells of the
germ-track may be compound, and consist of a number of
smaller units which become separated in somatic cells. But

1 “ Studies on Chromosomes,” iii, ‘Journ. Exp. Zoo.,’ vol. ii, No 1, 1906.

2 ¢ Zool. Jahrb.,’ vol. xiv, 1901, Anat. und Ontog., p. 573.

2 Boveri, ‘Ergebnisse tiber die Konstitution der Chromatischen Substanz
des Zellkerns.’ (Fischer, Jena.)

108 L. DONCASTER.

even if this is found to be a phenomenon of general occurrence
it does not necessarily affect the hypothesis of the individuality
of the chromosomes in any essential point.

CHROMOSOMES IN THE MaturATION Divisions or THE Eaa.

It has now been shown that in the spermatogonial and
oogonial divisions there are eight chromosomes, and that in
the spermatocytes these are reduced to four in the normal
heterotype manner. ‘These facts led me to re-investigate the
maturation divisions of the ege, since in my previous paper
(loc. cit.) I gave evidence that in both first and second polar
mitoses the number was eight.

The chromosomes in the maturation of the egg are much
less easy to observe than in the spermatogenesis, for there
are difficulties of technique to be overcome, and the egg has
to be preserved at exactly the right moment. But after
cutting some hundreds of eggs I have been able to convince
myself that while there are two types of maturation. In some
egos no reduction takes place, and eight chromosomes pass
into each of the four nuclei produced by the polar mitosis.
In other eggs four double chromosomes are found in the
equatorial plate of the second maturation division, and these
separate into their component halves sending four into each
daughter-nucleus (figs. 17—21).

I have never obtained a section of the first polar mitosis in
which it is quite certain that there are four “ gemini,” although
some figures strongly suggest this; but at the close of the
first division, when the chromosomes are arranging themselves
to form the equatorial plate of the second mitosis, four double
chromosomes are sometimes clearly visible (figs. 20, 21). I
have also several preparations which show only four when the
second polar mitosis is already begun. A comparison of figs.
17 and 19,18 and 20, respectively, will show the difference
between the reducing and equational types of maturation.

It must therefore be concluded that in some eggs pairing

GAMETOGENESIS AND FERTILISATION IN NEMATUS RIBESII, 109

(synapsis) of chromosomes takes place before the maturation
divisions, resulting in the separation of complete chromosomes
at one of the mitoses, while in other eggs no pairing takes
place, and each chromosome undergoes two equational divi-
sions. In connection with this it is noticeable that in the
egos having the equational type the eight chromosomes are
about half the size of the four seen in reduced eggs.

IT have found the reduced type in eggs from both virgin
and impregnated females, so that the view which first sug-
gested itself, viz. that reduction only takes place in eggs
which contain spermatozoa, is not tenable.

Re-examination of my sections of Peecilosoma luteolum
confirms me in the belief that in that species, which yields
females from virgin eggs, and is normally not fertilised, there
are two equational divisions in all the eggs of which I have
suitable preparations. A Newalon /s
‘the somatic mitoses of fertilised
egos appear always to have eight chromosomes; a larger
number has never been found. ‘his is what would be
expected if only eggs which undergo reduction are capable
of fertilisation.

In virgin eggs commonly eight are found, but in some cases
the equatorial plate seems to have four only, showing that
reduced eggs when not fertilised can develop as far as the
blastoderm stage (fig. 22). The number of eggs which die
before hatching varies, in some batches being very small, in
others more considerable ; it is possible that the reduced eggs
are those which fail to develop to larvee. Since, however, it
has been shown by the mitoses in the ovary sheath that the
chromosomes are possibly compound, it may happen that
reduced eggs which are not fertilised restore the normal
number of chromosomes by division of the compound chro-
mosomes, as was asserted by Petrunkewitsch (loc. cit.) with
regard to the bee.

In the developing egg

The conclusion that the eggs of one species may either
undergo reduction, or may retain the full number of chromo-

110 L. DONCASTER,

somes, although in each case there are two polar mitoses, is
of considerable interest. I know of nothing quite parallel
with it hitherto observed in animals, but I think it not
unlikely that in the two generations of the Gallflies, one of
which is bisexual and the other purely female, a similar state
of things may be found to exist. That there may be two
types of egg, one of which is reduced and requires fertilisa-
tion, and the other not reduced and parthenogenetic, is of
course not infrequent, but in such cases the eggs generally
have obvious external differences, and the unreduced form
has only one polar body. A condition more nearly resem-
bling that found in N. ribesii has been observed by Rosen-
berg in Hieracium,' in which the egg-cell in some flowers on
a head is reduced and can be fertilised, in others on the same
head not reduced and parthenogenetic. But here again the
number of maturation divisions is probably not the same in
the two cases. In the bee, according to Petrunkewitsch, all
the eggs are reduced, but if not fertilised, the somatic
number of chromosomes is restored automatically.

The conclusions here reached may make it necessary to
reconsider the provisional hypothesis of sex-segregation
sketched in my previous paper, but until further facts are
obtained in other species it seems premature to discuss the
bearing of my results on the problem of the determination
of sex. I have not found it possible, owing to the minute
size of the chromosomes, to determine whether anything
comparable with Wilson’s ‘‘ heterotropic”’ chromosome exists
in Nematus. In some figures (e. g. the group represented
in fig. 18) only seven chromosomes are visible instead of
eight, but when they are so minute it is always possible that
two are superposed and not distinguishable apart.

In conclusion I take this opportunity of expressing my
gratitude to Mr. J. HE. 8. Moore for allowing me to compare
some of my preparations with his, and for valuable help in
elucidating my sections.

[Nore.—In a series of eggs all laid by one insect on one

1 Brit. Ass., York, 1906. Discussion on Fertilisation, Sects. D and K.

GAMBETOGENESIS AND FERTILISATION IN NEMATUS RIBESII. 111

day the polar mitoses are abnormal. The most extreme
case (fig. 23) shows the “polar protoplasm,” full of dots
arranged roughly in lines like iron-filings in a magnetic field.
At each pole of the figure is a group of more conspicuous
stained bodies which may be chromosomes. Some of the
other eggs show a somewhat similar appearance on a smaller
scale, and in others nothing is clearly distinguishable in the
polar protoplasm. In all the eggs the peripheral protoplasm
is narrower than usual, and in the most markedly abnormal
egos it is practically absent. I have occasionally found
appearances of the same kind, but much less pronounced,
in eggs laid by other insects, but have not sufficient cases to
be able to throw any light on their meaning. |

SUMMARY.

1. True fertilisation (conjugation of male and female
pronuclei) may take place in N. ribesii, and the behaviour
of the polar nuclei is slightly different in fertilised and virgin
eges,

2. In the spermatogenesis there are eight chromosomes in
spermatogonial divisions; four “gemini” appear at the
beginning of the maiotic phase, and by heterotype and homo-
type mitoses distribute four chromosomes to each spermatid.

3. In the oogenesis eight chromosomes appear in oogonial
mitoses, but in divisions of nuclei in the ovary sheath more
than eight are found, suggesting that the chromosomes of
the germ-cells are compound. »

4. In the polar mitoses of the egg two types of maturation
are found. In some eggs there are successive equation
divisions so that the egg nucleus and each of the three polar
nuclei contains eight chromosomes. In other eggs normal
reduction takes place, separating entire chromosomes from
one another, and only four are found in each of the daughter
nuclei.

5. It is probable that only such reduced eggs are capable

Nigh 2 L. DONCASTER.

of fertilisation, but when unfertilised they may continue to
develop at least as far as the blastoderm stage.

Birmingham University;
November, 1906.

EXPLANATION OF PLATE 8,

Illustrating Mr. L. Doncaster’s paper on ‘‘ Gametogenesis
and Fertilisation in Nematus ribesii.”

All figures are drawn with an oil-immersion lens, but are not exactly on
the same scale. Those illustrating spermatogenesis are more highly magnified
than the remainder. AlJl represent Nematus ribesii except figs. 12, 13,
14.

Fie, 1.—Conjugation of male and female pronuclei. Three polar nuclei
near the edge of the egg.

Fies. 2, 3, 4.—* Polar protoplasm” of fertilised eggs showing chromo-
some groups derived from polar nuclei.

Fie. 5.—Nucleus of spermatogonium.

Fre. 6.—Spermatogonial mitoses. (a) Metaphase, side view; (B) Equ-
torial plate.

Vie. 7, a, B, c.—Spermatocyte: three prophases of heterotype mitosis.
(A) Showing 8 chromosomes; (B and Cc) Pairing to form 4 double chromatin
masses,

Vic. 8.—Heterotype mitosis, equatorial plate. (a) Pole view; (3) Side
view.

Fic. 9, A, B.—Heterotype anaphases.

Fic. 10.—Homotype. (a) Pole view of equatorial plate; (8, c) Anaphase,
side view.

Fie. 11.—Spermatid.

Fic. 12.—Young oogonium, N. lacteus.

Fies. 13, 14.—Stages of growth of oogonium, N. lacteus pupa.

Fic. 15.—Oogonial mitoses, larval ovary. (A) Pole view; (8) Side view.

Vic. 16, a—r.—Mitosis in ovary sheath with more than 8 chromsomes.
(a) Equatorial plate, pole view; (B, c) Similar stage seen from side and
obliquely ; (p, B, F) Anaphases.

GAMETOGENESISAND FERTILISATION IN NEMATUS RIBESII. 115

Fic. 17.—Second polar mitoses, equational type, with 8 chromosomes.

Fic. 18.—Second polar mitosis, equatorial plate in pole view, with 7 chro-
mosomes, some preparing to divide.

Fie. 19.—Second polar mitoses, metaphase; reduced type, with 4 chro-
mosomes.

Fie. 20.—Equatorial plate of reduced type, showing 4 double chromo-
somes.

Fic. 21.—Stage between first and second maturation divisions, reduced
type, with 4 double chromosomes each end.

Fic. 22.—Two blastoderm mitoses, each with 4 chromosomes and con-
spicuous centrosomes.

Fic. 23.—Abnormal polar mitosis.

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THE MOLLUSCAN RADULA. tS

The Molluscan Radula: its Chemical Composition,
and Some Points in its Development.

By
Igerna B. J. Sollas.

With Plate 9.

History.

THe molluscan radula, or dental ribbon, has been the sub-
ject of research for at least a century and a half. Aristotle (8),
though he speaks of teeth in Limax, alludes apparently
to the ridges on the jaw, and there is no evidence that he
knew of the existence of the radula: but it is interesting to
find that the great naturalist was well aware of the fact that
whelks bore holes in shells with the proboscis, although he
cannot have fully understood the process. Poli made a jest
of the tale as a fable, but Osler re-affirmed it in 1832 without
knowing of previous work, and is now credited with having
been the first to observe this interesting habit.

Swammerdam is the discoverer of the radula: he gives a
description of both the radula and jaw of the snail (Helix
aspersa), in Dutch and Latin, in his ‘Biblia Nature,’
Leyden, published posthumously. His death, as we are told
by Boerhaave in the Life of the author prefixed to this work,
occurred in 1680. The work is now too antiquated to possess
more than an historica! interest.

In 1757 Adanson (1) described radule from various
gastropods of Senegal: the teeth are “infinitely small, hardly

116 IGERNA B. J. SOLLAS.

visible, though sometimes perceptible to the touch. Looked
at with the microscope ... the pointed ends of the teeth
are turned towards the stomach like those of the tongue of
the lion or eat.” Adanson observed the regular arrange-
ment of the teeth, and in some cases counted them, finding
20,000 teeth in 200 longitudinal rows in a bulimoid land-
snail which the natives call “ Kambeul,”’ and 200 in 10
longitudinal rows in Patella.

Poli (18), in 1791, was the first to give a clear figure of a
radula in his magnificent work ‘ Testacea utriusque Siciliz.’

Troschel (22), in 1836, first established the radula as an
organ of great systematic importance. Curiously enough, in
the same year van Beneden, in a paper written in 1835, and
not quoted in the literature of this subject, points out the
possible value of the radula in determining the reality of
doubtful species. Troschel’s work attracted the interest of
zoologists to the radula; after an interval, in which Lebert,
Allman, and particularly Lovén, worked along the new lines,
Troschel published his ‘Gebiss der Schnecken’ (1856-1863)
—a general and masterly work now well known. His interest
was not restricted to the form of the teeth, but extended to
their chemical composition. Though Troschel was the first
to make the suggestion—thrown out apparently as a shrewd
ouess—that growth takes place at the posterior end of the
radula to make good the waste going on in front, yet he did
not follow it up by closer study, nor did he investigate the
development of the organ. It has been one of the chief
problems of later workers, but they have arrived at some-
what conflicting results. By combining a study of the
chemical composition with that of the development some of
the difficulties which have arisen may be removed.

CHEMICAL COMPOSITION.

In 1845 Hancock and Embleton (6), in a study of the
anatomy of Holis, state that the radular teeth consist of silica.
They base their conclusion on the partly mistaken observation

THE MOLLUSCAN RADULA. i fa ars

that the teeth do not dissolve in either acetic or nitric acid,
while hydrofluoric acid corrodes them. No particulars of
their experiments are given. ‘The same authors investigated
the teeth of Buccinum and came to the same conclusion.

In 1852 Leuckart (9), being interested in the distribution
of chitin in the animal kingdom, examined, among many
other objects, the radule of Gastropoda and Cephalopoda,
and pronounced them to be chitin. He emphasised the fact
that his identification of chitin rested entirely on two
characters—one its resistance to caustic alkali, the other
its solubility in boiling nitric acid. He adds: ‘It is possible
that in this sense chitin is a collective conception, and that
many special modifications will be discovered later. Perhaps
we may conclude this from the varying behaviour of chitin
when treated with alkali,’ and he expresses a wish that
chemists would investigate the matter. About the same
time Bergh (4), without knowing of Leuckart’s paper, con-
futed Hancock and Embleton’s view, and demonstrated the
absence of silica in three species of Prosobranchiate
Gastropods. Bergh’s is the first exact investigation ; we are
indebted to Troschel for a German translation of an extract
of his paper, which is written in Danish. Bergh showed that
in Buccinum antiquorum (Triton nodiferum), and in
Strombus gibberula most concentrated acids bring about
corrosion of the radula in the cold, and eventually complete
solution on boiling, while dilute hydrofluoric acid does not
alter the teeth in form, but renders them more transparent.
Incinerated ribbons of Marsenia perspicua gave no silica.
The radula of Buccinum antiquorum gave the reactions
of iron and calcium phosphate.

Troschel was dissatisfied with what he considered the con-
tradiction in the results of Leuckart and Bergh,! and there-
fore undertook with Bergemann experiments which combined
and reconciled the results of both these investigators. Helix,

1 This remark seems hardly fair to Leuckart, who nowhere states that

chitin is the sole constituent of the radula and is not interested in the ash; of
Bergh’s paper | have only read the extract given by Troschel.

118 IGERNA B. J. SOLLAS.

Patella, and Dolium were chosen for study, attention being
directed both to the jaw and radula. The radula of these
three forms was found to behave in a similar manner and
consists of an organic constituent, chitin, together with the
inorganic constituents, iron, calcium, carbonic, and phosphoric
acid. It was further shown that the radula of Helix
nemoralis contains 5 per cent. of ash, that of Dolium
galea 6 per cent.

Koehler’s paper, published (7) in the same year as Troschel’s
‘Das Gebiss der Schnecken,’ deserves a word of mention,
since this observer also affirmed the presence of both an
organic and an inorganic constituent, and suspected the
occurrence of calcium.

Later workers continue to make divergent statements in
describing the chemical composition of the radula. Sollas (19)
in 1885, when studying the nature of the silica in organisms
generally, made use of measurements of the refractive index
and specific gravity; he concluded that in the molluscan
radula silica was present and that, as in so many organisms,
it was in the form of opal (silica hydrate), but he does not
mention the species on which his observations were made.
Bloch (5) and others speak of the radula as chitin, but their
views do not appear to be based on original observation.
Similarly some modern text-books refer to this organ as com-
posed of chitin or conchiolin, others speak of it as siliceous.
Huxley and Ray Lankester, with more caution, do not commit
themselves on this point. It thus seemed worth while to look
once more for definite evidence of the presence or absence of
silica and of chitin in the radula. It will conduce to brevity
if I state at once the general results I have obtained. I find
that in all the odontophorous Mollusca the radula has an
organic basis of chitin; the Docoglossa are unique among
Mollusca in the composition of their teeth, of which the most
important constituent is silica hydrate or opal. All the other
groups, including the Rhipidoglossa, form a second type in
which the radular chitin is hardened superficially by deposits
containing calcium, iron, and phosphoric acid, which, together

THE MOLLUSCAN RADULA. 119

perhaps with an additional organic substance, form that
outer covering so long known as the enamel layer but
hitherto unexplained. I have not been able to confirm
Troschel’s statement that carbonic acid is present, and though
I have made repeated attempts, I have failed to determine
whether magnesium is one of the mineral constituents. These
points, therefore, must still be left to chemists. The Chiton-
idz present us with a deviation from the second type and
stand alone among the forms I have examined. In this
family ferric oxide is the most important mineral constituent
and is the cause of the dark colour of the teeth.

With the partial exception of Helix aspersa, the ash
of the radula preserves the form of the teeth.

In the first type of radula or that of the Docoglossa the
mineral matter may form as much as 27 per cent. of the
whole ribbon, this is the case in Patella vulgata; while in
the second type it may contribute only 2°4 per cent., as in
Helix aspersa, though in this species it sometimes rises to
3°3 per cent.; in Dolium galea it amounts, according to
Troschel’s analysis, to 6 per cent.

It is interesting to find that the Docoglossa, which are so
well-defined a group in other respects, differ so widely, not
only from the Pectinibranchiata, but also from the Rhipido-
glossa in the composition of the radula.

We may commence our more detailed account with the
Docoglossa, and first with

PATELLA.

If the radular ribbon of Patella is boiled in strong nitric
acid, the organic parts are completely dissolved, and there
sinks to the bottom of the test-tube a coarse-grained insoluble
residue. Microscopic examination shows that this consists
of the dark red brown cusps or free biting ends of the lateral
teeth (fig. 1), together with some thin, colourless, transparent
pieces ; some of these latter are free, some remain attached
to the cusps, showing that they are the skeleton of the basal

120 IGERNA B. J. SOLLAS.

part of the lateral teeth. This last point may be confirmed
by performing the solution in the cold, when all the hard
parts are left in their natural relative positions. Prolonged
boiling with nitro-hydrochloric acid or hydrochloric acid and
potassium chlorate in an open test-tube failed completely to
remove the dark red-brown colour of the cusps; to accomplish
this the teeth must be placed with one of these solvents in a
sealed glass tube and heated in a water bath for some days.
After this treatment some of the cusps become completely
freed from their iron content and appear perfectly colourless
and transparent; others, however, after digestion for an
entire week retain some of their red-brown colour.

The specific gravity of the teeth thus prepared was
ascertained by means of a diffusion column,! in which the
majority were found to float in a dense zone at a level corre-
sponding with a specific gravity of 1:98, though numerous
examples ranged on each side of this to 1°87 on the one
hand and 2°08 on the other. ‘his variation in specific
gravity corresponds with a difference in the degree of hydra-
tion of the silica: experiments made on colloid silica show
that when this is prepared from water glass it possesses a
specific gravity of 1:86 and contains 16°3 per cent. of water ;
when obtained from silicon fluoride, its specific gravity is
1:98, and the water amounts to 9°85 per cent.; while sponge
spicules with a specific gravity of 2°04 contain 7 per cent. of
water. Thus it would appear that the water in the siliceous
basis of the Patella teeth varies in amount from 7 to 16 per
cent. with a mean of 9°85 per cent.

It is interesting to observe in this connection that the
association of water and silica in the silica hydrates occurs
without any change of volume in either of the constituents,
and thus the proportion of water in the hydrate can be
directly calculated from the specific gravity. In the follow-
ing table the results of such a calculation are given for a

1 For an account of this method see Sollas, W. J., “ Physical Characters

of Calcareous and Siliceous Sponge Spicules,” ‘Proc, Roy. Soc. Dublin,’
vol, iv, p. 878, 1885.

THE MOLLUSCAN RADULA. Leal

small number of cases, the specific gravity of amorphous
silicon dioxide being taken as 2°21.

Si0,, H,O, water 23 per. cent., sp. gr. 1°73 (SiO,)3, (H,0),,
water 16°6 per cent., sp. gr. 1°85.

(Si0,),, H,O, water 13 per cent., sp. gr. 1:9; (S8i10,), H,O
water 9-1 per cent., sp. gr. 2°0.

(Si0,),, H,O, water 7 per cent., sp. gr. 2°04.

Under the microscope the teeth which have been treated
as described above present a faint brown granular appearance
when seen by transmitted light and a bright milky white by
reflected light: in this behaviour, which results from the
abundant presence of minute pores, they resemble common
opal. Between crossed Nicols they exhibit faint but evident
double refraction with undulose extinction; the same
character is sometimes presented by mineral opal, and is
attributed to internal stress, the existence of which in the
teeth is suggested by their lability to spring apart along
fractures parallel to their length.

The refractive index of the siliceous residue of the teeth
was determined by Becke’s method and found to be closely
approximate to 1°45; the fluids used were mixtures, one of
10 parts, the other 20 parts of Price’s glycerine, to 1 part
of water. Taking the refractive indices of these mixtures to
be 1:449 and 1:454, the above result is obtained.

Teeth from which the iron has not been completely ex-
tracted are distinguished by a relatively high specific gravity ;
under the microscope the ferric hydrate or oxide presents a
blood-red colour by transmitted light and marked double
refraction.

The ribbon of Patella, when soaked for some hours in
strong hydrofluoric acid, becomes colourless or nearly so
throughout, but the forms of the teeth are perfectly pre-
served. If it is next boiled in nitric acid it dissolves com-
pletely, as might be expected, since the siliceous matter has
already been removed by the hydrofluoric acid. On incinera-
tion the ribbon retains its form with a surprising completeness.
The ash proves to contain, in addition to silica already

122 IGERNA B. J. SOLLAS.

mentioned, a noticeable quantity of calcium, iron, magnesium,
and phosphoric acid.

The radula after treatment with hydrofluoric acid proves
very resistant to prolonged boiling in caustic potash (5 per
cent. to 40 per cent.) ; this suggests that it is composed of
chitin. With a view to testing this, radule were treated
with hydrofluoric acid, boiled for a considerable time in a
5 per cent. solution of caustic potash, which was frequently
changed, and were finally extracted with water, absolute
alcohol, and ether. The specific gravity of the organic
portion of the radula which remained after these processes
was the same as that of chitin obtained from the carapace of
Astacus, and subjected to the same treatment—viz. 1°40.
The specific gravity was determined by a diffusion column
formed of chloroform and absolute alcohol. I find that chitin
from various sources has a specific gravity differing but
slightly, if at all, from that of Astacus, and I hope to publish
details on this subject shortly. The refractive index of this
organic basis of the radula was found to lie between the same
limits as that of chitin, viz. 1°550 and 1°557.

There is an interest of a general nature attaching to the
fact that the radula of molluses has a chitinous basis, since
Bloch has brought forward special arguments (5) to prove
that this membrane is not produced by the direct metamor-
phosis of the formative cells, but by secretion. Bloch is able
to cite a number of workers who share his view, and he
refutes the arguments of Wiren, the only supporter of
Trinchese in the suggestion that the radula is formed by
direct metamorphosis of cell protoplasm. Chatin, on the other
hand, in 1892, maintained that the chitin of Libellulid larvee
is formed by direct transformation of the chitinogenous cells.

After soaking in hydrofluoric acid the entire ribbon and
teeth stain with borax carmine and other preparations, though
with differences in intensity in the various parts. When the
fresh radula is treated with the ordinary stains this is not
the case,as may be seen from fig. 8. The ribbon in this
figure has been stained first with Bethe’s stain and afterwards

THE MOLLUSCAN RADULA. 123

with borax carmine. Bethe’s stain acts readily on those
parts which resist borax carmine and remain unstained in
ribbons treated with borax carmine only.!

The action of hydrofluoric acid renders it possible to make
sections of the radula; these reveal the organic matter as a
solid basis having the form of the complete structures. With
the ordinary stains the cusps are less deeply coloured than the
roots of the lateral teeth (text-fig. 1), with Bethe’s stain the
bases of the roots of these teeth and parts of the marginals

TExt-Ficure 1.—A longitudinal section of the radula of Patella
vulgata after treatment with strong hydrofluoric acid and staining
with iron hematoxylin. ¢. s. m. Tensor superior muscle. 4, e. Basal
epithelium, c¢, Organic basis of the cusps. 7. e. Roofing epithelium.

1 Bethe’s method: the object is placed in a 10 per cent. aqueous solution
of anilin hydrochloride to which one drop of fuming hydrochloric acid is
added for every 10c.c. of water. After washing thoroughly the object is
transferred to a 10 per cent. solution of potassium bichromate. It will be seen
that at the young end of the Patella ribbon each row of teeth is uniformly
coloured by the carmine stain, the youngest teeth are paler than those imme-
diately in front of them. In front, again, of the darker red teeth the roots
of the laterals and centrals (inner laterals) are coloured green by Bethe’s
stain, their cusps being still red like the marginals. Finally, as we pass
forwards, the cusps become golden-yellow and then red-brown owing to the
presence of iron oxide, and the innermost marginal bears a band of green.
The contrast in staining properties between the marginals and remaining teeth
is very striking. The basal membrane itself is red, but in the preparation it
has been removed as far as possible, as it obscures the differentiation in
specimens mounted whole. These differences in staining properties are to be
found with greater or less deviations in the radula of other odontophorous
molluscs,

124 IGERNA B. J. SOLLAS.

alone are coloured; so that in this case there is a curious
reversal of the usual relative behaviour of these stains. I
have not found any other case in which Bethe’s stain will
colour in section a structure which has an affinity for the
ordinary staining reagents, though this is commonly the case
in working with material in bulk.

The above description refers to Patella vulgata; it is
equally applicable to P. pellucida, as well as to a number
of other species of Patella sent me by the kindness of Pro-
fessor Mitsukuri, and probably to all species of Patella. To
Professor Mitsukuri I am also indebted for specimens of
Nacella, the teeth of which, isolated by boiling in fuming
nitric acid, are shown in Fig. 6. The marginals, as in
Patella and all Docoglossa examined, do not contain a sili-
ceous skeleton; they dissolve completely in nitric acid and
are consequently not represented in the figure.

CRYPTOBRANCHIA.

My thanks are due to Mr. Rathbun, of the United States
National Museum, for specimens of Cryptobranchia con-
centrica. The lateral and central teeth possess a skeleton
of siliceous pieces which are fused in each row into a single
plate, but it is possible in this plate to detect outlines which
seem to mark the limits of once separate teeth. Apparently
there were three laterals and one central, the central tooth
being much reduced. In the darkly coloured cusps the line
of demarcation between the outer and inner laterals is clear,
but the two inner laterals of each side are closely fused
together (text-fig. 2).

ACMAEA,

Specimens of Acmaea virginea were obtained from
Plymouth. I owe to the kindness of Dr. Harmer examples of
Acmaea saccharina, as well as of many other molluses.
Marginals being absent in this genus the greater part of the
radular substance is silica; the solution of the organic matter

THE MOLLUSCAN RADULA. 15

sets free from the portion corresponding to each row of teeth
a pair of substantial siliceous plates of rectangular outline
when seen en face. On each of these are seated the red-
brown cusps of the lateral teeth. The cusps are detached
from the plates by further boiling. The basal pieces of each

Text-Ficurs 2.—A siliceous basal plate and the cusps carried by
it, isolated from the radula of Cryptobranchia concentrica by
the action of boiling nitric acid. ss’ Surfaces of attachment of the
cusps aud plate to one another.

side of the radula, though free from those of the other side,
are closely fused among themselves, leaving no trace of the
outlines of separate pieces.

The teeth, after treatment with nitric acid, have a refrac-
tive index which closely approaches that of chloroform; 1:4
being a little lower in the case of A. virginea, and a
little higher in that of A. saccharina, there is a corre-
sponding difference in the value of the specific gravity, which
was found to be 2:1 in the latter and 20 in the former species.
It is open to doubt, however, whether this difference is con-
stant, for the examples investigated were far from numerous,

126 IGERNA B. J. SOLLAS.

LEPETA.

I take this opportunity of thanking the Rev. Professor
Gwatkin and Dr. O. Nordgaard for examples of this genus.

Lepeta coeca.—The siliceous basal pieces of each row
are united into a single piece as in C. concentrica, and as
in that species the basal plate is divided by longitudinal lines
into six areas, whereas the coloured cusps appear to be
formed by the union of four pieces. In this case, however,
the double basal piece belongs to the outer cusps.

RAIPIDOGLOSSA.

Classified according to the chemical composition of the
radula, these forms belong to the second type mentioned
above (p. 118), in which the proportion of mineral matter in
the radula is small and does not include silica.

In all the forms belonging to the second type which I have
investigated the teeth can be isolated from the membrane by
cold nitric or hydrochloric acid. Teeth of Trochus zizi-
phinus freed in this way and washed were found to have a
refractive index lower than 1°557, and higher than 1°550.
The same is true of all the radule of this type. The
refractive index of chitin hes between the same limits.

Fig. 12 shows the result of staining the radula of Trochus
ziziphinus with hematoxylin; it is necessary to isolate the
teeth by teasing, as the membrane stains darkly and the teeth
are so close-set that it is impossible to make out details in the
radula stained intact. All the teeth take the stain at their
roots; in the series of marginals the length which takes the
stain increases as we pass outwards along a transverse row,
while, finally, on the outer side of the marginals is a paddle-
shaped piece made up of five flat pieces, placed edge to edge
(the flabelliform teeth). This piece stains completely, and
contrasts with the teeth lying to its inner side. This contrast
is most marked in the younger parts of the radula. The

THE MOLLUSCAN RADULA. 127

marginals pass by many gradations of form into the lateral
teeth. These outermost pieces, on the other hand, present a
sudden change in the series of marginals, being longer than
their neighbours, and agreeing in their staining properties
with the marginals of Patella. In fact, the reactions to stains
almost tempt us to suggest that the teeth generally regarded
as marginals are multiplied laterals, and that the marginals
are represented by these deeply staining teeth at the outer-
most edge of each row.
Haliotis and Emarginula give somewhat similar results.

TNIOGLOSSA.

Littorina littorea furnished the chief and most con-
venient material for the investigation of this group. The
radule were dissected out from a thousand individuals of
the species, and dried at 100° C.; they weighed 0:430 grm.,
and afforded, on incineration, 0°0158 grm. of ash, or 3°7 per
cent. This was found to contain iron, calcium, and mag-
nesium. A second experiment was made on the ribbons of
several thousand individuals, from which 0:0508 ash was
obtained ; this yielded 0°0083 of phosphoric acid, or rather,
of P,O;, corresponding to 16°3 per cent. or to 35°6 of calcium
phosphate. A contrast between the staining reactions of the
marginals and the central and lateral teeth exists here as in
the case of Patella, though it is less marked (fig. 9), and the
teeth run through the same stages as regards staining
capacity during their growth as in that genus, but in the
long radula sheath it is noticeable that the teeth retain for a
long time their affinity for the common stains, and only quite
near to the mouth-cavity become green under the action of
Bethe’s stain. Their great hardness is discovered when an
attempt is made to cut sections of the buccal mass. Previous
treatment with nitric acid is necessary for success in this
process.

128 IGERNA B. J. SOLLAS.

RHACHIGLOSSA.

In Buccinum undatum, as there are no marginal teeth,
the ribbon, doubly stained by the above method, does not
become marked out, as in previous cases, lato a green median
region, bordered on each side by a red marginal band; none
of the teeth in the fully developed parts of the radula are
coloured by protoplasmic stains, but all take Bethe’s stain.
It is noticeable that the teeth are very rapidly matured—a
necessary property in a tongue which must be quickly worn
out. In a specimen of Purpura lapillus I found every
one of the teeth in the mouth-cavity with cusps broken off.
It would seem as though the molluscan radula had become
adapted to hard work in two distinct ways: in the one case
it becomes very resistant through a long-continued process
of development—witness the long radula sac—in the other
a less strong radula is rapidly worn out and as speedily
renewed.

PULMONATA.

Helix aspersa.—A remarkable discrepancy was found to
occur in the behaviour of the radula of this species when
incinerated on different occasions. Several lots containing
from twenty to one hundred and fifty radule were heated, in
some cases with an ordinary Bunsen burner in a platinum
crucible, in other cases with a Herapath. These experiments
were all made in winter-time, and all gave the following
result: the radula at first preserved its form in ash, but soon
fused with continued heating: it gave an exceedingly minute
fusible drop, which solidified to an enamel-like glass, yellow
when hot, white when cold. In winter, also, the ash was
always found to contain some silica: the ash from some 150
ribbons was boiled in fuming nitric acid, in this reagent it
proved only partially soluble; the insoluble residue was

THE MOLLUSCAN RADULA. 129

washed in water and returned to the platinum spoon and
again heated: it was then infusible and was presumably
silica. In a quantitative analysis the amount of silica was
found to be about one third of the total ash. In one case, in
the month of April, 500 radule were incinerated with a
Bunsen and the ash afterwards ignited in a Herapath with a
dissimilar result from the preceding; no fusion of the ash
occurred, and in the analysis which followed no silica was
found. On the other hand, the presence of a large quantity
of phosphoric acid was indicated: the ash, which weighed
0°0101 grm., yielded 0:0036 germ. of P,O;, or no less than
30°6 per cent.

DEVELOPMENT.

The history of the study of the development of the radula
has been so excellently set forth by Réssler that on this point
I cannot do better than refer to his paper (15).

It has already been stated that there is a certain amount of
disagreement among those who have studied the growth of
the radula. The radula passes backwards into the radular
sac, which is the seat of its renewal ; 1t exhibits a continuous
forward movement due to growth from behind taking place
simultaneously with wear of the front end which is in use.
The radular sac is an evagination of the wall of the foregut ;
it consists of a basal epithelium and a roofing epithelium ;
these two epithelia pass into each other by way of a heap
of cells at the posterior end of the sac. In front of this cell
aggregate, immediately behind and below the last formed
tooth, are a set of cells, the odontoblasts, which may be few
and large or more numerous and small. The roofing epi-
thelium extends between the young teeth at the posterior end
of the sac, fitting closely into the spaces between them ; it,
in many cases, secretes a cuticular substance in the anterior
portion of the sac which likewise fits into the spaces between
the teeth, so that the teeth lie in pits of this secretion. All

voL. 51, PART 1.—NuEW SERIES. 9

130 IGERNA B. J. SOLLAS.

are agreed that the odontoblasts give origin to the teeth, but
some would have it that the roofing epithelium secretes a kind
of enamel or hard outer layer which is spread over the
surface of the tooth originally laid down by the odontoblast.
Among those who take this view are Réssler (15), Riicker (14),
Sharp (18), and Bloch (5). Bloch describes the enamel layer
as consisting of specially hard cuticular substance. Two
writers, more recently—Rottmann and Schnabel—deny the
presence of any such enamel, and maintain that the teeth are
laid down from the first in their definitive form and size, and
that the roofing epithelium contributes no substance whatever
to the radula, and is not to be regarded as secretory. There
is further difference of opinion as to the exact method by
which the forward movement is brought about, and on other
points.

The facts which have been already stated in dealing with
the Docoglossan and other radulee place beyond all doubt the
importance of the roofing cells of the radular sac in all
cases; in the case of Patella the lateral teeth as_ first
formed are soft and colourless, and it is only those situated at
a considerable distance from the odontoblasts which betray
by their yellow colour the presence of iron oxide, and this
must have come from the roofing cells. The other changes in
the maturing teeth all point to the secretory nature of the
roofing epithelium, a function which is strongly suggested
& priori by the conspicuous accuracy with which the cells
of this epithelium fit-in between the teeth, leaving not the
minutest portion of tooth or membrane surface untouched.
Schnabel and Rottmann were working with material other
than Docoglossa, namely with Gastropoda and Cephalopoda.
Therefore it will be worth while to consider at any rate one
of these cases in greater detail. That some considerable
changes do take place in the teeth after their first formation
is already clear, but the changes as seen in microscopic
sections are interesting and afford some further lght.

The young teeth, as we know from dissection are soft; they
take protoplasmic stains but slightly, and agree in this with the

THE MOLLUSCAN RADULA. 1)

part of the membrane on which they are seated. They show
lamination, the laminze running parallel to the matrix cell
(Réssler’s). Soon, as we pass forwards along the ribbon,
there is a sharp contrast in staining properties between the
young teeth and the underlying part of the basal membrane
(text-fig. 3), but this contrast is not maintained, the increased

Text-Ficure 3.—Longitudinal sections of the radula of Helix
aspersa, stained with hematoxylin, showing four stages in the
development of the teeth. @, the youngest tooth of the radula; d,
a tooth from the mouth-cavity; 4 and ce, intermediate stages.
(Drawn with Zeiss camera lucida, Zeiss obj. D., eyepiece 4.)

staining power spreading downwards through the thickness
of the membrane but always leaving a thin layer which
preserves the original resistance to stains. At the same
time that the membrane darkens the teeth, which nearer the
origin were uniformly darkly stained, acquire a lighter peri-
phery, the core remaining darkly stained; finally the surface
layers become quite colourless and only some scattered small
spheres of darkly staining matter remain in the interior.
These spheres are arranged with a certain definiteness in rows
which converge towards the posterior basal part of the tooth.
Such are the appearances seen in sections treated with a
protoplasmic stain only, whether hematoxylin, safranin,
borax carmine, or carmalum. If we first apply Bethe’s stain
and then safranin or carmalum, the whole section will be
coloured pink with the exception of the cores of the adult
teeth, which are green, and the surface layers of the same,
which are colourless. These outer layers are Sharp and
Rossler’s enamel. Réssler made the interesting note that
they are not doubly refracting, while the other parts of the

132 IGERNA B. J. SOLLAS.

radula are so. But they are evidently not formed, as Réssler
suggested, as a special secretion apposed to the outer surface
of the tooth as first formed, but rather by intussusception.
With regard to other doubtful points, Réssler assumed that
the same odontoblasts secrete all the teeth that are produced
during life; but later writers have thought that these cells
must be periodically replaced, for, as Bloch points out, “unless
we suppose the odontogenous cells to be replaced from time
to time we cannot understand how larger teeth are formed
with the growth of the animal, for it is unlikely that cells
continually engaged in active secretion can also grow”
(p. 381). The new odontoblasts, according to Bloch, are
formed out of the same cell complex from which the upper
epithelium is renovated. This assumption, he thinks, con-
tains no improbability “since the upper epithelium and the
odontoblasts have the same task, namely, the secretion of
chitin.’ But from what has preceded it is clear that the
functions of the roofing epithelium and of the odontoblasts
are very different; this need not, however, impair Bloch’s
main argument that the odontoblasts are replaced; there is
no reason why a single cell complex should not give off
chitin-secreting cells on the one hand and cells secreting
mineral matter on the other. ‘The single apical cell of the
stems and roots of various plants does more than this.
Again, Réssler considered that the radula must glide forward
to a certain extent over the basal epithelum, which he said is
delayed by the action of the retractor muscle (tensor superior
muscle of Amaudrut). Though I do not agree with his view,
Rossler, in alluding to the action of the muscle, seems to me to
have touched on an important problem which has been quite
neglected by some authors. To this I shall return presently.
Bloch objects to Réssler’s view: “ Ich kann mir nicht denken
wie die Basalplatte, die’ die Zihnchen tragt, und nach den
Praparaten mit ihrem Epithel in innigen Verbindung zu sein
scheint, tiber diesen Zellschicht hinweggleite. Da ist nur
eine Moglichkeit niimlich die Zelle bewegen sich mit der
Basalplatte nach vorn,” In the paragraph which follows he

THE MOLLUSCAN RADULA. 133

seems to say that the two structures keep pace in the younger
end of the radular sheath, but that later the epithelium may be
delayed. This admission amounts to granting that the epi-
thelium throughout its length cannot keep pace with the
membrane except by stretching—if this word can be permitted
to stand for the conversion of the high columnar cells of young
basal epithelium into lower cells. For if equal increments of
epithelium and of membrane are added in the radular sac, then
clearly there will be no gliding of one structure over the other
in any part of their length: they will keep pace with each
other. If, however, the increments of epithelium are less, then
cells of the epithelium must “stretch” or increase in volume
in order to keep pace with the overlying membrane. It is
highly probable that the increments of epithelium are less than
those of the membrane, for in the mouth-cavity the epithelium
is generally lower than in the sac; in Helix aspersa one cell
in the basal epithelium in the mouth-cavity covers as great a
length of membrane as four cells in the sac. Consequently if
we were to assume that equal lengths of epithelium and of
basal membrane were added in the sac, this would involve a
considerable relative movement of the epithelium and membrane
in the mouth-cavity, the epithelium moving more quickly than
the membrane, and this is wholly unlikely.

That the odontoblasts are replaced by fresh cells derived
from the cell aggregate at the extremity of the radular sac
seems most probable and is, I think, the view which has gained
acceptance; at the same time, investigators differ among them-
selves as to whether the replacement is gradual, so that each
group of odontoblasts secretes several teeth before it passes on
into the basal epithelium, or, more sudden, each odontoblast
group only secreting once before it is relieved by recruits.
On full consideration this view will be found to involve
a somewhat remarkable life-history for the odontoblasts.
Starting from the indifferent cell mass from which they arise
by cell-division, they become elongated and form a set of cells
which possess as a whole a definite and constant shape. They
secrete chitin first for the teeth, next for the basal membrane

134 IGERNA B. J. SOLLAS.

and are then described as exhausted. But they now pass on and
become the youngest cells of the basal epithelium, shortening
till they are of a uniform height with their neighbours, They
then travel forward, adhering to the basal membrane and
gradually shorten still further. As they continue their course
they encounter, as they leave the radular sheath to enter the
mouth-cavity, the superior tensor muscle of Amaudrut (2).
Now they have to play anew and arduous role: they must
adhere to the radular membrane on the one hand and make
connection with the tensor muscle on the other, and their
tensile strength must be at least as great as that of the pull
from the muscle. Strange to say, they next become liberated
from the muscle again and pass forwards, now as a low epi-
thelium, until they encounter another muscle, the inferior
tensor, when some of them become connected with it. After
this they once more move forwards to form part of the walls
of a groove (the sublingual groove of Réssler) which is the
natural outcome of the mode of growth of the radula and which
allows of the free play of the buccal cartilages in eating.

In conclusion I wish to express my indebtedness to Professor
Sollas for much suggestion and help in carrying out the work
contained in this paper, particularly in connection with the
observations on the relation between the specific gravity of
silica hydrate and its degree of hydration.

BIBLIOGRAPHY.

. Apanson.— Histoire Naturelle du Sénégal,’ 1757.

. AMauprut.— Ann. Sci. nat.,’ (8), vii, 1898, pp. 1-291.

. ARISTOTLE.—‘ De Animalibus Historia,’ iv, 4, 7, 8, 9.

. Bercu.—Extract in Troschel (28) from ‘ Konigl. Danske Videnskabernes
Selkabs Skrifur,’ 5th Raekke, 3 Bind.

. Brocu.— Jena Zeitsehr.,’ xxx, 1896, p. 350.

. Hancock anp Empieton.—‘ Ann. Nat, Hist.,’ 1845, xv, p. 9.

. Konuter.— Zeitschr. Naturw.,’ 1856, viii, p. 106.

. Lesert.—‘ Miiller’s Archiv,’ 1846.

, Levckxart.—‘ Arch, Naturg.,’ 185], i, p. 25.

POND

OoOnN DD A

THE MOLLUSCAN RADULA. 135

10. Lovin.—‘ Ofv. Ak. Forh.,’ 1847.

ll.v .MippEnporr.—‘ Mem. Ac. St. Petersb.,’ 1847.

12. W. Ostur.—‘ Phil. Trans.,’ 1832, p. 97.

13. Pott.—‘ Testacea utriusque Sicilie,’ 1791.

14, RuckEer.—‘ Ber. Oberhess. Ges.,’ 1883.
15. Rosstur.— Zeitschr. wiss. Zool.,’ xli, 1885, p. 447.

16. Rorrmany.—Ibid., Ixx, 1901, p. 651.

17. ScunaBeEt.--Ibid., Ixxiv, 1903, p. 651.

18. SHare.—‘ Inaug. Dissert. Wiirzburg,’ 1883.

19. Sottas.—‘ Proe. Dublin Soe.,’ 1885, n.s., vol. iv, p. 374.
20. SterKi.—‘ Proc. Amer. Phil. Soc.,’ 1893.

21. SwamMERDAM.—‘ Biblia Nature,’ Leyden, 1737; ‘Bibel der Natur

Leipzig, 1752; ‘ Book of Nature,’ 1757.

22. TroscnEL.—‘ Arch. Naturg.,’ i, 1836.

23. TroscueL.—‘ Das Gebiss der Schnecken,’ 1856-1863.
24. Van BenepDEN.—‘ Ann. Sci. nat.,’ 1835.

EXPLANATION OF PLATE 9,

Illustrating Miss Igerna B. J. Sollas’ paper on “The Molluscan
Radula: its Chemical Composition, and Some Points in
its Development.’

Tic. 1.—Lateral teeth of Patella vulgata, isolated by nitric acid. 1,
Two inner laterals (with broken cusps), showing the overlap of the basal
pieces ; 2, an outer lateral; 3, an inner lateral; 4 and 5, basal pieces from
which the cusps have become detached.

Fic. 2.—Lateral teeth of Patella vulgata which have been subjected to
the action of nitro-hydrochloric acid in a sealed tube.

Fig. 3.—A portion of the ash of an incinerated radula of Patella vul-
gata.

Fig. 4.—a, A siliceous basal plate from the radula of Acmaea virginea
bearing three cusps, isolated by nitric acid. 4, c, The same less magnified and
showing the three cusps separated from the basal piece, by further action of
nitric acid, and adhering together.

Fic. 5.—A siliceous basal plate from the radulaof Acmaea saccharina
bearing cusps.

136 IGERNA B. J. SOLLAS.

Fic. 6.—Lateral teeth of Nacella sp. a, Outer; b, inner lateral tooth.
The portion a’ becomes isolated (by boiling acid) from a, leaving a’.

Fic. 7.—A lateral tooth of Chiton sp. isolated from the radula by the
action of strong cold nitric acid, showing the hollow chitinous basal portion
and the brown cusp which contains iron oxide.

Fic. 8.—Portions of the radula of Patella vulgata in order of succession
(a—d) from the radula sac to the mouth cavity. Bethe’s stain and carmalum.

Fic. 9.—Portions of the radula of Littorina littorea in order of suc-
cession (a—d) from the radula sac to the mouth-cavity. Bethe’s stain and
eosin.

Fic. 10.—Portions of the radula of Buccinum undatum in order of
succession (a—d) from the radula sac to the mouth-cavity. Bethe’s stain and
eosin. |

Fic. 11.—A rough sketch of a strip of the radula of Trochus ziziphinus
including the marginals and laterals of three rows of one side. Stained with
carmalum.

Fic. 12.—Isolated teeth teased out of a radula of Trochus ziziphinus
which had been previously stained with hematoxylin.

Fic. 13.—Hight teeth from two rows of the radula of Helix aspersa, in-
cluding two centrals. Bethe’s stain and eosin ; surface view.

Fic. 14.—Two teeth and the underlying basal membrane of Helix
aspersa in longitudinal section. Bethe’s stain and safranin.

Fic. 15.—Three teeth and the underlying basal membrane of Helix
aspersa in longitudinal section. Stained with safranin only.

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 137

Observations on Tooth-Development in
Ornithorhynchus.

By
J. T. Wilson,
Professor of Anatomy, University of Sydney, N.S.W.,

and
J. P. Hill,
Jodrell Professor of Zoology, University College, London.

With Plates 10—12.

(1) Inrropuction.

Sivce the appearance of Professor E. B. Poulton’s first
announcement (1) of his discovery of true teeth of mammalian
type ina mammary-feetal specimen of Ornithorhynchus, several
papers have appeared dealing with the dentition of this animal.
Only one of these (2), embodying a more detailed account,
by Poulton himself, of his own investigations, concerns the
developing teeth prior to their eruption. The extreme
difficulty of procuring the necessary material has hitherto
stood in the way of further research in this direction. The
stage investigated by Poulton is accordingly the only one in
which, so far as we are aware, there is any record of the non-
erupted and immature dentition.

The observations herein set forth concern two specimens
of mammary foetus of Ornithorhynchus. One of these is
practically identical in its grade of development with Poulton’s

VoL. 51, PART 1.—NEW SERIES. 10

138 J. T. WILSON AND J. P. HILL.

stage, or perhaps very slightly younger; the other belongs
to a distinctly earlier period.

In this communication we do not propose to enter into any
detailed discussion of the original literature dealing with the
dentition of Ornithorhynchus. This, indeed, consists merely
of Poulton’s two papers (1, 2), and of papers by Mr. Oldfield
Thomas (4) and Professor Charles Stewart (5), on the
characters and condition of the erupted teeth in the adol-
escent animal. Such points as require attention will receive
special notice in the course of the paper.

The material which Professor Poulton had at his disposal
seems to have represented three distinct specimens from the
collection of Professor W. K. Parker, but of these two were
represented only in a fragmentary way. All the specimens
would seem to have belonged to the same period of develop-
ment.

In the stage described by him Poulton found that there
were present in the upper jaw “‘ three considerably developed
and large teeth.” These were already multicuspidate, with
the principal cusps calcified. In addition to these, and
immediately behind and to the inner side of the posterior
one, he found another tooth-rudiment ina very early stage of
development.

In the lower jaw he found corresponding tooth-structures,
except that corresponding to the anterior tooth of the upper
jaw. The actual presence or absence of the lower opponent
of the latter could not be definitely ascertained owing to
imperfection of the material, but its existence was inferred
as highly probable. The presence of four teeth was thus
regarded as established, with certainty in the upper jaw, and
with great probability in the lower.

Subsequently Oldfield Thomas (4) and later Charles Stewart
(5) described the appearance of the fully-developed teeth in
the half-grown animal. From Stewart’s description and
figures it appears that three teeth are erupted in the lower
jaw, of which the .anterior two are very large and multi-
cuspidate, whilst the posterior one is very much smaller.

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 139
The latter, it is clear, must derive its origin from that small,
papillated enamel-organ which Poulton described and figured
in his mammary-feetal stage. The two large anterior teeth
in Stewart’s specimen are, of course, the same as the two
large and partly calcified enamel-organs present in Professor
Poulton’s specimen. In the upper jaw Stewart found “a
small, oval, soft papilliform elevation, which apparently
corresponded with the most anterior of the three pairs of
teeth found by Mr. Poulton,” whilst the second and third
very large foetal teeth were represented by large multicuspi-
date teeth, like the two anterior teeth in the lower jaw, to
which teeth they appear to correspond. On the other hand,
in the upper jaw there appears to be no representative, in the
adolescent animal, of the enamel-organ representing the
fourth tooth stated by Poulton to be present “in a very early
stage ” or condition of development, and corresponding to the
early papillated enamel-organ which he figures from the lower

.

jaw.
Professor Stewart concludes that ‘the complete dental
3—3
formula would, as far as at present known, be =——., as sur-
d >) 3 Bre 9?

mised by Mr. Poulton.” These last words are surely due to
an oversight, for Professor Poulton clearly expresses the
4— 4°

Nevertheless, the above analysis of Professor Stewart’s
contribution indicates that it is far from certain that the
actual teeth of the upper jaw in the adolescent animal corre-
spond, each to each, with the three teeth of the lower jaw.
It is obviously rather improbable either that the large anterior
tooth of the lower jaw should be the genuine opponent of the
small anterior tooth of the upper jaw, or that the small
posterior tooth of the lower jaw should, similarly, correspond
to the very large tooth which is the posterior one in the upper
jaw of the adolescent animal. And we have also to take
account of Poulton’s definite statement that he found behind
the second large tooth of the upper jaw a tooth-rudiment in

surmise that the complete dental formula should read

140 J, Te WILSON AND ‘J. P. HILL.

a very early stage of development, and corresponding with
the one he actually figures in the lower jaw.

From an investigation of the dentitional condition of a
foetal Ornithorhynchus of a stage closely resembling that
examined by Mr. Poulton, we are enabled, as we believe, to
throw some additional light on the subject under consideration.

This specimen (designated as “ Beta” in our collection)
measured 250 mm. along the dorsal curvature of the body
from the tip of the snout to the tip of the tail, and 107 mm.
measured in a straight line from the vertex of the head to
the dorsal convexity of the tail. This latter measurement is,
however, practically worthless for comparison, since it depends
on the acuteness of curvature of the specimen, and this is by
no means constant. Poulton’s specimen was stated to be
83 mm. long “in the curled-up attitude in which it had been
received,” fixed by the alcoho]. From the published drawing
of the specimen (8, Pl. XV a) we estimate its dorsal contour
line from tip of snout to tip of tail to have been practically
the same as our own, viz. 250 mm.

Our specimen was in excellent condition. Its head was
removed and divided into two portions shghtly to one side
of the median plane by an incision which was not quite
parallel to the latter. The major (left-hand) portion was
imbedded in celloidin and decalcified in nitric alcohol. The
smaller (right-hand) portion was also decalcified and imbedded
in paraffin. Serial transverse sections from both sides of the
head were thus available for examination.

In addition to this specimen “ Beta” we have also investi-
gated the dentitional condition of a younger “ mammary
foetus,” whose external characters were minutely described
and figured by one of us a number of years ago (9). The
dorsal contour-line of this specimen (catalogued as “ Delta”
in our collection) measured 80 mm. from tip of snout to tip
of tail. Both sides of the head of this specimen have been
examined in complete transverse sectional series. ‘The
sections from one side are thick, and were cut in celloidin
and stained in picric-hematoxylin. ‘Those from the other

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 141

side are thin (11) and were cut in paraffin and stained in
borax-carmine.

(2) Description or Tar PHAse or TootH-DEVELOPMENT SEEN
IN THE YOUNGER Specimen (“ Delta’’).

The phase of tooth-development which we find to be
manifested in our younger specimen is one of which no
account has hitherto been available for Ornithorhynchus. It
is characterised by the presence of a continuous dental lamina

Trxt-Fic. 1.

w x
WwW

dv

z= rN :
y ’ . ,
\ 1 aN :
' , = :
y ' :
1

A oy
dv Ww x

throughout a considerable extent of both jaws. The lamina
begins, in the upper jaw, about 5°3 mm. behind the tip of the
snout, and extends backwards for a distance of 2°6 mm. The
lamina in the lower jaw begins about half a millimetre in
front of the plane of its commencement in the upper jaw, and
extends backwards to end at a distance of 2°8 mm. further
back.
Text-fig. 1 represents a scheme of the organisation of the
dental lamina in the two jaws at a magnification of about
18 diameters. From this it is evident that the lamina in
each jaw shows two definite papillated enamel-organs. These
are in the early stage at which practically the entire thickness
of the lamina is involved in their production, prior to the

142 ; J. T.. WILSON AND J. P. HILL.

emancipation from them of a “residual dental lamina”? or
“ Hrsatzleiste ”’ (Pl. 10, figs. 1, 2, 3).

The two enamel-organs in each jaw occupy very unequal
lengths of the dental lamina. As shown in text-fig. 1, the
anterior of the two enamel-organ (“‘w”’) is considerably less
elongated antero-posteriorly than the one bebind it (“x”).
There is also a slight difference in height in favour of the
posterior enamel-organ, but there is very little difference
between their dimensions measured in the coronal plane—i. e.
transversely to the length of the dental lamina. The form of
the anterior enamel-organ further shows a difference from
that of the posterior one. Its papilla is relatively more elon-
eated and is more pointed than that of the posterior enamel-
organ. This characteristic of the anterior tooth-germ is
illustrated (for the lower jaw) in fig. 5. In this figure it
will be seen that the “residual dental lamina” (‘‘ Ersatz-
leiste’’) is just beginning to emancipate itself, by further
growth, from: the medial aspect of the enamel-organ. In
front of the anterior enamel-organ, in each jaw, the dental
lamina is prolonged for a considerable distance as a narrow
ridge of no great vertical height (text-fig. 1). In one place
it exhibits a small localised thickening (“dyv’’) lying about
midway between the anterior enamel-organ and the plane
of the anterior disappearance of the much reduced lamina.
Apart from this slight bulbous thickening, the cross-section
of this anterior segment of the dental lamina exhibits a
slightly flask-shaped, though attenuated form, right up to
its point of disappearance anteriorly. It therefore here
lacks the broad and shallow character which one is accus-
tomed to associate with the true and original anterior, or
rostral, extremity of the dental lamina. Its actual form here
is suggestive rather of the suppression of a formerly more
extensive anterior segment of the lamina. Nor is this merely
a surmise. For, in the course of this attenuated anterior
segment of the lamina, lying in front of the more anterior of
the two evident enamel-organs, we find a calcified vestigial
toothlet. This is present in both jaws in our series of thin

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 143

sections from the left side of the head, stained in borax-
carmine (Pl. 10, fig. 4). It is also well seen in the upper
jaw in the hematoxylin-stained series from the opposite side
(fig. 5). It is, however, only doubtfully present in the lower
jaw of this series. Where it is visible the vestigial denticle
in each case projects partly into the mouth-epithelium, partly
into the labial aspect of the neck of the slender dental lamina,
so that it is partially surrounded by the epithelial cells of
these structures, which form a cap for its more superficial
portion. Its deeper or root-portion is simply imbedded in the
connective tissue. The dentine forms a shell partially enclos-
ing a more irregularly arranged dentinal mass (fig. 4).

It is in relation with this vestigial tooth that the attenu-
ated anterior segment of the dental lamina shows itself slightly
enlarged and swollen (‘ kolbig’”’) on cross-section. This some-
what bulbous character is not well illustrated in the sections
reproduced, since it only reaches its maximum in the sections
immediately in front of those figured. In the figures, how-
ever, there is some indication of a capsular arrangement of
the connective tissue around the lamina. The enlargement
of the lamina is too insignificant in amount to permit of its
accurate proportional representation in the schematic text-
fer 1.

Behind the large posterior enamel-organ the dental
lamina is continued backwards for some little distanee as
shown in the text-fig. 1. This posterior segment of the
lamina is relatively plumper and more massive than’ the
anterior segment, in accord with its more fertile and pro-
gressive character; for, as we shall subsequently show, this
segment gives rise to one of the large teeth of the later stage.
Already the advent of this tooth is heralded by a more
localised bulbous swelling, which is illustrated, in the lower
jaw in Fig. 2. This swelling shows a slight flattening of its
fundus (though not in the section depicted) which represents
the commencement of the process of cupping or papilla-
formation. In these respects the condition is practically
identical in both jaws of this specimen.

144 J. T. WILSON AND J. P. HILL.

(3) Description or THE PHAse oF T'ooTH-DEVELOPMENT SEEN
IN THE OLDER Specimen ‘ Bera.”

In the specimen of our older stage (text-fig. 2) there are
present in each jaw two very large enamel-organs, each with
a number of calcified cusps with whose special arrangement
we are not now concerned, These enamel-organs are without
doubt those of the two large multicuspidate teeth which are

Trxt-F ie. 2.

ice tas (ttt a ae | sera | 1 ie

found in the adolescent animal. Provisionally, and for the
present purpose, we designate the more anterior of these as
“x” and the posterior as ‘‘y” in the case of the upper teeth,
and as “x”? and “y”’ in the case of the lower (text-fig. 21).
In front of “x” in the upper jaw is a very much smaller
conical enamel-organ with one main, prominent, well-calci-
fied cusp. This cusp lies above the anterior end of the large
tooth “x” in the lower jaw, and we distinguish it by the
symbol ‘‘w.” Some distance in front of the lower tooth
“x” we find a calcified tooth of very small dimensions which
we regard as the true morphological opponent of “w” in the

1 In this schematic figure no attempt has been made to indicate the dental
lamina. Only the actual tooth-germs are shown. Magnification about 8 diam.

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 145

upper jaw, bearing in mind the fact that in both of our stages
the entire dental lamina and its derivatives in the lower Jaw
are uniformly in advance, as regards position, of the corre-
sponding structures in the upper. This small tooth, then, we
indicate by the symbol “w
of the tooth ““w” in the upper-jaw the deeper portion of the
dental lamina shows a small and rudimentary, but distinctly
papillated, enamel-organ. ‘This we designate as “v.” It
lies a short distance in front of the plane of “w” of the lower
jaw. No trace of any lower opponent to “v” is to be found.
It is, however, obvious that the anterior region of the dental
lamina in Ornithorhynchus is that which is most under
the influence of those factors which have brought about the
special modification of the mouth and jaws. It is, accord-
ingly, in this region that we might expect to meet with
evidences of suppression. And since, as we have already
pointed out, the dental lamina in the lower jaw is topo-
graphically in advance of that of the upper jaw, it is not

surprising that no trace of a lower “v” is to be met with in

.’ Now, some distance in front

this relatively late stage.

Passing now to the posterior region of the dental lamina of
our older specimen, we find that the posterior extremity of
the tooth “‘y” projects behind the plane of its opponent
“y”’ Behind the latter the dental lamina shows a small, but
well-developed, papillated enamel-organ, uncalcified, and
just beginning to emancipate itself from the residual dental
lamina. This enamel-organ we here designate as “z.”
Opposite to this in the upper jaw we still find the hinder
extremity of “y.” When the posterior end of the latter is
reached, the upper dental lamina continues for a short
distance (0°15 mm.) as a thick and bulky structure and then
suddenly stops without showing any definite differentiation
into an enamel-organ. ‘This terminal thickened portion of the
lamina may, however, be regarded as the anlage of a potential

igs 22

upper “z,”’ and Poulton would appear to have found it
actually papillated, although it apparently never erupts.

146 J. T. WILSON AND J. P. AILL.

(4) Synrueric CoMPARISON OF THE CONDITIONS MET WITH IN
THE STaGE OF 'TOOTH-DEVELOPMENT LAS DESCRIBED, (a)
WITH THE ADOLESCENT CONDITION, AND (b) WITH THE
HaruieR “Foran” Stace.

(a) With the adolescent condition.

When we compare the condition above described with that
of the adolescent animal as described by Oldfield Thomas and
Stewart, it is evident that in the upper jaw three of the tooth-

representatives which we have referred to undergo eruption,

9d 66 3) 2)
’ .

viz. our “w x,’ and “y In the lower jaw also three

= a

teeth cut the gum, but these are our ‘‘x,” “y,” and ‘z.”

(b) With the earlier fetal stage (i.e. our specimen
“ Delta’’).

When we now institute a comparison of the condition we
have described as occurring in our older stage with that met
with in the younger of our two specimens, the conclusions
which we are disposed to draw are not those which at first
suggest themselves, and which for a time we actually enter-
tained.

It has been shown that in the younger specimen “ Delta”
there are two well-developed enamel-organs present in each
jaw, but these are of widely different dimensions, the posterior
being much more elongated than the anterior.

Moreover, the shape of the anterior is different from that
of the posterior, its papilla, as seen in coronal section, being
more slender and pointed.

In the later foetal stage “ Beta,’ on the other hand, the

“x” and “x’’) of the two large enamel-

more anterior (
organs in each jaw are considerably more elongated than the

posterior (“y” and “y”). For this and other reasons we

TOOTH=-DEVELOPMENT IN ORNITHORHYNCHUS. 147

are constrained to regard the relatively elongated enamel-
organs which are posterior in both jaws of the younger

stage as the representatives, not of the teeth “y” and “y,”

but of “x” and “x” respectively in the jaws of the older
stage, “ Beta.’ It will be observed that both in the younger
and in the older ‘‘x” is substantially more elongated than
“x,” and is also situated in a somewhat more advanced posi-

tion.
(es ob}

The above identification of 5; in the younger specimen
x

necessitates the assumption that, between this stage and the
older one there has occurred a tolerably rapid growth and
differentiation of the posterior portions of the dental lamin
of the younger stage (v. text-fig. 1). This is precisely what
one might expect to take place during the process of differ-
entiation of a molar series; and it has already been shown
that, not only is the hinder segment of the dental lamina in
the earlier stage relatively plump and well-developed, but
that there is actually present in it the early rudiment of an
actual posterior tooth. Further, it may be stated that the
increase in absolute length of the head which has occurred
in the later stage is very considerable, allowing of marked
elongation of the posterior region of the jaw and of the molar
lamina. And, in fact, comparative measurements show that
the total length of the lamina in the older stage is not merely
absolutely but relatively greater than in the younger.
Comparison of the two schemes in text-figs. 1 and 2 (which,
though schematic, are nevertheless drawn to scale) will show
that in the older specimen the enamel-organ “ x” is not only
larger than its upper opponent, but is actually the largest
tooth-rudiment in either of the jaws. On the other hand, the

a

lower anterior enamel organ, “w,” in the younger specimen,

is appreciably smaller than its upper opponent, “ w,’’ and this

early inferiority of “w” already foreshadows the marked
reduction of “w,’’ as compared with “w,” in the older foetal
specimen, as well as its eventual entire absence in the

adolescent animal,

148 J. T. WILSON AND J. P. HILL.

For these reasons we have no hesitation in identifying the
anterior of the two prominent papillated enamel-organs in
the upper and lower jaws of our younger specimen with those
developing teeth in the older which we have marked as
“w” and “w” respectively.

In the course of our description of the older stage we have
noted the presence of a small cupped and somewhat im-
perfectly formed enamel-organ in front of the conical calcified
tooth “w.” The enamel-organ in question is quite deeply
placed and is obviously in series with the more fully developed
teeth behind it. We have designated it as “v.” Again, in
the younger specimen we have shown that there is present,
in the precisely corresponding coronal plane, a small and
already strongly calcified vestigial tooth (figs. 4 and 5). This
actually indents the deep aspect of the mouth epithelium at
the labial side of the attachment to the latter of the dental
lamina.- This, therefore, cannot be the enamel-organ “v”
of the older specimen. But we have also seen (p. 145) that,
in the immediate neighbourhood of this vestigial tooth, the
dental lamina is somewhat enlarged and bulbous. It seems
tolerably evident that this bulbous enlargement of the
adjacent part of the dental lamina must be the genuine
representative of the later enamel-organ (‘‘v’’) (text-fig. 2).
We, therefore, hold that this latter tooth-rudiment, “ v,”
must be regarded as the true morphological successor to the
small calcified vestigial tooth which is present in our smaller
specimen. The latter vestigial tooth we accordingly indicate
by the symbol “ dv,’ as being the deciduous predecessor of
the enamel-organ “v” of the later stage.

In the lower jaw of the older specimen no representative
of a lower “‘v” was met with. But in the younger, as we
have already stated, there is found, on one side certainly,
and more doubtfully on the other, a structure which, in its
situation and its relations to other structures, exactly corre-
sponds to the small calcified upper tooth “dv,” and thus
represents a lower “dv.” In the neighbourhood of this
vestigial tooth-element the dental lamina is slightly enlarged,

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 149

but, as we have just seen, no more mature product of this
enlargement appears in the later stage of development. This
is doubtless to be accounted for by the advanced position in the
jaw occupied by the tooth-germ in question.

If we now take account of the combined conditions set
forth in the schematic text-figs. 1 and 2, it will be evident
that they tend to establish the view that representatives of
five quasi-permanent teeth are developed in each jaw during
the phases of tooth-development under consideration. These
include the recognised teeth of the adolescent animal. (‘The
most posterior member of the series in the upper jaw (‘ 2”)
is not indicated in the scheme.) In addition to these there
have also been indicated vestigial representatives of deciduous
predecessors to the most anterior of these five tooth-elements.

We shall presently adduce evidence to prove that the
deciduous vestiges already described are not the sole repre-
sentatives of an earlier tooth-generation occurring in our
specimens. Meanwhile we may observe that the dentitional
characters above set forth seem to afford evidence of the
operation of factors which determine early suppression and
abortion of the more anterior segment of the dental lamina
and its derivatives in Ornithorhynchus.

(5) ConcerninG THE EprrnEniAL NopULES FORMERLY DESCRIBED
BY Proressor PouLTON IN CONNECTION WITH THE ENAMEL-
ORGANS OF ORNITHORHYNCHUS.

In the course of the account given by Professor Poulton
of the developing teeth in Ornithorhynchus, he described a
number of epithelial nodules situated ‘almost immediately
over the apex of each calcified cusp of the second and third
tooth.” He states that ‘“‘nothing of the kind could be made
out in the case of the first upper tooth” (i.e. our tooth “w”).
In these nodules he found that “the inner cells appear to be
corneous and collected into a dense central mass, between
which and the outer fusiform cells is a space containing

150 J. T. WILSON AND J. P. HILL.

loosely packed cells resembling the former in character.”
Their position was “at the extreme edge of the stellate
reticulum,” and from his figures they all appear to lie inside
the enamel-organ as defined by its outer epithelial layer.
“In some cases,” he notes, “ there was the appearance of an
epithelial cylinder extending from the nodule towards, or
perhaps reaching the stratum intermedium or enamel cells
over the apex of the cusp.” Further, there was always a
nodule above each of the chief cusps, while they were never
found elsewhere.

We have found the same nodular structures to be present
in our older specimen “ Beta,’ which nearly corresponds with
the specimens which formed the subject of Poulton’s investi-
gation, and we are therefore in a position to supplement his
account of these interesting structures.

(1) Relation of the nodules to the enamel-
organs.—In the first place, we find that, although the
nodules may appear to be included within the large enamel-
organs, they are really morphologically outside of them.
They have originally lain in contact with the exterior of the
enamel-organ and have been gradually enveloped through the
relatively enormous expansion of the latter. In many sections
the inclusion may seem to be complete, and, indeed, is so,
so far as such sections are concerned. But when the series
of neighbouring sections is carefully examined, we find that
an opening or depression in the surface of the environing
enamel-organ is discoverable, through which the engulfed
nodule is still in touch with the connective tissue of the tooth-
sac. In certain sections, indeed, the nodule appears to lie in
a comparatively shallow recess or bay in the surface of the
enamel-organ (Pl. 11, figs. 6 and 7). In other cases it is
difficult to be quite certain that such a communication with
the exterior as we have described is demonstrable. But in no
case is it indubitably absent, and its undoubted presence in
other cases is presumptive evidence that the condition in all
is originally identical.

Wealso have observed the appearance of epithelial cylinders

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 151

or strands (fig. 13) in relation to the tips of cusps in the
vicinity of nodules in the position indicated by Poulton. We
find, however, that where they are present their connection
with the tip of the cusp is the constant and characteristic
relation, whilst their relation to the concentric epithelial
nodule, if the latter be present, is inconstant and variable.
We find (a) that there may be a cylindriform or strand-like
prolongation from the tip of a cusp, either inner or outer,
without any nodule being present in relation with it; and (0)
such an epithelial strand is absent in some cases in which a
nodule is present in the vicinity of the apex of a cusp. Thus
there are no epithelial cylinders in connection with the cusps
to which the nodules marked “dx!” and “dx?” in text-fig. 2
are related. Nor has the presence or absence of the epithelial
strand anything to do with the extent to which the nodule is
imbedded in or recessed into the main enamel-organ; in the
case of “dx!” the nodule lies largely outside of the enamel-
organ in a very shallow bay of its surface-contour (figs. 6 and
7). On the other hand, the nodule “dx*” is largely surrounded
by the tissue of the main enamel-organ, but here, again, no
epithelial cylinder is present. We therefore believe that the
relation of the epithelial cylinder to the nodule is a mere
accident of the common relationship which both these
structures seem to possess to the prominence of the cusp.
But while the relationship between the nodule and the point
of the cusp is not necessarily an intimate one, as text-fig. 2
will show, the relationship between the point of the cusp and
the epithelial cylinder is fundamental. ‘he cylinder appears
to be, in fact, nothing but a prolongation of the inner enamel-
epithelium of the apex of the cusp, accompanied by a sheath,
composed of those cells of the stratum intermedium which
form a more condensed layer in contact with the inner enamel-
epithelium. In certain cases it is true that the epithelial
strand so constituted does appear to reach and come in
contact with the outer shell of the concentric nodule, but
this relationship is not an invariable one, and inall probability
is of no essential significance. In fig. 13 there is reproduced

$52 J: T. “WILSON “AND 5.0P BILL:

a photomicrograph of one of the epithelial strands, in which
its real nature as a prolongation of the epithelium of the tip
of the cusp is manifest. High-power examination has also
shown us, in the case of a few sections through such astrand,
that the inner enamel-epithelium is actually prolonged in the
core of the cylinder in the form of two closely-applied layers
of cells. Between these layers a line, indicative of a potential
lumen, is just recognisable, and we regard the cylinder as an
abortive prolongation of the apical part of the pulp-cavity,
within which pulp-cells have failed to penetrate, or have wholly
disappeared, so that neither dentine nor odontoblasts have
been differentiated within it. The epithelial cylinders thus
represent, in our view, portions of the cusps which have
undergone ontogenetic reduction.

(2) Position and arrangement of nodules with
reference to cusps.—Poulton states that there was
always a nodule over the apex of each of the chief cusps of
the two large teeth, while they were never found elsewhere.
We likewise find a nodule in relation with each of the two
chief cusps of these teeth—i. e., over the series of internal
cusps in the upper, and of the external cusps in the lower jaw
(text-fig. 2).

The photomicrographs herewith reproduced in figs. 7, 8,
and 10 illustrate the topographical relationship of several of
the chief cusps to their corresponding nodules as seen in the
section-series from one side of our specimen ‘ Beta.’ In
figs. 8 and 10 the nodules appear to lie inside the enamel-
organ, whilst in fig. 7, as already stated above, it only
partially indents the superficial aspect of the enamel-organ
(cf. also fig. 6 from opposite side).

It will further appear from an examination of text-fig. 2,
which also illustrates the condition met with on the same side
of the head, that in the case of the anterior chief cusp of the
enamel-organ ‘‘y,” there are two nodular structures present
in the vicinity of the cusp. ‘The more anterior of these is
seen in the photomicrograph in fig. 8. It presents the typical
characters of the nodules as described by Poulton and as we

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 153

find them exemplified in the majority of instances (cf. fig. 12)
The more posterior of the two nodular structures, however,
differs in a highly significant manner from the general type of
nodule. Its structural characters are well shown in the
photomicrographs in figs. 9 and 11. It will be seen that
within a concentrically arranged connective-tissue capsule
there is a pale zone, which on high-power examination
(fig. 11) shows radially arranged cells identical with those of
the internal epithelium of the main enamel-organ as shown in
fig. 9. Within this epithelium is a deeply stained ring of
dentine, and within this, again, is a connective-tissue core or
pulp. We have, therefore, here present all the essential
structures of a typical tooth, and there can be no hesitation in
regarding it as a vestigial tooth. Yet in its position and
relations to the enamel-organ, whose superficial aspect it
slightly indents, it exactly resembles the concentric epithelial
nodules with which it is in series (cf. figs. 8 and 9, and 11
and 12).

In the case of the lower teeth (text-fig. 2) quite similar
relations obtain in respect of the presence of epithelial nodules
in the vicinity of the principal cusps, which are in this case
the external cusp series. Here, again, there are two nodules
which are more or less in the neighbourhood of the anterior

chief cusp of “y.” ‘The more posterior of these is placed in
p y p p

advance of the prominence of the cusp with which it is pre-
sumably associated. In fact, it lies just in front of the
anterior limit of calcification of the cusp, instead of over its
prominence. ‘The second and more anteriorly placed nodule
in this region is much smaller and more superficially placed,
and it lies wholly in front of the calcified anterior cusp of
the tooth “y.” Indeed, so far as its position is concerned it
is placed rather in a vertical relation to the hinder portion of
the enamel-organ of the more anterior tooth “x,” though
behind the plane of the posterior calcified external cusp of
the latter. ‘The idea of its possible relation to the tooth “x ”
is further supported by the fact that it lies more labially than
the larger nodule behind it, which is related to the anterior

voL. Ol, PART 1.—NEW SERIES. ai!

154 J. T. WILSON AND J. P. HILL.

cusp of ‘‘y,” the anterior end of the enamel-organ of “ y ”
overlapping the hinder end of “x” on its mesial aspect.

In other respects the character and position of the nodules
in the lower jaw require no further illustration than that
which is afforded by text-fig. 2.

That the above arrangements are not merely fortuitous is
further testified to by the examination of the section-series
from the opposite side of the head of the same specimen. The
condition there existing is practically identical with that set
forth in text-fig.2. The only noteworthy deviations from
this scheme are: (a) that the well-calcified vestigial toothlet
in the upper jaw, illustrated in figs. 9 and 11 (and which we
have indicated by the symbol “dy*” in the scheme in text-
fig. 2), is on this side represented by a concentric epithelial
nodule of precisely similar character to the other nodules
with which it is in series, and (b) that the anterior of the
two nodules (“dy!”), which in text-fig. 2 are seen to lhe
opposite the adjacent ends of the two large lower enamel-
organs “x” and ‘‘y,” is absent on this side. It has already
been stated that it is comparatively small and insignificant on
the other side, so that its absence on the side now under con-
sideration is the less surprising.

(3) The morphological character of the nodules.—
From the foregoing account it will already be apparent that
we regard the series of epithelial nodules as constituting a
series of vestigial representatives of an earlier tooth genera-
tion. That they must be so regarded has already been
surmised by Marett-Tims (7) on the basis of Poulton’s
original account of them. ‘Tims was apparently led to this
interpretation of the structure in question through his own
observations (6) of the presence of corresponding structures
in connection with the dentition of other mammals, structures
which seemed to him to demand a like explanation.

The evidences in favour of such an interpretation may be
summarised as follows :

(a) The nodules are not fortuitously distributed, but form a
more or less regular series both as regards number and posi-

'

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 155
tion in both jaws and on both sides of the jaw in our older
specimen “ Beta.” They would appear to have had a closely
similar arrangement in Poulton’s specimen.

(b) In our specimen the typical concentric epithelial nodules
are in series with the undoubted vestigial toothlet which we
designate as “dy*”’; and on one side of the upper jaw “dy*”
is represented by a typical concentric epithelial nodule.

(c) Some of the typical nodules, though devoid of dentine,
show traces of other of the elementary tooth constituents.
Thus the nodule which we designate as “dy!” shows traces
of a stellate enamel-epithelium around the condensed con-
centric lamine of epithelium; and several of the nodules
exhibit in their central core a collection of deeply-staining
nuclei similar to those which form the obvious pulp of the
genuine denticle “‘dy*” (v. fig. 12, illustrating the structure

of a typical nodule).

(d) The nodules appear to be identical in character with
those present in various other mammals which Tims has
shown to require, in all probability, a like interpretation.

(4) Serial homology of the nodules with the
vestigial teeth “dv” and “dv” present in the
younger specimen “ Delta.”—In the course of our
description of the younger of our two _ specimens,
“Delta,” we have demonstrated the presence (figs. 4 and
5) of a vestigial tooth, “dv,” in connection with the seg-
ment of the dental lamina lying in front of the more anterior
of the two large enamel-organs there present (i.e. our tooth
“w”). We have previously given our reasons for regarding
this vestigial toothlet, “dv,” as belonging to an earlier tooth
generation from that to which the more posteriorly placed
enamel-organs ‘‘w” and “x” belong. It now only remains
to indicate that in our opinion the strongly and precociously
calcified degenerative toothlet, “‘dv,” is in series with, and
homologous to, the nodular vestiges present in the older
specimen in relation with the teeth “x” and “y.’ The
successor, “v,” of the vestigial “dv” is represented in the
younger specimen by a swelling of the dental lamina which,

156 J. T. WILSON AND J. P. HILL.

in the older specimen, has taken shape as the sie ip still
uncalcified enamel organ shown in text-fig. 2 as “v,” im front
of the calcified tooth “ w.”

The view of the homology of the small vestigial tooth “dy”
of the younger specimen with the nodular series of the older
is further strengthened by the fact that its lower opponent,
“dy,” is in a more advanced stage of involution than upper
«“ dy.’ It lacks the definite dentinal character of the latter,
its structural constituents show concentric lamination, and
altogether its appearance is strongly suggestive of the
structure of the nodules of the older stage.

(5) Origin of the nodules.—It might have been ex-
pected that the possession of a younger stage for comparison
with that represented by our specimen “ Beta” would have
enabled us to elucidate the mode of origin, or at least some
definite part of the early history of the nodular vestiges, since
these are obviously in process of involution in the older stage.
The two stages, however, prove to be too remote from one
another to yield positive and conclusive evidence as to the
mode of origin of the nodules. A priori one might have
expected to find the vestigial teeth represented in the younger

specimen by precocious enamel-organs. But although the
)

ce
large permanent teeth ye have not yet come into existence

w” ce +3
ns
as distinct enamel-organs, and both 7—;; ew? and’ = wy» are in the

condition of well-developed enamel-organs, there are no
definite enamel-organs to represent the early phase of the
nodular vestiges of the later stage.

Nevertheless we find that there are present, at intervals
along the labial aspect of the dental lamina, a succession of
peculhar structural differentiations which there is small room
for doubting to be the beginnings of the nodular structures.
These structural differentiations are of the nature of a series
of invasions or deep indentations of the neck of the dental
lamina, on its labial aspect, near the level of its continuity
with the deep surface of the mouth-epithelium. They occur

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 157

only in those regions of the lamina which constitute the
enamel-organs of the future teeth. ‘They therefore appear to
be appended to the enamel-organs in question. The later
relationship of the actual nodules to the main cusps of the
teeth is evidence that the early relation of these precursors of
the nodules to the enamel-organs is not a chance one.

In fig. 1 there is reproduced a photomicrograph of a section
through the enamel-organ of the upper tooth “x.” Here
there is seen a typical example of the structural arrangement
of one of these early “ nodular” differentiations. Imbedded
in the neck of the dental lamina, at its junction with the
enamel-organ, is a small group of cells occupying an other-
wise clear central area. This is surrounded by a zone of
enamel-epithelial cells, which show signs of differentiation
into layers, and the entire mass appears as an appendage of
the main enamel-organ. The group of central cells is meso-
dermal, and is in the next section seen to be continuous with
the cellular tissue of the surrounding tooth-sac.

Another example of the structures in question, less striking
but essentially identical, taken from the opposite side of the
jaw, is represented in fig. 2. Here the continuity of the
mesodermal core of the rudimentary nodule with the tooth-
sac tissue is more obvious, but even here it is nearly surrounded
by enamel-cells. It is plain that were the included pulp-cells
seen in fig. 1 to assume an odontoblastic function there would
result just such a shell of dentine as we figure in fig. 11 from
our older stage, whilst the surrounding enamel-cells would
account for the outer concentric layers of the fully-developed
nodules.

Since no differentiation of the future cusps has occurred at
the stage under consideration, no reference to the later con-

dition in this respect is relevant. And, since the enamel-
ce 9

organ of the future tooth wy? has not yet come into

existence as such, posteriorly, the rudimentary stage of the
interesting nodules connected with the upper tooth “y”
is likewise in abeyance,

158 J. T. WILSON AND J. P. HILL.

(73 9
é - eee F
In connection with the tooth @yn im the older specimen

“ Beta” we have seen that two nodules are present, “dx!”
and “ dx?’ and that there was a possibility that the anterior
of the three nodules in relation with the enamel-organ of the
lower tooth “y” is really related to “ x.’ There is not the

slightest doubt that there are multiple rudimentary nodules
ce 3)

in connection with the large enamel-organ of ay in the

younger specimen. But whether these are two or three in
number it is difficult to say. In the upper jaw we believe
that there are three, but the most posterior is at the hinder
extremity of the enamel-organ of “x,” behind which we have
the thick dental lamina from which “ y” subsequently arises,
so that we are unable to entirely exclude the possibility that
the last rudimentary nodule may really belong to “y.”
We incline to the contrary opinion. ‘There is abundant
evidence of the entire disappearance of some of the nodular
rudiments originally present in the fact that in the younger
specimen there appears to be two such differentiations of
typical character present in relation with the enamel-organ of

the tooth —

(79 33?
WwW

noted by Poulton, destitute of any vestigial nodule.

whilst this tooth in the latter stage is, as

(6) Genzrat Discosston or Toorn-HomoLocy Nn ORNITHO-
RHYNCHUS.

The facts set forth in the foregoing account seem to estab-
lish the existence in Ornithorhynchus of teeth belonging to
at least two dentitional series. In the series to which the
large multicuspidate teeth of the adolescent animal belong we
believe that we are justified in reckoning five members. In
the case of the upper jaw we lack positive evidence of the
actual formation of the most posterior member of the series,
and in the lower jaw, again, we believe that we are justified
in supposing that the most anterior member has undergone

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 159

suppression. Of the five teeth which we take to be the full

complement of members of the quasi-permanent series of
“ce ?

: Vv
which we have evidence, we hold the first and second Tar

€¢ 3)

and to correspond to premolars. This judgment is

ce Ww

based largely upon the relative size and simplicity of the

a9 3)
tooth

—, which is the best developed of these (?) pre-
V

molars, in both the earlier and later stages at our disposal.
The proportions may be judged of by the schemes in
text-figs. 1 and 2, and the form by a comparison of fig. 3 with
figs. 1 and 2.

¢ d)

SW,
If the tooth Cnr be premolar, then the molar formula may

be expressed, in accordance with our views and nomenclature, as
xy — (2) ”
ce i y = F
these teeth as molar.

But if we are also right in our recognition of the “ nodular ”’

9

series, including the obvious denticle “dy?,” as vestigial
ee I)
Cie?
then we have before us in Ornithorlhynchus a demonstration
of the presence of a whole series of precursors of the molar
teeth. And it is not the least remarkable aspect of this
demonstration that it exhibits these vestigial deciduous pre-
decessors of the large molar teeth in the form of a much more
numerous series of simple tooth-rudiments, each on the whole
corresponding with one of the cusps of their multicuspidate
molariform successors. The admission that there are excep-
tions to this general correspondence of deciduous nodule to
permanent cusp, as seen in text-fig. 2, can hardly succeed in
invalidating the idea of a deep-seated and definite corre-
spondence which is otherwise suggested by the facts. It is
not very difficult to imagine some explanation of the duplicity
“c dy! = dy* 9
ce dy! mae dy’ 23

>: There can be no hesitation in identifying

teeth in series with the undoubted vestigial teeth

of the nodules on the lines of a theory of the

160 J. T. WILSON AND J. P. HILL.

suppression or other modification of cusps. It is to be
remembered that it is only the series of primary cusps which
appear to have nodular correspondents.

It must also here be recalled that the examination of the
younger specimen ‘ Delta” has shown us that the early
differentiation of nodules is not confined to the region to be
occupied by molar cusps. Not only is there a vestigial quasi-
nodular predecessor, “dv,” to the enamel-organ “v,” but
there are one or perhaps two rudimentary labial nodular
structures in relation to the neck of the enamel-organ “ w,”
which disappear before the stage of specimen “ Beta” is
reached.

It cannot be denied that these facts tend towards the
establishment of a doctrine which may be regarded as
coming under the category of concrescence theories. In
some sense or other it would appear that the molariform
successors of the deciduous vestigial series represent com-
pound structures corresponding to two or three of their
simple (probably homodont) predecessors. It is not neces-
sary to suppose that any ontogenetic fusion occurs. The
mode of development of the successional molars in our
younger stage is decisive against the occurrence of any
fusion process. But the relation of the two series in the
molar region cannot but be regarded as suggestive of some
sort of phylogenetic substitution of a small number of com-
pound teeth for a large number of simple teeth—a process
which must be reckoned as covering the fundamentai idea of
concrescence.

It might well be accepted that a given segment of the
dental lamina, whose constitutive material gave origin in
one phylogenetic phase to a discrete series of simple teeth,
might, in another and later one, lose its discrete or particulate
character. On such a supposition the same morphological
material would in its later history be concerned in the pro-
duction of a larger composite structure within whose limits
the individual elements of the pre-existing series might be
only imperfectly preserved or represented.

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 161

An interpretation, practically identical with ours, of the
concentric epithelial nodules and of their relations to, and
significance for, the molar dentition in Ornithorhynchus, has
been repeatedly advanced by Marett Tims (7). Thus on
p. 135 of his memoir on “ The Evolution of the teeth in the
Mammalia” he says: “In the concentric epithelial bodies
of Cavia, Canis, Gymnura, and Ornithorhynchus we have, I
believe, the last traces of a vanishing dentition which must
have preceded the cheek-teeth on account of their labial
position. ‘These bodies remain quite distinct from the teeth
themselves and show no tendency to become fused.” Again,
in reference to the more general question of molar fusion he
states (ibid.) that “an antero-posterior fusion of the teeth of
the same dentition appears to me now to be the only solution
of the difficulty in accounting for the duplex condition of the
true molars of the greater number of mammals and of the
complex cheek-teeth of the rodents and fossil multi-tuber-
culata. The repetition, so to say, of the development of the
anterior and posterior halves of the rodent molars seems to
me to render this highly probable, though I have not yet
seen any actual fusion of enamel-germs. It may quite well
be that this early stage may have become slurred over in the
recapitulatory history, until it is entirely lost at the present
day. Possibly the same may be true of the ungulates and
proboscidians.” :

So far as Ornithorhynchus is concerned, this writer had
only the data supplied by Poulton on which to base his
interpretation, and his judgment must have been determined
mainly by his experience of the occurrence of similar con-
centric epithelial bodies in the other forms specified. It is,
therefore, noteworthy that with our wider opportunities of
experience of the condition in the monotreme form we have
been led to conclusions which do not differ in any important
respect from those arrived at by Marett Tims. That view,
then, which for him could not in the nature of the case be
more than a surmise, as regards Ornithorhynchus, has through
our observations received more or less definite confirmation.

von. 51, part 1,—NEW SERIES, 12

162 J. T. WILSON AND J. P. HILL.

Tn this connection we may note that we cannot now admit
the validity of the further surmise of the same writer that
the concentric epithelial cell-nests formerly described by us
as occurring in Perameles are of the same order or signi-
ficance as those we describe and figure in Ornithorhynchus.
In our previous paper (8, p. 518) we have ourselves expressed
the opinion, subsequently endorsed by Tims, as to their resem-
blance to the nodules of Ornithorhynchus. ‘This, however,
was prior to our study of the latter. We are now perfectly
confident that the cell-nests we encountered in Perameles
belong to a totally different category of structural differentia-
tion. These latter are purely epithelial degeneration products
formed in, or in connection with, the mouth epithelium over-
lying teeth which are about to undergo eruption; they are
quite irregular in their occurrence and in their arrangement ;
they make their appearance, for the first time, in comparatively
late stages of tooth-development—i.e. shortly before erup-
tion, and they contain at no time any trace of structural
elements save the concentrically arranged epithelial cells
themselves, having no elements referable to an origin from
the mesoderm as is the case with the genuine “nodular”
structures in Ornithorhynchus. ‘hey are, in fact, entirely
similar to the epithelial “ pearls’? which are met with in
various situations, as, for example, in the neighbourhood of
the median raphé of the palate ; whereas we have shown that
the nodules in Ornithorhynchus when fully developed show
obvious traces of the typical structure of an enamel-organ.

A further conclusion from the view of tooth-development
and tooth-homology in Ornithorhynchus set forth by us is
that no support is to be derived from it for any theory which
would seek to establish a serial distinction between the true
molar teeth and those which appear, prima facie, to belong
to the same series in front of them. It is quite true that the
suppression of the antemolar teeth has proceeded so far that
it would be rash to base any theory of serial homologies
between molars and antemolars on the condition obtaining
in Ornithorhynchus. All that we here affirm is that, so far

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 163

as they go, the facts rather point in the direction of serial
continuity between the teeth of the molar series and their
putative serial companions in front, meagrely represented in
“vy —w”
“(v) —w”
Finally, in several of the figures illustrative of the condition
in the older specimen (figs. 8, 9, 10) it will be observed that
a tolerably substantial “residual dental lamina” remains pre-
served along the lingual side of the large molar enamel-organs.
We have not so far had an opportunity of observing the sub-
sequent fate of this ‘‘ residual lamina,” but there is no reason
for belheving that it differs in any way as regards its destiny
from the residual lamina so often found in a similar situation
beside the developing molars of other mammals. Its presence
here, therefore, need not be regarded as expressive of any
special significance beyond that of representing the formal
potentiality of “ post-permanent ” molar successors, the pos-
sibility of the occurrence of which cannot be excluded from
the ordinary mammalian scheme of tooth-genesis.

our older specimen ‘‘ Beta” by the teeth

List oF REFERENCES.

1. Poutron, E. B.—< True Teeth in the young Ornithorhynchus para-
doxus,” ‘Proc. Roy. Soc.,’ vol. xliii, 1888, pp. 353-356.

2. Poutroy, HE. B.—*The True Teeth and the Horny Plates of Orni-
thorhynchus,”’ ‘Quart. Journ. Micr. Sci.,’ N.S., vol. xxix, 1889,
pp. 9-48.

3. Poutton, EK. B.—“The Structure of the Bill and Hairs of Orni-
thorhynchus paradoxus: with a Discussion of the Homologies
and Origin of Mammalian Hair,” ‘Quart. Journ. Mier. Sci.,’ N.S.,
vol. xxxvi, 1894, pl. xva.

4, Tuomas, O.—“On the Dentition of Ornithorhynchus,” ‘Proc. Roy. Soe.,’
vol. xlvi, 1890.

5. Stewart, C.—‘On a Specimen of the True Teeth of Ornithorhynchus,”
‘Quart. Journ. Mier. Sci.,’ N.S., vol. xxxiii, 1892, pp. 229-231,
pl. viii.

6. Marerr Tims, H. W.—“ Tooth-Genesis in the Caviide,” ‘Journ. Linn,
Soc. Zool.,’ vol. xxviii, 1901, pp. 261-286.

7. Maret Tims, H. W.—‘‘ The Evolution of the Teeth in the Mammalia,”
‘Journ, Anat. and Phys.,’ vol. xxvii, 1903, pp. 131-149.

164 J. T. WILSON AND J. P. HILL.

8. Witson, J. T. and Hitz, J. P.—“ Observations upon the Development
and Succession of the Teeth in Perameles, etc.,”’ ‘Quart. Journ. Micr.
Sci.,’ N.S., vol. xxxix, 1896.

9. Witson, J. T.—* Description, with figures, of a young specimen of
Ornithorhynechus anatinus from the Collection of the Australian
Museum, Sydney,” ‘ Proc. Linn. Soc., N.S.W..,’ ser. 2, vol. ix, p. 682.

EXPLANATION OF PLATES 10—12,

Illustrating Professors Wilson and Hill’s paper, ‘ Observa-
tions on Tooth-Development in Ornithorhynchus.”

All the figures are from photomicrographs of transverse sections. The
symbol +|: indicates the labial side.

Fic. 1.—Delta. Section of the enamel organ and dermal papilla of tooth
“x” of the upper jaw, right side, with nodule rudiment on the labial side of
neck of enamel organ. x 78.

Fic. 2.—Delta. Tooth “x” of left side, upper jaw, also with a labially-
situated nodule rudiment. In the lower jaw the dental lamina is swollen club-
like to form the rudiment of the lower tooth ce Pe eet B.

Fic. 3.—Delta. Tooth ‘‘w” of lower jaw, right side. Note the narrow
pointed character of the dermal papilla as seen in cross-section and compare
with that of “x”’’ as seen in figs. 1 and 2. On the lingual side the residual
dental lamina is commencing to free itself from the enamel organ. xX 78.

Fie. 4.—Delta. Section through the dental lamina of the upper jaw, left
side, about mid-way between its anterior extremity and the enamel organ “ w,”
showing the rudimentary toothlet ‘dv ” indenting the oral epithelium to the
labial side of the attachment of the lamina. x 175.

Fie. 5.—Delta. Corresponding section through the vestigial toothlet “dv ”
on the right side of the upper jaw. x 175.

Fie. 6.—Beta. Section through the upper tooth “x” passing through its
main antero-internal cusp and the nodule “dx!” related thereto. x 78.

Fie. 7.—Beta. Corresponding section showing the tooth “x” and the
nodule “dx!” of the opposite side of the head. x 78. = =

Fre. 8.—Beta. Section through the antero-internal cusp, upper tooth-
“y,” and its related nodule “ dy'.”. x 78.

Fic. 9.—Beta. Section shortly behind the preceding, showing the tooth-
let ‘‘ dy?” underlying the posterior region of the antero-internal cusp of upper
*‘y,”’ and well to the labial side of the residual dental lamina.’ The toothlet lies

directly behind and in series with the nodule “‘dy’” (cf. text-figure 2). x 78.

TOOTH-DEVELOPMENT IN ORNITHORHYNCHUS. 165

Fic. 10.—Beta. Section through the postero-internal cusp of upper tooth
“‘y,”? showing the nodule “ dy?” apparently imbedded in the stellate tissue
of the enamel organ to the labial side of the massive residual dental lamina.
x 78.

Fig.11.—Beta. High-power view of the toothlet “dy?” showing its central
connective-tissue core or pulp enclosed by a distinct ring of dentine, a distinct
layer of columnar cells representing the enamel epithelium and a peripheral
capsule of concentrically arranged connective tissue. x 345.

Fic. 12.—Beta. Section through the nodule “ dx*” to illustrate nodular
structure. Note the central cellular core enclosed by concentrically arranged
flattened cells forming a compact zone. xX 216.

Fic, 13.—Beta. Section through an epithelial strand to demonstrate its
continuity with the enamel epithelium covering the apex of the cusp. x 345.

von. 51, parr 1.—NEW SERIES. 13

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CONTENTS OF No. 202.—New Series.

MEMOIRS:

The Origin and Nature of the Green Cells of Convoluta roscoff-
ensis. By Freprerick Kersuz, M.A., Sc.D., University College,
Reading, and F. W. Gampiz, D.Sc., Manchester University.
(With Plates 13 and 14) :

On the Development of the Plumes in Buds of Cephalodiscus.
By W. G. RipEewoop, D.Sc., Lecturer on Biology at St. Mary’s
Medical School, University of London. (With 11 Text-figures)

On the Structure of Anigma xnigmatica, Chemnitz; a Contribu-
tion to our Knowledge of the Anomiacea. By GirpertC. Bourne,
M.A., D.Sc., F.L.S., Fellow of Merton College; Linacre Professor
of Comparative Anatomy in the University of Oxford. (With
Plates 15—17, and 2 Text-figures) . : :

On the Chromatin Masses of Piroplasma bigeminum (Babesia
bovis), the Parasite of Texas Cattle-Fever. By H. B. Fantuam,
B.Sc.Lond., A.R.C.S., University College, London, and St. Mary’s
Hospital Medical School. (With Plate 18, and 44 Text-figures) .

The Skin, Hair, and Reproductive Organs of Notoryctes. Contribu-
tions to our Knowledge of the Anatomy of Notoryctes typhlops,
Stirling —Parts IV and V. By Georcina Sweet, D.Sc., Mel-
bourne University. (With Plates 19 and 20, and a Text-figure)

Parorchis acanthus, the Type of a new Genus of Trematodes. By
Wituiam Nicott, M.A., B.Sc., Gatty Marine Laboratory, St.
Andrews. (With Plate 21) ‘ ‘ :

WAY 31 1907

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 167

The Origin and Nature of the Green Cells of
Convoluta roscoffensis.

By
Frederick Keeble, M.A., Se.D.,
University College, Reading,
and
F. W. Gamble, D.Se.,

Manchester University.

With Plates 13 and 14,

ConrTeENTS.
PAGE
Section J. Introduction 4 167
» II. Proof of the Origin of the Green Cells by infection 172
», III. The Isolation of the Infecting Organism and the

Synthesis of the Green Convoluta . a9
» IV. The Life-history of the Infecting Organism 5 Le
os V. The Normal Course of miteotion : 191

» VI. The Significance and the Consequences of the
Association of Animal and Green Cell 99
» VII. General Summary ; ‘ . 208
Tables I—VI ‘ ‘ ; ; » 210
Literature ‘ : , o OG
Explanation of Plates : : : ely,

Secrion I. Inrropucrion.

XANTHELLZ and Chlorelle are widely distributed among
the members of the basal groups of the animal kingdom. In
VoL. 51, PART 2.—NEW SERIES. 14

168 F, KEEBLE AND F. W. GAMBLE.

some animals they are present constantly, in others they occur
sporadically.

Green, yellow, or brown cells have been described in
representatives of every division of free-living Protozoa, in
certain sponges, in most anthozoan Ccoelenterates, in a few
Hydrozoa and Scyphozoa, and in accelous and rhabdoccelous
Turbellaria. Their occurrence in the higher groups is rare—
e. g. Zoobothrium (Polyzoa), Elysia (Mollusca), and Hehino-
cardium (Hchinoderms).

The association is obligate in Convoluta paradoxa,
C. roscoffensis, and in Hydra viridis; it is facultative
in the Protozoa, Anthozoa, and rhabdoccel Turbellaria.

In the former cases every individual of a species possesses
green or yellow or brown cells; in the latter cases only
certain individuals contain them.

Facultative association may exhibit itself in another manner:
in one part of its range all the individuals of a species may
exhibit the association, in another part the coloured cells
may be absent from all the individuals. Thus Noctiluca is
colourless in the North Atlantic and green in the Indian
Ocean. British Aleyonium have no zoochlorelle, whereas the
closely allied A. ceylonicum possesses them (Pratt, 1905),
It seems probable, indeed, that the maximum development
of these associations occurs in the warmer seas. According
as the association is facultative or obligate, it gives rise to a
less or greater modification in the behaviour and in the
structure of the animal.

Convoluta roscoffensis exhibits in a striking degree
such modifications (Gamble and Keeble, 1903). It lives
gregariously at mid-tide level on the beach and exposes
itself to the bright light of mid-day. Its colourless young
are as strongly phototropic as the green adults. With the
advent and multiplication of the green cells it ceases to ingest
solid food. Structural changes described in the body of this
paper also follow consequent upon the association of animal
and green cell.

Similar phenomena are exhibited by other animals. Thus

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 169

Cassiopeia, a medusa of warm seas, has adopted, as a conse-
quence of the association with itself of green cells, a posture
the reverse of that maintained by its allies. On certain reefs
it may be seen fixed by its aboral surface, exposing its oral
arms, and so the coloured cells contained therein to the light.

Various reef corals, richly provided with zoochlorelle,
show marked decrease in the size of their tentacles; in this
respect, and also in the number and degree of development
of their digestive filaments, they present a sharp contrast
with their more freely feeding allies (Pratt, 1905). Facts
such as these lead naturally to the hypothesis that the
association of animal and green cell results in a supply of
nutriment to the former from the latter.

Admitting, as it is reasonable to admit, this trophic hypo-
thesis, it still remains to inquire—as we do in the case of
Convoluta roscoffensis in the following sections—what
food-substances are transferred from green cell to animal and
how this transference is effected.

It is important to know how this hypothesis stands with
respect to such cases of voracious feeders as anemones,
Hydra, Convoluta paradoxa, and many green rhab-
doccels. Here it is clear that the animal is not subdued to
the working of the “ plant” within it.

It may also be asked how this at present vague hypothesis
meets the cases of facultative association. One of the two
main purposes of this paper is to put forward a supplemen-
tary hypothesis (Section VI, page 205), which we think
throws new light on the significance of chlorella and xanthelle
in animals, The other purpose of this paper is to provide
absolute proof of the source of origin of the green cells in
Convoluta roscoffensis.

The view generally held, that the green or brown cor-
puscles of animals are of algal nature, is based rather on
probability than on certain evidence.

The usual arguments in favour of this view are as follows :

The corpuscles in question have been shown in certain
cases to be capable of photosynthesis. In the light, gases

170 F, KEEBLE AND F. W. GAMBLE.

containing a high percentage (15-50) of oxygen may be
evolved from the animals. Starch may be present in the
corpuscles. They have been shown to contain chlorophyll,
patent or masked by other pigment. Structurally the
chlorellee resemble certain algal cells. A wall of cellulosic
or pectose substance, absent in some cases, may be present.
The corpuscles may contain a nucleus, a pyrenoid, and occa-
sionally an eye-spot. ‘his circumstantial evidence is strong,
but cannot be said to amount to proof.

Evidence of the origin of the green or brown cells to be
final must be of a like nature to that demanded by patholo-
gists in the case of a micro-organism suspected of pathogenic
properties ; the organism must be isolated in pure culture
and the infection-test applied.

So in the cases of animals infected with green cells ; these
cells must be isolated, and, by introduction into the body of
an animal previously free from them, be shown to give rise to
the normal green animal. In other words, the final proof of
the algal nature of the green cells can be provided only by
the synthesis of the green organism from its algal and animal
components. ‘This synthesis has not as yet been effected in
an indisputable manner in any single case. The green or
brown cells of Turbellaria, Coelenterates, or sponges have not
as yet been isolated and cultivated.

Beijerinck (1890) and Entz (1881-1882), it is true, made
cultures of green hydra, but owing to the proneness of the
cultures to infection from within and from without both the
authors and their critics have regarded the results with sus-
picion, especially in view of the fact that attempts at synthesis
were unsuccessful.

Among green protozoa two or three cases of alleged isola-
tion of the corpuscles have been recorded. By macerating
the bodies of Stentor, Paramecium, and Frontonia, Famintzin
(1889-1891), Dantec (1892), and Dangeard (1900) have
obtained colonies of alge. ‘The results are, however, some-
what discrepant, for whereas the first two authors regard the
alga of Paramecium as a true chlorella, the third considers it

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 171

to be a chlamydomonas. Schewiakoff (1891) successfully
fed colourless Frontonia on macerated green specimens, and
states briefly that multiplication of the green cells within the
host occurred. Dantec obtained a like result with Para-
mecium.,

Brandt (1883), after depriving sea anemones of their
yellow cells by long confinement in the dark, caused the
reappearance of these cells by placing the animal in fresh
sea-water in the light. But from experiments made by
ourselves, we venture to express some doubt as to whether
the apparently colourless animal had lost absolutely all its
yellow cells.

Haberlandt (1891), whose admirable work on Convoluta
roscoffensis is referred to in the text of this paper, was
unsuccessful in his attempts to cultivate the green cells of
this animal. Pending more exact information it seems to us
that Lankester (1882 and 1890) has done valuable service by
his championship of the opposed view, that of the intrinsic
nature of the corpuscles under discussion. For his view com-
pels those who hold the “algal” theory to investigate each
case separately and to vindicate their view by the synthesis
of the green animal.

When this has been done in such cases as those in which
cell-wall and nucleus are present in the corpuscles, there will
remain others—e. g. Hydra and Spongilla, whose green
cells are devoid of definite nuclei, and after these, such
puzzling instances as Vorticella campanula (Kngel-
mann, 1883), with its diffuse chlorophyll, as well as
Pelomyxa viridis (Bourne, 1891), the green corpuscles of
which seem difficult of explanation except on Lankester’s
hypothesis.

Influenced by the forementioned considerations, we have
investigated the origin of the green cells of Convoluta
roscoffensis. The results of this investigation are set forth
in Sections II—YV.

The experimental work recorded in this paper has been
carried on in the laboratory at Trégastel, Cotes-du-Nord,

172 F. KEEBLE AND F. W. GAMBLE.

France; in the Zoological Department, Victoria University,
Manchester, and in the Botanical Laboratory, University
College, Reading.

We acknowledge with gratitude the assistance we have
derived from a grant made by Section D of the British
Association for the purposes of this investigation.

Section II. Proor or toe ORIGIN OF THE GREEN CELLS BY
INFECTION.

The dark spinach-green colour of Convoluta roscoff-
ensis is due, as is well known from the descriptions of
von Graff (1905-1906), Geddes (1879, 1879 a, 1882), Haber-
Jandt (1891), and ourselves (1903), to dense layers of green
cells. These green cells are distributed with great uniformity
in the body, and extend from just below the epidermis into
the deeper tissues. Only in the anterior end of the body, in
front of the otocyst and rudimentary eyes, are the green cells
so few in numbers as to reveal the whitish colour of the
animal,

The general appearance of an adult Convoluta, when
examined microscopically, is not unlike that of the mesophyll
of a green leaf. In surface view the green cells are flat,
somewhat variously shaped bodies, now of rounded outline,
now drawn out at one end into long tail-like extensions which
appear to connect cell with cell.

The individual cells, described by Haberlandt (1891) in his
important contribution to our knowledge of the green cells
of Convoluta, are naked protoplasts, the larger part of each
of which consists of a more or less cup-shaped chloroplast
containing a large polygonal or irregular pyrenoid. The
small remaining part of the protoplast is colourless, and lies
either in the hollow cup-like invagination of the chloroplast
or, when the shape of this latter is irregular, excentrically.

Though often free, or almost free, from starch, the green
cells may, under certain conditions, contain considerable
quantities both of pyrenoid starch—that is, starch distributed

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 178

in the layer surrounding the pyrenoid (the starch sheath)—
and also of minute rods or granules scattered irregularly
throughout the substance of the chloroplast.

Two views as to the nature of the green cells have been
expressed—one, that they are alow which have been ingested,
and which have developed within the animal body, the other,
that they are integral parts of the animal tissue.

The observations of Georgevitsch (1899) that the just
hatched young are colourless lend support to the former of
these views, but do not suffice to establish it. For, since the
animals raised by Georgevitsch—hatched in filtered water—
only survived for three days, it is conceivable that ante-
cedents of green cells were present in a colourless form, but
that, owing to the short period during which the animals
lived, these colourless antecedents failed to give rise to the
green cells,

Our own observations (Section V) that the earliest recog-
nisable stages of the future green cells may be colourless
justify this reservation, lending no more support to the infec-
tion-hypothesis than to the suggestion of Haberlandt that the
green cells may be derived from colourless plastids, the per-
sisting remnants of once independent alg now transmitted,
as are the plastids of green plants, by the egg-cell.

In order to determine finally the origin of the green cells
of Convoluta, it is necessary, in the first place, to maintain
newly hatched animals under such conditions that infection
—if infection there be—cannot occur, and, in the second
place, if the result of the foregoing is the production of
colourless Convolutas, to expose the animals to infection
and to determine whether this exposure brings about the
development of green cells.

As we state in the Introduction to this paper, this rigorous
proof of the intrusive nature of the green or yellow cells of
animals has not hitherto been obtained in any single case,
we therefore describe in detail experiments which fulfil these
conditions, and which have led to a decisive result in the
instance of Convoluta roscoffensis.

174, F. KEEBLE AND F. W. GAMBLE.

In our previous work (1903), where strong, but not decisive,
evidence in favour of the infection-hypothesis was given, we
showed that it was possible to maintain young Convoluta
alive and colourless for several weeks. The freshly laid egg-
capsules containing the groups of fertilised eggs were—in
these experiments—removed from the neighbourhood of
adult animals and placed in sea-water which had been filtered
by means of a Pasteur-Chamberland filter. The animals
hatched out in the course of two or three days as colour-
less larvee and remained colourless for two or even three weeks
(Table II, Columns A and D). Samples of these colourless
animals taken at any time during this period and placed in
fresh, unfiltered sea-water became green in three or four days.

So far the result of this experiment pointed most definitely
to the environment as the source of the green cells; but this
conclusion was rendered less certain by the subsequent
behaviour of the animals reared in filtered sea-water. Certain
among these, sometimes few, sometimes many, ultimately
became green, and on microscopic examination were found
to be possessed of normal green cells (Table II, Columns B
and C). The sporadic and tardy appearance of green indi-
viduals among the Convoluta hatched in filtered sea-water
could be accounted for best on the supposition that small
numbers of the infecting organism had been introduced with
the egg-capsules into the filtered water, and that the three
or four weeks which elapsed before infection took place were
required for the multiplication of this organism. Microscopic
examination of the egg-capsules showed the high probability
of this supposition, for they were found to be infested, even
a day or so after they had been laid, and before the young
had escaped from them, with numberless green and colourless
cells of various kinds.

To eliminate this source of error it was necessary therefore
to obtain capsules as clean as possible, and to take the pre-
caution of isolating the young from this source of infection.
This we succeeded in doing by the following somewhat
laborious process.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 175

Batches of Convoluta were collected in watch-glasses from
the shore, care being taken to exclude particles of sand and
other foreign matter. The animals so obtained were washed
a number of times in filtered sea-water, and were then
allowed to descend into a large glass dish containing filtered
sea-water. This dish was illuminated unilaterally, and as
soon as Convoluta had taken up its markedly positive photo-
tropic position the dish was tilted, the water drained away,
and the bottom of the dish cleaned. A fresh lot of filtered
sea-water was added, and the dish so turned that the animals
were impelled to crawl across to the opposite side. As soon
as they had taken up their new light-position the tilting
and cleaning were repeated, filtered sea-water was again
added, and the vessel turned once more. In some experi-
ments these processes were repeated as many as eight times.
When the Convolutas in the dish began to lay, the egg-capsules
were picked out daily and placed in glass vessels containing
filtered sea-water. As a result of these precautions the
capsules were much freer from those organisms which, when
no precautions are taken, habitually infest them ; and larger
numbers of Convolutas hatched out under these conditions
remained colourless. Nevertheless, even with these pre-
cautionary measures some green animals ultimately appeared
among a great majority of colourless Convolutas. Hence
an additional safeguard from possibility of infection had to
be employed—namely, to separate the young as nearly as
possible at the moment of hatching from the egg-capsules.
This, though a tedious, was not a difficult, operation owing
to the fact that when on the point of hatching, three or
four days after the capsules have been laid, it suffices,
in order to release the larve, to take up the capsule in
a fine pipette and to eject it with some slight force into
the water. Numbers of animals were obtained in this
manner, free or almost free from capsule-remnants. Absolute
freedom it is almost impossible to obtain, owing to the
extreme tenuity and transparency of the capsules at the time
of hatching.

176 F, KEEBLE AND F. W. GAMBLE.

As Table III shows, we have, by adopting this procedure,
succeeded in obtaining batches of Convoluta which remained
absolutely colourless and uninfected.

The several columns of this table give the results obtained
in the cases of:

(i) Animals left in association with their capsule-remnants.

(2) Animals removed from their capsule-remnants.

(3) Animals which, having been so removed, were subse-
quently submitted to the risk of infection by placing them in
fresh unfiltered sea-water.

(4) Animals hatched without any precautions in ordinary
sea-water.

The animals hatched in unfiltered sea-water became
uniformly green after two or three days. Among those left
with capsule-remnants infection occurred less rapidly and
more sparingly. Thus, as Columns 3, 4, 5 of Table III show,
eleven individuals out of fifty-nine became infected during
the eight days which followed after general infection had
declared itself among the animals reared in unfiltered sea-
water, After seventeen days, infection had become general
in all these cases (Columns 3, 4, 5).

The contrast between this result and that set forth in
Columns 1 and 2 of the same table is emphatic and conclusive.
In those cases (Columns 1, 2, lable I11) in which the animals
had been separated at the time of hatching from their capsule-
remnants the total numbers of animals showing infection
were, in the one experiment (Column 1), five out of forty-four
animals examined, and in the other none out of forty-seven
examined. We conclude, therefore, that the green cells of
Convoluta are of intrusive origin, or, to use the terms
employed already, that they arise as the result of an infection
from the water of the sea. From a single infecting cell, or
at most from two or three, are produced the vast numbers of
the green cells of the adult Convoluta: infection taking place
normally during the first three days after hatching.

Experiments made in the course of this inquiry enable us
to answer two other questions—viz. May the green cells of an

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 177

infected Convoluta escape from the body, live freely again,
and again infect a young animal? and what is the origin of
the cells which in the preceding experiments developed on
the capsule and subsequently infected the larval Convolutas ?
are they derived from the interior of the body of the egg-
laying parent or from the environment?

With respect to the first question, we have failed, as
Haberlandt failed before us, to cultivate in nutrient media
green cells liberated from the body of the adult. But more
convincing than these negative results are those set forth in
Table I (Column A) and Table III (column headed “ Filtered
sea-water with adults”). In these places are recorded the
results of experiments in rearing young animals in association
with large numbers of adults which had been washed many
times in filtered water. Under these circumstances the larve,
though surrounded by great numbers of adults, many of
which, having ruptured in the course of egg-laying, had dis-
charged large numbers of green cells, failed for twenty days
to show sign of infection. It thus appears certain that the
ordinary green cells, as they exist in the body of one animal,
are incapable on their discharge from that animal of infecting
another, Indeed, under no conditions known to us do the
green cells of a Convoluta ever escape alive from the body.
Each Convoluta leaves the body of the mother as a colourless,
uninfected animal; as such it hatches out from its capsule,
and the cells which then infect it are derived from the
environment, neither they nor their direct ancestors ever
having been before within the body of a Convoluta. We
return to this matter again in Section V, where we give an
account of the histological changes which the green cells
undergo in the course of their development in the body of
Convoluta, and show that these changes account for the at
first sight extraordinary facts just described.

There remains to be considered the second question—as to
the origin of the green cells which make their appearance on
the capsules and which served in various of the foregoing
experiments as sources of infection to larval Convolutas,

178 F. KEEBLE AND F. W. GAMBLE.

These cells must have been derived from one or other or both
of two sources—viz. from the body of Convolutas disintegrated
during egg-laying or from the environment. In the former
case they must be special cells which, unlike the great
majority of the green cells of the adult body, have undergone
no degenerative changes, and so retain the capacity for
development.

The evidence points to the environment as the place of
origin of the green cells of the capsules. In the first place,
such cells are by no means uniformly present on the capsules,
and in the second place they are present less often on capsules
laid by animals which have been freed in some measure by
repeated washings from their associated flora and fauna. The
effect of this repeated washing is not to render the surfaces
of the animals free from extraneous organisms, but to reduce
their numbers and to confine them to such as attach them-
selves most tenaciously to the slime which covers the surface
of Convoluta. Chief among these most intimate associates
are the infecting alga, certain other minute unicellular alge,
and various diatoms. Repeated washings, then, reduce the
number of competitors for place on the capsules, and though
some of the infecting organisms may themselves be swept
away those which remain find their tenancy when they succeed
in reaching a capsule less disputed than is the case under
more natural conditions.

In the third place, as we show (Section 1V), the infecting
alga does not depend on chance for its association either with
the surface of the animal or with the egg-capsule. It is
attracted chemotactically thereto, and hence, though the
numbers of infecting alge in the water should be but few,
they will inevitably distribute themselves upon some of the
capsules.

Summary of Section II.

(1) Convoluta roscoffensis commences life as a colour-
less (non-green) animal.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 179

(2) At this stage it has no germ of infection within its body.

(3) Infection occurs normally within three days of “birth.”

(4) Water taken from the shore—not necessarily in the
neighbourhood of Convoluta-patches—contains the infecting
organism in such numbers as to induce wholesale infection in
large batches of larvee.

(5) The infecting aiga habitually settles down and de-
velops on the egg-capsules, which therefore serve as sources
of infection (see also Keeble and Gamble, 1905).

(6) Infection does not take place directly or indirectly from
the body of the parent.

(7) The green cells of an adult Convoluta are incapable of
life apart from the body of the animal.

(8) Consequently the association must be regarded, not as
a symbiosis, but as a case of parasitism, the host being the
green cells and the parasite Convoluta (see also Section VI).

Section III. Tue Isonarron or tae Inrectincg ORGANISM
AND THE SYNTHESIS OF THE GREEN CONVOLUTA.

It is natural that, in seeking to isolate the infecting
organism of Convoluta, effort should be directed first toward
the cultivation of green cells obtained from the body of the
green animal. Haberlandt (1891) was the first to attempt
this. His efforts were unsuccessful. We have attempted it
again and again, but have failed. Miss Harriette Chick, who
has had much experience in such work, was good enough to
make in the laboratory at 'Trégastel in 1905 a large number
of culture experiments, using a great variety of nutrient
substances in liquid and solid media. The experiments gave
no positive result. These failures make it in a very high
degree probable that the task is impossible, and so lend some
support to the conclusion arrived at in the preceding section
that the green cells of Convoluta, once developed within
the body of that animal, are no longer capable of separate
existence.

It became necessary, therefore, if the search for the

180 F, KEEBLE AND F. W. GAMBLE.

infecting organism was to have a successful termination, to
begin at the other end, namely, to seek for the organism
before its entrance into the body of Convoluta.

Our observation that infection may take place, not only
from fresh sea-water, but also from the remnants of egg-
capsules laid in filtered sea-water suggested a mode whereby
this search might be prosecuted with some hope of success:
the mode being the isolation and observation of egg-capsules
from which the larvee had escaped. The isolation was neces-
sary because, if left with the young animals, the capsules
disappeared, either being torn to fine shreds by the frequent
entrances and exits of the larvee or, perhaps, being devoured
by these larve. Accordingly, numbers of egg - capsules
obtained from well-washed animals were put into filtered
sea-water, and as soon as the young had emerged from them
the transparent, gelatinous remains of the capsules were
removed to another vessel of filtered sea-water. There they
were kept under daily observation. After seventeen days
(Table III, columns 3 and 5) several green spherical bodies
of about the size of the egg-capsules made their appearance.
Microscopic examination showed that these spherical bodies
were composed each of a pure culture of vast numbers of a
unicellular green organism. During the examination the
slight pressure of the cover-glass sufficed to burst the delicate
membrane of the green spherule and a swarm of active,
flagellated cells emerged, leaving behind the recognisable
remains of an egg-capsule (figs. 1, 2, 3, 4, Pl. 13). These
flagellated cells, a detailed description of which is given in
Section IV, presented so many features in common with the
green cells of Convoluta roscoffensis as to leave but
little doubt that they represented a free stage of these cells:
the cup-shaped chromatophore containing a polygonal pyre-
noid, the colourless part of the protoplast occupying, as is
sometimes the case in the green cells of the animal, the
narrow cavity of the cup, the red, lateral-lying eye spot, also
to be met with in the green cells of recently infected young
Convolutas, all pointed to this conclusion.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 181

It only remained, therefore, to apply the inoculation test—
that is, to submit colourless, uninfected animals reared in
filtered water to the chance of infection by these flagellated
cells. A batch of such animals was divided accordingly into
three groups. One, serving as a control, was maintained in
filtered water ; another, also a control, was placed in unfiltered
sea-water in order that its capacity for infection might be
tested; the third was put into filtered sea-water to which
numbers of the flagellated organism had been added. The
result proved conclusively that these flagellated cells are a
stage in the life-history of the infecting organism. Group 1
in filtered sea-water remained uninfected; Group 2 in
unfiltered sea-water showed the susceptibility of the animals
to infection: they became green in the course of three days ;
Group 3, exposed to infection by the flagellated cells, were
observed to ingest these cells, to tolerate their active division,
and to become in consequence normal green Convolutas.
Subsequently, when we had perfected our procedure so as to
be able to obtain fairly constant supplies of the flagellated
cells, we repeated the experiment frequently, and in all cases
with the same result. Thus Convoluta has been synthesised
from its elements the colourless animal and the green,
flagellated cell.

Summary of Section III.

(1) We have isolated and cultivated outside the body of
Convoluta its infecting organism.

(2) It is not from the green cells of the body of Convoluta
that the infecting organism may be isolated, but it may be
obtained readily from the remnants of the egg-capsules.

(3) The infecting organism which occurs sporadically on
the egg-capsules is derived, not from the green cells of the
body of the parent, but from free cells frequenting the surface
of the body at the time of egg-laying.

(4) If to filtered sea-water containing colourless Convoluta
the infecting alga is added the synthesis of the green animal
results.

182 F. KEEBLE AND F. W. GAMBLE.

Section [V. Tae Lire-History oF THE INFECTING ORGANISM.

The green spherules from which the swarms of flagellated
cells issued as described in the preceding section served as a
starting-point for the cultivation of the green alga which, as
just shown, is the source of the green cells of Convoluta.
The securing of material for this purpose was rendered com-
paratively easy owing to the well-marked positive photo-
tropism of the alga in its motile stage. Issued from the
egg-capsule, the flagellated cells swarm toward the more
brightly illuminated side of the vessel in which they are con-
tained ; there they settle down sooner or later, either singly
or in pairs, along and just above the water-line. Thus the
position of the algz is marked by a visible green patch.
This patch consists of numbers of flagellated cells, and also
of many which, having withdrawn their flagella, have sur-
rounded themselves with a well marked and often stratified
wall. A sample from such a patch was transferred by means
of a platinum loop to a vessel containing filtered sea-water, to
which a little potassium nitrate had been added, and in which
had been placed a number of empty egg-capsules. After
some days a green streak along the water-line made its
appearance on the brighter side of the vessel. ‘The vessel
was taken from Trégastel to England (Reading) in September,
1905, placed in the light in a cool incubator, and kept under
observation. The green scum gradually disappeared, and it
was feared that the organisms had died. Toward the end of
May, 1906, the vessel was placed on a bench in the laboratory
in a good light, and within a fortnight a green scum re-
appeared on the illuminated side of the vessel. Microscopic
examination showed the identity of the organisms constituting
this scum, with those added to the water the previous year.
Beside the green layer on the side of the vessel loose masses
of pale green mucilage, floated up to the surface by reason of
included gas-bubbles, made their appearance. Imbedded in
these mucilaginous masses were numbers of quiescent green
cells, lying singly, in pairs, or in groups.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 183

Subcultures in various media were made from this material.
Some of these were taken back again to Trégastel in the
summer of 1906, and proved sufficient for the infection of
colourless young Convolutas. ‘The cultures have also served
to demonstrate that the alga which infects Convoluta ros-
coffensis has a very varied life-history. In the first place,
the active flagellated cells are dimorphic. The macrocytes,
16 » in length (figs. 4, Pl. 13 and 12 4, Pl. 14), are nearly
double the size of the microcytes (4: 2°5) (figs. 38, Pl. 18
and 128, Pl. 14). Except in point of size the large and small
cells are similar in their histological details.

Both the large and small cells have four equal flagella
arising from the anterior colourless part of the protoplast.
The flagella in both bear the same relation to the length of the
body (2:1). In both the chloroplast is cup-shaped, the pyrenoid
sinele, the eye-spot lateral and situated in the anterior half of
the cell. In both the wall is extremely delicate and gives no
cellulose reaction—e. g. with sulphuric acid and iodine or
with Schultze’s solution or calcium chloride-iodine ; but with
zine chloride-iodine it gives a faint rose-colour (chitin reaction).

The nucleus—a description of which is given in Section V,
where a comparison between the free and imprisoned cells is
drawn—lies, in both large and small cells, in the colourless
part of the protoplast which fills the hollow of the cup-shaped
chloroplast. It suffices to say here that the nucleus is sus-
pended in a layer ef the protoplast from which run strands,
two downwards, serving as slings for the pyrenoid, and
others outward through the chloroplast at regular intervals to
meet the thin, colourless layer of protoplasm which forms a
pellicle around the exterior of the chloroplast.

Large and small cells are alike equally phototropic, and
both settle down after a period of activity, withdrawing their
flagella and surrounding themselves with amore or less thick
mucilaginous wall. The wall may form with great rapidity,
so that encysting macrocytes are often to be met with whose
flagella may be seen in undulating movement within the
enclosing wall.

voL. 51, pART 2,—NEW SERIES. 15

184. F. KEEBLE AND F. W. GAMBLE.

The resting-cells (Pl. 14, figs. 11 and 16) vary consider-
ably in form and in behaviour. ‘Thus, single flagellated cells
may come to rest, withdraw their flagella and, without form-
ing a thick wall, undergo longitudinal division into two or
four cells contained within the wall of the mother-cell. ‘These
daughter-cells, at first without a distinct wall, organise
flagella, form a delicate cell-wall, and escape from the de-
liquescent mother-wall as active flagellated cells. Again, in
the case of the macrocytes, the active cell comes to rest,
surrounds itself with an extremely thick stratified wall, takes
on a spherical shape, and becomes green throughout as
though the whole cell were filled with small, polygonal, green
granules. This appearance is due to the colourless proto-
plasm, which in the active stage isin large measure confined
to the cup-like hollow of the chloroplast, now radiating out
in all directions through the chloroplast to the outer wall and
so demarcating the green chloroplast into polygonal areas.
In these rounded resting-cells the pyrenoid is at first recog-
nisable, but later breaks up into a number of pieces, and
finally into many granules. ‘The eye-spot may take the form
of a circular plate or ring lying near the periphery of the cell,
or it may disappear altogether,

These rounded thick-walled cells (PI. 14, fig. 11), after a
period of rest, may organise four daughter-cells of oval shape
and having all the characters of the active cells except that
no pyrenoid is visible at first and no flagella are as yet
developed. <A third form of resting-cell resembles that just
described, except for the fact that the green colour dis-
appears. ‘his colourless resting-cell appears, in surface view,
to be divided up into small, regular, polygonal areas, and in
this cell pyrenoid and eye-spot may be indistinguishable.

Yet, again, paired resting-cells are met with. ‘hese con-
sist of two thick-walled cells, whose broad apposed surfaces
are flattened as by mutual pressure (Pl. 14, fig. 16 4). Such
paired cells are either green or colourless. Tinally, yet
another form of resting stage occurs. In tlis the resting-
cells are paired, but one of the two cells is smaller than the

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 185

other ;in short, a series of stages are met with—pairs of equal
sized cells, pairs in which one cell is of normal and the other
of reduced size, half normal, less than half, and even to a mere
speck (PI. 14, figs. 16 a, B, c).

This phenomenon we refer to again when dealing, at the
end of the section, with the systematic position of the infect-
ing organism. The flagellated cells which settle down, with-
draw their flagella and surround themselves with a thick
mucilaginous wall do not necessarily pass through a period
of rest, nor do they necessarily divide up subsequently to
two or four green or colourless cells as described previously ;
but the life-history is lable to be short-circuited in the
following manner: a green cell may encyst itself temporarily
and then cast off the cyst and escape once again as an active
cell. The organism under consideration exists also in a
colonial form (Palmella condition). he cells in the colony
form plates imbedded in masses of mucilage produced by
the coalescence of the outer layers of the walls of the indi-
vidual cells. The cells in the colonial state are rounded,
uniformly green, and possessed of a pyrenoid less refractive
than that of the active cell (figs. 14 and 15, Pl. 14). The
cells constituting the colony undergo rapid division, being
rather budded off from the parent-cell than produced by
equal division of that cell. Here and there among the mass
mature cells occur. In these an eye-spot is visible, and the
protoplast is differentiated into green chloroplast and colour-
less neck as in the active cells. The mature cell may be seen
moving inside its walland every now and then escaping from
the wall as an active flagellated macrocyte. Hence, scattered
among the green cells of the colony are empty cysts consist-
ing of the walls left behind by escaped active cells (figs. 14.¢
and 15, Pl. 14). The green spherules from which we first
obtained the infecting alga consist of such colonial stages
which have developed within, and come to fill completely, the
ege-capsule of Conyoluta. Under other conditions the
colonial form is made up of cells which show the reticular
type of structure already described as occurring in resting-

186 F. KEEBLE AND F. W. GAMBLE.

cells. It is probable, therefore, that the constituents of a
colony may, under certain conditions, all pass into the resting
stage.

Another curious colonial form also occurs. Oval green
cells showing the characters of the active cells, differentiated
chloroplast, eye-spot, and pyrenoid, are attached to one
another by sleeve-like branching columns of _ stratified
mucilage (fig. 8, Pl. 13).

One of the most striking points in the life-history of this
alga is the occurrence in approximately equal numbers of
green and colourless resting-cells. These cells may be—as
already stated—isolated or in pairs. The single green cells
undergo, after a period of rest, division into four, green,
oval cells which resemble active cells except that their walls
are extremely thin and that flagella are lacking. The four
daughter-cells are extruded together from the envelope of
the mother-cell and undergo further division or become at
once active cells.

Similarly the green paired cells divide to eight oval cells.
The colourless cells behave in a precisely similar way, giving
rise to four or eight daughter-cells which are extruded through
a circular opening in the wall of the mother-cell. The colour-
less daughter-cells consist each of a highly vacuolated foam-
like protoplast, including a large refractive mass which
appears to correspond with pyrenoid and chloroplast of the
green cell. These cells may either undergo further division
or assume gradually and without further division a green
colour, becoming first faintly yellow, then quite yellow, and
finally green.

The simultaneous formation by the alga of green and
colourless cells is certainly a curious phenomenon.

It is well known that the zygotes of various algee lose their
chlorophyll, but we know of no case where resting-cells are
alternatively colourless or green. ‘I'he division of the green
cell into a green group of daughter-cells and the division of
the colourless cell into a corresponding group of colourless
cells appears to indicate that a true dimorphism exists,

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 187

Among the Euglenz and also among Diatoms it is known
that green (or brown) cells may lose their pigments, and,
assuming a colourless state, exchange a holophytic for a
saprophytic existence.

From the fact that when infection of Convoluta occurs
normally from sea-water the earliest recognisable stage of the
infecting organism in the body of the animal is a colourless
or faintly yellow cell it would appear that the colourless
phase of the alga described above represents a_ perfectly
normal stage of its life-history.

The suggestion may be hazarded that the formation of
colourless cells capable of a saprophytic mode of hfe is an
adjustment which widens enormously the range of distribution
of the alga. The green, active cells swarm toward the light,
and so have their distribution determined by the light factor
of their environment. The colourless forms, living sapro-
phytically, increase by division even in darkness. Thus the
alga may be enabled to live, not only in the surface waters,
but also below the surface of the sand wherever organic
débris provides material for its support. Such a divided
habit would undoubtedly be of the utmost service to the alga,
for, attached to the mucilaginous film which surrounds the
body of Convoluta or to the gelatinous egg-capsule, it under-
goes immersion deep in the sand at every tide. As each tide
arrives at the patch of sand covered by the vast colonies of
Convoluta these latter descend, as we have described in an
earlier paper, out of reach of the disturbance caused by the
moving water. The eggs of Convoluta, moreover are laid,
not on, but beneath the surface of the sand, so that an
organism which adjusted its habits so as to become an associate
of Convoluta would be compelled to pass many hours of the
day, and not infrequently, when attached to the egg-capsules,
many days,in darkness. This it could support only if it were
capable of a saprophytic habit.

That the infecting alga is capable of such a habit cannot
be doubted, for, apart from the colourless stage being regarded
as evidence, the active green cells themselves give corrobora-

188 F. KEEBLE AND F. W. GAMBLE.

tive testimony, since, in cultures containing disintegrating
Convolutas, they take up positions among the breaking-down
ereen cells of the animal aud increase greatly in numbers.
Again, the rapid increase of the alga which follows after
infection is strong evidence of saprophytic powers of nutrition.
Finally, we have proved by comparative cultures that the
infecting alga increases by vegetative division quicker when
supplied with organic nitrogen than when the nitrogen is in
the form of nitrates. The details of these experiments are
given in ‘lable IV, which summarises the results of a series
of cultures of the alga in sterile sea-water, to which nitrogen
in different combinations was added. The most rapid increase
took place in the culture containing uric acid, next in
that containing urea, the increase in sea-water contaiming
potassium nitrate being less than in either of these culture-
fluids. The infecting organism, then, has even in its green-
celled stage marked saprophytic tendencies—tendencies which
appear to find full expression in the colourless stage and
which offer the key to the origin of the remarkable associa-
tion of green cell and Convoluta.

Two questions remain—(1) Is there any special adaptation
in the habits of the infecting organism which brings it into
close association with Convoluta, or is its prevalence in the
surface slime of the animal and in the empty egg-capsules a
matter of chance? (2) To what systematic position is this
organism to be assigned ?

To the first question the following experiments provide
a decisive answer. If to a bulk of filtered sea-water con-
taining egg-capsules which have been laid under the cleanest
possible conditions a number of the flagellated cells are
transferred by means of a platinum loop, then within a few
hours one or more of these cells will be found to have
settled down on each capsule. Yet more striking results
are obtained if to a hanging-drop containing an egg-
capsule with its group of developing eggs a number of
the flagellated cells are added. In such «a preparation
the motile green cells are seen to approach tle capsule, to

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 189

swarm about it, to press in close ranks into the soft
gelatinous wall and so to imbed themselves in the envelope
(tigs.6, 75, Pl. 13):

We conclude that the egg-capsule exercises a chemotactic
influence on the active cells, and that the constant presence
of one or more green cells of the infecting alga on egg-
capsules contained in water to which a platinum loopful of
the active cells has been added is thus accounted for. The
behaviour of the cells which settle on the capsule proves that
they find this a favourable medium for growth. Within a
few hours each cell, having withdrawn its flagella, increases
considerably in size, and whilst retaining its green colour
takes on a granular appearance ; the eye-spot and pyrenoid
become fainter and the cell undergoes division. ‘This division,
like that of the temporary resting-cells already described,
is in most cases longitudinal, but occasionally instances occur
in which the division is transverse.

The mode of division is possibly determined by the state
of symmetry of the cell; as long as the colourless proto-
plasmic plug occupies the cavity of the cup-shaped chromato-
phore the division will be longitudinal, but when the protoplasm
becomes distributed radially and uniformly transverse division
may occur, and this in turn may give place to the budding of
the spherical units of the palmella stage.

The daughter-cells produced by the division of the green
cells settled on the ege-capsule undergo a continuous series
of divisions, and so give rise to a loose colony, which colony
constitutes the green spherules, the discovery of which formed
the starting-point for this description.

The second question, that of the systematic position of the
infecting organism, remains to be cousidered. ‘The assem-
blage of characters presented by this alga—its firm wall, its
single cup-shaped chromatophore containing in its hollow the
colourless protoplasm and nucleus, its pyrenoid and lateral
eye-spot far removed from the bases of the flagella, its power
of starch-formation—indicate that it must be assigned to a
position in that primitive group of green alge the Chlamydo-

190 F. KEEBLE AND F. W. GAMBLE.

monadew ; and the possession by the cells of four flagella
would indicate the genus Carteria.

Nevertheless we would wish to assign it only provisionally
to this genus for the following reasons :

First, the infecting alga never has contractile vacuoles, two
of which are described in the known species of Carteria.

Second, though like some species of Carteria—e. g.
C. multifilis (Fresen)—it possesses cells of two sizes (West,
1904); yet, whereas in C. multifilis the small cells are
gametes and the large, vegetative cells, neither the large nor
the small cells of our organism appear to be obligate
gametes. Numerous experiments which we have made of
bringing together microcytes from different cultures, micro-
cytes and macrocytes, and macrocytes from different cultures
have given but meagre and extremely rare evidence for any
sexual fusion. Leaving aside as unexplained the phenomenon
already described in which one of a pair of resting-cells
gradually decreases in size and finally disappears, we have
only once obtained evidence of what may be a gametic union.
Pl. 14, fig. 11 p, shows the case in point. Here two motile
cells have come together, their walls have united, and the
cells appear in process of fusion.

If this be a true case of sexual fusion, it presents features
of great interest. For, as Blackman (1900) has pointed out,
the Chlamydomonadex exhibit a remarkable series of modes
of gametic fusion. In some forms the walls are thrown off
whilst the gametes are approaching one another, in others
at the moment of meeting. In Chlamydomonas multi-
filis, a four-ciliate form, partial fusion takes place first, so
that the walls are left fused at one spot. Blackman adds
that the series might be completed at its lower end by a
form which fused without losing its walls. The figure just
referred to appears to indicate that this primitive form of
sexual union is exhibited by the infecting alga of Convoluta.,

A third character which seems to separate it from Carteria
is the peculiar branching habit which it sometimes presents.
In this condition (Pl. 18, fig. 8) inverted green cells are

THE GREEN CELLS OF CONVOLUYLA ROSCOFFENSIS. 191

borne at the ends of branching gelatinous stratified stalks.
This habit occurs, according to Oltmanns (1904), in other
genera of the Chlamydomonadee, viz. Chlorangium (Stein),
and Physocytium (Borzi). The nearest approach, however,
to this habit of the infecting alga is exhibited by species of
the genus Prasimocladus of the family Chlorodendracew,
which stands, according to Oltmanns, between the Chlamydo-
monadez and the Polyblepharidez.

We conclude that the infecting organism is a true alga
and a primitive member of a primitive group, the Chlamydo-
monadeew; and that whilst presenting many features cha-
racteristic of the genus Carteria, its possession of certain other
characters, facultative gametes, branching as well as _plate-
like colonial form and its lack of contractile vacuoles, makes
its assignment to that genus doubtful.

Summary of Section IV.

y

The infecting organism of Convoluta is an alga belonging
to the Chlamydomonadee.

Tn its free stage it bears four equal flagella and possesses
the general characters of members of this family.

The active cells are of two sizes, but neither large nor small
cells appear to be obligate gametes.

The organism is capable of a saprophytic as well as of a
holophytic existence ; in the former state it may be colour-
less.

The active cells are attracted chemotactically to ege-
capsules of Convoluta. ‘They settle down and undergo active
vegetative division in the capsules, and are finally liberated
as a swarm of four-flagellated active cells.

Section V. THe Norma Course or INFECTION.

We have demonstrated in Section III that the active
flagellated cells of the infecting alga may be ingested by
Conyoluta and, dividing in the body of the animal, give rise
to the green-celled “ tissue” characteristic of the adult.

192 F. KEEBLE AND F. W. GAMBLE.

When, however, infection takes place from ordinary sea-
water, when, for example, young Convolutas raised under
sterile conditions are placed in a few cubic centimetres of
sea-water brought fresh from the shore, it is not the green
flagellated cell which constitutes the first stage of infection.
The first sign of the presence of the infecting alga under
these normal conditions is afforded by a larger or smaller
colourless body lying in the central vacuole of the animal.
The large body consists of a pair of closely apposed resting-
cells, such as have been described already (Section IV). ‘The
small body is a single resting-cell of the infecting alga. Soon
after ingestion the mucilaginous wall which surrounds the
resting-cell swells considerably and the contents divide, in
the case of the smaller cell into four, in that of the larger
cell into as many as eight, colourless daughter-cells. ‘These
colourless cells are 15-16 in Jength—that is, of about the
same dimensions as those of the macrocytes. They escape
or are discharged from the central vacuole, and take up
fairly definite stations in the body of Convoluta, two right
and left of and a little behind the otocyst, and two on either
side about the middle of the body. ‘These cells, which are to
form the starting-points from which the green tissue of the
adult animal will be developed, lie each, like the mother-cell
which gave rise to them, in a clear vacuole. The colourless
cell at this stage has very granular contents, and a pyrenoid
which is large and somewhat oily-looking. Even in this
stage the subsequent differentiation of the protoplast into
chloroplast and colourless protoplasm may be indicated by a
plug or core of clearer protoplasm lying in the hollow of a
more granular chloroplast. The cell now develops rapidly,
an eye-spot makes its appearance, and a yellow tinge becomes
visible in the leucoplast; at first extremely faint, the yellow
becomes more marked, and is succeeded by a green colour
which pervades the whole chloroplast. ‘The cell is bounded
by no well-marked wall, the limiting layer does not give a
cellulose reaction, and is so delicate that the shape of the cell
changes with the movements of the aniunal.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 198

The infecting organism, though without flagella, now
resembles in other respects the active flagellated free stage.
It is, in fact, as both its histological features and its develop-
ment show, a daughter-cell produced by the division of a
colourless resting-cell. Such resting-cells on resuming
activity undergo division into four (the paired cells into as
many as eight) daughter-cells, which, as described in Section
IV, are discharged together or severally through a circular
opening in the thick mucilaginous wall of the cyst. Sub-
sequently these cells surround themselves, in the free state,
each with a thin wall. In the body of Convoluta the forma-
tion of a defiuite wall is suppressed.

The small] colourless or faintly yellow cell, which is often
the first recognisable sign of infection, is a daughter-cell
which has been produced outside the body of the animal
by the division of such a resting-cell as that just described.

Convoluta is then infected normally either by a resting-cell
or by non-motile daughter-cells produced by the division of
a resting-cell, though, as already shown, the organism in its
flagellated phase may be taken up and give rise to normal
infection.

We have now traced the infecting organism to its place in
the body of Convoluta. At certain, fairly constant, stations
of the body, several naked green cells he, each in a clear
vacuole. At about this stage, or after their further division,
the green cells lose their regular oval outline, and the colour-
less part of the protoplast becomes more or less excentrically
placed with respect to the chloroplast. It is easy to cause
free active cells to undergo similar changes. All that is
necessary to effect this is to transfer them from sea-water to
a fluid—e. @. diluted sea-water—of lower osmotic pressure.

Hence we infer that the peculiar and variable shapes of the
ereen cells of adult Convolutas are due to the osmotic pressure
of the vacuolar fluid in which they lie being lower than that
of sea-water. Incidentally, this observation serves to clear
up another point. Haberlandt makes the suggestion that the
colourless, excentrically-lying part of the green cell is in

194 F. KEEBLE AND F. W. GAMBLE.

reality an animal cell or part of an animal cell which has
associated itself with the green cell, and that the nucleus
lying in this colourless part belongs to this animal cell. From
the preceding it follows that this suggestion cannot be main-
tained ; for the colourless part of the green cell is nothing
other than the plug or core of colourless protoplasm which
in the free, active cell occupies the cavity of the cup-shaped
chromatophore, and which in a fluid of osmotic pressure lower
than that of sea-water becomes displaced excentrically. The
question of nucleus we reserve (see p. 196). We turn now to
consider the further development of the green cells which,
as the result of infection, are planted about in the body.

The cells increase, sometimes very considerably in size, and
undergo division. The process of this division in cells which
have not yet become distorted is as follows: The colourless
“neck ”’ of protoplasm elongates, extending toward the base
of the cell, where come to be placed pyrenoid and eye-spot.
A vertical fold appears in the pyrenoid which later cleaves
into two. The eye-spot degenerates during the division of
the cell, taking on the form of a broken hoop of dull red
colour. The eye-spot does not reappear in the danghter-
cells which arise by longitudinal division. In the case of
the larger originally colourless cells the colourless protoplasm
occupies the middle of the cell. When about to divide a
pyrenoid makes its appearance toward either end of the cell,
and what appears to be a transverse division occurs.

The daughter-cells in either case separate from one another
and, lying each in a clear space, undergo further divisions.
These subsequent divisions are, like those which take place
in the palmella state, more of the nature of budding than of
equal division ; hence are produced rows of cells of gradually
decreasing size, which run from the periphery into the
deeper tissues of the body (figs. 9 and 10, Pl. 13). The re-
semblance between the green tissue of loosely associated cells
which occupies the body of Convoluta and the palmella stage
of the alga is noteworthy.

The appearance presented by an abnormal Convoluta—

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 195

unique among the many thousands examined—lends support
to the view which we put forward that the “ Convoluta stage”
of the algais to be regarded as consisting of an hypertrophied,
senescent palmella.

The animal in question presented an appearance under the
low power of the microscope very like that of a fern-pro-
thallus. Its body was lobed posteriorly, so as to present a
conventionally heart-shaped: form, and its green cells, so
closely packed as to obscure all other elements of the body,
were rounded and uniformly green, quite like those of the
typical palmella-stage, which stage at the time of these
observations was unknown to us.

To return to our consideration of the green cells of the
normal animal. .'The rapid division of the green cells in
recently infected Convoluta is accompanied by significant
changes in the nucleus, changes which since they render
intelligible the loss of power of independent existence on the
part of the green cells of the adult animal we now describe in
some detail.

The nucleus of the flagellated cell (fig. 12, P]. 14) hes in the
colourless part of the protoplast, about the middle of its depth,
equidistant from the bases of the flagella and the pyrenoid.
Its general appearance is that of aspherical uniformly staining
body, from the periphery of which radiating branches run,
two upward towards the points of insertion of the cilia and
two downward toward the pyrenoid. These branches, though
mainly cytoplasmic, show by their staining reactions with
nuclear stains (e.g. Benda’s iron hematoxylin) that they also
contain nuclear material.

In other flagellated cells the nucleus presents the appear-
ance described by Dangeard (1899) as occurring occasionally
in the Chlamydomonadez, of a clear spherical area, in the

centre of which lies a deeply staining “ nucleolus.”

Again, in
other specimens the nuclear substance consists of three fairly
large granules lying either side by side or else pyramid-wise;
the granules may be spherical or elongated, of equal or

unequal size. Occasionally specimens show two vertical rows,

196 F. KEEBLE AND F. W. GAMBLE.

each of three granules. So far we have met with no indica-
tions of mitotic division except this separation of the nucleus
into rows of granules.

In the division stages of the non-motile cells, groups of
rods and granules may be made out and appear to represent
phases of nuclear division.

The cells of the colonial stage (Fig. 15, pl. 14) present two
types of nucleus. The resting nucleus, lying slung in cyto-
plasm in the centre of the cell, consists of a homogeneous,
deeply-staining central body, surrounded by a clear area,
the limits of which cannot be sharply distinguished from
those of the cytoplasmic envelope containing it. he dividing
nucleus consists of a diffuse group of fine granules, occupying
a circular or oval area of the cytoplasm. The granules
sometimes form two groups at opposed ends of a cell.

Thus the nuclei of the flagellated stage and of the actively
developing palmella stage are, though small, distinct and
readily recognisable. So, too, is the nucleus of the green or
colourless cell, as it lies in the body of a larval. Convoluta
immediately after ingestion, After the first division in the
body the nucleus of each daughter-cell has the appearance of
that just described as characteristic of the actively dividing
cells of the colonial stage—namely of an oval area occupied
by diffuse granules (Fig. 15, pl. 14). As division of the
infecting cells proceeds the presence of nuclear granules in
the resultant cells is made out with increasing difficulty, till
finally, in the adult animal, whose body is densely packed
with green cells, it is often difficult to pick out any in which
remnants of nuclear granules remain, ‘I'he great majority of
the green cells of Convoluta are not complete cells, but cells
which show all stages of diminishing nuclear substance
(Figs. 9 and 10, pl. 15). Thus the conclusion to which our
physiological investigations led us that the body of Convoluta
is the grave of the green cell receives histological confirmation.
If this degeneration of the nucleus begins almost immediately
after the ingestion of the infecting cells some light is perhaps
thrown on the fact to which attention has frequently been

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 197

drawn—that the green cells of Convoluta are without cell-
walls, for it is well known that the nucleus of the plant-cell
presides over cell-wall formation. An enucleated fragment
of a plant-cell, though capable of performing certain vital
processes—e. g. photosynthesis—is unable to form a new wall.

We may therefore regard the suppression of cell-wall as the
first result of a progressive nuclear degeneration. Enough,
however, of the nucleus is left to preside over cell-division,
which proceeds rapidly after infection; as this division goes
on less and less nuclear material remains, till finally partial
cells, incapable of independent life and probably also incapable
of further division, alone are left.

It is noteworthy that we have come across no evidence of
mitosis in the dividing green cells of the animal.

A significant phenomenon is revealed by a reference to
Pies. 9 and 10, pl. 15, and to the description thereof. Here,
as at (Nw., ge.), are isolated green cells, in each of which a
nucleus is recognisable; in other parts of the section are
ingrowing rows of cells budded off from the outermost cell,
and of these rows only the outermost cells contain a distinct
nucleus; others contain only deep-staining granules; and
others, again, no nuclear substance whatever (Fig. 9, pl. 15).

A parallel suggests itself between the green cells of Con-
voluta and the red blood-cells of the higher vertebrates. As
the red discs are enucleate partial cells budded off from the
nucleated red cells, so may the green cells be regarded as
enucleate partial cells budded off from the outermost row of
nucleated green cells, and as the red dises are of limited life
and specialised (respiratory) function, so are the green cells
of limited life and of specialised (photosynthetic) function.

The green cell devoid of a nucleus of its own would not,
however, appear to be shut off from all nuclear influences.
For the enucleate green cell may be connected by a finely
drawn process with another green cell still possessed of
nuclear substance (Pl. 13, fig. 9). Moreover, such green
cells as are without nuclear material are accompanied each
by a large attendant nucleus of animal origin; this close

198 F. KEEBLE AND F. W. GAMBLE.

association of “attendant nucleus ” and green cell is shown
in Pl. 13, fig. 9, Nw. Mes. Further investigation of this
phenomenon is required ; but such observations as we have
made point to the belief that these attendant nuclei are those
of wandering cells which lie in wait, as it were, for enucleate
green cells and at a subsequent stage bring about their
destruction by digesting them.

The foregoing. interpretation of the series of facts described
offers some support in favour of the hardy suggestion, due
originally to Schimper,' that the higher green plants are an
association of two organisms—one a colourless organism, the
other originally a green alga but now represented by the
chloroplasts and by them alone.

For an adult Convoluta is a complex of two organisms—one
the colourless animal, the other the chloroplast remainders
of the original green alga; but in this case, unlike that
imagined for the green plant, the synthesis is not a perma-
nent one. It endures but for the lifetime of the animal and
has to be recommenced in every larval Convoluta.

Summary of Section V.

(1) The colourless phase of the infecting alga normally
supphes the cells which develop in the body of Convoluta
into the green-celled tissue, which tissue is comparable with
the palmella-stage of the alga.

(2) But infection may also result from the ingestion of the
green active or green temporarily resting-cell.

(3) Ingestion is followed by the active division of these
cells which develop transient eye-spots, etc., but which do
not form a cell-wall.

(4) The distorted shapes of the green cells in the animal
are due to osmotic conditions,

(5) The nucleus of the green cell dividing in the animal
undergoes progressive degeneration and finally disappear,
leaving the photosynthetic machinery intact.

1 * Bot. Zeit.,’? 1883, p. 112, footnote.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 199

(6) The green partial cells which result are incapable of
independent existence.

Section VI. Tue SIGNIFICANCE AND THE CONSEQUENCES OF
THE ASSOCIATION OF ANIMAL AND GREEN CELL.

The relation between Convoluta and its green cells has
sometimes been regarded as an example of parasitism, the
parasite being the green cell and the host the animal. That
this is an erroneous view we have already demonstrated.

More usually it is assumed that both partners in the
association receive advantage therefrom, and consequently
the relation is considered to be symbiotic.

It is obviously important, if such a term as “symbiosis” is
to retain any value whatever, that a detailed examination of
each case which appears to fall into this category should be
made before it is relegated thereto. This is the purpose of
the present section. In it we show that the relation between
Convoluta and its green cells is both intricate and variable
in the sense that the nature of the relation changes as the
association becomes more intimate.

We show that it is only by taking the widest view of the
case that it can be brought within the category of symbiotic
phenomena.

The inquiry as to what is the significance and what are the
consequences of the association of green cells and Convoluta
resolves itself in large measure, but not entirely, into an
examination of the modes of nutrition of infecting alga and
of animal. The mode of nutrition of the infecting alga in its
free state must be first described.

The fact that we have succeeded in obtaining luxuriant
cultures of the alga in artificial sea-water to which traces of
potassium nitrate were added proves that it is capable of a
typical plant-like (holophytic) mode of nutrition. The alga
under these conditions photosynthesises carbohydrates—
storing the surplus as starch—and utilises the nitrogen of
‘the nitrates in the formation of its proteids.

volt. 51, PART 2.—NEW SERIES. 16

200 F. KEEBLE AND F. W. GAMBLE.

But, as we have already shown in Section IV, the alga in
its free state grows more actively when enabled to obtain its
nitrogen from organic compounds (uric acid and urea) than
when “inorganic nitrogen” (nitrate) is supplied.

Next, as to the mode of nutrition of the alga in its “ Con-
voluta stage.”

Geddes (1879-1881) was the first to demonstrate that
Convoluta gives off oxygen when exposed to lght. He also
proved the presence of starch in the green cells, stating that
this substance is present always in small quantities. Our
own experiments complete the proof that the green cells of
Convoluta photosynthesise carbohydrates. Thus, we have
shown that the green pigment of these cells is true chlorophyll
(op. cit., p. 377), and that starch, present in the green cells,
disappears in darkness and reappears when the animals are
brought into the light. The photosynthetic activity of the
green cells having been demonstrated, the questions remain,
Does the animal receive a share of the elaborated carbo-
hydrate ? and, if so, How is the transference of this substance
from green cell to animal tissue effected ?

Geddes has stated that Convoluta survives only a few
days’ exposure to darkness, the implication being that when
photosynthesis is arrested death from starvation ensues. But
according to our experiments (op. cit. p. 375), if care is
taken with respect to the water supply Convoluta may live in
darkness for several weeks.

Again, Geddes’ statement that starch occurs in Convoluta
only in small quantities might be interpreted as meaning that
the product of photosynthesis is passed on to the animal as
soon as it is formed, and that only a small residue is accumu-
lated in the green cell. The observation, however, is incorrect,
for the green cells of Convoluta at certain times contain so
much starch as to give to the body when stained with iodine
a deep blue or blue-black colour.

We have endeavoured to ascertain to what conditions the
variable amount of starch in the green cells is due.

For this, samples of animals were collected twice daily

THE GREEN OELLS OF CONVOLUTA ROSCOFFENSIS. 201

during a lunar month from a certain patch of Convoluta.
‘The samples were taken just after the tide had left the patch
—1i. ce. immediately after the sojourn of the animals in dark-
ness below the surface, and again after their spell of isolation,
just as the tide was about to cover them.

The animals were fixed on glass slides, decolorised, stained
with potassium iodide-iodine, and the amount of starch esti-
mated by the resulting coloration.

The records obtained (‘Table V) during August, 1905, exhibit
a bi-monthly periodicity in the amount of starch present in the
animal. The amount is large during the spring tides and falls
off during the slack tides. The result is perhaps susceptible of a
simple explanation. Jor during the spring tides low water, and
consequently exposure of Convoluta to light, occurs about the
middle of the day (and night), whereas during the neap
tides the exposure to light takes place during the early
morning and late afternoon. Consequently, though the
number of hours of daylight to which Convoluta is exposed
may be actually greater during the siack tides than during
the spring tides, the animals are exposed to a higher light
intensity (of the mid-day) during the spring tides than during
the neaps. Hence it seems reasonable to suppose that photo-
synthesis will be more active during the spring tides, and
that this greater activity will be recorded by the larger
deposit of starch. It is worth noting—though aside from
the question under consideration—that the periodicity of
ege-laying by Convoluta receives some explanation from the
foregoing; for the egg-laying periods, occurring soon after
the spring tides, follow closely on periods of abundant
nutrition. Returning to the question, Does the animal
receive a share of the carbohydrate elaborated by the green
cells? we have just seen that on this hypothesis periodicity
of egg-laying receives a simple explanation. But conclusive
evidence is derived from observation of the rates of growth
of recently infected Convoluta. The rapid increase in the
numbers of green cells in the body—from one or two to
thousands—is accompanied, not by any diminution of the

202... F. KEEBLE AND F. W. GAMBLE.

animal’s tissues, but by the marked growth of the animal.
Whilst uninfected Convoluta, as described presently, remain
diminutive, those which become infected resume growth and
increase rapidly in size.

It appears probable on several grounds that the animal
cells are unable to lay hold of the starch contained in the
green cells. Thus, though young Convolutas fed on starch-
grains ingest them readily, they digest them not at all.
Again, when adult Convolutas are kept in darkness the starch
of the green cells disappears with extreme slowness—even
after eight days some starch may still be present. This
slowness of disappearance would seem to indicate that it
takes place according to the requirements of the green cell,
and not according to those of the animal. It is therefore
likely that the animal cells obtain the product of photo-
synthetic activity directly as sugar. Just as from the green
cells of a plant exposed to light there is a constant osmotic
streaming of photosynthesised sugar to the colourless cells,
so from the green cells of Convoluta sugar passes to the
colourless animal cells. In general, the demand in adult
Convolutas for food material is so great that but little starch
accumulates in the green cells. It is only during the mid-
day exposure to high light-intensity at the spring tides that
photosynthetic activity considerably exceeds that of trans-
location. At all other periods during the adult life of the
animal the photosynthesised carbohydrate passes away as fast
as it is formed from the green cell to the animal. The sugar
thus obtained is stored ultimately in the animal tissues or in
the egos in the form of fat.

We have next to consider the system, green cell and
animal, with respect to nitrogen metabolism. ‘To do this
requires a review of the facts known with respect to the
nutrition of the constituents of the system.

Von Graff (1905-1906) was the first to draw attention to
the absence of any vestiges of solid food from the body of
Convoluta. He concluded that this animal does not take up
solid food. Geddes’ observations, which, however, as we

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 208

have shown, require considerable modification, led him to
conclude that the animal relies on the green cells for its
food supply.

Georgevitsch (1899), finding that colourless larvex die, if
uninfected, within two or three days, seemed thereby to
establish the existence of a yet closer dependence of animal
on green cell.

We have shown in a former paper that von Graff’s state-
ment is too absolute, and that of Georgevitsch inaccurate.

We may summarise our own observations as to the ingestion
of food by Convoluta.

A well-marked mouth—already described by von Graff in
adult animals—is present in just hatched animals. This
mouth, capable of a wide gape, is situated on the under
surface, about the middle of the body. When open the mouth
is connected by a short, ciliated, ectodermal invagination
with an axial mass of highly vacuolated tissue which con-
stitutes a rudimentary gut.

By means of this mouth a young Convoluta takes up almost
any objects which it can encompass; diatoms and unicellular
green alge form the staple diet in the open, while in the
laboratory starch-grains, litmus, lamp-black, and sand-grains
may be swallowed.

If colourless uninfected Convolutas are cultivated in water
devoid of the infecting alga but rich in other organisms, such
as diatoms, they continue to feed actively for some time—for
a week or more. But after this period they become increas-
ingly inert and ultimately cease altogether to take up food.

In this state they await, as it were, the infecting alga. If
the latter is supplied they ingest it, and, as it develops, the
animals resume their activity. If, on the other hand, the
infecting organism is still withheld, the lethargic condition
becomes more pronounced, and undergoing gradual diminu-
tion in size, Convoluta finally dies though lying in the midst
of food of a kind which, at an earlier stage, it took up and
digested readily enough, and on which young infected Con-
volutas are still feeding. It is to this remarkable behaviour

204 F. KEEBLE AND F. W. GAMBLE.

that we attribute the failure of all our attempts to raise a
colourless race of Convolutas.

The infected young animals continue during the period of
development of their green cells to ingest solid food. But
when this period is complete and the reproductive organs are
mature all ingestion of solid food ceases.

The only food materials now available are such substances
synthesised by the green cells as may pass in solution from
ereen cell to animal cell.

This mode of nutrition of the animal is of short duration.
It is soon succeeded, or rather overlapped, by another which
consists in the digestion by the animal cells of whole groups
of green cells. During the adult life of Convoluta large or
small aggregates of green cells in all stages of disintegration
are to be met with, lying in vacuoles in the axial tissue which
constitutes the gut of the animal.

From this stage onward the ultimate significance of the
relation between animal and green cells stands more and
more clearly revealed. The animal is now and henceforth
parasitic on its green cells, As a result of this habit old
animals are not infrequently to be met with in laboratory
cultures whose bodies are half green and half colourless.

If in order to summarise these conclusions we employ the
terms used by plant physiologists in describing the principal
modes of nutrition, and if we consider for this purpose a
green Convoluta as an organism, then we may distinguish
the following styles of nutrition :

(1) In the pre-infection stage the animal feeds hetero-
trophically—i. e. typically animal-wise.

(2) Young infected stage; the green animal is nourished
mixotrophically, viz. by (solid) ingested food, and by
photosynthesised food-substances.

(3) Mature green Convoluta; the mode of nutrition is
holophytic—typically plant-like. The green cells are the
photosynthetic agents, the animal’s colourless cells receiving,
just as do the colourless cells of plants, the products of
photosynthetic activity.

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 205

(4) Old green Convoluta; the only term that can be apphed
is autotrophic; the green animal behaves to its green cells
just as any animal or plant may behave to any reserves of
food-material ; it digests them.

If we consider the same facts from the point of view of the
animal Convoluta; then stages two, three, and four represent
the progressive parasitism of the animal on its contained green
cells.

We have next to consider the sources of the supplies of
nitrogen compounds to the green cells and to the animal.

The facts already described, proving that the infecting
alga thrives better on organic than on inorganic nitrogen
compounds suggest that the habit of this alga of settling
down on the egg-capsules of Convoluta originated in obedience
to these nitrogen-requirements.

The following considerations and observations lend sup-
port to this view. Whereas most Turbellarians have a well-
developed excretory system of flame-cells, Convoluta
roscoffensis has none. Nor do we find in this species
those granular accumulations, often appearing as localised
refractive bands or patches, which are constant features in
allied species and which von Graff regards as being of an
excretory nature. The fruitlessness of our prolonged search
for flame-cells justifies us in holding that none exist in
Convoluta roscoffensis. Moreover, green Convolutas
show no signs of any excretory substances. The green cells
we know are capable of utilising such excretory nitrogen
compounds as uric acid and urea. It seems, therefore, pro-
bable in a high degree that the green cells of Convoluta are
its excretory system.

The following observations lend a certain measure of
support to this hypothesis. If larval Convolutas are protected
from infection and kept without food, their large stores of
reserves gradually disappear, and vacuoles charged with long
acicular crystalline bodies make their appearance. ‘The
numbers of these vacuoles and of their contained crystals
increase till they form one of the most striking features of the

206 F. KEEBLE AND F. W. GAMBLE.

body. We have not yet succeeded in determining the chemical
nature of these crystals; they disappear when the ‘‘ vacuole”
containing them is destroyed, as it is by every fixative or
reagent we have used. Nevertheless it cannot be doubted
that these crystals are products of metabolism, and it seems
likely that they are of the nature of excretory nitrogenous
substances. Now, these acicular crystals do not occur in the
infected green animal.

Admitting the argument here set forth, the conclusion
follows that the green cells, on gaining entrance to the body,
take over the business of disposing of the waste products of
nitrogenous metabolism. We have learned already that the
life of the animal depends on the occurrence of infection,
that failing infection the animal ceases to feed and dies,
possibly as the result of auto-intoxication by the accumulated
waste products. Thus the closeness of the relation between
animal and green cells is such that they together constitute,
in the green Convoluta roscoffensis, one organism.

The conclusions concerning the réle of the green cells in
utilising the nitrogenous waste of the animal as material for
proteid synthesis appear to us to throw light on the last
phase of the nutrition of the animal. That phase is charac-
terised by the digestion of the green cells. In the early
stages of its development Convoluta offers to the green cells
ample supplies of waste nitrogen compounds, the products of
its proteid metabolism the materials for which were derived
from the reserves in the egg and from the products of
digestion of solid food. On these excretory nitrogen com-
pounds, and on the product of its own photosynthetic activity,
the green cell flourishes, and in turn furnishes soluble carbo-
hydrates to the animal. But when ingestion of solid food
ceases there follows a shortage of nitrogenous waste sub-
stance. The green cell, adjusted to utilise organic nitrogen,
now has only available for proteid synthesis the soluble
nitrogen compounds of the sea-water. A dearth of nitrogen
compounds available for anabolic processes subvenes. This
dearth falls earliest and acutest on the animal. Urged by its

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 207

nitrogen hunger, it turns upon the green cells and raids such
stores as they contain by digesting them. ‘These stores are
but limited, and when exhausted, death, both of the animal
and of its green cells, inevitably follows.

The election by the infecting alga of the egg-capsules and
the surface of the animal as a normal habitat may also be
regarded as a symptom of nitrogen hunger ; so, too, is the
ultimate digestion of the green cells by the animal.

Taking the broadest view of the whole relationship, includ-
ing therein, not only the green cells inhabiting the body of
Convoluta, but also the free green cells living on soluble
organic waste contained in the egg-capsules, we may classify
it as a symbiotic relationship, for it is now only by reason of its
infection by the green cells that Convoluta roscoffensis,
the species, persists, and though the green cells which enter
the animal never escape alive, this is, as it were, but the price
which the species has to pay for its lodging.

But if we confine our attention to a green Conyoluta, not
looking beyond the association of green cells and animal, then
the relation constitutes a case of parasitism, the host being the
green cells and the parasite the free-living animal.

The extraordinary restriction of the range of the species
C. roscoffensis we may regard as the result of this peculiar
and economically unsound attempt on its part to solve the
“nitrogen question.”

Summary of Section VI.

(1) Convoluta exhibits four phases of nutrition, passing
from the typically animal to the completely parasitic.

(2) The infecting alga shows specialisation in the direction
of saprophytism.

(3) This habit enables the green cells to utilise the products
of the animal’s nitrogenous metabolism, and so to develop
rapidly within the body, where they serve as an excretory
system to the animal.

(4) he habit of the infecting alga, in its free state, of fre-

208 F. KEEBLE AND F. W. GAMBLE.

quenting the egg-capsules and also the surface slime of the
body of Convoluta is to be ascribed to its nitrogen require-
ments; this habit developed originally with no reference to
Convoluta (being shared with various Chlamydomonadee ;
e.g. Carteria subcordiformis, Wille n. sp. (1903)), was
nevertheless an essential preliminary to the association of
animal and green cell.

(5) The association entails ultimately the death of the
green cell and of Convoluta; but whereas the former dies
without issue the latter first produces one or more batches
of eggs.

(6) The consequences of the association are: To the green
cell hypertrophy, nuclear degeneration, premature senescence,
and death. ‘'l'o the animal: suppression of excretory system,
cessation of feeding, resignation of power of existence apart
from the green cells—i.e. obligate parasitism; adaptations
facilitating the photosynthetic activities of the green cell—
e.g. marked positive phototropism identical with that dis-
played by the infecting alga in its free state.

Section VII. GENERAL SUMMARY.

Convoluta roscoffensis hatches out from its egg-
capsule as a colourless animal whose body contains neither
green cells nor antecedents of green cells.

Infection takes place neither directly nor indirectly from
the body of the parent, but from the sea-water or from the
egg-capsules to which the infecting organism is chemo-
tactically attracted and on which it habitually settles down
and develops.

Experiment shows that the green cells of adult Convoluta
are incapable of life apart from the body of the animal ;
histological examination, proving that the development of the
green cell within the body is accompanied by degeneration of
its nucleus, supplies the explanation.

The infecting organism has been isolated, and, by the addi-

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 209

tion of it to colourless Convoluta, the green animal has been
synthesised.

In its free stage the infecting organism shows the essential
characters of the Chlamydomonadee ; its four equal flagella
point to its inclusion in the genus Carteria ; certain peculiari-
ties suggest that the assignment of the infecting organism to
this genus should be only provisional. The infecting organism
is capable of a saprophytic as well as of a holophytic mode of
life and occurs in both colourless and green forms. — Its
saprophytic habit leads to its association with the egg-
capsules and constitutes a first physiological step toward its
association with the body of Convoluta.

The green cells serve as an excretory system to the animal.

The relation between green cell and animal changes with
their development, passmg from a symbiotic relation to ove in
which the animal is parasitic on the algal cells.

The association leads to marked changes of habit on the
e. g. to its ceasing from the ingestion of

part of the animal
food—and is best interpreted as an economically unsound
attempt on the part of both green cell and animal to solve
the “ nitrogen problem.”

F. KEEBLE AND F. W. GAMBLE.

210

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218

NSIS.

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GREEN OELLS OF CONVOLUTA ROSCOFFI

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F, KEEBLE AND F, W. GAMBLE.

TastE V.—Daily Estimate of Starch-content of Green

Cells of C. roscoffensis during Twenty-eight days.
Trégastel, 1905.
Recorded day-period
Date. Tide. airing eich Can Starch.
uncovered.

Aug. 18 82 decimetres 11—38.30 Much.
tee? (i) 80 - 11.20—4.15 se
je) 78 ao 11.30—4.15 E
Aneel 75 sts 11.45—4.50 mr
pe) 72, 3 12.30—6 5
se OR} 68 Ee 16.15
os | 67 “4 1.50—6.40 Little,
20 68 s 3.30—7 4

= daylight—9.30
— i { 4.30—dusk } ae
ee 74: 2 { tee } Fair amount.
2808 79 :; 6.50—12 50 Much.
ed) 83 is 7.50—1.30 a
sit) 85 a §.30—2.30 -
S34 Git 6), 9-33.15 if

Sept. 1 9] 9.45—4 om
ce 90 mi 10.15—4.15 a
BND) 88 x 11—4.40 a
sare A 84 5 _12—6 Less
Tk 79 s 1—6.40 _

—7
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—8.15
” 7 71 9 { 5 30 } ”
—9 More but still
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ne —10 e
sg og 73 ” { 5.30—11.20 } Little.
6.50—12 noon
» 10 oe: j 6.30—12 night } v
ol 79 a 7—1.10 Much.
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aS 84 + §.30—2 "
14 84 8 10.80—3 —

215

GREEN CELLS OF CONVOLUTA ROSCOFFENSIS.

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GTA I,

17

voL. O01, part 2.—NEW SERIES.

216 F. KEEBLE AND F. W. GAMBLE.

14.

15.

LITERATURE REFERRED TO IN THE TEXT.

. 1879. Guppgs, P.—“ Observations on the Physiology and Histology of

Convoluta Schultzii,” ‘ Proc. Roy. Soe.,’ vol. xxviii, 1879.

. 18794. Geppes, P.—“ Sur la Chlorophylle animale et sur la Physiologie

des Planaires Vertes,” ‘Archiv de Zool. Exp. et Générale,’ ser. i, vol.
viii, 1879.

. 1881. Gepprs, P.—“The ‘Yellow Cells’ of Radiolarians and Calen-

terates,” ‘ Proc. Roy. Soc. Edin.,’ 1881.

. 1881, 1882. Enrz, G.— Ueber die Natur der Chlorophyllkorperchen

der Thiere,” ‘ Biol. Centralb.,’ vols. i and ii, 1881, 1882.

. 1882. Lanxester, BH. Ray.—® On the Chlorophyll-corpuscles and Amy-

loid Deposits of Spongilla and Hydra,” ‘Quart. Journ. Micros. Sci.,’
vol. xxii, 1882, p. 229.

. 1883. Branpt, K. — “Ueber die morphologische u. physiologische

Bedeutung des Chlorophylls bei Thieren II,” ‘ Mitt. Zool. Stat.
Neapel.,’ vol. iv, 1888, p. 191. (Contains a full list of references up
to date.)

. 1883. Excetmann.— Ueber tierisches Chlorophyll,” ‘Pfiiger’s Archiv

f. d. ges. Phys.,’ vol. xxxii, 1883, p. 80.

. 1889, 1891. Faminrain, A. — ‘ Beitrige z. Symbiose v. Algen u.

Thieren,” ‘Mém. de PAcad. Imp. d. Sci. St. Petersburg,’ vols. xxxvi
and xxxvili, 1889, 1891.

. 1890. Berertnck, M. W.—‘ Kulturversuche mit Zoochlorellen,” etc.,

‘ Bot. Zeit.,’ vol. xlviii, 1890, p. 725.

. 1890. Lankester, IW. Ray.—“ Protozoa.” Zoological articles reprinted

from ‘ Encycl. Brit.,’ 1890, p. 5.

. 1891. Bournr, A. G—‘On Pelomyxa viridis,” ‘Quart. Journ.

Micros. Sci.,’ vol. xxxii, 1891, pp. 857—364.

, 1891. Hapertannt, G.—“ Bau u. Bedeutung d. Chlorophyll-zellen von

Convoluta roscoffensis.” Appendix to ‘Die Organisation der.
Turbellaria accela,’ by von Graff, Leipzig, 1891.

. 1891. ScugwraKorr, A.—(Infection in Frontonia), ‘ Biolog. Centralb.,’

vol. xi, 189], p. 475.
1892. Danrec, F. Lu.—* Recherches sur la Symbiose des Algues et les
Protozoaires,”’ ‘ Ann. de |’Instit. Pasteur,’ vol. vi, 1892, p. 190.

1899. Danegearp, P. A.—“‘ Mémoire sur les Chlamydomonadinées,” ete.,
‘ Le Botaniste,’ 6th ser,, fase. 2—6, 1899, p. 66,

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 217

16. 1899. Georcnyitscn, J.— tude sur le Developpement de la Con-
voluta roscoffensis,” ‘ Archiv. d. Zool. Exp. et Gén.,’ ser. iii, vol.
vii, pp. 843—361,

17. 1900. Buackman, F. F.—‘‘ The Primitive Algae and the Flagellata,”’
‘Ann. of Bot.,’ xiv, 1900.

18. 1900. Dancrarp, P. A. —‘‘Les Zoochlorelles d. Paramecium,” ‘ Le
Botaniste,’ 7th ser., fase. 8, 4, 1900.

19. 1903. GamBie, F. W., and Kersie, I'.—‘‘ The Bionomies of Convoluta
roscoffensis, ete,” ‘Quart. Journ. Micros. Sci.,’ vol. lvii, 1903.

20. 1903. Witte, N.—‘ Algologische Notizen,’ vols. ix—xiv, ‘ Nyt. Magazin
for Naturvidenskaberne,’ vol. xli, 1903.

21. 1904. Orrmanns, F.—‘ Morphologie u. Biologie d. Algen’ (Jena, 1904),
vol. i, p. 189; see also p. 374, vol. ii, 1905, for literature on ‘* Zoo-
chlorellen.”

22. 1904. West, G.—‘ The British Fresh-water Alge,’ Cambridge, p. 188.

23. 1905—1906. Grarr, L. von.— Turbellaria,”’ in Bronn’s ‘ Thierreich,’
vol. ii, 1905—1906.

24. 1905. Krrsue, F., and Gamsie, F, W.—“The Isolation of the infecting
Organism of Convoluta roscoffensis,” ‘Proc. Roy. Soc.,’ ser. B,
1905 (an abstract of Section IIT of this paper).

25. 1905. Pratt, KH. M.—‘‘ Zoochlorelle and Digestive System of Aleyo-
naria,” ‘Quart. Journ. Micros. Sci.,’ vol. 49, 1905. Ceylon Pearl
Oyster Fisheries, Suppl. Rep. xix.

EXPLANATION OF PLATES 13 & 14,

Illustrating the paper by Dr. Keeble and Dr. Gamble on
“The Green Cells of Convoluta roscoffensis.”

REFERENCE LETTERS.

Bact. Bacteria on the caps. (egg-capsule). 2.1. Basement membrane.
C. Empty cyst left by an escaped flagellated cell from the palmella state. CZ.
Chloroplast. Cé/. Cilia. C. Pl. Cytoplasmic plug forming the “neck”’ of the
green cell. Zp. Hpidermal layer. G.c. Green cells. Mes. C. Mesenchyme
(probably phagocytic) cells of Convoluta. MM/w. Mucilage of wall of green
cell. Nu. G.C. Nucleus of green cell. Nw. Mes. Nucleus of mesenchyme
cell associated with the green cell. Ov. Eggs of Convoluta in the common
capsule. Pyr. Pyrenoid. St. Stigma (eye-spot).

218 I. KEEBLE AND F. W. GAMBLE.

PLATE 13.

Fia. 1.—Egg-capsule of Convoluta filled with a dense mass of flagellated
green cells forming a pure culture of the infecting organism. X35.

Fic. 2.—The same capsule under slight pressure. The flagellated cells are
swarming actively and escaping through the ruptured wall.

Fic. 3.—The smaller green cells (microcytes) of the infecting organism in
the active phase, showing the delicate wall, four flagella, chloroplast, stigma
and clear “neck” of cytoplasm in which the nucleus is lodged. (For
histology see also Fig. 12 B, Pl. 14.)

Fic. 4.—The larger green cells (macrocytes). (No contractile vacuoles in
either form of green cell.) Cf. Fig. 12.

Fie. 5.—A portion of the egg-capsule; the green cells have come to rest
and lie in pairs.

Fic. 6.—The infection of an egg-capsule by the green cells chemotactically
attracted thereto (see pp. 188, 189 of text).

Fic. 7.—Infected capsule, more highly magnified. Green cells (and
bacteria) seen on the outer edge of the capsule. Other green cells have made
their way through the capsule to the egg (ov.).

Fre, 8.—Peculiar colonial form of the green-celled infecting organism (for
regular palmella stage see Figs. 14 and 15, Pl. 14). Non-flagellated green
cells in inverted position borne at the ends of branching columns of stratified
mucilage.

Fra. 9.—Dorsal portion of transverse section through the body of an
adult Convoluta roscoffensis showing the structure, arrangement, and
relations of the green cells. The majority of the green cells have no nuclei,
some mere granular traces. ‘The green cells are arranged in rows running
inward from the periphery ; the outermost green cell of a row has a nucleus
(red). Indications of continuity between green cells are seen and the close
relation between them and the mesenchyme cells of Convoluta is shown.
x 750.

Fic. 10.—Another more highly magnified section through the body of
Convoluta (fixed with Fleming, stained with safranin). Four green cells
suspended by slender processes from the basement membrane and separated
by mesenchymatous cells. ‘he green cell on the left hand has a nucleus
(Nu. ge.), the others contain only granules which stain deeply with safranin ;
similar deep-staining granules are also plentiful inthe subjacent. mesenchyma
(Cam. luc., oc. 12, obj. 45).

THE GREEN CELLS OF CONVOLUTA ROSCOFFENSIS. 219

PLATE 14,

Fie. 11.—Life-history of the green-celled infecting organism in the free
state. A, Eneysted colourless macrocytes, resting and dividing into colourless
daughter-cells. 3B, Microcyte dividing. c, Eneysted green macrocytes,
resting and dividing into green daughiter-cells. »p, Apparent fusion of two
macrocytes (see text, p. 190). », Apparent fusion of two unequal colourless
cells.

Fic. 12.—a, A typical free macrocyte. 3B, A typical free microcyte. The
nucleus is homogeneous, and in A sends processes towards the bases of the
flagella and also tuwards the pyrenoid. The reticulate surface-cytoplasm is
shown, the more internal chloroplast being drawn in optical section.

Fic. 13.—Green cells from the body of just-infected larval Convolutas.
The large nucleus of the recently ingested green cell contrasts markedly with
the degenerate nucleus of the daughter green cell budded off later from such
a mother-cell. (Cam. luc., oc. 12, obj. ;4; Zeiss.)

Figs. 14 and 15.—The infecting organism in the palmella (colonial) stage.
A, B show green cells of the palmella organising active cells. c, The empty
cyst left after the escape of a flagellated cel]. Fig. 15 illustrates the origin
of new cells of the palmella by budding.

Fic. 16.—a, B, c, the gradual disappearance of one of a pair of apposed
resting-cells (see text, p. 184). Dp, 5, Colourless and green resting-cells.
F, G, Formation of colourless daughter-cells within a colourless mother-cell
H, F, Formation and discharge of green daughter-cells.

PLUMES OF CEPHALODISCUS. 221

On the Development of the Plumes in Buds of
Cephalodiscus.

By

W. G. Ridewood, D.Se.,
Lecturer on Biology at St, Mary’s Medical School, University of London.

With 11 Text-figures.

PAGE
Introduction. ; . ; . 221
Methods 223
Development of the Plumes. in Bae of Genlalouiecns

hodgsoni : : 224
Development of the Plumes in Buds of Genhelodisens

dodecalophus . : 230
Development of the Plumes in Bade of Genlmlodisene

nigrescens ; 235
Development of the Eee, in Bude of enialadivons

gilchristi : : . 242
General Remarks on the Collar and its Appendage ; . 245
Summary : 251
Explanation of the Muprevialions plored in Text- fears Q,

3, 4,6,7,and9 . : ; : ; > 252

INTRODUCTION.

During the progress of an investigation on the anatomy of
three recently discovered species of Cephalodisecus (C.
nigrescens, ©. hodgsoni, and C. gilchristi), kindly
entrusted to me for description by Prof. H. Ray Lankester,
F.R.S., Director of the Natural History Museum, London, I
noticed that in the development of the buds there is no
torsion of the first and second plume-axes such as is de-
scribed by Masterman as occurring in buds of C. dodeca-

222 W. G. RIDEWOOD.

lophus,! and that the last plumes do not develop between
the earlier plumes and the buccal shield, as recorded by the
same writer, but on the side of those plumes remote from the
buccal shield. His figures 64—68 are reproduced as text-
fies 1.

I was unable to settle the point satisfactorily in time for
the introduction of my results into the two papers describing
the structure of the polypides and tubaria of the new species,”
but since the completion of those papers I have made an
exhaustive study of the growth of the plumes in buds of

M1
" : . See = es May we)
ee a a

TreXxT-FIGURE 1.—Diagrammatic transverse sections of plumes and
buccal shield of Cephalodiscus dodecalophus at five stages of
development. Copied from A. 'T. Masterman’s paper in the ‘Trans.
Roy. Soc. Kdin.,’ vol. xxxix, part 3, 1898, plate 4, figs. 64—68.
The original numbering has been retained.

Cephalodiscus hodgsoni, C. nigrescens, and C. gil-
christi, and having made from the abundant material at
my disposal a well-graded series of buds of each species, I
was enabled to confirm my earlier conclusions,

I finally prepared a series of buds of the “ Challenger ”
species, Cephalodiscus dodecalophus, with the result
that I am satisfied that the buds of this species agree with

1 «Trans. Roy. Soc. Edin.,’ vol. xxxix, part 3, 1898, p. 521.
2 <«iscovery” Expedition Reports,’ vol. ii, 1907, and ‘ Marine Investi-
gations in South Africa,’ vol. iv, 1906,

PLUMES OF CEPHALODISCUS. 223

those of the other three species. The torsion described by
Masterman does not occur, but the grooves of the first and
second pairs of plumes, developed in such a manner that
they face ventrally towards the buccal shield, continue to
face the shield. The last two pairs of plumes are not deve-
loped between the first two pairs and the shield, but on the
dorsal side of them, i.e. on the side remote from the shield.
The grooves of these last plumes (fifth and sixth pairs) are
not directed towards the shield, but away from it.

Although the four series of plumes of buds were prepared
for the purpose of deciding the points raised by Masterman,
they serve to show a number of other interesting features,
such as (1) the difference of plume development in the buds
of the four species; (2) the relation which the last-formed
pair of plumes bear to the edge of the post-oral lamella;
and (3) the conversion, in the later stages of growth, of the
line of the bases of the collar-outgrowths (plumes and post-
oral lamella) from a circle to a double crescent, or two
incomplete ellipses. I have selected from each of the four
series a number of preparations to illustrate these points ;
they are reproduced in text-figures 2, 3, 4, 6, 7, and 9, and
are described under headings of the respective species.

Merruops.

In the case of the youngest stages the whole bud was
mounted in diluted glycerine, and gold size was run round
the edge of the cover-glass to keep it firmly in position, and
to prevent the glycerine from accumulating dust. Most of
the buds were dissected, the shield being first removed by
tearing through its stalk by the aid of fine mounted needles,
and then the collar region, with plumes and post-oral lamella
(e.g. text-fig. 2, 3), was removed by carefully manipulating
the needles between these parts and the “ body” of the bud.
The three parts, shield, collar region, and “ body ” with its
stalk, were then mounted on the same slide in dilute glyce-
vine.

22.4 W. G. RIDEWOOD.

he relation which the collar region, as dissected out, bears
to the whole bud will be made clear by reference to text-
fig. 5, A, a diagrammatic side view of a bud with three pairs
of plumes, and text-fig. 10, a diagram of a longitudinal
section of the adult polypide; in the latter figure the limit
of the collar region is marked by two lines of heavy dashes.

The results here recorded and illustrated are based upon
135 preparations of buds made and mounted as above ex-
plained. In some cases (e.g. text-fig. 6, r) the three parts—
“body,” collar appendages, and shield—were drawn sepa-
rately on tracing paper, and the perfect bud was recon-
structed by a superposing of these transparent sheets.

No staining fluids were used. The dissections were made
under a Greenough binocular erecting microscope magni-
fying 20 and 40 diameters; the drawings were made by the
aid of a compound microscope with Zeiss apochromatic 8 mm.
objective and No. 6 compensating ocular with eye-piece
micrometer. The figures in text-figs. 2, 3,4, 6, 7, and 9 are
all reproduced to the same scale of enlargement, viz. 62
diameters.

DEVELOPMENT OF THE PLUMES IN Bubs or CEPHALODISCUS
HODGSONI.

In the earliest phase of development the bud is ovoid or
pyriform (text-fig. 2, a) without signs of buccal shield or
plumes. ‘The shield and the first pair of plumes make their
appearance simultaneously (B and c). The plumes are at first
small mounds, later hemispherical (p), and still later longer
than broad (n, F, etc.). The second pair of plumes appear
on the external side of the first pair (p, 2), and in contact
with them. ‘The two plumes of the first pair are not in con-
tact with one another at their bases. The third pair of
plumes appear lateral to the second pair (8, 3), and in con-
tact with them.

The bud increases considerably in size before the fourth

PLUMES OF CEPHALODISCUS. 225

plumes appear; the buccal shield assumes its definitive shape
by becoming flattened, and by the differentiation of the pos-

TEXT-FIGURE 2.—Cephalodiscus hodgsoni. Figures illustrating
the development of the plumes of the buds. C and L are side views,
the others are dorsal views. J represents the collar and its appendages
(plumes and post-oral lamella) dissected from a bud a little older than H.
The first pair of plumes are just appearing in B and C, the second in
D, the third in E, the fourth in H, and the fifth in K and L. The
series is continued in text-fig. 3. All figures enlarged 62 diameters.
For explanation of the lettering see end of paper.

terior lobe. In G@ and u the dotted line marks the position
of the free edge of the posterior lobe. ‘The fourth plumes

226 W. G. RIDEWOOD.

appear when the bud has reached the stage of development
represented in H. The structure which in G is marked po.l.
might at first glance be taken for the developing fourth
plume; it is the lateral part of the post-oral Jamella.

Figure J represents the collar region and its appendages
(the plumes and post-oral lamella) of a bud alittle older than
that shown in figure H; the buccal shield has been dissected
off from the one side, and the “body” and stalk from the
other. The position of the mouth is indicated in the figure,
but this region is always more or less damaged in making
such a dissection as that described. The torn stalk of the
buccal shield in this preparation—as also in the similar pre-
parations shown in text-fig. 3, N, 0, and Rr, and in other of
the text-figures—occupies a position intermediate between
the mouth and the bases of the first pair of plumes. In
figure J the fourth plumes are hemispherical projections at
the bases of the third plumes, touching these on the one side,
and in continuity with the post-oral lamella on the other.
Traces of pinnules are appearing on the first and second
plume-axes, and the terminal end-bulbs of these same plumes
are swelling out.

In x the fourth plumes are now longer than broad, and
the fifth pair are making their appearance. UL is a side view
of a bud of about the same stage as K, as may be determined
by comparing the development of the fourth and fifth plumes
in the two buds. Pinnules are now appearing on the third
plumes, and it will be noticed, in the case of the first, second,
and third plumes, that the pinnules are not laterally placed
on the plume-axes, but ventro-laterally, and the groove
between the two rows of piunules of each of the plumes in
question opens ventrally, or ventro-laterally, towards the
dorsal or stalked side of the shield.

The figures in text-fig. 3 continue the series shown in
text-fig. 2. mis a little more advanced than 1 of text-fig. 2;
the fifth plume is now hemispherical in shape; pinnules are
appearing on the fourth plumes, and the end-bulbs are
becoming differentiated. The end-bulbs of the first, second,

!pxT-FIGURE 3.—Cephalodiseus hodgsoni. Figures illus-
trating the development of the plumes of the buds; series continued
from text-fig. 2. _M shows the left side of a bud in which the fifth
pair of plumes are swollen knobs; the other figures are dorsal views
of the collar and appendages (plumes and post-oral lamella) of older
buds, represented in whole (N and R) or in part only (O, P, Q and
S). The fifth pair of plumes have the form of clavate knobs in N,
and the sixth in R. All figures enlarged 62 diameters, For ex-
planation of the lettering see end of paper.

228 W. G. RIDEWOOD.

and third plumes—the last concealed in the figure by the
second plume—are showing refractive beads (7.b.), and their
surface is no longer smooth, but warty; the pinnules are
larger and more numerous than in L. ‘lhe figure m, like 1,
shows how the ciliated groove between the two rows of pin-
nules of each of the plumes 1, 2, and 3 faces towards the
dorsal surface of the buccal shield.

Figure N is a dissection made in the same manner as that
shown in figure J, by the removal of the buccal shield on the
one side of the collar and the ‘‘ body” of the bud on the
other. The preparation is viewed from the dorsal surface, so
that the grooves of the plumes are facing away from the
observer. The fifth plumes are seen to be continuous with
the bases of the fourth plumes, and with the post-oral
lamella; they are unequally developed on the right and left
sides of the body.

In 0 the pinnules on the first plumes are now finger-like
processes, those on the fourth are in the same stage of
development as the pinnules of the first plumes were in the
stage represented in figure wm. ‘There are twelve or thirteen
pairs of pinnules on the plumes of the first pair, and about
nine on the fourth plumes, but the numbers later become
increased by the development of new pinnules at the basal
end of each series. The fifth plumes are larger than in the
last stage, and show an indication of pinnules, but as yet they
have no end-bulbs. There is no sign of a sixth plume
between the fifth plume and the post-oral lamella. 0, like n,
is a dorsal view, and the ciliated grooves of the plume-axes
are not seen; the grooves of the first and second plumes are
facing ventrally, those of the third ventro-laterally, and
those of the fourth postero-laterally.

Figure p shows an unequal development of the right and
left plumes of the fifth pair. On the right side the plume
has about eight pairs of pinnules and a moderately differen-
tiated end-bulb, whereas on the left side the length of the
plume is only a little greater than the width. The groove
on the right-hand plume is facing posteriorly.

PLUMES OF CEPHALODISCUS. 229

Figure Q shows the developing sixth plume, wedged in
between the base of the fifth and the antero-lateral edge of
the post-oral lamella;! it is already pear-shaped, and with a
narrowing base. ‘lhe preparation here drawn is presenting
its dorsal surface to the observer, and the fifth plume is
showing its groove facing postero-dorsally.

In figure r the fifth plumes are larger and with better
developed pinnules than in Q, but the sixth plumes are not
more advanced; indeed, on the left side the sixth is less
developed than it is in Q. It is worthy of special remark
that, while in the case of the first and second plumes the
development of the right and left plumes of each pair is
regular and symmetrical, in the fifth and sixth plumes the
one is very frequently more advanced than the other of its
pair. The grooves of the fifth plumes should be facing
posteriorly or postero-dorsally ; by the pressure of the cover-
glass upon the preparation the plumes have twisted round
somewhat. For the same reason the first plumes have their
erooves facing to the left, while the third plume on the
right side is twisted right round so as to present its grooved
surface to the observer. The pinnules on the first plumes
are now fairly long, and the pinnules of the third and fourth
plumes are finger-like, and resemble those of the first plumes
in the stage shown in figure o.

s differs from rin that the sixth plumes are more advanced,
the right hand one showing signs of pinnules. The fifth
plume shows its groove facing posteriorly. The bud from
which the preparation shown in figure s was made was a very
late bud, so large, and with such great development of the
visceral mass, bringing the base of the stalk from a posterior
to a ventral position, that, if it was not still in connection
with a full-grown polypide by means of its stalk, one might
easily mistake it for an adult polypide.

1 The close relation obtaining between the base of the last-formed plume
and the edge of the post-oral lamella was, I believe, not known until it was
pointed out by Harmer two years ago (‘Pterobranchia of the ‘Siboga”’
Expedition,’ 1905, p. 36),

230 W. G. RIDEWOOD.

Possibly in some cases the sixth plumes are permanently
arrested in their development on one side, or on both sides,
or they are not developed at all, for some polypides of
Cephalodiscus hodgsoni of full size and with mature
gonads have five pairs of plumes only. In the cases of those
polypides which are found with eleven plumes the explana-
tion may just as likely be that the sixth plume has failed to
develop on one side as that one of the plumes has been lost
by injury.

As regards the development of the pinnules on a plume-
axis, about five pairs appear simultaneously, and at the time
of their origin they occupy nearly the whole, or, at all events,
more than half of the length of the plume-axis behind the
end-bulb. The later pinnules are developed a pair at a time
at the basal end of the series.

DEVELOPMENT OF THE PLUMES IN Bups or CEPHALODISCUS
DODECALOPHUS.

The bud of Cephalodiscus dodecalophus in its
earliest phase (text-fig. 4, 4) resembles that of C. hodgsoni
except in being somewhat smaller. The first pair of plumes
and the buccal shield begin to differentiate simultaneously (B
and c). By the time the second plumes appear (8) the first
pair are relatively longer than in C. hodgsoni. ‘The third
plumes develop before the second pair have grown very
large (F and @).

In buds with three pairs of plumes present the size of the
first pair varies considerably, as may be seen by reference to
Handa. Although u is a larger bud than 4, it is not much
more advanced in its development; the second and third
plumes are only slightly more developed than in G, and there
is no sign of the fourth plumes. Yet the first plumes are
very large in u, and have refractive beads in the terminal
end-bulbs, whereas the corresponding plumes in @ are

PLUMES OF CEPHALODISCUS. 231

no larger in proportion than in C. hodgsoni (cf. text-fig.
2, Gt) 6

In 3 the fourth plumes are appearing, and the gradation
from the first plumes to the fourth is fairly uniform; refrac-
tive beads are now visible on the second plumes. On

TEXT-FIGURE 4.—Cephalodiscus dodecalophus. Figures
illustrating the development of the plumes of the buds. J, K and
L are ventral views of the collar and appendages (plumes and
post-oral lamella); B and F are side views of complete buds; the
remaining figures are dorsal views of complete buds. The first pair
of plumes are just appearing in B and C, the second are fairly large
in E, the third are appearing in F and G, and the fourth in J; in K
the fourth plumes are moderately well developed, but there are no
fifth plumes; in L the fifth plumes are fairly well developed. All
figures enlarged 62 diameters. For explanation of the lettering see
end of paper.

comparing J of text-fig. 4 with 3 of text-fig. 2 it will be seen
that in C. dodecalophus the lateral lobes of the post-
VOL, 51, PART 2,—NEW SERIES. 18

232 W. G. RIDEWOOD.

oral lamella are more prominent as free flaps than in C,
hodgsoni.

Pinnules first appear in the stage represented in J, i.e. in
the stage when the fourth plumes are appearing, or possibly
a little earlier (H). They are at first confined to the first
pair of plumes, and they appear as five or six pairs of small
eminences arising simultaneously; additional pairs develop
in succession at the basal end of the series. In x there are
seven pairs, and int eleven or twelve pairs of pinnules on
the plumes of the first pair. The pinnules on the second,
third, and subsequent plumes arise later than those on the
first, as might be expected. About four pairs of pinnules
are just appearing on the second plumes in J.

Masterman,! in figuring a bud about as advanced as G and
H of text-fig. 4 of this paper, shows four pairs of long slender
pinnules on the first plumes. There is nothing comparable
with this in my preparations. My observations also fail to
accord with those of Dr. Masterman in the matter of the
development of the first few pinnules on the plumes. In
figs. 69—74 of his Plate 4 he gives figures illustrating the
growth of a plume—he does not say which. In fig. 71 there
are no pinnules, in fig. 72 one pair, in fig. 73 the first pair
have elongated considerably, and there is a new pair on their
proximal side, in fig. 74 there are four pairs of pinnules
shown. My own observations go to show that the first five
or six pairs of pinnules arise simultaneously on the first
plumes; on the second and third plumes the first four or five
arise simultaneously, and on the fourth pair (text-fig. 4, K, 4)
the first three or four. At a stage when any one plume has
but four pairs of pinnules I have not seen the pinnules nearly
so long and slender as those shown in Masterman’s fig. 74.

In 1 of text-fig. 4 the fifth plumes have attained consider-
able size and exhibit about four pairs of pinnules. The pin-
nules on the first and second plumes are now long, except the
youngest of the series near the base of the plume-axis. End-
bulbs with refractive beads (7.b.) are present on the first four

1 «Trans. Roy. Soc. Edin.,’ vol. xxxix, 1898, pl. 2, fig. 24.

PLUMES OF CEPHALODISCUS. 233

pairs of plume-axes. Both x and 1 are ventral views, and
the groove between the two rows of pinnules of each plume-
axis is directed towards the observer.

It will be noticed that in J, x, and 1 the right and left
plumes of the first pair are separated by a small interval, in
which is situated a slight mound of the basal part of the
stalk of the buccal shield, obliquely torn in the making of
such preparations as those here figured; but the several
plumes of the same side of the body are in close contact
with one another at their bases, and the last developed plume
is wedged in, with acertain amount of over-lapping, between
the last plume but one and the antero-lateral edge of the
post-oral lamella.

Except in distorted specimens the last developed plume
lies slightly dorsal to the edge of the post-oral lamella, and
lies slightly ventral to the last plume but one (x). It must
be noted, however, that in stages such as that shown in 1,
and in later stages, the line of plume bases and base of the
post-oral lamella does not lie in a plane, and a certain amount
of distortion or crumpling is bound to result from any attempt
at mounting the dissection between two pieces of glass.
Note the wrinkle in the middle of the posterior edge of the
post-oral lamella int. In such a stage as is shown in J the
line of plume-bases and attached edge of the post-oral lamella
is nearly in one plane, a plane slightly oblique to the long
axis of the bud. The fourth, fifth, and sixth plumes, however,
are not developed in this plane, but more dorsally, and this
is how it comes about that in the adult polypide the bases of
the six plumes are set in a circle or ellipse in one oblique
plane of the body, while the attached edge of the post-oral
lamella hes in another oblique plane, which intersects the
first plane at the points of contact of the sixth plumes with
the edge of the post-oral lamella. This will be made clear,
I think, by reference to text-fig. 5, and its accompanying
legend. See also text-fig. 8, a diagram of Cephalodiscus
nigrescens.

234 W. G. RIDEWOOD.

Masterman has stated! that the first three pairs of plumes
arise with their grooved faces directed towards the buccal

TEXT-FIGURE 5.—Diagrams showing that the later developed
plumes (4, 5 and 6) have their bases set more dorsally than the
earlier. The heavy dots mark the several plume-bases, the heavy
line the line of attachment of the post-oral lameila. In A, a side
view of a bud with three pairs of plumes, the plume-bases and the
post-oral lamella are in one plane, a plane nearly parallel with the
buccal shield. In B the base of the fourth plume is more dorsally
placed than the others, and the line of the post-oral lamella rises at
its anterior end. Similarly with the fifth plume and the post-oral
lamella in figure C. In D, a diagram of an adult polypide, the base
of the sixth plume is not only dorsal to that of the fifth, but is ante-
rior also; the right and left plumes of the sixth pair are thus fairly
close together; the line of attachment of the post-oral lamella is
produced forward so that the end of the line is, as before, close to
the base of the last-formed plume. In D the pinnules are not
shown, and the plume-axes are represented very diagrammatically.

a. Anus. c.p. Collar pore. g.d. Gonad duct. g.s. Gill slit.
st. Stolon. Other lettering as before.

shield, but later rotate upon their axes so that the grooves
face away from the shield ; further, that the last three pairs
1 ¢Trans. Roy. Soc. Edin.,’ vol. xxxix, 1898, p. 521.

PLUMES OF CEPHALODISCUS. PRS)

of plumes arise between the first three pairs and the shield
and have their grooves directed towards the shield (see text-
fig. 1 of this paper). Iam ina position to confirm Harmer’s
opinion! that this statement is without foundation, and that
Masterman’s earlier enumeration of the plumes? is the
correct one. In Cephalodiscus dodecalophus, as in
C. hodgsoni, the grooves of the first and second pairs of
plumes, developed in such a position that they face the
buccal shield, continue to face the shield, and the last two
pairs of plumes are not developed between the first two
pairs and the shield, but on the side of the first two plumes
remote from the shield, i.e. to their dorsal side; and the
grooves of the last-formed plumes are not directed towards
the shield, but away from it. The grooves of the third and
fourth plumes face somewhat laterally.

The six pairs of plume-bases are set on an ellipse, and the
erooves of the twelve plumes face away from the foci of the
ellipse. The six grooves of the right side lead down into
the space between the shield and the post-oral lamella on the
right side of the mouth; similarly the six grooves of the left
side lead into the left side of the mouth (see text-fig. 8,
C. nigrescens).

DEVELOPMENT OF THE PLUMES IN Buns or CEPHALODISCUS
NIGRESCENS.

While in buds of Cephalodiscus hodgsoni and
Cephalodiscus dodecalophus the first pair of plumes
begin to appear at about the same time as the buccal shield,
in those of Cephalodiscus nigrescens the buccal shield
is well differentiated, and with a well-defined posterior lobe
(text-fig. 6, A) before there is any sign of plume development.
When the first pair of plumes make their appearance (8, 1)
they are small as compared with the bud as a whole, and
when the second plumes begin to develop (c, 2) the buccal

1 ¢ Pterobranchia of the “ Siboga”’ Expedition,’ 1905, pp. 36, 37.
2 *Quart. Journ. Mie. Sci.,’ vol. 40, 1897, p. 346, pl. 26, fig. 36.

Gants r.e.

Es
ap +>
b: oe

I,
! “sp.l
' J
oe gritos har Sanit e
re. i's 3

TEXT-FIGURE 6.—Cephalodiscus nigrescens. Figures illus-
trating the development of the plumes of the buds. A, B, C, D
and F are dorsal views of complete buds; E shows the plumes dis-
sected from a bud a little earlier than F. The first pair of plumes are
just appearing in B, the second in C, the third in D, and the fourth
in F. The series is continued in text-fig. 7. All figures enlarged
62 diameters. For explanation of the lettering see end of paper.

PLUMES OF CEPHALODISCUS. UB

shield is of remarkably large size. Compare text-fig. 6, c,
with text-fig. 2, D; and text-fig. 4, E.

The third plumes appear to the outer side of the second,
and are in contact with them at their bases (p, 3). The two
plumes of the first pair are widely separated from one
another (p, 1). Whenin buds of Cephalodiscus hodgsoni
and Cephalodiscus dodecalophus the third plumes
make their appearance (text-fig. 2, n, 3, and text-fig. 4, G, 3)
they are directed strictly laterally, whereas in Cephalo-
discus nigrescens (text-fig. 6, p and x, 3) they point more
anteriorly than laterally. This doubtless is connected with
the relatively late origin of the plumes, and the relatively
small size of the plumes during the early stages of their
development.

A, B, and p illustrate a peculiarity of many buds and adults
of Cephalodiscus nigrescens, the bending forward of the
posterior lobe of the buccal shield. The occurrence of the pos-
terior lobe in this position may be due to an exceptional con-
traction of the muscles of the shield brought about by the
irritating properties of the formalin solution in which the
animals were killed, but whether this be so or not, it indicates
considerable mobility of the organ in question in normal con-
ditions of existence.

Text-fig. 6, r, shows the dorsal view of a bud in which the
fourth pair of plumes are making their appearance. The
“body ” of the bud here figured was placed so far forward as
compared with the stalk of the shield that the plumes, in-
stead of being set on the anterior edge of the “body” as in
D, lie in a deep groove between the “‘ body ” of the bud and
the dorsal wall of the buccal shield. The plumes are drawn
in a dotted line to signify that they would be seen in the
positions they occupy in the figure if the “body” were
transparent; as a matter of fact the “body” is so black
that the employment of the usual clarifying reagents fails to
make it transparent. The bud in question was drawn as a
whole, with no plumes visible; then the “body” was dis-
sected off and the characters and positions of the plumes

238 Ww. G. RIDEWOOD.

noted. This relation of the “body” and shield whereby
the plumes are not visible except by dissection is a common
one; it is only exceptionally that the “ body” is so much
drawn back as to expose the developing plumes as com-
pletely as in text-fig. 6, p, and in fig. 66 of Plate 7 of the
‘“ Discovery ” Expedition Reports,’ vol. 11, 1907.

The post-oral lamella is not well defined until after the
fourth plumes have developed. In text-fig. 7, a, the two

2.

Text-FricguRE 7.—Cephalodiscus nigrescens. Figures illus-
trating the development of the plumes of the buds; series con-
tinued from text-fig. 6. G, H and J represent the collar and
appendages (plumes and post-oral lamella) as seen in dorsal view ;
K and L are single plumes. The fifth pair of plumes are appearing
in H, and the seventh in J. All figures enlarged 62 diameters.
For explanation of the lettering see end of paper.

lateral flaps are complete, but the free edge connecting the
two flaps behind the mouth is not yet entire, and so in making
such a dissection as is shown in G and H this part is left with
a ragged edge (u, 7.e.). The plumes of the first three pairs
are elongating and are swelling out so that the base of each

PLUMES OF CEPHALODISCUS. 239

appears comparatively narrow (Gand H). The fourth plume
in @ is set between the base of the third plume and the
anterior part of the free edge of the post-oral lamella, and so
when the fifth plume appears (H, 5) it would seem as though
that plume must have arisen from the edge of the post-oral
lamella itself. Similarly also with the sixth and seventh
plumes.

I have not met with a bud showing the early development
of the sixth plumes, so that there is a greater interval than I
could have wished between 4H, with fifth plumes appearing,
and J, with seventh plumes appearing.

Up to the time when the fifth plumes are beginning to
develop, the size of the plumes diminishes from the first to
the fifth (a), but when the seventh is making its appearance
(s) the plumes first formed have failed to maintain their
initial superiority in size, and the first five pairs of plumes
are nearly of equal size.

Pinnules arise rather late. In text-fig. 7, 3, the seventh
plumes have already appeared, and yet the pinnules of the
first plumes are not more than hemispherical projections.
On the plumes of the first pair the first ten or twelve pairs of
pinnules arise simultaneously, the later ones are added at the
basal end of the series. In Cephalodiscus hodgsoni
(text-fig. 3, 0) and Cephalodiscus dodecalophus (text-
fig. 4, L) the pinnules on the first plumes are already finger-
hike projections at the time when pinnules first make their
appearance on the fifth plumes, and before the axes of the
last plumes (the sixth) have shown signs of development.
In Cephalodiscus nigrescens, however (text-fig. 7, J),
pinnules begin to appear simultaneously on the first five
pairs of plumes, and are very little advanced at a time when
the last plumes (the seventh in this species) have already
come into existence. The bud from which fig. 3 was drawn
is of the same size and general appearance as that repre-
sented in fig. 67 of Plate 7 of the ‘ Antarctic “ Discovery ”’
Expedition Report on Cephalodiscus,’ 1907.

In text-fig. 7,5, the first five plumes have elongated con-

240 W. G. RIDEWOOD.

siderably since the stage shown in H, and the axes are less
bluntly tipped. The interval between the right and left
plumes of the first pair is not relatively, but absolutely less
than in earlier stages—all these figures are drawn to the
same scale of magnification. The exigencies of pictorial
delineation demand that the structures under consideration
be drawn flat; it is well to bear in mind, therefore, that,
while the structures represented near the median line of the
figure are on the antero-ventral surface of the “ body ” of the
bud, just above the buccal shield, the lateral parts (plumes 6
and 7, and lateral parts of the post-oral lamella) are set high
up the sides of the “body.” ‘The figure if cut out and bent
back would give approximately the correct relation of the
parts.

In the adult the right and left seventh plumes are as close
together as are the right and left first plumes. The line of
plume-bases in the adult, when viewed from the front, is an
ellipse, incomplete on the upper side; and the line of the
attached edge of the post-oral lamella is another ellipse
similarly incomplete. The two ellipses join where they are
incomplete (see text-fig. 8). The parts seen are, of course,
at very different distances from the observer; the base of
the fifth plume is in the middle distance, the base of the
first plume is nearest to the observer, and the mid-ventral
part of the post-oral lamella farthest away. The stalk of the
buccal shield and the mouth lie in the middle distance
between the two ellipses, and the anus dorsal to both (com-
pare text-fig. 8 and text-fig. 5, D).

The seventh plumes of the adult, and in some polypides
the sixth plumes also, do not arise directly from the ‘‘ body ””
of the polypide, but are processes of a paired lophophoral
upgrowth, which is connected with the “ body” at the base
of the fifth plume and the more ventral parts of the collar-
outgrowths (see “Cephalodiscus,” ‘ ‘* Discovery ” Expedi-
tion Reports,’ 1907, p. 31, last paragraph).

Late buds of Cephalodiscus nigrescens are extremely
rare in the material at my disposal. Possibly after the stage

PLUMES OF CEPHALODISCUS. 241

of development represented in figure J has been reached the
buds migrate. Whatever be the explanation the fact remains
that from an abundant supply of buds of various stages I
have only discovered one which shows pinnules in a further
stage of development than that drawn in figure 3. The bud
is figured in the ‘ Report on Cephalodiscus’ (“ Discovery ”
Expedition, Pl. 7, fig. 68). It has fourteen plumes, of
approximately the same size and development, standing for-
ward ina bunch, parallel with one another; the stalk of the
bud is no longer terminal, but projects from the ventral
surface of the “body.” Two of the plumes of this bud are
shown in text-fig. 7, kK and L; K isa nearly dorsal view, L a

TEXT-FIGURE 8.—Diagrammatic representation of the view ob-
tained on looking backward at an adult polypide of Cephalodiscus
nigrescens after cutting short the plumes and post-oral lamella,
and removing the buccal shield. The cut bases are represented in
black. po.d. The edge left after clipping away the post-oral lamella.
1, 2,3, 4,5,6 and 7. The stumps of the several plume-axes, showing
the grooves facing away from the middle of the ellipse upon which
they are set. 4.8. Stalk of the buccal shield. c.z.m. Position of
central nerve mass. m. Mouth. a. Anus. The arrows indicate
the direction of the food currents from the grooves of the plumes
into the mouth. This figure is in the main similar to Harmer’s
figures of C. levinseni (‘ Pterobranchia of the “ Siboga’’ Expe-
dition,’ 1905, pl. 12, figs. 158—160), but it is treated more dia-
grammatically.

nearly lateral view. The plume-axis is very massive and
pigmented, and has a smooth hemispherical extremity ; some
of the other plumes of this bud, however, have the ends less
rounded, more like those of 5. There are about seventeen or

242 W. G. RIDEWOOD.

twenty pairs of pinnules, and the middle ones of the series
are the longest.

DEVELOPMENT OF THE PLumMES IN Bups oF CEPHALODISCUS
GILCHRISTI.

Cephalodiscus gilchristi resembles Cephalodiscus
nigrescens, and differs from Cephalodiscus dodecalo-
phus and Cephalodiscus hodgsoni, in the tardy deve-
lopment of the plumes in comparison with the rapid growth
of the buccal shield. The bud, at first pear-shaped (text-fig.
9, A), soon becomes differentiated into a buccal shield and a
“body,” with stalk (B). Even at this early stage of develop-
ment the posterior lobe of the buccal shield is well defined
(zB, p.l.). A pair of mounds appear on the right and left
sides of the “‘ body,” near its junction with the middle of the
shield (c and pb), and these develop into the first pair of
plumes. It is to be noted that these mounds are strictly
lateral structures, and do not project forward as do the first
plumes of the other three species under consideration during
the early stages of growth.

The second pair of plumes arise postero-ventrally to the
bases of the first (e and F, 2), and in a dorsal view such as
that shown are partially concealed by the first pair. When
the third plumes begin to develop (c, 8), the first pair project
antero-laterally instead of strictly laterally, possibly owing
to the pressure put upon them by the second and third
plumes, which arise posteriorly to their bases. Hach of the
six plumes now present is wider at the middle than at its
base.

When the fourth plumes make their appearance (un, 4) the
length of the first and second plumes is more than twice
their width at the base; and before there is any sign of the
fifth plumes pinnules begin to differentiate upon the first two
pairs of plumes (5, 1 and 2). The first plumes are now
directed more anteriorly than laterally, and the post-oral
lamella is clearly distinguishable (J, po.l.).

TEXT-FIGURE 9.—Cephalodiscus gilchristi. Figures illustrating
the development of the plumes of the buds. J is a dorsal view, and
K and L ventral views of the collar and appendages (plumes and
post-oral lamella); the other figures are dorsal views of complete buds.
The first pair of plumes are just appearing in D, the second in H, the
third in G, the fourth in H, and the fifth in K. All figures enlarged
62 diameters. For explanation of the lettering see end of paper.

244. W. G. RIDEWOOD.

At a time when the fifth plumes are just appearing (x, 5)
the first plumes point well forward and not laterally, the
fourth plumes are twice as long as broad, and pinnules are
making their appearance on the third plumes. It would
seem that the fifth plumes grow very slowly, for in the
specimen figured in text-fig. 9, 1, the fifth plume on the one
side is not much longer than broad, and on the other side it
is even less developed, and yet the pinnules on the first pair
of plumes are digitate processes, twice as long as broad, and
pinnules have already made their appearance on the fourth
plumes. ‘The first four pairs of plume-axes are now almost
of the same length.

In all of the buds of Cephalodiscus gilchristi, except
the very young ones, the surface of the “‘ body ” is closely
studded over with refractive beads similar to those which
occur on the end-bulbs of the plumes of Cephalodiscus
dodecalophus and Cephalodiseus hodgsoni. In the
present species the refractive beads occurring on the plumes
are negligible; the beads occur mostly on the general
surface of the body, and, in the adult particularly, on the
upper surface of the anterior margin of the buccal shield
(see description of Cephalodiscus gilchristi, ‘Marine
Investigations in South Africa,’ vol. 4, 1906, p. 184). The
refractive beads are not shown in text-fig. 9.

Buds in which the fifth plumes are more advanced than
in t, and buds in which the sixth plumes are developing I
have been unable to find. This is not without significance
when taken in conjunction with the fact that in the material
of Cephalodiscus nigrescens I found only one late bud
showing the plumes more advanced than is represented in
text-fig. 7, 5, and with the fact that in Cephalodiscus
hodgsoni the later stages in plume development, showing
the development of the sixth plumes, are not rare. The
species gilchristi and nigrescens have tubaria in which
the polypides are isolated, and live in separate tubular
cavities, which open individually on the surface, and do not
communicate the one with the other. The species hodgsoni,

PLUMES OF CEPHALODISCUS. 245

like dodecalophus, has a tubarium in which the polypides
live in a common cavity, which opens by several ostia to
the exterior. In my report on Cephalodiscus in the
«« Discovery ” Expedition Reports’ (pp. 23, 24) I ventured
to suggest that, in the species of the former kind (i.e. species
of the sub-genus Idiothecia) the late buds severed their
connection with the parental stolon, and wandered over the
surface of the colony in order to settle down in some con-
venient situation, usually the apex of a branch, and to secrete
tubes of their own. ‘The fact now recorded, namely, the
inability to discover in the material of Cephalodiscus
gilchristi any individuals intermediate in growth between
buds with small fifth plumes and full-grown polypides with
well-developed sixth plumes, supports that suggestion, for
the late buds, while developing their fifth and sixth plumes,
would be migratory forms on the surface of the colony, and
would be brushed off while the specimen was in the trawl.
In the species of the sub-genus Demiothecia (e.g.
Cephalodiscus hodgsoni and C. dodecalophus) the
late buds would complete their development within the
common cavity of the tubarium, and while being drawn up
in the trawl, and while undergoing fixation in preservative
fluids, would not be more likely to be lost than the younger
buds and the adults of the colony.

GENERAL REMARKS ON THE COLLAR AND ITS APPENDAGES.

Harmer in his recent ‘Monograph on the Pterobranchia
of the “Siboga” Expedition’ (p. 30) lays stress on the rela-
tions of the plumes (or “arms”’) to the post-oral lamella (or
“ operculum”) in Cephalodiscus. He points out that
“in Balanoglossus the anterior margin of the collar forms
a projecting fold encircling the base of the proboscis-stalk.
The ventral half of this fold may be regarded as constituting
a lower lip, while the dorsal part is connected, in the middle
line, with the anterior neuropore. In Cephalodiscus the

246 W. G. RIDEWOOD.

neuropore is not represented, and the collar forms no projec-
tion in the median dorsal line above the base of the pro-
boscis. Except for this interval the whole of the anterior
margin of the collar forms a strongly-developed fold, split
up dorsally to form the arms, and ventrally constituting the
operculum.”

My observations on the development of the plumes and
post-oral lamella of buds of Cephalodiscus entirely bear
out this contention. While, however, Harmer finds it neces-
sary to insist that the post-oral lamella is a derivative of the
anterior edge of the collar, and not of its posterior edge as

as

iy

mI

TEXxT-FIGURE 10.—Diagram of the collar and adjacent parts of
Cephalodiscus as seen in a longitudinal section of the polypide.
b.s. Thick ventral wall of the buccal shield. ¢.c. Anterior part of
collar cavity. c.c’. Posterior part of same. c.p. Position of right
collar pore, in distance. e. Torn edge of body-wall of right side.
gs. Internal opening of gill slit. m. Mouth. zo. Notochord.
p. Opening of the cavity of the plume-axis into the collar cavity.
p.a. Axis of first plume of right side. p.c. Proboscis cavity.
ph. Dorsal wall of pharynx. pi. Pinnules. po./. Median part of
post-oral lamella. se. Wall or septum between collar cavity and
trunk cavity. sp. Space between the posterior flap of the buccal
shield and the post-oral lamella, leading into the mouth (m.).
¢.c. Dorsal part of trunk ceelom. ¢.c’. Ventral part of same.

he formerly supposed,' the point does not appear to me to be
of special consequence. The post-oral lamella is really a
pair of ventro-lateral flaps united together behind the mouth,

1 «“Challenger”’ Reports,’ vol. xx, part 62, 1887, p. 43.

PLUMES OF CEPHALODISOUS. 247

the connection being but a mere hollow ridge. The ridge is
certainly connected with the hinder edge of the collar (text-
fig. 10, po.l.), and the middle part of each lateral flap with
the middle of the length of the collar, i.e. not with the front
edge nor the hind edge; in the region of the collar canal,
which is set on the postero-dorsal edge of the collar, about
midway between the dorsal and ventral surfaces, the base of
the flap is near the front edge. But the relations of the
plumes to the post-oral lamella can be clearly understood in
spite of this; and it is a very significant circumstance, in
connection with the view that the post-oral lamella and the
plumes belong to one and the same system, that in the
development of the buds of Cephalodiscus each new
plume arises between the last-formed plume and the end of
the post-oral lamella; one might almost say that each new
plume is differentiated from that part of the post-oral lamella
immediately in contact with the last-formed plume. The
post-oral lamella may thus be regarded as composed of
postero-ventral plumes which fail to differentiate as separate
plumes.

In the adult the food-grooves of the axes of the first and
second pairs of plumes face ventrally,! i.e. towards the dorsal
wall of the front part of the shield; the grooves of the
third and fourth pairs face laterally, and those of the fifth
and sixth pairs dorsally or dorso-laterally. Tracing these
grooves basally one finds that those of the right-hand plumes
bend round and converge to the right side of the mouth, and
those of the plumes of the left side of the body to the left
side of the mouth, the food current being in all probability

1 Tt is to be noted that Harmer orientates the polypide of Cephalodiscus
in such a way that the antero-posterior axis of the body is a line passing from
the middle of the buccal shield through the central nerve mass, and ending
on the rectal side of the body a little below the anus, so that the visceral
mass is regarded as a ventral downgrowth (‘ Pterobranchia of the ‘‘ Siboga”’
Expedition,’ 1905, p. 23). In the present paper the ordinary orientation is
adopted, namely, plume-apices anterior, stolon and face of shield ventral,
intestine dorsal, rounded end of visceral mass posterior.

voL. 51, part 2,—NEW SERIES. 19

248 W. G. RIDEWOOD.

guided into the mouth and prevented from escaping by the
free edge of the post-oral lamella being at the time in close
contact all round with the posterior flap of the buccal shield.
The lateral lobe of the post-oral lamella thus keeps the food
current distinct from the exhalent current through the gill
slit and from the water passing in or out of the collar canal,
both the gill slit and the collar pore being situated posterior
to the lateral lobe of the post-oral lamella.

If Phoronis is justly to be regarded as a member of the
Hemichordata, and the tentacles are collar-structures com-
parable with the plume-axes of Cephalodiscus, one must
admit, with Harmer,! that what is in Cephalodiscus known
as the post-oral lamella is in Phoronis produced into a row
of tentacles instead of a paired flap. On this admission there
is no great difficulty in effecting a comparison between the
plume-systems of the two.

The line of the bases of the tentacles of Phoronis runs
in the double spiral well shown in Benham’s figure of the end
view of the animal with the tentacles cut short.” This figure,
if inverted, is closely comparable with text-fig. 8 of this
paper. Below the mouth is a row of tentacles, corresponding
with the post-oral lamella of Cephalodiscus, continued on
each side to the centre of the spiral, which point is equivalent
to the uppermost limit of the post-oral lamella in text-fig. 8.
Here it becomes continuous with the tentacles of the supra-
oral series, as in Cephalodiscus the extremities of the base
of the post-oral lamella are continuous with the terminal
members of the series of plumes. The notch in the supra-
oral series of tentacles, marked # in Benham’s figure, is the
equivalent of the space between the right and left plumes of
the first pair in Cephalodiscus.

Not only has this correspondence between plumes and post-
oral lamella of Cephalodiscus with the two series of
tentacles of Phoronis been indicated by Harmer, but that

1 ¢ Pterobranchia of the “ Siboga” Expedition,’ 1905, pp. 116, 117.
2 ‘Quart. Journ. Mic. Sci.,’ vol. 80, 1889, pl. 10, fig. 7.

PLUMES OF CEPHALODISCUS. 249

author also directs attention! to a still more remarkable
similarity between the processes of the lophophore and the
lower lip of the Sipunculoid Phymosoma, described by
Shipley,” and the plume-axes and _ post-oral lamella of
Cephalodiscus. The ends of the lower lip of Phymosoma
are continuous with the ends of the series of finger-like pro-
cesses of the lophophore, and Shipley’s figure 32 on Plate 4
of his paper bears a close similarity to my text-figure 8. It
may be not without significance that, when viewed from the
front, the nephridiopores of Phoronis and of Phymosoma
occupy the same positions with regard to the anus and other
parts as do the gonadial apertures of Cephalodiscus.

While the position of the epistome of Phoronis does not
militate against the view here favoured of an affinity between
Phoronis and Cephalodiscus, it is to be noted that,
according to the observations of de Selys Longchamps,* the
pre-oral lobe of the larva Actinotrocha, which is homologous
with the proboscis of Balanoglossus and the buccal shield
of Cephalodiscus, disappears during the metamorphosis,
and does not persist as the epistome of the adult Phoronis.

Although the occurrence of the five divisions of the ccelom
in Actinotrocha, corresponding with the proboscis cavity,
two collar cavities and two trunk cavities, claimed by
Masterman,! has been contested by Goodrich,’ Ikeda,°® and
de Selys Longchamps,’ these last three writers agree that
there is a division of the ccelom into a pre-septal and a post-
septal part, the septum in question following the course of
the tentacles of the larva. It is still possible, therefore, that

IL. ¢., p. 119.

2 ¢Quart. Journ. Mic. Sci.,’ vol. 31, 1890, figs. 1 and 2.

3 «Développement Postembryonnaire et Affinités des Phoronis,” ‘Mém.
Classe Sci. Acad. Roy. Belgique,’ i, 1904, p. 73.

SSAPrOCs Roy. Soc. Edinb.,’ vol. xxi, 1896, pp. 62, 63, and 130; also
‘Quart. Journ. Mic. Sci.,’ vol. 43, 1900, p. 395.

5 «Quart. Journ. Mic. Sci.,’ vol. 47, 1904, p. 111.

6 * Journ. Coll. Sci. Imp. Univ. Tokyo,’ xiii, part 4, 1901, pp. 540 et seq.
7 Loe. cit., pp. 15, 16. |

250 W. G. RIDEWOOD.

the pre-septal cavity may represent the pair of collar cavities
of Balanoglossus and Cephalodiscus.! The obliquity
of the coelomic septum of the Actinotrocha is exactly similar
to the obliquity of the boundary between the collar cavities
and trunk cavities of Cephalodiscus (see the upper line
of dashes in text-fig. 10), the dorsal part being much more
anterior than the ventral.

A comparison of such published figures of buds of
Rhabdopleura as show developing plumes, with the buds
of Cephalodiscus described in the earlier part of this paper,
confirms the general impression that the two plumes of the

TExt-FIGURE 11.—Two buds of Rhabdopleura normani,
dorsal aspect; copied from Lankester, ‘Quart. Journ. Mic. Sci.,’
vol. 24, 1884, pl. 39, fig. 8, buds 5 and 7. &. Body of the bud.
b.s. Buccal shield. p.a. Plume-axis, pi. Developing pinnules,
s. Stalk.

adult Rhabdopleura are equivalent to the first pair of
plumes of Cephalodiscus. The plumes of Rhabdopleura
arise as a pair of digit-like processes (see text-fig. 11), close
together, and dorsal to the shield; later on the pinnules
develop in pairs, and in the adult the pinnules are directed
ventrally towards the shield, exactly as are the pinnules of

1 See Fowler, ‘ Festschr, 70ten Geburtstage R. Leuckarts,’ 1892, p. 297,
and Harmer, ‘ Pterobranchia of the ‘‘Siboga”’ Expedition,’ 1905, p. 116.

PLUMES OF CEPHALODISCUS. 251

the plumes of the first pair in Cephalodiscus. The second
and later plumes of Cephalodiscus are not represented in
Rhabdopleura. Besides the figures of buds of Rhabdo-
pleura published by Lankester and reproduced here as text-
fig. 11, those by Allman?! and Schepotieff ? may be consulted
with advantage.

SUMMARY.

1. The torsion of the axes of the first and second plumes
of the buds of Cephalodiscus described by Masterman
does not take place. The grooved faces of the axes of these
plumes are in the first instance directed towards the dorsal
face of the buccal shield, and they maintain this relation
through life.

2. The last two pairs of plumes do not arise between the
first two pairs of plumes and the buccal shield, as described
by Masterman, but they arise on the dorsal side of those
plumes, the side remote from the shield. ‘Their grooves are
directed, not towards the shield, but away from it.

3. The evidence derived from a study of the buds of
Cephalodiscus bears out the contention of Harmer that
the series of plumes and post-oral lamella are continuous.
The plume-axes develop in pairs successively, the median
pair first, then a pair lateral to these, and so on. When the
post-oral lamella appears, usually at the time when the third
or fourth pair of plume-axes are beginning to develop, its
edge is in contact with the last-developed pair of plume-axes.
The fifth plume-axis appears between the fourth plume-axis
and the end of the margin of the post-oral lamella, the sixth
between the fifth and the end of the post-oral lamella, and, in
the case of Cephalodiscus nigrescens, in which there are
seven pairs of plumes in the adult, the seventh arises
between the sixth and the end of the margin of the post-
oral lamella.

1 «Quart. Journ. Mic. Sci.,’ vol. 9, 1869, pl. 8, figs. 7 and 8.
2 «Zool. Anzeiger,’ vol. xxvili, 1905, p. 802, fig. 6.

252 W. G. RIDEWOOD.

4, The line of plume-bases in the adult, when viewed from
the front, is an ellipse, incomplete on the upper side (text-
fig. 8) ; and the line of the attached edge of the post-oral
lamella is another ellipse, similarly incomplete. The two
ellipses join where they are incomplete.

5. Separate accounts are given in this paper of the plume-
development in buds of Cephalodiscus hodgsoni, C.
dodecalophus, C. nigrescens, and C. gilchristi.

6. The plumes develop relatively later in buds of C.
nigrescens and C. gilchristi than those of C. hodgsoni
and C. dodecalophus.

EXPLANATION OF THE ABBREVIATIONS EMPLOYED IN TEXT-FIGURES
9, 3, 4, 6, 7; and 9.

1, 2, 3, 4, 5, 6, 7. The first, second—seventh pairs of plumes of the buds.
b. The “body” of the bud. 4.s. Front lobe or main portion of the buccal
shield. m. Mouth, or position of mouth so far as can be ascertained in the
dissected preparations, this region being much disturbed during the dissection.
p. Pinnules. p.a. Plume-axis. y./. Posterior lobe of buceal shield. po.v.
Post-oral lamella. 7.4. Refractive beads in the swollen ends of the plume-
axes in C. hodgsoni and C. dodecalophus. se. Ragged edge caused by
dissection of the part figured from the rest of the bud. 7./. Red line of the
buccal shield. s. Stalk of the bud.

ON THE STRUCTURE OF ENIGMA MNIGMATICA. 2538

On the Structure of Ainigma enigmatica,
Chemnitz; a Contribution to our
Knowledge of the Anomiacea.

By
Gilbert C. Bourne, M.A., D.Sc., F.L.S.,
Fellow of Merton College; Linacre Professor of Comparative Anatomy
in the University of Oxford.

With Plates 15—17, and 2 Text-figures.

In a recent paper in the ‘ Arbeiten aus dem zoologischen
Instituten der Universitit Wien,’ Sassi (16) has given a full
account of the anatomy of Anomia ephippium, and has
elucidated several hitherto obscure points relating to the
kidneys and the reno-pericardial orifices in that species.
Before Sassi’s paper had come into my hands I had made
some observations on the allied genus Ainigma, a tropical
member of the Anomiacea, the principal outcome of which
has been to confirm Sassi’s results. At the same time I have
found certain differences between the genera Anomia and
finigma which are of sufficient importance to make it worth
while publishing a short account of the latter genus.

Iam indebted to Mr. R. Shelford, of Emmanuel College,
Cambridge, and of the Hope Department of Zoology at
Oxford, for the specimens of Ainigma enigmatica,
Chemnitz, which form the subject of this memoir. They
were found living under conditions described by previous
authors, on the roots and branches of Nipa (a palm of the
family Phytetephantine) at Sarawak, and Mr. Shelford tells

254 GILBERT C. BOURNE.

me that the specimens he collected were uncovered not only
at low tides, but also at high neap tides, so that they re-
mained exposed for days together to the rays of a tropical
sun, but nevertheless always remained moist and fresh. The
power of resisting desiccation for so long a time is unusual
among lamellibranchiate molluscs, and indicates some struc-
tural adaptations enabling the animal to survive in such a
habitat. I shall show in the course of this paper that the
thickened mantle lobes and the presence of certain irregular
passages and channels connected with the mantle cavity may
be regarded as modifications for the retention of a sufficient
supply of moisture, but in the essential features of its
anatomy Ajnigma is very similar to Anomia ephippium.

Afnigma, like other Anomiacea, is firmly fixed by a stout
byssus passing through a large sinus or circular perforation
in the right valve of the shell. The specimens given me by
Mr. Shelford have been carefully preserved for histological
purposes, and the right valve has in every case been wholly
or partially removed. I am therefore unable to give an exact
account of it, but from the fragments remaining in several
specimens, I can confirm the descriptions given in concho-
logical works, namely, that the upper extremities of the sinus
through which the byssus passes curve round above the
byssus and meet one another above the ligament, but do not
fuse together as in adult specimens of Anomia ephip-
pium.

The left valve varies very much in size and shape, and its
form seems to be moulded to some extent upon the substra-
tum to which the animal is attached. It is generally more
or less elongate-oval in shape, of a dark purple colour, nacre-
ous and iridescent internally. It is very thin and more or
less translucent, especially when wet, this latter character
being apparently of some importance to the economy of the
animal, as will appear later on.

There are certain points of difference between the left
valve of Ainigma and that of Anomia which require some
little explanation. Jn the latter genus the left valve is more

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 205

or less symmetrical ; the umbo is close to the dorsal margin
and is more or less median in position and the ligament is
immediately within and below the umbo. In Avnigma, as
is clearly shown in the text-figure 1, the left valve is
asymmetrical; the umbo is prominent and obliquely curved,
so that it points toward the anterior and upper margin, and
it is situated at a distance of some 3—5 millim. from that
margin, but is connected with it by a narrow slit or fissure.
Internally this fissure extends as far as the ligament, and is
more or less at right angles to it. The ligament, which is
3—4 millim. long in large-sized specimens, is situated obliquely

Text-Fic. 1.—The left valve of Ainigma enigmatica; internal
view (left-hand figure) and external view (right-hand figure). a, B.
The true dorso-ventral axis of the shell. c,p. The apparent dorso-
ventral axis. ad. Impression of the adductor muscle of the valves.
a.r.p. Impression of the anterior retractor pedis muscle. 6.m. Im-
pression of the byssus muscle. p/.m. Impression of the branchio-
pallial muscle. p.7.p. Impression of the posterior retractor pedis
muscle.

above the umbonal fossa, and a line, aB, drawn at right angles
to it indicates the original dorso-ventral axis of the shell. Itis
obvious that an inequality of growth, clearly indicated by the
growth lines on the outer surface of the valve, has produced
a secondary symmetry, and that the apparent dorso-ventral
axis has been rotated through an angle of 40° in a postero-
anterior direction. Sassi has shown that in Anomia ephip-
pium the animal has undergone a similar rotation to an
extent of 90°, that is to say through an angle twice as great

256 GILBERT C. BOURNE.

as that in Ainigma, but there is also this difference between
the two genera; that whereas in Anomia the rotation has
affected the position of the animal with respect to its shell,
the latter retaining its symmetry, in Ainigma the inequality
of growth has affected the shell as well as the contained
animal, and both are asymmetrical to the same degree, as
may be seen by a comparison of pl. 15, fig. 1, with the text-
figure. Notwithstanding the change of symmetry, it will be
convenient to describe the upper and nearly straight margin
of the shell as dorsal, the lower convex margin as ventral,
and the two ends as anterior and posterior respectively.

The impressions of the posterior adductor muscle, the
anterior and posterior extractor muscles of the foot (here
serving as retractors of the byssus) and of the large byssus
muscle are shown in the text-figure and do not require special
description. But it should be noticed that there is a fifth
muscular impression, pl.m., which has been overlooked in
previous descriptions of this genus, but Woodward (17)
describes and figures a similar muscle under the name of the
branchio-pallial muscle in Placuna placenta. This impres-
sion marks the point of attachment of a specialised band of
pallial muscles running forward and downward in the left
mantle lobe.

GENERAL ANATOMY.

Fig. 1, pl. 15, is a representation of the animal lying in
the left valve of the shell. The right mantle lobe has been
largely cut away, and the left mantle lobe is obviously con-
tracted by the action of preservative fluids; otherwise the
animal is shown in its natural position. As has been explained
above, Ainigma has not undergone so great a degree of
rotation as Anomia, and retains its original symmetry with
regard to the shell. We find accordingly that pallial cavity
or sinus lying dorsad of the (posterior) adductor muscle, and
containing the posterior recurved free ends of the gills, is

ON THE STRUCIURE OF ANIGMA ANIGMATICA. 257

smaller than in the latter genus, and the dorsal sutural union
of the mantle edges is considerably longer, extending from
the posterior edge of the ligament to the ventricle between
the points marked a, # in fig. 1.

Before proceeding to a description of the different organs
it is necessary to point out that, in addition to the postero-
anterior rotation described above, the anterior half of the
body of Ainigma (and of all other Anomiacea) has been
twisted round from left to right in connection with the
peculiar development of the byssus and its retractor muscles.
The nature and effect of this torsion has already been
described by de Lacaze Duthiers (8) and Sassi (16); but a
repetition is not out of place, if only to save the reader the
trouble of reference to their papers. In a normal Lamelli-
branch the byssus cavity and groove are situated on the
posterior margin of the foot, and the retractor muscles of the
byssus are paired and symmetrical, passing dorsally to their
insertions on the right and left valves on either side of the
hinge. In the Anomiacea the byssus, instead of passing out
between the valves, passes through the well-known hole or
sinus in the right valve, and drags the posterior margin of
the foot over to the right. The retractor muscle of the
byssus, instead of being paired and attached near the hinge
line of both valves, is single and is attached near the centre
of the left valve. As a consequence of these displacements,
the whole of the anterior half of the body is twisted over to
the right in such a way that the paired organs of the right
side come to lie above the byssus muscle, and those of the
left side below it; the visceral nerve commissures and the
kidneys being specially affected. When this torsion of the
foot and the lower part of the visceral mass is kept in mind,
much that is puzzling in the anatomy of the adult is made
clear.

The Mantle.—The edges of the mantle are free for nearly
the whole of their extent, and are only united dorsad of the
visceral mass between the points marked z, x in fig. 1. The
left mantle lobe is entire, and covers in the whole left side of

258 GILBERT C. BOURNE.

the animal, its continuity being broken only by the surfaces
of attachment of the adductor muscle, the retractor muscles
of the foot and byssus, and the special slip of pallial muscle
referred to above. The edges of both mantle lobes are
thickened and bear small pallial tentacles covered by a
columnar epithelium. There are also a few much larger
tentacles on the posterior lower margin of the right mantle
lobe. ‘There are no marginal pallial eyes or pigment spots,
but at the bottom of the groove formed in the thickened
pallial margin a track of columnar cells, supplied by twigs of
the circumpallial nerve, can be distinguished. In the region
of the visceral mass, that is to say in its upper half, the left
pallial lobe is very thin; but in its lower and posterior half,
where it covers the large recurved gills, it is greatly thick-
ened by the abundant development of lacunar tissue. The
slip of pallial muscle, attached to the left valve of the shell,
is Shown at pl.m. in fig. 2. It coincides in position with the
attachment of the axis of the left gill to the mantle lobe, and
must function as a retractor of the gill. The left gonad
extends backward to the level of the anus in the left pallial
lobe, and in the same lobe there is a large anterior extension
of the left gonad, forming a conspicuous sausage-shaped
swelling in front of the mouth (figs. 1 and 2, go.a.).

The most remarkable feature in the left pallial lobe is the
presence of a number of deeply pigmented spots, arranged in
an irregular oval at a considerable distance from the pallial
margin. These eye-spots, as I must call them, vary in number
_in different individuals. In the specimen shown in fig. 2
there are twenty-three. They are, however, very constant in
position: the most anterior eye-spot is always situated just
behind the surface of attachment of the anterior retractor
pedis muscle, and the most posterior close to the attachment
of the pallial muscle. Hach eye-spot consists of a ring of
black pigment surrounding a central opaque white area. ‘The
histology of these organs will be described in the latter part
of this paper; but I may say here that their minute structure
leaves little doubt that they are sensory in function and

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 259

adapted for visual functions. As they are situated at a
greater or less distance from the edge of the mantle, they
must always be covered by the shell, and the existence of
visual organs in such a position is somewhat extraordinary.
But, as I have pointed out, the left valve of the shell is thin
and translucent enough to allow a considerable amount of
light to pass through. As Ainigma spends a large part of
its existence uncovered by the sea, with its valves tightly
closed to prevent evaporation, it is probable that these eyes
are efficient in informing the animal of the duration of day-
light, or, at any rate, of the incidence of direct sunlight. It
is probable enough that after sunset the valves of the shell
are slightly opened to admit of the aeration of the water con-
tained in the pallial chamber, and are kept tightly closed to
prevent evaporation during the heat of the tropical day.

The right mantle lobe is very irregular in shape, and
presents a large anterior sinus corresponding with the sinus
of the right valve of the shell, and serving for the passage
of the byssus. In the anterior part of the body the right
mantle lobe is attached by a very narrow band of tissue to
the visceral mass, the line of attachment running above and
nearly parallel to the upper edge of the byssus cavity. In
this part of the body, indeed, the viscera are thrust over to
the left side of the body and the visceral mass is adherent to
the left pallial lobe. But in the hinder part of the body the
rectum and caecum of the crystalline style, passing respectively
above and below the adductor muscle, cross over from the
left to the right side, and are here adherent to the right
mantle lobe and embedded in the mass of the right gonad
and right kidney (compare figs. 11 and 13). The lower and
posterior part of the right mantle lobe is exceedingly thick,
and its inner surface is pitted and folded in a very irregular
manner. As the animal lies with its right valve lowermost
these folds and pits must serve for the retention of water
during the long periods in which it is uncovered by the
tides.

The mouth, as is shown in fig. 1, lies asymmetrically on the

260 GILBERT C. BOURNE.

right side. It is concealed in a labial groove formed by two
deep folds of the integument, which are nothing else than
the greatly enlarged and modified labial palps. The extent
and relations of these labial folds in Anomia were first
described by de Lacaze Duthiers (8) and more fully by
Sassi (16); they have very much the same relations in
fnigma as in that genus. The external labial fold passes
round in front of the mouth forming a hood and the internal
fold passes behind the mouth, the two enclosing between
them a groove of varying depth lined by a high columnar
ciliated epithelium, which contains numerous gland-cells in
the region of the mouth. On the left side of the body the
two folds, as they pass backward from the mouth, become
very deep and prominent, and enclose between them a deep
groove or gutter whose walls are thrown into numerous
vertical folds covered by a ciliated epithelium. At a short
distance behind the mouth the folds hang far down in the
mantle cavity on the left side of the foot and are suspended
from the visceral mass above by a thin sheet of tissue.
Towards the posterior end of the foot the groove becomes
shallower, and its walls are less folded, and eventually the
external labial fold unites with both the direct and the
reflected lamella of the left external demibranch, and the
internal labial fold with the direct lamella of the left internal
demibranch. Thus the left labial groove becomes continuous
with the inter-branchial chamber of the left side (see text-
figure 2,4 ands). On the right side the two labial folds
run back above the byssus cavity, parallel with and below
the line of attachment of the right mantle lobe to the visceral
mass. Dorsad of the byssus the labial folds are inconspicuous,
the labial groove contained between them is shallow, and the
epithelium lining is ciliated and glandular, but not ridged.
Towards the posterior end of the byssus the groove turns
downward and backward, and the labial folds enclosing it
increase rapidly in vertical depth. At the same time the
walls of the now very deep groove are thrown into numerous
vertical ridges, and the epithelium of the ridges is richly

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 261

ciliated but not glandular. The outer right labial fold, con-
tinuing to increase in vertical depth, eventually passes into
the upturned reflected Jamella of the right external demi-
branch, and the internal right labial fold becomes continuous
with the thin membrane by which the direct lamella of the
right internal demibranch is attached to the body-wall below
the byssus muscle. The right labial groove thus becomes
continuous with the inter-branchial chamber of the right side,
but it is continued backwards as a cul-de-sac for some dis-
tance beyond the point of union with the gill, and in sections
the right branchia appears to be suspended from the lower
wall of this cul-de-sac, as is shown in text-figure 2, E.

The foot (fig. 1, f.) is reduced to a flat muscular projection
at the anterior angle of the byssus cavity. In most of the
spirit-preserved specimens it is contracted to a small, lanceo-
late, muscular mass, but in the individual figured it is unusu-
ally long and ribbon-shaped. The extremity of the foot is
always pointed, and bears a single small tentacle, similar in
all respects to the marginal tentacles of the mantle. There
is no infundibuliform cavity at the end of the foot as in
Anomia ephippium, but the right (morphologically the
ventral) surface is grooved and covered with numerous trans-
verse, ciliated ridges. The mass of the foot is highly muscular
and contains numerous mucous glands, and it is evident that
this organ is very extensile. It seems probable that it can
be protruded some distance beyond the shell, and that it is
auxiliary to nutrition, minute particles being swept by ciliary
action along the groove on its right surface, and thence to
the right labial groove. .

_ The principal muscles connected with the foot have not
undergone as much modification in Ainigma as in Anomia
owing to the lesser degree of rotation in the former genus.
The two tapering muscular bands running respectively for-
wards and backwards from the great retractor muscle of the
byssus to their surfaces of attachment on the left valve, are
clearly the homologues of the anterior and posterior retractors
of the foot of other Lamellibranchia (fig. 2, a.7.p. and p.r.p.).

262 GILBERT C. BOURNE.

But here they have shared in the torsion of the other organs
of the anterior part of the body, have become twisted round
to the right, have lost their primitive connection with the
right valve, and are inserted on the left valve only, serving
rather as accessory retractors of the byssus than as retractors
of the foot. It will be observed that both the anterior and
posterior retractors of the foot arise from double origins
above and below (morphologically right and left) of the
retractor muscle of the byssus, but that each is formed into
a single strand shortly before its attachment to the shell.

The retractor muscle of the byssus is a large, coarsely-
fasciculated muscle, nearly circular in section, and passes
transversely from its attachment to the left valve to the
byssus cavity. It probably represents the right and left
retractors of the byssus of symmetrical Lamellibranchia, but
betrays no sign of its primitive paired origin. De Lacaze
Duthiers (8), however, considers that the right retractor
byssi muscle is aborted in Anomia, but it should be observed
(fig. 10) that the muscle is equally well developed above, that
is on the morphological right, and below, that is on the
morphological left of the byssus cavity.

The byssus cavity is large and shallow, more or less oval
or lozenge shaped, and bordered above and below by muscular
lips, which are really the posterior continuations of the right
and left margins of the foot. The bottom of the cavity is
lined by a number of close-set, parallel folds or lamellz
running fore and aft in the direction of the long axis of the
shell. These lamellae form the byssus gland, the histology
of which will be described in the latter part of this paper.
But I may state here that the byssus of Ainigma is not
calcified like that of Anomia, but consists of a number of
parallel plates of the byssus substance, secreted by the
epithelial cells covering the ridges of the byssus gland.
These lamelle are fused externally into a plate which is
directly and firmly fixed to the substratum to which the
animal is attached.

The Gills.——The natural position of the gills, as seen

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 263

from the right side is clearly shown in fig. 1. They are
relatively even larger than in Anomia, and, as in that genus,
their posterior ends are recurved and form the posterior
boundary of the dorsal or supra-branchial chamber into which
the anus opens, and in which the ventricle of the heart lies.
In their microscopic as well as their macroscopic structure
the gills of Ainigma are singularly like those of Anomia
ephippium as described by Ridewood (15), to whose paper
the reader is referred for details. Thus the reflected filaments
of the right and left internal demibranchs are fused together
and the whole series of filaments are in organic continuity
along the line of union, and are traversed by a blood-vessel.
The upper ends of the reflected filaments of the right and
left external demibranchs are secondarily reflected downwards
to form the so-called velar fold or flap, and at the angle of
reflection the whole series of filaments are united in organic
continuity, and are traversed from end to end by a blood-
vessel (text-figure 2, b.v.). The lower ends of the velar fila-
ments are, however, independent, and have an arrangement
of cilia which appears to have been overlooked by previous
observers. As is shown in fig. 6 these velar filaments are
sub-triangular in section, and the chitinous lining of their
cavities is thickest on the internal or morphologically ventral
side, this being the reverse of what obtains in the direct and
reflected limbs of the filaments. The outer (morphologically
dorsal) side of the velar filament is broad and flat, and is
covered by a cubical epithelium bearing a number of short
stiff cilia. ‘The opposite face of the filament is narrowed and
covered by longer columnar epithelial cells bearing long fine
cilia, continuous with the frontal cilia of the reflected or
ascending limb of the filament. There can be little doubt
that the short stiff cilia borne on the pallial faces of the velar
filaments have the function of ciliated discs, and give a suffi-
cient amount of friction against the inner surface of the
mantle to prevent the whole of the reflected lamella from
shipping down. One might expect to find a corresponding
ciliated ridge, or row of ciliated discs, on the mantle, but I
VOL. 51, PART 2.—NEW SERIES. 20

264. GILBERT C. BOURNE.

can find no trace of them, and am certain that they do not
exist. It should be noted that the velar flap on each side
abuts on the thickened and corrugated lower moiety of the
mantle lobe, so that ciliated discs on that side are un-
necessary.

Asin Anomia ephippium, there are no ciliated discs on
either the direct (descending) or reflected (ascending) limbs
of the filaments, but there is a well-developed single row of
discs at the angle of reflection, the details of which are
shown in fig. 5. There is, asin A. ephippium, a very low
inter-filamenter septum close to the angle, but beyond this
no other interlamellar junctions whatever. A transverse
section of two filaments, showing the frontal and lateral cilia
and other details, is given in fig. 7. The attachments of the
gills to the body-wall and mantle are affected to a considerable
extent by the asymmetry of the anterior half of the body
and by the fact, mentioned above, that the visceral mass is
adherent to the left mantle lobe in the anterior part of the
body but to the right mantle lobe in the posterior part. As
these relations have not been sufficiently fully described in
Anomia, I will enter into them somewhat closely in
/Anigma. ‘The posterior recurved ends of the gills lie free
in the mantle cavity and are not attached to the mantle. In
the more anterior part of their courses the right and left
branchie are differently affected by the asymmetry due to
the twisting of the byssus and foot over to the right. The
relations of the left branchia are on the whole those of a
normal symmetrical Lamellibranch.* At a point nearly ver-
tically below the anus its axial fold becomes attached by a
deep suspensory fold to the left mantle lobe. In this fold
run the muscular fibres which have already been described
as a specialization of the pallial musculature forming a
retractor muscle of the branchia (figs. 2 and 13). Passing
forward the axial attachment of the left branchia becomes
shifted more and more towards the middle line, largely in
consequence of the interposition of the fibres of the posterior
retractor pedis muscle between it and the mantle (fig. 12).

ON THE STRUCTURE OF @NIGMA AINIGMATICA. 265

In front of the adductor muscle the axis of the left branchia
is suspended in the mantle cavity by a deep suspensory fold
attached to the lower surface of the visceral mass just below
the lower limb of the left kidney, and has lost all connection
with the left mantle lobe. These relations are continued for-
ward as far as the posterior edge of the foot, where the direct
and reflected lamellee of the outer demibranch become con-
tinuous with the external left labial fold and the direct
lamella of the inner demibranch passes into the internal left
labial fold as described above (p. 260).

The fold formed by the united upper ends of the reflected
filaments of the right and left inner demibranchs acquires
no attachment till it reaches the anterior end of the great
extractor byssi muscle. Here it is pushed over to the left
and unites with the body wall close alongside of the attach-
ment of the direct lamella of the left inner demibranch, and
its blood-vessel passes into the blood sinus lying below the
recurrent limb of the left kidney, this sinus discharging its
blood into the left auricle (text-figure 2, B and ¢, v).

As may be seen in fig. 1, the anterior end of the right
outer demibranch curves round dorsally behind the byssus
cavity. As explained above (p. 261) its reflected lamella
becomes continuous with the right external labial fold, and
its direct lamella with the right internal labial fold behind
the byssus (see text-figure 2, p and x). The anterior fila-
ments of both lamelle of the right outer demibranch are
very short, but those of the direct lamella of the right inner
demibranch are very long and, as may be seen in fig. 1, d.b’,
extend below and to the left of the byssus cavity as far
forward as the foot. As is shown in text-figure 2,4 to E,
and in fig. 10, these elongated anterior filaments of the right
inner demibranch are connected with the ventral surface of
the body, below the byssus muscle, by a thin membrane, 2,
which stretches across to the left side and eventually, as in
A, becomes attached to the left inner labial fold. Thus, as a
consequence of the displacement of organs several times
referred to, the inner demibranch of the right side is carried

266 GILBERT C. BOURNE.

\\\

N

S\
Ss \\

oy
SE}
mY

Wy

=|
. R-

Trxt-FIG. 2.—Transverse sections through Ainigma enigma-
tica to show the attachments of the gills to the body wall. a. Part
of a section through the anterior edge of tlie foot. 3B. A section
taken a short distance behind a. c. A section through the middle of
ihe byssus muscle. oD. A section through the posterior edge of the
byssus muscle. xz. A section taken through the heart. ¥. The
tenth section of the series behind B. dm. Byssus muscle. /. Foot.
go. Gonad. kk. Left kidney. k’. Right kidney. Zi. Liver. (9.0.
Left labial groove. /.g.7. Right labial groove. /.m. Left mantle
lobe. Z.pg. Left pericardial gland. 7.m. Right mantle lobe. 2.
Anterior venous branch of the left auricle; the pericardial gland
forms a thickening of its outer wall. wz. Membranous attachment
of the right inner demibranch to the lower, morphologically the left,
side of the foot and byssus muscle. z. Spaces serving as reservoirs
for water. 1. Reflected lamella and—ir. Direct lamella of the left
outer demibranch. 1. Direct lamella and—rv. Reflected lamella
of the left inner demibranch. v. Sutural union of the reflected
lamella of ‘the right and left inner demibrancls. v1. Reflected
lamella and—vir. Direct lamella of the right inner demibranch.
viul. Direct lamella and—rx. Reflected lamella of the right outer
demibranch.

ON THE STRUCTURE OF MNIGMA ANIGMATICA. 267

across the body and attached to what is morphologically the
left side of the foot.

Toe ALIMENTARY TRACT.

The mouth leads into a narrow, and, for a Lamellibranch,
relatively long, cesophagus, lined by a ciliated glandular
epithelium, whose characters will be described in the latter
part of this paper. The cesophagus passes into a capacious
stomach occupying the greater part of the visceral mass
dorsad of the foot. The roof of the stomach is thin, and
lined by a few ciliated but non-glandular columnar epithe-
hum, which extends down for some distance on the right
wall, especially in the anterior half of the stomach. The
right wall and floor, and in the posterior half of the stomach,
the left wall are, on the contrary, lined by an epithelium
consisting of very long attenuated ciliated cells, intermixed
with which are numerous elongated claviform gland-cells
filled with yellow granules. The floor of the stomach is also
thrown into longitudinal folds, and is covered by a thick
cuticular layer, the “fléche tricuspide”’? of Pol, which is
apparently secreted by the yellow gland-cells. ‘This question
will be discussed more fully in the latter part of this paper.
The stomach is embedded in the liver, which opens into it
by several large ducts. One of these ducts is dorsal, and
communicates with the superior lobe of the liver; the
remainder are posterior and ventral, and some of them run
far back in the posterior mass of the liver before breaking
up into branches (figs. 9 and 10, lu. d.). Posteriorly the
stomach presents a dorsal caecum, into which one of the
largest of the posterior liver ducts opens. Ventrally it
narrows in diameter, and gives off the intestine and sac of
the crystalline style. .

The intestine opens into the stomach by an aperture
common to itself and the sac of the crystalline style. The
entrance to the intestine is guarded by a number of promi-

268 GILBERT C, BOURNE.

nent longitudinal ridges covered by ciliated epithelinm.
The intestine itself is a narrow tube running back for some
distance close to and on the right of the sac of the crystalline
style. In this part of its course its lumen is narrowed by the
projection of four prominent ciliated ridges into its interior.
Just anterior to the heart the intestine enlarges somewhat
suddenly in diameter, the four internal longitudinal ridges
disappear, and it turns sharply upwards, makes a single
complete turn, bends up at a sharp angle towards the
ventricle, and then runs a straight course below the ventricle
to the anus. In the last section of its course there is a
distinct typhlosole in the large intestine. The rectum is
very short and funnel-shaped. Its epithelium differs entirely
from that of the large intestine, consisting of clear, attenuated,
ciliated cells with deeply-staining nuclei, indicating that it is
a proctodeum. The histology of the alimentary tract will be
described further on.

_As in Anomia, the sac of the crystalline style is exces-
sively long. At first nearly median in position (fig. 10), it
passes over to the right and runs back, closely attached to
the right mantle lobe, below the adductor muscle. In the
posterior part of its course it lies parallel to and above the
attachment of the right branchia, and finally it curves

forward and ends blindly (figs. 10—13, ery.).

Tue Circulatory SYSTEM.

The ventricle of the heart, as in all Anomiacea, lies free in
the dorsal bay of the mantle cavity, and is not enclosed in a
pericardial sac. Situated dorsally to the intestine, it sits, so
to speak, astride of the latter organ, the auricles passing
down on either side of it like a rider’s legs. ‘lhe walls of
the ventricle are very thick and muscular, as also are the
walls of the auricles, but the latter not to so great a degree
as in Anomia. ‘I'he aorta arises from the antero-ventral
angle of the ventricle (its aperture is guarded by a valve),

ON THE STRUCTURE OF ASNIGMA AENIGMATICA. 269

and, running towards the right dorsad of the rectum, it at
once divides into three branches. The median branch
penetrates the liver mass, and soon breaks up into branches
and disappears. The left-hand branch constitutes the anterior
aorta. ‘This vessel runs forward to the right of and above
the intestine, passes through the coil of the intestine to the
left upper side of the visceral mass, and runs forward over
the stomach to the cesophagus, where it divides into numerous
branches. The right-hand branch of the aorta runs down on
the right of the intestinal coil towards the cecum of the
crystalline style, and then bends back to take a posterior
course, supplying the gills and right mantle lobe with blood.

The course of the veins is similar to that described in
Anomia ephippium by Sassi. The left auricle divides
below the level of the intestine into two branches. Of these
the posterior runs down to the outside of the large upper
posterior lobe of the left kidney, and, turning back, may be
traced as far as the visceral ganglia, where it receives several
vessels from the gills and left mantle lobe. The anterior
branch courses forward in close connection with the upper
limb of the left kidney, and its outer wall is thickened by an
abundant glandular tissue, which will be described in con-
nection with the excretory organs. ‘he right auricle dilates
to form a large thin-walled sac lying to the right of and
partially above the intestine. The walls of this sac are
locally thickened by the same glandular tissue that accom-
panies the anterior branch of the left auricle, which, from
its relations to the reno-pericardial funnels, must be regarded
as the representative of the pericardial gland. The venous
cavity may be traced back along the right side of the upper
lobe of the right kidney, and in the posterior part of its
course it receives numerous accessions from irregular sinuses
and vessels bringing back blood from the right mantle lobe,
the right branchia and the sac of the crystalline style.

The right and left auricles communicate with one another
by a sinus passing ventrad of the intestine, immediately
below the hinder end of the ventricle. A similar vessel

270 GILBERT C. BOURNE.

occurs in Anomia ephippium, and was interpreted by
Pelseneer (13) as a relic of the pericardium, a mistake which
anybody might be excused for falling into, seeing how
peculiar are the relations of the kidneys, venous channels,
and remnants of the pericardium in that genus.

Tue Excrerory ORGANS.

Sassi (16) is the first author who has given a correct and
intelligible account of the kidneys in Anomia ephippium.
Fig. 4, which is a reconstruction from one of my series of
sections, shows that these organs are extremely similar in
finigma. ‘The chief difference between the two genera
consists in the fact that, whereas in Anomia the left
kidney forms a complete loop behind the byssus muscle, in
Ainigma the upper and lower limbs are free from one
another posteriorly. In the right kidney the upper limb
extends much further back along the intestine in Ainigma
than it doesin Anomia. In all other respects I can fully
confirm Sassi’s observations. In Ajnigma, as in Anomia,
the right and left kidney sacs communicate by a wide
opening situated below the auricle (fig. 4, ap.). The left
renal sac runs forward, dorsad of the byssus muscle, as far
as the foot, and then turns sharply back, running just above
the attachment of the axis of the left branchia as far as the
visceral ganglion, where it turns upward and opens into the
suprabranchial chamber somewhat in front of the right renal
aperture. The left reno-pericardial canal is, as in Anomia,
situated far forward near the anterior bend of the renal sac
(/. rp. in fig. 4), and the left gonaduct opens into the renal
sac by a distinct ciliated funnel situated close above and
nearly opposite to the left reno-pericardial canal. The right
renal sac is roughly crescentic in shape, the concavity of the
crescent embracing the adductor muscle. The right reno-
pericardial funnel (7. rp. in figs. 4 and 11) is situated imme-
diately below the right auricle, and the right gonaduct opens

ON THE STRUCTURE OF ANIGMA ANIGMATICA. DAA

into the right wall of the kidney sac nearly opposite to, but
a little further back than, the reno-pericardial funnel. So
far the only difference between the structure and position of
these organs in AJnigma, and those in Anomia, as described
by Sassi, consists in the fact that whereas that author says
there are no ciliated funnels to the gonaducts in the latter
genus, there are very distinct gonaducal funnels, with a
well differentiated ciliated columnar epithelium (fig. 15) in
Ajnig ma,

In respect of the relations of the reno-pericardial funnels
to the remnants of the pericardium, there is, however, a
great difference between the two genera. Sassi has shown
that in Anomia ephippium the reno-pericardial canals
lead into a system of short and slightly branched tubules
ending in glandular diverticula, and these he identifies, no
doubt correctly, as the remnants of the pericardium. I have
already shown that Avnigma is in several respects a less
specialised form than Anomia, and in this particular matter
of the reno-pericardial ducts and their connections it has
clearly undergone less modification than the latter genus.
Fig. 14 is a drawing of a section through the left reno-
pericardial canal of Adnigma. ‘The canal is lined by a
columnar epithelium, each of whose constituent cells bears a
long flagellum. ‘he upper end of the canal is lined by a
flatter non-ciliated epithelium, and passes into a mass of
reticulate glandular tissue containing many inter-cellular
spaces. ‘Though it is not particularly well shown in the
section figured, the communication between these spaces
and the reno-pericardial canal can be easily traced in ihe
series of sections. Some few sections further back the canal
appears to open, by an aperture so distinct that it might
almost be described as a ciliated funnel, into a central lumen
in the mass of glandular tissue, and this as it is traced back-
wards breaks up into a number of irregular branches or
channels communicating with the spaces in question. From
its relation to the reno-pericardial canal there can be no
doubt that this lumen together with the irregular channels

DAP PH GILBERT C. BOURNE.

opening into it represents the pericardial cavity, nor can
there be any doubt that the branching canals are homologous
with the structures described by Sassi in Anomia ephip-
pium. If the lumen represents the pericardial cavity, the
glandular tissue clearly is the homologue of the pericardial
gland, and the chief point of difference between Ainigma
and Anomia is that in the former the pericardial gland is
well developed, and retains not only its function as an ex-
cretory organ, but also its connection with the heart. As is
shown in figs. 4 and 14, and in text-figure 24 and B, l. pg.,
the left pericardial gland is of considerable vertical extent in
the immediate neighbourhood of the left reno-pericardial
canal. Posteriorly it narrows to a thin band of glandular
tissue, which may be traced backwards, lying to the outside
of and above the upper limb of the left renal sac, and in
close connection with the anterior of the two veins into which
the left auricle divides, as far as the muscular wall of the
left auricle itself. As it approaches the heart the peri-
cardial gland spreads out over the walls of the auricle and
becomes intimately fused with them, extending, as I shall
show further on, into the thickened walls of the ventricle.

On the right side the right reno-pericardial canal (figs. 4
and 11,7. rp.) leads into the lumen of a similar but larger
mass of glandular tissue, occupying the right wall and floor
of the dilatation of the right auricle described on p. 269.
This dilatation of the blood-vessel has no apparent analogue
inAnomia. The glandular sac forming part of the thick-
ness of its walls is of course the right pericardial gland, and
its histological structure as well as its relations to the upper
posterior lobe of the right renal sac and to the right auricle
are analogous to those of the left pericardial gland. ‘The
extent of the two glands and their relations to the kidneys
and heart are indicated by the spaces enclosed between the
black lines in fig. 4.

The structure and distribution of the pericardial glands of
the Lamellibranchia has been worked out in great detail by
Grobben (6). Without entering into the details of his careful

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 273

and voluminous research on this subject, I may say briefly
that he has shown that in a large number of Lamellibranchs
belonging to different families the epithelial walls of the
pericardium are glandular and have an excretory function.
In many species, and among the Filibranchia in the Arcide,
Mytilide, and Pectinide, the glandular tissue is localised
on the auricles and in some other forms, e.g. Venus
verrucosa (see Grobben, loc. cit., fig. 15), it extends to
the ventricle of the heart. The characteristic histological
elements of these glands are oval or somewhat irregular cells
with a spherical nucleus, alveolar protoplasm containing a
few granules, and in the latter a distinct brown concretion.
Though the shape, size, and appearance of these cells vary in
the various species examined by Grobben, they are present
in the pericardial glands of all, and are very distinct in
character from the concentric concretions found in the
kidney. The structure of the pericardial gland in Ainigma
is shown in fig. 14. The tissues have doubtless undergone
contraction, and are otherwise altered by the action of spirit,
but it is clear enough that the bulk of the gland in the
vicinity of the reno-pericardial canals is formed by a mass of
branching cells, whose processes unite to form a reticulum.
Or the structure might be otherwise described as a mass of
vacuolated protoplasm containing numerous oval nuclei,
smaller, and with a denser chromatic network than the
nuclei of the kidney cells. ‘This tissue may be traced along
the upper margin of the kidney from the reno-pericardial
funnel to the roots of the auricles on either side of the body,
and on arriving at the thickened muscular walls of the
auricles below the intestine it seems to thin out and dis-
appear. In the spaces or vacuoles of this tissue are olive-
brown concretions, which vary in size and appearance with

1 The concretions in the kidney are commonly said to consist of uric acid ;
but Letellier (9) states that no uric acid is excreted by any Lamellibranchiate,
and that the urinary concretions consist of calcium carbonate and acid phos-
phates of lime and magnesium. According to this author the Lamellibranch
kidney excretes wea, the Gastropod kidney uric acid,

274 GILBERT C. BOURNE.

the metabolic condition of the animal. In some of my series
of sections the concretions are small and are contained in
oval cells, as shown in fig. 16, a. These cells are almost
identical with the pericardial cells of Venus verrucosa
and Cardium edule figured by Grobben (loc. cit., figs. 53
and 54). In another of my series the concretions are much
larger, and are either surrounded by a thin cell-envelope
with the nucleus lying to one side, as in fig. 16, b, or the
cell structure is no longer distinguishable. Similar conditious
are figured by Grobben for divers Lamellibranchs.

The presence of these highly characteristic cells and
concretions not only enables us to identify the above-de-
scribed tracts of tissue as pericardial glands, but also to trace
the latter beyond their apparent limits. The pericardial
glands appear to thin out on the auricular walis, and to stop
short of the heart. But an examination of the walls of the
heart with high powers of the microscope reveals the fact
that they are penetrated by the glandular tissue. The two
auricles, embracing between them the intestine, lie, like the
ventricle, in the pallial cavity, and have relatively thick
muscular walls, covered externally by a columnar epithelium
continuous with the external epithelium of the body.

The muscle-fibres, both of the auricles and ventricle, cross
one another in all directions, forming a sort of sponge-work
with numerous spaces. A high power of the microscope
shows that the tissue of the pericardial glands runs through
these spaces in the muscular walls of the auricles, and extends
into the ventricle. ‘The characteristic cells containing olive-
brown concretions can be distinguished even with a low
power in the inter-muscular spaces of both auricles and
ventricle (fig. 19). Furthermore, a careful examination of
the columnar epithelium of the heart shows that the inner
ends of its component cells do not rest on a basement
membrane, but are prolonged internally into fine processes
which run in between the muscular fibres. In other words,
the external epithelium of the auricles and ventricle is fused
to and partly immersed in the subjacent muscular tissue,

ON THE STRUCTURE OF ANIGMA ANIGMATICA, 275

much as the epidermal cells of many Platyhelmia are immersed
in the subdermal muscular and connective tissue.

With these facts before us we have a ready explanation of
the hitherto unsolved problem of the fate of the pericardium
in the Anomiacea.

In a typical Filibranch with a well-developed pericardium
we should find the following layers in transverse section :—
1. The external epithelium. 2. The outer epithelial wall of
the pericardial cavity. 38. The inner glandular wall of the
pericardial cavity adhering to the auricles or ventricle. 4.
The muscular wall of the auricles or ventricle. It is clear
that in Ainigma the pericardial cavity has disappeared in
the vicinity of the heart, and that the external epithelium,
the outer and inner walls of the pericardium, and the mus-
cular wall of the heart itself have become more or less inti-
mately fused together; in particular the glandular tissue of
the inner pericardial wall has become incorporated with the
muscular wall of the heart. None the less all these forms of
tissue—epidermic, glandular, and cardiac muscular—can be
recognised by careful microscopical examination. It is
obvious that the disappearance of the pericardial cavity and
the fusion of its walls with those of the heart is in some way
correlated with the torsion of the body produced by the
excessive development of the byssus muscle, and the attach-
ment of the latter to the centre of the left valve of the shell.
The result of this is, that the left kidney has been dragged
forward to pass round the byssus muscle and the left reno-
pericardial canal has been shifted forward far from its typical
position, involving a great anterior extension of the left peri-
cardial cavity. The corresponding structures on the right
side have not suffered so much displacement, but the strain
to which the whole system of organs must have been sub-
jected during the passage from the larval to the adult condi-
tion is sufficient to account for the obliteration of the peri-
cardial cavity, except in the immediate proximity of the
reno-pericardial canals. Sassi has suggested that the whole
of the venous vessel from the ventricle to the left reno-

276 GILBERT C. BOURNE.

pericardial aperture is the representative of the left auricle,
and if we regard all that vessel as auricle which is covered
with pericardial glandular tissue, his suggestion is correct.
But as the muscular coat of the afferent vessels of the heart
does not extend as far in AJnigmaas in Anomia, and as
the left afferent vessel divides close below the intestine into
an anterior and a posterior vessel, I have preferred to restrict
the term auricle to those muscular vessels which embrace the
intestine.

The gonads.—The sexes are separate. The ovaries and
testes occupy the same position in the two sexes, so a descrip-
tion of one will apply equally well to the other. The left
gonad is much the larger of the two, and extends for a long
distance in front of and behind the gonopore. Its anterior
moiety has the same relations as in Anomia, that is to say,
it runs forward below the stomach and liver, and just above
and to the right of the left labial groove to the mouth,
where it passes to the right of the attachment of the anterior
retractor pedis muscle, and passing into the mantle forms
the large preoral mass which is so conspicuous a feature
when the animal is opened. The posterior moiety runs back
as a fine canal, bearing a few slightly branched diverticula,
below the cecum of the crystallme style and above the
byssus muscle; its course being here on the right rather
than on the left side of the body. Behind the byssus muscle
it passes over to the left side of the body, and divides into
two branches running respectively along the upper and lower
edges of the posterior retractor pedis muscle. Behind the
attachment of this muscle the gonad passes into the left
mantle lobe, and there forms a large follicular mass extend-
ing back for some distance beyond the level of the anus, as
shown in figs. 2,12, 13. The left gonad does not extend
into the mantle in Anomia, and Pelseneer (14) has stated
that it does not do so in any of the Anomiacea, a somewhat
rash generalisation which must now be corrected. ‘The right
gonad in the anterior part of its course lies dorsad of the
stomach and liver on the upper side of the visceral mass,

ON THE STRUCTURE OF ANIGMA AMNIGMATICA. DEL

where it forms a large lobe above and to the right of the
intestinal loop. From this lobe two main branches pass
backward. ‘I'he one passes to the dorsal side of the caecum
of the crystalline style, and accompanies the cecum for the
whole of its course in the right mantle lobe, and forming a
large mass above its extremity, as shown in fig. 15. The
other branch, into which the gonaduct opens, maintains a
more dorsal position, and, passing above the adductor muscle,
forms a considerable follicular mass to the right of and above
the posterior part of the intestine.

The gonaducts, as has been described above, open by
distinct ciliated funnels into the renal sacs close to the reno-
pericardial apertures of the sides of the body to which they
belong. A gonaducal funnel of a male is shown in fig. 15.
There is no difference in the histological characters of the
funnel in the two sexes. ‘he gonaducts, that is to say those
sections of the ovarian or testicular tubules that are lined by
a low cubical instead of a germinal epithelium, are extremely
short. The histology of the ovaries and testes does not call
for special description, but in one of my series of sections the
ovary was penetrated throughout by a number of green
filaments, whose nature I could not satisfactorily determine.
They have the appearance of filamentous algw, and may
possibly be symbiotic or parasitic within the molluse. But
they are not of constant occurrence, for I could find no trace
of them in two other females examined.

The Nervous System.—As may be seen in fig. 3, the
neryous system is of the usual lamellibranchiate type, and
the modifications it has undergone are attributable only to
the torsion which has affected these in common with all the
other organs of the anterior moiety of the body. ‘Thus the
right cerebro-pleural ganglion lies above and somewhat
behind the left. The cerebro-pedal connectives are relatively
short, and the right pedal lies above the left pedal ganglion.
The right visceral connective passes above, and the left
visceral connective below, the byssus muscle and the right
visceral ganglion is somewhat above and in advance of the

278 GILBERT C. BOURNE.

left. But beyond this distortion which it shares with the
other organs of this region, the nervous system presents few
features requiring special description. ‘The otoliths lie a
short distance behind the pedal ganglia, and are connected
with the latter by short nerves. The two stout nerves
passing from the pedal ganglia, that from the right ganglion
distributed to the upper and that from the left ganglion
distributed to the lower surface of the byssus muscle are
worthy of mention, because de Lacaze Duthiers (8) describes
a large nerve passing from the visceral ganglia to the
byssus muscle in Anomia. No such nerve is present in
Ainigma, the byssus muscle being wholly innervated from
the pedal ganglia. The branchial and pallial nerves issuing
from the right and left visceral ganglia should be noted.
The branchial nerve of either side runs straight down into
the suspensory fold of the gill axis, and there enters a
distinct branchial ganglion (fig. 8, br.g.), from which fine
nerves run forwards and backwards in the gill axis. The
pallial nerve issues separately from the branchial nerve on the
right side, but the two have a common origin from the left
visceral ganglion. The branches of the two viscero-pallial
nerves diverge in the mantle lobes and unite in each mantle
lobe with a distinct circumpallial nerve, which forms a
complete ring in the thickened margin of the mantle, and
forms a connection anteriorly with nerves issuing from the
cerebro-pleural ganglia.

Histology.—The specimens of Ainigma collected by
Mr. Shelford were so well preserved that I have been able
to work out the histology of some of the organs in some
detail, but I do not propose here to do more than give an
account of some of the more striking features that came
under my notice. A detailed account of the histology of
various members of the Lamellibranchia, including the
Anomiacea, is indeed a desideratum, but for such a task
fresh specimens are necessary, and where they are not avail-
able it is inexpedient to attempt more than a description of
such characters as can be accurately studied in sections.

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 279

Pallial Organs.—The lobes of the mantle were so much
contracted in spirit that I have been unable to make out the
details of the histology of the marginal tentacles to my satis-
faction. The tentacles are covered by a high columnar
epithelium, and receive an abundant nerve supply from the
circumpallial nerve. There are some specially large tentacles
on the lower posterior edge of the right pallial lobe (fig. 13,
t.), and near the bases of some of these there are ganglionic
enlargements on the circumpallial nerve. The thickened
edges of the mantle present the three reduplications com-
monly occurring in the Lamellibranchia, and in the bottom
of the groove formed by the two outermost reduplications
there is, in the hinder part of both mantle lobes, a tract of very
definite columnar cells supphed with twigs from the circum-
pallial nerve. These cells are probably sensory in function.

As may be seen in the sections, figs. 9—13, the lower
moieties of the mantle lobes overlapping the gills are very
much thickened, and their inner surfaces are thrown into a
number of folds, which are specially prominent in the right
lobe. ‘These foldings are no doubt exaggerated by contrac-
tion in spirit, but there can be little doubt that they exist in
the fresh state, and serve to retain water when the animal is
uncovered for long periods by the tide. The thickened lobes
of the mantle are nearly entirely composed of a lacunar
tissue, traversed by very few branching muscle-fibres. The
greater part of the musculature of the mantle lies imme-
diately below the external layer of epithelium, and all within
this is a spongy lacunar tissue, which must serve as a reservoir
for the retention of fluid.

In this connection it may be observed that the asymmetrical
arrangement of the organs, and in particular the narrow
attachments of the mantle lobes to the visceral mass except
in the regions of the attachments of the muscles to the valves
of the shell, induces a certain laxity and independence of the
organs, which can be better understood by an examination of
figures 11—13 than by any description. As a consequence
there are numerous irregular. spaces communicating with the

VoL. 51, PART 2.—NEW SERIES. |

280 GILBERT C. BOURNE.

suprabranchial or the pallial cavities, and some of them, such
as those marked z in figs. 11 and 13, end in veritable culs-
de-sac. The entrances to these culs-de-sac is guarded
by two ridges, whose thickened borders are covered by a
columnar epithelium furnished with stiff cilia. A transverse
section of one of these ridges is shown in fig. 8. There can
be little doubt that when the thickened borders of these
ridges are in apposition the cilia interlock, and thus close
the entrance to the cul-de-sac, which then serves as a
reservoir for water. The largest of these reservoirs are
situated between the hinder part of the right labial groove
and the visceral mass (fig. 11, z.), between the right kidney
and the right mantle lobe, below the level of the heart (text-
figure 2, I’, z.), and between the hinder part of the intestine
and the right mantle lobe (fig. 13, z.). It is evident, then,
that Ainigma is well provided against the danger of
desiccation when exposed for days together to the rays of
the sun.

The most remarkable of the pallial organs are the pigment-
spots or eyes of the left pallial lobe, whose position has been
described on p. 258. The structure of these organs is alto-
gether peculiar in that two epidermic layers, namely, those
of the outer and inner face of the mantle, share in their
formation. As is shown in fig. 20, each eye comprises a
cornea, a lens, a vitreous body, and a layer of deeply-
pigmented cells forming a rudimentary retina. The cornea
is formed by a local modification of the external epithelium
of the mantle, which, elsewhere formed of columnar cells
with an admixture of glandular cells, here becomes flatter ;
its component cells are transparent and vacuolated, and the
cell outlines are scarcely distinguishable, but the nuclei are
distinct and oval, with scattered chromatin granules. ‘The
lens (fig. 21) is a biconvex lenticular mass lying below the
cornea, but separated from it by a thin sheet of tissue
continuous with the vitreous body. The lens, in spirit
specimens, is of an opaque white colour, and no definite
structure can be discerned in it. It is a vacuolated mass of

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 281

finely granular substance containing a few nuclei, and as the
latter are identical with the nuclei of the cornea, it is
probable that the lens is formed by a proliferation of the
epithelial cells. The lens is embedded in a vitreous body
consisting of a mass of finely granular, vacuolated proto-
plasm containing numerous nuclei which are smaller and stain
more deeply than the nuclei of the lens or cornea. This
vitreous body,in which no cell outlines are distinguishable, is
evidently a local modification and concentration of the sub-
epidermal tissue. The retinal layer, as is clearly shown in
fig. 20, is formed by the epithelium lining the inner face of
the mantle. Some of the eyes are borne on very thin parts
of the mantle, and in these, as the vitreous body occupies the
whole thickness of the mantle, the retina simply consists of a
modification of the cells lining the inner face of the mantle.
They become very large and columnar (fig. 21), and are thickly
loaded with black pigment granules, but there are no chitin-
ous rods or rhabdomes, such as are commonly found in
retinal cells. On the other hand they bear a close resem-
blance to the retinal cells of the visual organs of the siphons
of Mya arenaria, Solen vagina, and Dreissensia poly-
morpha, figured by Sharp (17). In the case of those eyes
situated on thickened portions of the mantle, the internal epi-
thelium is deeply invaginated, as is shown in fig. 20, and the
extremity of the invagination spreads out below the vitreous
body to form a two-layered optic cup which in section bears
some resemblance to the optic cup of a developing vertebrate
eye. The cavity of the optic cup is in free communication
with the pallial cavity by the “optic stalk,’’ if we may give
this name to the stalk of invagination. In most cases the
whole of the invaginated cells are pigmented, those of the
stalk as well as those of the cup, and both layers of the cup
are always pigmented, but it is only the cells of the anterior
layer of the cup, that is to say, those in contact with the
vitreous body, that are enlarged and columnar. The hinder
wall of the cup and the walls of the stalk are composed of
low cubical or flat epithelial cells and the transition between

282 GILBERT C. BOURNE.

these and the large columnar pigmented cells at the margin
of the cup is well shown in fig. 21. The preservation of my
specimens was not good enough to allow of my working out
such delicate details as the nerve supply of these organs. In
every case a nerve derived from the circumpallial nerve could
be detected in close proximity to an eye, and in fig. 20 two
such nerves are seen in section, one on each side of the optic
stalk. But I have been unable to trace nerve-fibres running
into those columnar pigmented cells which, because of their
characters and position I have called retinal. Further inves-
tigations are necessary before the exact nature and function
of these “eyes’’ can be determined with certainty. But
their structure points to their being photoscopic, or possibly
thermoscopic. I have already alluded to their somewhat
paradoxical position and suggested that they may serve to
warn the animal to keep the valves of the shell closed during
the day-time.

The Byssus and Byssus-gland.—In looking through
the literature of the subject one notes with surprise the
controversy about the nature of the byssus in Anomia
ephippium. ‘The calcareous plate or ‘“ ossicle” was a
stumbling-block to many of the older authors, who regarded
it as a third valve of the shell, and, though de Lacaze
Duthiers gave cogent reasons for regarding the ossicle as a
calcified byssus, and Moore (12) described the fixation of the
young forms and the modification of the left valve produced
by the asymmetrical attachment of the byssus, neither of
these authors gave any account of the microscopical structure
of the byssus gland, and as recently as 1878 von Jhering
emphatically denied any homology between the “ Falten-
organ” of Anomia and the byssus-cavity of other Lamelli-
branchia.

The question was finally decided by Barrois (1), who
showed that the macroscopic and microscopic structure of
the “Faltenorgan” is in all essential particulars identical
with the byssus cavity of Arca tetragona. But Barrois
contented himself with two very diagrammatic woodcuts of

ON THE STRUCTURE OF ANIGMA ZNIGMATICA. 283

the byssus cavity of the two genera, and I have been unable
to find any accurate drawing or description of the minute
structure of the byssus of the Anomiacea. The “ ossicle”
being absent in 4inigma, the byssus and byssus-gland of this
genus is a more suitable object for microscopical study than
the corresponding organ in Anomia, and its resemblance to
the byssus gland of Arca tetragona as described by
Boutan (4) is obvious. In the latter genus the foot is very
small, and has a groove on its hinder or posterior surface ;
the byssus cavity is very large, and the byssus is a stout oval
structure consisting of a number of lamelle which, as Boutan
says, overlie one another like the coats of an onion. The
byssogenous gland consists of some twenty to twenty-five
parallel epithelial folds or lamin, which traverse the interior
of the byssus cavity, and the chitinoid lamelle of the byssus
are formed from, and their inner ends are contained between,
these laminz. Following Boutan, I will describe the internal
divisions of the byssus itself as ‘‘lamelle” and the epithelial
folds of the byssogenous gland as “ lamine.”

The extent of the byssus-cavity in AJnigma and its
relation to the massive byssus-muscle are shown in the small
scale drawing fig. 10, and a transverse section through both
byssus and byssus-gland as seen under a higher magnification
in fig. 17. The left-hand part of the latter figure shows that
the lamelle of the byssogenous gland are folds of the epi-
thelium lining the byssus cavity, each fold having in its
centre a core of connective tissue. The figure, though drawn
under a magnification of 600, is on too small a scale to show
histological details clearly, but it may be seen that the edge
of each lamina is covered by large columnar cells filled with
granules. To the right of the figure the byssus itself is seen
in siti. It consists of a number of chitinoid lamelle lying
between the byssogenous epithelial lamine. ‘The dark line
down the centre of each lamella shows its double origin from
the walls of adjacent laminz. ‘The outer ends of the lamellz
are united to form a plate, and it is evident that this plate
increases in thickness by the addition of material secreted by

284. GILBERT C. BOURNE.

the large glandular cells on the edges of the lamine. This
plate is firmly attached to the substratum on which the
animal rests. There is no trace of calcification in it, and in
several of my specimens the bark of the root to which it was
fixed remains adherent, showing that there is no question of
the existence of an “ ossicle,’’ which has been torn off when
the animal was detached. A comparison of this drawing
with Boutan’s figures (loc. cit., pl. 14, figs. 18, 21, and 22)
leaves no doubt as to the identity, in all essential particulars,
of the byssus and byssus-gland of Ainigma with the
corresponding structures in Arca tetragona. The laminze
are much more numerous and the byssus cavity is relatively
wider and shallower in the former genus, that is all.

Fig 18 is a very highly-magnified drawing of the outer end
of a single byssogenous lamina lying between two lamellz of
the byssus of Ainigma. The sides of the lamine are clothed
by a clear, generally-cubical, ciliated epithelium. I have no
doubt that this is a ciliated epithelium, and that it corrre-
sponds with the ciliated epithelium lining the byssus cavity
of other Lamellibranchia, as, for instance, in Cyprina
islandica (Carriére, 5), Dreissensia polymorpha
(Horst, 7), and Jousseaumia (Bourne, 3). Boutan, how-
ever, is of a very different opinion. He says, of similar cells
in Arca, “Au-dessus de Vepithelium, en contact avec le
produit sécrété, on apergoit une striation trés nette qu’on
serait tenté de prendre, au premier abord, pour des cils
vibratiles ; en réalité, ce ne sont que des petits batonnets de
matiére sécrétée, absolument immobiles.” Immobile they
may possibly be, as I suspect that their function is to afford
sufficient surface friction to prevent the byssus lamelle from
slipping out of place, but that they are true cilia is shown by
their insertion on a striated border of each epithelial cell, by
their correspondence with the cilia undoubtedly borne by
similar cells in other Lamellibranchia, and by the fact that
they are present where the secretory activity is in abeyance,
but absent where it is still in progress, which is the reverse

ON THE STRUCTURE OF MNIGMA ZXNIGMATICA. 285

of what would be the case if they were “little rods of
secreted material.”

Between the epithelial walls of the lamina is a plexus of
connective-tissue cells, among which there are elongate pyri-
form or spindle-shaped masses of granules in which no nuclei
ean be distinguished. Deeper down in the lamine a few
glandular cells loaded with granules are scattered through
the connective-tissue core, but there is no compact mass of
byssogenous cells, such as is usually to be found in other
Lamellibranchia. The elongated strings and globules of
granules must be identified with the streams of granules
which I have described in Jousse aumia (8) as travelling by
intercellular paths from the byssus gland to the byssus cavity.
I am of the opinion that the byssogenous cells break up, and
that the secretion travels between the irregular spaces of the
connective tissue, and that there are not definite ducts as
described by Horst (7) in Dreissensia. Boutan figures
irregular branching ducts in Arca tetragona (4, pl. 13,
fig. 12), but he does not enter into histological details, and
his figure might equally well be interpreted according to
Horst’s views or my own. Perhaps the most remarkable
feature in Ainigma is the cap of granular columnar cells on
the edge of each byssogenous lamina. These cells are clearly
continuous with the ciliated epithelium of the sides, but they
are not ciliated, and are filled with byssogen granules. It
may be inferred that they have taken up these granules from
the intercellular channels of the connective tissue, and that
they secrete them again at their free surfaces, thus adding to
the thickness of the byssus plate. The concentric lines in
the latter (fig. 18) clearly indicate that there has been a con-
tinuous addition of fresh matter from the large granular cells
capping the edge of the lamina. There is no possibility of
confusing the byssogenous with the mucous cells in Ainigma;
the latter are, indeed, numerous in the foot and in the lips of
the byssus cavity, but they never penetrate into the lamina,
and are easily distinguished by their oval or polygonal shape
small nuclei and clear contents.

286 GILBERT C. BOURNE.

I will conclude with a few remarks on the histology of the
labial grooves and alimentary tract.

The right and left labial grooves pass without any distinct
line of demarcation into the mouth. The right groove as
stated on p. 260 is shallow and smooth for a large part of its
course posterior to the mouth; the left groove, on the con-
trary, soon becomes deep, and is thrown into numerous
vertical ridges. In both grooves the vertical ridges are
covered by a very high, ciliated, columnar epithelium, in
which no gland cells can be distinguished. But the smooth
portions of both grooves are lined by a characteristic epithe-
lium shown in fig. 23. The ciliated columnar cells are very
distinct, and havea doubly refractive border. Between them
are two kinds of gland cells, elongated granular, and ovoid
clear cells. The former are elongated and occupy the spaces
between the ciliated cells, their free ends reaching to the
surface. They are filled with fine yellow granules, and their
nuclei are to be found in the inner third of their length. As
these nuclei are identical with those of the ciliated cells it is
probable that the granular gland cells are modifications of
ciliated cells. The ovoid clear cells are very large, with clear
contents staining pink in picro-indigo carmine; their nuclei
stain uniformly dark red in borax carmine. From their
staining properties these ovoid cells appear to be mucous
cells, and they are similar in size and appearance to the
mucous cells of the foot, but, unlike the latter, are not
rendered polygonal by mutual pressure.

The epithelium of the labial grooves passes into the
cesophagus, but the finely-granular cells soon disappear, and
their place is taken by large coarsely-granular gland cells.
The mucous cells at the same time disappear. The cesophagus
is surrounded by a very distinct layer of subepithelial
muscular fibres. The epithelial lining of the cesophagus
passes gradually into that of the stomach. In this cavity the
elandular cells, as has been already described on p. 267, are
restricted to the side walls and floor; the roof is thin and
lined by moderately long ciliated cells only. It is noticeable

ON THE STRUCTURE OF HNIGMA @NIGMATICA. 287

that the thick, cuticle-like lining of the stomach, the “ fléche
tricuspide ” of Poli, corresponds exactly in extent to the area
in which the yellow glandular cells occur, and is not present
in the roof and upper portion of the left wall where these
cells are absent. A series of transverse sections shows that
the thick glandular tract of the stomachal epithelium is con-
tinued posteriorly into the cecum of the crystalline style,
while the ciliated non-glandular tract of the roof and left side
passes into the small intestine, and at the entrance into the
latter is thrown into a number of stout, ciliated ridges which
form a straining apparatus, and are continued into the four
prominent ridges projecting into the lumen of the intestine.
The transition from the glandular epithelium of the floor of
the stomach into the characteristic epithelium of the cecum
of the crystalline style is a gradual one; the ciliated cells of
the stomach gradually become shorter and stouter, the yellow
gland cells gradually become scarcer, until shortly after its
origin from the stomach the cecum is lined exclusively by
the epithelium shown in fig. 22. I have shown (8) that in
Jousseaumia there is a similar localised tract of glandular
cells in the stomach passing through a similar transitional
epithelium into the cecum of the crystalline style, and I have
suggested that the last-named structure is secreted by the
gland cells in question.

Our knowledge of the structure and functions of the
“fléche tricuspide” and the crystalline style is due to the
researches of Barrois (2), List (10) and Mitra (11). Thanks to
the last author, we know that the style consists of a proteid
material belonging to the globulin class, and that it contains
an active amylolytic ferment. He supposes, without giving
any very cogent reasons for his conclusions, that the sub-
stance of the style is secreted by the liver, and is stored up
as a flexible solid in the czecum, or in some forms, in a special
compartment of the stomach or intestine. Barrois has care-
fully investigated the structure of the “ fléche tricuspide” of
the stomach and the crystalline style in Donax trunculus,
and has entered much more fully into the histology of the

288 GILBERT C. BOURNE.

tissues in contact with these structures than has Mitra.1 He
describes the ‘‘ fléche tricuspide” of Donax as forming a
complete lining of the cavity of the stomach, and as being
more or less adherent to the anterior end of the crystalline
style. He does not recognise any gland cells in the epithe-
lium of the stomach, but suggests that the “ fléche tricuspide”
is formed from a granular mass detached from the ends of the
epithelial cells, and gives some not very satisfactory figures
in support of his statement. He further suggests that the
function of the “ fléche tricuspide”’ is to protect the walls of
the stomach from injury. In his description of the cecum of
the crystalline style of Donax, Barrois calls attention (and
as far as I can determine he is the only author who has done
so) to the existence of a groove lined by a modified epithe-
lium, running the whole length of the right side of the caecum
of the crystalline style, from its origin from the stomach to
its extremity. There is a similar groove along the right side
of the cecum of Ainigma, showing the same histological
characters as those described and figured by Barrois in
Donax. I have given a careful drawing of this groove and
the adjacent parts of the wall of the cecum in fig. 22. As
regards the characters of the epithelial cells, it corresponds
so exactly with Barrois’ description that I need give no
further account of it, except to call attention to the extremely
long cilia borne by the short cells lining the bottom of the
groove, and the short and fine cilia borne by the tract of
modified columnar cells on the upper side of the groove.
These are not described by Barrois, who says on the contrary,
“toute la surface épitheliale est tapissée de cette épaisse et
forte couche de cils vibratiles d’ont j’ai parlé & maintes re-

1 Mitra appears to have been very imperfectly acquainted with the researches
of his predecessors on this subject, and his quotations of literature are mostly
derived from text-books. Had he read Barrois’ memoir, he would have found
that as early as 1686 v. Heide suggested that the crystalline style was sub-
servient to digestion: ‘aliquendo cogitavi hunc stylum suppeditari alimini
fermentum.” Barrois gives a very interesting account of the various views
that have been held on the origin and function of the crystalline style.

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 289

prises, et sur d’excellentes préparations au carmin aluné, j’ai
pu la suivre aussi bien au niveau de l’épithelium modifié que
sur la reste de la section.” I think Barrois must have been
mistaken on this point, but however that may be, the modified
cilia are very conspicuous in Ainigma, ‘Their arrangement
leaves no doubt that the groove has a function analogous
to that of the endostyle of an Ascidian, and that its cilia
sweep a current of liquid or viscous matter (I could find no
solid particles in it) along the length of the czecum.

From a consideration of these histological details, I suggest
that the material of the crystalline style is secreted by the
yellow glandular cells of the epithelium of the stomach, and
that the so-called cuticular lining of the stomach, or “ fléche
tricuspide”’ of Poli, is nothing more than the coagulated
viscous secretion of these glands. This viscous secretion,
I suggest, is swept into the ceecum by the action of the cilia
of the ciliated groove, and is there moulded and solidified into
the substance of the crystalline style which, as Mitra has
proved,is a globulin containing an amylolytic ferment. During
the process of digestion, the style as a whole is moved forward
into the cavity of the stomach by the action of the stiff brush-
like cilia of the normal cecal epithelium, and is gradually dis-
solved, liberating the amylolytic ferment. It is true that
several authors, including Barrois, have asserted that the
anterior end of the crystalline style is continuous with the
so-called ‘‘fléche tricuspide,” but this confirms rather than
contradicts my suggestion. These same authors assert, and
I agree with them, that the substance forming this cuticular
coat or “fléche tricuspide”’ is identical with the crystalline
style. When one examines this substance carefully, one
finds that it forms a lining to the wall of the stomach, thin in
some places, thicker in others, and where it is thicker its
inner surface (that is the surface farthest from the epithelium)
passes insensibly into a mass of granular coagulum, in which
the anterior end of the style appears in some cases to be
imbedded. But there is no real continuity between the two.
The anterior end of the style is in all cases much reduced in

290 GILBERT C. BOURNE.

diameter, and is evidently undergoing dissolution. It can-
not at one and the same time be losing material and receiving
additions from the substance which has been called the
cuticle or the “ fléche tricuspide.”’ My interpretation is that
the crystalline style is added to at its hinder end, and that
the material for its renewal is carried down the czecum by
the ciliated groove. List (10) has given an interesting
account of the formation of the crystalline style in specimens
of Mytilus fed with Indian ink, which is not inconsistent
with my suggestion. He does not, however, appear to have
subjected the style to the same careful chemical analysis as
Mitra.

The numerous follicles of the liver are formed of two kinds
of cells. Large, coarsely-granular, clear cells with distinct
nuclei, and wedged in among the outer ends of these a smaller
number of deeply-staining, finely-granular cells resembling
the demilune cells of mixed salivary glands of mammals.

The intestine, in the first part of its course where it is of
narrow calibre, with four ridges projecting into its lumen, is
lined by an epithelium consisting of attenuated ciliated cells
with deeply-staining, densely-crowded nuclei, among which
are a few goblet cells, smaller, and with more finely-granular
coutents those occurring in the stomach. In the loop of the
intestine the ridges die out, the goblet cells disappear, and
the attenuated ciliated epithelium alone remains, In the
straight part of the intestine this is replaced by a columnar-
ciliated epithelium with a clear, somewhat granular cytoplasm
and pale oval nuclei containing a sparse chromatic reticulum
(fig. 24). There are no gland cells in this region, but the
cytoplasm of the epithelial cell stains bluish-green in picro-
indigo-carmine, and is very distinct from the section of the
intestine preceding it and from the proctodeum. It is usual
to call this part of the intestine the “rectum.” But it is so
sharply marked off from the terminal portion of the alimentary
tract that I prefer to describe it as the large intestine, and
to restrict the name rectum to the short, somewhat enlarged
section of the gut which opens to the exterior by the anus

ON THE STRUCTURE OF HNIGMA ZNIGMATICA. 291

This rectum, which is clearly proctodzal in origin, is lined by
an epithelium composed of clear and very attenuated ciliated
cells with deeply-staining nuclei.

The whole of the large intestine, and a section of the small
intestine preceding it, is infested by sporozoan parasites,
whose characters are shown in fig. 24. They are not suffi-
ciently well preserved to admit of careful description, but
they are evidently the trophozoites of a Coccidian, some of
which are forming cysts containing sporoblasts. They have
some likeness to the genus Klossia, but as I have not been
able to discover the spores, or to trace the various stages of
the life-history of this parasite, it will be better to record its
existence without conferring upon it a new and probably a
misleading name.

Summary oF Resutts.

J. Ainigma, though modified in the same direction as
Anomia, has undergone a less degree of torsion, and has
retained more of the typical features of a normal Lamelli-
branch.

2. There is, on the left side, a specialised pallial muscle,
attached to the left valve, and acting as a retractor of the
left gill.

3. A ring of eye-spots, of peculiar structure, is found on
the left mantle lobe, at a considerable distance from the edge
of the mantle.

4, Adaptations for resisting desiccation during long ex-
posure to the sun and air are found in the thickening and
corrugation of the lower moieties of the mantle lobes and in
the existence of cecal extensions of the pallial cavity, which
can be closed by the apposition of the ciliated edges of ridges
developed on the mantle and body-wall.

5. The structure of the byssus gland is of the same type as
that of Arca tetragonaand Anomiaephippium. There
is no calcified ossicle.

6. The inner demibranch of the right gill is attached to

292 GILBERT C. BOURNE.

what is morphologically the left side of the foot. The minute
structure of the gills is curiously similar to that of Anomia
ephippium. The velar filaments of the external demibranchs
bear special cilia.

7. The intestine is coiled.

8. The kidneys and the openings of the reno-pericardial
ducts and gonaducts into the kidneys are similar to those of
Anomiaephippium. The gonopores have ciliated funnels.

9. There are extensive remnants of the pericardial gland.
The wall of the pericardial cavity is shown to be incorporated
with the walls of the ventricle and auricles.

10. An internal ciliated groove runs along the whole length
of the ceecum of the crystalline style.

11. The left gonad extends far back into the left lobe of
the mantle.

List OF THE PRINCIPAL AUTHORS REFERRED TO.

1. Barrots, To.—Sur la structure de ’Anomia ephippium,” ‘Bull.
Sci. Dép. Nord’ (2), ii, 1879.

2. Barrots, Tu.—“ Le stylet crystallin des Lamellibranches,” ‘ Rev. biol.
Nord France,’ i, 1890.

3. Bourne, G. C.—*On Jousseaumia, a new genus of Eulamellibranchs,”
‘Report on the Pearl Oyster Fisheries and Marine Biology of the Gulf
of Manaar,’ part v, Roy. Soc., 1906.

4, Boutan, L.—‘ Recherches sur le Byssus des Lamellibranches,” ‘ Arch.
Zool. expér. et gén.’ (3), ili, 1895.

5. CarriErg, J.—* Die Driisen im Fusse der Lamellibranchiaten,” ‘ Arb.
Zoo). Inst. Wurzburg,’ v, 1879.

6. GropBeN, C.—‘ Die Pericardialdriise der Lamellibranchiaten,” ‘ Arb.
zool. Inst. d. Univ. Wien.,’ vii, 1888.

7. Horst, R.—‘‘Ist die Byssus eine Cuticularbildung ?”’ ‘Tijdschr. Ned.
Dierk. Vereen,’ (2), ii, 1889.

8. Lacaze Duruiers, H. pe.— Mémoire sur l’organisation de l’Anomie
(Anomia ephippium),” ‘Ann. des Sci. Nat.,’ 4e ser., ii, 1854.

9. Lereriier.— Etude sur le fonction urinaire chez les Mollusques acé-
phales Lamellibranches,” ‘Arch. de Zool. expér. et gén. (2), v bis,
1887.

10. List, Tu.—“ Die Mytiliden,” ‘ Fauna u. Flora des Golfes von Neapel,’
xxvii, 1902.

ON THE STRUCTURE OF ANIGMA ANIGMATICA. 293

11. Mirra, S. B.—“ The Crystalline Style of Lamellibranchia,” ‘ Quart.
Journ. Micr. Sci.,’ N. 8., xliv, 1901.

12. Morse.—“ Remarks on the Relations of Anomia,” ‘ Proc. Boston Soe.
Nat. Hist.,’ xiv, 1871.

18. PELSENEER, P.—‘‘ Contribution a l’étude des Lamellibranches,” ‘ Arch.
de Biol.,’ xi, 1891.

14, PELSENEER, P.—‘ Mollusca,” Lankester’s ‘Treatise on Zoology,’ part v,
1906.

15. Rriprewoop, W. G.—‘On the Structure of the Gills of the Lamelli-
branchia,” ‘ Phil. Trans. B.,’ exev, 1903.

16. Sasst, M.—‘“‘Zur Anatomie von Anomia ephippium,” ‘Arb. zool.
Inst. d. Univ. Wien.,’ xv, 1903, p. 81.

17. SHarp, B.—‘‘On the Visual Organs in Lamellibranchiata,” ‘ Mitth. Zool.
Stat. Neapel,’ v, 1884.

18. Woopwarp, S. P.—‘ Ann. Mag. Nat. Hist.,” xvi, 1855.

EXPLANATION OF PLATES 15—17,

Illustrating Prof. G. C. Bourne’s paper on ‘The Structure
of Ainigma enigmatica, Chemnitz: a Contribution
to our Knowledge of the Anomiacea.”

LETTERING IN ALL THE FIGURES.

ad. Adductor muscle. az. Anus. ap. Aperture between the right and
left renal sacs. a.7.p. Anterior retractor pedis muscle. é7.g. Branchial
ganglia. éy.c. Byssus cavity. dy./. Lamella of byssus. dy./m. Laminz of
byssogenous gland. Jdy.pl. Byssus plate. c.cm. Cerebral commissure. c.d.
Ciliated discs. c.f. Latero-frontal cilia of gill filaments. ¢.fr. Frontal cilia.
c.s Chitinous skeleton of gill filaments. ¢.v. Ventral cilia on velar filaments.
cen. Cornea. cry. Cecum of crystalline style. dd. Right external demi-
branch. dé*. Right internal demibranch. 4%. Left internal demibranch.
dv‘, Left external demibranch. ep.ex¢. External epithelium. ¢.s. Pallial eye
spots. /. Foot. gl.c. Gland cells. go.a. Anterior lobe of left gonad. go./.
Posterior lobe of left gonad. go.r. Right gonad. iz¢. Small intestine. zé'.
Large intestine. 4.cz. Concretions in cavity of kidney. J/.cp. Left cerebro-
pleural ganglion. J.ep. Left excretory pore. Uf.r. Right labial folds or
palps. d.gd. Left gonoducal opening into kidney. /a./. Left labial groove.
Ig.r. Right labial groove. /i. Liver. J/i.d. Hepatic ducts. /.k. Left kidney.
ix. Lens of eye. J.p.g. Left pericardial gland. /.rp, Left reno-pericardial

294, GILBERT C. BOURNE.

aperture. m. Mouth. mf. Muscle fibres. mt. Left mantle lobe. — mé!. Right
mantle lobe. @. Gisophagus. p.c.c. Pericardial cells with endoplastic con-
cretions. pd.g. Pedal ganglia. pl.m. Branchio-pallial muscle. pr.p. Poste-
rior retractor pedis muscle. 7. Rectum. r.cp. Right cerebro-pleural ganglion.
rep. Right excretory pore. 7k. Right kidney. spy. Right pericardial
gland. rp. Right reno-pericardial aperture. rt. Retina. sé. Stomach.
v. Ventricle of heart. v.cm.d. Left visceral connective. v.cmr. Right
visceral connective. vel. Velar fold of gill filaments. v.g. Visceral ganglia.
vi. Vitreous body. v.m. Visceral mass. «. The dorsal pallial suture. 2.
Cavities between various parts of the body, ending blindly and serving for
storage of water. 1. Reflected lamella of left external demibranch. 11. Direct
lamella of left external demibranch. 111. Direct lamella of left internal
demibranch. iv. Reflected lamella of left internal demibranch. v. Union
between the reflected lamella of the right and left internal demibranchs.
vi. Reflected lamella of right internal demibranch. vir. Direct lamella of
right internal demibrancb. itt. Direct lamella of right external demibranch.
1x. Reflected lamella of right external demibranch.

PLATE 15.

Fic. 1.—View of the animal lying in the left valve of the shell, after
removal of the right mantle lobe. x about 23.

Fic. 2.—The animal removed from its shell and viewed from thie left side,
showing the ring of pallial eye-spots. ‘The principal organs of the left side
are seen through the transparent left mantle lobe.

Fic. 3.—A reconstruction from a series of sections to show the course of
the alimentary canal and the nervous system.

Fie. 4.—A reconstruction from a series of sections to show the renal
organs and the extent and relations of the pericardial glands. The right
kidney is represented in a darker, the left kidney in a lighter tone. The
spaces enclosed by the black lines represent the pericardial glands.

Vic. 5.—<A horizontal section through the angle of the direct and reflected
lamelle of one of the demibranchs, showing the single row of ciliated dises,
ed. Highly magnified.

Fic. 6.—A transverse section through three velar filaments, showing the
stiff cilia, ¢.v., on the ventral faces of the filaments. Zeiss’ ;4; hom. imm.
comp. oc. 4.

Vic. 7.—A transverse section through two adjacent gill filaments. Zeiss’
jz hom. imm., comp. oe. 4.

Fig. 8.—A transverse section through a pallial fold guarding the entrance
to one of the water reservoirs, showing the ciliated ridge, ¢.7., on the edge of
the fold. x. A branch of the pallial nerve.

ON THE STRUCTURE OF MNIGMA ANIGMATICA. 295

PLATE 16.

Fre. 9.—A transverse section through the body at the level of the foot
Str. The ‘fléche tricuspide ” of Poli.

I'ic. 10.—A transverse section passing through the posterior half of the
byssus muscle.

Fre, 11.—A transverse section passing through the ventricle of the heart.

Vic. 12.—A transverse section passing through the middle of the adductor
muscle.

Fic. 138.—A transverse section passing about 2 millim. in front of the anus.

Fie. 14.—A section through the right reno-pericardial canal and the
adjacent portion of the pericardial gland. Highly magnified.

Fie 15.—A section through the ciliated funnel of the opening of the left
gonaduct of a male into the kidney. The columnar ciliated cells of the funnel
are conspicuous. go. A follicle of the testis. sp.d. Sperm duct.

Fie. 16.—a. A group of pericardial cells containing small concretions.
6. A group of larger concretions from the pericardial gland of another
specimen of Ainigma.

PLATE 17.

Fic. 17.—A transverse section through the byssus and byssogenous lamine.
x 600.

Fic, 18.—A transverse section through the distal end of a byssogenous
lamina and two adjacent lamelle of the byssus. cé/. Ciliated epithelial cells
at the sides of the lamina. gv. A mass of byssogen granules in the connective
tissue core of the lamine. Zeiss’ =; hom. imm., comp. oc. 4.

Fic. 19.—Portion of a section through the wall of the ventricle of the
heart, showing ep. ewt., the external epithelium; m.f, the branchial cardiac
muscle fibres; pe.c., the characteristic pericardial gland cells with endoplastic
concretions.

Fic. 20.—A section through a pallial eye, showing the deep invagination of
the epithelium lining, the inner face of the mantle, and the modification of the
deeper invaginated cells to form a retinal layer.

Fic. 21.—A portion of another section through a pallial eye. Zeiss’ 35
hom. imm., comp. oc. 4.

Fic. 22.—A portion of a section through the caecum of the crystalline style,
showing the ciliated groove, g¢., and the tract of modified epithelium on the
dorsal side of it. The section is reversed in the drawing, so that the veutral
side is uppermost.

Fie. 23.—A section through the right labial groove, close to the mouth.

Fie. 24.—A section through the epithelium of the large intestine. spor.
Sporozoan parasites.

VoL, 51, PART 2,—NEW SERIES. 22

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM.

2

9

4
(

On the Chromatin Masses of Piroplasma
bigeminum (Babesia bovis), the Parasite

of Texas Cattle-Fever.

By

H. B. Fantham, B.Sc.Lond., A.R.C.S8.,

University College, London, and St. Mary’s Hospital Medical School.

With Plate 18, and 44 Text-figures.

ContTENTS.

]. Introduction
II. Methods

IIT. Previous observations on ‘ihe Chromatin of Pir oplas ma

bigeminum and P.canis .

1V. The Chromatin Masses of P. hime nnn um in (a) double
pyriform, (8) double ovoid, (y) single pyriform, (6)
rounded, («) groups of more than two, (Z) dividing, and
(m) other intra-corpuscular forms; also in (@) free

parasites
V. Note on the Cytoplasm oe Pe Bieennnin

VI. The Probable Significance of Nuclear Dimorphism in

Piroplasma .
VIL. Summary of Results
VIII. References to Literature
IX. Explanation of Plate

I. Inrropuction.

PAGE
297

299

300

302
317

318
320
323
324

Some time ago, while looking at a preparation of Piro-
plasma bigeminum lent to me for examination, I was at
once somewhat surprised to find that many of these very
small parasites distinctly exhibited what has been called
“nuclear dimorphism,” for quite typical and non-dividing

298 H. B. FANTHAM.

“nucleus ” or

forms showed the presence of more than one
chromatin mass. ‘This feature of “nuclear dimorphism” is
of great interest at present in elucidating the probable close
affinities of the Heemosporidia and Hemoflagellates.

As is well known, the micro-organism Piroplasma
bigeminum (Babesia bovis) is the pathogenic agent of
Texas Fever (Red-water) in cattle. The preparation exa-
mined and now described was a blood-smear, made imme-
diately after the death of the Bovine, labelled “ scraping
from heart-muscle,” and came from Australia. There have
been serious ravages among cattle in Queensland, due to
Red-water. A few other preparations of the parasite were
also examined.

In this particular blood-film from heart-muscle about 90
per cent. of the red corpuscles were infected, one corpuscle
usually containing two pyriform parasites, especially charac-
teristic of this, the type-species of Piroplasma, and which
led to the specific name bigeminum (Smith and Kilborne).

All members of the genus Piroplasma are small, usually
occurring in pairs inside various mammalian erythrocytes, so
that the diameter of the host-cells only averages 7 u, and the
parasite is considerably smaller, about 34 by l'5yu. The
minute character of the parasite has been a source of great
difficulty in the present research. Great care has been
necessary with the illumination, and high magnifications have
been used.

For the photo-micrographs, from which the figures in Plate
18 were drawn, I am greatly indebted to Dr. N. H. Alcock,
Lecturer on Physiology at St. Mary’s Hospital Medical
School, assisted by Mr. G. R. Lynch. ‘To these gentlemen
I would tender my best thanks for the trouble they have
taken in photographing this exceedingly difficult object.
Since it was found that the photo-micrographs, which clearly
exhibited the “nuclear dimorphism” in question, were too
delicate to sustain clear reproduction in the number of copies
required for circulation, the photographs have been drawn
(twice the size of the original) by the lithographic artist.

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 299

II. Meraops.

he blood-smear examined had been fixed and stained by
the Romanowsky method. The resulting film was a thin,
even, and good one, and was covered and mounted in Canada-
balsam. ‘he stain of the host-cells (red blood-corpuscles)
was somewhat faint, which was afterwards found to be a
distinct advantage, but the parasites were still perfectly well
stained, and exhibited the red or purplish-red colour in the
chromatin and blue in the cytoplasm characteristic of the
Romanowsky coloration.

In order to eliminate, as far as possible, sources of error
incidental to stained preparations—errors more especially
applicable, however, to the shape and structure of the cyto-
plasm of the parasite—the slide was examined under various
kinds of illumination. hese, in brief, were—first, critical
illumination, using as a source of light the sharp edge of a
parafiin flame ; second, monochromatic light (green or
yellowish-green was best) ; and thirdly, less critical illumina-
tion from a Welsbach burner or an electric lamp. The last
mentioned should, on the whole, be rejected as somewhat
untrustworthy and uncomfortable in a research of this kind,
where so much depends on the correct determination of the
colour of the chromatin masses. The monochromatic light is
comfortable and useful for structural detail, but a source of
white light, such as is obtained from a paraffin flame, is
necessary for the determination of colour, especially of the
looser chromatin. In all cases, however, the chromatin could
be distinguished, either absolutely or relatively, from the
cytoplasm of the parasite, but with very bright (white)
sources of light, the relative sizes of the chromatin masses to
each other were sometimes misleading, and there was, in
such cases, a lack of finer detail. Wrong impressions were
also apt to be obtained with the very powerful sources of
light, concerning the size and condition of the vacuoles
mentioned hereafter. Daylight was also used when possible
(winter).

300 H. B. FANTHAM.

The objectives employed were Zeiss’ 2 mm. and 3 mm.
apochromatics, aperture 1:40, and compensating oculars 8, 12,
and 18, the various combinations of these all giving, in the
main, precisely similar results, with little or no increase in
detail, for a maximum amount of detail is obtained with
compensating ocular 8 and 3 mm. apochromatic homogeneous
immersion objective.

Lastly, it seems quite certain that relatively pale-stained
preparations are much to be preferred to more deeply stained
ones. Nuttall and Graham-Smith (8, p. 588) lay stress on
the necessity of differentiation with methylated spirit after
coloration with comparatively dilute solutions of Giemsa’s
stain. It seems to me probable that, in the past, the frequent
deep-blue staining, together with the more or less blue
coloration of the enclosing blood-corpuscle, usually obtained
by the various modifications of the Romanowsky method,
using strong solutions, after one quarter to half an hour’s
staining, has perhaps obscured the finer chromatic details
and masked the looser chromatin in such small endoglobular
forms as Piroplasmata, and has also hidden the fiuer structural
details of the cytoplasm, vacuolated or otherwise.

III. Previous OBseRVATIONS ON THE CHROMATIN OF PrRO-
PLASMA BIGEMINUM AND P. CANIS.

The parasite of Texas fever was discovered by Smith and
Kilborne, and carefully described by them in 1893 (10),
though they saw the parasite in 1889 and apparently ‘noticed
[it] in the spleen of a case as early as 1886” (10, p. 213).
Their classical monograph is perhaps, nowadays, hardly con-
sulted as muchas it still deserves to be, though the memoir is
not always easy of access, unless one procures a copy direct
from Washington. The chief stain used by the American
investigators was the alkaline methylene blue of Léffler, and
the presence of a nucleus in the parasite is not specifically
mentioned by them. However, there can be little doubt that
the nucleus, as described later by Laveran and Nicolle (8),

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 301

was really seen by Smith and Kilborne, for on page 215 of
their monograph they remark, regarding fresh specimens,
that: “The smaller forms [of the parasite] are as a rule
homogeneous ; the larger forms are very frequently observed
to be provided, in the rounded end of the pyriform body,
with a very minute spherical body probably not more than
O-l to 0°24 in diameter, which contrasts dark with the body
itself .... In the largest pyriform bodies there was seen
in the centre of the enlarged end a somewhat larger round
or oval body which seemed to take the place of the smaller
body or else be associated with it. This second body was
from 0°5 to 1 win diameter. It changed its appearance with
the focus . . . . One or both of these bodies were observed
in some of those forms undergoing amoeboid changes.”
Judging by the latter part of this quotation Smith and
Kilborne even may have seen “nuclear dimorphism” in
parasites occurring “in fresh blood of the acute disease
during life.”

Laveran and Nicolle, apparently, were the first definitely to
describe and figure! the nucleus of P. bigeminum in
1899 (8), from stained specimens treated with Borrel blue
and eosin, according to Laveran’s modification of the
Romanowsky method, after fixation by heat and corrosive
sublimate. In twin intra-corpuscular forms they found a
spherical or oval karyosome at the blunt end of the parasite,
measuring 0°74 to 0°9 u in diameter, with a clear zone sur-
rounding the karyosome, which clear area they regarded as
the peripheral part of the nucleus. Laveran and Nicolle also
mention the frequent presence of numerous, easily stained
granules at the pointed extremity of the parasite. ‘These
granules, they state, might easily be mistaken for a second
karyosome in deeply stained specimens, but, on decolourising
a little, are seen to be really only “agglomerations of
granules.” Personally, I consider that at the pointed end of
the parasite loose chromatin is not infrequently seen (see
p. 303).

' These figures are reproduced in Minchin’s ‘ Sporozoa’ (7), p. 269, fig. 80.

302 H. B. FANTHAM.

It should also be noted that Ziemann (12), in 1898,
very briefly mentioned the occurrence of chromatin in P.
bigeminum without figuring it.

Schaudinn (1904), in a brief note at the end of his remark-
able memoir on 'l'rypanosoma noctue and Spirochaeta
ziemanni (9, p. 438), points out the presence of nuclear
dimorphism in P. canis and P. bigeminum. Liihe (4, 5),
in 1906, confirmed the observation as regards P. canis, and
further considered the question. Schaudinn and Liihe
described a large mass of chromatin, the “ principal” nucleus,
and a smaller, dense, punctiform mass of chromatin, which
Schaudinn suggested was homologous with the blepharoplast
of a Trypanosome, to which Liihe agrees.

Lastly, Nuttall and Graham-Smith (8), in October, 1906,
also saw these two chromatin masses in P. canis, and, in
addition, a third mass of loose chromatin, described by
them for the first time.

My own observations on the chromatin of P. bigeminum,
as seen more especially in blood from the heart-muscle, may
now be set forth.

IV. ‘Tae CHoromatin Masszs or P. BIGEMINUM.

The observations to be described were always made on
those parts of the preparation which appeared well fixed
aud properly stained, especially in so far as the parasite was
concerned.

It may be stated at the outset that to give a general
account of the distribution of the chromatin, which might
be taken as the average or type, is difficult, as considerable
variation was noticed. This variation has been already notified
by Nuttall and Graham-Smith (8) in the case of the chromatin
of P. canis, a species larger than P. bigeminum. It would
seem best, then, to illustrate, by accurate diagrams, as many
different forms as possible, at the same time noting their
relative frequency of occurrence.

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 305

(a) The Chromatin in Pairs of Pyriform Intra-
corpuscular Parasites.

These were the most numerous forms observed, and
often in each parasite two distinct masses of chromatin
were seen, situated on the sides of the body of the micro-
organism at about the middle of its length (text-figs. 1,
2,3). For convenience, this position may be termed “the
equatorio-lateral”—a somewhat cumbrous term, perhaps,
and not very correct geometrically—but the position of the
chromatin masses in ovoid forms has also to be considered,
and in such cases the term will be rather useful. Of these
lateral chromatin masses one is nearly always larger than
the other (text-figs. 1, 2, 3, etc.), though it cannot always be
stated that the smaller chromatin mass is merely “ puncti-
form,” as it often appears relatively larger than the idea
of size conveyed to my mind by such a term, and may be
surrounded by a somewhat deeply-staining area of cytoplasm.
Sometimes the lateral chromatin masses are very nearly
equal in size (text-fig. 13), Besides these lateral chromatin
masses there is not infrequently a third rather loose mass
of chromatin, more mesh-like or woolly in character, often
situated at the pointed end or apex of the parasite in the
midst of rather deeply-staining granular cytoplasm (text-fig.
2). Laveran and Nicolle (8) mentioned the occurrence of
numerous granules, easily stained, at the pointed end of the
parasite, as we have already briefly noted. They did not con-
sider that these granules formed a “ second karyosome ”—that
is, that they were composed of chromatin, as at first sight
appeared, because after slightly decolourising the preparation
—which, they state, appeared too deeply stained at first—it
could be seen that this apical mass consisted only of granules
(“une agglomération de granulations”). It seems to me
likely that, in decolourising the preparation a little, the
characteristic colour-reaction of the chromatin became very
faint or was lost, for among these granules of cytoplasm,
situated at or near the pointed end of the parasite, I feel

304. H. B. FANTHAM.

sure that there is also chromatin present, sometimes perhaps
only loosely packed—at any rate in many cases (PI. 18, figs. 1,
2, and 4 show position of this). Further, Koch depicts
chromatin as occurring at the apex of P. bigeminum from
Africa (1, taf. 1, figs. 1 to 4), and Nuttall and Graham-Smith
figure masses of apical chromatin in P. canis (8, Diagram 1,
figs. 3, 4, 14, 20; Diagram 3, figs. 4, 6, 8, 9, 18, 16, 19).

Ina Trypanosoma, carefully stained by the Romanowsky
method, as by Giemsa’s solution, the nucleus appears bright
red and the blepharoplast violet-red in colour. In Piro-
plasma, if the two lateral chromatin masses, more or less
unequal in size, be strictly homologous with the nucleus and
blepharoplast | of a Trypanosome, one would expect similar
differences in staining reaction. In the specimens which have
come under my observation it is difficult occasionally to dis-
tinguish in depth and colour of stainmg between the larger
and smaller chromatin masses of this minute parasite, as
sometimes the one, sometimes the other, may appear slightly
more deeply stained, and a difference in tint, as regards
violet, is exceedingly difficult to determine in this case.
However some specimens certainly showed the blepharoplast
more violet in tintafter staining. Furthermore, Piroplasma
is generally intra-corpuscular, and relatively not often free in
mammalian blood, and is much smaller than any 'Trypanosome,
while the exact method of staining and mounting the prepara-
tion, together with its age, are of the utmost importance in
determining the finer staining reactions of chromatin.

As regards the structure of the chromatin masses, it is very
difficult to make out any details in such small objects. The
principal nucleus and blepharoplast appear compact, and
separate granules of chromatin cannot be made out in them
with any degree of certainty, though, as a rule, the nucleus

2 The term ‘ blepharoplast,” as relating to Piroplasma, is used in this
memoir for convenience, and without prejudice to its literal meaning as a
uuclear body related to a flagellum. In the present state of our knowledge it
is impossible to state definitely whether a flagellate stage occurs in the life-
history of Piroplasina or not (vide Sect. V1).

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 309

is rather less dense than the blepharoplast. The loose
chromatin varies somewhat in texture in different forms.
It often consists of relatively coarse, chromatic granules,
closely associated and touching each other, yet loosely
packed compared with the principal chromatin masses, and
staining less deeply than these; and this structure may be
well seen under monochromatic light, after one has carefully
determined the loose chromatin area under white light.

The foregoing description applies to the distribution and
structure of the chromatin masses as seen in the most
commonly-occurring parasites (about one-fifth of the whole).

Another fairly common form of distribution of the chro-
matin in pyriform parasites may be termed the “ polar”
distribution (text-figs. 5, 6, 7, and 8). In these forms there
is a large nucleus near the blunt end of the parasite, and a
at some
distance from it, sometimes near the apical end (text-figs.
5, 6, 7). ‘The position of the nucleus in these parasites
corresponds with that often noted in the past before the

small mass of chromatin—the so-called blepharoplast

blepharoplast was observed. Occasionally, however, the
relative position of these two nuclear masses may be reversed
(text-figs. 11 part,! 14 part), and the larger chromatin mass
then appears near the pointed end.

The nucleus and so-called blepharoplast occasionally occur
close together (text-figs. 10, 9 part, 18 part).

Forms in which the chromatin masses appear much alike,
both as regard size and density, are sometimes found (text-
figs. 13, 15).

One of the parasites represented in text-fig. 18 has much
chromatin of varying density, along one side (cf. Koch [1,
tia. 1, figs. 1,2, 4:]).

Other types, such as are drawn in text-figs. 9, 12, and 16,
are rare, especially the latter, where the parasites possessed
only a paucity of chromatin.

Smaller, and presumably younger, intra-corpuscular pairs

‘Part’ here refers to the fact that only one member of the pair of
parasites shows the particular feature.

306 H. B. FANTHAM.

of parasites usually exhibited two nuclei already differentiated ;
it was somewhat rare to find only one “
forms, and then the single chromatin mass was polar, as
figured by Laveran and Nicolle (8).

Having noted the principal types of chromatin distribution

nucleus” in small

Text-FIGs. 1—9.

oe
gee

In these and following text-figures the dense chromatin masses of a
parasite are represented black; the loose chromatin is shaded. ‘The
cytoplasm is diagrammatically indicated by small dots, more closely
aggregated and deeply marked where the cytoplasm was more deeply
stained. Very faintly-stained areas and vacuoles are left clear.
Intra-corpuscular parasites are figured inside more or less circular
areas, representing the outlines of infected red blood-corpuscles.

%

in intra-corpuscular pyriform pairs, a few words are necessary
regarding the inter-relation of the three chromatin masses
already mentioned. ‘There is sometimes a bridge of some-

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 307

what loose chromatin connecting the nucleus and blepharo-
plast (Pl. 18, fig. 1; and text-fig. 2 part).

In many cases a marked cytoplasmic band stretches
across the parasite equatorially between these two “ nuclei”?

(text-fig. 1 part).

‘Trext-ries. 1O—1S8.

The loose chromatin sometimes surrounds one or other of
these denser chromatin masses (text-fig. 3 part, 12 part,
18 part, see also text-fig. 54), or forms a rod in relation with
one of these masses (text-fig. 15 part).

Attention may be drawn to the small forms shown in
text-fig. 17, where the blepharoplast occurs at the apical end,
and the principal chromatin mass stretches across the parasite

near the blunt “polar” end. Loose chromatin cannot be

308 H. B. FANTHAM.

definitely distinguished as a separate mass in these small
and young forms.

Usually the shape of the principal chromatin mass is ovoid,
especially when occurring laterally. At other times, but less
commonly, it is round, as in the case of “ polar” distribution.
Occasionally it is seen to be irregular (text-fig. 10 part).
The so-called blepharoplast is usually round, occasionally
ovoid. Rarely, the principal chromatin mass is in the form
of a curved rod (text-figs. 8 part, 16).

These different forms and appearances, more especially the
varying distribution of the chromatin masses, whether
‘‘equatorio-lateral ” or “ polar,” may be explained, at any
rate in part, by regarding the parasite as viewed from
different aspects, as Christophers has pointed out in connection
with the Leishman-Donovan body. It may also be that, as
the parasite grows older, the blepharoplast becomes further
removed from the nucleus. The intra-corpuscular pyriform
parasites are almost certainly flattened to a greater or less
extent, as Nuttall and Graham-Smith (8, p. 639) have shown
in the case of the larger form, P. canis, from observations on
living blood.

These observers also mention the occurrence of an achro-
matic halo around the nucleus or blepharoplast in some cases
(8, p. 592). I have very rarely seen this in P. bigeminum,
and then somewhat imperfectly (cf. text-fig. 9, part).

On the whole, in the case of pairs of parasites, each member
of the pair is usually very like its fellow in size, shape, and
distribution of chromatin. Variations, however, occur (e. ¢.,
text-figs. 9, 15), which are comparatively few in proportion to
the large number of parasites examined. Reference has
already been made, incidentally, to these variations, whenever
it has been necessary to use the word “ part” in referring to
a figure, thereby indicating variation among the individuals
of a pair.

Minute isolated masses or dots of chromatin, other than the
three masses already mentioned, were rarely, if ever, seen
with certainty in these minute parasites. Further, no really

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 309

definite cases of the entire absence of chromatin from a
parasite were encountered.

Regarding the relative positions of the nucleus and ble-
pharoplast, it may be noted that although the latter is fairly
often seen in close proximity to the former, and even con-
nected with it by loose chromatin, yet I have never seen the
blepharoplast actually on the nucleus, or directly budded off
from it, as stated by Lithe in P. canis. Also, the relative sizes
of these two chief chromatin masses can only be determined by
careful differentiation and sharp focussing from the granular
cytoplasm, sometimes surrounding one or both of them.

(8) The Chromatin in Pairs of Ovoid Intra-
corpuscular Parasites.

Pairs of strictly ovoid parasites, in which each
member of the pair is truly ovoid, are rare. Under this
heading it will be convenient to consider cases of pairs of
parasites where one member is strictly ovoid in shape while
the other is more or less so, yet still shows something of a
pyriform contour and may be rather fusiform (text-fig. 24),
for the gradation in shape between the two forms is pro-
gressive. Such pairs of parasites merit separate consideration
as a group after the large number of more strictly pyriform
types already discussed.

In these cases we have the same broad distribution of
chromatin as before. The “ equatorio-lateral”” arrangement
of the nucleus and so-called blepharoplast preponderates.
This distribution of the chromatin, together with the ovoid
contour of the micro-organism, recalls, superficially, the
appearance of the Leishman-Donovan body.

A few interesting forms showing the connecting bridge of
chromatin between the nucleus and blepharoplast were seen
(text-fig. 20 part). Three masses of chromatin in each para-
site of a pair are shown in text-fig. 19, which masses appeared
stained about the same colour and intensity, while the mass
which might probably be termed the blepharoplast was in

310 H. B. FANTHAM.

each case triangular in surface view, with the apex tapering
towards the centre of the parasite and surrounded by loose
chromatin in one case. The principal nucleus of the more
pyvriform parasite (text-fig. 19) was rather dumb-bell-shaped,
suggesting division, but the general facies of this parasite did
not lend support to this view. In text-fig. 24, where each
parasite is seen to be somewhat pointed at the ends, there
were well-marked masses of loose chromatin at one end. In

Trext-Fies. 19 —24.

24

text-fig. 25 two pale-staining parasites are drawn, with peri-
pheral, flattened chromatin. One of them showed a pointed
process of cytoplasm, rarely seen in stained preparations
apart from spleen-blood. In text-fig. 22 a rather unusual
difference in size between the individuals is shown, the larger
one being bean-shaped. Both exhibited nuclear dimorphism.

(y) The Chromatin of Single Pyriform Intra-
Corpuscular Parasites.

These forms were not numerous, and no new disposition of
the chromatin masses was found, '‘l'ext-fig. 25 represents a

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 311

typical parasite, possessing nucleus and blepharoplast, with,
in this case, a rather well-marked bridge of looser chromatin
joining them (cf. preceding text-figs. 2,20). The presence of
such a connecting bridge or strand of chromatin in forms of

Trxt-FIGs. 25 AND 26.

P. canis is mentioned and figured by Nuttall and Graham-
Smith (8, p. 594, fig. 12).

In text-fig. 26 a large pyriform parasite is represented with
less well-marked masses of chromatin, the larger of which is
irregular in shape.

The loose mass of chromatin was not always to be found in
these single intra-corpuscular forms.

(0) The Chromatin of Rounded Intra-Corpuscular
Parasites.

These are not very common, and the parasites may occur
either singly or in pairs—usually only one in a corpuscle.

TExt-FIGS. 27 AND 28.

They generally exhibit a clear, very faintly stained, or quite
unstained, area in the centre, and the chromatin is, in some
VoL. 51, PART 2.—NEW SERIES. 23

312 H. B. FANTHAM.

cases, not easily identified. However, chromatin is always
present, and is situated strictly peripherally, the chromatin
masses being flattened against the circumference of the
parasite. One, two, or three (text-fig. 28) masses of
chromatin were seen in various specimens. In text-fig. 27
there are really two masses of chromatin, as a blepharoplast
occurs close to the nucleus.

These parasites are truly rounded forms, not merely pyri-
form ones seen blunt-end-on, as could be determined by
focussing, and from the fact already mentioned, that the
central area was clear; also the corpuscle-hosts are only thin,
bi-concave discs. Some of these parasites are ring-like.

(c:) On the Chromatin of Endoglobular Parasites
occurring in Groups of More than Two in a
Corpuscle.

Cases of three or four parasites, more especially four,
occurring together within a corpuscle are fairly common.
These may result from multiple infection or from two binary

Sop

31

TEXxT-FIGS. 29—31.

G

29

divisions after a single infection. All the members of such a
group usually exhibit the same or similar distribution of the
chromatin elements. In text-figs. 29—31 there are slight
differences in the chromatin masses among the members of a
group, and it is for this reason more especially that they are
figured.

Of the group represented in text-fig. 29, one of the parasites
only possessed a mass of loose chromatin. In text-fig. 30

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 313

two easily distinguished pairs are shown, which would appear
almost certainly to have resulted from a multiple (binary)
infection. In text-fig. 31 a group of three parasites is repre-
sented, all differing to some extent. Such groups are rare.

The general distribution of the chromatin in groups of
parasites conformed to that already given.

(¢) The Chromatin of Intra-corpuscular Parasites
suggesting Division.
Parasites in process of division were not common. In
text-fig. 52 is represented a typical, heart-shaped form in
rocess of longitudinal division. Such forms have been
Ig 8

Trxt-FIGs. 32—35.

figured previously, but without showing -the blepharoplast
in the partially separated forms. In this dividing parasite
two nuclei, “polar”? in position, and two blepharoplasts,
“apical” in position, were clearly seen.

In text-fig. 83 nuclear division is depicted in a large, ovoid
form. ‘lhe principal nucleus is here drawn out into a some-
what curved rod, with slightly enlarged ends, the outline of

314 H. B. FANTHAM.

the rod in its middle portion being not quite sharp, but a
little irregular. Nothing in the nature of chromosomes could
be definitely distinguished, nor reduction division with
certainty. The blepharoplast (text-fig. 33) had divided into
two dots, lying side by side and still touching, at the peri-
phery of the parasite, away from the dividing nucleus. A
vacuole was present near the latter.

Text-figs. 34 and 35 represent ovoid forms, suggesting
parasites in process of division. Hach contains several
chromatin masses of varying size, and more or less peripheral
in position. In text-fig. 34 the relative sizes of the four
chromatin masses suggest the presence of two nuclei and
two blepharoplasts.

(n) Other Intra-corpuscular Forms.

In text-fig. 36 is shown a rare form, consisting of two intra-
corpuscular parasites, closely approximated at one end, which

Trext-Fic. 36.

36

may be termed rod-shaped, but are really more like a dumb-
bell or spoon in outline. Bacillary or rod-like forms of
P. bigeminum are known, but from immune cattle (Theiler).
The parasites drawn in text-fig. 36 may possibly be pyriform
ones seen edge-on. ‘Two masses of chromatin, one larger than
the other, were seen in each parasite situated near the ends.
Irregular forms are very rare. Nuttall and Graham-Smith
(8) describe intra-corpuscular parasites showing processes in
stained preparations and also in the living condition, and
remark (more especially concerning free parasites) that cyto-
plasmic processes seen in living parasites are often not found

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 315
in stained preparations, owing to retraction at death or to the
preparation of the film or smear and the difficulty of staining
such fine processes. I have only seen one good case of @
stained parasite with a protoplasmic process (text-fig. 23),
already mentioned.

(0) The Chromatin of Free Parasites.

Iixtra-corpuscular or free parasites are not very numerous,
and are found chiefly, as might be expected, at the edges of
the smear. Regarding epi-corpuscular forms, emphasised by
Liihe (4, 5) in the case of P. canis, I have only seen one or
two doubtful cases, not sufficiently definite to merit detailed
description, and my observations have been strictly confined
to stained specimens.

The free parasites are nearly always more or less pyriform
in shape (text-figs. 37 to 42), occasionally rounded (text-
figs. 43 and 44), and are usually slightly larger in size than
the corresponding intra-corpuscular forms.

The distribution of chromatin in free forms on the whole
closely corresponds with that found in pyriform intra-cor-
puscular parasites. Common cases are shown in text-figs,
37 and 39, where nucleus and blepharoplast are distinguished,
and in some cases an apical mass of looser chromatin (text-
figs. 87 and 38) is seen. In text-figs. 38 and 39 the strictly
pyriform contour is modified, approaching the fusiform.

Occasionally the pyriform outline is seen to be modified in
the direction of its long axis. We then have interesting
forms like those shown in text-figs. 40 to 42. These are long
and thin, and sometimes exhibit a prolongation of the pointed
extremity into a short, pointed cytoplasmic process (“ flagel-
lum”?! of some authors). Such processes, however, hardly
suggest great motility. These forms always possessed a
nucleus and blepharoplast, the latter at the pointed end.
Rarely, smaller chromatoid granules could, with difficulty,

be distinguished (text-fig. 42). These forms bear at least
1 Unfortunately this term (“ flagellum’) is used, in the Protozoa, for both
a cytoplasmic process and a chromatic process rather indiscriminately.

316 H: B. FANTHAM.

a superficial resemblance to Crithidia (Léger) and some
species of Herpetomonas (vide Woodcock [11, p. 268,
fig. 38]). Similar forms have been observed in different
species of Piroplasma, often without “nuclear dimorphism”
having been noticed therein. Such free forms of Piroplasma
bigeminum are certainly very interesting, more especially
to those parasitologists who attach great weight to the obser-
vations of Schaudinn ([9], p. 488) on blood-films prepared by
Weber at night from a cow kept in the dark, and dying of

V'ext-Fics. 37—44.

43 44,

piroplasmosis, wherein a small Trypanosome was found
accompanying P. bigeminum. ‘'Trypanosome-like (trypani-
form) bodies were also found in old films of Kossel and
Weber made from the contents of the digestive tract of ticks,
which had fed on cows suffering from piroplasmosis. How-
ever, the inference that Piroplasma possesses a trypaniform
stage is not in the least supported by the researches of Koch
on P. bigeminum and P. parvum (1) from the gut of
ticks, nor by the work of Kleine (2) on culture forms of
P. canis. Kleine also carefully searched for flagellate stages
in the blood of infected dogs, without success.

Regarding the chromatin of free rounded forms (text-
figs. 43, 44) it closely resembles in distribution that of
similarly shaped intra-corpuscular forms, and needs no further
comment,

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 317

V. Nore on rHe Cyroptasm or P. BIGEMINUM.

As judged by the distribution of the stain, the cytoplasm is
more especially peripheral, and often a clear area is seen in
the centre. Such a clear area may be regarded as a vacuole,
though one must beware of such appearances, sometimes
deceptive, when observations are confined to stained prepara-
tions. However, vacuoles have been described in Piroplas-
mata by various observers while examining the parasites in
the living condition. The vacuolated condition, as determined
from stained specimens, is well shown in text-figs. 1, 2, 3, 9,
12, 21, 26, 27, and 32. A vacuole with a sharp and definite
edge is shown in text-fig. 33. It does not always follow that
the clear space inside a parasite, as diagrammatically repre-
sented in the text-figures, is a vacuole, for when one examines
the preparation under monochromatic light a slight shadow
may sometimes appear therein, suggesting a continuous cyto-
plasm within such parasites, which may be more clear and
hyaline in the centre, or less permeable to stain than that at
the periphery. The vacuoles, of which more than one may
occur in a parasite, usually lie between the chromatin masses,
and appear only in the larger parasites. Two vacuoles may
occur in a parasite separated by a marked cytoplasmic
strand (text-figs. 3 part, 21 part, 22 part).

The cytoplasm shows a granular structure, more especially
at the apical end of some forms (text-figs. 3, 12 part). The
very finely-granular character of the protoplasm is difficult to
represent in drawings. The very small dots in the text-
figures, which figures are semi-diagrammatic, only show the
limit of the obviously stained area, yet that is almost the limit
of certainty in such small and delicate (endocorpuscular)
objects as Piroplasmata.

The central clear area seen in most rounded parasites
would appear to be a vacuole.

I was not able to obtain any further evidence as to the
character of the cytoplasm from an extended examination of
the stained free forms.

318 H. B. FANTHAM.

It may be stated at this point that the dimensions of the
larger intra-corpuscular parasites examined was broadly about
3 by 15, and agreed well with the measurements given by
former observers, though variations occur. ‘lhe nucleus
(largest dense chromatin mass) was from 0°34 to nearly 1
in long diameter in some forms, and exhibited variations in
size in different specimens. ‘The so-called blepharoplast was
sometimes only about half that in diameter.

VI. Tse Propaste SiGniricANce or NucieaArR DimorpHisM
IN PIROPLASMA.

As far as evidence goes at present—that is, considering the
observations of Schaudinn (9), Liihe (4, 5), aud Nuttall and
Graham-Smith (8), as well as those herein set forth, it would
appear that there is some justification for the terms “ nucleus”
and ‘ blepharoplast,” as applied to the larger and smaller
dense chromatin masses respectively in Piroplasmata, at any
rate, as a “ working hypothesis ” (Schaudinn), and so sug gest-
ing affinities of this rather aberrant Heemosporidian genus
with the Hemoflagellates.

The loose, reticulate chromatin, it seems to me, might
similarly be tentatively compared with the chromatoid
granules often recorded in ‘Trypanosomes, though the loose
chromatin of Piroplasma appears to be relatively of greater
bulk.

Mention has already been made of Schaudinn’s observation
on Weber’s blood films taken at night from a diseased cow,
and Kossel and Webev’s films from the gut of ticks. It is
much to be regretted that the trypanosome-like organisms
occurring in these preparations were not subjected to a
lengthy and detailed examination and carefully figured. Even
then the question of a double infection would not have been
disposed of satisfactorily.

Also, it has been mentioned that Koch’s (1) and Kleine’s (2)
researches do not at all support, so far as known at present,
the hypothesis of a trypaniform phase in the life-history of
Piroplasma. However, in Koch’s large pyriform parasites

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 319

from the gut of ticks, with protoplasmic radiations at the
blunt (“polar ’’) end, there is a larger nucleus at this end and
a smaller blepharoplast-like body nearer the other (‘ apical ”’)
end. ‘Three chromatin masses are seen in the zygote-like
forms, with radiations at opposite poles. It is remarkable
that Kleine (2) obtained similar results by cultural methods
in the case of P. canis.

The superficial resemblance of Piroplasmata, exhibiting
well-marked nuclear dimorphism, to the Leishman-Donovan
body is striking. At one time it occurred to me that, perhaps,
the loose chromatin of Piroplasma, in some forms of its
distribution, might be compared with the “tail”?! of chromatin
in the Leishman-Donovan body, though this ‘“ tail,’’? when
present, seems to be always attached to the blepharoplast of
the Leishman-Donovan body, and stains rather darkly, and
the resemblance to loose chromatin is none too well marked.
Laveran and Mesnil would seem to be perhaps justified in
placing this body in the genus Piroplasma (as P. donovani),
considering superficial appearances only, but the
difference of habitat must be borne in mind, and the Leishman-
Donovan bodies are rarely, if ever, intra-corpuscular. The
chromatin masses of the Leishman-Donovan bodies, too, are
well defined. Flagellate stages most probably do occur in the
life-cycle of these bodies (c f. Rogers, Christophers, Leishman,

and others on “

cultures.’’).

I will venture, however, to make a suggestion on the
“nuclear dimorphism” of Piroplasma from a_ rather
different point of view, namely, its relation and interpretation
in terms of the distribution of chromatin in the Protozoa
generally (vide Mesnil [6]). From this standpoint it is
probable that the nucleus (large chromatic body) of Piro-

1 This ‘ tail,” which is stated to occur in some of the larger forms of the
Leishman-Donovan parasite, is perhaps, on the other hand, only the developing
flagellum of the flagellate stages first found by Rogers in cultures, though one
hardly gathers this from the published accounts of the formation of the
flagellum, This chromatin ‘ tail” merits further consideration by workers
with the necessary material, for a flagellum does not occur in parasites in the
human spleen.

320 H. B. FANTHAM.

plasma consists of vegetative chromatin, and is, indeed, a
trophic nucleus, while the blepharoplast, according to the
upholders of the flagellate affinities of Piroplasma, would
be a kinetic nucleus—that is, a specialised and separate
portion of vegetative chromatin. But the so-called blepharo-
plast, usually denser and more deeply staining, may really
consist of generative chromatin (cf. the micro-nucleus of
Paramecium). ‘I'he loose chromatin would be a reticulum
of extra-nuclear chromatin or chromidia. Whether this
loose chromatin consists of tropho-chromidia (chromidia
properly so-called) related to the cell-metabolism of Piro-
plasma, or whether the loose chromatin consists of idio-
chromidia and represents a reserve of generative chromatin,
is difficult to say at present. The significance of the so-called
blepharoplast at the moment is most problematic, and pre-
cludes more definite statements.

Nuttall and Graham-Smith (8, pp. 641-643) consider the
various hypotheses which have been advanced at different
times regarding the development of Piroplasma. ‘They
reject them all, after careful studies on the living parasites.

It would appear, then, premature and useless to discuss
further the possible life-cycle and affinities of Piroplasma;
there have been, recently, it seems to me, too many “ phylo-
genies ” of such problematic organisms, often founded on
slender evidence, and the complete life-history of Piro-
plasma, and even Trypanosoma, has yet to be ascertained.

Lastly, as definite and further pronouncements’ on the
developmental stages of Piroplasmata in ticks may be expected
shortly, it is better to wait for these, when we may learn
whether a flagellate stage really does occur in the life-cycle
of Piroplasma, and what is the true significance of Koch’s
forms, so difficult to interpret alone.

VII. Summary or ReEsvutts.

(1) Allthe specimens (stained) of Piroplasma bige-
minum, which were examined, showed the presence of a
1 See Addendum.

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 321

chromatin mass or masses. Usually there was more than one
chromatin mass in each parasite (see pl. 18, figs. 1 to 7).

(2) In the pyriform and ovoid parasites there are usually
present (a) a rather large and dense chromatin mass—the
nucleus; ((3) a second, somewhat smaller, usually denser
mass of chromatin—the blepharoplast, which is sometimes
only punctiform; and many parasites possess in addition (y) a
rather looser mass of chromatin, of a woolly or mesh-like
structure (chromidial reticulum).

(3) Variations occur in the relative positions of these
chromatin masses, and less frequently in their relative sizes.
These variations may be due in part to the parasite being
viewed from different aspects.

(4) The loose chromatin mass is often relatively well-
marked in P. bigeminum.

(5) Free parasites were sometimes seen in which the
pyriform contour was slightly modified, and the apical end
prolonged into a short cytoplasmic process. Such forms
possessed a nucleus and blepharoplast, and somewhat re-
sembled Heemoflagellates (text-figs. 40 to 42).

(6) In round forms nuclear dimorphism also probably
occurs, for there is usually more than one chromatin mass
present (text-figs. 27, 28, 45, and 44). Amoeboid forms were
not available for examination.

(7) The cytoplasm of P. bigeminum appears to be
vacuolated in character. Unfortunately, the observations
were, of necessity, confined to stained preparations.

(8) The possible significance of these results is_ briefly
discussed in the preceding section. More definite pronounce-
ments are premature, pending further knowledge of the
developmental stages of Piroplasma in the tick.

December 31st, 1906.

ADDENDUM.

Since writing the foregoing, two papers have appeared
relating to Piroplasma (Babesia) canis, one by Chiris-

o22 H. B. FANTHAM.

tophers (18) on the developmental forms of the parasite in the
dog-tick (Rhipicephalus sanguineus) of Madras, and
the other by Kinoshita (14) on the forms of the parasite in
the dog’s blood and in sodium-citrate cultures.

The paper by Christophers, though only a preliminary
account, is most important. Most of the forms mentioned by
Koch in the cases of P. bigeminum and P. parvum in
the gut of adult ticks were seen by Christophers, except the
markedly radiate forms. -Stages in the development of
P. canis were traced in the tick-egg, in the nymph, and in
the developing adult of the dog-tick. ‘lhe life history
appears to be simple. Loose chromatin was seen in several
stages, but no mention of any flagellate form occurs, nor is
such figured. Also the presence of a blepharoplast is not
mentioned. ‘'Thisabsence of a flagellate stage in-the develop-
mental cycle in the tick is most important, and Christophers’
fuller memoir will be awaited with the keenest interest.

Kinoshita’s paper is interesting, though to me it is a little
dificult to summarise. ‘The author gives excellent figures
of nuclear dimorphism in the comparatively large form,
P. canis, the largest species of Piroplasma, and sufficiently
large to provide finer morphological detail often invisible in
the smaller P. bigeminum. He describes schizogony and
gametogony, and considers the blepharoplast to consist of
“animal” chromatin. He figures two forms, each with a
chromatic appendage arising from a large blepharoplast,
which he considers to be microgametes. Kinoshita himself,
apparently, does not think that a flagellate stage normally
occurs in Piroplasma, though in an editorial footnote,
p. 306, additional and independent evidence for the presence
of a flagellate stage is set forth.

It is most probable, indeed certain, that a flagellate stage
does occur in the life-cycle of the Leishman-Donovan body,
and may be expected in the alimentary tract of a blood-sucking
Arthropod, namely, the bed-bug, as suggested by Rogers, and
now being worked out by Patton (vide ‘Ind. Med. Gaz.,’ 1906,
p. 302). On the other hand, a flagellate stage appears to be

° G20) a Slt -

——

9

THE CHROMATIN MASSES OF PIROPLASMA BIGEMINUM. 328

absent in the case of Piroplasma. The pathogenic agents
of kala-azar and hemoglobinuria would not appear, then,
to belong to the same genus. However, the evidence at
present available is conflicting, and further discussion is
premature.

February 14th, 1907.

VIII. Rererences To LITERATURE QUOTED IN THE TEXT.

1. Kocu, R., 1906.—‘ Beitrage zur Entwicklungsgeschichte der Piro-
plasmen,” ‘ Zeitschr. f. Hyg.,’ liv, July 26th, 1906, pp. 1-7, 3 pls.

2. Kieins, F. K., 1906.—“ Kultivierungsversuche der Hundepiroplasmen,”
‘ Zeitschr. f. Hyg.,’ liv, pp. 8-16, 2 pls.

3. Laveran, A, et Nicontz, M., 1899.—‘‘ Contribution a l’étude de
Pyrosoma bigeminum,”‘C, R. Soe. Biol.,’ li, pp. 748-751, 15
text-figs.

4. Line, M., 1906.—‘‘ Zur Kenntniss von Bau und Entwicklung der
Babesien,” ‘ Zool. Anzeiger,’ xxx, pp. 45-52.

5. Linz, M., 1906.—‘ Die in Blute schmarotzenden Protozoen und ihre
nachsten Verwandten,” in Mense’s ‘ Handbuch der Tropenkrankheiten,’
Leipzig, Bd. ili (‘ Babesia,’ pp. 193-202, taf. 8).

6. Mesnit, F., 1905.—‘‘ Chromidies et questions connexes,” ‘ Bull. Inst.
Pasteur,’ ill, pp. 312-322.

7. Mincnin, E. A., 1903.—‘‘Sporozoa,” in Lankester’s ‘Treatise on
Zoology,’ pt. i, fase. 2, pp. 150-360 (‘ Piroplasma,’ pp. 269-270,
fi. 80).

8. Nurtaut, G, H. F., and Granam-Smitu, G. 8., 1906.—‘ Canine Piro-
plasmosis.—V. Further Studies on the Morphology and Life-History of
the Parasite,’ ‘Journ. Hygiene,’ vi, No. 5, pp. 586-651, 3 pls.

9. Scuaupinn, F., 1904.—‘* Generations- und Wirtsweclisel bei Trypano-
soma und Spirochete. Vorlaufige Mitteilung,” ‘Arb. a. d. Kaiserl,
Gesundheitsamte,’ vol. xx, pp. 387-439, 20 figs. (see p. 438).

10. Smiru, T., and Kizzoryg, F., 1893.—‘ Investigations into the Nature,
Causation, and Prevention of Texas or Southern Cattle-Fever,”
‘Kighth and Ninth Ann. Rept., Bureau of Animal Industry,’ pp. 177-
304, with 10 pls. Washington, U.S.A.

11. Wooncock, H. M., 1906.—* The Hemoflagellates : a Review of Present
Knowledge relating to the Trypanosomes and Allied Forms,” part ii,
‘Quart. Journ. Mier, Sci.,’ N.S., 50, i1, June, 1906, pp. 283-331. (See
p. 244, footnote 3; pp. 253-266 [fig. 37] and p. 269. Also ‘‘ Biblio-
graphy on Leishman-Donovan Bodies,” p. 330.)

B24 H. B. FANTHAM.

12. Ziemann, H., 1898.—‘* Neue Untersuchungen iiber die Malaria und
den Malariaerregern nahestehende Blutparasiten,” ‘Deutsche med.
Wochenschr.,’ xxiv, pp. 123-125 (especially p. 125).

References quoted in Addendum.

13. Curistorners, S. R., 1907.—“ Preliminary Note on the Development
of Piroplasma canis in the Tick,” ‘ Brit. Med. Journ.,’ No. 2402,
Jan. 12th, 1907, pp. 76-78, with 26 text-figs.

14. Kinosuita, K., 1907.—‘ Untersuchungen tiber Babesia canis (Piana
und Galli-Valerio),” ‘Arch. f. Protistenkunde,’ viii, pp. 294-320, tafs.
12 and 13.

IX. EXPLANATION OF PLATE 18.

Illustrating Mr. H. B. Fantham’s paper, “ On the Chromatin
Masses of Piroplasma bigeminum (Babesia bovis),
the Parasite of Texas Cattle-Fever.”

Figs. 1-7.—Lithograph drawings made from photo-micrographs of Piro-
plasma bigeminum (Babesia bovis) from heart-smear, showing chromatin
masses and their distribution. These photo-micrographs were taken under
monochromatic (yellowish-green) light, using a Thorpe’s grating. Zeiss’s
3mm.-apochromatic homog. immers. objective was used, with compensating
oculars 8 or 12.

The lithograph drawings are twice the size of the photographs.

These minute intra-corpuscular parasites were very difficult to focus for.
photography in order to bring out detail successfully, and so the relative sizes
of the nuclear masses do not appear always precisely correct.

The red blood-corpuscles were in all cases only very faintly stained.

Fic. 1.—Shows bridge of chromatin between nucleus and blepharoplast in
parasite to left. Note also pair of parasites on right.

Fre. 2:—Pair of large pear-shaped intra-corpuscular parasites, showing
nucleus and blepharoplast in each, and loose chromatin at apex of one.
Magnification greater than in other figures.

Fia. 3.—Blood-corpuscles showing rather better than in other figures, onc
pair of parasites distinct.

Ftc. 4.—One parasite shows three chromatin masses, and chromatin bridge.
Fic. 5.—One parasite, on right, shows chromatin bridge faintly.

Fic. 6.—Several pairs of parasites, on left, show nuclear dimorphism some-
what faintly.

Fic. 7.—Parasites at top left-hand part of field show chromatin masses.

SKIN AND REPRODUCTIVE ORGANS OF NOTORYOCTES. 325

The Skin, Hair, and Reproductive Organs
of Notoryctes.

Contributions to our Knowledge of the Anatomy of Notoryctes
typhlops, Stirling—Parts IV and V.

By
Georgina Sweet, D.Sc.,
Melbourne University.

With Plates 19 and 20, and a Text-figure.

INTRODUCTION.

The following is a continuation of the work on
Notoryctes typhlops, of which Part I, on the Nose and
Jacobson’s Organ; Part II, on the Blood-vessels ; and Part
ITI, on the Eye, have already been published [11, 12].

It has been thought well to make the record of the
anatomy of Notoryctes more complete by incorporating here
a fuller account of the skin and hairs, and also of the repro-
ductive organs and associated parts, than has previously been
given.

Professor Baldwin Spencer has therefore kindly handed to
me the drawings and notes he had made in connection with
these structures ; for this, as also for access to his stock of
animals, I wish to record my indebtedness and thanks.

As stated previously in another place no embryonic material
is forthcoming, so that no facts concerning the development
of these parts can be given.

326 GEORGINA SWEET.

Part IV.—The Skin, Hairs, and Certain Associated Structures.

The skin forming the surface of the body generally, con-
sists of two or three layers of cells, covered by a thin corneous
layer (Pl. 19, figs. 3, 4, 12, e.). Over the snout and tail
the epidermis becomes most highly developed forming the
strong horny coverings to those parts. The skin undergoes
modification, also in the region of the special “ ischiotergal
patch,” on the hinder part of the back, overlying the ischio-
tergal slip of muscle previously referred to by Dr. Stirling
[10, p. 159], Professor Baldwin Spencer [8, p. 46], and Pro-
fessor Wilson [18, p. 6]. The diameter of this patch varies
as measured in sections, from 9 mm. to 10 mm., 9°6 mm.
being an average width. It can always be seen from the
exterior by its darker golden-brown colour.

The varying thickness of the skin in different parts is seen
in the following table :

: ' Chin and Side of | General |Centre of Ischi-
TUG Gee: SM Head. Surface. | otergal Patch.

Stratum Corneum “13 mm.| ‘009 to'014 mm. |:040 mm.|018 to ‘021 mm.
x lucidum |04 mm.)). L ‘014 mm.|'007 to 012 mm.
» Malpighi 071 mm. } oF to-021 mm. § 034 mm./|'050 to ‘063 mm.

Total thickness of |
Epidermis . 244 mm. *019 to 085 mm. |:088 mm.|'075 to ‘096 mm.

Total thickness of
Dermis . lpia) am °85 mm. 52 mm.|1:54 to 2°45 mm.

The thinness of the dermis in the snout is due to the
absence of hairs in that part, while in the chin and general
surface of the body, and especially in the ischiotergal patch,
these structures are highly developed. Immediately below

SKIN AND REPRODUCTIVE ORGANS OF NOTORYOTES. 327

the level of the hair-roots in this modified region, is a thick
layer of fat cells. The greater thickness of the dermis here
does not end abruptly, but gradually decreases from the
centre of the area till it reaches the margin, where there is a
small sudden decrease in thickness, marking the boundary of
the patch [cf. 8, pp. 45, 46].

The difference in thickness of the corneous layer in the
modified patch, and over the general body surface, is occa-
sioned by the compactness of this layer in the former case,
and its looseness in the latter.

Hairs.

As in Ornithorhynchus [cf. 7 and 9] and Echidna (cf. 9]
the hairs are of two kinds, large and small, arranged in
bundles (Pl. 19, figs. 1 and 3) containing usually one large
and a greater number of small hairs [cf. 8, pp. 45, 46]. The
hairs of each bundle have a common neck and opening (figs.
2-4, c.f.o.) to the surface, where the individual follicles are
absent.

The bundles are furthermore arranged in groups of three
(Pl. 19, fig. 1), or sometimes four or even five, in more or
less straight lines, each bundle, however, having its own
follicular opening not communicating with that of the other
bundles in the group (see Pl. 19, fig. 2), so that in trans-
verse sections near the surface, the group arrangement is
often lost.

The arrangement at deeper levels is shown in PI. 19,
fig. 1, where we have a typical group of three bundles from
the skin of the back. The number of small hairs in each
bundle varies from nine to twenty, the most numerous
examples showing from eleven to nineteen. Occasionally
the large hair seems to be absent. As will be seen, there
are in the group figured—

Bundle 1. 1 large hair and 15 small hairs.

Group l. por ee ge |e i LS 55

3) 3. 1 3) 92 12 3)

VoL. OL, PAKY 2.—NuW SERIES, 24

328 GEORGINA SWEET.

Other bundles contain—
Bundle 1. 1 large hair and 12 small hairs.

Grovp 2. meet Peat on ees Sigil +
” 3 if 3) +) 22 )

Bundle 1. 1 large hair and 21 small hairs.
Group 3. so Mae ee es a) agility -
3) 3. 1 PP) 3) 19 Pe)

Bundle 1. O large hair and 21 small hairs.
Gnowr 4 Te Ly Sai 2lcplee

Successional hairs are only occasionally seen.

Each bundle is separated from those around it by a deeply
staining, more or less compacted fibrous tissue. In the
ischiotergal patch, and on the chin and side of the head, the
bundles are more closely packed together than on the
shoulder, thigh, and body generally.

The large hair grows in varicus positions in the bundles,
generally posteriorly, but by no means always so.

The muscles connected with the hairs are as usual] unstriated,
both in the ischiotergal patch and elsewhere.

Glands.

In the modified patch, around the cloacal opening and on
the head, the sebaceous gland mass often almost entirely
surrounds the bundle, lying both between the latter and its
muscles, and between this bundle and its fellow. The glands
in this modified area, and those around the cloacal opening
are also very long, extending well down towards the hair-
roots, and even below them (PI 19, fig. 4), but on the side
of the head and under the chin, though the glands often
extend round the bundles, they are very much shorter than
in the ischiotergal patch specially.

On the shoulder and thigh, as well as the general body
surface the gland mass is very much smaller and shorter (see
Pl. 19, fig. 4). The main duct of the gland is found most
often just behind the large hair of a bundle, and should the

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 329

large hairs of the three bundles in a group vary in size, as
often happens, the largest hair and gland duct is generally
to be found in connection with the most central bundle.

The greatly marked glandular development of the ischio-
tergal region, and the peculiar structure of the ends of the
hairs, as described below, no doubt account for the usually
matted character of the hair-covering over this area.

Structure of the Hairs.

The ordinary hairs, both large and small, forming the fine
silky golden covering of Notoryctes vary considerably in
diameter below the epidermis as seen in the following table:

General Surface.

Large hair. Small hair. Large hair. Small hair.
‘021 mm. ‘005 mm. | 027 mm. 005 mm.
a2) 55 ‘OO79 © 5s 034 ,, “006: 4:5
045, sD0D: # 048, “O07 5;
“055. 55 "009 “056. —., 009 .,,

Ischiotergal Patch.

The average size for the shaft enclosed in the skin is for
the large hair ‘040 mm., and for the small hair ‘007 mm.

In depth below the surface of the epidermis we find
variations according to the region; thus over the body
generally a typical large hair may extend for ‘49 mm. below
the stratum corneum, while in the ‘ ischiotergal”’ patch it is
commonly 1°66 mm. in depth, though it may be more.

Such difference in length may easily be found only 9 or
10 mm. apart. The hair roots are also very long in the head
region, but not so much so as in the modified patch.

In structure [cf. 8, pp. 45, 46] the large hairs are not
unlike, though much smaller than, those of the Ornitho-
rhynchus [cf. 7 and 9]. They have a rapidly tapering
smooth tip (PI. 19, figs. 5 and 6). This is succeeded by a

330 GEORGINA SWEET.

wide flattened part, which in a typical hair at a distance of
1 mm., from the tip is ‘09 mm. wide. From this point down-
wards we find a diminution in size till at its exit from the
follicle it is round, and ‘03 mm. in diameter. The tip is
solid, but in the wider shield and long smooth shaft, the
medulla is much vacuolated; the air cavities become regu-
larly arranged, so as to give the ladder-like appearance at
about 2 mm. below the extreme point. There is no pigment
in the older hairs, though in the successional large hairs
there is pigment in the centre of the flattened shield.

In the ischiotergal patch, however, these large hairs are
much pigmented right to the free end, causing the darker
colour of this area. The tip, moreover, instead of being
smooth and pointed as elsewhere, is deeply split (Pl. 19, figs.
9 and 10), giving rise to a somewhat brush-like appearance
at the end of each hair. These are frequently ‘1 mm. in
width just below the division.

The small hairs are imbricated all over (Pl. 19, fig. 7), and
taper gradually from base to tip over the general surface,
but in the ischiotergal region (Pl. 19, fig. 8) they are irre-
gularly waved near the tip, somewhat resembling a badly
made spear.

The roots of both large and small hairs are situated at
about the same level in the dermis. In minute structure the
hair sheaths, as well as the hairs, are normal.

The long straight hairs found around the cloacal opening
in both male and female are in distinction to those of the
rest of the body surface, not disposed in bundles, but are
isolated. In diameter they average ‘047 mm. to ‘05 mm.

Special Cutaneous Structures.

When examining the epidermis over the region of the
rudimentary eye, I found curious modified groups of epidermic
cells with a more or less definite arrangement; and, on further
investigation, they are met with over most of the head region,

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 331

over the modified ischiotergal patch, and in the pouch region.
The epidermis over the head region, i.e. behind the snout,
consists of at most three layers of cells, except at special
points (Pl. 19, fig. 12). Here the epidermis is much thicker
(Pl. 19, fig. 12, s.o.), having four or five layers of cells,
causing the Malpighian layer to become depressed into the
dermis, and the stratum corneum to become slightly raised.
Sometimes the whole structure resembles a raised platform,
often like a much-truncated cone in outline. In horizontal
diameter the top of the modified area varies from ‘11 mm. to
‘15 mm.; while in vertical depth, excluding the corneous
layer, it is ‘(04 mm. to (06 mm. The whole area of the
Malpighian layer involved may have a diameter of 2 mm.
The slightly larger examples were found in the ischiotergal
patch, though these structures are much more conspicuous in
the region of the head and neck, since there, as previously
stated, the whole normal Malpighian layer may be only ‘01
mm. thick. These structures are normally separated by a
space of 2°5 mm.

The loose outer part of the corneous layer, which so
readily becomes detached elsewhere, tends to adhere more
firmly to the underlying part over these areas. The cells of
the Malpighian layer are much elongated and thinner, and,
in addition to sloping upwards and inwards, they are much
crowded horizontally.

A tendency to become arranged around a central core is to
be noted in some cases. Their nucleiare also much elongated
and larger. In the modified “ischiotergal” patch the whole
epidermis is, as previously stated, much thickened, but these
cutaneous structures are equally, if not more commonly,
developed there than in the skin of the head region. Over
the shoulder and thigh regions, as over most of the body,
these cutaneous organs do not appear to be present. The
structure is often pierced by a group of hairs, or it may be
flanked by such a group, while, on the other hand, they
would seem often to exist without any relation to hair-
groups.

don GEORGINA SWEET.

- Beneath the modified epidermic areas, there can generally
be seen a group of more numerous connective tissue cells,
not unlike those found in a developing hair-papilla. Some-
times there is a group of about a dozen oval cells staining
less deeply than the cells above, and with two or three well-
defined nucleoli, I have not been able to detect either nerve
or blood supply to these structures, though in a few cases
the oval cells appeared to give off processes towards the
Malpighian layer, the cells of which immediately above are
very pointed, and almost spindle shaped, penetrating the
second layer of more cubical cells.

The general appearance of these structures is that of a
taste bud with the stratum corneum continuous over it, and
not unlike a developing cutaneous sense organ of Triton.

Of the sensory function of these structures I can therefore
offer no conclusive evidence, but it is reasonable to suppose
that in the absence of the sense of sight and pari passu
with the apparently increased olfactory function, there might
exist some special organ or organs of tactile sense in Noto-
ryctes, as in some other forms. It is at least possible that
such may be the function of these special cutaneous struc-
tures. They cannot be early stages of developing hairs from
the fact that they are present in the fully-grown animal
alongside well-developed bundles of hairs and often pierced
by such bundles.

Further they are quite different in structure and size from
those very early stages of a developing hair which at first
sight they slightly resemble.

So that, although direct proof of such nervous character is
wanting, one is led to consider it probable that they are some
form of tactile sense-organ, which must be of special use to
such a burrowing, sightless animal as Notoryctes.

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 333

Part V.—Reproductive Organs.

Urinary AND MALE ReEpropUCTIVE ORGANS.

My observations confirm those of Dr. Stirling as to the
position, appearance, structure and relations of the testes,
epididymis, vasa deferentia, and their associated blood-
vessels; also as to the absence of any external scrotum. The
following additional points are worthy of note.

Around the base of the thick muscular-walled bladder
(Pl. 20, figs. 13—16, blr.), and extending over the origin and
anterior part of the urethra, is a well-marked glandular part
evidently prostatic in character, and enclosed by a strongly-
developed circular muscle coat. Within each of the large
muscular bulbs attached to the root of the penis is a well-
developed corpus cavernosum with thick fibrous walls. These
corpora cavernosa leave the muscular masses and approach
each other posteriorly in the mid-ventral line beneath the
urethra, forming the “cylindrical body ” described by Dr.
Stirling [10, p. 182], though, owing to the absence of sections,
he was not able to determine the presence of the corpora
cavernosa. At the plane of coalescence of the median walls
of the corpora cavernosa (P]. 19, fig. 18, c.c.), the corpus
spongiosum (PI. 19, fig. 18, c.s.) appears ventral to them again,
and with them, extends posteriorly into the penis. This organ
lies in a preputial sac ventral to the rectum, and on its dorsal
side some distance from the tip (which contains only a blood-
vessel) the urethra opens by a long slit-like opening.

In this region the rectum loses its typical glandular char-
acter, the cloaca being lined by a very thick stratified epithe-
lium (‘095 mm. in thickness), which has a thick, corneous
layer. When seen in section, it is very conspicuous when
contrasted with the epidermis which consists of two or three
layers only (028 mm. thick) in this region. The lining epi-
thelium of the cloaca is also very much folded, and_ rests
upon a thick layer of submucous connective tissue. Hxtending

334 GEORGINA SWEET.

along the length of the cloaca, to near its external opening
there are as in Perameles [4, p. 57] a number of much-coiled
branching diffuse tubular glands lying amongst the muscle
bundles of the cloacal wall, and in the connective-tissue
surrounding it, especially dorsally and laterally. These
gland tubes, which may be found also to a less extent around
the rectal wall (PI. 19, fig. 18, g.¢.), have very thin walls,
composed of cells, which are small and distinctly non-
elandular in appearance, and yet the lumen always contains
secretion.

Their ducts, in number about thirty, open along the whole
length of the cloaca, dorsally, ventrally, and laterally, though
most numerous dorsally.

_ On either side of the anterior half of the cloaca, or even
more anteriorly still, hes an “anal gland” (PI. 19, fig. 18,
a.gl.) similar to those of some other Marsupials, a single
duct from each [contrast Dr. Stirling, 10, Pl. 9, fig. 5]
running backwards in the adipose tissue outside the muscle-
layers of this part to enter the cloaca laterally, close to its
hinder end [cf. Perameles, 4, p. 57]. These are readily
distinguishable in sections from the diffuse gland ducts by
their size and structure, while the other two pairs of duct-
like structures described and figured by Dr. Stirling are seen
to be simply fibrous bands anchoring the gland in position.
This anal gland is most curious in structure, as indicated by
Professor Hill in Perameles. It consists in Notoryctes of an
almost spherical hollow ball, 2-4 mm. in external diameter,
with a fibrous capsule, from which processes pass inwards,
just comparable to the trabecule of a lymphatic gland, and
forming a more or less complete network. Unlike a lym-
phatic gland, however, the centre of the ball is hollow (1°4
mm. in diameter), and the whole gland appears to be sur-
rounded by striated muscle-fibres [cf. 1, pp. 157, 161, and
contrast 4, p. 57]. Between the trabecule the alveoli are
filled with long oval cells, those nearest the capsule contain-
ing protoplasm with deeply-staining nuclei. As they pass
inwards towards the central cavity, however, these cells lose

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 835

their nuclei and become disintegrated, so that the ball
contains a greater or less quantity of broken-down material,
which is passed to the exterior by the duct and cloaca.

In the region of the cloacal opening are numerous solitary
large hairs. Associated with the bundles of hairs are also
very greatly developed sebaceous glands, which may extend
below the hair-roots, but do not occur unless in connection
with the hairs. ‘They are only found around the hinder end
of the cloaca, and always lie outside, and generally separated
from, the muscular wall of the cloaca.

We have in Notoryctes, therefore, representatives of three
of the types of glands, described by Professor Disselhorst
and others as found in connection with the reproductive
organs in Marsupials, viz. in Cuscus orientalis, Smin-
thopsis crassicaudata, Macropus robustus, etc. [cef.
1, pp. 154—163], viz.:

(1) The tubular glands lined by a single layer of cylin-
drical epithelial cells.

(2) The rectal or anal gland with a duct unbranched, as in
Sminthopsis, and Cuscus, and Antechinomys, leading into the
cloaca,

(3) Hair glands—large complicated sebaceous glands
which do not here, however, lose their close relationship to
the hair-follicles, as they do in Sminthopsis, etc., where
they come into connection with the tubular and the rectal
glands, and lie amongst the muscles of the cloacal wall
[1, p. 161, fig. 159]. Nor is there here any special develop-
ment of muscles around these enlarged sebaceous glands, as
described by V. Den Broek for Cuscus orientalis [1,
p- 161].

As to the morphology of the rectal or anal gland which
VY. Den Broek has considered to be a highly developed
modified sebaceous gland [1, p. 163], this seems to me most
probable from the somewhat similar general internal arrange-
ment of some of the larger groups of sebaceous glands
found around the cloacal opening of Notoryctes—though
these have not in this form a striated muscular investment

336 GEORGINA SWEET.

such as surrounds the anal gland; and also from the fact
that the secretion of both is necrobiotic involving the death
of the cells [2, p. 29]. Of the fourth type of gland
described by V. Den Broek [1, p. 163] there is no special
representative in Notoryctes.

FremaLe REPRODUCTIVE ORGANS.

It is of especial interest to note the relations and compara-
tive morphology of these organs in all Marsupials since
Professor Hill’s valuable work on those in Perameles.

Hence to the admittedly incomplete description and some-
what erroneous figure given by Dr. Stirling, and the brief
comments of Professor Spencer, further details are here
added, together with the necessary figures. The whole of
these organs have been examined by means of several sets
of serial sections, and in addition by dissection. |

The various relations herein described may be better
understood by reference to the accompanying text-figure, in
which the parts are represented as seen from the ventral
surface, and to scale as far as is possible for serial sections.

In Notoryctes, as in some other marsupials, there are in
addition to the ovaries, oviducts, uteri, and vaginz proper,
two lateral vaginal canals, with vaginal ceca, and a median
vaginal apparatus. The bodies of the uteri (text-figure
ut. b.) are much twisted, and their internal surface is folded
and glandular. They lie ventrally to the rectum with their
mesial surfaces almost touching in the middle line anteriorly,
while posteriorly they are separated by the anterior end of
the bladder (ant. b. of blr.). Their posterior ends approach
each other almost transversely to the length of the animal,
to enter the uterine necks (wt. .), which are much smaller
and lie close together ventrally to the rectum, and dorsally
to the fundus of the bladder (see Pl. 20, fig. 13, wt. n.).
The convoluted bodies of the uteri may in some extend for a
short distance alongside the bladder, and ventral to the

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 337

necks of the uteri, the long axis being parallel to that of the
animal. In some of these respects Notoryctes resembles
Myrmecobius fasciatus [5, p. 520] rather than Pera-
meles obesula. The uterine necks, the internal surfaces of
which are much folded, pass backwards often in contact
with each other, and embedded in connective tissue, to open
into the vagine on a small papilla (see text-figure, and

==. Utsb=

No ant. bof bln

SECTION 39.

TEXT-FIGURE,

Pl. 20, fig. 14, wé. m.) as in ‘l'arsipes [5, p. 523] and Acrobates
[5, p. 525].

Passing backwards from these openings are two large
median vagine (text-figure, and Pl. 20, figs. 14, 15, med.
v. c.), separated for some distance back by a septum which
is absent, however, at the extreme posterior end, leaving a
small opening, apparently a natural one, by which the two

338 GEORGINA SWEET.

median vaginal canals may communicate with one another.
In the possession of this communication Notoryctes is quite
unlike the young Perameles [4, p. 54], young Dasyurus
[6, p. 371], Myrmecobius [8, p. 526], or the young Tricho-
surus [95, p. 528], etc., and more like Petaurus [5, p. 526] or
Acrobates [5, p. 525].

It is, however, normal in the termination of the median
vagine “blindly in the connective tissue between the
posterior ends of the lateral vaginal canals, and in compara-
tively close proximity to the urogenital sinus.” The anterior
limb of each vagina (text-figure, and Pl. 20, fig. 14, a.v.c.)
passes forwards from the opening of the uterine neck. It is
very short, but wider and thinner walled than either the neck
of the uterus or the lateral vagina. It lies laterally to the
uterine neck of its own side, and very soon opens into a fair-
sized expansion (text-figure, and Pl. 20, fig. 13, vag.c.)
corresponding to the much larger vaginal ceecum on either
side in Perameles, its anterior border in Notoryctes being
situated at about half the length of the uterine necks. In
Notoryctes, moreover, in contradistinction to Perameles, the
vaginal ceeca being smaller, never lose their close relationship
to the uterine necks. Hach lateral vagina (text-figure,
and P]. 20, figs. 14—16, lJ.v.c.) runs back as a small,
thick-walled, straight, or slightly-curved tube laterally to the
anterior limb of the vagina and the median vaginal canal,
and separated from the latter by the ureter (text-figure,
and Pl. 20, figs. 14—16, wr.), which comes here to lie more
ventrally than in its anterior part. Thus the whole of these
tubes in this region, i.e. the median vaginal canals, the
ureters, and the lateral vagine, lie in the same horizontal
line ventral to the rectum and dorsal to the bladder (PI. 20,
figs. 14 and 15),

As in other allied forms, there is here a urogenital strand
which contains anteriorly the necks of the uteri, the anterior
limbs of the vagine, the vaginal czeca, and the lateral vagina,
and more posteriorly the median vaginal canals, the ureters,
and the lateral vagine, with the hinder half of the bladder

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCIES. 339

and the whole length of the urethra. Indeed, so closely
bound together are these parts that a true conception of their
relations cannot be obtained by dissection. Notoryctes then,
in this respect, also may be compared with such forms as
Perameles, Tarsipes, and Acrobates, and contrasted with the
free coiled lateral vaginze of Myrmecobius.

Posteriorly to the median vaginals, and occupying the
short space between it and the anterior extremity of the long,
narrow, urogenital sinus, there lies a mass of dense deeply-
staining connective tissue (text-figure, and Pl. 20, fig. 16,
s.c.t.) exactly similar in appearance and position to that in
Perameles [4, p. 54, 55], and other forms, through which in
Perameles birth takes place. Careful examination of this
part in Notoryctes has failed to show any indication of a split
such as occurs in other forms, such as Perameles, Tarsipes,
_Macropus, Trichosurus, and Dasyurus, but it is open to ques-
tion whether this means that the animals examined had not
borne any young, as might possibly be indicated by the
absence of spermatozoa and their accompanying secretion in
the ducts, and by the general appearance of the parts, or, that
parturition having taken place similarly to Perameles, the
epithelium has been completely renewed, as in Perameles and
Dasyurus, and even the split in the connective tissue has been
quite closed, as in Dasyurus also.

Certainly, unless these animals were virgins, there is here
no permanent pseudo-vaginal passage [cf. Acrobates], and
even were they not so, the connection between the two median
vaginal canals, shows no trace of any such forced opening as
occurs at birth in older forms of Perameles and Acrobates,
so that we may infer that a median vaginal connection is
normally present, as in Petaurus.

At the same time, I should judge that birth takes place, as
in Perameles, through a pseudo-vaginal cleft in the connective
tissue, and not through the lateral vaginal canals.

We may notice particularly in concluding this part—

(1) The sharply marked distinction between the uterine
and vaginal parts of the ducts.

340 GEORGINA SWEET.

(2) The large size of the median vaginal canals in compari-
son with those of other forms and with its own vaginal cecum,
and the intercommunication of the two median vaginal
canals.

(3) The presence of the urogenital strand which encloses
the anterior vaginal canals as well as the other ducts, as in
Perameles, Peragale, etc., and that of the dense connective
tissue anterior to the long urogenital sinus.

Notoryctes then, like Perameles, seems to be more primi-
tive in type,.as far as its female reproductive organs can
show.

Pouch.
As previously described by Professor Spencer [8, p. 49]

the pouch is present in both male and female forms, as
in some other Marsupials. In the male it is but slightly,
though unmistakably, developed, while in the female, it is
naturally much larger. My observations confirm the state-
ments hitherto published [8 and 10] as to the position and
relations of the pouch and mamme, etc. The general appear-
ance is shown in PI. 19, fig. 17, herewith.

The epidermis of this region is similar to that of the rest
of the body, but has, here and there, some of the special
“sense organs” described in Part IV. ‘lhe dermis is dense
and deeply staining around the pouch area, least so on the
roof of the pouch cavity. ‘The sphincter marsupii is present,
and lies deep down in the very thick layer of adipose tissue
in the walls of the pouch. This tissue also contains numerous
bundles of non-striated fibres, mostly transverse, with others
longitudinal to the body. The dermis has a very abundant
blood-supply.

The groups of hairs are less closely packed in this area,
especially on the roof of the pouch cavity, and usually consist
of three bundles each. The hairs are chiefly of three average
sizes—the largest yellow-brown in colour ‘018 mm. thick,
the smaller ones, which are colourless and much imbricated,
being ‘006 mm. to ‘009 in diameter.

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 341

The sebaceous glands are very well developed, especially

around the circumference of the mammez. Beneath the
mamme the dermis extends even more deeply than elsewhere,
and contains numerous mucous ducts. The mammary ducts,
three to four in number, open close together on the apex of
each mamma, and have, in a marked degree, the characters
of sudoriparous glands, as opposed to sebaceous glands ; so
that, as in Monotremes [2, p. 40] the mammary glands and
sweat glands are apparently homologous in Notoryctes also.

10.

BIBLIOGRAPHY.

. DissrLHorst.—‘ Lehrbuch der Vergleichenden Mikroskopischen Anatomie

der Wirbelthiere,’ Vierter Teil, pp. 154—163.

. KGcEtinc.—“ Ueber die Hautdriisen der Monotremen,” ‘Anat. Anzeiger,’

Bd. xviii, 1900, pp. 29—42.

. Hir.—* Placentation of Perameles,’’ ‘Quart. Journ. Mic. Sci.,’ vol. 40,

N.S., pp. 385 et seq.

» Hirir.— ‘Contributions to the Morphology and Development of the

Female Urogenital Organs in the Marsupialia :”
**T, Perameles,” ‘Proc. Linn. Soc. N.S.W.,’ 1899.

- Hitu.— “II. Myrmecobius fasciatus,” ‘ Proc. Linn. Soc. N.S.W.,’

1900.
“TIT. Tarsipes rostratus,” ‘ Proc. Linn. Soc. N.S.W.,’
1900.
“IV. Acrobates Ste mee ae Linn. Soc. N.S.W..,’
Petaurus breviceps,” 1900.
“V. Trichosurus vulpecula,” ‘ Proc. Linn. Soc. N.S.W..,’
1900.

. Hitt.—** Foetal Membranes, Placentation, and Parturition of Native Cat

(Dasyurus viverrinus),” ‘Anat. Anzeiger,’ Bd. xvii, 1900.

. Poutton.—“ Structure of the Bill and Hairs of Ornithorhynchus

paradoxus,” ‘Quart. Journ. Mic. Sci.,’ vol. 36, p. 143.

. SpenceR.— Report on the Work of the Horn Scientific Expedition to

Central Australia, 1896,’ ‘‘ Mammalia,” pp. 45 et seq.

. SPENCER and SweetT.—“ The Structure and Development of the Hairs

of Monotremes and Marsupials,’ ‘Quart. Journ. Mic. Sci.,’ vol. 41,
pp. 549 et seq.

Srirtinc.—‘ Description of a New Genus and Species of Marsupialia,
and Further Notes on the Habits and Anatomy of Notoryctes

342 GEORGINA SWEET.

typhlops,” ‘Trans. Roy. Soc. S,Aus.,’ 1891, pp. 154 et seq., and
pp. 283 et seq.

11. SwrrT.—“ Contributions to our Knowledge of the Anatomy of Noto-
ryctes typhlops,” Parts I and II, ‘ Proc. Roy. Soe. Vic.,’ vol. xvii,
N.S., 1904.

12. SWEET. Part ILI, ‘Quart. Journ. Mic. Sci.,’ vol. 50, 1906.

13. Witson.— On the Myology of Notoryctes typhlops,” ‘Trans. Roy.
Soc. S. Aus.,’ 1894.

EXPLANATION OF PLATES 19 anp 20,

Illustrating Miss Georgina Sweet’s paper on “The Skin, Hair,
and Reproductive Organs of Notoryctes.”

REFERENCE LETTERS.

a. Artery. a.gl. Anal gland. a.¢. Adipose tissue. a.v.c. Anterior vaginal
canal. d/r, Bladder. c.c. Corpora cavernosa. c¢.f.0. Common follicular
opening. c¢.s, Corpus spongiosum. c/.o. Cioacal opening. d. Dermis.
e. Epidermis. gf. Gland tubes. 7.4. Large hair. /.v.c. Lateral vaginal
canal. m. Muscles of hair. med.v.c. Median vaginal canal. 7. Rectum.
r.s. Root sheath of single hair. s.c.é. Dense connective tissue, s.4. Small
hair. s.g. Sebaceous gland. s.g.d. Sebaceous gland duct. s.0. Sense organ.
ur. Ureter. ura. Urethra. ur.gs. Urogenital sinus. w¢.b. Body of the
uterus. w/.z. Neck of the uterus. o. Vein. vag.c. Vaginal cecum.

(All figures except Fig. 17 were drawn by the aid of the camera lucida.
For Figures 1—1]1 and 17 I am indebted to Professor Spencer.)

Puate 19, Figs. 1—12 and 17—18; Puate 20, Figs, 13—16.

Fie. 1.—Horizontal section through the skin of the back, across a typical
group of three bundles of hairs, showing large (/.4.) and small hairs (s.4.),
sebaceous gland (s.g.), and duct of same (s.g.d.). Zeiss C, oc. 2.

Fic. 2.—Portion of stratum corneum, showing two bundles of hairs, each
emerging from common follicle (¢,/:0.), and showing distinct root sheaths
(r,s.) below. Zeiss C, oc. 2.

Fic. 3.—Longitudinal vertical section through two bundles of hairs in
ischiotergal region, showing slightly thickened epidermis (e), large (/./.) and

SKIN AND REPRODUCTIVE ORGANS OF NOTORYCTES. 543

small hairs (s.4.), with separate roots and sheaths below (7.s.), and common
follicular opening (¢,f.0.) above. Also gland (s.g.) and muscles (m.). Zeiss C,
oc: 1.

Fic. 4.—Small bundle of hairs from the side of the ischiotergal patch,
showing greatly lengthened sebaceous gland, and also diminishing length of
base of hairs. Zeiss C, oc. 1.

Fic. 5.—Shows length and shape of pointed large hairs found over general
surface of body, with root attached. Zeiss A*, oc. 4.

Fie. 6.—Same hair as in Fig. 5, under higher magnification, showing solid
tip and broad shield. Zeiss ©, oe. 2.

Fic. 7.—Small hair from general body surface. Zeiss C, oc. 2.

Fie. 8.—Small hair from ischiotergal patch, showing irregular surface.
Zeiss C, oc. 2.

Fic. 9.—Large hair from ischiotergal region, showing outline and length.
Zeiss A*, oc. 2.

Fies. 10 and 11.—Two large hairs from ischiotergal patch, showing the
brush-like, deeply cleft extremity in contrast to the pointed tip of Fig. 6.
Zeiss C, oc. 2.

Fic. 12.—Section through epidermis of side of head, showing “sense
organ” (s.o.), with bundle of hairs cut transversely. Zeiss C, oc. 4.

Fics. 13—16.—Transverse sections from a complete series taken through
the female urogenital ducts, their position being indicated by section ines 1 in
Text-figure (page 337). All drawn under Zeiss A*, oc. 4.

Fig. 13.—Section No. 27 through the bladder (d/r.), vaginal ceca
(vag.c.), necks of the uteri (wé..), ureters (w.), and rectum (7.).
(Contrast Hill, 4, fig. 3.)

Fig. 14.—Section No. 19 cut obliquely, showing on one side the
opening of the uterine neck (w/.z.) into the anterior vaginal canal
(a.v.c.) to form the median vaginal canal (med.v.c.), while on the other
side the section is slightly anterior to this opening. (Cf. Hill, 4, figs. 5
and 6.)

Fig. 15.—Section No. 14 cut through septum between the two
median vaginal canals (med.v.c.) just anterior to their fusion, showing
ureters (w.) in same horizontal plane as lateral (/.2.c.) and median
(med.v.c.) vaginal canals, (Cf. Hill, 4, fig. 7.)

Fig. 16.—Section No. 10 taken through dense connective tissue
(s.c.¢.) connecting the walls of the median vaginal canals with the wall
of the urogenital sinus, showing ureters (wr.) passing ventrally to open
into the base of the bladder (/r.) close to the opening from it of
the urethra. (Cf. Hill, 4, figs. 11, 12.)

VOL. 51, PART 2.—NEW SERIES. 25

344 ~~ GEORGINA SWEET.

Fic. 17.—View of pouch cut open ventrally, showing position of the two
mamme, and the relation of the pouch to the cloacal opening (c/.0.). Xx 2.

Fic. 18.—Section through rectum of male, showing anal glands (a.g/.),
urethra, corpora cavernosa, corpus spongiosum, and gland tubes in rectal
wall. Zeiss A*, oc. 2.

PARORCHIS ACANTHUS, A NEW TREMATODE. 345

Parorchis acanthus, the Type of a new Genus
of Trematodes.

By

William Nicoll, M.A., B.Sc.,
Gatty Marine Laboratory, St. Andrews.

With Plate 21.

TE form with which I am about to deal is one possessing
several distinctive features of importance, and for that
reason I have thought it worthy of special consideration.
The following account is by no means exhaustive, but it is
sufficient to place the species beyond any doubt of recog-
nition, and to establish its claim to be regarded as the type
of the genus to which it belongs.

Parorchis acanthus, mihi.
1906. Zeugorchis acanthus, gen.andsp.n. Nicoll,
Ann. and Mag. Nat. Hist. (7), xvii, p. 519—
522, pl. xii, figs. 4, 5; xiii, figs. 6, 7.
1907. Parorchis acanthus, Nicoll. Ibid (7), xix,
p. 1268.

The original description of this form as Zeugorchis
acanthus was made from two preserved specimens found in
the bursa Fabricii of a Herring-gull (Larus argentatus).
It is incomplete and erroneous in parts. I have since

346 ... WILLIAM . NICOLL.

obtained numerous examples from the same situation, and in
the only common gull (Larus canus) which I have had an
opportunity of examining an adult individual occurred in the
rectum. The habitat is invariably the bursa Fabricii and
the rectum, never the coeca or the intestine proper. The
infection is not very numerous, there being seldom more
than a dozen parasites in one host. In several instances,
especially in young birds, examples of all sizes were found
ranging from small immature forms measuring ‘45 mm. to
fully developed adults over 4 mm. long. When removed
from the host the parasite displays considerable vitality,
several having been kept alive in distilled water over twenty-
four hours, and doubtless if a proper temperature were
maintained they would survive much longer.

The general outline of the body is roughly oval, narrower
in front and rounded behind. In the extended state the
breadth is comparatively uniform, but on contraction a
characteristic shape is assumed, like that depicted in my
original representation of the species. Three well-marked
regions are then differentiated, a small head, a stout neck,
and a broad flat posterior part. The head is always distinct,
and possesses a raised ridge surrounding the oval sucker.
The ridge forms a shoulder-like prominence on each side of
the sucker, and its ends, which do not meet ventrally, are
tucked up towards the mouth. On the edge of the ridge is
a single row of regularly-arranged spines, about sixty in
number, and having a fairly uniform length of ‘037 mm,
The ant-acetabular region or neck is thick and muscular.
Its ventral surface is flattened and beset with numerous
strong spines; the dorsal surface is more convex and devoid
of spines, except for one or two near the extreme edge of the
body. On the ventral surface there isa sort of ridge between
the genital papilla and the ventral sucker, which extends a
short distance round the sucker on each side. The post-
acetabular region is flatter and more delicate in structure.
It is not nearly so muscular nor capable of so much con-
traction as the ant-acetabular region. Its ventral surface

PARORCHIS ACANTHUS, A NEW TREMATODE. 847

has a few spines near the ventral sucker, but the dorsal
surface has none.

The usual length of adult individuals is 3—5 mm. Sexual
maturity is seldom attained below a length of 3 mm. The
various measurements which will follow have reference to an
individual of about 4mm. length. The breadth of the head is
fairly constant, ‘82—°87 mm. In the extended state the
breadth of the rest of the body is 1:2—1:4 mm., but on
contraction the posterior acetabular region may be as much
as 3 mm. broad. The average breadth is about one third
of the length of the body. In young examples the cephalic
breadth is proportionately greater than in adults. The
thickness of the body varies from ‘4 mm. in the neck to
‘8mm. at the ventral sucker. The post-acetabular region
has an average thickness of ‘5 mm.

The suckers are globular in shape and extremely muscular.
The apertures are circular. The oral sucker may attain a
diameter of ‘5 mm., while the ventral sucker is usually rather
more than twice as great. In young specimens the proportion
between the diameters of the two suckers more nearly approxi-
mates 2:3, the oral sucker being proportionately larger.

The cuticle is well developed and has an average thickness
of -005—-007 mm., although in contracted parts it may be
twice as thick. ‘The spines, where they occur, are imbedded
deeply init. The anterior spines have a length of ‘019 mm.
Passing backwards they increase in size, and may be found
as long as ‘031 mm. with a base measurement of ‘012 mm.

The alimentary system is very well developed. There is a
short thin-walled pre-pharynx, ‘11 mm. long (Plate 21, fig. 2,
p-ph.). The pharynx is muscular, and measures ‘24 by ‘17 mm.
On contraction of the animal the pharynx is thrust up inside
the pre-pharynx, and the walls of the latter instead of con-
tracting are bent down round the pharynx. The cesophagus
is about three times as long as the pharynx. Its walls are
crinkled so that numerous small dilatations are formed. In
section its dorso-ventral diameter is much greater than its
transverse diameter. ‘The epithelium of its lumen is well

348 WILLIAM NICOLL.

developed, usually in one layer, sometimes in two or three.
The bifurcation takes place a short distance in front of the
ventral sucker, almost on the same level as the genital aper-
ture. The diverticula pass round on either side of the
sucker ; behind it they are directed in towards the middle
line of the body, pursuing a more or less zigzag course.
Further back they again bend out to pass round the outer
border of the testes, and their terminal part, which is usually
somewhat dilated, again approximates the middle line. Like
the cesophagus the diverticula are wider dorso-ventrally than
from side to side. The characteristic features of the alimen-
tary system are thus its shape, the slightly sacculated condi-
tion of its walls, and the latteral flattening.

The excretory system is one of the most peculiar features
of this Trematode, and differs very much from the types
usually found. The vesical proper (Plate 21, fig. 1, Hv.) is not
of large size, but is remarkable for its shape. The outline is
very irregular, there being on each side about four short
unsymmetrical branches, which may be bifurcated at their
ends. It is situated at the posterior end of the body; the
aperture is not terminal, but is a little forward on the dorsal
surface. The excretory system comprises, in addition, two
median and two lateral trunks, which communicate pos-
teriorly with the vesicle. The median trunks are wide,
compressed dorso-ventrally, and occupy a dorsal position.
The lateral trunks are more irregular, and fill up the side-
areas of the body. Their lumen is traversed by numerous
septa, which divide it, as it were, into a system of anasto-
mosing vessels. Viewed from externally the appearance
produced is that of a mosaic of irregular patches, amongst
which the excretory fluid circulates. The Jumen of the
median trunks is not so much divided. Behind the ventral
sucker they begin to branch, and communications between
the two trunks of one side, as well as between the two median
trunks, are not infrequent. At the level of the ventral
sucker the median trunks each send out a branch, which
forms anastomosing connections almost completely surround-

PARORCHIS ACANTHUS, A NEW TREMATODE. 349

ing the sucker. In sections the sucker has thus the appear-
ance of being separated from the rest of the body by a cavity
(Plate 21, fig. 2). In front of the ventral sucker the four
trunks are no longer distinguishable, but are represented by
a number of smaller vessels, which still retain their dorsal or
lateral position. A few of these small vessels extend as far
forward as the head.

The system which has just been described probably cor-
responds to the usual simple Y-shaped or V-shaped excretory
vesicle. The remainder of the excretory apparatus does not
differ from that commonly met with. On each side of the
body there is a long narrow unbranched collecting tubule,
having a ventral situation not far to the outer side of the
intestinal diverticula. They are circular in section, and are
lined with flagella throughout the greater part of their
course. ‘Their walls are distinctly marked, and differ in this
respect from those of the excretory trunks, which have the
appearance of being mere sinuses in the connective tissue.
The fluid in the vesicle and trunks is limpid, and contains
comparatively few granules. In the living animal it can be
seen to be driven to and fro by the movements of the body.

I am not aware of any Distomid which possesses an ex-
cretory system exactly corresponding to this type. Cases in
which the limbs of the excretory vesicle are put into com-
munication with each other by means of anastomosing vessels
are not uncommon, but they are not of the same nature as
the present instance. A condition displaying more resem-
blance is found in Mesometra (Monostomum) orbicu-
laris (Rud.),! in which there is on both dorsal and ventral
surfaces a system of anastomosing canals, mapping out little
polygonal masses of parenchyma. ‘The form of the vesicle,
however, in this species differs entirely from that of
Parorchis.

The musculature is not essentially different from that of
other forms already described. Beneath the cuticle there is
a single layer of circular muscles. This is continued round

1 Cf. ‘ Bronn’s Thierreich,’ 1V, Vermes Ii, p. 650, pl. xxxi, fig. 3.

350 WILLIAM NICOLL.

the outside of the oral sucker, but is apparently absent from
the ventral sucker. Within this is a much thicker layer of
muscles having mainly a longitudinal direction. Numerous
transverse fibres stretch across the body, especially in the
anterior region.

The genital organs exhibit many features of peculiar
interest. The testes are two comparatively large bodies
measuring ‘55—'60 mm. in diameter, situated not very far
from the posterior end of the animal. They are placed side
by side, almost, in some cases quite, touching each other,
and practically on the same level. The obliquity, if any, is
very slight, the left testis being possibly a little in advance
of the right. ‘They are compressed dorso-ventrally; the
outline is roughly circular or polygonal, there being from six
to eight distinct, though not very deep, lobes on each. ‘The
ovary lies a short distance in front of the testes, almost
median or very slightly to the left of the middle line. Its
outline is a regular oval, the long axis being transverse; its
section is also oval, the long axis again being transverse. It
is smaller than the testes, from which it is separated by part
of the uterus, and measures ‘33 by*25 mm. The yolk glands
are not of very great extent. They consist of a series of
unequal-sized follicles on each side of the body, extending
from the ventral sucker to the testes. Anteriorly they are
situated close to the outer side of the intestinal diverticula.
They retain this position throughout the greater part of their
extent, but posteriorly they cross the diverticula ventrally,
and a few follicles are to be found on the inner side near the
testes. From this point the yolk-ducts run obliquely towards
the middle line in a narrow strip of connective tissue lying
between the testes and the median trunks of the excretory
system. ‘I'he shell gland is of large size, lying close to the
ovary, dorsal and behind it. ‘here is a small globular recep-
taculum seminis situated between the testes. Laurer’s canal
is present.

The vasa deferentia occupy a somewhat dorsal position in
the body. They are not symmetrically situated, one being

iE ee

PARORCHIS ACANTHUS, A NEW TREMATODE. 351

nearer the middle line and more dorsal than the other. They
unite in a fairly small, pear-shaped vesicula seminalis. This
is a simple structure, not included in the cirrus pouch, and
lying dorsal to the posterior half of the ventral sucker,
behind which it extends a short distance. It is near the
middle line of the body. The ductus ejaculatorius runs
dorsal to the ventral sucker; its termina] third is surrounded
by numerous prostatic glands. ‘The penis, in its retracted
state, is represented by a short dilated sinus, the wall of
which is beset with numerous comparatively large spines.
The penis and prostate are enclosed in a thin membrane
forming the cirrus pouch. ‘The genital aperture is situated
a short distance in front of the ventral sucker on a con-
spicuous oval papilla in the middle line of the body.

The uterus has its beginning in the triangular area bounded
by the ovary and testes. After executing several windings
between these organs it proceeds to form convolutions run-
ning transversely from one side of the body to the other.
These convolutions extend some distance to the outer side of
the intestinal diverticula. They overlap the testes to a
slight extent, but do not pass behind them. Anteriorly they
are bounded by the ventral sucker. The vagina is well
developed, situated to the left of the ductus ejaculatorius
and dorsal to the ventral sucker. It opens on the left of the
male duct at the genital aperture.

The ova are large and numerous, arranged in single file in
the uterus. They are elliptical in shape, and the shell is of
a light yellow colour. Near the ovary the ova are darker,
and present the appearance of being in the process of seg-
mentation. They measure ‘081—:095 mm. by *040—:044
mm. Development takes place rapidly, however, so that
towards the middle of the uterus the almost fully formed
larva can be observed within the egg-capsule. The latter
has by this time increased in size to about -106—:113 mm.
by °(056—-062 mm. Shortly after the extrusion of the ova,
which process I observed on one occasion,! the capsule is

1 The previously-noted occurrence of ova in the ventral sucker was, I

VOL. 51, PART 2,—NEW SERIES. 26

352 WILLIAM NICOLL.

burst open at the blunt pole and the larva is set free. This
is a small actively-moving Miracidium (Plate 21, fig. 6), not
differing much from the usual type. It measures ‘18 by ‘05
mm., and the body is differentiated into two distinct parts, a
head and a posterior part. The surface is completely covered
with long cilia. Near the centre of the head is situated a
large, dark, usually five-lobed pigment-spot (fig. 6 e.s.).

I shall now proceed to define the genus of which this
species is to be regarded as the type.

Genus Parorcuis, mihi, 1907 (= Zeuagorcnis, mihi, 1906).
Body of moderate size and roughly oval outline, in the
contracted state differentiated into three regions, head, neck,
and hinder part, varying considerably in breadth. Anterior
part of ventral surface beset with strong spines and oral
sucker surrounded by an incomplete row of spines (or spines
entirely absent ?). Anterior part of body muscular, posterior
part more delicate. Suckers well developed; ventral sucker
much larger than oral sucker. Intestine with short pre-
pharynx, powerful pharynx, cesophagus of considerable length
with irregularly sinuate walls, and diverticula which pass
round the outer side of the ventral sucker, then bend in
towards the middle of the body, and, after bending out
again, extend nearly to the hinder end; usually somewhat
dilated at their termination. Hxcretory system consisting of
a small median, irregularly-shaped vesicle posteriorly, into
which open two median and two lateral excretory vessels ;
the latter are divided by septa into numerous lacune, and as
they pass forward branch into numerous smaller vessels.
Genital aperture median in front of ventral sucker. Cirrus-
pouch includes only the penis and the pars prostatica. Penis
beset with spines. Vesicula seminalis at some distance to
the rear, small, oval, extending a short way behind the
ventral sucker. Vagina well developed. ‘Testes distinctly
lobed; situated side by side, on approximately the same
level near the hinder end of the body. Ovary transversely
believe, purely accidental. I have not observed them in that situation
again,

PARORCHIS ACANTHUS, A NEW TREMATODE. 353

oval, pretty close in front of the testes, almost median.
Shell gland well developed. Receptaculum seminis small,
between the testes. Laurer’s canal present. Yolk-glands
not extensively developed, composed of unequal follicles ;
situated for the most part close to the outer side of the
intestinal diverticula, and extending between the ventral
sucker and the testes. Uterus also confined between the
latter limits; convolutions fairly numerous, and having a
transverse direction from side to side of the body. Eggs of
large size, with light yellow shell, containing even at some
distance from the genital aperture a fully developed Miraci-
dium larva.

Habitat.—The terminal portion of the intestine of birds.

Ty pe.—Parorchis acanthus, mihi.

In my previous description of the type I made mention of
the remarkable similarity which it bears to Distomum
pittacium, Braun, apart from the fact that the latter form
entirely lacks spines. Further investigation of Parorchis
acanthus has convinced me that the two species are very
closely related, and, indeed, differ only in minor details,
The outstanding feature of distinction is the absence of
spines in Distomum pittacium. I am inclined to believe,
however, that this difference does not actually exist, and that
the spines have been removed as a result of the method of
preservation, and their traces unnoticed by Braun. As far
as I can gather, his description is based on a single specimen
from the Vienna Museum collection, and he has had no
opportunity of examining the living animal.

Immersion in a weak acid solution for even a compara-
tively short time causes the spines to disappear wholly or in
great part, and something of this nature has possibly occurred
to Braun’s specimen. The circum-oral collar, of which one
would have expected at least a trace to remain, is not repre-
sented in his figure, but, in the absence of spines, it might be
apt to be passed over. As sufficient ground for venturing
the foregoing supposition I would adduce the remarkable
similarity in the internal structure of the two forms. It is

354 WILLIAM NICOLL.

needless to recapitulate the features of resemblance; I shall
rather point out the particulars in which they differ. Braun’s
specimen measured 3°5 mm., so that it is about the same size
as an average example of Parorchis acanthus. In the
former the oral sucker is much smaller than the ventral
sucker, the diameters having a ratio of 1:3; in the latter the
ratiois nearly 1:2. In P. acanthus the pharynx is slightly
larger than that of D. pittacium, and in the latter the
testes are much smaller. As a consequence the yolk ducts
pass in front of the testes. The yolk glands do not quite
reach the ventral sucker anteriorly, but the uterus has several
convolutions on either side of the sucker as far forward as
the middle of it, and, in addition, it extends very far back,
passing even beyond the testes. The convolutions extend to
the extreme edge of the body, and thus the whole organ is
more extensive than that of Parorchisacauthus. Braun
doubts the existence of a true cirrus-pouch, but marks it in
his figure. Owing to what is, no doubt, an oversight, he
represents an aperture in the centre of the cirrus pouch (c.b.),
while on the right side of it the male duct appears to open
separately. No mention is made of the excretory system.

From the foregoing considerations there can be little
hesitation in including Distomum pittacium, Brn., in the
genus Parorchis, which, therefore, comprises P. acanthus
as type and P, pittacius (Brn.).

With regard to the systematic position of the genus I have
previously made some remarks. It displays much affinity
with the genus Pygorchis, Looss, a member of the sub-
family Philophthalmine. It might, though with some diffi-
culty, be included under this sub-family, and is, in any case,
very nearit. It agrees in the size and muscular nature of
the body, the relative sizes and development of the suckers,
the well-developed alimentary system, the position of the
genital aperture and genital glands, the situation and small
extent of the yolk glands, and the condition of the uterus
and ova. The differences consist in the spines, the cephalic
ridge, the long cesophagus, the weakly-developed cirrus

PARORCHIS ACANTHUS, A NEW TREMATODE. 355

pouch, and the symmetirical-lobed testes. Looss does not
describe the form of the excretory system in the sub-family.
The genus is thus brought into close relation with a sub-
family, from which it differs externally, and affords a good
illustration of the point which Looss! insists upon, namely,
that external character is often a fallacious guide to the
systematic position of a form.

EXPLANATION OF PLATE 21,

Illustrating Mr. William Nicoll’s paper on “ Parorchis
acanthus, the Type of a new Genus of Trematodes.”

The following letters apply to all the figures. Bs. Ventral sucker. D.St,
Yolk glands. zr. Hxcretory system. J. Intestinal diverticula. A.S¢.
Ovary. X.G. Oviduct. Z.C. Laurer’s canal. M.S. Oral sucker. Oe.
(Esophagus. PP. Penis. P.G. Genital aperture. PA. Pharynx. P.Ph.
Pre-pharynx. Pr. Prostate glands. 7. Testis. Rs. Receptaculum seminis,
S.D. Shell gland. U¢. Uterus. Vy. Vagina. V.S. Vesicula seminalis.

Fic. 1.—Parorchis acanthus, extended, slightly compressed. Ventral
aspect. Pa. Pre-acetabular ridge. Hzv', Lateral excretory trunk.

Fie. 2.—Sagittal section through anterior part of body, a little to one side
of the middle line. Pa. Pre-acetabular ridge.

Fic. 3.—Transverse section through genital aperture.
Fic. 4.—Sagittal section through ovary and shell gland; almost median.

Fic. 5.—Transverse section through receptaculum seminis and testes;
somewhat oblique. §.&. Collecting tubule.

Fic. 6.—Miracidium larva. e.s. Pigment spot.

1 ¢ Zool. Jahrb.,’ Syst. xii, p. 596.

VoL. 51, PART 2,—NEW SERIES. 27

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CONTENTS OF No. 203.—New Series.

MEMOIRS:

PAGE
The Chetognatha, or Primitive Mollusca. With a Bibliography. By
R. T. Guntur, F.L.S., F.R.G.S., Fellow of Magdalen College,
Oxford. (With 10 Text-figures) . : ‘ . B57

The Structure, Development, and Bionomies of the House-fly, Musca
domestica, Linn.—Part I. The Anatomy of the Fly. By
C. Gorpon Hewitt, M.Sec., Lecturer in Economic Zoology,
University of Manchester. (With Plates 22—26) : . 395

Trichomastix serpentis, usp. By C. Cuirrorp Dosett, B.A.,
Scholar of Trinity College, Cambridge. (With Plate 27, and 2
Text-figures) . ‘ 7 ' 3 . 449

Notes on Common Species of Trochus. By H. J. Freure and
Muriet M. Gerrines, University College, Aberystwyth. (With

Plate 28) 459

Note on the Formation of the Skeleton in the Madreporaria. By
Marta M. Ocitvir Gorpon, D.Se.(London), Ph.D.(Munich),

F.L.S. 473

Studies in Spicule Formation. WVII.—The Scleroblastic Development

of the Plate-and-Anchor Spicules of Synapta, and of the Wheel
Spicules of the Auricularia Larva. By W. Woop.ianp, The
Zoological Laboratory, King’s College, London. (With Plates 29
and 30, and 6 Text-figures) ' , ; :

SEP 17 1987

9

THE CHA TOGNATHA, OR PRIMITIVE MOLLUSCA. 307

The Chetognatha, or Primitive Mollusca.

With a Bibliography.

By

R. T. Gunther, E.L.S., F.R.G.S.,
Fellow of Magdalen College, Oxford.

With 10 Text-figures.

Iv is the purpose of the present paper to demonstrate that,
so far as our present knowledge of the Chetognatha goes,
more numerous and more cogent reasons can be given for
allying them with the Mollusca than with any other group.
We shall endeavour to show that no organ of importance
has been described in Chetognath anatomy which is not
closely paralleled by similar and, we believe, homologous
organs among the Mollusca. Indeed, we believe we can go
further and demonstrate that the divergences of structure
between the Chetognatha and the Mollusca are slighter than
those known to exist between different orders belonging to
the latter phylum. The theory of the Molluscan and Cheto-
enath affinity explains in a simple manner many facts in the
anatomy and developmental history of both groups, while
there are no well-established facts which are inconsistent
with it.

HistToricAL SKETCH.

It is not necessary to attempt here a complete review of
the stages by which our knowledge of the Cheetognatha has
VOL. 51, PART 5. —NEW SERIES. 28

358 R. T. GUNTHER.

been accumulated. Several of our predecessors have already
published such historical résumés, and so it will suffice to
refer the reader to the works of Hertwig, Langerhans, and
Grassi for fuller details than we give here.

In his original description Martin Slabber (1775) expressed
the opinion that the ‘ arrow-shaped worms” should consti-
tute a sub-division of the Linnean group of Vermes. Quoy
and Gaimard (1827), after examining the large Sagitta
bipunctata from Gibraltar, were uncertain as to whether
it was Zoophyte or Mollusc. D’Orbigny (1834), however,
decided in favour of the Mollusca, and associated the arrow-
worms with the Heteropoda, an association in which he was
followed by Milne-Edwards (1845), Troschel (1845), Siebold
(1848), and Burmeister (1856). Milne-Edwards, indeed,
went so far as to point out that the “prépuce”’ together
with the head of Sagitta were the equivalents of the Mol-
luscan head.

The anatomical work of Krohn (1844) marks a new epoch.
Having been fortunate in obtaining specimens of large size
at Messina, Krohn was able to investigate many hitherto
undiscovered details of the internal organisation, and
amongst others the nervous system, testes, and buccal
apparatus. The result was an alliance of the Arrow-worms
with the Annelida. Five years later Oersted (1849) sug-
gested that Sagitta should rather be considered as a Nema-
tode, an association favoured by Leuckart (1854) and by
several writers of zoological text-books (e.g. Claus). The
comparison rests principally upon a certain similarity of
arrangement of the musculature.

Passing over a suggestion of relationship with the Tardi-
erada and lower Arthropods by Huxley (1852 and 1878),
which has been reconsidered by Grassi (1883) on account of
fancied resemblances between the cerebral ganglia, we come
to the more serious proposal that a new subdivision of the
worms should be constituted for the reception of the Sagittee ;
and of the names proposed, that of Leuckart (1854)—Cheeto-
enatha—has taken precedence of Oesthelminthes of

THE CHETOGNATHA, OR PRIMITIVE MOLLUSCA. 399

Gegenbaur (1856) and of Pterhelminthes of Harting
(circ. 1865).

In 1857 a fantastic theory of Vertebrate affinity was put
forward by Meissner. It has, however, failed to find further
support than that accorded to it by Haeckel. Metschnikoff
(1867) included the Chetognatha in his new group of
Ambulacrata, together with the Echinodermata, Brachiopoda,
and Enteropneusta. This view has been supported by
Biitschli, who, while believing the Chetognatha to have some
affinity to the Annelida, considered that the method of the
development of the hypoblast is a sufficient justification for
regarding them as being near akin to the Echinodermata
and to the Tunicata; still more recently (1876) he has
proclaimed the Brachiopoda to be their nearest relations.

A revival of the Molluscan theory is due to Langerhans
(1878), who re-investigated the nervous system, and dis-
covered the circumcesophageal nerve ring.

In more recent times, while the Annelidan theory has
found some support, notably by Giard (1875) and Gourret
(1884), it has also found a vehement opponent in Grassi,
who maintains that among the Chetognatha there is not to
be found any trace either of the metamerisation of the body,
or of sete, or of parapodia. On turning to recent zoological
text-books we find that the majority of writers content them-
selves with expressing their uncertainty concerning the
exact position of the group in the Zoological System, but,
on the whole, there is a distinct feeling in favour of an
association with the “ Vermes.”

INTRODUCTORY.

The theory that the Chetognatha are primitive Mollusca
is on first thoughts open to the criticism that they do not
possess features which have hitherto been generally accepted
as essential to members of the Molluscan phylum, and
that the obvious points of resemblance are the results of
convergence rather than indications of phylogenetic affinity.

360 R. I. GUNTHER.

We shall endeavour to meet these objections, and to show
that the resemblances are shared by all the chief organs in the
Chetognath anatomy, namely, by the nervous, alimentary,
genital, and other systems; that also in their development
the Chetognath approaches very close to the primitive
Molluscan type, and that in the case of those features in
which the Chetognath organisation appears not to be of the
Molluscan type, interesting parallels are to be found within
the limits of the Molluscan phylum itself.

At the same time it is not proposed to merge the Cheeto-
gnatha in any one of the existing Molluscan classes; they
are rather to be regarded as modern and modified repre-
sentatives of a free-swimming ancestor from which the
Molluscan phylum has sprung. They represent, in fact,
Archimollusca modified for an active swimming life in the
open waters of the ocean, with affinities to all existing
classes of the phylum. ‘They have, to our mind, more
primitive features than the “ Archimollusc” of Lankester,
which we shall term the creeping Archimollusc to distinguish
it from our swimming type of archimollusc ; and to empha-
size this relationship we propose to consider the Chetognatha
as belonging to the lowest class in the Molluscan phylum
from which the others have sprung.

EXTERNAL ORGANISATION,

The external configuration of the Chetognath body has
justly earned for them their common name of “ Arrow-
worms.” Human ingenuity has not got further in striving
after the form best adapted to fast swimming beneath the
surface of the water. ‘he lines of the Sagitta and the
Whitehead torpedo are the same. Comparatively few of the
Mollusca, which have taken to a swimming mode of life,
have reached such perfection of outline; in all the diffi-
culty of disencumbering themselves of unwieldy shells and
feet and mantle flaps has had to be overcome, and even
when these have all gone, as in the languid Phyllirhoe, the

pacts

S2teerse

Sececs

\ =
y
4

E | \ 2
p+ - -4---- -- VISC.g: ~---- -
| : =

YM NAAN)

(6
Wotofircknss,
We era

Whee

Text-Fic. 1—General view of Chetognath anatomy. a. Side
view. B. Dorsal view of anterior region. c. Side view of
head of Sagitta macrocephala! showing the reduced hood

(Ff).

1 From a specimen captured by Mr. G. Murray on November 21st, 1898,

in lat. 52°—18/°1 N., long. 85°—53’'9 W., in 1510 fathoms.

362 R. T. GUNTHER.

result is not satisfactory. The Pteropods are the butterflies
of the sea rather than its dragon-flies; and the elaborate
hydraulics of the Cephalopods effect locomotion by a totally
different process, which we are not considering here.

The separate regions of body merge gently into one
another, and all projections unnecessary for functions of
swimming or feeding have been smoothed down. For this

Text-FIG, 2.—Grimalditeuthis (from Pelseneer, after Joubin).
f', fi’, lateral and caudal fins.

reason the visceral sac is all contained within the cigar-
shaped contour of the body-wall. In Mollusca the integu-
ment of the visceral sac is typically folded near its junction
with the head and foot, but no such mantle-flap or pallium is
distinguishable in Sagitta. The pallial ectoderm is, however,
drawn out in the horizontal plane to form the lateral and
caudal fins of many of those Dibranchiate Cephalopoda (text-
fig. 2) in which the shell is most degenerate, and the resem-
blance is strengthened by the deposition of a chitinous or

THE CHHZTOGNATHA, OR PRIMITIVE MOLLUSCA. 3638

conchyolinous substance within the fold either in the form of
fin-rays or of a continuous lamina.

If we may be permitted to imagine the alterations of
structure which a typical Molluse would undergo if it were to
become adapted for an active, free-swimming, predacious
life; I think that, judging by living examples, we should
find that they would be in the direction of the structure of
the Cheetognatha.

That foot, visceral hump, shell, and mantle cavity might
disappear, is clearly shown by the structure of Phyllirhoe.
But the Chetognatha differ from Phyllirhoe in their smaller
size, and in the far greater activity of their swimming move-
ments. ‘To these two factors we attribute the relatively large
development of certain organs, such as the longitudinal
muscular system and the non-existence of others, e.g. separate
vascular and respiratory systems respectively.

It may be premised at once that there is nothing in the
Cheetognath anatomy or development which we have felt able
to homologise with the shell or shell-gland of the Mollusca.
We cannot, however, consider that the absence of this pallial
organ is very material to the main issue, for even among
Mollusca of very moderate swimming powers the shell is
usually the first structure to dwindle and disappear (Hetero-
pods, Phyllirhoe, etc.), and might therefore be expected to be
altogether absent in a Molluse built upon such perfect lines
for swimming as Sagitta. But it may be argued, if the
ancestor of Chetognatha had a typical Molluscan shell, surely
some trace of a shell-gland would be noticeable in the younger
stages of their growth, it being so conspicuous a feature in
Molluscan developments. For its absence two explanations
may be given: firstly, by our hypothesis, the Chetognatha
are presumed to have become modified for their pelagic life
at a very early period, before the Molluscan phylum and
the Molluscan shell had been in existence for a long time, in
other words, the Cheetognatha are a far older group than any
of the shell-less Mollusca.

Tf the loss of all trace of the shell-gland may have been the

364 R. T. GUNTHER.

effect of time, its loss has no doubt also been accelerated by
the marvellous rapidity with which Cheetognath development
takes place.

SEGMENTATION.

Although the body cavity appears to be divided into
three paired cavities by septa, there is no justification
for regarding the Chetognatha as exhibiting any real
metameric segmentation at all. In the first place the two
transverse septa cannot be regarded as homologous with
one another, or with the septa which separate the meta-
meres of Annelida, because in time, mode, and purpose
of origin they are absolutely different. | Embryological
examination shows that the anterior septum is produced by
the meeting and fusion of the somatic and splanchnic meso-
blast very early in embryonic life, at a time when differentia-
tion of tissues has not begun and the mesoblast is still
continuous with the hypoblast; and that the posterior septum
appears in close connection with the genital cells, and is not
formed by the whole thickness of the mesoderm, but probably
only by the cellular envelopes of the genital cells, and cer-
tainly by the splanchnic layer exclusively.

In the light of our new knowledge concerning these
developmental phenomena, the theory of a close relationship
of Chetognatha to an unsegmented group of animals of a
high grade of organisdtion is in no way prejudiced by the
division of the body into three sections which have the guise
but not the reality of metameres.

INTEGUMENT.

The Chetognatha differ from the majority of Mollusca in
the absence of a more or less extensive ciliated epithelial
covering of the body both in the larvalandadult stages. But
though devoid of a general investment of cilia which might

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA. 3865

impede their active movements in the water, most of them
possess a circular or oval ring of ciliated cells on the dorsal
side of the head and behind the eyes. This ciliated organ is
innervated directly from the cerebral ganglion, and is believed
to have an olfactory or gustatory function. It will be re-
ferred to again as the homologue of the velum or preoral
circiet of cilia of the 'Trochophor larva of the Mollusca (text-
fig; l,.).

The external epithelium of the Cheetognatha, however,
agrees with that of the Mollusca in the presence of gland-
cells, which are especially abundant upon the ventral surface
of those littoral species, which live among algz, such as
Spadella cephaloptera; and similar cells, though less
well developed, have been noticed in Sagitta darwinii,
Grassi, and to less extent in Spadella marioni by Gourret.

The two large glands (text-fig. 1, gl.) on the upper side of
the head have been described by Gourret in Spadella
marioni. They are covered by the “hood,” and may be
variously regarded as mucous glands for the lubrication of
the hood, as poison glands for the spines, or as extra-buccal
glands. It is unlikely that their function is excretory, as
Gourret suggests.

SuUB-EPITHELIAL TISSUE.

In some species of Cheetognatha, e.g. Spadella draco, the
sub-epithelial tissue presents a peculiar vesicular condition
which is particularly noticeable in the neck region anterior
to the lateral fin. Similar cells occur in the sub-epithelial
(mesodermal) connective tissue of many Mollusca.

Foor.

There is no distinct organ in the Chetognatha which can
be compared with the Gastropod or Lamellibranch foot, but
it must be remembered that the Chetognatha are not creep-

366 R. T. GUNTHER.

ing animals, nor is there any reason to suppose that their
recent ancestors (like those of the Heteropoda) were s0, or,
indeed that they have ever had a creeping ancestor at all.
On the other hand the circumoral hood, “ Kappe” or
“ prépuce,” situated, as it is, between the head and visceral
regions, occupies the same relative position as the “foot” of
a Dibranchiate Cephalopod, or the lateral lobes of Nautilus,
and to these structures we desire to compare it. The method

Trxt-F1¢. 3.—Tremoctopus (from Pelseneer, after Joubin).
Note the interbrachial membrane.

of innervation would, no doubt, settle the question of the
homology of these organs easily were the hood a more power-
fully developed muscular organ than it is. According to our
theory the Chetognath “hood” is the Cephalopod circum-
oral foot in its most primitive condition, or else it represents
that organ in a reduced condition as a mere reduplica-
ture of the ectoderm into which the body cavity extends but
a short distance. It is therefore to be compared with the
interbrachial membrane of many of the Cephalopoda (Histio-

THE CHETOGNATHA, OR PRIMITIVE MOLLUSCA. 367

teuthis, Tremoctopus [text-fig. 3], Leioglossa) rather than
with the arms themselves. In the deep-sea form Sagitta
macrocephala it is ina much reduced condition (text-fig.
1,c,f.). In development, its bilateral origin already indicated
by the notch in the mid-ventral line is very clear, for it is
formed from two separate ectodermal thickenings, one on
either side of the mouth (Doncaster).

The two glands near its dorsal attachments may secrete
mucus of the inner surface of the hood (text-fig. 1, a, B, gl.).
According to the theory that hood and Cephalopod inter-
brachial membrane are homologous organs, these hood-glands
and the aquiferous pores of Tremoctopus and other Cephalo-
poda, cannot be regarded as homologous,

MuscuLATURE AND SKELETON.

The musculature of the Chetognatha, which has so often
been cited as their point of resemblance to the Nematoda, is
as distinct as possible from our ordinary conception of
Molluscan musculature, but among the varied members of the
Molluscan phylum there are some which present features
closely resembling the Chetognath condition.

Further, the Cheetognath muscle fibres are striped, whereas
Molluscan fibres are as a rule unstriped.! But were striped
muscle entirely unknown in living Mollusca we should not
feel inclined to attribute much importance to this difference,
believing that muscle striation is merely an indication of
efficiency from the point of view of rapidity of contraction.
Molluscs are slow in their movements, the Chetognatha are
quick, that is all. However, Spillmann has shown that well-
striated muscle may occasionally appear among the Mollusca,
for instance, in the hearts of Turbo rugosus and Acmea
virginea.

The arrangement of the longitudinal musculature in a

1 Several text-books, e.g. those by Lang and Sedgwick, are in error in
stating that true striated muscle does not occur in the Molluscan phylum,

368 R. T. GUNTHER.

sheath surrounding the viscera is an adaptation for swimming
similar to that in Phyllirhoe, whose ancestors, no doubt, pos-
sessed both foot and shell. The subdivision of the muscle

\

ml

Text-ric. 4.—Transverse sections showing musculature.
A. Chetognath (after Hertwig). 3B. Cheetoderma (after von Graff).

bands into four quadrants finds an exact counterpart in
Cheetoderma (von Graff, text-fig. 4). The homology of these

TExt-F1G. 5.—Endocephalic skeletons.
A. Spadella marioni (after Gourret). 32. Nautilus (after Lankester).

muscles with the four longitudinal retractor muscles of the
head of the Cephalopoda will be noticed later.

THE CH®TOGNATHA, OR PRIMITIVE MOLLUSCA. 369

The occurrence of three pairs of supporting plates in con-
nection with the spines on the surface of the buccal mass is
an important feature in Chetognath anatomy, but of far wider
significance is the complicated chitinous (?) skeletal piece
with its large lamella and two lateral wings serving for the
attachment of the retractor, extensor, and other muscles in
the head. his internal cephalic skeleton we believe to be
the homologue of the cartilaginous cephalic skeleton of the
Cephalopoda, and bears a superficial resemblance to that of
Nautilus (text-fig. 5). Its presence in addition to the many
other points of resemblance between the two groups should
be something more than a mere case of analogy.

Nervous System.

The remarkable resemblance of the Chetognath and
Molluscan nervous systems was recognised as long ago as
1844 by Krohn, the discoverer of the nervous system in
Sagitta hexaptera, and again by Langerhans, who dis-
covered the buccal ganglia. The two most recent accounts
by Hertwig and Gourret are not entirely in agreement, but
both may be readily referred to the Molluscan plan. Indeed,
the resemblance is so close that, in a description of the
Cheetognath nervous system, the Molluscan terminology may
be appropriately employed.

Certain characteristics which are primitive among the
Mollusca are retained in the nervous system of the Cheeto-
gnatha, but these co-exist with others which appear to be
directly associated with the more active mode of life of the
latter. In two features, namely, in the widely distributed
nerve plexus which enwraps the body, and in the close
adhesion of the central nervous system to the ectoderm, the
Cheetognatha are more primitive than the Mollusca; the
ladder-like arrangement of the nerves that occurs in
Amphineura, for instance, is not a Chetognath feature,
although the nerves issuing from the ventral ganglion are

370 R. T. GUNTHER.

highly suggestive of it. The uniform sheathing of ganglionic
cells, indicating lack of concentration, which gives peculiar
interest to primitive nerve-cords, as in the Chitonide, is not
to be found in Cheetognatha, whose active movements demand
a more perfect apparatus for their co-ordination, aud in

1. CHA TOGNATHA

Visc.

2. NAUTILUS. Opt.

Ped. Visc.

Text-Fic. 6.—Nervous systems. Bucc. Buceal ganglion; Ph.
Pharyngeal ganglion; Ped. Pedal ganglion; Vise. Visceral or
ventral ganglion of Chetognatha.

which a more centralised nervous system has accordingly
been evolved.

The cerebral ganglion, consisting of a median group of
nerve-cells and of two lateral ones joined by commissural
fibres, gives off posteriorly a pair of rhinophoral and a pair
of optic nerves to the olfactory organs and eyes respectively,

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA. 371

and the great cerebro-visceral connectives which lead back
to the ‘‘ ventral” or visceral ganglion. Lateral extensions of
the cerebral ganglion give off two nerves, which are, at any
rate, partly motor, to the cephalic or buccal muscles, and a
pair of lateral nerves to the integument of the head.

To demonstrate the existence of ventral commissures below
the cesophagus is not always an easy matter. Langerhans
definitely affirmed the presence of a circumcesophageal ring
completed by a subcutaneous commissural nerve just behind
the mouth, a statement which Hertwig was not able to
confirm. My own observations on Neapolitan material have
led me to believe that there is a commissural plexus of
ganglion cells beneath the skin, which, if more concentrated,
might give rise to a commissural nerve strand such as
Langerhans figures. Such a nerve-loop is to be compared
with the stomatogastric loop of the Mollusca, the commissural
nerves of which are often extremely attenuated, and, owing
to their position on the buccal muscles, difficult to demon-
strate; in Nautilus, for example, the completion of the
buccal nerve-loop has only been recently proved by Graham
Kerr. The co-existence of two pairs of ganglia, which may
be termed buccal and pharyngeal in Cheetognatha, in many
Gastropoda (e.g. Patella), and in Nautilus is a significant
feature.

The descriptions of Hertwig and of Gourret differ in
regard to the mode of origin of certain nerves from the
cerebral mass, but they may be reconciled if we suppose the
cerebro-buccal connectives and motor nerves to the muscles
of the mandibles, to run side by side for a short distance
after leaving the cerebral ganglion in Sagitta hexaptera
(Hertwig), but to be separate from the start in Spadella
marioni (Gourret).

If it could be established that the buccal ganglia of the
Cheetognatha are derived from the stomodzal ectoderm the
resemblance to the Mollusca would be complete.

Concerning the existence of the posterior or visceral loop
there can be no doubt. The visceral ganglia appear early as

372 R. T. GUNTHER.

ectodermal thickenings of relatively enormous size. Though
at first lateral in position, they soon become approximated,
and give rise to the “ventral ganglion,’ which, however,
never loses its primitive character of lying immediately
beneath the ectoderm.

The pedal ganglia and their commissural loop do not exist
in any conspicuous form, nor should we expect to find them
in the absence of a well-developed and muscular foot. It
might be possible to trace their homologues by making a
more minute study of the innervation of the hood; but at
present we merely suggest that cells corresponding to those
of Molluscan pedal ganglia may be merged in the lateral
expansions of the cerebral ganglion already mentioned.
Von Jhering, on the other hand, believed that the ‘ ventral
ganglion” consisted of visceral and pedal ganglia united.

Pedal ganglia and nerves excepted, the Chetognath
nervous system admits of a close comparison in point of
detail with that of Mollusca, but the particular type to which
it exhibits the closest affinity is that of Nautilus, as will be
seen by reference to the diagrams (text-fig. 6).

A peculiar feature in Nautilus is the undivided nature of
the cerebral ganglion, which is also found in Chetognatha,
though unusual among Mollusca. The chief differences are
the absence of the pedal loop, the more centralised arrange-
ment of ganglionic cells on the visceral loop, the close
adhesion of the nervous system to the ectoderm and the
superficial nerve plexus.

SENSE ORGANS.

The tactile organs of Chetognatha consist of isolated
neuro-epithelial cells terminating in tactile hairs, and of
groups of tactile bristles which belong to a type of sense
organ which is widely distributed among Mollusca.

The “olfactory”’ (?) ring of ciliated cells and the area within
it (text-fig. 1, v.) is undoubtedly an organ of great importance.

Tima

THE CHETOGNATHA, OR PRIMITIVE MOLLUSCA. 378

It seems to be the homologue of the velum and the rhino-
phoral organs enclosed within it of the Mollusc, for the
innervation from the cerebral ganglion by two nerves lying
just within the optic nerves, is precisely similar. It is
possible that the minute organs which Weiss has described
in certain Oigopsid Cephalopoda are to be compared with
them. And, in this connection, both the tentacles of
Spadella cephaloptera and the olfactory spoon-shaped
organs of Chiroteuthis should be made objects of further
study.

The eyes are undoubtedly of the Molluscan vesicular type.
The retinal cells contain rods; the crystalline lens is secreted
within an invagination of the ectoderm, which has become
overgrown by a flattened epithelium. The fact that in the
Molluscan eye the pigment forms a complete cup to the
retina, but is mainly restricted to one side of the ocellus in
Sagitta, is a minor point resulting from the peculiar method
of the grouping of Chetognath eyes in threes.

As in the Amphineura, otocysts are unknown.

ALIMENTARY CANAL.

If our hypothesis of the origin of the Mollusca be correct,
it follows that the straight and symmetrical alimentary canal
of the Chetognatha is in a more primitive condition than
that of any other Mollusc. Excepting for the absence of the
radula, an organ better adapted to quiet browsing than to
predatory Cheetognath habits, the buccal armature is singu-
larly like the armature which predatory Molluscs develop.
The resemblance both in position and structure between the
lateral groups of mandibles of a Gymnosomatous Pteropod,
such as Clio (text-fig. 7, h.) and the “hooks” of Sagitta, is very
close. No chitinous teeth are developed in the mid-ventral
line, but the repeated series of spines in marginal and lateral
groups above the entrance to the buccal cavity suggests a
possibility of the successional development and replacement

VoL. 51, PART 3,—NEW SERIES. 29

374 R. T. GUNTHER.

of chitinous elements near the mouth. And if this were to
happen in the mid-ventral line a radula would result. At

Trxt-Fic. 7.—Buccal armature of Clio borealis.
h. “Hooks.” 7. Radula.

Trext-Fic. 8.—Buccal armature of Chetognatha and Amphineura.
a. Krohnia hamata (after Fowler). 4. Sagitta serrato-dentata.
ce. Sagitta bipunctata (after Fowler). d. Lepidomenia (after
Kowalewsky). e. Proneomenia (after Hubrecht),

the same time it must be remembered that the radula is
absent in several Amphineura and in Lamellibranchia, in the

THE CHETOGNATHA, OR PRIMITIVE MOLLUSCA. 375

Leioglossal Cephalopoda, and that it even tends to disappear
in certain groups of carnivorous Gastropoda.

A comparison between the forms of teeth characteristic of
Chetognatha and of Amphineura is instructive (text-fig. 8).

Compare the saw-like hooks of Krohnia hamata and S.
serrato-dentata (a, b) with a tooth of Lepidomenia (d),
and the teeth of S. bipunctata (c) with those of Proneo-
menia (e).

No special salivary glands have been noticed, but the
alimentary canal of Sagitta cephaloptera is furnished
with two (liver?) diverticula (text-fig. 1, a, h.).

GENITAL ORGANS.

The genital organs of the Chetognatha are situated in the
hinder part of the visceral sac. The gonads are paired and
hermaphrodite, but the male and female gonads develop in
separate cavities, and pass to the exterior by separate paired
ducts. ‘The Mollusca too are typically hermaphrodite, and
although there are some reasons for regarding the dicecious
condition as primitive in the group, both ova and sperma-
tozoa are derived from the epithelial lining of the same
cavity; but in Mollusca belonging to very different groups
there is a tendency for the male and female gonads to
develop apart, either in separate male and female acini
(Pleurobranchide, Nudibranchs, Cardium oblongum,
etc.), or in separate regions of the same gland (Cycladide),
or in different glands (Anatinacea and Septibranchia). The
longitudinal partition between the paired gonadial cavities
of the Chetognatha reminds us of the division between the
paired gonads of the Aplacophora, and is typical, there is
good reason to believe, of the primitive condition in Mollusca
(text-fig. 10).

Thanks to the minute observations of Doncaster we are
able to draw attention to an astonishing similarity between
the behaviour of the gonads both of the Mollusca and of the
Cheetognatha, which we believe may throw a new light upon
the morphology of the transverse septum of the latter group.

376 R. T. GUNTHER.

The following are the details of the process as they have
been described among Mollusca :

In Chiton, Haller observed the young ova to sink below the
ovarial epithelium, and then growing larger bulge it out, so
as to form a follicle. By a process of growth each egg-cell
gets carried out into the perigonadial ccelom on the end of
a stalk made by the follicular epithelium.

In the Cephalopoda the same process obtains. In Nautilus
the result is exactly as in Chiton. In Octopus, Argonauta,
and others a higher grade of complexity is reached by the
formation of branches from the simple egg-stalks, resulting
in the production of ‘‘egg-trees.” In the Oigopside the
region that bears the egg-stalks projects right across the
genital cavity as a “ spindle-shaped body beset all over with
stalked eggs.”

In Sagitta the process is as follows: The genital cells in
the embryo lie close to the longitudinal partition, bedded in
mesoblast, one behind the other (Doncaster, figs. 14, 17).
At a particular stage they become enclosed in a sort ef
cellular envelope of mesoderm, which does not yet form a
definite epithelial sheath; a condition to be compared with
the stage in Chiton and other Molluscs before stalk-formation
has commenced. Fig. 15 of Doncaster’s paper shows some
of these follicular nuclei closely adpressed to a genital cell.
About the fourth day genital cells which have lain quiet for
some time ‘‘ move slowly across the coelomic cavity until they
reach the body-wall on each side.” While traversing the
colom the male and female cells on each side move together,
and during their progress the transverse septum is formed
between them by the cells, which we homologise with
the follicular cells of Mollusca. ‘The process of the forma-
tion of the transverse septum is therefore absolutely distinct
and unlike the process of the growth of septa in Annelids,
and although it is impossible to say whether the genital cells
themselves or the follicle cells which lie about them are the
vere cause of the movement, yet the fact of its occurrence
in the two groups we are considering is significant.

THE CHETOGNATHA, OR PRIMITIVE MOLLUSCA. 377

The origin of the genital cells side by side from the walls
of the same cavity, and the subsequent division of this
cavity into anterior and posterior parts by a septum of quite
peculiar growth, may be given as reasons against the view
that the so-called trunk and tail cavities of Cheetognatha are
indications of metamerism.

Again, if the trunk and tail cavities were homomeric, we
should expect to find their ducts also homomeric. ‘This is
certainly not the case, for the efferent genital ducts of the
Cheetognatha are essentially different both in structure and

/

°
SETTING

Ore: Soe \

PS

STLULA WTO
WV Aerzerea
i
\ oe ry
AA at a
penny Liane é

TrExt-FIG. 9.—Ova and follicle cells.

A. Chetognath (after Hertwig). 3B. Lamellibranch (after Pelsencer).
in development. ‘he sperm-ducts being ciliated, and the
oviducts being devoid of ciliation, was clearly established by
Hertwig in 1880, and should have made later writers hesitate
before adopting a theory of metameric segmentation; but
quite recently the embryological studies of Doncaster have
indicated that the two ducts are derived from different germ-
layers—the sperm duct from epiblast, and the oviduct from
mesoblast.

We suggest that the oviduct of Chetognatha is merely a
gonoceel, partly lined by the follicular epithelium covering
the egg-cells, which has acquired an opening of its own to
the exterior (text-fig. 9).

378 R. T. GUNTHER.

In the absence of complicated copulatory organs the
Cheetognatha resemble the Amphineura.

The spermatozoa are pin shaped, as in many Mollusca.

When we come to compare the arrangement of body
cavities, genital organs, and their ducts in the Cheto-
gnatha with the various arrangements of these organs among
the Mollusca, we find that the same structural plan is
common to both groups, and that in the Chetognatha certain
primitive features are retained (text-fig. 10).

The ancestor common to both Chetognatha and Mollusca
had, we imagine, paired but unsegmented ccelomic spaces.
Gonad mother-cells developed in each half, and were passed
to the exterior through paired ccelomoducts, which also
served for the discharge of renal products. This ancestor
we believe to have been hermaphrodite (text-fig. 10, a).

In the Aplacophoran Proneomenia, Chetognatha, and
Cephalopoda certain of these primitive conditions persist,
but not all in the same form.

In Proneomenia the ccelom has become restricted to a
relatively small portion of the whole body. The anterior
ends of the paired ccelomic spaces remain separate as
gonoceels; the posterior portions have become confluent,
forming the pericardium, which communicates with the
exterior by right and left coelomoducts, which serve the
double purpose of genital and renal ducts (text-fig. 10, c).

In Cephalopoda the posterior portion of the ccelom is
the gonocoel; it is undivided, and communicates with the
exterior by a pair of gonad ducts (Dp). The anterior portion of
the ccelom is paired in Octopoda, and forms the two pericar-
dial spaces, each of which has its own duct to the exterior (8).

In Chiton the gonadial ccelom has become separated from
the posterior pericardial coelom, and each has its own pair of
ducts to the exterior (F).

The universal occurrence of paired ducts in these primitive
types is, we think, explained by the theory that in the
common ancestor the coelomic spaces were divided by a
median longitudinal partition.

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA.

ae

U)
pare
ey]

ae

C. APLACOPHORA. D. Nauticus.

cc

E. @ OCTOPODA. F. POLYPLACOPHORA.

Trxt-F1G. 10.—Body cavities.

The dotted line represents the ccelomic epithelium. ge. Pericardial

celom ; gc. Perigonadial ccelom.

379

380 R. T. GUNTHER.

In Sagitta this primitive independence of right and left
coelomic spaces is retained, but the male and female portions
of the hermaphrodite gland have become separated by the
transverse genital septum, and a second pair of gonad ducts
has come into existence in consequence (text-fig. 10, B).

See Addendum, page 394.

DEVELOPMENT.

It now remains to be demonstrated that the developmental
histories of the Chetognatha and Mollusca are not so diverse
as has been usually supposed, for many close comparisons
are possible between them.

In the larval stage the Chetognatha have no velum or
other trace of external ciliation, and, in this respect, they
resemble the Cephalopoda. The late appearance of cilia may
be accounted for by the early development taking place inside
an egg-shell, and by the larva, on hatching, being already so
advanced as to have its adult swimming organs sufficiently
developed. The typical Mollusc, on the other hand, hatches
at an earlier stage, and goes through a period of swimming
by cilia before settling down to the method of locomotion
peculiar to its adult condition. So, although we have no
evidence that the Chetognath ancestor was provided with any
ciliary swimming organ, such a thing is very probable; and
Grassi’s suggestion that the ciliated olfactory ring is the sur-
vival of a larval organ (although he did not indicate the right
one) may not be far from the truth.

We will now pass to the chief features of resemblance in
the embryology. The invaginate gastrulais common to both.
The mouth has been proved to develop by a stomodaal
invagination at the pole opposite the blastopore both in those
Gastropoda which have little food-yolk and in the Cheeto-
gnath, and the same statement is true of the Scaphopoda and
Lamellibranchia and of Dondersia, though not of Chiton.

In Mollusca the mesoblast cells usually arrange themselves
in two lateral masses, in which paired ccelomic cavities
subsequently appear as schizocoelic spaces. Hrlanger was

=e are=>

EE

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA. 381

certainly in error in attributing an enteroccelic origin to
them in Paludina vivipara, and his statement has been
refuted by Ténniges—and this is practically identical with
what happens in Sagitta, for even here the lateral meso-
dermal pouches lose their enteroceelic lumen, according to
Doncaster, and the permanent coelomic spaces appear as
splits at a later stage. ‘The only important point of differ-
ence is that the paired lateral ccelomic spaces of Sagitta
remain separate throughout life, the splanchnic mesoblast
giving rise to the dorsal and ventral mesenteries, whereas in
the Mollusca they generally become confluent, although in
every case indications of their paired origin may be perceived
in the fact that all organs derived from them are typically
paired.

The four large cells which he at the posterior end of meso-
blastic pouches in a young Sagitta larva remind us of the
first large mesoderm cells which are so conspicuous in the
larvee of Chiton, Teredo, and other Mollusca. Both ulti-
mately give rise to the genital cells, but in the Chetognath
these cells do not form anything but ova and certain ovarian
structures (perhaps the oviduct). In the Mollusca the large
cells give rise to other mesodermic structures as well.

The resemblances between Chetognath and Molluscan
nervous systems have already been described; it remains
but to observe that in their development they are absolutely
identical. ‘he dorsal and ventral ganglia appear as inde-
pendent local thickenings of the ectoderm in which a
considerable proliferation of nuclei takes place. A fact of
importance is that the “ventral ganglion” of Sagitta, like
the visceral ganglia of Mollusca, is paired in its origin,
being derived from two cell-bands on the ventro-lateral
regions of the body (Doncaster).

The buccal nervous system was said to be mesodermal by
Hertwig, but this statement needs further confirmation. The
development of the buccal ganglia from the stomodeal
ectoderm has been demonstrated in Paludina, and a similar
origin is possible in Sagitta.

382 R. T. GUNTHER.

The origin of the hood from two lateral ectodermal thicken-
ings on either side of the mouth has already been described.

The fate of the mesoderm and of its paired cavities is of
the highest importance. In Sagitta, after a transient stage,
in which the body cavities are entirely obliterated, two bilate-
rally symmetrical pairs of cavities appear as schizoccels, the
head cavities and the gonadial cavities respectively. The
chief function of the mesoblast of the anterior or head pair of
cavities is apparently to give rise to the musculature of the
pharyngeal apparatus. The cavities themselves during the
process of growth become extended into the base of the hood
thus proving this organ not to be purely ectodermal in origin,
a fact which is of considerable importance from the point of
view of homology with the interbrachial membrane of the
Cephalopoda.

The fate of the perigonadial coelomic cavities is very similar
in Chetognatha and Mollusca, and, in our opinion, the
Cheetognath type of ccelom, gonads and their ducts represents
the ancestral Molluscan condition with greater exactitude
than any scheme hitherto proposed.

In spite of the great variations in the arrangement of the
Molluscan ccelom it is generally agreed that the most primi-
tive condition is that in which the gonadial and reno-pericardial
cavities are united as in the Cephalopoda and in the Aplaco-
phoran Amphineura, as in a modified degree in the more
archaic Gastropoda (Trochus) and Lamellibranchia (Soleno-
mya), and as is indicated by the origin of the gonads from the
wall of the pericardial space in Paludina and Dreissensia.

This view has been expressed by Dr. Pelseneer in a series
of diagrams indicating the probable transformations of the
genital duct (Mollusca, fig. 5 bis, p. 14), but in delineating
the “ancestral hypothetical form’? Dr. Pelseneer has not
carried the history as far back as is now possible, for he has
omitted to emphasise the real cause of the bilateral symmetry
of the parts, namely, the primitive independence of right and
left coelomic cavities, necessitating the development of inde-
pendent right and left ducts whether genital or renal.

ree ea
— ——

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA. 383

When, as is typically the case in Mollusca, the right and
left coelomic cavities become confluent, one of the original
pair of ducts may be dispensed with. Confirmatory evidence
for the primitive separation of the ccelomic cavities into right
and left among Mollusca is to be found in the paired structure
of the gonads of Aplacophora and Lamellibranchia, in the
paired origin of the pericardial ccelom in Paludina, and in
the very widely-distributed, paired, renal cavities. In the
Chetognatha the primitive division of ccelomic spaces into
right and left halves is retained throughout life.

Thus far our comparison lies on certain ground. The next
point toe be considered is the significance of the division of
the perigonadial ccelom by the transverse genital septum in
the Chetognatha, and this is capable of more than one inter-
pretation. If we regard this genital septum as a division
between two homomeric segments we shall adopt the simplest
explanation. The condition of transversely-divided coelomic
spaces in Chiton would be evolved from the Cheetognath con-
dition on the assumption that the posterior cavity has lost
the power of developing gonads, and that its ducts have
become purely renal in function.

The genital ccelom of Chiton would, on this theory, be the
homologue of the anterior pair of body cavities of Sagitta.

Unfortunately the observed facts of development do not
support the view of a metameric repetition of these cavities.
The genital rudiments in Mollusca first appear as ridges on
the pericardial walls, and therefore in a cavity which is not
metamerically repeated, and grave doubts have been recently
thrown upon the view that the genital septum of Sagitta is
an indication of metameric segmentation by the above-
mentioned observations of Doncaster.

We are thus led to the conclusion that Sagitta and primi-
tive Mollusca, like the Aplacophora, had but a single pair of
gonadial-renal-ccelomic cavities, into which the hermaphrodite
gonads were dehisced and from which they passed to the
exterior by a single pair of ducts, together with the nitro-
genous waste. By the differentiation of the walls of this

384 R. T. GUNTHER.

cavity and its ducts into excretory and gonad-bearing portions
we arrive at the condition typical of the higher types of
Mollusca in which a second pair of ducts is developed in con-
nection with the discharge of the genital products. Such is
the condition in the Cephalopoda in which the gonadial peri-
cardial cavity remains undivided, but in the Polyplacophora
the gonadial ccelom has become completely separated from
the renal celom. In the Chetognatha the gonadial coelomic
spaces have become separated for the purpose of dividing
the male and female sexual cells (text-fig. 10).

Both Cheetognatha and Mollusca are hermaphrodite. The
parent germ-cells originate at the same time, and are indis-
tinguishable. It might be suggested that the determining
factors of sex only come into operation after the germ-cells
have migrated into separate cavities in which different nutri-
tive conditions prevail.

CONCLUSIONS.

The very numerous and close resemblances which exist
between the Chetognatha and every class of Mollusca show
that there is no single structure, or absence of structure, of
importance in the Chetognath anatomy which is not capable
of being developed somewhere within the limits of the
Molluscan phylum, and that many apparently insignifi-
cant features have their exact counterparts therein. In
short, granted similar conditions of life, a Chetognath type
might be expected to arise from the Molluscan stock. On
the other hand, the claims which have been put forward in
favour of the alliance of the Chetognatha with the Annelida
or Nematoda are not capable of being supported by so
extensive or consistent an argument.

Can the Chetognatha be definitely associated with any
one class of Mollusca, or are their relationships more
general ?

The following morphological characters are among those
obviously of importance :

THE CH@TOGNATHA, OR PRIMITIVE MOLLUSCA. 3885

1. The original bilateral symmetry of the Mollusca is
presented by the Chetognatha in its most perfect form,
especially in respect of the body cavities.

2. The Chetognatha resemble many Mollusca of undoubt-
edly primitive type, in the absence of apparent segmenta-
tion.

3. The vermiform shape of the body, recalling that of the
Amphineura aplacophora.

4, No extensive hemoccel has been hitherto identified.

5. The alimentary canal is straight; the anus opens in
front of part of the visceral sac.

6. There is no evidence of a radula, either in the Cheto-
enatha or in their ancestors. ‘The buccal armature is other-
wise very like that of many Mollusca.

7. The nervous system is of the Molluscan type.

8. The “hood,” the suggested homologue of the circum-
oral Cephalopod “ foot.”

9. The growth of the genital cells within a follicular
epithelium and upon stalks. Hermaphroditism,.

10. The two pairs of openings from the perigonadial
coelom to the exterior. These are believed to be the homo-
logues of the two pairs of ducts leading from the pericardial-
gonadial ccelom of the more primitive Mollusca.

11. The cephalic endo-skeleton and lateral fins.

12. The preoral ciliated ring ; the suggested homologue of
the velum.

The characters of the Chestognatha are, on the whole,
just those of the more archiac types of Mollusca rather than
of the Gastropoda or Lamellibranchia. Their affinity to the
Aplacophoran Amphineura is indicated by the vermiform
shape and bilateral symmetry of the body, by the straight
alimentary canal, by many negative characters, such as
absence of shell, foot, and radula. The Amphineura may
have a more primitive nervous system, but in the Cheeto-
enatha the paired arrangement of body cavities is the more
primitive. Molluscan nephridia and Chaetognath sperm-ducts
would appear to be homologous structures, but the genital

386 R. T. GUNTHER.

ducts of Chiton may only be the analogues of the oviducts
of Sagitta.

The resemblance to certain Cephalopoda is also surpris-
ingly close. The body-cavity of the Chetognatha is in the
more primitive condition, in that it remains divided longi-
tudinally throughout life, but owing to the fact that the
Cephalopoda are dicecious, there is not the same need for a
separation of the male and female gonads by a genital
septum that there apparently is among Chetognatha. The
nervous system of Nautilus and Chetognatha are both modi-
fications of the same plan. Endocephalic skeletons, germ-
cells mounted on stalks, and last, but not least, the bilateral
“ foot,” hood-like and surrounding the mouth, are common to
both Nautilus and Cheetognatha.

Another very remarkable fact is that the peculiar charac-
teristics of the Leioglossal Octopods—no radula, arms united
by a membrane, fins developed on the sides of the body—
are all characteristics of Sagitta.

Although a few naturalists have already suggested that
the nervous system of the Mollusca and the Chetognatha
may indicate relationship, no one has hitherto pointed out
how perfect is the resemblance between the other organs of
the two groups.

We have in the Chetognatha the key to many Molluscan
mysteries, not the least important of which is the develop-
ment of the Cephalopoda, which, owing to the presence of a
large mass of inert food-yolk, has been profoundly modified.
In fact, in the matter of developmental history, Sagitta
would seem to bear the same relationship to the Cephalopoda
that Amphioxus bears to the higher Vertebrata.

If our theory be correct the Chetognath type of develop-
ment may be that of the Pre-Silurian ancestors of Nautilus,
and probably of the earlier straight-bodied Cephalopoda,
such as the Orthoceratidee and the Bactritide.

A further inference of far-reaching importance is that
the crawling Archimolluse of Lankester was not the common
ancestor of the entire Molluscan phylum, but only of the

THE CHETOGNATHA, OR PRIMITIVE MOLLUSCA. 387

Polyplacophor-Gastropod-Lamellibranch section, and _ that
the Cephalopoda have never had a creeping ancestor at all ;
and, in our opinion, the Creeping Archimolluse itself has
been derived from a Swimming Archimolluse from which
the Amphineura, Aplacophora, and Cephalopoda have been
independently evolved, and of which the Chetognatha are
the nearest living representatives.

Several groups of Mollusca descended from the creeping
Archimolluse have again taken to a predatory life among
the Necton and Plankton, and have apparently thrown back
to the earlier swimming Archimolluscan ancestor in several
respects. It is thus that characteristics common to the
Cheetognatha and the Pteropoda are to be explained. The
hereditary taint of asymmetry, however, is never lost ; even
in Phyllirhoe the anus and the genital organs remain on one
side of the body.

In the light of the undoubtedly primitive nature of the
Chetognatha, the negative characteristics of the Aplacophora
may well be reconsidered. Pelseneer and others consider
that the Polyplacophora present the most archaic characters
among the Amphineura, but until we have good evidence to
the contrary there is at least equal justification for the view
that certain structures in the Aplacophora are in an incipient
rather than in a reduced condition. Foot, radula, and shell
may never have attaimed to. a higher grade of development
among their ancestors than that which they have reached
among the living Aplacophora.

The association of the Chetognatha with the Mollusca does
not throw much hght on the position of Rhodope. The
absence of heart, shell, radula, and foot are not, in our
opinion, insuperable obstacles to including this curious minute
form inthe Molluscan phylum, but the fact of the presence
of flame cells opens a vista of new difficulties.

PHYLOGENY AND CLASSIFICATION.

By the inclusion of the Cheetognatha within the Molluscan
phylum no new suggestion is indicated as to the connection

388 R. T. GUNTHER.

of the phylum with any other division of the animal kingdom,
but the classes within the phylum will require some re-
adjustment. ~

Our phylogenetic speculations are influenced by the follow-
ing facts :

A. Mollusca typically pass through a_ free-swimming,
bilaterally, symmetrical, “ veliger” stage.

B. In creeping and sessile forms the foot and shell reach
their highest development.

c. In free-swimming marine forms the shell tends to atrophy,
and the creeping foot tends to become either a swimming
organ or to disappear.

It may therefore be fairly argued thatif a race of Mollusca
had always been free swimming, either foot or shell might be
absent.

The Chetognatha may therefore be fairly regarded as the
living adult representatives of the phyletic stage indicated
by the veliger larva, and it is from such an ancestor that we
conceive the creeping Polyplacophora, the worm-like A placo-
phora, and the swimming Cephalopoda to have been inde-
pendently derived.

A scheme of classification which would represent this view
would be—

Phylum.—MOLLUSCA.
Grade a.—Nectomalacia or Mollusca natantia,
Mollusca in which the primitive free-swimming habit
has been retained : “ foot” circumoral, a propodium
developed from paired lateral Anlage.
Class 1. Cheetognatha.
Without shell.
», 2. Cephalopoda.
With shell.

Grade n.—Herpetomalacia or Mollusca reptantia.
Mollusca in which a creeping habit has been developed :
foot postoral, a metapodium, developed as an
unpaired median structure.

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA. 3889

Class 5. Amphineura Aplacophora.
Without shell.
», 4. Amphineura Polyplacophora.
Shells octuple.

Or

. Lamellibranchia.
Shell bivalve.
. Gastropoda.
Shell single.
. Scaphopoda.
Shell single, tubular.

3)

o>

a)

J

a)

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1897.

1897.

THE CHATOGNATHA, OR PRIMITIVE MOLLUSCA. 393

LanceRuANS, P.—‘‘ Die Wurmfauna von Madeira,” ‘Zeitschr. wiss.
Zool.,’ xxxiv, pp. 132-6, pl. vi, figs. 50, 57, 60.

Hertwic, O.—“ Die Chetognathen,” ‘Jen. Zeitschr. Nat.,’ xiv, pp.
196—3811, pls. ix—xiv.

Grassi, B.—“‘Intorno ai Chetognati,” ‘Att. R. Ist. Lomb.,’ xiv,
fase. 6.

“T Chetognati,” ‘Fauna u. Flora des Golfes von Neapel,’ v.

Levinsen, G. M. R.—‘‘ Systematisk geografik Oversigt over de nordiske
Annulata, Gephyrea, Chetognathi og Balanoglossi,” ‘ Vid. Med.,’
pp. 160—251.

Gourret, P.—‘‘Considérations sur la faune pélagique du Golfe du
Marseille suivies d’une étude anatomique et zoologique de la
Spadella marioni,” ‘Ann. Mus. Hist. Nat. Marseille,’ ii.

* Recherches sur Anatomie et |’Histologie de la Spadella
marioni,” ‘C. R. de l’Inst.,’ xevii, pp. 861-4.

“Ta cavité du corps et les organes sexuels de la Spadella,”’
ibid., pp. 1017-9.

Jourpain, M.S.—‘‘On the Embryogeny of Sagitta,” ‘ Comptes Rendus,’
exiv, pp. 28, 29; abstract in ‘Ann. Nat. Hist.,’ ix, 1892, pp. 415-16.

StroptMann, S.—‘*‘ Die Systematik die Chetognathen und die Ver-
breitung der einzelnen Arten im Atlantischen Ocean,” ‘ Archiv f,
Naturg.,” lviii, p. 333.

Béraneck, E.—‘‘ Les Chétognaths de la Baie d’Amboine,” ‘Rev. ~
Suisse,’ iii, fase. i.

Conant, F. S.—* Description of two new Chetognaths (Spadella
schizoptera and Sagitta hispida),” ‘Johns Hopkins Univ.
Cire.,’ xiv, pp. 77,78; ‘Ann. Nat. Hist.,’ xvi, pp. 288—292, 2 figs.

AuRiviuius, C.—“ Das Plankton der Baffins Bay und Davis Strait,”
‘ Festskrift for Liljeborg,’ p. 188.

Conant, F. S.—‘‘ Notes on the Chetognaths,” ‘Johns Hopkins Univ.
Cire.,’ xv, pp. 82—85; ‘Ann. Nat. Hist.,’ xviii, pp. 201—214.

Fowter, G. H.—* On the Plankton of the Faeroe Channel,” ‘Proc.
Zool. Soc.,’ 1896.

Strmuaus, O.— Die Verbreitung der Chetognathen im siidatlan-
tischen und indischen Ozean,” ‘ Inaug. Dissertation,’ Kiel.

Aina, T.— Chetognaths of Misaki Harbour,” ‘ Annot. Zool. Japon,’
1; p: 1d.

Cnun, C.—‘ Beziehungen zwischen dem arktischen und antarctischen
Plankton,’ 8vo, Stuttgart.

394,

1900.

1900.

1902.

1903.

1903.

1903.

1905.

1905.

R. T. GUNTHER.

Doncaster, L.—‘ Chetognatha,” ‘Fauna Maldive and Laccadive
Archipelagoes,’ i, p. 209.

Sremvuaus, O.— Cheetognathen,” ‘ Hamb. Magelhaens Sammelreise,’
Vv.

Hessz, R.—“ Untersuchungen iiber die Organe der Lichtempfindung
bei niedere Thieren; viii, B, Die Sehorgane von Spadella hexa-
ptera,” ‘ Zeitschr. wiss. Zool.,’ Ixxti, p. 572.

Doncaster, L.—‘‘ On the Development of Sagitta; with Notes on the
Anatomy of the Adult,” ‘Quart. Journ. Mier. Sci.,’ vol. 46, 1903.
Gintuer, R. 'T.—“On the Distribution of Mid-water Chatognatha
in the N. Atlantic during the Month of November,” ‘Ann. Mag.

Nat. Hist.,’ ser. 7, xii, pp. 334-7.

Krumaacn, T.—“ Die Greifhaken der Chetognathen,” ‘Zool. Jahrb.,’
Xvili, p. 579.

Fow.er, G. H.—‘ Biscayan Plankton collected during a cruise of
H.M.S. ‘Research,’ 1900,” ‘Trans. Linn. Soc.,’ x, pp. 55—87,
pls. 4—7.

StroptmMann, S.—“ Die Chetognathen,” ‘Nordisches Plankton,’ iii,
x, pp. 10O—17, Kiel.

ADDENDUM TO Pace 380.

Conant states that there is no longitudinal partition be-
tween the spermatic chambers in Spadella schizoptera,
and that the right and left oviducts are in communication
with one another by branches which meet and form a small
blind tubule lying on the mid-ventral line beneath the intes-

tine.

This union is believed to be a means for helping a

single copulation, affecting one side only, to fertilise the ova
of both sides of the body. (May, 1907.)

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 395

The Structure, Development, and Bionomics of
the House-fly, Musca domestica, Linn.

Part I—The Anatomy of the Fly.
By

C. Gordon Hewitt, M.Se.,
Lecturer in Economic Zoology, University of Manchester.

With Plates 22—26.

ContTENTS.

PAGE
T. Introduction . 3 : : : - aude
II. Methods : , : : : - o99
III. External Structure—1. Head ; ; . 400
° 2. Thorax : 5 . 406
3. Abdomen . : . 414
IV. Internal Structure.—1. Muscular System. . 415
2. Nervous System. oe ALG
3. Alimentary System . . 420
4. Respiratory System . 424

5. Vascular System and Body
Cavity . ; - 429
6. Reproductive System . 430
V. Internal Structure of Head . 3 : . 435
VI. Summary 4 ‘ ‘ ; : . 439
VII. Literature’. 3 ; : ; . 442

I. Inrropvuction.

THIs paper is intended to be the first of a series of three
dealing with the anatomy, development and bionomics of the
House-fly, Musca domestica, L. The second part will

396 C. GORDON HEWITT.

include an account of the anatomy of the larva, its develop-
ment and the breeding habits of the fly; the series will be
concluded with an account of the bionomics of the fly with
special reference to its relations with man.

The term “ House-fly ” to the zoologist refers only to one
insect—Musca domestica of Linneus, but to the popular
mind it includes insects, not different species only, but differ-
ent families of Diptera. The Root Maggot fly (PI. 22, fig. 2),
Anthomyia radicum, L., sometimes occurs commonly in
houses. Homalomyia canicularis, L. (fig. 3), often
called the Small House-fly, is a very common inhabitant of
houses. The latter species is smaller than M. domestica,
and on this account they are frequently supposed to be young
specimens of the latter species by persons who are ignorant
of the fact that growth takes place during the larval stage
and not after the exclusion of the imago. Stomoxys
calcitrans, L. (fig. 4), is found in houses, especially in the
autumn. It is frequently mistaken for M. domestica, and
as it is one of the blood-sucking species (See Austen,
1906), the pernicious habit is attributed to the harmless
M. domestica either on account of the supposed ill-nature
of the latter or the influence of some change in the
weather.!

In addition to these, other species of flies occur in houses
but these will be considered in a. later part. Reference has
been made here to the various species inhabiting houses to
show that the term ‘‘ House-fly”’ as ordinarily used is rather
an inclusive one.

The House-fly has received some attention from naturalists
in all ages. Reaumur (1738), De Geer (1752-78) and Bouche
(1834) have all included a short account of this insect in their
classical memoirs. They do not contribute much to our
knowledge of the anatomy and development of the fly. The

1 Stomoxys calcitrans can be readily distinguished from M. domes-
tica by the awl-like proboscis which projects forwards from beneath the head.
It has a more robust general appearance, a dark spotted abdomen, and its
flight is more steady.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 397

most complete of these early accounts is that of Keller
(1790) which is illustrated by several striking plates. He
gives an interesting account of the development and breed-
ing habits, but in attempting to describe the anatomy he was
not so successful as exemplified by his mistaking the brown
testes for kidneys. In 1874 Packard wrote what is up to the
present time the most complete account of the development
of this species, and in 1880 Taschenburg, in his ‘ Praktische
Insektenkunde ? gave a good popalar account of the insect.
Howard has more recently (1898 and 1902) contributed to
our knowledge of the developmental history.

No complete account of the anatomy of this insect has yet
been published. A short popular account by Samuelson and
Hicks (1860) though interesting is very superficial, and con-
tains much that is inaccurate. Macloskie (1880) has published
an account of the proboscis of M. domestica, and the foot
has been made an object of study by several workers, chief
of whom are Hepworth (1854), and Merlin (1895 and 1905),
who correctly described the glandular hairs of the pulvilli.
Wesche has recently (1906) described the genitaha of both
sexes, but his description and figures are inaccurate. An
interesting account of the copulation of the fly has been pub-
lished by Belese (1902), in which he briefly describes the
reproductive organs, his work will be referred to later.
Lowne’s monograph (1895) on the Blow-fly (Calliphora
erythrocephala), which is an elaboration of his previous
memoir (1870) is the only complete account which has been
published on Muscid anatomy. The result of my study of
the anatomy of M. domestica, which was begun in 1905,
and is being continued in the Zoological Laboratories of the
Manchester University, has been to make it apparent that
much of Lowne’s work needs confirmation.

Musca domestica was first described by Linneeus (1758),
his description is as follows :—

“ Antennis plumatis pilosa nigra, thorace lineis 5 obsoletis
abdomine nitidulo tessellato: minor. Habitat in Europe

398 C. GORDON HEWITT.

domibus, etiam Americe. Larve in simo equine. Pupe
parallele cubantes.”

Later Fabricius described it more fully in his ‘Genera
Insectorum.’ The House-fly, together with the Blowfly, and
the blood-sucking flies Stomoxys and Glossina belongs to
the family Muscide, which is characterised by having the
terminal joint of the antenna—the arista always combed or
plumed and by the absence of large bristles or macrocheete
on the abdomen. The Muscide, together with the Antho-
myide and Tachinide constitute the group Muscide
calypterate are characterised by the possession of squame,
small lobes at the bases of the wings which cover the halters.
In the acalyptrate muscids the squamez are absent or rudi-
mentary. These two groups belong to the suborder Cyclor-
rhapha, one of the two primary divisions of the Diptera.
The Cyclorrhapha have coarctate pupe, the pupal case
being formed by the hardening of the last larval skin, and
the flies escaping through a circular orifice formed by the fly
pushing off the end of the pupa by means of an inflated sac-
like organ—the ptilinium which is afterwards withdrawn into
the head, its presence being marked by a frontal crescentic
opening the lunule. The other sub-order the Orthorrhapha
have obtected pupe.

The most complete specific description of Musca domes-
tica has been given by Schiner (1864), of which the follow-
ing is a free translation :—

“ Frons of male occupying a fourth part of the breadth of
the head. Frontal stripe of female narrow in front, so broad
behind that it entirely fills up the width of the frons. The
dorsal region of the thorax dusty grey in colour with four
equally broad longitudinal stripes. Scutellum grey, with
black sides. The light regions of the abdomen yellowish,
transparent, the darkest parts at least at the base of the
ventral side yellow. The last segment and a dorsal. line
blackish brown. Seen from behind and against the light the
whole abdomen shimmering yellow, and only on each side of
the dorsal line-on each segment a dull transverse band, The

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 399

lower part of the face silky yellow, shot with blackish brown.
Median stripe velvety black. Antenne brown. Palpi black.
Legs blackish brown. Wings tinged with pale grey with
yellowish base. The female has a broad velvety black, often
reddishly shimmering frontal stripe, which is not broader at
the anterior end than the bases of the antenne, but becomes
so very much broader above that the hght dustiness of the
sides is entirely obliterated. The abdomen gradually be-
coming darker. ‘The shimmering areas on the separate
segments generally brownish. All the other parts are the
same as in the male.”

The mature insects measure from 6-7 mm. in length and
13-15 mm. across the wings. Flies which have been starved
during the larval stage or subjected to adverse conditions are
generally smaller in size.

II. Meruops.

All the details of the anatomy which are about to be
described have been studied by means of dissections. The
dissections were made on both fresh and preserved material
under a Ziess’ binocular dissecting microscope with magnifi-
cations varying from 25-65 diameters. Serial sections have
been made to confirm the dissections and to study the histo-
logical details.

Perfect series of sections of the whole fly were hard to
obtain on account of the somewhat brittle nature of the in-
ternal chitinous structures. These internal chitinous skeletal
elements caused the greatest trouble as they were apt to
damage the internal anatomy. Celloidin sections were not a
great improvement on those cut in paraffin. The best results
were obtained by fixing the flies from 12-24 hours in
Henning’s solution, which is—Nitric acid 16 parts, chromic
acid (‘5 per cent.) 16 parts, corrosive sublimate saturated in
60 per cent. alcoho] 24 parts, picric acid saturated in water
12 parts, and absolute alcohol 42 parts, washing out with
iodine alcohol. This not only fixes, but to a certain extent,
though not completely, softens the chitin. They should not

4.00 C. GORDON HEWITT.

be imbedded too long or the chitin becomes brittle again.
Serial sections made of recently emerged imagines before the
chitin has hardened give good results. Other fixing agents
used were Perenyi, Rabl’s Chromoformic, Picro-formal
(Boum’s solution), Glacial acetic acid, and absolute alcohol.
Of the various stains which were used the most satisfactory
were Heidenhain’s Iron-hematoxylin, Brazilin,! and Dela-
field’s Heematoxylin. With the last stain perfect results
were obtained by overstaining and differentiating with acid-
alcohol.

The structure of the thoracic ganglion was studied by
means of reconstructions. The method employed was as
follows :—The sections were drawn by means of the camera
lucida on Bristol board of a thickness proportional to the
macnification. They were afterwards cut out and seccotined
together. The resulting model was trimmed and soaked in
melted paraffin, taken out and dipped several times till a thin
coating of paraffin covered the model. This was then trimmed
down to the original size, all the interstices having been
filled by the paraffin. After a coating of graphite it was
electrotyped with copper. In this way a permanent model
was obtained.

III. External Structure.

1. The Head Capsule.

The head capsule of M. domestica presents great modi-
fications when compared with the typical insect head. Con-
siderable difficulty is experienced in explaining its structure
in the morphological terms employed in the simpler orders
of insects. Lowne did not lessen the difficulty in describing
the head of the blowfly by the invention of new terms of
little morphological value. ‘The head of the fly is strongly
convex in front (Pl. 23, fig. 1), the posterior surface being
almost flat and slightly conical. For the sake of clearness the

1 See Hickson, 8. J., “ Staining with Brazilin,” ‘ Quart. Journ. Mier. Sci.,’
vol, 44, pp. 469—471, 1901.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 401

composition of the head capsule will be described from
behind forwards. The occipital foramen occupies a median
slightly ventral position on the posterior surface. It is
surrounded by the occipital ring, the inner margin of which
projects into the cavity of the head. From the sides of the
inner margin of the occipital ring two short chitinous bars
bend inwards and approach each other internally, forming a
support—the jugum for the tentorial membrane. On each side
of the occipital ring below the jugum a small cavity occurs
into which a corresponding process from the prothorax fits,
forming a support for the head.

The occipital ring is surrounded by the four plates, which
make up the sides and back of the head capsule. On the
ventral side, between the occipital ring and the aperture
from which the proboscis depends, a median basal plate, the
gulo-mental plate, represents the fused gula and basal por-
tions of the greatly modified second maxille. The occipital
segment is bounded laterally by the gene (Lowne’s para-
cephala) and dorsally by the epicranium. ‘These parts have
been divided by systematists into so many regions that a
somewhat detailed description will be necessary to make
their boundaries clear.

The genz bear the large compound eyes which occupy
almost the whole of the antero-lateral regions of the head.
On the posterior flattened surface of the head the gene are
flat, and extend from the gulo-mental plate to the epicranial
plate, the sutures of the latter being vertical. On the dorsal
side each sends a narrow strip between the inner margin of
the eye and the epicranium; this strip surrounds the eye
and meets the ventral portion of the gena ; it is of a silver to
golden metallic lustre. On the ventral side below the eye
each gena bounds the proboscis aperture laterally ; a number
of stout bristles arise from this margin and also from its
antero-lateral region, which is often spoken of as the “ jow].”
In the anterior region, where the genz are in contact with
the clypeus, there are two prominent ridges bearing strong
sete ; these are usually known as the “ facialia.”

402 C. GORDON HEWITT.

The epicranium (epicephalon of Lowne) on the posterior
surface of the head is flat. On the anterior surface it is
convex, and divided into a number of regions. On the top
of the head between the eyes it is called the vertex. This
contains the three ocelli situated on a shghtly raised ocellar
triangle, which is surrounded by a second triangle, the
vertical triangle. The median region in front of and below
the vertex is the frons. In the middle of this there is a
black frontal stripe. In the male the eyes are only narrowly
separated by the frontal stripe. Im the female the frontal
stripe widens out on the vertex. This character provides a
ready means of distinguishing the male from the female, as
the result of it is that in the male the eyes are close together
on the dorsal side being separated by about one fifth of the
width of the head, whereas in the female the space between
the eyes is about one third the width of the head. The
edges of the genx bordering on the frons bear each a row of
stout setee—the fronto-orbital bristles. The antenne arise
from the lower edge of the frons. Hach antenne consists of
three joints and the arista. The two proximal joints are
short and compose the “scape.” The third joint, the
flagellum, is longer, somewhat cylindrically prismatic in
shape, and hangs vertically in front of the clypeus. It is
covered with sensory sete, and contains two pits of sensory
function (olfactory, I believe). From the upper side the
plumose arista arises. ‘This probably represents the terminal
three joints of the antenna. The lower edge of the frons
represents the anterior margin of the epicranium.

The rest of the facial region is composed of the clypeus or,
as it is usually called, the face—a convenient term, but one
which hides its true morphology. The face is depressed,
and is covered by the flagelle of the antenne. Between the
upper and lateral edges of the face and the lower edge of
the epicranium a crescentic opening, the lunule, marks the
invagination of the ptilinium. The epistomium is a narrow
strip below the face bounding the anterior edge of the
proboscis aperture.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 403

The Skeleton of the Proboscis.—The proboscis of
M. domestica is very similar to that of the blowfly, which
has been described by Kraepelin (1880) and Lowne (1895),
though the results of these authors differ in many details.
My study of M. domestica confirms Kraepelin’s results, and
as Lowne’s is the only complete account of the muscid head
a full description of its internal and external anatomy will
be given in this paper.

Lowne regards the greater part of the proboscis as being
developed from the first maxille and not from the labium
or fused second maxille, which is the usually accepted view
and one which I support on morphological grounds. On
account of his exceptional conclusion he refuted the com-
monly accepted terms for the various parts and invented
new ones. It will be necessary for the sake of descriptive
clearness to refrain from constant reference to these or any
discussion as to their value.

The proboscis consists of two parts, a proximal mem-
branous conical portion—the rostrum and a distal half the
proboscis proper which bears the oral lobes. The term
haustellum is also used for this distal half (minus the oral
lobes), and as a name it is probably more convenient, as the
term proboscis is used for the whole structure—rostrum,
haustellum, and oral lobes.

The rostrum (fig. 13, Ros.) is attached to the edges of
the proboscis aperture, that is to the epistomium, gene, and
the gulo-mental plate. It has the shape of a truncated cone,
and bears on the anterior side a pair of palps, which bear
sensory sete of two sizes.

The haustellum (fig. 18, H.), or proboscis proper, is
attached to the distal end of the rostrum. The posterior
side is formed by a convex, somewhat heart-shaped sclerite
—the theca (figs. 1 and 3, th.) which probably represents
a portion of the labium. ‘The lower angle of the theca
is incised by a semicircular sinus. By means of this the
theca rests on a triradiate chitinous sclerite—the furca, which
consists of a median, slightly convex rod (fig. 1, f.), from the

4.04. C. GORDON HEWITT.

anterior end of which two arms diverge and form the chief
skeletal structures of the oral lobes. The lower end of the
theca rides on this structure, the bottom of the sinus resting
on the median rod, and the two-pointed lateral terminations
of the theca rest on the arms. In this manner these pro-
cesses, in a state of repose, keep the arms of the furca closely
approximated. The result of this will be seen later in study-
ing the musculature of the proboscis.

The sides of the haustellum are membranous. On its
anterior face, in a groove formed by the overlapping mem-
branous sides, lie the labrum-epipharynx and labium-hypo-
pharynx. The labrum-epipharynx (figs. 1 and 3, l.ep.) is
attached at its proximal end to the membranous rostrum, but
is incapable of a labral-like movement on account of its close
connection with the labium-hypopharynx. ‘Two slightly-
curved, hammer-shaped apodemes (fig. 1, ap.) are attached
to the proximal end of the labium-epipharynx. They assist
in folding the proboscis during retraction, as will be shown
later. The labium-epipharynx is shaped like a blunt arrow-
head; the external surface is somewhat flattened. It is
composed of two pairs of sclerites, an outer pair enclosing an
inner pair, which form the pharyngeal channel. The edges
of the inner tube are connected by a groove with the hypo-
pharyngeal portion of the labium-hypopharynx, as shown
in fig. 8. The labium-hypopharynx (fig. 8) represents the
fusion of the hypopharynx with the greatly modified and
fused second maxille or labium. It consists of a sclerite,
curved in section, having the chitinous hypopharyngeal tube
(fig. 3, hp.) fused to it along the upper half of its length.
The edges of the hypopharyngeal tube cngage with those of
the inner pair of sclerites of the labium-epipharynx, as men-
tioned before. Distally the hypopharyngeal tube becomes
free from the labium, as shown in fig. 3, and ends in a point
where the lingual salivary duct opens.

Down each side of the labium-hypopharyngeal sclerite a
rod-like thickening runs. Distally these thickened margins
(paraphyses of Lowne) articulate with the discal sclerites.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 405

The discal sclerites (fig. 1, ds.) are united at the posterior
end to form, when the oral lobes are expanded, a U-shaped
structure, with the limbs constricted in the middle where the
ends of the thickened margins of the labium-hypopharynx
articulate. They are sunk in deeply between the two oral
lobes at the base of the oral pit with the free ends of the U
anterior, these being spatulate and curved anteriorly.

The two oral lobes are normally connected by a bead and
groove attachment along their anterior edges, but under
pressure the connection is severed, and the oral dise presents
a heart-shaped instead of the normal oval appearance. The
oral lobes are covered on their upper aboral surfaces by
sensory sete, the large marginal sete being different in
structure from the rest. On the lower or oral surface a large
number of channels, the pseudotrachee (fig. 1, ps.) run from
the internal margins of the oral lobes to the external borders.
The channels of the pseudotrachee are kept open when the
lobes are extended by means of small incomplete chitinous
rings, which give the channels a tracheal appearance, hence
their name. Hach of these incomplete rings has one end
bifid, and as the bifid ends alternate the opening into the
channel has a zigzag appearance. ‘lhe number of pseudo-
tracheze on each lobe is generally thirty-six, and they are
grouped in three sets. The anterior set of twelve all run
into a single large pseudotracheal channel running along the
anterior inner margin of the lobe, and a posterior set of
twenty-one all run into a channel running along the posterior
inner margin; between these two sets three pseudotrachez
run direct into the oral aperture. The oral aperture lies at
the base of the small oral pit, which is a space kept open
between the oral lobes by means of the discal sclerites. The
pseudotracheee do not extend as far as the discal sclerites,
but on entering the oral pit the rings cease and the sides of
the channels are covered by overlapping teeth, which extend
back to the discal sclerites. Between the pseudotracheze the
membranous surface of each oral lobe is thrown into two
longitudinal sinuous ridges, and projecting up from the

VOL. 51, PART 3,—NEW SERIES. dl

406 C. GORDON HEWITT.

bottom of the furrows are several papille, generally four
or five to each interpseudotracheal area, of a gustatory
nature—the gustatory papille (figs. 1 and 18, gp.).

The Fulcrum.—tThis chitinous portion of the pharynx
(fig. 1, F.) lies on the lower part of the head and in the
rostrum. Kraepelin describes it as being shaped like a
Spanish stirrup iron. Its structure will be best understood
by referring to the figures. It consists of an outer portion,
which is U-shaped in section; the basal portion, which is
posterior and forms the floor of the pharynx (which Lowne,
unfortunately, terms the hypopharynx) is vertical when the
proboscis is extended. This basal portion is evenly rounded
at both ends, and at the sides of the upper end there is a
pair of processes—the posterior cornua (fig. 1, pc.) which
serve for the attachment of muscles. The sides of the
fulerum are somewhat triangular in shape; their upper
anterior portions are produced to form the anterior cornua
(a.c.); here the sides bend inwards at right angles, and
meet below the epistomium, upon which the fulerum is
hinged. The fulcrum is therefore quadrilateral in section at
the upper proximal end, and trilateral at the lower distal end.
The basal portion (fig. 2, b.p.) forms the floor of the pharynx;
the roof of the pharynx is formed by another chitinous piece
(r.p.) with a median thickened raphe. This roof les parallel
with the basal piece, and is fused with the sides of the
fulcrum. On the membranous wall of the pharynx, between
the labium-hypopharynx and the fulcrum, a small chitinous
sclerite (fig. 1, k.) is developed, which Lowne terms the
hyoid sclerite. It is U-shaped in section, and serves to keep
the lumen of the pharynx in this region distended.

2.. The Thorax.

As in all Diptera the possession of a single pair of wings
has resulted in the great development of the mesothorax at
the expense of the other thoracic segments, consequently the
thorax is chiefly made up of the sclerites composing the

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 407

mesothorax. The prothorax and metathorax compose very
small portions on the anterior and posterior faces respec-
tively. Seen from above the thorax is oviform with the
blunt end anterior and slightly flattened. Three transverse
sutures on the dorsal side mark the limits of the prescutum,
scutum, and scutellum of the mesothoracic segment; the
mesothoracic scutellum forms the pointed posterior end, and
slightly overhangs the anterior end of the abdomen.

The Prothorax.—The prothoracic segment has been
reduced to such an extent that it is hopeless to attempt to
homologise all the separate sclerites with those of a typical
thoracic segment. ‘lo obtain a complete view of the pro-
thorax it 1s necessary to examine it from the anterior end
after the removal of the head. ‘The following sclerites can
then be recognised. The prosternum is a median ventral
plate, quadrilateral in shape having the anterior end rounded
and broader than the posterior end. It does not occupy the
whole of the prosternal area, but is bounded by the prosternal
membrane. Internally a ridge runs to the posterior end of
the prosternum and bifurcates, each ridge running to the
posterior corners, to which two strong processes (the hypo-
tremata of Lowne) are attached. In front of the prosternum
there is a small saddle-shaped sclerite which, on account of
its position, may be called the interclavicle (the sella of Lowne).
Two lobes at its anterior end are covered with small pro-
cesses, probably sensory in function. A pair of small sclerites
is situated in front of these lobes; these sclerites with the
interclavicle no doubt belong to the prosternum. The inter-
clavicle is ventral to the cephalothoracic foramen. The
jugulares (3me jugulaires of Kunckel d’Herculais) are two
prominent pocket-shaped sclerites lying one on each side of
the cephalothoracic foramen, and having their convex faces
external. Lying immediately below each of the jugulares is
a small rod-like sclerite—the clavicle. The dorsal region of
the prothorax the pronotum (fig. 6 pr.) is formed by two
sclerites united in the median line, their dorsal sides being
curved, From the ventral side of the pronotum a pair of

408 C. GORDON HEWITT.

chitinous apodemes project into the thoracic cavity. The
lateral regions of the pronotum are in contact with the
humeri and the prothoracic episterna. ‘The humeri (hw.) are
a pair of strongly convex sclerites situated in the antero-
lateral region of the thorax. They are bounded above by the
prescutum of the mesothorax, internally and below by the
episterna of the prothorax, and externally by the lateral plate
of the mesosternum and the anterior thoracic spiracle. Its
inner concave surface serves for the attachment of the muscle
of the prothoracic coxa. The episterna (eps.’) (epitrochlear
sclerites of Lowne) are comparatively large sclerites forming
the lateral regions of the prothorax. They overhang the
attachments of the prothoracic limbs. The internal skeleton
of the prothorax consists of the two stout hollow apodemes—
the hypotremata mentioned previously. They arise from the
postero-lateral edges of the prosternum, and run obliquely
across the ventral edge of the anterior thoracic spiracle
where the hypotreme divides, the posterior branch runs up
the posterior margin of the spiracle, between the lateral plate
of the mesosternum and the peritreme (the chitinous ring
surrounding the spiracle), the anterior branch fuses with the
prothoracic episternum.

The Mesothorax.—The notum of the mesothorax occu-
pies the whole of the dorsal side of the thorax. It is com-
posed of the four sclerites to which Audouin (1824) gave the
name of prescutum, scutum, scutellum, and postscutellum.
The prescutum (prs.) forms the anterior part of the dorsal
region of the thorax. Its anterior portion bends down almost
vertically to unite with the pronotum. ‘The anterior edge of
the prescutum is inflected after the pronotal suture, and is
produced in the median line into a small bifurcating process.
The prescutum is bounded laterally by the humerus and a
membranous strip —the dorso-pleural membrane. The scutum
(se.) is the largest of the mesonotal plates. It occupies the
whole of the median dorsal region of the thorax. Anteriorly
itis bounded by the prescutum, laterally by the alar membrane
and the lateral plate of the postscutellum, and posteriorly by

STRUCTURE, DEVELOPMENT, AND BLONOMICS OF HOUSE-FLY. 409

the scutellum. From the lateral region of the scutum a pro-
cess projects forwards and downwards, and articulates with
the posterior portion of the wing-base (the metapterygium).
The scutellum (sctl.) is a triangular pocket-shaped sclerite
which overhangs the postscutellum and the base of the abdo-
men. ‘The posterior surface of the thorax is chiefly composed
of the large postscutellum. ‘This is made up of three pieces,
a median escntcheon-shaped plate (mpsc.) strongly convex to
the exterior, and two convex lateral plates (lp.sc.). The lateral
plates are bounded below by the metasternum and spiracles,
and anteriorly by the pleural region of the mesothorax.

The mesosternum is a sclerite of considerable size and forms
the keel of the thorax. It consists of a median ventral por-
tion (ms.) which is produced laterally to form two large
lateral plates (/p.). The median portion is bounded in front
by the prosternum and tbe foramina of the anterior coxe, and
behind by the median coxal foramina. A short distance
behind the anterior end a depression in the mid-ventral line
extending to the posterior edge indicates a median inflection
forming the entothorax. The lateral regions of the posterior
margins of the mesosternum are inflected on each side to form
the entopleura. The lateral plates of the mesosternum form
the whole of the anterior portion of the pleural region ; each
is bounded in front by the humerus, spiracle, and prothoracic
episternum, and above by the dorso-pleural membrane, and
behind by the mesopleural membrane. ‘The ventral side of
the lateral plate is continuous in front with the median plate
of the mesosternum, and behind is united by means of a
suture. The remaining portion of the mesopleural region is
made up of the episternum, epimeron, and two small sclerites
connected with the wing-base—the parapteron and costa.
The episternum (eps.”) is situated behind the mesopleural
membrane and below the alar membrane, below and behind
it is bounded by the epimeron. Its surface is marked by two
convexities, the ampulle, the upper of the two corresponding
to Lowne’s great ampulla of the blowfly. ‘The dorsal side of

410 C. GORDON HEWITT.

the episternum is intimately connected with the sclerites' of
the anterior portion of the wing-base.

The epimeron (ep.’) is a triangular sclerite, and is bounded
below by the mesosternum and metasternum, behind by the
lateral plate of the postscutellum, and above by the episternum
and alar membrane. The parapteron (pt.) is a sclerite situ-
ated at the top of the mesopleural membrane. The greater
portion of it is internal, only a small triangular portion can
be seen externally. Internally this is continued as a cruri-
form sclerite to which are attached important muscles con-
trolling the wings. The costa (ca.) is asmall sclerite situated
on the dorsal margin of the epimeron. The internal skeleton
of the mesothorax consists of the entothorax, entopleura,
mesophragma, and the inflected edges of the episterna and
epimera. The entothorax is composed of a median vertical
plate subtriangular in shape, on the top of which a median
plate produced laterally into wing-like processes rests. On
this structure the thoracic nerve-centre lies. The entopleura
and the inflected edges of the episterna and epimera all serve
for the attachment of wing muscles. The mesophragma
(mph.) is a convex sclerite fused with the lower edge of the
postscutellum. Its posterior edge is incised in the middle
and forms the dorsal arch of the thoraco-abdominal foramen.

The Metathorax.—The largest sclerite of the greatly
reduced metathorax is the metasternum (mts.). It is a wing-
shaped sclerite with the narrow transverse portion situated
between the coxal foramina of the median and posterior pairs
of legs; the expanded lateral portions form the wall of the
thorax above the insertions of these legs. The edges of the
narrow transverse strip are inflected, and unite the lateral
portions of the metasternum. A trough-shaped longitudinal
fold—the metafurca rests on the narrow transverse portion of

1 In this account the individual sclerites which compose the wing base will
not be described. Lowne has described them at great length for the blowfly,
and although the wing-base sclerites of M. domestica differ slightly in shape
from those of Calliphora, Lowne’s description of their relations holds good
for the former insect.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. A411

the metasternum. The posterior end of the metafurca bends
downwards and articulates with the posterior coxe on each
side. The metafurca serves for the attachment of the thoraco-
abdominal muscles. The pleural region of the metathorax is
a narrow triangular space situated behind the lateral portion
of the metasternum and the posterior coxe. It is composed
of a narrow triangular episternum and epimeron. ‘The former
(eps.”’)is bounded in front by the metasternum, the posterior
thoracic spiracle and the base of the haltere, below by the
posterior coxal foramen, and behind by the epimeron. The
epimeron (ep.”) isalso bounded below by the coxal foramen and
behind by the narrow dorsal arch of the metathorax and the
first abdominal segment, its apex comes in contact with the
base of the haltere. The dorsal region of the metathorax has
practically disappeared, all that can be recognised as meta-
notum is a narrow chitinous strip (mn.) on each side between
the apex of the metapleural area and the dorsal edge of the
first abdominal area.

Wings.—The wings are situated at the sides of the
scutum on the alar membrane, to which are attached the
sclerites of the wing base. They are covered with very fine
hairs.

In describing the neuration of the wings the nomenclature
proposed by Comstock and Needham (1898) for the wings of
the whole group of insects will be employed.

The nervures of the wing are ocreacous. The anterior
edge of the wing (fig. 16) is formed by a stout nervure, the
costa (Cj.), which is very setose. The second longitudinal
nervure, the subcostal (Scj.), joins the costal about half way
along its length. A small transverse nervure, the humeral (h.),
divides the costal cellinto costal (C.) and first costal (1 C.) cells.
The next main nervure—the radial—divides into a number
of branches (in the typical insect five) ; some of these have
coalesced in the fly. A nervure joining the costal just past
the middle is the first radial (R,.) cutting off the subcostal
cell. The next nervure, which joins the costal on the apical
curve, represents the fused second and third radial nervures

412 C. GORDON HEWITT.

(R.2+3). This cuts off the first radial cell (1 R.). ‘The last
nervure, which joins the costal almost at the apex of the
wing, represents the fused fourth and fifth radial nervures
(R. 4+5), and so cuts off the third radial cell (3 R.). The
fourth main longitudinal nervure is the median, which, in the
typical insect, divides into three, but in the fly the nervures
have undergone coalescence, as will be shown. The first and
second median nervures have coalesced (M.1+2), and do
not run direct to the margin of the wing, but bend forwards
and almost meet R. 4+ 5 on the costa. About half way across
the wing a transverse nervure, the radio-medial (7m.) unites
R.4+5 and M. 1+ 2, and cuts off the fifth radial cell (5 2.)
from the radial (/?.). The next longitudinal nervure repre-
sents the coalesced third medial and cubital nervures (JM.
3+Cu. 1). It runs to the posterior margin of the wing
about half way along the length of the latter. The nervures
M. 1+2 and M. 3+Cu. 1 are united by two transverse
nervures. The proximal nervure—the medio-cubital (i.cw.)
cuts off the small triangular medial cell (I.) ; the distal trans-
verse nervure (m.) cuts off the first second medial cell (2 M.')
from the second second medial cell (2 M.”). The last longi-
tudinal nervure—the anal (4,.)—is undivided, and does not
reach the margin of the wing, thus incompletely separating
the first cubital (1 Cw.) and anal (4.) cells. A small trans-
verse nervure, the cubito-anal (cu.a.), slightly more proximal
than the medio-cubital, cuts off the small triangular cubital
cell (Cw.) from the first cubital cell (1 Cu). Running parallel
with, and posterior to, the anal longitudinal nervure, there is
apparently another nervure. This, however, is not a true
nervure, but is merely a chitinised furrow giving additional
strength to the posterior angle of the wing. The posterior
edge of the base of the wing is divided into a number of
lobes. These are the anal lobe, and, as Sharp (1895) pro-
posed, the alula, antisquama, and squama. ‘he squama is
thicker than the rest, and is attached posteriorly to the wing
root between the mesoscutum and the lateral plates of the

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-ELY. 413

postscutellum. It covers the haltere, as in all “ calyptrate ”
Muscidee.!

The Halteres.—The halteres or balances (fig. 6, hal.) are
generally considered to represent the rudimentary meta-
thoracic wings. They are covered by the squame, and are
situated on the sides of the thorax above the posterior
thoracic spiracles. Hach consists of a conical base on which
are a number of chordonotal sense-organs, and on this base
is mounted a slender rod, at the end of which a small spheri-
cal knob is attached. The wall of the distal half of this
sphere is thinner than the proximal half, and in preserved
specimens is generally indented. Experiments show that the

1 The nomenclature of Comstock and Needham has not yet been adopted
by dipterologists in general; but, on account of its great morphological value,
it will no doubt in course of time replace the present confused system. It
may therefore be useful if the nomenclature employed in the foregoing
description be compared with those most usually employed.

Longitudinal nervures.—-C,. Costal. Sc,. Mediastinal, auxiliary. 2,.
Subcostal, Ist longitudinal, &. 2+ 8. Radial, 2nd longitudinal. 2. 4+ 5.
Cubital, 3rd longitudinal; ulnar (Lowne). J/. 1+ 2. Median, 4th longitu-
dinal; discal (Verrall). J. 3 + Ca. Submedian, 5th longitudinal; postical
(Verrall). 4,. Anal, 6th longitudinal. Pseudonervure, axillary, 7th longi-
tudinal.

Transverse nervures.—d. Humeral, Ist transverse; basal cross vein
(Verrall). zm. Discal, 2nd transverse; middle cross vein (Verrall); medial
transverse; anterior transverse (Austen). m-cw. Auterior basa! transverse
(Austen) ; lower cross vein (Verrall); postical transverse (Lowne). m. Pos-
terior transverse (Austen); postical cross vein (Verrall); discal transverse
(Lowne). cw-a. Posterior basal transverse (Austen); anal cross vein (Verrall) ;
anal transverse (Lowne).

Cells.—C. Costal. 10. Second costal. Sc. Third costal (Lowne cor-
rectly calls this ‘‘sub-costal”). 12. Marginal. 32. Sub-marginal; cubital
(Lowne). 5. First posterior cell (Austen); sub-apical (Lowne and Verrall).
21°. Second posterior cell (Austen); apical. 1Cw. Third posterior cell
(Austen and Verrall); patagial (Lowne). 24/1. Discal (this term is used also
in Lepidoptera, Trichoptera, and Psocoptera, and in each family refers to a
different cell!). #&. Anterior basal cell (Austen); upper or Ist basal or
radical (Verrall); prepatagial (Lowne). JZ. Posterior basal cell (Austen) ;
middle or 2nd basal or radical (Verrall); anterior basal (Lowne). Cu. Anal
cell (Austen) ; lower or 3rd basal or radical (Verrall) ; posterior basal (Lowne),

4.14 C. GORDON HEWITT.

halteres are organs of a static function. ‘They are not
balancing organs in the sense that they are equivalent to the
balancing pole of a rope-walker. ‘They also have probably an
auditory function. They are innervated by the largest pair
of nerves in the thorax.

The Legs.—The three pairs of legs are composed of the
typical number of segments. Hach consists of coxa, trochanter,
femur, tibia, and tarsus. The coxe are the only segments
which show any considerable difference in the three pairs of
legs. The anterior coxze are comparatively large and boat
shaped, the intermediate coxe are smaller and their separate
sclerites more marked; the coxal plates of the intermediate
cox are shown in fig. 6 (cp.). The coxal joints of the pos-
terior pair of legs are almost similar to those of the inter-
mediate pair. The anterior femora are shorter and stouter in
the middle than those of the intermediate posterior pairs of
legs. The anterior tibie are also shorter than those of the
succeeding legs. The anterior tibiz are covered on their
inner sides with closely-set, orange-coloured sete which serve
as a comb by means of which the fly removes particles of dirt
adhering to the setee which clothe its body ; the first tarsal
joints of the posterior legs are also similarly provided. The
tarsi consist of five joints, the terminal joints bearing the
“ feet.” ‘These organs about which so much has been written
consist of a pair of curved lateral claws or “ ungues” which
subtend a pair of membranous pyriform pads—the pulvilli.
The pulvilli are covered on their ventral sides with innumer-
able, closely-set, secreting hairs by means of which the fly is
able to walk in any position on highly polished surfaces. A
small sclerite lies between the bases of the pulvilli. The
tarsal joints and the other segments of the legs are covered
with a large number of setee.

3. The Abdomen.
The abdomen is oviform with the broad end basal. ‘The
total number of segments which compose the abdomen is eight
in the male and nine in the female. ‘The visible portion con-

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 415

sists of apparently four segments in the male and female, in
reality there are five as the first segment has become very
much reduced, and has fused with the second abdominal seg-
ment forming the anterior face of the base of the abdomen
(see fig. 8). The segments succeeding the fifth are greatly
reduced in the male, and in the female they form the tubular
ovipositor which, in repose, is telescoped within the abdomen.
The second, third, fourth, and fifth abdominal segments are
well developed, and consist of a large tergal plate, which
extends laterally to the ventral side. The sternal plates are
much reduced, and form a series of narrow plates lying on
the ventral membrane along the mid-ventral line. The
spiracles are situated on the lateral margins of the tergal
plates. The sclerites of the abdomen which are exposed are
strongly setose, especially the fourth and fifth dorsal plates,
but they do not bear macrochebe.

IV. InvrTernNAL StRuctTURE.
1. The Muscular System.

The muscular system of the fly is similar to that of
Volucella, described by Kunckel d’Herculais (1881), and of
the Blow-fly, described by Lowne and Hammond, and conse-
quently they will be but briefly described. The muscles may
be divided into the following groups: 1. Cephalic, 2. Thoracic,
3. Segmental, 4. Those controlling the thoracic appendages,
and 5. Special muscles.

1. The cephalic muscles will be considered in the detailed
description of the head.

2. The thoracic muscles are enormously developed and
almost fill the thoracic cavity. They are arranged in two
series. The dorsales (figs. 13 and 15, do.) are six pairs of
muscle-bands on each side the median line, attached posteriorly
to the postscutellum and mesophragma, and anteriorly to
the prescutum and anterior region of the scutum. The
sternodorsales (st.do.) are vertical and external to the dorsales
and are arranged in three bundles on each side. The first

416 C. GORDON HEWITT.

two pairs have their upper euds attached to the prescutum
and scutum, and their lower ends inserted on the mesosternum,
the third pair is attached dorsally to the scutum and ventrally
to the lateral plate of the postscutellum above the spiracle.
As Hammond has shown in the blowfly (1881) all these
muscles are mesothoracic. The dorsales by contraction
loosen the alar membrane and so depress the wing, the
sternodorsales have the opposite effect.

3. The segmental muscles. These muscles, which are so
prominent in the larva, have almost disappeared in the imago.
They are represented by the cervical muscles, certain small
thoracic muscles, the thoraco-abdominal muscles, and the
seomentally-arranged abdominal muscles together with the
muscles controlling the ovipositor and male gonapophyses.

4, The muscles controlling the thoracic appendages, the
wings, legs, and halteres. There is an elaborate series of
muscles controlling the roots of the wing, but in order to
avoid too much detail they will not be described here. The
flexor muscles of the anterior coxe have their origin on the
inner surfaces of the humeri, a fact supporting the pro-
thoracic nature of these sclerites; the flexors of the middle
pair of legs have their origin on the sides of the posterior
region of the prescutum. The internal muscles of the leg are
similar to those of the blowfly and Volucella.

5. Special muscles. These are the muscles controlling the
spiracular valves, the penis, and other small muscles.

2. The Nervous System.

The central nervous system (fig. 11) consists of (1) the
brain or supracesophageal ganglia which are closely united
with the subcesophageal ganglia, the whole forming a compact
mass which I propose to call the cephalic ganglion (fig. 1,
C.G.), perforated by a small foramen for the passage of the
narrow cesophagus, and (2) the thoracic compound ganglion
which is composed of the fused thoracic ganglia with the
abdominal ganglia. The two compound nerve-centres are

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 417

united by a single median ventral cord running from the
subcesophageal ganglia to the anterior end of the thoracic
nerve-centre.

The cephalic ganglion consists of the supracesophageal
ganglion and the subcesophageal ganglia so closely united
that the commissural character of the cireumcesophageal con-
nectives is quite lost. Hxternally, on the dorsal side of the
brain three longitudinal fissures can be seen, a median fissure
and two lateral fissures marking the origin of the optic lobes.

The supracesophageal ganglia. The characters of the ganglia
composing the brain are hidden by the sheath of cortical
cells which fills up the spaces between the ganglia, the
characters of these can be ascertained by the serial sections.
The median mass the procerebrum is formed by the fusion of
the procerebral lobes. These are united before and behind,
and enclose a central ganglionic mass—the central body.
Behind the procerebrum two pairs of fungiform bodies arise.
On the anterior face of the procerebrum the antennal or olfac-
tory lobes which represent the deutocerebrum are situated
laterally. Hach sends a nerve (figs. 1 and 11, an.n.) to the an-
tenna. Above these and on the dorsal side are a pair of lobes
—the frontal lobes contiguous with each other in the median
line—these belong morphologically to the tritocerebrum.
Posterior to these in the median dorsal line of the cerebrum
a single median nerve, the ocellar nerve (figs. 1 and 11,
oc.n.), arises; this runs vertically to the ocelli. A pair of
lobes which correspond to Lowne’s thalami of the blowfly are
situated external to and between the frontal and antennal
lobes. The peduncles of the optic lobes have their origins
from the sides of the procerebrum. Hach optic peduncle (fig.
11, O.P.) contains three ganglionic masses which Hickson
(1885) has termed from the brain peripherally the opticon,
epiopticon, and periopticon (fig. 1, P.O.) respectively.

The subcesophageal ganglia (fig. 1, S.0.). The commissures
uniting the supracesophageal ganglia to the cesophageal
mass cannot be recognised as such, owing to the extreme
state of cephalisation of the cephalic ganglia. They are

418 C. GORDON HEWITT.

represented by the regions lateral to the cesophageal foramen,
and from the anterior side of each of them arises a pharyn-
geal nerve (figs. 1 and 11, ph.n.). From the ventral side of
the subcesophageal gangliaa pair of nerves—the labial nerves
(fig. 1, /b.n.)—arise and run down the proboscis, innervating
the muscles of that organ; on reaching the oral lobes they
bifurcate and branch freely, supplying the numerous sense
organs in those structures. The cortical cells (Leydig’s
“Punktsubstanz”), which fill up the spaces between the
egangla and form an investing sheath round the whole gang-
ionic mass, are of two kinds. The smaller cells are rounded,
their nuclei are large in proportion to the protoplasm, and
their protoplasmic fibres anastomose with each other. Among
these smaller cortical cells, and also occasionally in the
ganglionic substance, larger ganglionic cells occur, their
protoplasm taking the stain very readily. Unipolar, bipolar,
and tripolar ganglion cells are found.

The eyes. Hach eye contains about 4000 facets. They
are similar in all respects to the eyes of the blowfly, which
have been fully described by Hickson (loc. cit.), whose
results my study confirms; consequently, a description of
their structure will not be given. It should be noted that, in
spite of the fact that Hickson corrected many mistaken views
held by Lowne in his memoir (1884), these are repeated in
his monograph of the Blowfly.

The cephalo-thoracic nerve cord (fig. 11, ¢.7.) unites the
cephalic and thoracic ganglha. Near its junction with the
thoracic ganglion a pair of cervical nerves (cer.n.) arise,
innervating the muscles of the neck.

The thoracic ganglion (figs. 12 and 14) is pyriform, with
the broad end anterior, and rests on the entothoracic skele-
ton of the mesothorax. As in the cephalic ganglion, the
component ganglia are ensheathed in a cortical layer, which
is of the same nature. The nerves of the three pairs of legs
(pr.cr., ms.cr., mt.cr.) arise from three large ganglia, which
are the prothoracic (P7.G.), mesothoracic (Ms.G.), and meta-
thoracic (Mt.G.) ganglia. These are united by a median

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 419

longitudinal band of nerve tissue, which runs dorsal to them,
and behind the metathoracic ganglia swells out into a gang-
honic mass (4.G.), which represents the abdominal ganglia.
In this median dorsal band there is a median dorsal fissure
stretching posteriorly from above the middle of the meso-
thoracic ganglia. The dorsal regions of the mesothoracic
and metathoracic ganglia show ganglionic swellings. From
the antero-dorsal sides of the prothoracic ganglia a pair of
prothoracic dorsal nerves (pr.d.) arise and supply the muscles
of that region, including those of the anterior thoracic
spiracle. The nerves supplying the mesothoracic legs
(ms.cr.) arise from the postero-ventral sides of the meso-
thoracic ganglia. Between the mesothoracic ganglia there
is a median ganglionic mass, situated slightly dorsal, from
the middle region of which the nerve-fibres of the large pair
of dorsal mesothoracic nerves (m.s.d.) arise; Lowne, in the
blowfly, calls these prothoracic. The roots of these nerves
are broad dorsoventrally. These nerves innervate the
sterno-dorsales muscles of the middle region. In_ this
median mesothoracic nerve centre, posterior to the origin of
the dorsal mesothoracic nerves, the fibres of a pair of nerves,
the accessory dorsal mesothoracic nerves (ac.ms.), have their
origin; these appear externally to arise dorsal to the roots of
the mesothoracic crural nerves. he dorsal metathoracic
nerves (mt.d.), which innervate the halteres, and are the
largest pair of thoracic nerves, have their origin from the
median dorsal band in front of the metathoracic ganglia, so
that they appear to be almost mesothoracic in origin. The
metathoracic crural nerves (mt.cr.) arise from the posterior-
ventral sides of the metathoracic ganglia. Posterior to these
a pair of slender nerves, the accessory dorsal metathoracic
nerves, have their origin, and innervate the muscles at the
posterior end of the thorax.

The dorsal band becomes much thinner posterior to the
abdominal ganglion, and runs into the abdomen as a median
abdominal nerve (ab.n.). In the thorax two pairs of abdo-
minal nerves arise. In the abdomen the abdominal nerves

420 C. GORDON HEWITT.

arise alternately and irregularly from the median abdominal
nerve. The median abdominal nerve finally terminates in
the genitalia.

3. The Alimentary System.

The alimentary canal of the house-fly is shorter than that
of the blowfly, and also than that of Glossina described by
Minchin (1905), and slightly longer than the alimentary tract
of Stomoxys described by Tulloch (1906). It serves as a
good example of the Muscid digestive canal. It is of a
suctorial character, and consists of pharynx, cesophagus,
crop, proventriculus, ventriculus or chyle stomach, proximal
and distal intestine and rectum.

The pharynx has already been described, and will be
further referred to in the detailed description of the head.
At the proximal end of the fulerum, where the cesophagus
arises, there is usually a small mass of cells, which Kraepelin
has described as glandular, but which I believe to be simply
fat-cells.

The oesophagus (figs. 1, 17, 20, @s.) commences at the
proximal end of the pharynx, and describes a curve before
passing through the cesophageal foramen in the cephalic
ganglion, where it narrows slightly. It then passes through
the cervical region into the thorax in the anterior region, of
which it opens into the proventriculus (figs. 17, 20, Pv.),
continuous with, and in the same line as the cesophagus, the
duct leading to the crop (fig. 20, d.er.) passes along the
thorax dorsal to the thoracic nerve-centre, and entering
the abdomen it leads into the crop, which lies on the ventral
side of the abdomen. The cesophagus has a muscular wall,
enclosing a layer of flat epithelial cells, and is lined by a
cuticular intima, which is thrown into several folds at the
anterior end.

The crop (fig. 17, Cr.) is a large bilobed sac, capable of
considerable distension, and, when filled with the liquid food,
it loses its bilobed shape, and occupies a large portion of the

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 421

antero-ventral region of the abdomen. Its walls exhibit
muscular (unstriped) fibres; the flat epithelial cells have a
very thin cuticle.

The proventriculus (Pv.) is circular and flattened dorso-
ventrally. Its structure will be understood by reference to
fig. 20. In the middle of the ventral side it opens into the
cesophagus, and on the dorsal side the outer wall is continued
as the wall of the ventriculus (Ven.). The interior is almost
filled up by a thick circular plug (Pv.p.), the cells of which
have a fibrillar structure, and it is pierced through the
centre by the csophagus. The neck of the plug is sur-
rounded by a ring of elongate cells, external to which the
wall of the proventriculus begins, and, enclosing the plug at
the sides and above, it merges into the wall of the ventriculus.
I do not agree with Lowne in regarding the proventriculus
as “a gizzard and nothing more,” but its structure suggests
a pumping function and also that of a valve. On the dorsal
side of the cesophagus, at its junction with the proventriculus,
a small ganglion, the proventricular ganglion (Pv.g.), lies,
communicating by a fine nerve with the cephalic ganglion.

The ventriculus, or chyle stomach (figs. 17, 20, Ven.),
represents the anterior region of the mesenteron, the posterior
region of the latter being formed by the proximal intestine.
It is narrow in front, and widest in the posterior region of
the thorax, where it again narrows in passing through the
thoraco-abdominal foramen into the abdomen to become the
proximal intestine. Except in the anterior and posterior
regions, where columnar cells compose the digestive epi-
thelium, the walls of the ventriculus are thrown into a
number of transverse folds, which are again subdivided
longitudinally, the result being the formation of small crypts
or sacculi, which are lined by large cells. These sacculi
correspond to the digestive cceca of other insects.

The proximal intestine (figs. 17, 21, p.tnt.) is the
longest region of the gut. It varies in length considerably.
In the normal-sized condition its course is as follows :—
Beginning at the anterior end of the abdomen it runs dor-

VoL. 51, PART 3.—NEW SERIES, 32

422 C. GORDON HEWITT.

sally beneath the heart to the posterior region, where it
curves downwards, turns to the left, and runs forward for a
short distance, curving to the right, where it doubles back
transversely to the left. Here it doubles sharply back to the
right, from whence it runs forward for a little way, and
crosses over to the left. Curving, it runs posteriorly to
become the distal intestine. Its walls are lined by an
epithelium of large columnar cells.

The distal intestine (d.wnt.). The junction of this
with the proximal intestine is marked by the entrance of the
ducts of the malphigian tubes. It runs posteriorly, and
curves dorsally and forwards to become the rectum, from
which it is separated by a cone-shaped valve—the rectal
valve, the position of which is marked externally (fig. 21, X.).
The epithelium of the distal intestine consists of small
cubical cells, which project into the lumen, and are covered
by a fairly thick chitinous intima. The epithelial wall of
the distal intestine is thrown into usually about six longi-
tudinal folds.

The rectum (rect.) is composed of three parts, an anterior
region, an intermediate region which is swollen to form the
rectal cavity, and a shorter region posterior to this which
opens externally by the anus. The anterior region is lined
by cubical cells, whose internal faces project into the lumen
of the rectum, and give the chitinous intima a tuberculated
structure. The intermediate region which forms the rectal
cavity contains the four rectal glands (rect.gl.). Its walls are
lined by a thin cuticle supported by a flattened epithelium.
The posterior portion of the rectum is short, and has thick
muscular walls. ‘The cuticular intima is continuous with that
of the external skeleton.

Salivary Glands.—There are two sets of salivary glands
—a pair of labial and a pair of lingual glands. The structure
of the labial glands will be described in the account of the
anatomy of the head.

The lingual glands (fig. 17, sl.g.), though considerably
longer than the total length of the body, are of the simplest

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 4238

tubular type. They are of uniform width throughout their
whole length, except the slightly swollen blind termination.
These blind ends he one on each side of the ventral and
posterior region of the abdomen, generally embedded in the
fat-body. ‘hey take a sinuous course forwards through the
abdomen into the thorax, where they run alongside the ven-
triculus. At the sides of the proventriculus they are thrown
into several folds, which appear to be quite constant in cha-
racter. They pass forwards at the sides of the cesophagus
and on entering the cervical region the ducts lose their
glandular character, and assume a spiral thickening ; before
leaving the cervical region the two ducts unite below the
cesophagus, and the single median duct enters the head ven-
tral to the cephalothoracic nerve cord, and runs direct to the
proximal end of the hypopharynx, at the end of which it
opens. A short distance before entering the hypopharynx
the salivary duct (fig. 1, sal.d.) is provided with a small
valve controlled by a pair of fine muscles (s.m.), which
serves to regulate the flow of the salivary secretion. The
glands are composed of glandular cells (fig. 22), which are
convex externally, and have a fibrillar appearance in section.
No vacuoles have been found in the cells.

The Malpighian Tubes.—A pair of malpighian tubes
(fig. 21, malp.) arises at the point of junction of the proximal
and distal intestines, that is, where the mesenteron joins the
proctodeum. Hach malpighian tube shortly divides at an
angle of 180° into two malpighian tubules. The malpighian
tubules are very long and convoluted, and intimately bound
up with the diffuse fat-body, so that it is a matter of consider-
able difficulty to dissect them out entire. They have a
moniliform appearance and are of uniform width throughout ;
never more than two cells can be seen in section. They are
generally yellowish in colour. As in most insects they are
undoubtedly of an excretory nature, as the contents of the
cells and tubules show. Lowne’s view that, in the blowfly,
they are of the nature of a hepato-pancreas is untenable
morphologically and physiologically.

424, C. GORDON HEWITT.

The Rectal Glands.—The four rectal glands (rect.gl.)
are arranged in two pairs, two on each side of the rectal
cavity. Hach rectal gland (fig. 25) has a conical or pyriform
apex with a swollen circular base. Itis composed of a single
layer of large columnar cells (r.gl.), the papilla being hollow,
with the cavity in communication with the general body
cavity. It is covered externally by a perforate chitinous
sheath (sh.), which is continuous with the intima of the rectum.
A number of trachee (t7.) enter the cavity of each gland, and
fine tracheze may be seen penetrating the wall. The cavity
of the gland is filled with a loose tissue of branching cells.
As the gland is capable of pulsation there is no doubt a
constant interchange of blood between the cavity of the gland
and the body cavity (which is a hemoccel). By this means
waste products may be extracted from the blood by the
large gland cells and excreted into the rectum through the
pores on the external sheath of the gland. The rich supply
of tracheze probably assists the cells in the process of excre-
tion, as we find the trachez very numerous, and intimately
connected with the malpighian tubules.

4. The Respiratory System.

The respiratory or tracheal system is developed to a very
great extent in the fly and occupies more space than any
other anatomical structure. Only by dissection of the freshly-
killed insect can one obtain a true conception of its impor-
tance. It consists of tracheal sacs of varying size having
extremely thin walls and trachez which may arise from the
sacs, or, in the case of the abdominal trachezx, independently
from the spiracles.

The Anterior Thoracic Spiracles (figs. 6 and 13, a.th.).
—KHach is alarge vertical opening behind the humeral sclerite
and above the anterior legs. It is surrounded by a chitinous
ring, the peritreme and the opening is guarded by a number
of dendritic processes which prevent the entrance of dust
and other foreign bodies. It leads into a shallow chamber or

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 420

vestibule which communicates with the rest of the spiracular
system through a valvular aperture.

The anterior thoracic spiracles supply the whole of the
head, the anterior and median regions of the thorax, the
three pairs of legs, and by means of the abdominal air-sacs
a large part of the viscera.

Internal to the valve the tracheal system divides. The
tracheal sacs springing from the posterior side are as follows:
Ventrally a rather narrow tracheal duct leads into a sac—
the anterior ventral thoracic sac (fig. 13, a.v.s.) situated at
the side of the thoracic ganglion which it supplies. Above
the origin of this another tracheal duct leads to a vertical sac
supplying the anterior sterno-dorsales muscles. Dorsally
the ducts of two sacs take their origin; the smaller and more
dorsal is a flat sac closely appcsed to the anterior ends of the
dorsales muscles (do.) which it supplies ; the more ventral of
the two is one of the two most important branches of the
anterior thoracic spiracle (the other being the branch
supplying the head). In the thorax it takes the form of an
elongated sac lying below the dorsales muscles, and by side
of the alimentary canal. From the dorsal side of this the
longitudinal thoracic sac (l.¢r.s.) a number of branches arise
which supply the lower dorsales muscles. It is constricted
about the middle of its length and anterior to the constric-
tion; a branch is given off which supplies the ventral portion
of the median sterno-dorsales muscles. In the posterior
region of the thorax another ventral branch is given off from
which branches arise, one supplying the ventral portions of
the posterior sterno-dorsales muscles, the other opening into
the posterior ventral thoracic sac (p.v.s), which supplies the
intermediate and posterior legs. The longitudinal thoracic
sac then narrows, and passes through the thoraco-abdominal
opening into the abdomen. In the adomen it immediately
dilates to form one of the large abdominal air-sacs (a.b.s.).
The pair of abdominal air sacs in some cases occupy about
half the total space of the abdomen. When the fat-body is
not greatly developed they occupy almost the whole of the

4.26 C. GORDON HEWITT.

basal portion of the abdomen. They give off internally a
large number of trachez which ramify among the viscera and
provide a large portion of the contents of the abdomen with
alr.

From the anterior side of the anterior thoracic spiracle a
flattened sac arises. On its ventral side this gives off a
branch which supplies the muscles of the neck and the ante-
rior leg. The sac then narrows into a rather thick-walled
cervical tracheal duct (c.tr.), which passes through the neck
alongside the cephalo-thoracic nerve-cord and enters the head.

Tracheal Sacs of the Head.—The tracheal sacs of the
head occupy the greater portion of the head capsule. They
entirely fill up all the space which would otherwise be hemo-
coel. ‘hese tracheal sacs are supplied by the cervical
tracheal ducts which, on entering the head capsule, curve
dorsally behind the cephalic ganglion. Before curving up-
wards each gives off a large ventral duct (fig. 4), which
spreads out beneath the cephalic ganglion forming a structure
of a tentorial nature upon which the ganglion rests. The
dorsal cephalic ducts unite behind the cephalic ganglion
above the cesophagus. From the point of junction three
ducts arise, two lateral ducts and a median dorsal duct. The
median dorsal duct (m.d.) opens into a large bilobed dorso-
cephalic sac lying on top of the ganglion, and occupy-
ing the dorsal region of the head capsule. It gives off
branching tracheal twigs supplying the antero-dorsal portion
of the optic ganglion (periopticon). Hach of the lateral
ducts (fig. 4, l.d.) supplies the posterior cephalic sacs. It
first communicates with a sac (fig. 13, p.c.s.) lying behind
the dorsal portion of the optic ganglion to which it gives off
a large number of tracheal twigs. This sac opens into an
elongate vertical sac which occupies the ventro-posterior
region of the head capsule. The remaining tracheal sacs of
the head are supplied by the tentorial tracheal ducts (tr.d.),
which spread out beneath the cerebrum in a fan-shaped
manner, and are bilaterally distributed. Hach half, in addi-
tion to giving off internally tracheal twigs to the optic

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSH-FLY. 427

ganglia, communicates with two tracheal sacs. An internal
duct leads into a large spherical sac, the anterior cephalic
sac (a.c.s.) situated in the anterior region of the head dorsal
to the fulcrum. From the dorsal side of this sac a branch
is given off which supplies the antenna of its side; the ven-
tral side is continued down the fulcrum as a narrow tracheal
sac. The lateral portion of the tentorial tracheal duct opens
into the ventro-lateral cephalic sac (v.c.s.) situated posterior
to the optic ganglion. The lower end of this sac gradually
narrows as it enters the rostrum which it traverses, giving
off half-way along its length a trachea which supplies the
palp of that side. On reaching the haustellum it takes the
form of a trachea proper, having annular thickenings.
Shortly after entering the haustellum it gives off two branches
to the muscles of this region. The main trachea is continued
into the oral lobe of its side where it divides into anterior
and posterior branches, and these again divide into numerous
small trachez running to the edges of the oral lobes. Lowne,
in his description of the tracheal system of the blowfly,
describes and figures the tracheal supply of the proboscis as
being of the nature of tracheal sacs and capable of distension ;
he also describes a trefoil-shaped tracheal sac at the base of
the oral lobes giving off very regular branches, the dilation
of which causes the inflation and tension of the oral lobes.
The mechanism of the proboscis will be discussed later
(p. 439), but it may be noticed here that in M. domestica
there is no trace of a trefoil-shaped sac at the base of the
oral lobes, and that all the tracheal structures of this the
haustellum region are definite annular tracheew, and there-
fore incapable of distension.

The posterior thoracic spiracle (figs. 6 and 15, .th.)
is triangular in shape and guarded by dendritic processes.
It possesses a vestibule which leads into a distributing tracheal
sac. The tracheal sacs of this system (fig. 15) have not the
extended range of those supplied by the anterior thoracic
spiracle, but are confined to the thorax, chiefly in the median
and posterior regions which are not «rated to any great

428 C. GORDON HEWITT.

extent by those of the other system. They supply chiefly
the large muscles of the thorax. Laterally a series of sacs
(l.th.s.) extends antero-dorsally in an oblique direction, ex-
ternal to the sterno-dorsales muscles to the humeral region.
From the first of these sacs a large number of tracheal twigs
arise and supply the muscles of the wing and the anterior
sterno-dorsales muscles. Ventral to this sac a large sac
(m.v.s.) penetrates internally between the anterior and median
sterno-dorsales muscles and supplies the lower dorsales
muscles. From the dorsal side of the distributing sac a
number of sacs arise, some of which penetrate between the
sterno-dorsales muscles and supply the upper dorsales mus-
cles. A more posterior set supplies the posterior regions of
the dorsales muscles, ramifying between them in a very
extensive manner, some ultimately terminating in the tracheal
sacs beneath the scutum and the scutellar sac (sc.s.).

The abdominal spiracles differ in number in the two
sexes. In the male there are seven pairs of abdominal
spiracles; in the female I have only been able to find five
pairs. In both sexes each of the large tergal plates which
cover the abdomen has near its lateral margin a small circular
spiracle. ‘The first abdominal segment which has fused with
the second has a pair of small spiracles (see fig. 8) slightly
anterior to those of the second (apparent first) abdominal
segment. In addition to these the male possesses two pairs
of spiracles in the membrane at the lateral extremities of the
rudimentary sixth and seventh abdominal segments (see
fig. 5). In the female I have been unable to find any addi-
tional spiracles. Hach of the abdominal spiracles is provided
with a vestibule and atrium which are separated by a valve
controlled by a minute chitimous lever. All the spiracles
of the abdomen communicate with tracheze which ramify
among the viscera and fat-body; there are no tracheal sacs
in connection with these spiracles.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 429

5. The Vascular System and Body-cavity.

By the great development of the tracheal sacs in the head,
the muscles in the thorax, and the fat-body and air sacs in
the abdomen, the hemoceelic space in the fly is greatly
reduced. The blood is colourless, and is crowded with cor-
puscles, mostly containing substances of a fatty nature.

The fat-body varies greatly in the extent of its develop-
ment. In some cases it may almost fill the body-cavity,
pushing the intestine back into a postero-dorsal position:
this is generally the case in flies before hibernating; in other
cases it may be only moderately developed. The fat-body
receives a very rich tracheal supply, and stores the products
of digestion which are conveyed to it by the blood with which
it is bathed. It consists chiefly of very large cells, both
uninucleate and multinucleate; the fat-cells of the head are
not so large.

The dorsal vessel or heart lies in the pericardial chamber,
immediately beneath the dorsal surface. It extends from the
posterior end to the anterior end of the abdomen, and four
large chambers, corresponding to the four visible segments,
and a small anterior chamber can be recognised; the last
represents the chamber of the first abdominal segment. ‘The
chambers are not separated by septa, but each has a pair of
dorso-lateral ostia situated at its posterior end where the alar
muscles of the pericardium arise. The walls of the heart are
composed of large cells. The pericardium contains fat-cells
and trachez, and its floor is composed of large cells of a
special nature. The alar muscles run laterally in the floor of
the pericardium to the sides of the dorsal plates where they
are inserted. The anterior end of the heart is continued as a
narrow tube (fig. 20, d.a.) along the dorsal side of the ven-
triculus, where it terminates in a mass of cells (/.g.), which
are usually considered to be of a lymphatic nature.

430 CG. GORDON HEWITT.

6. The Reproductive System.

The two sexes are slightly different in size, the females
being larger than the males; the sexual dimorphism of the
width of the frontal region of the head has already been
noticed (p. 402). There does not appear to be any great
disparity in the numerical proportions of the sexes; near
breeding places there is naturally a preponderance of
females.

The Female Reproductive Organs.—The generative
organs of the female consist of ovaries, spermathece or
vesiculee seminales, accessory glands and their ducts.

The ovaries, when containing mature ova, occupy the
greater part of the abdominal cavity (fig. 23, ov.). They lie
ventral to the gut, occupying the whole of the ventral and
lateral regions, the gut resting on the V-shaped hollow
between them. Hach ovary contains about seventy ovarioles,
in each of which ova in various stages of development can be
seen. ‘The two short thin-walled oviducts (ov.d.) unite on
the ventral side of the abdomen to form the common oviduct
(c.o.d.). The walls of the common oviduct are muscular, and
when the ovipositor is in a state of rest, retracted into the
abdominal cavity, the oviduct curves forwards and dorsally
to enter the ovipositor (ov.p.) ventral to the rectum (rect.).
Here it swells slightly to form a sacculus (fig. 26, sac.) which
leads into the muscular vagina (vag.). The vagina opens into
the ventral side of the ovipositor immediately behind the
sub-anal plate.

The spermathecee (sp.) or vesicule seminales are three in
number, two on the left side, and a single one on the right.
Each consists of a small, black, oviform, chitinous capsule, the
lower half of which is surrounded by a follicular investment
continuous with the cellular wall of the duct, the whole havy-
ing the appearance of an acorn with a long stalk. The ducts
of the spermathece are lined by a thin chitinous intima con-
tinuous with the chitinous capsule, and they open at the pos-
terior end of the sacculus on the dorsal side.

STRUCTURE, DEVELOPMENT, AND BLONOMICS OF HOUSE-FLY. 431

There isa single pair of accessory glands (ac.g.), which are
fairly long, and on nearing the vagina they become narrower
to form a slender duct, which opens on the dorsal side of the
vagina immediately behind the ducts of the spermathece.
The accessory glands are closely united with the fat-body.
They probably secrete the adhesive fluid which covers the
eges when they are laid, and causes them to adhere to each
other and to the material upon which they are deposited.
Behind the accessory glands there is a pair of thin-walled
transparent vesicles (tasche dell’ ovidutto of Berlese),
which I propose to name the accessory copulatory vesicles
(a.c.v.) on account of the part they take in ensuring firm
coitus with the male during copulation, during which process
they expand to a much greater extent.

The ovipositor (fig. 8). The terminal abdominal segments
of the female are much reduced to form a tubular ovipositor,
the chitinous sclerites being reduced to form slender chitinous
rods. When extended it equals the abdomen in length. It
is composed of segments vi, vil, vill, and ix, each being sepa-
rated from the adjacent segments by an extensible inter-
segmental membrane, which is covered with fine spines.
When the ovipositor is retracted (fig. 25, ovp.) it lies in the
interior of the posterior end of the abdomen, the segments
being telescoped the one within the other, so that only the
terminal tubercles are visible from the exterior. The dorsal
arch of the sixth abdominal segment is reduced to a A-shaped
sclerite (vi, d.), lying on the dorsal side of the segment.
The ventral arch of this segment is reduced to a slender
chitinous rod (vi, v.) in the mid-ventral line. The dorsal
arch of the seventh segment is represented by two slightly-
curved sclerites (vii, d.), with their concave faces opposite ;
the ventral arch (vii, v.) is similar to that of the sixth
segment. At the junction of the posterior ends of the
sixth and seventh segments with the inter-segmental mem-
branes succeeding them there are several setose tubercles
arranged more or less in pairs, but they vary in development
in different individuals. The dorsal arch of the eighth

432 C. GORDON IIEWITT.

segment consists of two parallel and slender sclerites (viii, d.),
not so narrow as those of the two preceding segments. A
pair of slender sclerites (vil, v.) also represents the ventral
arch. The terminal anal segment, which I consider repre-
sents the reduced ninth segment, has a dorsal chitinous
sclerite, the sub-anal plate (sw.p.), which is triangular in
shape, and a ventral sub-anal plate of the same shape. ‘The
female genital aperture is situated at the anterior end of the
latter plate, between the eighth and anal (ninth) segments.
A pair of terminal setose tubercles is situated laterally at the
apex of the anal segment.

The Male Reproductive Organs.—The male repro-
ductive organs (fig. 24) are situated ventral to the alimentary
canal, and lie within the fifth abdomimal segment. They
consist of a pair of testes, vasa deferentia, ejaculatory duct
and sac, and the terminal penis. There are no accessory
genital glands in the male.

The testes (te.) are a pair of brown pyriform bodies, with
their long axes placed transversely, and their pointed ends
facing. In young males they have a bright red appearance.
They are covered with a follicular investment of cells, which
varies in thickness apparently according to age. The thin
brown chitinous capsules contain the developing spermato-
zoa. ‘Ihe pointed end of each testis is continued as a fine
vas deferens (v.d.), which meets that of the other testis in the
median line, where they open into the common ejaculatory
duct (d.e.). This runs forwards for a short distance, and then
bends to the left ventrally, and, after several convolutions on
the left ventral side of the abdomen, the duct narrows con-
siderably, forming a narrow ejaculatory duct. This crosses
over the dorsal side of the rectum to the right side, where it
runs forwards for a short distance and then curves back in
the median ventral line, opening into a pyriform ejaculatory
sac (e.s.). The walls of this ejaculatory sac are muscular,
longitudinal muscles, giving the walls a striated appearance.
It contains a phylliform, chitinous sclerite—the ejaculatory
apodeme (e.a.), Which has a short handle at the broad end.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 433

This sclerite is, no doubt, of great assistance in propelling
the seminal fluid along the ejaculatory duct during copula-
tion. A short distance behind the ejaculatory sac the duct
opens into the penis.

The Male Gonapophyses.—The extremity of the
abdomen in the male (fig. 10) has undergone considerable
modification in the formation of the external genitalia. The
visible portion of the abdomen, as seen from above, consists
of the first five abdominal segments; the remaining three
seoments are slightly withdrawn into the fifth segment, and,
on looking at the abdomen from the posterior end, only the
terminal segment, the eighth, surrounding the anus, can be
seen. The sixth and seventh segments have been greatly
reduced. The sternal portion of the fifth segment consists
of a cordiform sclerite (V.v.), the apex of which is directed
forwards, and each of the lateral margins of the base is
produced to form a short process, swollen at the tip—these
lateral processes form the primary forceps (p.f.), and le at
each side of the aperture of the male genital atrium (g.a.), of
which the posterior edge of the sclerite forms the lower or
anterior lip. The dorsal plates of the sixth and seventh
segments lie on the membrane, which is tucked underneath
the posterior edge of the fourth abdominal segment. The
dorsal plate of the sixth segment (vi, d.) is a narrow, trans-
verse sclerite ; its lateral edges, which do not extend down
the sides, are slightly produced anteriorly. The ventral
plate of the sixth segment (vi, v.) is asymmetrical, and, with
the dorsal plate of the seventh segment, produces a _pro-
nounced asymmetry of the posterior end of the male abdo-
men. It consists of a spatulate plate on the left side, the
anterior or ventral side of which is produced into a narrow
bar extending across the ventral side of the aperture of the
genital atrium, its distal extremity bifurcating. The dorsal
plate of the seventh segment (vii, d.) is asymmetrical. It
consists of a narrow sclerite, which, on the dorsal side, is
similar to the sixth dorsal plate, but the left side (see fig. 5)
extends down the side, and broadens out into a somewhat

434 CO. GORDON HEWITT.

triangular-shaped area; the anterior edge of this is incised,
and receives the seventh spiracle (vii, a.sp.); the ventral
edge is internal to the spatulate portion of the sixth ventral
plate. The ventral arch of the seventh sclerite has been
completely withdrawn into the abdomen, and consists of
a pair of curved sclerites (fig. 9, vil, v.), somewhat rhom-
boidal in shape, lying dorsal to the fifth ventral arch and
ventral to the penis (P.); they form the secondary forceps.
Their lateral edges, which are thickened articulate with the
alar processes of the body of the penis (c.pe.), and with the
dorsal arch of the eighth abdominal segment (viii, d.). Their
inner edges are curved, and almost meet in the mid-ventral
line. The dorsal arch of the eighth and last abdominal
seement (viii, d.) forms the apex of the abdomen. It consists
of a strongly convex sclerite, deeply incised on the ventral
side; in this incision the vertical slt-like anus (fig. 10, an.)
lies. The ventral portion of the segment is completed by a
pair of convex sclerites (viii, v.), which are united in the mid-
ventral line, forming the ventral border of the anal membrane
and the dorsal side of the entrance to the genital atrium.

All the sclerites of the posterior segments except the sixth
and seventh are setose.

Berlese (1902) in his account of the copulation of the
House-fly describes the genitalia. From his account of the
male genitalia he appears to have missed the narrow dorsal
arch of the sixth segment, or, what is very probable, he may
have mistaken it for the fifth dorsal arch, as he terms the
seventh dorsal arch the sixth, and describes what I have
called the ventral arch of the seventh as the dorsal arch of
that segment. This mistake in nomenclature has probably
arisen from the fact that he considered the visible portion of
the abdomen as consisting of four segments instead of five,
in which case the narrow dorsal arch of the sixth segment
would naturally be taken for that of the fifth."

1 Berlese describes a sinistral asymmetry of the posterior segments, but
his figures show a dextral asymmetry, a mistake probably in the reproduction
of his figures which has escaped the author’s notice.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 439

The penis (figs. 7 and 9) lies internally on the ventral side
of the abdomen, dorsal to the ventral arches of the fifth and
seventh segments. It is composed of several sclerites. A
median sclerite (¢.pe.), the anterior and ventral edge of which
is roughly semicircular in outline, forms the body of the
penis. his is produced laterally to form two alar processes ;
at the bases of these processes the lateral extremities of the
dorsal arch of the eighth segment articulate with the body of
the penis; the extremities of the processes are attached to
the lateral extremities of the ventral sclerites of the seventh
segment, the secondary forceps. ‘The penis proper consists
of a hollow cylindrical tube, the theca, which receives the
ejaculatory duct. The theca articulates with the body of the
penis by means of a pair of small chitinous nodules (“ cor-
netti’’ of Berlese); posterior to the attachment the theca is
constricted slightly. Below the aperture for the entrance of
the ejaculatory duct, the theca is produced into a ventrally
directed curved process, the inferior apophysis (i.ap.) ; above
the aperture a short cylindrical process, the superior apo-
physis (s.ap.), arises. The anterior end of the theca is con-
tinued as a slightly inflated hyaline structure, the glans
(p.gl.), at the curved extremity of which the ejaculatory duct
opens.

V. Tue Internat Srructure oF tHe Heap.

The skeletal framework and tracheal system of the head
have already been described. It remains, therefore, to give
an account of the musculature of the head and pharynx, and
also an account of the oral lobes.

The posterior region of the head (fig. 1) not occupied by
tracheal sacs is usually filled up with small multinucleate fat-
cells (f.c.), which are also occasionally found in the proboscis.
The frontal sac or ptilinium (P¢.) fills up the anterior portion
of the head not occupied by air-sacs. Its crescentic opening,
the lunule, has already been described. It is attached to the

436 C. GORDON HEWITT.

wall of the cephalic capsule by muscles which vary consider-
ably in the extent of their development. In recently
emerged flies the muscle-supply of the ptilinium is consider-
able, as they have served to retract the sac after it has been
inflated to assist the exclusion of the imago, but in older
specimens it becomes less. The walls of the ptilinium are
muscular and lined by a chitinons intima covered with small
broad spines.

The Musculature of the Proboscis.—The chief
muscles controlling the movements of the pharynx and pro-
boscis are these :

The Dilators of the Pharynx (figs. 1 and 2, d.ph.)—
This pair of muscles occupies the interior of the fulcrum.
Nach muscle is attached to the antero-lateral regions of the
fulcrum and inserted into the dorsal plate of the pharynx
(r.p.). These muscles are the chief agents in pumping the
liquid food into the cesophagus, and in drawing it up through
the pharyngeal tube.

The Retractors of the Fulcrum (fig. 1, 7.f.).—These
muscles are attached to the internal anterior edges of the
gene, and are inserted into the posterior cornua (p.c.) of the
fulcrum. Their contraction causes the rotation of the fulcrum
on the epistome as a hinge in the retraction of the proboscis.

The Retractors of the Haustellum (7.h.).—These
muscles have their origin on the dorso-lateral regions of the
occiput. They are long and narrow, and running on each
side of the common salivary duct are inserted into the dorsal
margin of the theca.

The Retractors of the Rostrum (r.7.).—This pair of
muscles has its origin at the sides of the occipital foramen,
and is inserted into the posterior side of the membranous
rostrum about half-way down its length. In the retraction
of the proboscis these muscles draw in the rostrum,

The last two pairs of muscles acting together assist in the
retraction of the whole proboscis.

The Flexors of the Haustellum (fh.) have their
origin close to that of the retractors of the rostrum at the

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 437

sides of the occipital foramen. They are inserted into the
base of the labral apodeme (ap.), and serve to flex the
haustellum on to the anterior face of the rostrum.

The Extensors of the Haustellum (ex.h.)—Hach of
these muscles arises from the distal cornu of the fulerum, and
is inserted into the head of the labral apodeme.

The Accessory Flexors of the Haustellum (a,f.h.)
are attached to the lower (distal) anterior margin of the ful-
crum, and inserted with the extensors into the head of the
labral apodeme.

The Flexors of the Labium-epipharynx (f.l.).—
These muscles have their origin on the anterior and upper
edge of the fulcrum, and are inserted into the proximal end
of the labium-epipharynx. The first pair of the last three
sets of muscles serve to extend the haustellum in the exten-
sion of the proboscis, and the remaining two pairs assist in
the retraction of the proboscis by flexing the haustellum on
to the rostrum.

A pair of very fine muscles (s.m.) have their origin at the
base of and internal to the posterior cornua of the fulcrum.
They are inserted into the dorsal side of a small valve (s.v.)
on the common salivary duct which regulates the flow of the
secretion of the lingual salivary glands.

The muscles of the haustellum are—

The Retractors of the Furea (r.fw.).—A pair of
muscles having their origin on the upper part of the theca.
Each is inserted along the upper proximal half of the lateral
process of the furca. When the muscles contract the lateral
processes of the furca, which, in a state of repose are brought
together by the elasticity of the ventral cornua of the theca,
are diverged, and thus cause the divergence and opening of
the oral lobes.

The Retractors of the Discal Sclerites (7.d.s.).—
These muscles have their origin on the lateral edges of the
upper part of the theca, and are inserted upon the sides of
the discal sclerites. They work together with the retractors

VOL, 51, PART 3,—NEW SERIES, 30

438 C. GORDON HEWITT.

of the furca, their contraction causing the divergence of the
discal sclerites, and the consequent opening of the oral pit.

The Dilators of the Labium-hypopharynx (dil.).
—These fan-shaped muscles arise in the middle region of
the theca on either side the median line, and diverging
are inserted in the lateral edges of the labium-hypopharyngeal
sclerite. By their contraction they will widen the channel of
the labium-hypopharynx.

The Dilators of the Labium-epipharynx (di.l.)—
These form a series of short muscles attached to the anterior
and posterior walls of the labium-epipharynx. The size of
the pharyngeal channel will be regulated by these muscles.

The Oral Lobes.—The external structure of the oral lobes
has already been described. Their internal structure and
histology will be given here, as it seemed preferable to do so
rather than postpone it to a future communication.

The setigerous cuticle and the pseudo-trachez lie on a
hypodermis of cubical cells (fig. 18, hy.). Beneath the hypo-
dermis of the aboral surface is another layer of cells contain-
ing a large amount of dark pigment. Hach of the large
marginal sensory bristles (g.s.) of the aboral surface has a fine
channel running down the whole length of the seta. This
channel communicates with the cavity of a pyriform mass of
nerve-end cells (s.p.), consisting of five or six cells. These
masses of cells occupy a large part of the interior of the oral
lobes. As these gustatory bristles are exposed and directed
ventrally when the proboscis is retracted, they may assist the
fly in testing the nature of its food before extending its pro-
boscis. On the oral side of the oral lobes the nipple-like gus-
tatory papille (figs 1 and 18, gp.) have already been descrided.,
The aperture at the end of the papilla leads into a fine duet,
which ends in a pyriform sensory bulb (s.g.p.). The trachez
(t.) can be seen running through the cells, some of which con-
tain several nuclei, and from their appearance are probably
derived from the fat-body. No tracheal sacs could be found
either in the oral lobes or at their bases, but the annular
tracheee are continuous with those of the proboscis, The

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 439

heemoccel of the oral lobes is well developed. This supports
the view set forth by Kraepelin, and with which I agree that
the inflation of the oral lobes is due to the blood. I consider
that the extension of the proboscis is due to the inflation of
the tracheal sacs of the head. The proboscis having been pro-
truded the oral lobes are then diverged by the contraction of
the retractor muscles of the furca and discal sclerites, and dis-
tended by the inrush of blood which keeps them turgid, and
causes the openings into the pseudo-tracheal channels to
remain open.

The Labial Salivary Glands (figs. 19 and 1, Jb.sl.).—
These salivary glands lie in the haustellum at the base of the
oral lobes. The glands, which are spherical in shape, are com-
posed of a large number of gland cells somewhat triangular
in shape. Hach gland cell is 40, in size, and possesses a
large nucleus (12 4), and internal to this a permanent circular
vacuole (vac.), which is 16 4 in size, and is lined by a thin
chitinous intima. The duct of each gland cell opens into the
side of the vacuole (od.). The ducts (ic.d.) are intracellular,
and run from the centre of the gland, some of them uniting,
to form a number of fine ducts on the ventral sides of the discal
sclerites, which unite and open into the oral pits by a median
pair of pores. Kraepelin, in his description of the proboscis
of the blowfly, described the labial glands and their ducts
(but not their histology) of that insect, his description being
similar to the condition I find in M. domestica. Lowne,
however, states that in the blowfly he traced the ducts of the
gland cells through the oral lobes to the apertures of the
gustatory papillae, which he regarded therefore as the aper-
tures of the labial salivary glands.

The secretion of the labial salivary gland serves to keep
the surface of the oral lobes moist.

VI. Summary.

1. The exoskeleton of the head capsule and of the pharynx
is described in detail; the relations of the parts in the terms

44.0 C. GORDON HEWITT.

generally employed by dipterologists to the morphological
divisions of the insect head capsule are shown. On morpho-
logical grounds, the view that the distal portion of the
proboscis represents the modified second maxille or labium
is adopted, as opposed to that of a first maxillar derivation
put forward by Lowne for the blowfly.

2. After a detailed description of the external and in-
ternal skeletal structures of the thorax, the neuration of the
wings is described in the terms proposed by Comstock and
Needham in their valuable memoir; and to facilitate their
more general adoption for the wings of the Muscide and
other Diptera, a comparison is made between their nomen-
clature and the several systems employed in describing the
muscid wing.

3. The abdomen is shown to consist of eight segments in
the male and nine in the female, in both cases the first five
segments form the visible portion of the abdomen; the
external genitalia of the two sexes are described under
another section.

4. As the muscular system does not differ from that of
Volucella described by Kunckel d’Herculais and the blow-
fly described by Hammond and Lowne, it is briefly described.
The cephalic muscles, however, are fully described in the
detailed description of the head (V).

5. The nervous system, which is of the normal muscid type,
is described, but for the sake of clearness a very detailed
description of the composition of the cephalic ganglion is not
given. The structure of the optic tract is similar to that of
the blowfly as described by Hickson. The structure of the
thoracic nerve-centre is found to differ slightly from that of
the blowfly as described by Lowne.

6. The alimentary canal is similar in its structure to those
of Stomoxys and Glossina, only differing in a few details.
The mesenteric region, which is represented by the ventri-
culus or chyle, stomach, and proximal intestine, is well
developed. The lingual salivary glands, rectal glands, and

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 441

Malpighian tubes are described ; the function of the rectal
glands is believed to be of an excretory nature.

7. As the tracheal systems of the Diptera have not received
much attention a detailed account of the tracheal system is
given. There are two thoracic spiracles, the first of which
supplies the whole of the head, the anterior and median
regions of the thorax and the three pairs of legs, and by
means of a pair of large abdominal air-sacs a large part of
the viscera. The posterior thoracic spiracle supplies the
muscles of the median and posterior region of the thorax,
especially the large dorsales muscles. There are seven pairs
of abdominal spiracles in the male and five pairs in the female
all of which are connected with tracheee only.

8. The dorsal vessel or heart is found to consist of five in-
complete chambers, each with a pair of ostia. The anterior
end is continued forwards along the dorsal side of the ventri-
culus, and terminates in a glandular mass in the anterior
margin of the proventriculus.

9. The reproductive organs of the male are simple, con-
sisting of a pair of testes, vasa deferentia, and common
ejaculatory duct; there are no accessory glands such as are
found in many other Diptera. The terminal abdominal
segments of the male exhibit a sinistral asymmetry.

The ovaries of the female, when mature, occupy the
greater portion of the abdominal cavity. There are a pair
of accessory glands (probably of a “gum” or “glue”
nature), three spermathecew, and a pair of vesicles used
during copulation. The ovipositor is about as long as the
abdomen, and is composed of segments six to nine.

10. The musculature of the head is described in detail, and
it is found that the House-fly agrees with the blowfly in the
number and relations of its cephalic muscles, though in a few
cases the attachments are slightly different. In the haus-
tellum and oral lobes of the House-fly no tracheal sacs similar
to those described and figured by Lowne for the blowfly
occur, but only annulated tracheee are found, and, as these
are incapable of distension, the view that the oral lobes are

44.2 C. GORDON HEWITT.

distended by the action of inflated air cannot be held. The
extension of the proboscis I believe is due to the inflation of
the tracheal sacs of the head and rostrum, and I agree with
Kraepelin that the distension of the oral lobes is effected by
blood-pressure.

Two kinds of gustatory sense-organs are found on the
margin of the aboral and on the oral surfaces respectively.
The latter were described in the blowfly by Lowne as the
openings of the ducts of the labial salivary glands, but
Kraepelin’s correct description of their structure in the
blowfly is confirmed by this study of the House-fly. The
labial salivary glands are described in detail. They consist
of large cells containing permanent vacuoles, which com-
municated with intracellular ducts. These open by a pair of
pores into the oral pits, the secretions of the glands serving
to keep the surface of the oral lobes moist.

VII. Livreratvre.

The following is not intended to be a full list of the litera-
ture relating to M. domestica. Further references will be
given in the succeeding parts.

1824. Anpouin, V.—“ Récherches Anatomiques sur le thorax des Animaux
Articules et celui des Insectes Hexapodes en particuliére,” ‘ Ann.
Sci. Nat. Zool.,’ vol. i.

1906. Austen, KE. E.—‘ Illustrations of British Blood-sucking Flies,’ 74 pp.,
34 col. plates. Brit. Mus. (Nat. Hist.), London.

1902. Brrieszk, A.—‘‘ L’accoppiamento della Mosca domestica,” ‘Rev.
Patolog. vegetale,’ vol. ix, pp. 8345—3857, 12 figs.

1834. Boucuer, P. Fr.—‘ Naturgeschichte der Insekten,’ Berlin.

1898.—Comsrock, J. H., and Neepuam, J. G.—‘* The Wings of Insects,”
‘Amer. Nat.,’ vol. xxxii, p. 43, etc., and through the vol. into

vol. Xxxili.
1752-78. De Grrr, Cart.— Mémoires pour servir a |' Histoire des Insectes,’
Stockholm.

1881. Hammonp, A.—“On the Thorax of the Blowfly (Musca vomi-
toria),” ‘Journ. Linn. Soe.’ (Zool.), vol. xv, pp. 9—31, 2 pls.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 445

1854.

1885.

1902.

Hepworth, J.—‘On the Structure of the Foot of the Fly,”
*Q. J. M.S.,’ vol. 2, pp. 158—160.

Hickson, S. J.—‘‘ The Eye and Optic Tract of Insects,” ‘Q. J. M.S.,’
vol. 25, pp. 1—39, 3 pls.

Howarp, L. O.—‘ House-flies” (in ‘ The Principal Household Insects
of the United States,’ by L. O. Howard and C, L. Marlatt), United
States of America Dept. of Agriculture, Washington. Division of
Entomology. Bull. No. 4, N.S., revised ed., pp. 43—47, and figs. ;
aud (1898) Circular No. 35, 2nd series, pp. 1—8, and figs.

Ketuer, J. C.—‘Geschichte der gemeinen Stubenfliege,’ 32 pp.,
4 pls., Nurnberg.

KraEpetin, K.—‘‘ Zur Anatomie und Physiologie des Russels von
Musca,” ‘Zeit. f. wiss. Zool.,’ vol. xxxix, pp. 683—719, 2 pls.

1875-S1. Kuncxket v’Hurcunats, J.—‘ Récherches sur l’organisation et le

1874.

1738.

1860.

Développement des Volucelles,’ Paris.
Liyyevs, C. pr.—‘ Systema nature’ (10th ed.), vol i, p. 596; and
‘Fauna suecica,’ ed. ii, Holmie, 1761.

Lowne, B. T.—‘* The Anatomy and Physiology of the Blowfly (Musca
vomitoria),’ 121 pp., 10 pls., London.

“On the Compound Vision and the Morphology of the Eye
in Insects,” ‘Trans. Linn. Soe.’ (Zool.), vol. ii, pt. 11.

‘The Anatomy, Physiology, Morphology, and Development
of the Blowfly (Calliphora erythrocephala),’ 2 vols., London.

Mactoskiz, G.— The Proboscis of the House-fly,” ‘Amer. Nat.,’
vol. v, pp. 153—161.

Meru, A. A. C. H.— The Foot of the House-fly,” ‘Journ. Quekett
Club’ (2), vol. vi, p. 348.

** Supplementary Note on the Foot of the House-fly,” ‘Journ.
Quekett Club’ (2), vol. ix, pp. 167, 168.

Mincuin, E, A.—* Report on the Anatomy of the Tsetse-fly (Glos-
sina morsitans),” ‘Proc. Roy. Soc.’ (Ser. B), vol. Ixxvi, pp. 531
— 547, and figs.

Packarp, A. 8.—“ On the Transformations of the Common House-
fly, with Notes on allied forms,” ‘ Proc. Boston Soc. Nat. Hist.,’
vol. xvi, pp. 136—150, 1 pl.

Reaumur, R. A. F. pp.—‘ Mémoires pour servir 4 |’Histoire des
Insectes,’ Paris (vol. iv).

SAMUELSON, J., and Hicks, J. B.—‘ The Earthworm and the Common
House-fly,” ‘Humble Creatures,’ pt. i, 79 pp., 8 pls., London.
‘The House-fly,’ pp. 26—79, pls. 3—8.

444, C. GORDON HEWITT.

1862. Scuiner, J. R.—‘ Fauna Austriaca: die Fliegen.’ 2 vols., Wien.

1895. Snare, D.—* Insects,” part ii, ‘Cambridge Nat. History,’ London.

1906. Tuttocu, F.—‘ The Internal Anatomy of Stomoxys,” ‘Proc. Roy.
Soc.’ (Ser. B.), vol. Ixxvii, pp. 523—531, 5 figs.

1906. Werscurt, W.—‘‘The Genitalia of both Sexes in the Diptera, and

their relation to the Armature of the Mouth,” ‘Trans. Linn. Soc.,’
vol. ix, pp. 339—386, 8 pls.

Tue UNIVERSITY,
MANCHESTER.

EXPLANATION OF PLATES 22—26,

Illustrating Mr. C. Gordon Hewitt’s paper on “ The Structure,
Development, and Bionomics of the House-fly (Musca
domestica, Linn.). Part I. Anatomy of the Fly.”

PLATE 22.

Vic. 1.—Musca domestica. Female.

Fic. 2.—Anthomyia radicum. Female.

Fic. 3.—Homalomyia canicularis. Male.

Fic. 4.—Stomoxys calcitrans. Female. The halters of this species
have been drawn too far back, and in this and the other species the nervures
of the wings have been made thicker than they naturally are.

These figures are not drawn to the same scale.

PLATE 23.

Fie 1.—Interior of the head of M. domestica. In this figure the left
side of the head capsule and of the proboscis have been removed and the
compound eye of the same side, leaving the optic ganglion (periopticon). All
the tracheal structures have been omitted.

a.c. Anterior cornu of fulcrum. aff. Accessory flexor muscles of hau-
stellum. ap. Apodeme of labrum. az.n. Antennal nerve. C.G. Cephalic
ganglion. di.J. Dilator muscles of labium hypopharynx. d.ph. Dilator
muscles of pharynx. ds. Discal sclerite. ea.4. Extensor muscle of hau-
stellum. J. Fulcrum. f. Furea. fc. Fat-cells. 4. Flexor muscle of
haustellum. 2. Flexor muscle of labrum-epipharynx. y.p. Gustatory
papille of oral lobes. & Hyoid sclerite of pharynx. /b.n. Labial nerve.
lé.sl. Labial salivary gland. ZAp. Labium-hypopharynx. /.ep. Labrum-
epipharynx. map. Maxillary palp, @s. Gsophagus. oc.n. Ocellar nerve.
phn. Pharyngeal nerve, p.c. Posterior cornu of fulerum. 2.0. Periopticon.
ps. Pseudotrachea. Pé, Ptilinium. 7.d.s. Retractor muscles of the discal

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 440

sclerites. 7,f. Retractor muscle of the fulcrum. 7,fz. Retractor muscle of
the furea. 7.4, Retractor muscle of haustellum. 7.7. Retractor muscle of
rostrum, §.0, Sub-csophageal ganglion. sal.d. Common duct of the lin-
gual salivary glands. s.v. Valve of the common salivary duct. s.m. Muscle
controlling the valve of salivary duct. ¢h. Theca.

Fic. 2.—Transverse section through the lower portion of the head-capsule,
showing the muscles and tracheal sacs in this region and the fulerum in
section. (Camera lucida drawing.)

bp. Floor of pharynx. r.y. Roof of pharynx. é7.s. Tracheal sac. Other
lettering as in Fig. 1.

Fic. 3.—Transverse section through the lower half of the haustellum,
where the hypopharynx (4p.) has become free from the labium. (Camera
lucida drawing.)

dil. Dilator muscles of the labium-epipharynx. ¢r. Trachea. Other
lettering as in Fig, 1,

Fic. 4.—Posterior view of the tracheal ducts which supply the cephalic
sacs and trachee.

ctr. Cervical trachere which fuse above the cesophagus on the posterior
side of the cephalic ganglion, 7d. Lateral duct. m.d. Median dorsal duct.
¢n.d. Yentorial tracheal ducts which spread out beneath the cephalic ganglion,

Fic. 5.—Lateral view of the terminal segments of tle abdomen of the male
after their removal from the fifth segment.

vi, a.sp. and vii, a.sp. Sixth and seventh abdominal spiracles. Lettering as
in Fig. 10.

Fic. 6.—The thorax seen from the left side. The insertions of the larger
setz are shown; for the sake of clearness the sclerites of the wing- base are
omitted.

a.th. Anterior thoracic spiracle. ca. Costa. cp. Intermediate coxal
plates. ep’., en’. Epimera of the meso- and meta-thoracic segments. eps’.,
eps’’., eps’, Episterna of the pro-, meso-, and meta-thoracic segments. Aad.
Haltere. Aw. Humerus. Jp. Lateral plate of mesosternum. /p.se. Lateral
plate of postscutellum. mph. Mesophragma. mpsc. Median plate of post-
scutellum. mz. Metanotum. ms. Mesosternum. més. Metasternum. p.th.
Posterior thoracic spiracle. pé. Parapterm. pr.z. Pronotum. prs. Pre-
scutum of mesothorax. sc. Scubum. scé/. Scutellum.

Fic. 7.—Penis seen from the right side after it has been removed from
within the terminal abdominal segments.

i.ap. Inferior apophysis. ¢th.p. Theca of penis. p.g/. Glans. s.ap. Supe-
rior apophysis. Other lettering as in Fig. 9, ete.

Fie. 8.—Abdomen of female showing the extended ovipositor.
V, d@. to ix, d. Fifth to ninth dorsal arches or plates of the abdomen. V, x.

44.6 C. GORDON HEWITT.

to vill, v. Fifth to eighth ventral plates or arches. sw.p. The suranal plate
(ninth dorsal arch).
The anus is situated between the two lateral terminal tubercles.

Fic. 9.—Dorsal view of the penis and the ventral half of the terminal
abdominal segments. The median portion of the eighth dorsal arch has been
removed, leaving the lateral portions attached to the body of the penis (c.pe.)
and the ventral arch of the seventh segment (vii, v.).

Lettering as in Fig. 10.

Fic. 10.—The posterior end of the abdomen of the male seen from behind,
showing the pronounced sinistral asymmetry.

v, @. to vill, d. Fifth to eighth dorsal plates or arches. _v, v. to viii, v. Fifth
to eighth ventral plates or arches. az. Anus. g.a. Aperture of genital
atrium. p.f. Primary forceps.

PLATE 24.

Vie. 11.—Nervous system. The very fine nerve which runs along the
dorsal side of the cesophagus to the proventricular ganglion (Po.g., Fig. 20)
has been purposely omitted.

ab.m. Abdominal nerve. ac.ms. Accessory mesothoracic dorsal nerve. ac.mt.
Accessory metathoracic dorsal nerve. cer.z. Cervical nerves. cz. Cephalo-
thoracic nerve cord. O.P. Optic peduncle. pr.cr., ms.cr., mi.cr. Pro-, meso-,
and meta-thoracic crural nerves. pr.d., mis.d., mt.d. Pro-, meso-, and meta-
thoracic dorsal nerves.

Fie. 12.—Thoracic compound ganglia. Left aspect.
Lettering as in Figs, 11 and 14.

Fie. 13.—The tracheal sacs supplied by the anterior thoracic spiracle (@.d/.).
In this figure the tracheal sacs supplied by the posterior thoracic spiracle and
the sterno-dorsales muscles of the left side have been removed. The left side
of the head and proboscis have also been removed. ‘The first abdominal seg-
ment has been removed to show the large abdominal air sacs (ad.s.) and an
abdominal trachea which is supplied by the second abdominal spiracle (a.sp.).

a.c.s. Anterior cephalic sac. a.v.s. Anterior ventral thoracic sac. ¢.ér.
Cervical tracheal duct. d.c. Dorsal cephalic sac. do. Dorsales muscles.
H. Haustellum. J/.¢7.s. Longitudinal tracheal sac. p.c.s. Posterior cephalic
tracheal sacs. p.v.s. Posterior ventral thoracic sac. p.op. Periopticon.
Ros. Rostrum, v.c.s. Ventral cephalic sac.

Fic. 14.—Thoracic compound ganglion after the removal of the cortex.
Seen from the ventral side. This and Fig. 12 were drawn from models re-
constructed from sections.

Pr.G., Ms.G., Mt.G. Pro-, meso-, and meta-thoracic ganglia. 4.G, Abdo-
minal ganglion. Other lettering as in Fig. 11.

Fie. 15.—The tracheal sacs supplied by the posterior thoracic spiracle.

STRUCTURE, DEVELOPMENT, AND BIONOMICS OF HOUSE-FLY. 447

In this figure the left side of the thorax has been removed, together with the
wing muscles aud the posterior sterno-dorsales. It must be imagined that
this figure is superimposed on Fig. 18.

do. Dorsales. /..s, Lateral thoracic sac. m.v.s. Median ventral sac.
9.th. Posterior thoracic spiracle. sc.s. Scutellar sac. s¢.do. Sterno-dorsales.

Fie. 16.—Wing. The nervures are drawn slightly thicker than they
naturally are.

an. Anal lobe. al. Alula. as. Antisquama. 4. Anal cell. 4,. Anal
nervure. Cz, Cubital cell. 1 Cw. First cubital cell. ev-a. Cubito-anal trans-
verse nervure. C,. Costa. @C. Costal cell. 10. First costal cell. JZ. Medial
cell. m.cw. Medio-cubital transverse nervure. m. Medial transverse nervure.
2M, 2 M?. First and second second medial cells. J21+2. Medial longitu-
dinal nervure. J23+Cu. Medio-cubital longitudinal nervure. &. Radial
cell. £1 to R445. Radial longitudinal nervures. Sc. Subcostal cell.
Sc,. Subcosta.

PLATE 25,

Fic. 17.—The alimentary canal as it is seen on dissection from the dorsal
side. ‘The malpighian tubes have been omitted, and also the distal portion of
the lingual salivary gland (s./g.) of the right side. The duct of the crop (Cr.)
is shown by the dotted line beneath the proventriculus (Pv.) and veutriculus
(Ven.).

p.iat. Proximal intestine. d.z¢. Distal intestine. rect. Rectum.

Fic. 1$.—Portion of a transverse section of the oral lobes, showing the two
types of gustatory sense organ, ete.

gs. Gustatory seta. g.p. Gustatory papilla. Ay. Hypodermis under which
lies a pigmented layer. p.s. Pseudo-trachea in section. s.g.p. Sensory bulb
of gustatory papilla. sp. Sensory bulb of gustatory seta. ¢r. Trachea.

Fie. 19.—Transverse section of labial salivary gland, to show the structure
of the gland cells (g.c.). (Camera lucida drawing.)

hy. Hypodermis. ic.d. Intracellular duct. y.s. Pseudo-trachea., od.
Opening of intracellular duct into the permanent vacuole (vac.) of the gland
cell.

Vie. 20.—Section through the proventriculus and the anterior end of the
ventriculus, to show the structure of the proventricular plug (Pv.p.) and the
ducts of the cesophagus (@s.) and crop (d.cr.). (Camera lucida drawing.)

Fic, 21.—The posterior region of the alimentary canal, to show the rectal
glands (rect.g/.) with their tracheal supply, the origin of the malpighian tubes
(malp.), and the position of the rectal valve indicated at x.

Fie. 22.—Transverse section of the lingual salivary gland, showing the
fibrillar character of the gland cells. x 220. (Camera lucida drawing.)

448 C. GORDON HEWITY.

PLATE 26.

Fig. 23.—Female reproductive organs in sitti; the left ovary and the
viscera have been removed. The ovipositor (ovp.) is shown retracted, in
which state the common oviduct (c.o.d.) is doubled back.

ac.g. Accessory gland. a.c.v. Accessory copulatory vesicle. ov. Ovary
composed of about seventy ovarioles, and containing ova in various stages of
development. ov.d. Oviduct. re¢r.m. Retractor muscles of the ovipositor.
Sp. Spermathece or vesicule seminales.

Fic. 24.—The male reproductive organs. They have been slightly spread
out, and the rectum (rect.) has been turned over to the right side.

d.e. Hjaculatory duct. e.a. Hjaculatory apodeme. e.s. Bjaculatory sac.
te. Testis. v.d. Vas deferens,

Fic. 25.—Vertical section of one of the rectal glands, to show its struc-
ture. xX 56. (Camera lucida drawing.)

sh. Perforate chitinous sheath. .g/. Gland cell. ¢r. Trachea.

Fie, 26.—Terminal region of the female reproductive organs, showing the
accessory glands, ete.

sac. Sacculus. vag. The muscular vagina which evaginates during eopula-
tion; a pair of retractor muscles are shown. Other lettering as in Fig. 23.

TRICHOMASTIX SERPENTIS. 449

Trichomastix serpentis, n.sp.

By
C. Clifford Dobell, B.A.,
Scholar of Trinity College, Cambridge.

With Plate 27, and 2 Text-figures.

THE subject of this paper is a parasitic flagellate which I
have obtained from the rectum of Boa constrictor, L.
Owing to the kindness of Mr. W. A. Harding, of Histon,
Cambridge, I was enabled to examine one of these snakes
shortly after it had died of canker of the mouth. In the
rectum there was about 30 c.c. of a brownish, almost odour-
less, alkaline fluid, containing many organic particles in
suspension, This fluid was transferred to a glass dish, and
prevented as far as possible from evaporating by covering
with a thick glass plate fixed down by vaseline. It was by
examination of this culture from time to time that the obser-
vations here recorded were made.

The first examination was made upon October 30th, 1906,
one day after the death of the snake. Trichomastix was
found to be present in small numbers. The parasites in-
creased in numbers in the culture, and reached a maximum
at the beginning of December, 1906. A decline in the
number of the organisms present in the fluid then followed,
accompanied by several curious changes in the animals
themselves. At the end of February, 1907, there were very
few specimens to be found after a very careful search, and
by March 2nd, 1907, all the parasites had died out. Alto-

450 C. CLIFFORD DOBELL.

gether I was able to keep the organisms alive in this manner
for a period of 120 days. All attempts to grow the organ-
isms in other fluids (solutions of peptone, albumen, etc.)
were unsuccessful. The death of the culture was not due to
increase in the number of bacteria or to a change in the
reaction of the medium, both of which remained very much
as they were in the beginning. The numbers of the bacteria
were somewhat reduced however. Death resulted, I believe,
from a too great increase in the amount of katabolic
products.

The method of examination was as follows:—A few drops
of the culture were drawn up in a fine pipette (used exclu-
sively for this purpose), and examined fresh either in a
hanging drop preparation or under a coverslip with wax
feet, and waxed round the edges. In this latter anzrobic
condition I have been able to keep the animals alive and
active for thirteen days. For examining the living animals
I used almost exclusively a 2°5 mm. apochromatic water
immersion objective by Zeiss, with compensating oculars 2,
6, 12, and 18. Most of the observations here recorded were
made from the living animal. Good permanent preparations
were exceedingly hard to obtain owing to the small numbers
of the parasites and the large amount of gritty foreign
matter in the fluid. However, a few successful preparations
were made, the stains employed being Delafield’s hama-
toxylin, Heidenhain’s iron hematoxylin, and Giemsa’s stain.
Observation of the living animal was often facilitated by
intravitam staining with neutral red. Brillantcresylblau
(Griibler) and methylene blue were also tried, but proved to
be of but little use.

From the morphological characters of this parasite there
can be no doubt that it is properly referable to the genus
Trichomastix, Blochmann. Hitherto this genus has been
represented by but a single species, T. lacertz, Biitschli.
The life-history of this form has been fully worked out by
Prowazek. It is, I think, highly probable that the form
which I am about to describe will prove to be quite common

TRICHOMASTIX SERPENTIS. 451

in Ophidia generally. Unfortunately I have not been able
to obtain suitable material for deciding this point up to the
present. I venture, however, to give the specific name
serpentis to this parasite.

It is possible that the form under consideration is identical
with that described by Grassi under the name Monocer-
comonas coronelle from the gut of Coronella aus-
triaca. The description given is, however, not sufficiently
accurate for the identity to be established. The same may
be said of an organism described by Hammerschmidt in the
cloaca of Tropidonotus natrix, and named by him Cer-
comonascolubrorum. This form appears to be the same
as Monocercomonas colubrorum (Hammerschmidt),
Doflein, and Bodo colubrorum (Hammerschmidt), Saville-
Kent.

STRUCTURE, ETC.

Trichomastix serpentis is usually of a more or less
oval or pyriform shape. It is subject to considerable varia-
tions however. Some of the more commonly occurring
modifications of its form are seen in text-fig. 1 a—g.

TExtT-FIc. 1.

Most of these shapes are only temporary, the animal
usually returning after a time to a condition more or less

452 C. CLIFFORD DOBELL.

closely corresponding with that depicted in Pl. 27, fig. 1.
This shape may therefore be considered as ‘‘normal.” The
leneth varies from about 8, to 17, an average-sized
animal being about 14. At the anterior end [see PI. 27,
fig. 1] are inserted three anteriorly directed flagella, in
length about one and a quarter times that of the body. From
the same basis arises another and longer flagellum, which is
directed backwards (Schleppgeissel). he spot from which
these flagella arise appears in the living animal as a refrac-
tive granule, situated immediately above the nucleus, which
is placed anteriorly. In stained preparations this basal
granule is seen to be composed of a chromatic substance
which stains like the nucleus. Running through the whole
length of the body of the animal is a somewhat flexible axial
rod (Achsenstab), which terminates anteriorly in the basal
granule of the flagella, and posteriorly is drawn out into the
caudal process of the body. The axial rod either traverses
the nucleus, in order to reach the base of the flagella, or else
lies in close contact with it. It is impossible to be quite
certain of the exact relationships of these structures. ‘The
rod is, I believe, skeletal in function. Its general aspect
and relations to the nucleus recall the axial rods of the
pseudopodia of the Heliozoa. Near the base of the flagella
is to be seen a well-marked cell mouth or cytostome.

The creatures exhibit great activity of movement, which
does not appear to be affected in the very least by light, as is
the case with some flagellates. Warming appears to increase
their activity, but no critical experiments on the effects of
temperature were made. ‘The food consists of the small
micro-organisms abounding in the medium.

Division.— Division is, as in most flagellates, longitudinal,
and presents certain features of interest. Briefly, the pro-
cess is as follows [see Pl. 27, figs. 2—10] :—An ordinary
individual [fig. 2] becomes more or less globular [fig. 3].
At the same time the axial rod is absorbed, that is to say, it
disappears. ‘The flagella are now seen as a very rapidly
vibrating bunch, springing from a refringent spot—the

TRICHOMASTIX SERPENTIS. 453

basal granule. This latter loses its connection with the
nucleus, and divides, so that two bunches of flagella are
formed. It has been found impossible to count the flagella
at this stage, or to determine how the new ones arise. Con-
comitantly the nucleus becomes dumb-bell shaped [fig. 4].
The organism then assumes a roughly triangular appearance,
but is rapidly drawn out, and becomes somewhat sausage
shaped. A constriction appears in the middle [fig. 6], and
it is now possible to make out that there are four flagella at
either end, one in each case directed away from the other
three. The nucleus is still dumb-bell shaped, and situated
mesially. The daughter-cells now rapidly draw apart [fig. 7],
and remain connected by only a small bridge of protoplasm,
at either end of which is a nucleus, the daughter nuclei
being still connected by a very fine protoplasmic strand,
Further separation now takes place, finally resulting in the
snapping of the connecting strand of protoplasm [figs. 8, 9].
In the daughter individuals which are thus separated, it will
be seen that the nuclei lie at the extreme posterior end of
the body, and there is no axial rod. Up to this stage the
process usually occupies about twenty minutes. If one of
these daughter cells be carefully watched, it will be seen that
the nucleus travels forwards, and as it does so the axial rod is
seen to be developed in the region immediately posterior to
it. The rod appears to be formed by the nucleus in its track
as it passes forward [figs. 9, 10]. After some time—usually
one to two hours—the nucleus reaches the anterior end of the
body, and enters once more into relation with the basal
granule of the flagella. In some way the axial rod becomes
connected with the basal granule, but it is impossible to see
how this is effected. The cytostome appears to be lost prior
to division, and to be re-formed in each of the daughter cells,
but I am not quite clear on this point.

This account of the division of T. serpentis differs from
that of 'T. lacerte as described by Prowazek. If I under-
stand this author correctly, the axial rod would appear to
function as a kind of division centre; for it retires towards

VOL. 51, PART 3,—NEW SERIES, 34

454 C. CLIFFORD DOBELL.

the nucleus, arranges itself there at right angles to its
original position, and the chromatin passes along it forming
a nucleus at each end.

Unfortunately, in spite of much careful observation, I
was unable to ascertain whether conjugation occurred or not.
In T. lacerte only autogamy occurs, and has been very care-
fully worked out by Prowazek in his excellent paper on
parasitic flagellates. I have frequently seen two individuals
apply themselves to one another for a short time, but they
always separated without any conjugation taking place. In
some cases a very delicate cyst wall was formed, the animal
becoming globular, and a number of refractive bodies were
developed inside. In the course of a few hours, however,
these forms always disintegrated.

DEGENERATION AND DEATH.

As already mentioned, it was found possible to keep the
organisms in cultures for 120 days, but no longer. The
animals all underwent degenerative changes, and finally died.
A very great variety of forms occurred among those animals
which were degenerating. ‘lhe following is the course of
events which usually took place, though many variations
were seen. First, the animals cease to move from place to
place. Movements of the flagella continue, but at a slower
rate, the animal remaining approximately in the same
position. At this period the posterior flagellum frequently
shows a tendency to become adherent to the body, a
delicate membrane uniting them together [see text-fig. 2, a].
In this condition the creature is almost indistinguishable
from a Trichomonas—a genus which differs from
Trichomastix in possessing an undulating membrane
(often with a short free flagellum) in place of the posterior
flagellum. This adhesion of the posterior flagellum soon
becomes more extensive, and finally the flagellum is com-
pletely merged in the protoplasm of the body. The move-

TRICHOMASTIX SERPENTIS. A455

ments of the anterior flagella are meanwhile becoming slower,
and they very frequently become completely fused into a
single finger-like process [text.-fig. 2, b], which continues to
contract in the same direction as did the flagella. This state
may continue for hours, or even days. The axial rod disap-
pears before long, and finally the whole organism becomes
an amoeboid mass of protoplasm [text.-fig. 2, ¢]. In this
condition no movement away from the original position takes
place, the protoplasm being simply thrust out and then
rapidly withdrawn. Undulating movements, involving the

Trext-Fic. 2.

whole amoeba, frequently and rapidly occur. The creature
remains in this condition sometimes for days. Before death
the movements become slower, and finally cease. The
animal then becomes rounded off, and, after a varying
period of time, disintegrates. Not uncommonly two or
more creatures, degenerating side by side, run together
and fuse before death. This is even more remarkable in
the case of the degenerate forms which attempt to divide.
All the stages of division up to that corresponding with
fio. 7, Pl. 27, may be gone through, when suddenly both
individuals, instead of separating, become amceboid and
fuse. Death follows, as in the case of the ordinary amoeboid
forms. In all cases disintegration of the nucleus appears to
occur before that of the cytoplasm.

456 C. CLIFFORD. DOBELL.

A very remarkable fact was sometimes observed in the
involution forms. An individual, instead of dividing when
it reached a certain size, continued to grow. In this way
giant individuals arose, which reached the enormous length
of 30, i.e. about twice the normal length. Very few of
these were observed, but quite a number reached a length of
20 w—24. In these exceptionally large forms I was able,
in an unexpected manner, to confirm my previous observa-
tions on the inter-relationships of nucleus, flagella, and axial
rod. Structures which had been made out with great difficulty,
and after many hours’ watching, were here seen very plainly
after an examination lasting a few moments. These giant
forms divided abnormally, commonly giving rise to three or
four daughter-cells [see fig. 14, Pl. 27]. Division rarely
became complete, the whole usually fusing into a large
amoeboid mass, which finally died. Unfortunately, owing to
insufficiency of material, I was never able to obtain stained
preparations of these stages. It could be seen in the living
animals, however, that each of the daughter individuals
possessed a nucleus, axial rod, and full complement of
flagella. Sometimes the old axial rod was seen sticking
out of the central mass of protoplasm [fig. 14].

A very similar atypical division has been described by
Prowazek in the allied form, Trichomonas lacerte, Prow.
—apparently as a normal occurrence. I cannot think,
however, that this is a normal process in Trichomastix
serpentis.

Before concluding I may call attention to the resemblance
between the basal granule of the flagellar apparatus and the
blepharoplast of a trypanosome. As I find that a detailed
comparison of these structures has been made already by
Laveran and Mesnil in the case of the closely allied form
Trichomonas, I refer the reader who may be interested in
such speculations to their original paper.

TRICHOMASTIX SERPENTIS. 457

LIvERATURE REFERENCES.

1. Brocumann, F.—“ Bemerkungen iiber einige Flagellaten,”’ ‘ Zeitsclir. f.
wiss. Zool.,’ xl, 1884, p. 42.

2. Bitscuxi, O.—* Protozoa, II,” in Bronn’s ‘ Tierreich,’ 1883-87.

3. Doriern, &.—‘ Die Protozoen als Parasiten u. Krankheitserreger,’ Jena
1901.

4. Grasst, B.—‘‘Sur quelques Protistes endoparasites,” ‘Arch. ital. de
Biol.,’ ii, 1882, p. 402, and iii, 1883, p. 23.

5. Hammerscumipt, C. f.—‘‘ Neues Entozoon im Harn der Schlangen,”’
Heller’s ‘ Arch. f. phys. u. path. Chem. u. Mikrosk.,’ 1844, p. 83.

6. Laveran, A., et Mesnix, F.—“ Sur la morphologie et la systématique
des Flagellés & membrane ondulante (genres Trypanosoma, Gruby,
et Trichomonas, Donné), ‘C. R. Acad. Sci. Paris,’ exxxiii, 1901,
p. 131.

7. Prowazex, 8.—* Untersuchungen tiber einige parasitische Flagellaten,”
‘Arb. aus dem Kaiserl. Gesundheitsamte,’ xxi, 1904, p. 1.

8. Savitie-Kent, W.— A Manual of the Infusoria,’? London, 1880—1882.

9. SriLEs, C. W.—‘‘ The Type-species of certain Genera of Parasitic Flagel-
lates, particularly Grassi’s Genera of 1879 and 1881,” ‘ Zool. Aunz.,’
xxv, 1902, p. 689.

EXPLANATION OF PLATE 27,

Mlustrating Mr. C. Clifford Dobell’s paper on “'l'richo-

mastix serpentis, n. sp.”

[The drawings from the living animal were all made under a Zeiss 2°5 mm.
water immersion apochromatic objective (apert. 1:25). Those from stained
preparations (Figs. 11, 12, 18, and 15) under a 3 mm. oil immersion (apert.
1:40) by the same maker. Compensating oculars, Nos. 2, 6, 12, and 18, were
employed. |

Fie. 1.—Living Trichomastix serpentis, showing details of structure.
At the upper end is seen the large vesicular nucleus, upon which lies the
basal granule of the flagella. The axial rod is seen supporting the basal
granule and flagella; and passing backwards, in relation to the nucleus, it
traverses the whole length of the body and terminates in the caudal process.

458 C. CLIFFORD DOPBELL.

To the left of the nucleus is seen the cystostome. Numerous food particles
are seen in the posterior part of the body. ‘This figure is drawn on a larger
scale than the others.

Figs, 2—10 show division as observed in the living animal. (For the
sake of clearness, the nucleus is represented as being more distinctly
outlined than is really the case.)

Fic. 2.—Animal before division.
Fig. 3.—Animal becoming rounded posteriorly. Axial rod disappearing.

Fic. 4.—Nucleus has become dumb-bell shaped, and there are now two
bunches of rapidly vibrating flagella, at the base of which a refringent spot is
visible, no longer attached to nucleus.

Fic. 5.—Flagella separating—likewise the nuclei. These latter are still
connected by a protoplasmic strand, however.

Fig. 6.—Body has become elongated, and a constriction has appeared.
Nuclei still connected, and flagella now distinctly seen to consist of two
groups of four each, one being in each case directed away from the other
three.

Fig. 7.—Separation of daughter-cells from one another. Nuclei still con-
nected, and lying between the two individuals.

Fie. 8.—Daughter-individuals nearly drawn apart.

Fic. 9.—One of the daughter-individuals soon after separation. The
nucleus, which lay at the extreme posterior end, is now making its way ante-
riorly, and the axial rod is seen behind it.

Fic. 10.—Later stage of same individual. The nucleus has not yet reached
the base of the flagella, and the growth of the axial rod is therefore not yet
complete.

Fic. 11.—Preparation, showing nucleus, basal granule, and flagella, ete.

Fre. 12.—Stage in division, corresponding with Fig. 4. The chromatin
masses at the bases of the groups of flagella are well shown.

Fic. 13.—Specimen at about the stage seen in Fig. 10.

Vic, 14.—Large individual dividing into three. Living animal.

Kig. 15.—Degenerate animal which has become ameeboid. ‘The nucleus is
breaking up, and there are, in addition, many food particles in the cytoplasm.

The specimens reproduced in Figs, 11, 12, 18, and 15 were stained by
Giemsa’s method.

NOTES ON COMMON SPECIES OF TROCHUS. A59

Notes on Common Species of Trochus.

By
Hi. J. Fleure and Muriel M. Gettings,
University College, Aberystwyth.

With Plate 28.

Wuite the present work was in progress a paper on
Trochus was published by Randles (10), and stated several
results to which our study had also led us. In view of these
published results, the present paper has been shortened as
far as possible, and includes only some supplementary
observations on special points not particularly studied by
Randles.

Our observations, in the main, confirm those he has
described. We find, for example, the pericardial communi-
cations of the kidneys just as he has stated, and are thus led
to the belief that the Rhipidoglossa possess, typically, a
pericardial canal for each kidney, the gonad communicating
with the pericardial canal of the right kidney.

Randles rightly refers to the unsatisfactoriness of Pilsbry’s
subdivision of the old genus Trochus or the family Trochidee
from the anatomical point of view, and we have found the
two commonest species of T'rochus of interest in this connec-
tion. In Pilsbry’s work Trochus lineatus, Da Costa, often,
and perhaps better, styled T. crassus, Montagu, becomes
Monodonta crassa, Montagu, and belongs to the group of
the Trochinine. In discussion we shall use for it the name
T. crassus, Montagu. Pilsbry makes T. umbilicaris,

4.60 H. J. FLEURE AND MURIEL M. GETTINGS.

Montagu, into Gibbula obliquata, Gmelin, belongmg to
the group of the Gibbuline. For the purposes of this paper
we callit T. obliquatus, Gmelin. The fault of the Pilsbry
sytem is that it is based too exclusively on conchological
differences, too little being known about detailed structure
to make an anatomical survey possible. In the definition of
the group Trochininz, however, we find that members of
this group do not possess jaws, and, as those structures are
present in T. crassus, Montagu, just as in Tl’. obliquatus,
Gmelin, the unsatisfactoriness of the system is further
demonstrated. ‘These jaws also show very interesting
adaptations in their structure and relations.

They are paired chitinous plates formed in the same way
as those of Haliotis (8). Here, as in Haliotis, the anterior
and downward-pointing ends of the jaw-plates do not lie
against the actual gut wall, but against mouthward-outgrowths
of the latter, so that the anterior edges of the plates are to
some extent free (fig. 1). The jaw-plates in these species
are much smaller and thinner than those of Haliotis and the
Docoglossa. In T. crassus, Mont., they are merely a pair
of dorso-lateral plates on projections of the wall of the buccal
cavity, continuous with one another across the median line.
The tissue beneath them, instead of being primarily muscu-
lar, as in Haliotis, has a structure (fig. 2) rather like that of
the superficial parts of the odontophore cartilages. It shows
muscle-fibres running in small strands through what is prac-
tically a mass of “ cartilage” of loose texture. ‘The contrasts
in structure point to functional differences between the jaws
of Trochus, and those of Hahotis and other Rhipidoglossa.
In Hahotis the jaws are. distinctly lateral, and their strong
brush-like free edges help actively in the work of bringing
food fragments into the mouth. In Patella the two jaws are
connected dorsally, and the edges form a strong arch which
leaves space enough beneath for the protrusion of the unusu-
ally broad and solid odontophore cushion; the dorsal part of
the jaws is fairly flexible, and the lateral edges are thus able
to help in cutting food when the animal is browsing on a

NOTES ON COMMON SPECIES OF TROCHUS. 461

seaweed. The jaws in the species of Trochus mentioned are
not strong enough to be useful in these directions. ‘They
probably protect and give firmness to the upper lip, leaving
the work of drawing in food particles to the odontophore
and the lip papillee.

Randles states that there is close agreement in anatomical
characters between the various species of Trochus studied by
him, and this agreement is very marked in the two species
already named. Still, there remain differences between
T. crassus, Mont., and T. obliquatus, Gmel., which it
seems possible to correlate with the differences in their habits.
This must not be taken to imply, however, any belief that
the two species are specially related to one another, we have
no evidence at present on this point.

T. obliquatus, Gmel., occurs in abundance where there is
a reasonable amount of plant life and adherent growth on
rocks washed by the tide, and it frequents both the upper
and the under sides of boulders, often retiring beneath when
it is exposed to strong tide wash, or to heat, strong light, or
possibilities of drought when the tide is away. It crawls
over and browses on a great variety of Algz, but has hitherto
been classed too exclusively as a vegetable feeder. It is
almost omnivorous, and in early summer may often be found
attacking the egg-fringes and chains of Nudibranchs and
other forms. Remains of eggs and other animal matter are
frequently seen in sections of the gut.

Tl’. crassus, Mont., is found, to some extent, with the pre-
vious species, but it lives, for the most part, nearer high-tide
level, so much so that specimens may remain for a considerable
time in corners washed only by high spring-tides. It crawls
over the rocks chiefly above half-tide level, but is more
lethargic than T. obliquatus, and less inclined to browse
on the larger Algae. During stormy periods, especially in
winter, numbers may be found huddled in sheltered nooks,
often with a number of Littorina littorea as companions.

As is well known, the spire of T. obliquatus is typically
much lower than that of T. crassus, and this is probably

4.62 H. J. FLEURE AND MURIEL M. GETYINGS.

correlated with the greater activity of the former in the
shore-zone where a high spire would give too much purchase
to a side blow from a wave.

BRANCHIAL CAVITY.

(a) Mucus Gland.—The mucus gland of T. obliquatus
is developed on the right side of the rectum in ridges and
furrows running towards the anus (fig. 5). This tissue is
evidently the equivalent of the mucus gland of the right side
in Haliotis and Pleurotomaria, and its disposition reveals its
primary function of coating rough excreted fragments
(expelled from the right kidney or the anus) with slime,
so that they may be less likely to injure the gill-leaflets
and other delicate organs before being washed away. There
is also a special aggregation of this tissue around the aper-
ture of the right kidney (fig. 5). The mucus gland of the
left side is not nearly so well developed in Trochus
obliquatus as in the more primitive Rhipidoglossa. It
forms ridges which coat the transverse pallial vein just
in front of the external aperture of the left kidney, and
stretches forward along the rectum to some extent; it is
developed also along the afferent axis of the ctenidium,
where this axis unites with the roof of the branchial cavity,
and especially at the front end of the uniting fold. This
indicates that its special function is to protect the main blood
channels of this region and the gill leaflets hanging in the
branchial cavity from the damage due to grit or hard
fragments which may have wandered into the cavity or may
have been expelled from the kidneys or the anus.

In T. crassus the mucus gland epithelium is found in the
corresponding places, but is also present on ridges in the
roof of the branchial cavity between ctenidium and rectum
to afar greater extent than in T. obliquatus (fig. 4). This
comparatively greater development of the mucus gland in a

NOTES ON COMMON SPECIES OF TROCHUS. 4.63

high tide form contrasts with the reduction of the gland
observed by Pelseneer (6) in the high tide Littorinas.
There can be no doubt, however, that the roof of the
branchial cavity in the latter, with the prolongation of
the ctenidial leaflets across it, is much more specialised
for respiratory purposes. The mucus gland of T. crassus
preserves the condition found in many other species of the
genus, and, in spite of its approach to a high-tide habitat,
there is greater need for damp protecting slime here than in
Littorina. T. crassus keeps its foot in contact with the
rock throughout a period of exposure, while a high-tide
Littorina withdraws deep into its shell, the edge of which
remains adherent to the rock by means of a dried film of
mucus. In other words, the branchial cavity of T. crassus
cannot be nearly so completely protected (by retraction)
from drying during a period of exposure as can that of
Littorina, and the greater development of mucus gland may
be a compensation for this. T. crassus is found in shadier
and less exposed corners than the high-tide Littorinas often
manage to occupy, and not usually so far up the shore.

(b) Gill.—tThe gill is built on the same lines in the two
species under discussion, but shows several specialisations on
the condition found in more primitive Rhipidoglossa. The
loss of the right gill and the migration of the anus and
excretory openings towards that side has been discussed by
Ainsworth Davis (1) as a device for more complete separa-
tion of incurrent and excurrent streams through the branchial
cavity. The surviving left ctenidium in Trochus remains
biseriate, as in Haliotis and Pleurotomaria, but the details of
its disposition are very different, as is well known. In those
more primitive types a median axis between the two series of
leaflets contains the longitudinal afferent and efferent blood
channels of the ctenidium, and that axis is attached to the
wall of the branchial cavity on the side of the efferent
channels only. Right at the back of the cavity the afferent
axis is also attached to the roof on either side (fig. 8), and
the basi-branchial sinus, which feeds the afferent ctenidial

464. H. J. FLEURE AND MURIEL M. GETTINGS.

channels, runs across the roof of the cavity in the region of
these attachments.

In Trochus this connection of the afferent side of the gill
axis with the roof of the cavity has extended much farther
forwards. This has raised the lower series of gill-leaflets so
that they hang in the cavity instead of almost, if not quite,
resting on its floor. In this new position they are more
efficiently bathed by the incoming water, and are less lkely
to impede its course, and they are also more easily kept from
“ packing together.” The dorsal series of leaflets is, however,
necessarily enclosed in a pocket through this development,
and, in the species considered, this series is reduced as
compared with the more freely hanging ventral leaflets.

In detailed structure the ctenidial leaflets of Trochus
resemble in the main those of Halhotis (fig. 6). ‘They are
epithelial folds with a foundation of elongated cells below
the epithelium of each surface, and bridges of cells across
the cavity, which is a blood space. Between the epithelium
and the cells which more or less line the blood space, we
find, as in Haliotis, a development of chitinous substance,
the ends of the chitin plates towards the “efferent” border of
ctenidial leaflet being thickened. Away from this thick part,
the chitinous layer thins out, and is soon no longer observa-
ble; but, where the efferent border of the leaflet meets the
efferent side of the gill axis, it can be seen that, as in
Haliotis, the chitinous plate of one side of a leaflet is
continuous with that of the opposite face of the next leaflet.
A nerve runs along each border of the leaflet beneath the
epithelium. ‘lhe epithelium (fig. 6) along the efferent border
is mostly ciliated, and it is composed of high and narrow
cells, some of which have basal nuclei, and apparently nerve
connections, so that they are very probably sensory cells.
A little way in from this border the epithelium is a good deal
higher, very regular, and close set, and the surfaces of these
extra high eells have a thick covering of uncertain nature
(fig. 6). In this way the height of the band is much
increased, and, as the bands of successive leaflets are opposed

NOTES ON COMMON SPECIES OF TROCHUS. 4.65

to one another, they must act as cushions to keep the leaflets
from packing together, the thickened chitinous plates already
mentioned contributing to stiffen the leaflet.

The surface of the leaflet 1s thrown into folds, which run
fairly parallel with the line where the leaflet unites with the
gill axis. The epithelium of this part of the gill varies very
much in appearance according to the exact direction in which
it happens to be cut, but it is not nearly so high as that
along the efferent border.

The afferent border of the leaflet is the one which is most
directly exposed in the branchial cavity. Along this edge
the epithelium is fairly high and regular, and includes
mucus-secreting cells; we do not think it is ciliated (fig. 6).
There are no supporting chitinous plates and no cushions
near this border ; if the topographically lower efferent edges
of the leaflets are stiffened and held apart that suffices to
keep the leaflets from packing against one another. The
mucus-secreting cells of the afferent border must help to
keep the leaflet from injury due to rough fragments rubbing
against this exposed part. In Trochus, but not in Haliotis,
we find the afferent border somewhat expanded (fig. 6), and
this may be a cushion arrangement, so the statement above
made perhaps needs modification. The appearance of odd
fragments in the mucus outside the expanded border suggests
that the swellmg may have the added value of hindering
the entry of these fragments into the chinks between the
leaflets.

Notes on tHE KiIpnNeys.

The kidneys of Trochus were described by Perrier (8) and
Haller (4), and Thiele (12) added the observation of a
“nephridial gland”’ along the left side of the left kidney.
Randles (10) and Pelseneer (7) have both described the peri-
cardial communications of the kidneys, and Randles has added
correct sketches of the cells typical of the right kidney. We

4.66 H. J. FLEURE AND MURIEL M. GETTINGS.

give sketches illustrating the types of cell found in both
kidneys (figs. 10—12), with the corresponding cells from
Haliotis (figs. 14 and 15) to show the close agreement between
these two types; our figures do not agree with those of
Perrier and Haller.

The nephridial gland of Trochus (cf. Perrier’s Glande
nephridienne of Monotocardia) is composed of a set of
branching tubules opening into the kidney cavity, and em-
bedded in a tissue rich in blood vessels (Perrier’s Glande
hématique of Monotocardia). The cells lining the tubules of
this gland (fig. 12) are much lower than those of the left
kidney. The function of this tissue, as, indeed, the function
of the whole left kidney, is quite unknown, but it is, perhaps,
important to notice that it occurs along that side of the
kidney from which blood channels go to join the efferent
ctenidial vein, and to form with it the left auricle. If
the nephridial gland of Trochus is really the homologue of
the nephridial gland in the Monotocardia, this is an argument
in favour of the view of Lankester (5), Pelseneer (7), and the
embryologists that the kidney of Monotocardia is equivalent
to the left kidney of the Diotocardia. Perrier, Woodward
(18), and one of ourselves have urged the opposite view on
other grounds which cannot be altogether set aside, and the
question must be further discussed after more comparative
and embryological research, some of which is now being
undertaken.

Circulation.—In its main features, the circulatory system
of the species of Trochus considered agrees with that
described by one of us for Haliotis (8). Blood leaves the
ventricle by the aorta, the communication being guarded by
asimple valvular flap. The aorta bifurcates soon after leaving
the heart, and one branch goes to the visceral hump while
the other runs forward and ensheathes the radular sac. This
is as in Haliotis, but here the blood channel is practically
embedded in the side of the shell muscle for some distance.
Still surrounding the radular sac it reaches the head where
a great deal of its blood seems to be directed into spaces

NOTES ON COMMON SPECIES OF TROCHUS. 467

surrounding the pedal ganglia and pedal cords. Thence
blood is distributed throughout the foot. It appears to
gather again in a median sinus above the nerve-cords, which
opens in front into the general cavity of the head. The blood
in the head is thus in part returned from the foot, and in
part supplied from the branch of the aorta which surrounds
the radular sac. ‘The muscles of the odontophore are probably
mainly supphed from the latter channel, while the blood from
the former source is very likely partly aérated in the head
when the animal is extended and active.

The blood from the general cavity of the head is collected
into a fairly definite channel which carries it back to the
right kidney. These species of T'rochus, however, differ
from Haliotis in that the anterior lobe of this kidney is much
reduced, so that the blood channel does not run nearly all
the way in the kidney wall, as is the case in Haliotis. The
right kidney also receives blood from the visceral hump
further back. In fact the blood flow from the head is by no
means so intimately connected with the right kidney as in
Haliotis. That organ must, in 'l'rochus, be mainly a purifier of
blood from the visceral hump.

An afferent channel from the right kidney takes blood from
these two sources into the roof of the branchial cavity at the
back, and runs across beneath the rectum to the left kidney,
but first gives off a channel into the branchial roof. We
look upon this last channel as a probable homologue of the
afferent channel of the right ctenidium of Haliotis which has
been lost in Trochus. ‘The efferent channel from the right
kidney thus corresponds with a part of the basi-branchial
sinus of that type.

The blood reaching the neighbourhood of the left kidney,
as just described, goes in part to that organ, but the main
flow runs forward in a subrectal sinus, and then turns to the
left, crossing the roof of the branchial cavity, as the trans-
verse pallial vein of most authors (figs. 4 and 5).

Before reaching the left kidney, however, it communicates
with the right auricle, which also receives a channel from the

468 H. J. FLEURE AND MURIEL M. GETTINGS.

branchial roof. Following Thiele, we homologise the latter
channel with the efferent vein of the right ctenidium (lost in
Trochus) of Haliotis.

In Haliotis the right auricle is supplied from the right
efferent ctenidial vein, and also connects with the _ basi-
branchial sinus, so conditions are essentially similar in
Haliotis and Trochus, allowance being made for the loss of
the right ctenidium in the latter.

The channel from the right to the left kidney in Trochus
together with the forwardly running subrectal sinus and the
transverse pallial vein, may be homologised with the basi-
branchial sinus of Haliotis. The changes in detail are con-
nected with the large size of the left kidney and its position
in the roof of the branchial cavity.

As already stated, the blood spaces beneath the epithelium
of the left kidney are supplied from the channel arriving
from the right kidney, and so some of the blood (particularly
that from the visceral hump) may be purified from nitrogenous
waste. That from the head is, however, by no means so com-
pletely purified as it has really only skirted the right kidney.

The transverse pallial vein shows interesting differences in
the two species considered. It is equally obvious on dissec-
tion in both, mainly owing to the covering ridges of the
mucous gland, but the blood space is relatively larger in
T. obliquatus, Gmel., than in T. crassus, Mont. In the
former, also, its anterior and posterior branches are essentially
afferent ctenidial channels sending blood through the gill
axis into the leaflets. In T. crassus the gill does get some
blood in this way, but a great part of the contents of the
transverse pallial vein goes out into the roof of the branchial
cavity which also receives blood from the sub-rectal sinus.
There seems, therefore, ground for the opinion that the roof
of the branchial cavity in this mid- to high-tide form is of
considerable importance in respiration, while the ctenidium
cannot have quite so much importance as in the other
species.

The blood from the ctenidium together with that brought

NOTES ON COMMON SPECIES OF TROCHUS. 469

by various channels from the roof of the branchial cavity
goes back along the efferent ctenidial channel to the left
auricle, which also receives important supplies from the left
kidney. The left kidney thus gets its blood from the
efferent channel of the right kidney, and passes it on to the
left auricle, but its blood spaces are intimately associated
with those of the neighbouring part of the roof of the
branchial cavity, so perhaps a good deal of what it con-
tributes to the left auricle has been aérated.

As the efferent channel from the right kidney communi-
cates with the right auricle before reaching the left kidney,
past which blood flows to the left auricle, it follows that
right and left auricles are not so very indirectly connected
with one another. This is another point of resemblance
between Trochus and Haliotis.

Reference has been made to the possible respiratory
activity of the roof of the branchial cavity, especially in
T. crassus, Mont., and it is interesting to notice that the
corresponding tissue seems to have acquired respiratory
functions in Patella, which is also often a mid- to high-tide »
form.

In a recent paper Spillman (11) incidentally mentions the
course of the circulation, but the vagueness of the state-
ments made renders a discussion of his views unnecessary.
He has evidently not given any special attention to this
particular subject.

Bionomical Notes.—T. crassus, Mont., is often found
in fairly large numbers huddled together in protected gullies
and corners during the winter, especially when the weather
is stormy. In summer it is interesting to watch these
animals, often following one another’s tracks as if the fresh
trail of mucus were a help in progression. ‘They also have
the habit of mounting on one another’s shells, where they
apparently browse the small Algee, etc., on the surface. As
Trochus has no mating. habits (in common species, at any rate),
these peculiarities in behaviour may be thought of as the
indistinct beginnings of that mutual recognition, perhaps

VoL. 51, PART 3.—NEW SERIKS. 30

470 H. J. FLEURE AND MURIEL M. GETTINGS.

mainly through chemical sense impressions, which must be
a first step in the evolution of the mating tendency.

Probably some of the ancestors of the Montocardia, which
have mating habits, went through somewhat similar stages
in the course of their evolution. Prof. Ainsworth Davis has
suggested to us that the egg-eating habit of certain species
of Trochus should also be considered in connection with the
development of mating habits.

Our material was obtained from the shores of Cardigan
Bay, south of Aberystwyth, which show for many miles as a
plane of marine denudation formed by fairly evenly worn
ridges of Silurian grits and slaty shale. The old valleys in
the Silurian rocks, of which most of the coast cliffs are
built, are filled in some cases by the boulder clay of the
glacial epoch. The small streamlets which run down from
the cliffs thus drain sometimes off the boulder clay, and
sometimes off the shales and grits. We find that in several
cases T’. crassus occurs in abundance where the shore is
washed by streams from the boulder clay, but disappears
sometimes almost abruptly when the boulder clay makes way
for the Silurian rock. The same thing is true, though in a
lesser degree, for Trochus obliquatus, and the fact seems
to be of sufficient interest to be chronicled, though we have
not yet been able to approach an explanation. The boulder
clay is found on analysis to be rich in salts of potash and
magnesium. ‘Trochus is not found much within three miles
of Aberystwyth at present ; the boulder clay in this imme-
diate neighbourhood has probably been carried by a different
set of glaciers from that flowing seawards further south.
The pollution of the Ystwyth and Rheidol rivers by lead
works may also be a determining factor.

List oF PAPERS QUOTED.
1. AinswortH Davis, J. R.—‘ Bionomical Considerations in Gastropod
Evolution,” ‘ Journal of Malacology,’ 1905.

2. Drummonp, Miss.—‘‘ On the Development of Paludina,” ‘Quart. Journ.
Mier. Sci.,’ vol. xlvi, 1902.

NOTES ON COMMON SPECIES OF TROCHUS. 471

3. Freure, H.J.—‘‘ Zur Anatomie und Phyiogenie von Haliotis,” ‘Jenaische
Zeitschrift,’ Bd. 39, 1904.

4, Haier, B.—‘Studien iiber docoglosse und rhipidoglosse Prosobranchier,’
Leipzig, 1894.

5. Lanxester, HE. Ray.—“ On the Originally Bilateral Character of the
Renal Organs of Prosobranchia,” ‘Ann. Mag. Nat. Hist.,’ ser. 5, vol.
vii, 1881.

6. PELSENEER, P.—‘‘ Prosobranches aériens et Pulmonés_ branchiféres,
‘Arch. Biol.,’ xiv, 1895.

7. PELSENEER, P.—‘‘ Les Mollusques archaiques,” ‘Mem. cour. de l’Acad.
Belg.,’ t. lvii, 1899.

8. Perrier, R.—“ Le Rein des Gastéropodes Prosobranches,” ‘ Ann. Sci.
Nat. Zool.,’ ser. 7, t. viii, 1899.

9. Pitspry-Tryon.—‘ Manual of Conchology,’ New Jersey, U.S.A. In
progress.

10. RanpiEes, W. B.—‘‘ Anatomy and Affinities of the Trochide,”’ ‘ Quart.
Journ. Micr. Sci.,’ vol. xlviii, 1904-5.

11. Spruuman, J.—‘‘ Herz u.s.w. der Diotocardier,” ‘Jenaische Zeitschrift,’
Bd. 40, 1905.

12. Tietz, J.—‘‘ Die systematische Stellung der Solenogastren und die
Phylogenie der Mollusken,” ‘ Zeitschrift f. wiss. Zool.,’? Bd. xxii,
1902.

13. Woopwarp, M. F.—‘* The Anatomy of Pleurotomaria,” * Quart. Journ.
Mier. Sci.,’ vol. xliv, 1901.

EXPLANATION OF PLATE 28,

Illustrating the paper by Dr. H. J. Fleure and Miss Muriel M.
Gettings on ‘ Notes on Common Species of Trochus.”

REFERENCE LETTERS.

Aff. Afferent side or axis of the ctenidium. CA. Chitinous plate. C#.
Ctenidium. C¢.4f. Afferent blood channel of the ctenidium. Ct. Effe-
rent blood channel of the ctenidium. #/. Hfferent side or axis of the cteni-
dium. JZ.dw. Left auricle. L.K.#/. Efferent channel from the left kidney.
L.K. Left kidney. Mue.gi. Mucus gland. N.G. Nephridial gland. Osph.
Osphradium. &. Rectum. #&.K. Excretory channel of the right kidney.
R.Au. Right auricle. §.8.S. Subrectal sinus. S¢. Strengthening tissue
beneath the jaw-plates.

472 H. J. FLEURE AND MURIEL M. GETTINGS.

Fic. 1.—The jaw-plates of Trochus crassus, Mont.

Fic. 2.—A section of the jaw-plates.

Fic. 3.—Cells from the strengthening tissue beneath the jaw-plates.

Fic. 4.—The roof of the branchial cavity in Trochus crassus, Mont.

Fre. 5.—The roof of the branchial cavity in Trochus obliquatus, Gmelin.

Fie. 6.—An oblique transverse section of a gill leaflet of Trochus.

Fic. 7.—Section of the roof of the branchial cavity and gill of Trochus.

Fic. 8.—Section of the roof of the branchial cavity and gills of Haliotis.

Fie. 9.—Section of the left kidney of Trochus in the region of the nephri-
dial gland.

Fie, 10.—Epithelium of left kidney of Trochus.

Fie. 11.—Epithelium of right kidney of Trochus.

Fic. 12.—Epithelium of nephridial gland of Trochus. -

Fic. 13.—Section of pericardium of Trochus showing the blood channels
which feed the left auricle.

Fic. 14.—Epithelium of the right kidney of Haliotis.

Fic. 15.—Epithelium of the left kidney of Haliotis.

eee ee a

FORMATION OF THE SKELETON IN THE MADREPORARIA, 473

Note on the Formation of the Skeleton in the
Madreporaria.

By
Maria WM. Ogilvie Gordon,
D.Se.(London), Ph.D.( Munich), F.L.8.

Wirutn the last few days there has been brought under
my notice a short account by M. Armand Krempf! of the
mode of origin of the calcareous skeleton in Madreporarian
Corals.

M. Krempf, whose observations were made on a species of
the genus “ Seriatopora,” writes as follows:

“Tt is easy to verify that the corallum (“ polypier”) of
this animal, in conformity with the investigations of Heider,
is formed by the imbrication of a multitude of small calca-
reous scales, themselves constituted by a small bundle of
fibres of lime carbonate.

“JT have been able, in addition, to demonstrate by means
of a decalcification conducted with great caution, the sub-
stratum of organic matter in the midst of which the calca-
reous part of this scale has taken solid form.

“ A very fine membrane limits each of these elements,
tending thus to give it the physiognomy of a small cell.
After suitable treatment of the fresh skeleton with decalci-
fying reagents, the whole of the delicate organic meshwork
(trames agglomérées) forms a light translucent mass, similar
to jelly and of extreme fragility.

1 A. Krempf, ‘Sur la formation du squelette chez les Hexacoralliaires a

polypier.”’ Note by M. Armand Krempf, presented by M. Yves Delage
(‘Comptes Rendus,’ 21st January, 1907).

474. MARIA M. OGILVIE GORDON.

“Thus there exists, contrary to the opinion of Gardiner
e]

(1900), who was not able to disclose it in his preparations, an

organic substratum in the skeleton of the ‘‘ Coralliaries.”

“ Contrary again to the opinion of Duerden (1904), who
admits the existence of a fundamental substance, but who
considers it as homogeneous, this substance has a histo-
logical constitution, well characterised, which realises all the
appearances of a cellular structure.

“These facts much depreciate in value Koch’s hypothesis
of extracellular secretion. ‘They seem to fully justify Heider,
who considers the ‘ polypier’ as formed by the accumula-
tion of calcified cells.

“At the same time this is not so; the calcareous scales
whose juxtaposition forms the coralJum are not the skeletons
of calicoblasts. They have not the value of cells.”

Before proceeding further with M. Krempf’s description a
few remarks may be made regarding these preliminary
statements.

The first statement that M. Krempf “finds it easy to
verify that the skeletal parts are formed by the
imbrication of a multitude of small calcareous
scales” is a corroboration of observations which were
unknown to zoologists previous to the publication of my
work on the “ Madreporaria.” !

That paper for the first time gave a description and
illustrative figures of the minute skeletal elements which
everywhere compose the corallum. I described these ele-
ments as “calcareous scales,” and showed the imbricated
manner of their apposition with one another, and their
various shapes and positions at different parts of the corallum
(l.c., pp. 114,115, 127; figs. 7a—c; 8 a—c, etc.).

The references made by M. Krempf to von Heider’s work
are somewhat ambiguous, as they lead one to infer that von

1M. M. Ogilvie, ‘‘ Microscopic and Systematic Study of Madreporarian
Types of Corals,” ‘Phil. Trans. Roy. Soc.,? London, vol. 187 (1896), B,
pp. 883—345. Also see my monograph on the “Stramberger Korallen”
(Paleontographica, Stuttgart, 1897, Suppl. ii, No. 7, pp. 73—282, pls. vii—
xvili).

—— ve

FORMATION OF THE SKELETON IN THE MADREPORARIA. 475

Heider had deciphered this structure in 1881. Full references
to the papers of von Heider and Koch are made in my work
(l.c., pp. 91—97). Von Heider made a special study of the
soft parts, and observed here and there certain cells in whose
organic contents small groups of calcareous fibres were
included, and the nuclei were either shrunken or vanished.
To these cells he gave the name of “ calicoblasts,” and thought
they occurred in the mesoderm, but must, in some way,
accumulate to build the corallum. As I explained in my
paper, Heider’s surmise was that the individual skeletal parts
in the Madreporaria were formed in a manner analogous with
the skeleton of Alcyonarians, but that in the former the
calicoblasts produced a solid structure at the outer limit of
the mesoderm, whereas the analogous cells in the Alcyonarian
polyp formed calcareous spicules, which remained isolated
throughout the whole hfe of the polyp (l.c., p. 93, and cf.
Heider, ‘‘ Korallenstudien,” 1886). Heider admitted he could
not trace the connection between the groups of fibres as he
observed them in cells of the embryonic skeletal disc, and the
various complex structures of the mature skeleton.

Subsequent observers said that the cells referred to by
von Heider were components of the ectodermal tissue, and
that they never contained inorganic material as he depicted ;
that the calcareous material of the corallum was originally of
the nature of a secretion thrown out by the cells of the ecto-
derm (ref. Ogilvie, 1896, 1. c., pp. 101-2).

This was the position when my work was published by the
Royal Society of London, and I there demonstrated that the
whole corallum was composed of a series of calcareous
lamellee, each of which was primarily an organic tissue com-
prising innumerable minute cellular parts, in each of which
lay a group of calcareous fibres. As these parts corresponded
in appearance to the ‘‘ calicoblast ” described by von Heider,
I applied this term and wrote: “Hach lamina (average
width ‘003 to ‘005 millim.) is a deposit of calico-
blasts, the wavy outline corresponding to origin-
ally separate cells” (lc., p. 128; pp. 114—117, etc.).

476 MARIA M. OGILVIE GORDON.

I demonstrated by descriptions and illustrations (p. 123,
124, 138; figs. 13, 14, 17, etc.) that several layers of calico-
blasts were shed from the ectoderm of the polyp during each
period of active growth, and that these adhered more closely
with one another than with the previously or subsequently
formed series of layers, yet that each layer was complete in
itself; farther, that each individual calcifying part originally
limited by organic walls (which I termed cell-walls), retained
its individuality during its transformation into a “ calcareous
scale,” and could be obtained apart from its neighbours as
an individual entity.

It creates rather a confusion of ideas when M. Krempf
implies that von Heider had held the view that the “ calcareous
scales” were the “skeletons of calicoblasts”—since von Heider
was not aware that the “juxtaposition of calcareous
scales formed the corallum,” and he could not possibly
have claimed for these skeletal elements, as I did, that they
were transformed cell-products, and represented the
“ fibre-containing cells,” which he had observed in the cellular
tissue, and termed calicoblasts (cf. Ogilvie, 1. c., p. 125).

The second part of M. Krempf’s account in the ‘ Comptes
Rendus’ describes a series of observations which bear upon
the relationship of the ectoderm to the layer of skeletal ele-
ments immediately external to it. Before quoting from
M. Krempf, I shall indicate the direction followed in my
investigations of this subject.

My sections of the soft parts corroborated the observations
of Dr. Fowler and Dr. Bourne (cf. Ogilvie, |. c., pp. 101-2).
Comparing my sections of the soft parts with my prepara-
tions from the corallum, I was able to observe farther that in
these positions, such as the periphery of the calyx, which
corresponded to the most closely nucleated and actively-
dividing parts of the ectodermal tissue, the calcareous scales
were most thickly piled in the corallum, and their organic
remnants (“dark points”) most closely grouped—sometimes
in little rigs, sometimes in rows.

“Tn all cases we have simply to do with centres and axes

FORMATION OF THE SKELETON IN THE MADREPORARIA. 477

of calcification, around which the calicoblasts are grouped in
the living polyp, and from which, therefore, similarly oriented
fibres ultimately radiate when complete calcification has taken
place” (1. c., p. 128; refer to p. 183, fig. 18; p. 148, fig. 29,
etc.).

The wider spacing or closer massing of the unit elements
in the skeletal layer was thus shown to depend upon localisa-
tion of “centres of calcification” in the ectoderm, and the
number produced in a single growth-period to be greatest
where fissional activity was most marked in the ectodermal
layer. Careful measurements conducted over many species
farther showed that the size of the “ calicoblast cells” in the
ectoderm was the same as the size of the unit elements in
the skeletal layer (p. 117, 136, etc.). Upon such grounds “I
took the nuclear fission to be associated with the separation
of the organic outer layer’’ containing the skeletal elements,
and concluded that, in virtue of fissional processes in the
cellular tissue of the ectoderm, calcifying calicoblasts were
constantly being eliminated, and the vitality of the ectoderm
itself renewed.

“The fibre-containing calicoblasts which le next the
skeleton are shed off, so to speak, from the polyp, new cells
constantly taking their place in the ectoderm by cell-divi-
sion”’ (Ogilvie, l. c., p. 102).

“At the beginning of any particular growth-period the
calcification goes on only locally at certain points of the
calicoblastic layer of the colony ” (id.,1.c.,p.131; cf. p. 143).

“The calicoblasts remain adherent to one another in dense
groups or may be more uniformly distributed. And in this
manner they are gradually left behind on the skeleton, and
completely calcify, while active cell-division develops con-
stantly new ectodermal cells. The calicoblasts adherent to
the skeleton represent such as were already in course of
losing living continuity with the polyp at the time when the
polyp was removed from the skeleton” (id., ]. c., p. 116).

“The scale-like arrangement on the surface presents irre-
gularities—sometimes like thicker zones, sometimes thicker

478 MARIA M. OGILVIE GORDON.

patches. Those are doubtless due to irregular disposition of
the calicoblasts, as they originally separate from the ecto-
dermal polypal layer” (id., l. c., p. 116).

Mr. Duerden’s work,! referred to by M. Krempf, brought
the first confirmation of my description of the primarily
organic nature of the layer external to the ectoderm in
which the skeletal elements developed, but Mr. Duerden
described it as homogeneous, and thought it took origin from
the ectoderm probably by a process of secretion, and that the
calcareous groups of fibres which made their appearance
must be regarded as “ ectoplastic” in origin. Against this
I wrote as follows:

“On Mr. Duerden’s interpretation, if I understand it
aright, we are to believe that the exceedingly particular
skeleton arises by a sort of crystallisation in an organic
cuticular matrix produced by, but distinct from, the ecto-
derm. I still uphold my opinion, supported by remarkable
correspondences of measurements, which cannot be mere
coincidences, that the individual ectoderm cells or nuclear
parts exert a determining individual influence on the origin
of the lime-forming skeletal units (= ‘ calicoblasts””? in my
work) in the cuticular product, which is, after all, a composite
product from many ectodermal cells, and persists in retaining
its originally particulate character.” (Quart. Journ. Micr.
Sci.,’ 1905.)

“Hach individual lime-forming part or ‘ calicoblast’ of

the skeletal layer derived its origin from an ectoderm cell in
virtue of divisional processes, part of the cell layer continuing
as ectoderm, part being shed as the layer of calicoblasts ”
(‘Quart. Journ. Micr. Sci.,’ vol. 49, pt. 1, Oct., 1905, pp. 207,
208).

M. Krempf now recognises the distinct partitioning of the
croups of calcareousfibres by organic strands, butsaysalthough
these simulate cell-walls in their appearance, the contained

1 J. E. Duerden, ‘‘ The Coral Siderastraea radians and its Post-larval

Development,” Washington, U.S.A., publ. by Carnegie Institution, Dec.,
1904.

FORMATION OF THE SKELETON IN THE MADREPORARIA. 479

portions have not morphologically the value of cells, as nuclei
can only be shown to be present in some of them. The fol-
lowing is M. Krempf’s description :

“In the ectoderm which carpets the skeleton, and which
consists of a protoplasmic layer with scattered nuclei, in
which it is somewhat difficult to define cellular limits, one
sees the skeletal element appear in one place and another
under the form of a small mass, readily stainable with nucleus
dyes.

“Tt presents distinct fibrous structure. Its fibres, clearly
individualised, are in general regularly parallel with one
another and most often are disposed perpendicularly to the
surface of the calicoblastic epithelium. Its shape varies
according to the points of the skeleton where one observes
it. Sometimes the shape is that of a small parallelepiped,
fairly regular and flattened, sometimes it recalls that of a
Lepidoptera scale, sometimes even that of a cup compressed
parallel with its vertical axis and hollowed by a not very deep
cavity.”

The setting of the fibres in the layer external to the ecto-
derm and in all the skeletal lamellae was shown in my illus-
trations and was very frequently described by me, as also the
varieties of form displayed by the skeletal elements at differ-
ent parts of the corallum (I. c., pp. 118, 121, 125, 128, 136,
137, 138, 144, ete.). To continue M. Krempf’s observations :

“There is always a small nucleus, homogeneous and highly
chromophil, placed at one of the sides of the mass.

“This element, arrived at its complete development, cal-
cifies entirely. It then ceases to form part of the still living
cell within which it is developed and adheres to the skeleton
with which henceforth it is embodied. But the nucleus of
the cell which formed it is not carried away with it. It with-
draws on the contrary into the midst of the protoplasmic
layer which remains living, and after a period of rest, the
duration of which I cannot fix, it presides at the elaboration
of a new element similar to the preceding.

“Tt is only after having assisted at the formation of a

480 MARIA M. OGILVIE GORDON.

certain number of these elements that it becomes embodied
in the last-formed, finally removed from the living layer and
incorporated in the skeleton where good preparations enable
it to be recognised. It has then almost completely lost its
sensitiveness to colour, but it has preserved almost without
alteration its characteristic shape and its dimensions.

“Tn the preparations which I have examined and which up
to the present only concern a single species, I have found an
average of 150 scales unprovided with nuclei for one nucleated
scale. If one is justified in supposing that none of the nuclei
disseminated in the skeleton can escape observation, the
necessary conclusion would be that one determinate calico-
blast can, during its life, produce a total of about 150 cal-
careous scales.”

M. Krempf, in the foregoing description, appears to reserve
the term “calicoblast” for that evanescent stage in the pro-
duction of a skeletal element which immediately precedes its
actual separation from the ectoderm, in fact to limit it to the
nucleated fibre-containing body in the ectoderm. It must,
however, be remembered that von Heider gave the term to
fibre-containing bodies in which, as a rule, the nucleus was
shrunken or vanished. Thus so far as terminology is con-
cerned, I was strictly accurate when in my work I applied
the term to organic, fibre-containing bodies, whether nucleated
or non-nucleated ; calcareous scales are most certainly what I
defined them to be —“ calcified calicoblasts.”’

It is another question whether each of them may be looked
upon as the morphological equivalent of a cell, or cellular
part.

Until M. Krempf’s evidence is given in full, it is impossible
to form any opinion regarding his statement of the oft-
repeated withdrawal of a nucleus into the ectoderm. Mean-
time, from the brief description he now puts before us, I see
no reason to infer that the presence of a spent nuclear body
in a skeletal element marks out that particular element as the
morphological equivalent of a cell, whereas the other skeletal
elements have not that morphological value. Each time that

FORMATION OF THE SKELETON IN THE MADREPORARIA. 481

there is a definite aggregation of protoplasmic material with
reference to the nucleus, a living cell takes form, and each
time that, by an act of fission, an organic, fibre-containing
unit is added to the skeletal layer, that unit may be termed
the structural equivalent of a cell, although it is not itself
living. Some proportion of the nuclear vitality has been
employed in the making of each fibre-containing unit. In a
word, each calcareous scale represents the product of a
nucleated body.

The most important morphological point in M. Krempf’s
description is his statement that the nucleated cell is finally
transferred “from the living layer to the skeleton.”
This has been the real point of controversy. ‘To quote from
my work in 1896 :—“ Fowler and Bourne have observed the
specialised character and large size of the calicoblastic cells
at the growing points of the skeleton in several corals,
among others Galaxea. Both these authors regard the calico-
blasts as ectodermal cells, which merely secrete calcareous
substance without being themselves calcified.” ... “I find
that the cells themselves become incorporated in the
skeleton.” ...

On M. Krempf’s own showing, this transferred nucleated
cell becomes a calcareous scale, undistinguishable from its
neighbours ; in the farther processes of calcification there is
no difference between nucleated and non-nucleated skeletal
elements.!

M. Krempf has given his attention especially to the histo-
logical processes, and we may expect a valuable addition to
our knowledge of these. The outstanding features in his

1 Tt may be of interest to add that in my preparations I sometimes observed
traces of nuclei with the organic remnants in calcareous scales ; in some, the
reaction to stains was quite marked; in others, very faint; and in still
others, no nucleus could be discerned by means of stains. I felt I could not
draw any secure conclusion as to the absence or presence of the nuclei,
and knowing how irregularly they were distributed in the ectodermal tissue,
I did not follow this line of observation, but in my published work limited
myself to the statement that each “scale ” contained organic remnants.

482 MARIA M. OGILVIE GORDON.

results are his recognition of the controlling agency of
ectodermal nuclei, and of divisional processes in ectodermal
tissue as the means of origin of the external organic layer in
which are contained the primary groups of calcareous fibres.
These, together with his observation of the marked in-
dividualisation of each calcareous group, confirm the inter-
pretation I gave of the relationship between the ectoderm
and the skeletal parts. .

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 483

Studies in Spicule Formation.

VII.—The Scleroblastic Development of the Plate-and-Anchor
Spicules of Synapta, and of the Wheel Spicules of the
Auricularia Larva.

By
W. Woodland,
The Zoological Laboratory, King’s College, London.

With Plates 29 and 30, and 6 Text-figures.

Part I.—The Spicules of Synapta inherens and §. digitata.

INTRODUCTORY.

Tur complete morphogenesis of the curious aggregate
(8) plate-and-anchor spicules of Synapta inherens has
been previously described and figured by Semon (6), but,
with the exception of my description of the disposition of the
scleroplasm associated with the adult spicules (7), no account
of the part played by the living tissues of the organism in the
production of these wonderful structures has hitherto been
published.

These plate-and-anchor spicules are so well known that it
is not necessary for me to do more than briefly mention their
more obvious characteristics. Hach spicule, as seen in the
integument of Synapta inherens, e.g., consists of two
parts—the anchor and the plate—and these two parts are
quite separate from each other. The anchor consists of a
shaft, a bow (with its two arms), and a handle—to use the
current terminology ; the plate, on the other hand, has several
large perforations, and is somewhat more pointed in shape
where it comes into apposition with the handle of the anchor
than at the opposite side (fig. 23). This more pointed region

484, W. WOODLAND.

of the plate I shall term the base, and the opposite, more
obtuse region the apex. In side aspect it can be seen that
the handle of the anchor and the base of the plate form a dis-
tinct joint, the one with the other (fig. 25), and the anchor
and the plate rotate upon each other at this joint, the angle
included between the two in consequence varying in magni-
tude (Ostergren, 4). It can also be observed in side view
that the arms of the anchor bow do not lie in the same plane
as the shaft, but in a plane shghtly more inclined towards the
dermal layer, i.e. more nearly parallel with the plane of the
plate (text-fig 1).

Respecting the general arrangement of the spicules, it is
an obvious and noteworthy fact that the major axes of these
plate-and-anchor spicules, as they lie in the body-wall, are
disposed transversely to the long axis of the animal, so that
in a transverse section of Synapta the spicules are seen in
side aspect (text-fig. 1). Further, although the spicules never
assume, as regards their major axis, other than a transverse,
or approximately transverse, disposition in the body-wall, it
is quite a matter of chance as to whether the bow of the
anchor and apex of the plate of each spicule he to the right
or to the left of the observer; in other words, they are disposed
pretty equally in both positions.

A few words concerning the preparation of Synapta material
for the study of the spicule development are necessary. My
material was obtained from Naples, and consisted of specimens
of the two species—Synapta inherens and 8. digitata,
My specimens of S. inhewrens averaged | em. to 1:5 em. in
length, and they were specially prepared for me by the osmic
acid and picro-carmine method described in Study IV, e.g.!

1 Tam indebted to Dr. P. Mayer for recommending me his picro-magnesia
carmine stain as a substitute for the Ranvier and Weigart preparations. The
objection to these latter is their inconstancy of composition—the free ammonia
evaporating and the carmine being precipitated ; an excess of free ammonia of
course tends to macerate the tissues. I must say, however, that with one
exception I have always used Ranvier and Weigart with perfect success, and
so long as free ammonia is absent there is little objection to them, at least in
practice.

a

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 485

I may mention that after fixation it is well to slit open the wall
of the Synapta so as to stain the integument from the inner side
as well as the outer. The integument is subsequently cut into
convenient pieces before mounting, and, like the Cucumarias
of Study IV, immersed for about five minutes in a saturated
solution of lichtgrun! in absolute alcohol, then washed well in
absolute, cleared in xylol, and mounted in balsam, the inner
side of the wall being placed uppermost on the slide. To
observe the spicules in side view, I cut transverse sections of
the undecalcified * body-wall about 12 u to 20 mw in thickness
or slightly thicker. Numerous sections must be cut, since

Trext-rig. 1.

Camera lucida drawing of a portion of a transverse section through the
body-wall of the adult Synapta inhewerens, showing the spicules in
position. The longitudinally-wrinkled condition of the dermal epi-
thelium and the dermal anchor-pockets are observable. These latter
are more apparent in a surface-view of the integument. The
thickenings of thedermal epithelium are the sense-organs. (Reprinted
from ‘ Quart. Journ. Micro, Sci.,’ vol. 49, p. 557.)

the majority of spicules are fractured during the process of
cutting, and it is only occasionally that the knife happens to
fall just on each side of the (usually young) spicule instead of
on to it.

My specimens of 8. digitata were considerably older,
none being less than 5 cm. in length; nevertheless, I have
been able to observe all stages in development (though

1 In the plates a grey tint has been substituted for green.
* Sections of the decalcified body-wall I found to be useless.

VoL. 51, PART 3.—NEW SERIES. 36

486 W. WOODLAND.

naturally a far greater amount of material had to be examined)
excepting the initial granule. I may here remark that the
numerous specimens labelled “Synapta digitata” which
have been forwarded to me differ considerably in their
spiculation—in the forms and sizes of the spicules—and I
cannot help suspecting that the S. digitata examined by
me includes several distinct species, or at least sub-species.
The specimens of 8S. inhwrens and S. hispida, on the
other hand, have always been constant in their spicule
characters. In most specimens of 8. digitata the spicules
differ considerably in size and form in the same animal
feature I have not noticed in the other two species.

a

Taz DEVELOPMENT OF THE SPICULE IN SYNAPTA INHMRENS.

The first sign of the future spicule is the multiplication of
the cells of the dermal or outer layer at one point. I say
“cells,” but, strictly speaking, it is a multiplication of nuclei
to form a syncytium, since cell-outlines are rarely, if ever,
distinguishable. his preliminary formation of a syncytium
is best seen in a section of the body-wall (fig. 1). It will be
observed in fig. 1 that in these young Synaptas the dermal
layer is already quite separate from the circular muscle layer,
the intervening space containing, at this stage, irregular
strands of nerve- and muscle-fibres. ‘These initial syncytia
are easily distinguishable in section from the numerous
thickenings of the dermal layer which form the sense-organs,
since the elongated cell-outlines and pigment deposits are
conspicuous features in these latter. It is also easy to dis-
tinguish them in surface view under a low magnification after
a little practice. The sense-organ thickenings are, needless
to say, much the more numerous.

The actual spicule first appears as a small spherical granule
situated in the centre of the syncytium, but more internally
than externally, so that the majority of the nuclei are situated
on the other side of the spicule when this is viewed from the
internal side of the body-wall (fig. 2). This spherical granule

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 487

next elongates on one side (figs. 3 and 4) in a direction

transverse to the long axis of the animal. Thus, remarkable
as it may seem, the minute rod which the granule gives rise
to, and which, in its turn, becomes the anchor of the adult
spicule, is from the very first orientated in the direction
assumed by the full-grown structure. The process formed
from the side of the initial granule (situated to the right or to
the left of the observer, as the case may be) continues to grow
(figs. 5-10) until there is produced a stick-like structure,
equal in length to the future anchor. One end of this stick-
spicule is somewhat swollen to form a knob, and represents
the unmodified half of the original granule—the other half
having grown out to form the stick. The stick is also usually
of slightly greater diameter mid-way in its length than
towards its extremities; in other words, the stick tapers
somewhat, both towards the knobbed and the pointed ends.
Also it possesses, at this stage, a distinct axial thread. The
nuclei of the syncytium, up to this period of growth, and until
the knobbed end of the stick has produced the recurved arms
of about half the adult length, remain, for the most part, on
the outer side of the stick—i. e. towards the external dermal
layer—although one or two may be situated more internally
(figs. 9, 10, 11). I may also mention that the syncytium
surrounding the spicule usually remains in connection with
the epithelium from which it originated by means of a few
irregular protoplasmic strands.

The next stage in the development of the anchor spicule is
the protrusion of both sides of the knobbed extremity of the
stick or shaft to form the arms of the future bow of the anchor
(fig. 10). These incipient arms elongate, and very soon
become recurved; at the same time this lateral extension of
the knobbed extremity stretches the substance of the syn-
cytium enveloping the spicule so as to produce the appearance
shown in figs. 16 and 18.

When the arms of the anchor have become half-grown a
very remarkable phenomenon occurs in connection with the
syncytial nuclei, situated a little bow-ward of the middle of

488 W. WOODLAND.

the shaft. A number of these (usually from six to ten), which
up to the present have been situated either at the sides of the
shaft or on its external aspect, now migrate on to its internal
side, and form a small cluster in that position (fig. 12). This
fact seems to me to be most remarkable and I am quite
unable to account for it, at least in an adequate manner.
This cluster of nuclei is always easily distinguishable from
the rest of the syncytium, and it is this specialised part which
gives rise to the plate spicule. Thus, although the anchor
and plate portions of the entire aggregate spicule are quite
separate, and without doubt equivalent to two spicule
individuals—to two echinoderm plate-spicules, to be precise—
yet they are both developed from the same syncytium, though
in distinct parts of it.

The plate-spicule, like other spicules, first originates as a
eranule, and this is situated in the centre of the internal
cluster of nuclei (fig. 18). This granule next elongates on
opposite sides and at right angles to the length of the
anchor-shaft (cf. the development of the plate-and-anchor
spicule of Synapta digitata described below) to form a
rod or rhabdus (figs. 14, 15, 16) which is at first pointed at
both ends. The nuclei of the cluster are situated on both
sides of this rod, and in fact the entire development of the
plate spicule is identical with that of the plate spicules of the
Cucumaridz described by me in Study IV, with the excep-
tion that the number of nuclei initially concerned is greater
in the present instance. The rod thickens at its extremities
(fig. 16), then bifurcates (fig. 18), these primary bifurcations
elongate and themselves bifurcate (fig. 20), and the processes
of bifurcation and fusion of the extremities continue (figs. 21,
22), as in the Cucumarian plate-spicule, until the adult plate
is produced (fig. 24). The Synapta plate-spicule is obviously
different in several respects from the Cucumarian plate-
spicule, but the plan of structure is the same. ‘lhe Synapta
plate possesses extremely large perforations, which diminish
in size towards the more pointed “basal” extremity, and the
edges of these perforations develop small processes which

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 489

add considerably to the picturesque effect of the whole. As
the spicule increases in size, the nuclei of the portion of
syneytium concerned in its formation distribute themselves
more uniformly over its area, Apparently very few, if any,
nuclei are subsequently added to those initially present in the
internal cluster which gives rise to the plate-spicule. It must
also be remarked that the special internally-situated portion of
the syncytium which produces the plate becomes almost quite
separated from the rest of the syncytium enveloping the
anchor as the growth of the plate proceeds, since the anchor
and the plate gradually diverge to include the angle pre-
viously mentioned (text-fig. 1) ; the syncytia of the anchor and
of the plate in fact alone remain continuous at the joint formed
between the handle of the anchor and the base of the plate,
and this continuity of the two syncytia in this region probably
serves to some extent both to keep the two structures in
apposition and to render the joint a true joint, 1. e. a mutual
centre of rotation (see fig. 44, in Study IV, 7).

To return to the later development of the anchor. Up to
the time when the arms of the anchor-bow are but half-grown,
the syncytium in this region is, as before mentioned, stretched
to form two “ patagia,” so to speak, on the two sides of the
shaft (figs. 16, 18). But on further elongation of the arms
the central portions of the patagia apparently give way, with
the result that the scleroplasm remains in the neighbourhood
of the shaft as a thin layer, and away from the shaft as “two
elongated strands of protoplasm containing many nuclei
which run on either side from the arms of the bow to the
handle ” (Study IV). When the arms are fully formed, these
two strands (figs. 20-24) are thickened at the bow end,
forming ‘‘ blobs,” and the nuclei at this end of the anchor are
almost entirely collected in these two thickenings. There is
also in each strand another such collection of nuclei situated
in the vicinity of the handle. Nuclei do occur in other parts
of the strand, but they are principally aggregated in these
two regions ; they also occur, of course, on the shaft, bow,
and handle, though not in clusters. Thus the formation of

490 W. WOODLAND.

these conspicuous strands of protoplasm joining the extremi-
ties of the arms with the handle is due to the presence of
these arms and not vice versa, as I suggested when I first
observed the scleroplasm associated with the fully-formed
spicule (7). The strands are not muscular and exercise no
“ tractive action” which can account for the recurved shape
of the arms, i. e. they do not exert an active pull producing
this effect, though it is possible that they have a slight
passive influence in this connection.

THe DEVELOPMENT OF THE SPICULE IN SYNAPTA DIGITATA.

As might be anticipated, the development of the plate-and-
anchor spicules of Synapta digitata proceeds on very
much the same lines as that of Synapta inherens, but
there is one difference which, though slight, is yet remarkable
on account of its striking incomprehensibleness. In the last
section I described the plate of the spicule as arising in the
form of a rod disposed transversely to the length of the
anchor-shaft (figs. 16,18, e.g.). In S. digitata the plate
also arises as a rod, but curiously enough it is disposed
parallel with the anchor-shaft and not transversely to
it (figs. 17,19). I have observed scores of young plates of
both species of Synapta, but I have never observed a single
exception to this rule, that the plate-rod of 8S. inhzrens is
disposed at right angles to, and the plate-rod of 8S. digitata
parallel with, the length of the shaft.

‘THEORETICAL CONSIDERATIONS.

The distribution and disposition of the plate-and-anchor
spicules in the body-wall of Synapta are subjects which first
demand our attention. Respecting the former there is little
to say, since the spicules are uniformly spread over the entire
area of the body-wall. However, it is worth remarking that
the production of these relatively few but huge spicules in
Synapta involves the concentration of numerous scleroblasts

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 491

in a limited number of centres—i. e. involves the formation
of syncytia, whereas in the Cucumariide, e.g. in which the
spicules are relatively numerous but small, the scleroblasts
either remain independent or only associate in pairs. In
other words, given a certain number of scleroblasts in the
organism, these can either aggregate in some degree to
produce a relatively few huge spicules, or can remain inde-
pendent and produce numerous small spicules. What factor
determines the choice of these alternatives in any given
instance I do not pretend to know, and I can only remark, on
Spencerian grounds, that the Synapta condition is the more
highly evolved.

With regard to the conspicuously definite disposition of
the spicules in the body-wall, the explanation of this feature
is, I believe, not difficult to find. In the first place it must
be remembered that the body-wall of Synapta is highly con-
tractile, the creature being able by means of its powerful
longitudinal muscles to contract itself with ease to a con-
siderable fraction of its normal length. This longitudinal
contraction must and does involve a transverse wrinkling of
the body-wall, and this contraction not being an infrequent
expression of the animal’s activity, it seems clear that the
initial granule of the spicule will elongate in the transverse
grooves formed in the body-wall during such contractions.
It is only necessary to mount and examine a contracted
portion of the body-wall of Synapta to see that rigid structures
like the spicules must lie in the grooves so formed, and no
objection can be taken to this view on the ground that the out-
growth of the initial granule is appropriately orientated from
the very first, smce the grooves may be assumed to be quite
capable of determining the direction of the initial as well
as of the later growth of the spicule. Whether the granule
elongates on one side or the other I believe to be purely a
matter of chance, the spicules being, as before-mentioned,
pretty equally disposed in both directions, as they should be,
in the absence of any determining factor. Why the granule
only elongates on one side and not on both I am unable to

492 W. WOODLAND.

say. The contractility of the animal is then in all probability
the cause of the definite transverse disposition in the body-
wall of the long axes of the anchors.

The shape of the anchor can also, I believe, be associated
to some extent with the contractility of the body-wall. The
body-wall of Synapta not only possesses bands of longitudinal
muscle, but also a continuous sheet of circular muscle, the
presence of which latter implies that the body-wall can
diminish in diameter. Now the body-wall consists in the
main of two layers: the external thin dermal layer and the
internal, comparatively thick, sheet of circular muscle, and
between these two layers the elongated spicules lie, on a bed
of fibres, in a transverse position. If the circular muscle-
layer be imagined to contract, then it is evident that the
dermal layer, which at first is uniformly attached to the
muscle-layer, will be thrown into longitudinally-disposed
folds (see text-fig. 1), and that the position of these longi-
tudinal folds will, to some extent, be determined by the
spicules. In other words, the dermal layer will adhere to the
contracted muscle-layer in those parts of the circumference
which are devoid of spicules, but that, where spicules are
present, the dermal layer will, by the inevitable protrusion of
elongated spicules situated transversely on a diminished
circumference, be separated from the muscle-layer in the form
of pockets pushed out by the anchors. Consider the
mechanical aspect of the matter. Rods lying between two
connected layers forming the wall of a cylinder and situated
transversely with regard to its long axis will, if their length
be adapted to the circumference of the cylinder, not project on
the surface at their extremities in any appreciable degree (text-
fig. 2). If, now, the inner layer only of the cylinder contract in
a considerable degree, it is evident that the extremities of the
rods will project on the exterior and that the outer layer will
be thrown into pocket-like folds enveloping the extremities
of the rods, but that in the portions of the circumference
situated between the rods it will still remain more or less
attached to the contracted inner layer, though in a longitu-

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 493

dinally-wrinkled condition (text-fig. 3). If, further, we suppose
that, in some way, the rods are caused to project on the
exterior by one extremity only, the pocket-like folds will be
rendered still more conspicuous (text-fig. 4). These condi-
tions are those found in the case of the adult spicules of
Synapta, the last possibly being brought about by the con-
tinued attachment of the knob end of the primary anchor rod
to the dermal epithelium from which it originated, and by
the connection of the handle extremity of the shaft with the

Trext-Figs. 2, 3, 4, and 5.

=.

4 5

These diagrams illustrate the formation of the dermal pocket which
envelops the anchor-bow described in the text. In fig. 2 the rod is
lying tangentially on the uncontracted circular muscle-layer of the
animal; in fig. 3 the circular muscle-layer has contracted, and in
consequence the outer dermal layer is thrown into longitudinal folds
and forms two pockets at the extremities of the rod; in fig. 4 the
anchor protrudes on the surface at one extremity only, with the
result that one large dermal pocket is formed; in fig. 5 the anchor
and dermal pocket enveloping it are viewed from the surface, though
they are supposed to be foreshortened.

plate, which les internally and parallel with the muscular
layer (see text-fig. 1)! The knobbed extremity of the anchor

1 IT cannot guarantee that the position of the spicules relative to the
dermal- and muscle-layers represented in figs. 11, 12, and 15 is exactly that
which obtains in nature, since the process of section-cutting is liable to shift
the spicules.

4.94. W. WOODLAND.

supporting a pocket-like protrusion of the dermal epithelium,
it seems probable that the lateral extension of the knob to
form the recurved arms of the anchor-bow is, after all, but
an illustration of the deposition of skeletal matter in the
direction of least resistance. It is an elementary fact
that the anchor-bow does lie in a closely-enveloping dermal
pocket, which is identical in general outline with the bow,
and as fresh calcareous matter has for some reason to be
deposited at the knobbed extremity of the anchor-shaft, the
suggestion that the form which this additional deposit of
calcareous matter takes is largely determined by the envelop-
ing dermal pocket is not a very bold one.

The objection that, were the form of this further deposit of
calcareous matter merely determined by mechanical pressure
this calcareous matter would simply assume a more or less
spatulate form and not the arms of an anchor, may be met
by the reply that the bow-form, which the calcareous matter
does assume, is not supposed to be solely due to the
mechanical contact of the dermal pocket. As is well known,
rod-structures in echinoderms generally have, for some
unknown reason, a tendency to bifurcate terminally, and
though the shaft of the Synapta anchor is abnormally large
owing to the large syncytium concerned in its production, yet
this is no reason why this rod should prove an exception to
the general rule. If this be the case, then the two arms of
the anchor-bow of Synapta must be regarded as the echino-
derm rod-bifurcations, which in this case have been secon-
darily reflected and otherwise modified by the special condi-
tions obtaining. Similarly, the handle of the anchor may be
regarded on this hypothesis as an abortive attempt of the
shaft to bifurcate at its internal extremity.

It seems useless to attempt any further explanations in
connection with the form of the Synapta spicule, since such
explanations must necessarily, in our present state of know-
ledge, be vague and unsatisfactory. ‘There is, of course, one
obvious question which every intelligent observer of these
spicules has asked himself, viz. why are the plates and

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 499

anchors developed in connection with each other? But to
assume that this question is capable of being answered implies
that we know the answers to several questions which natur-
ally precede it. Grant that the huge anchor-shaft owes its
size, shape, and disposition to the factors already mentioned,
grant also that the relatively small rod of the incipient plate
is small because of the small syncytium concerned, yet we are
quite ignorant either as to why the six to ten nuclei should
migrate in a bunch from the mass of the syncytium on to the
internal side of the shaft, or as to why this cluster of nuclei
when produced should give rise to a separate plate-spicule.
Why should not the internal cluster of nuclei merely deposit
an outgrowth of the shaft on its internal aspect in the same
manner as the gastral actinoblast of the calcareous sponge
deposits the gastral ray on the triradiate basis? Personally,
I am as yet quite unable to suggest solutions to these problems.

Before concluding I may mention that occasionally no plate
is developed in connection with the anchor, the anchor remain-
ing solitary (fig. 24). For some reason or other, the migration
of nuclei on to the internal surface of the shaft has (presum-
ably) not taken place. Whether plate structures are ever
developed in Synapta, apart from the anchor, I cannot say
for certain. I have observed several fully-formed solitary
plates, but, although no signs of disturbance were visible,
yet I suspect that the anchors had, in every case, been de-
tached. It is, indeed, hard to suppose that the plates would
assume the normal shape (which is modified in connection
with the anchor!) when developed by themselves. I have
also observed two or three instances of what appear to be
young plates developing on their own account, but, again, I
cannot be quite certain that they had not become detached
from anchor counterparts. Anchors can develop without
plates (I think the instances are too numerous to suppose
that the plates had merely become detached), but it is
very doubtful if normal plates can develop apart from
anchors.

1 This is conspicuously shown in the abnormalities figured by Hérouard (2).

4.96 W. WOODLAND.

Nove on some PointeD OsLonc BopiEs PRESENT IN THE WALL
OF SYNAPTA INHERENS.

While searching the internal surface of the body-wall of
S. inherens for young spicules, I not infrequently en-
countered the curious bodies depicted in fig. 26. ‘They were
stained a dark green, and were apparently in every case
enclosed each in a single cell, which was one of a cluster.
The surrounding cells (which often contained large vacuoles
not shown in the figures) did not appear to be in any way
connected with the cell containing the body, and, so far as I
observed, only one of these bodies was contained im each
cluster. I did not detect any internal structure in the interior
of these bodies. Beyond offering the somewhat trite sugges-
tion that they may be parasites, I am unable to explain their
nature.

SUMMARY.

(Ll) The first sign of the future spicule is the multiplication
of the nuclei of the dermal epithelium at one point to form a
syncytium.

(2) In this syncytium a calcareous granule is deposited on
its internal aspect.

(3) This granule elongates on one side either to the right
or to the left of the long axis of the animal to form the shaft
of the future anchor; the other side of the granule persists
for a while as the knobbed extremity. During development
the knobbed extremity remains in apposition with the dermal
epithelium, but the opposite end comes into connection with
the subjacent fibrous layer, so that the shaft is not situated
tangentially in the body-wall but is inclined. The shaft, like
the initial granule, lies on the internal aspect of the syncytium,
i.e., nearly all the nuclei lie on the external side of the shaft.

(4) When the shaft has attained its full length, and the

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 497

knobbed extremity has commenced by lateral growth to give
rise to the arms of the anchor-bow, six to ten nuclei of the
syncytium travel round in a cluster to the internal side of
the shaft about mid-way in its length, and there produce,
quite independently of the rest of the syncytium, a granule
which elongates and bifurcates and finally becomes the plate
much in the same way as that already described for Cucu-
marian spicules (Study IV). The shape of the Synapta plate
is modified in connection with the anchor. Also the portion
of the original syncytium devoted to the production of the
plate becomes entirely separate from the rest of the syncytium
except in the region of the anchor-handle and _ plate-base,
where the anchor and the plate are in contact and include a
joint.

(5) In Synaptainherens the rod producing the plate
lies transversely to the length of the anchor, whereas in
S. digitata it as constantly hes parallel. No explanation
is offered in connection with this curious fact.

(6) The arms of the anchor-bow are perhaps to be
homologised with the almost universal terminal bifurcations
of the echinoderm rod-spicule, and their recurved form is
possibly due to contact with the dermal epithelium which
forms a pocket for their reception. The syncytium in con-
nection with the anchor is stretched by the formation of the
arms into “patagia,” which subsequently form the con-
spicuous strands of protoplasm joining the extremities of the
arms with the handle. These strands are, as shown by their
development, not muscular, and exercise no -tractive function
which can account for the recurved shape of the arms.

(7) The transverse disposition of the Synapta spicules is
attributed to longitudinal contractions of the body-wall.

(8) The shape of the anchor is to be largely attributed to
contact with the body-wall, which, it must be remembered, is
contractile transversely as well as longitudinally.

(9) The very usual association of anchor and plate is a
physiological problem at present insoluble,

498 W. WOODLAND.

Part 2.—The Spicules of the Auricularia Larva.

INTRODUCTORY.

The morphogenesis of the Auricularian wheel-spicule has
been described and figured by Semon (5). An unillustrated
account of the scleroblastic development of the Auricularian
wheel has also been published by Chun (1) in 1892, but, as I
surmised in Study IV, Chun’s remarkable statements con-
cerning this subject are totally misleading, owing to the fact
that he unconsciously studied decalcified material. As will
be seen in the quotation given below, Chun described the
spicule as being deposited within a mould which is formed
for its reception, so to speak, in the substance of the sclero-
plasm. I am able to say that this supposed mould is noth-
ing but the space contained within the scleroplasm which
was occupied by the spicule before it was dissolved away by
acid reagents. JI have recently observed many of these
“ moulds” in improperly preserved (decalcified) material, and
I am able to make the above statement concerning their real
nature because I also possess properly preserved (undecal-
cified) material.

My material consisted of numerous Auricularia larve in
different stages of growth obtained at Naples and specially
fixed with absolute alcohol. I stained them for a week ina
saturated solution of Griibler’s safranin in absolute alcohol,
and washed out the superfluous stain by keeping the larve
in warm 90 per cent. alcohol! (several changes) for a week ;
they were then dehydrated, cleared in cedar-wood oil and
mounted in balsam in the ordinary way.

Tur DEVELOPMENT OF THE SPICULES OF THE AURICULARIA
LARVA.

The wheels and “ globes,” as I shall term the other class of

calcareous deposits found in the Auricularia larva, are found

1 Absolute alcohol with the required percentage of distilled water to ensure
neutrality.

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 499

in the pair of processes situated at the anal, i. e. lower,
extremity of the larva. Both wheels and globes abut against
the ectoderm of the larva, though they arise from cells
internally situated, 1.e. from the mesenchyme cells. But
before describing the development of the spicules I will
quote the relevant passages of Chun’s account of this subject,
which, though incorrect as regards the main point, yet contains
many true observations. “ At the time of the appearance of
the first calcareous wheels the cellular elements of the gela-
tinous substance (filling the interior of the larva) are sharply
differentiated into skeletogenous and connective-tissue cells.
The latter possess several long processes, which are much
ramified, and are interwoven almost after the manner of felt;
the skeletogenous cells, on the contrary, are spherical and
surrounded by a distinct membrane, in consequence of which
they emit no pseudopodia. The sharp histological differentia-
tion of the mesoderm cells, which was certainly preceded by
an indifferent stage, may be essentially due to the fact that
the calcareous bodies originate at a remarkably late period in
comparison with what is found to be the case in other Echino-
derm larve. The skeletogenous cells accumulate . . . close
beneath the ectodermal epithelium.” ‘ A richly vacuolate
plasma at once distinguishes the skeletogenous cells, the
average size of which is 0°01 mm. ‘They rapidly grow to
twice and thrice this bulk, while simultaneously the number of
the cell-nuclei increases. In the same Auricularia we meet
with all intermediate stages between uni- and multinucleate
cells, which at first still retain a rounded contour, but subse-
quently flatten out on one side and become cup-shaped. The
nuclei measure from 0:003 mm. to 0:004 mm. in length, and
originally (so long as only from two to four are present)
occupy a peripheral position; they afterwards increase to
from six to eight in the case of the Mediterranean Auricularie,
and to from twelve to eighteen in that of those from the
Canary Islands, and form a central nuclear cluster. When
the cells have attained a size of 0:03 mm. there appears within
the old cell-membrane a new one, which has an undulating

500 W. WOODLAND.

outline towards the circular margin and speedily assumes a
star-shaped form. ‘The tubular rays of the star which grow
out are equal in calibre and meet the external membrane,
arching forwards somewhat at the points of contact. The
longitudinal extension of the radially-arranged outgrowths
keeps pace with the increase in the size of the cell, and finally,
when the cell attains a size of from 0°06 mm. to 0:07 mm.,
the rays become united by a peripheral membranous ring. It
is now impossible to mistake the mould of the subsequent
calcareous wheel, prepared as it is by the complex folds of an
internal membrane ; the central portion with the cluster of
nuclei corresponds to the nave, the tubes running out like the
rays of a star represent the spokes, and the peripheral ring
takes the place of the circumference (the felly) of the future
calcareous wheel. Moreover the calx is actually secreted
into this organic matrix formed by the skeletogenous cell, as
into a mould, and in such a way that (as the older accounts
already teach us) calcification takes place first in the nave,
then in the spokes, and finally in the felly of the wheel. It
is likewise in accordance with the theories which have recently
been formulated as to the share of the nuclei in the vital
processes of the cell that, corresponding with the centrifugal
progress of the calcification, the majority of the cell-nuclei
also separate from one another in a centrifugal direction, and
in the case of the Auriculariz from the Canary Isles come to
lie in the acute angles between the spokes. In rare instances
they advance as far as the middle of the spokes, or even to the
periphery. No secondary multiplication of the spokes of the
wheel takes place; their number corresponds exactly with
that of the undulating evaginations of the newly-formed
internal membrane, which develop into radiating tubes. As
is well known, the number of the spokes varies; in the case
of the Auriculariz from the Canaries, we find from thirteen
to eighteen. Since the diameter of the fully-formed cal-
careous wheels is found to be from 0:09 mm. to O'l mm., it
follows that a ten-fold enlargement of the diameter of the
skeletogenous cells takes place, since the latter in the stage

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 501

with a single nucleus only measure 0°01 mm. Nevertheless,
after the secretion of the calcareous wheels they expand still
further; for if we examine the wheels in alcoholic prepara-
tions . . . we can distinguish a distinct periphery
formed by a delicate membrane, from which, alternating with
the spokes and almost equalling them in length, membranous
tubes arranged in the shape of a star run to the periphery of
the wheel, where they usually exhibit flask-shaped expan-
sions. On careful decalcification of the wheels by means of
weak chromic acid it is easy to show the nuclei and the
contour of the wheel in the shape of a delicate membranous
envelope within the skeletogenous cell. The above state-
ments as to the formation of the wheels in the Auricularia
reveal a mode of development which at first appears to be
unique. While the skeletal pieces of Echinoderms were
hitherto essentially regarded as intercellular structures, the
formation of which was due to several mobile amoeboid cells
(I am well aware that more recent observers are inclined to
attribute the shape of the skeletal elements without hesitation
to directly mechanical influences), we now find that the
form of the calcareous wheel is traced out within
a multinucleate cell by means of an organic mem-
brane which assumes complex folds, and that in
this definitely circumscribed mould the casting
of the hard parts ensues.”’

I have quoted Chun at length because his remarks show
how carefully most of his observations were made and how
purely accidental his mistakes were. I will now describe the
mode of formation of the wheels and globes in the Auri-
cularia larva as it really occurs. JI am unable to say for
certain as to whether the syncytium which deposits the
spicule arises from one cell solely by the simple multiplication
of its nucleus as Chun describes, or as to whether it is afso
formed in part by the fusion of originally separate scleroblasts.
I have certainly seen cells containing two and three nuclei, but
I have also seen numerous clusters of separate scleroblasts
which, unless they take on some other function, must coalesce

vot. 51, PART 3,—NEW SERIES. 37

502 W. WOODLAND.

with each other to produce the few spicules found ; probably
both processes occur. The syncytium of the Auricularia
larva differs from the syncytium of Synapta in that in the
former each nucleus is almost entirely surrounded by a dis-
tinct layer of protoplasm, 1. e. cell-outlines are to a large
extent visible, each nucleus occupying a distinct subspherical
portion of the syncytium, whereas in the latter the individual
cells are so fused together as to constitute merely a mass of
protoplasm containing numerous nuclei. The syncytium of
the Auricularian spicule contains, in the specimens I studied,
from five to nine nuclei.

The spicule first appears in this syncytium as an endoplastic
spherical granule (figs. 27, 28), which is usually situated on
the side of the syncytium next to the adjacent body-wall
(not indicated in most of the figures). This granule flattens
and gradually becomes disc-shaped with a slightly concave
internal surface, the centre of which at first bears a slight
eminence, and a convex external surface (figs. 31, 32) which
is in contact with the body-wall. All the scleroblasts (i. e.
the nuclei with their subspherical masses of protoplasm) are
situated on the concave side of the disc, i.e. remote from the
body-wall, and are clustered together (figs. 31,32, 34), though
of course the entire disc (and all later stages of the spicule) is
enveloped by the scleroplasm. The next step in the develop-
ment is the formation of processes (variable in number, rang-
ing from about fourteen to eighteen) at the margin of the
disc or nave of the future wheel (fig. 35), and these processes,
which vary in number in different spicules, elongate, and
ultimately form the spokes (figs. 86-38). By the develop-
ment of these spokes (the exact shape of which can be seen
in fig. 41) the convexo-concave disc assumes more the shape
of acup. Finally these spokes, by lateral extension, join up
to’ form the felly of the wheel (figs. 39-41). The adult
structure then, as shown by the figures, forms a cup-shaped
structure, in the concavity of which are lodged the sclero-
blasts—somewhat like eggs in a basket (fig. 41).

In every case the extension of the scleroplasm which is

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 903

necessary for the envelopment of the entire spicule is effected
by the growth of the spicule itself, the spokes, e. g. pushing
out the peripheral scleroplasm before them as they increase
in length. The syncytium no more enlarges itself for the
accommodation of the spicule, as Chun imagines, than an
adipose tissue corpuscle swells out to make room for the
secreted oil.

Unlike Chun [have not observed that “ corresponding with
the centrifugal process of the calcification, the majority of the
cell-nuclei also separate from one another in a centrifugal
direction.” On the contrary, in the Auriculariz observed by
me, they do not desert the nave portion of the wheel. In
short, the development of the Auricularian wheel resembles
that of all other echinoderm spicules in that they are endo-
plastic deposits, the form of which bears little or no relation
to the disposition of the nucleus or nuclei. The study of
spicule formation clearly shows that the proximity of a
nucleus is not essential to the deposition of skeletal material
at any given point, and that deposition goes on in any given
region of a cell independently of the distance of the nucleus
or nuclei from that region. Nuclear material is in all proba-
bility a sine qu& non where spicular deposition is con-
cerned, but, as in the political constitution, the exact position
of the governing centre is of little or no account in regulating
the activities of the various areas governed—work proceeds
in all quarters, and not merely in the vicinity of the nucleus
or parlament as the case may be.

The development of the calcareous globes of the Auricularia
is quite simple. The globe originates as a granule in the
syncytium and simply increases in diameter by the uniform
deposition of calcareous matter on its surface. The globe
protrudes on the surface of the body-wall, at least half of its
area being in contact with the wall, and the nuclei of the
syncytium, probably in consequence of the contact, become
restricted to the surface of the internal hemisphere (fig. 42).

504 W. WOODLAND.

THEORETICAL.

With regard to the cause or causes determining the form
of the Auricularian spicules, little can be said. It seems
probable that the flattened disc- and later cup-forms are at
least in part due to contact of the spicule with the body-wall,
unless these are indeed to be attributed to the presence of the
nuclei on the inner side of the plate only, the nuclei, contrary
to the usual assumption, preventing deposition in their
immediate vicinity.!

But whatever forms the spicules may assume, it is clear that
one of their inevitable attributes, viz. weight, may play anim-
portant part in the life of the larva. In the Auriculariz I have
studied, the wheels and globes are all situated, as before stated,
at the lowest extremity of the larva, and it seems certain that
this extremity is lowest because of the presence of the
relatively weighty spicules in this region. In other words,
the larva maintains a certain position in the water because it
is appropriately weighted. The so-called baguettes de
corps of the pluteus larva doubtless serve a similar
function.

SUMMARY.

(1) The spicule first appears as a granule contained in a
syncytium, in which, however, the scleroblasts retain their
individuality to some extent, which is not the case in the
syncytium of Synapta.

(2) The spicule becomes disc- and then cup-shaped,
develops the spokes as outgrowths from the margin of the
disc, and finally forms the felly of the adult wheel, the spicule
during the whole of its development being enclosed by the
syncytium in which all the nuclei (scleroblasts) are situated
on its internal side, i.e. away from the body-wall against
which the spicule lies.

1 I may point out in this connection that Hérouard (8) attributed, though

quite erroneously, the perforations of the holothurian plate to the presence
of nuclei. See Study IV.

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 9505

(3) The extension of the scleroplasm depositing the spicule
is determined by the growth of the spicule itself, and is not

Text-FIG. 6.

‘be abnormal siliceous spicule found inserted into the body of an Auricu-
laria larva. The whole spicule is enveloped in a large syncytium.
Nearly midway in its length is situated the cluster of curious
radiating spines with swollen bases attached to the main shaft. The
figure of the entire spicule is magnified just over 500 diameters.

the result of a mould-forming tendency on the part of the
protoplasm, as Chun asserted.

506 W. WOODLAND.

(4) The situation of the heavy wheel and globe spicules at
one (the lower) extremity of the larva determines the position
which the larva assumes in the water—the spicules weight
the larva.

Norte on A Curious Tyre or SILIcEouS SPICULE.

The curious spicular structure represented in text-fig. 6 was
found inserted for a short distance into the body of one of the
Auricularia larve which I examined. As it is not doubly
refractive under the polariscope, I conclude that it is siliceous.
It is well clothed in a thick layer of scleroplasm containing
numerous nuclei. Being quite unable to identify this spicule
I should say that it is probably a freak.

LITERATURE.

1. Cuun, C.—“ Die Bildung der Skelettheile bei Hchinodermen,” ‘ Zool.
Anzeig.,’ vol. xv, 1892; translated in ‘Ann, Mag. Nat. Hist.’ (6), vol.
ii, 1893.

2. Hirovarp, E.—“ Sur une Loi de Formation des Corpuscules Calcaires et
sur ’Homologie qui existe entre ces Corpuscules chez Ankyroderma et
Synapta,” ‘Bull. Soc. Zool. France,’ vol. xxvii, 1902.

3. Hiérovarp, E.—“ Recherches sur les Holothuries des Cotes de France,”
‘Theses,’ Paris, 1890.

4. OsTERGREN, Hs.—‘* Ueber die Function der Ankerfoérmigen Kalkkorper
der Seewalzen,” ‘ Zool. Anzeig.,’ vol. xx, 1897.

5. Semon, R.—‘ Beitrage zur Naturgeschichte der Synaptiden des Mittel-
meers,” * Mittheil. Zool. Stat. Neapel.,’ vol. vii, 1887.

6. Semon, K.—‘ Die Entwickelung der Synapta digitata, und ihre Bedeu-
tung fiir die Phylogenie der Echinodermen,” ‘ Jena Zeitsch.,’ Bd. xxii,
1888.

7. Wooptanp, W.— Study IV. The Scleroblastic Development of the
Spicules in Cucumariide ; with a Note relating to the Plate-and-anchor
Spicules of Synapta inhewrens,” ‘Quart. Journ. Mier. Sci.,’ vol.
xlix, 1906.

8. WoopLtanp, W.—“A Preliminary Consideration as to the Possible Factors
concerned in the Production of the Various Forms of Spicules,” op.
cit., vol. li, 1907.

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 507

EXPLANATION OF PLATES 29 & 30,

Mlustrating Mr. W. Woodland’s “Studies in Spicule Forma-
tion. VII.—The Scleroblastic Development of the Plate-
and-Anchor Spicules of Synapta, and of the Wheel
Spicules of the Auricularia Larva.”

Figs. 1, 9-25 x 800 diameters; Figs. 2-8, 26-42 x 1600 diameters,

PLATE 29.
The Development of the Synapta Plate-and-Anchor Spicules.

Fic. 1.—Portion of a transverse section through the body-wall of Synapta
nherens showing the multiplication of nuclei at one centre on the internal
side of the dermal epithelium to form a syncytial mass. To the right lies the
circular muscle layer and between this and the dermal epithelium some muscle-
and nerve-fibres.

Fic. 2.—A syncytium viewed from the internal side of the body-wall. The
initial granule is deposited on its internal aspect—i.e. the majority of nuclei
are situated on its external side. These syncytia have very definite outlines,
being quite distinct and usually distant from all surrounding structures. It
must also be remarked that, owing to difficulties of observation, the exact
number of nuclei figured cannot be guaranteed as having been the actual
number present. It can, however, be guaranteed that every nucleus figured
was carefully observed and correctly placed relative to the spicule, but, as is
self-evident, it is impossible to be certain that every nucleus present was
observed.

Fic. 3.—The granule has elongated slightly towards the observer’s left. It
is possible that the syncytium is in part formed by the fusion of at-first-
separate scleroblasts as well as by the multiplication of nuclei; this latter,
however, is the principal mode of formation of the syncytium. Some of the
nuclei are larger than others, a feature probably denoting approaching nuclear
division.

Fie. 4.—The initial granule has here elongated to the observer’s right hand.

Fies. 5—8 illustrate the further elongation of the granule to one side or
the other. The axis of the future shaft is first discernible when the full length
is nearly attained. The majority of the nuclei, it will be noticed, lie on the
external side of the shaft.

Fic. 9.—The nearly adult shaft.

508 W. WOODLAND.

Fic. 10.—The fully-elongated shaft. The terminal knob (representing the
unelongated portion of the initial granule) is now extending laterally. The
axis is conspicuous at this stage.

Fie. 11.—The fully-elongated shaft seen in a transverse section of the body-
wall. The external position of the majority of the nuclei relative to the shaft
is well shown.

Fie. 12.—Adult shaft, in which the arms of the anchor are about half
formed, seen in a transverse section of the body-wall. In this figure there is
shown in the clearest manner the external position of the majority of the
nuclei, and the internal position of the half-dozen or so nuclei which have
migrated internally to form a separate cluster in this position. All stages of
this migration can be seen in the actual preparations.

Fic. 18.—Spicule viewed from the internal aspect. A granule has been
deposited in the internally-situated cluster of nuclei. The formation of the
arms of the bow is distending the general syncytium in this region (formation
of the “‘ patagia’’).

Fic. 14.—The granule is elongating on both sides in a direction transverse
to the length of the anchor-shaft.

Fic. 15.—The same stage of growth of another spicule viewed in a trans-
verse section of the body-wall.

Fic. 16.—Spicule viewed from the internal aspect. The granule has become
a distinct rod with rounded extremities and swollen centre. In this species—
S. inherens—it is, as remarked in the text, situated transversely to the
length of the anchor-shaft.

Fic. 17.—The same stage in 8. digitata. It will be noticed that in this
species the rod is situated parallel with the shaft and not at right angles to it.
This difference between the two species of Synapta is quite constant.

Fic. 18.—S. inherens. The extremities of the transverse rod have
bifurcated. The “patagia” are well shown here.

Fie. 19.—S. digitata. The extremities of the parallel rod have bifurcated.

PLATE 30.

Fie, 20.—S. inherens. The bifurcated extremities of the transverse rod
have themselves bifurcated. The “ patagia” are giving place to the “strands ”
uniting the apices of the anchor arms and the handle.

Fies. 21—23 illustrate the further formation of the plate and the proto-
plasmic strands with their aggregates of nuclei.

Fic. 24 illustrates an anchor, in connection with which no plate has been
formed.

SPICULES OF SYNAPTA AND AURICULARIA LARVA. 9509

Fic. 25.—Side aspect of the anchor-handle and plate-base to show the joint
formed between the two.
Fic. 26.—The problematic bodies found in the body-wall.

The Development of the Auricularian Wheel-Spicule.

Fic. 27.—The initial granule in the syncytium. Its proximity to the body-
wall is indicated.

Figs. 28—30 illustrate the growth of this granule (surface aspect).

Fig. 31.—The plate-like form which this granule assumes and the clustering
of the nuclei on its inner aspect (the aspect remote from the wall).

Fre. 32.—The plate or dish in side view. The inner position of the cluster
of nuclei is well shown.

Fic. 33.—Plate viewed from its inner aspect.

Fie. 34.—Lateral aspect of full-sized plate.

Fic. 35.—Plate viewed from its outer aspect (nuclei situated on its remote
side). The edge of the plate is crenate—the first indication of the spokes.

Fics. 36—38.—Plate in all cases viewed from its inner aspect (nuclei on the
near side). The formation of the spokes and the corresponding distension of
the scleroplasm of the syncytium is shown. The nuclei with their accompany-
ing scleroplasm lie in the concavity of the plate.

Fie. 39.—The spokes extend circumferentially.

Fic. 40.—The felly of the wheel formed. The entire spicule is enveloped
by the scleroplasm.

Fic. 41.—The wheel viewed from the side in optical section showing the
“ egos-in-a-basket ”’ appearance.

Fig. 42.—One of the calcareous globes of the Auricularia larva with its
enveloping syncytium situated in a shallow pocket of the body-wall.

VoL, 51, PART 3.—NEW SERIES. 38

at min) al W4,'? ofl vetiek Mic wilh iNT Ril Ae tiers Eaten At hs
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7

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CONTENTS OF No. 204.—New Series.

MEMOIRS:

The Development of the Head-muscles in Gallus domesticus, and
the Morphology of the Head-muscles in the Sauropsida. By F. H.
Evcrwortu, M.B., D.Sc., Professor of Medicine, University
College, Bristol. (With 39 Text-figures) : : .

The Development of Ophiothrix fragilis. By E. W. MacBring,
M.A., D.8c., F.R.S., Professor of “Zoology in McGill University,
Montreal. (With Plates 31—36, and 4 Text-figures) 4

On the Segmentation of the Head of Diplopoda. By Marearet
Rozinson, University College, London. (With Plate 37, and
6 Text-figures) . . :

The Fixation of the Cypris larva of Sacculina carcini (Thompson)
upon its Host, Carcinus menas. By Grorrrey Smits, M.A.,,
New College, Oxford. (With 6 Text-figures) .

Physiological Degeneration in Opalina. By C. Ctirrorp DoBELt,
B.A., Scholar of Trinity College, Cambridge. (With Plate 38, and
2 Text-figures) . : : : F

Some Facts in the Later eee of the Frog, Rana tempo-
raria—Part I. The Segments of the Occipital Region of the
Skull. By Acwers I, M. Kunior, B.Sc., Associate of Newnham
College, Cambridge. (With Plates 39 and 40)

Wirtn Tittz, Contents, anp INDEX TO VoL. 51.

PAGE

511

557

607

625

633

647

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 511

The Development of the Head-muscles in Gallus
domesticus, and the Morphology of the
Head-muscles in the Sauropsida.

By
F. H. Edgeworth, M.B., D.Se.,
Professor of Medicine, University College, Bristol (from the Anatomical
Laboratory).

With 39 Text-figures.

, A FEW papers have been published on the early stages of

~ development of the head-muscles of Gallus and other birds,
and on the insertion of the mandibular muscles into Meckel’s
cartilage and the lower jaw; but as yet there has been no
description of the formation of all the muscles in any one bird
which might serve as a basis for a discussion of their morpho-
logy. The following is a contribution to the subject founded
on an investigation of Gallus.

I found it was impossible to understand these head-muscles
without comparing them with those of Reptiles, but the diffi-
culty of doing so was accentuated by the almost complete
absence of knowledge of their development; for although
Corning has described the early stages in Lacerta, the later
ones are not known, and no other Reptiles have been investi-
gated. Several friends, however, were good enough to lend
me sections or embryos of the later stages of Chelone, Alliga-
tor, Sphenodon, Agama, and Chameleon, and I was able to
procure those of Tropidonotus natrix, so that it was
possible to follow the growth and changes in the muscles
which take place and give each group its special charac-
teristics.

voL. 51, PART 4,—NEW SERIES. 39

512 F. H. EDGEWORTH.

The treatises of Gadow and Hoffman have been taken as
guides for the adult anatomy of the head-muscles of the
Sauropsida. Some changes in the nomenclature have been
made in an attempt to apply identical names to homologous
muscles, for examination of their development shows that
different names have been given to homologous muscles,’ and
the same name to muscles of different origins and morpho-
logy.2 A list of the terms employed is given in an appendix.

VISCERAL CARTILAGES.

The development of the visceral cartilages in the various
groups of the Sauropsida has already been described by

noto. _ped pl
UNM UnLSEZIN
Ooo, , SR

°
° 26

> dor.aor
dor. ceph.cel.
na

Be 90 >
ig eg meso

gE Spl meso

At cs At vent. ceph. coe/

Trext-Fic. 1.—Transverse section through embryo of Gallus, with
11 trunk somites. (For explanation of lettering see p. 555.)

Parker, Howes and Swinnerton, and by Broom; and I have
very little to add. An interesting point is, that in early
stages of Tropidonotus there is a long pterygoid process pro-
jecting forwards from the upper end of the quadrate (text-fig.
37, p. 543). By the time chondrification has taken place this
pterygoid process has atrophied, and there is only its proximal

1 E.g. the anterior part of the ventral longitudinal muscles has been
called “‘cerato-mandibularis”” (Sphenodon and Lacertilia vera), ‘“ genio-
hyoid”” (Chelonia and Rhiptoglossa), “ maxillo-hyoideus’”’ (Ophidia), and
“ anterior belly of maxillo-coracoideus’’ (Crocodilia).

2 KE. g. the name “cerato-hyoideus”’ has been given to a muscle developed
from the first branchial myotome of Sphenodon and Lacertilia vera, to
the hyoglossus in Crocodilia and Rhiptoglossa, and to a muscle developed
from the posterior mylohyoid in Birds.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 5138

end left—as a stump, which Parker (who, however, did
not see the earlier stage) erroneously homologised with the
“pedicle ” in the frog.

2 noto
dor aor phar

3enceph ' dorceph ce/
pher / tr som

Trext-FIG. 2.—Coronal section through embryo of Gallus, with
14 trunk somites ; outside the dorsal aorta, on either side, is seen
the upper lateral part of the pharynx (ep. text-fig. 3), and outside
this the upper part of the dorsal cephalic eeelom, which is con-
tinuous behind with the first trunk somite. (For explanation of
lettering see p. 555.)

In all the Sauropsida, consequently, there is a long ptery-
goid process to the quadrate in early stages of development.

- hyoid som
- dor ceph. cae/

SSS é °
som mesob/ Y / --+-+----\-. vent. ceph. coal
<7 ae
\ \ _. r.art
oe
\
ete)

hypob/, sp/ meso

TExt-Fic. 3 —Transverse section through embryo of Gallus, with
18 trunk somites. (For explanation of lettering see p. 555.)

In Sphenodon, Lacertilia vera, Rhiptoglossa, and Croco-
dilia this pterygoid process has a processus ascendens, or epi-
pterygoid, which is developed very early in Sphenodon (vide
Howes and Swinnerton), late in the Alligator (text-fig, 22,
p. 527).

514 F. H. EDGEWORTH.

There are three median hyobranchial cartilages in Gallus.
Parker called them the cartilago entoglossa, basihyal, and

\ i, Wi, oe
F } Le Wiae
\ ‘a V/ z g
SS a 4
Ds ee /gs

Trxt-Fic. 4.—Sagittal section through embryo of Gallus, with
2 fully formed and 1 partially formed gill-pouch. (For explana-
tion of lettering see p. 555.)

urohyal, and stated that the first named was formed by lateral
fusion of the lower ends of the ceratohyals. Geoffrey Smith

5, premand.seg.an.
mand. aor.ar-
i SX '/g.S.
~ Ay. aor. ar.
Hers \ 29:5:
=\ /braorar
CSELS

_dor.a@or

--@s

tr art
vent ceph ca/

Text-Fic. 5.—Sagittal section through embryo of Gallus, with
3 gill-pouches. From the anterior portion of the gut a hollow
pouch projects laterally, the wall of which is cut by this section
(the Anlage of the premandibular segment). (For explanation of

lettering see p. 555.)

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 515

subsequently stated that in a five-day-old chick each cerato-
hyal is continuous below with a “ branchial blastema.”
Transverse sections show that whereas the lower ends of

via&viil - Ss ‘ a
roto ale ene \b - gass 9g

premand segan ts Sj \)

Trxt-Fia. 6.—Transverse section through an embryo of Gallus,
showing fully formed Anlage of premandibular segment. (For
explanation of lettering see p. 555.)

the first branchial bars meet each other in the middle line, the
lower ends of the ceratohyals are separated from one another

ze
yO
WH
3g.S.--- “ } on J
/br 80r ar..- (|
Shr my. ----

é ae gl. 29.8.
ONES Ne Ayord my.

tr art € Zi i post my/o.

ven ceph cael -- == /

Trxt-Fic. 7.—Transverse section through an embryo of Gallus,
with 3 fully formed gill-pouches, in hyoid and first branchial seg-
ments. (For explanation of lettering see p. 555.)

by a median procartilaginous structure (text-fig. 12, p. 519),
which is continuous with a median structure in front and
behind. Chondrification takes place, the middle portion of

516 F. H. EDGEWORTH.

the ceratohyal disappears,! and the lower end forms a short
cornu to the cart. entoglossa, which is, therefore, a median
basihyal or glossohyal with ceratohyal cornua. The middle
piece (Copula i, of Gaupp) is a first basibranchial, and the
urohyal (Copula ii) a second basibranchial.!

This conclusion is supported by comparison with the condi-
tions found in Reptiles. In Sphenodon, Lacertilia vera,
Rhiptoglossa, Chelonia, and Crocodilia there is a continuous
basihyobranchial cartilage, and in the first four groups there
are ceratohyal cornua homologous with those in Gallus. In

dor aor

FgS Ec
3g.s..
ZIGtS ae
gs /- 8
5 enceph
mand aorar _

“~- 267 aor.ar.
> ven. ceph.coet.

Je) EY (Ne SS Gicteas e.
“/ breor.ar.

” Ay.gor ar.

trart

O.sup.an. mend my. w.

Text-Fic. 8.—Sagittal section through an embryo of Gallus, with
4 gill-pouches. (For explanation of lettering see p. 555.)

Sphenodon these ceratohyal cornua are formed by ceratohyal
bars which are continuous above with the hyomandibulars,
in Agama, Chameeleon, and Chelone they are the lower ends
of ceratohyal bars which, in embryonic stages, were continuous
with the hyomandibulars. In the Crocodilia there are no
ceratohyal cornua to the basihyobranchial, and the single

1 The small bit of cartilage which is described by Geoffrey Smith as
separating from the lower end of the infrastapedial on the eighth day is
probably homologous with that called “stylohyal”’ by Parker in Chelidon
urbica.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 517

embryo I had was too far advanced in development (chondri-
fication was complete) for one to see this stage—of a cerato-
hyal continuous from the hyomandibular above to the basi-
hyobranchial below.

A first branchial bar is present in all groups of the Saurop-
sida, a second branchial in Chelonia! (vide text-fig. 19, p. 525),
Sphenodon, and Lacertilia vera, but is absent in Birds, Croco-
dilia, Rhiptoglossa, and Ophidia.

The main differences, consequently, between Birds and

geng vi. gpetr x x! 2 cr ny.
- AV. x. \ Lenmy-; 3.tr.my

ant.card Vv.
ant.card v
2)
gtrx
39s
2 br my

: /g.s.-
mand.my

: 29. Ss:
hy my /br my.

Text-Fia. 9.—Sagittal section through an embryo of Gallus, with
4 gill-pouches. It shows (partially) the formation of the ventral
longitudinal muscles, which originate from the first (half) myo-
tome and the four succeeding ones. Owing to the spiral twist of
the embryo no single section shows all the five downgrowths, (For
explanation of lettering see p. 555.)

Reptiles in the state of the hyobranchial skeleton, are that in
the former a second basibranchial is developed® and the
median cartilages become jointed. ‘There are correlated

1 The suggestions of Gaupp in regard to the nature of the cart. entoglossa
in Birds and the existence of a second branchial bar in Chelone are thus

confirmed.
2 It is absent in some few Birds, e.g. Rhea, Platalea.

518 F. H. EDGEWORTH.

differences in certain muscles of the head which will be
described later on.
Parker, who did not investigate any embryonic stages,

4 -~ cerhyal c.

- Rext : quad &émean.
mand my.

buccav. wi

; ;
oy : Wes
' a]
eat
\ ie ,
4 ' \,
:
5

R.sup.

ui *Reext.

; _{hyomand.c.
__.cerhyalc

mand my. quad.& me an

Trext-Fias. 10: anv 11.—Sagittal sections through an embryo of
Gallus at the end of the fifth day. 10 is the more external. (For
explanation of lettering see p. 555.)

described the columella of the adult Chameleon as consisting
of stapedial, mediostapedial, extrastapedial, and supra-

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 919

stapedial portions, the extra- and supra-stapedial portions
lying internal to the quadrate. Study of the development,
however, shows that his “extrastapedial ” is the persisting
upper end of the ceratohyal which lies at first posterior to the
quadrate (text-figs. 32 and 33, p. 536) in the hyoid segment.
A true extrastapedial is absent.

The hyoid bar of Tropidonotus similarly lies at first in the
hyoid segment (text-fig. 37, p. 543). The “ tuberculum” (of
Parker) attached to it is probably a suprastapedial element ; it
is developed as an outwards and upwards projecting process of

gipetr (x

wi ---4-

car.art.

‘ 4 y Jf
: o post mylo,
' ‘ 7 ~ xu.

ven Car. zine VLM. BY.

}

Trxt-F1G. 12.—Transverse section through an embryo of Gallus
at the beginning of the sixth day. (For explanation of lettering
see p. 555.)

the hyoid bar (at a stage later than that shown in text-fig. 37,
p- 543), and only subsequently rotates forward. Parker stated
that the “tuberculum” became adherent to the stapedial plate,
but so far as my observations go it simply disappears. The
“ stylohyal,” of Parker, which becomes adherent to the inner
face of the quadrate is the separated lower end of the hyoid
bar.

In both Chameleon and Tropidonotus the hyoid bar thus
assumes a secondary position internal to the quadrate,

520 F. H. EDGEWORTH.

and, probably consequent on this, a suprastapedial is not
developed.

The first branchial bar of Tropidonotus! is not developed
as a dorsi-ventral bar which subsequently rotates into its
adult position with lower end forward, but as a longitudinal
one (text-fig. 38, p. 543). Its position and elongation back-
wards, together with the carrying back of the origin of the hyo-

yy - post.mylo
Tes

‘
4/88

Trxt-F1G. 13.—Transverse section through an embryo of Gallus
at the middle of the sixth day. (For explanation of lettering see
p. 555.)

glossus muscle, are evidently correlated with the development
of the (secondary) protrusible tongue.

On the Number of Vertebre coalescing with the
Skull.—Suschkin? described, in Tinnunculus, four vertebre
coalescing with the skull, each of which possessed, as tem-
porary procartilaginous structures, a pair of “ cranial ribs.”

1 It cannot be the ventral portion of the hyoid bar as Rathké (cited by
Gaupp) thought, for, on its first appearance as a procartilaginous structure,
it lies behind the second gill-pouch, i. e. in the first branchial segment.

2 Cited by Gaupp.

~HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 521

In Sphenodon there are similarly four vertebre visible (text-
fig. 26, p. 529). In the other Sauropsida investigated only
three were visible—probably due to a dropping out, in deve-
lopment, of the most anterior. In Tropidonotus, for instance,
the first two hypoglossus roots pass out together, i.e. the
witervening vertebra is not developed, even as procartilage.

tal processes or cranial ribs were only seen in Sphenodon
(text-fig. 26, p. 529), and then only over the hindermost coalesc-
ing vertebra; these persist, forming on each side the structure
known as the “proatlas.”! In this embryo chondrification

A. sup. R ext
E. \ pyraquani Igs. AV 14
u x v 'mandmyup <
\ . \ : gass g ‘
f : \\ Moe <, yu
3 \W IES oe : ~ \cerhyalc
i SS ___.-}Sstapm
\ ie Y Bees Soicery,
ul Ne,
dep mand

vy) quad.
Oinf Rink temp & mass an

TrExt-Fic. 14.—Sagittal section through an embryo of Gallus

at beginning of seventh day. (For explanation of lettering see
p- 555.) ‘

@

had already taken place in the basis cranii, so that possibly in
an earlier stage a greater number of costal processes was
present. A similar explanation probably holds for the
“ proatlas ” of the Alligator.

1 Howes and Swinnerton did not see the posterior part of the basis cranii
in stage P,and by stage Q the evidence of coalescence of vertebra has disap-
peared. They consequently did not express any opinion as to the nature of
the “ proatlas.”

522 Fr. H. EDGEWORTH.

MUSCLES.

The premandibular segments are formed in Gallus by
hollow lateral outgrowths from the anterior end of the fore-
gut, which, a little later, is constricted off, forming a canal
connecting the vesicles (text-figs. 5 and 6, pp. 514,515). The
lumen in the canal disappears, and the resulting column of
cells atrophies. The vesicle, on either side, becomes a solid

, sphen pn gang of Vi.

LO
Kaze We 0
00%.

7} mand. my.up

NE
pty pr of quad
~ pal pter bar

, /
ent.cardv °
temp.émaen \X

pty en
hngman

15

ant. my/lo gen hyoid

Trxt-ric. 15.—Transverse section through an embryo of Gallus
at beginning of seventh day. (For explanation of lettering see
p- 555.)

clump by proliferation of its lining cells, and develops into
the eye-muscles innervated by the third nerve.

Tn the Lizard! and Duck * the premandibular segment is also
formed from the hypoblast, though in a slightly different way
—by solid lateral outgrowths from the dorsal wall of the
foregut, which are cut off, and develop a cavity extending
from side to side. In the Lizard the eye-muscles begin to

1 Corning (loc. cit.).
2 Rex (loc. cit.).

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 9523

develop from the walls of each lateral vesicle before its
cavity is obliterated.

The muscles developed from the premandibular segment
are exceedingly uniform throughout the Sauropsida—a rectus
superior, rectus inferior, rectus internus, and obliquus inferior.
In Birds and Crocodilia a levator palpebre superioris is also
developed from the rectus superior.

An orbicularis palpebrarum is developed in Birds, Spheno-

dep palp.mf\_-
pty profquad. -~~ ~cerhyalc
Me. ~ =. Bri
-\--------/-- cons coll;

Text-Fic. 16.—Sagittal section through an embryo of Gallus at end
of seventh day. (For explanation of lettering see p. 555).

don, Lacertilia vera, and Rhiptoglossa—probably from the
mesoblast of the mandibular segment.

The myotomes of the head in Gallus are developed from
the epithelium lining the dorsal cephalic ccelom on either side
of the forepart of the alimentary canal (text-figs. 1 and 2,
pp. 512, 513). The layers disappear at the sites of the gill-
pouches, but thicken in the intervening portions, and form the
somites of the head—the corresponding head cavities (dorsal
cephalic ccelom) disappearing (text-fig. 3, p. 513).

524. F. H. EDGEWORTH.

The cells forming the somites are at first undifferentiated,
then those lying centrally become converted into the myo-
tomes, whilst those lying peripherally become part of the
general mesoblast. The lower ends of the myotomes are
continuous with the epithelium lining the ventral cephalic
coelom (text-fig. 7, p. 515). In the case of the mandibular
and hyoid segments the lower ends of the myotomes become
continuous with the mylohyoids, which are formed from the
epithelium lining the obliterated portions of the ventral
cephalic ccelom in those segments (text-fig. 7, p. 515). The
lower ends of the first and second branchial segments separate
from the epithelium lining the ventral cephalic ccelom.

The formation of the myotomes of the head of Gallus is

thyr hy- lary.cart.

' "\ lary.
7. 06 ento. 168. ee ae

set

i Penthy ees if sternothy
hyp gl rect. \ Serprhy.
ant cons colli “cons.colli

Trxt-Fic. 17,—Sagittal section through an embryo of Gallus at end
of eighth day. (For explanation of lettering see p. 555.)

thus similar to that of Scyllium. ‘The difference is that the
head cavities in Gallus are not nearly so marked as in
Scyllium. In Gallus, too, some of the general mesoblast is
proliferated from the epithelium (from both splanchnic and
somatic layers) before the formation of the head somites
(text-fig. 1, p. 512).

In Gallus four myotomes are formed—those in the mandi-
bular, hyoid, first and second branchial segments (text-fig. 4,
p- 514) ; and there are similarly fourin Lacerta.'. In reference
to this point, it should be remarked that Corning, following
v. Wijhe’s views, described them as “ Seitenplatten,” i.e. as
visceral structures. As, however, I have shown that Balfour’s

1 Corning.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 525

view—that the muscle plates of the head are serially homo-
logous with those of the trunk and somaticeis probably the

18.

ie
-ant card v
ers fi

gs

1 S
S --~-\-_fyomand c
-~--1- dep mand

' gant mylo
cer hya/c

Al 7 sternohy.

post my/lo.

/
gen. hyord

TEx?-F1e@s. 18 and 19.—Sagittal sections through an embryo of
Chelone, in “stage 4” of Parker. 18 is the more external. (For
explanation of lettering see p. 555.)

true one, I shall assume that those of Lacerta are also
somatic. It will be seen later that the changes undergone

526 F. H. EDGEWORTH.

by the myotomes of Lacerta and Gallus are closely similar to
those of Scyllium.

Myotomeof the Mandibular Segment.—The superior
oblique muscle of Gallus is developed as a forward and
upward directed off-shoot of the upper end of the mandibular
myotome (text-fig. 8, p. 516), just as in Scyllium. It never
containsalumen. In these respects it resembles the superior
oblique of Lacerta (Corning). In some Birds—Anas, Larus
(v. Wijhe), it has a transitory lumen—possibly owing to a
relatively earlier formation.

20.

gen gloss.

yr.
post.my!o.

‘TExt-Fic. 20.—Sagittal section through an embryo of Chelone,
in “stage 4”? of Parker. (For explanation of lettering see p. 555.)

In Scyllium the mandibular myotome (after separation of
the Anlage of the superior oblique) forms a vertical strip
lying across and outside the horizontal palato-pterygoid
process of the quadrate. It divides into an upper part above
the palato-pterygoid process, and a lower between this and
Meckel’s cartilage—the upper part forming the levator
maxilla superioris, and the lower the adductor mandibule,
A portion, however, of the strip does not so divide, and forms
the first dorsal constrictor.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 527

In the Sauropsida the mandibular myotome (after separa-
tion of the Anlage of the superior oblique) grows upwards to

al. pty.m. R. sup. gang.ofy,.

| mand.my.up. -

palpterbar > \ -.@
Me. ‘ ' \ sternohy.
mendmyup. ' post mylo.
ply. profquad.

Trxt-rics. 21 and 22.—Sagittal sections through an embryo of
Alligator, slightly younger than “stage 2” of Parker. The
pterygoid muscle can be seen extending upwards outside the
pterygoid process (which has as yet no processus ascendens) of the
quadrate, and excluding the upper part of the mandibular myo-
tome from its insertion into the pterygoid process. 21 is the more
external. (For explanation of lettering see p. 555.)

vou. 51, PART 4,—NEW SERIES. 40

528 F. H. EDGEWORTH.

abut on,! or even reach a higher level than (text-fig. 32, p. 536),
the Gasserian ganglion, and similarly forms a strip lying across
and outside the pterygoid process of the quadrate (text-figs.
10, 18, 32, 36, pp. 518, 525, 536, 541), and then divides into
an upper and a lower part.

-AYV.
- ant.cardy.
--/g.s.

- Cep.mand.

mend.my.U.p.

Jass.g.

‘

_pty.profquad.
SAW:

\ 7: cer hyal.c.
een 29 -dep.mand.

at, epipter.

Trxt-FiGs. 23 and 24.—Sagittal sections through an embryo of
Sphenodon, in “stage P-Q”’ of Howes and Swinnerton. 23 is the
more external. (For explanation of lettering see p. 555.)

In Gallus, which preserves a movable pterygo-quadrate,
the upper part of the myotome, after separation of a strip to
form the depressor palpebree inferioris (text-fig. 16, p, 523),

1 First described by Corning in Lacerta.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 529

develops into the orbito-quadratus—an elevator of the ptery-
goid process of the quadrate (text-fig. 16, p. 523)—though in
later periods of development the insertion shifts in part to the

x.
~_ \_ cerhyalc.
-24reorar

Me. | post.mylo. : SS vertv.
25 ent.mylo. sternohy.
“i parach.
eth.sph pl. \ érab. otosph.p/. I
‘ F /

'
\
i}
‘
1
'
'
1
'
‘
!
i)
'
f
‘
1

_-CoSt. prac

- hyog/oss.
-- cer.hyal.c.

=== Bri.
PB RE

-- 49.5

Trxt-Flas. 25 and 26.—Sagittal sections through an embryo of
Sphenodon, in “stage P-Q”’ of Howes and Swinnerton. 25 is the
more external. (For explanation of lettering see p. 555.)

body of the quadrate, and in part to the hind end of the
pterygoid bone.

In Reptiles the upper part of the myotome (after separa-

530 F. H. EDGEWORTH.

tion of a strip to form the depressor palpebre inferioris in
Sphenodon,! Agama, Chelone, and Alligator) either forms a
muscle tying the pterygoid process to the side of the skull, or
is inserted into the (membranous) palato-pterygoid bar or
atrophies. An insertion into a membrane bone must be a
secondary feature. Reptiles must therefore be descended
from forms which, like Selachians and Birds, had a movable
pterygo-quadrate. Birds preserved this feature, and with it
the primitive insertion of the upper part of the mandibular
myotome, whereas in Reptiles the pterygo-quadrate became

Sa
pty.profguad.-
V3 --- --|

Me.--\-~

phar

pty.an.

TExt-FiG. 27.—Transverse section through an embryo of Sphe-
nodon, in “stage P”’ of Howes and Swinnerton. The upper part
of the section is more anterior than the lower. (For explanation
of the lettering see p. 555.)

fixed, and the upper part of the myotome can be seen, during
development, undergoing secondary modifications.

In Alligator (text-figs, 21,22, p. 527) and Chelone the upper
part of the myotome atrophies without ever having had an
insertion into the palato-pterygoid bar; the pterygoid process,
either in continuity with the quadrate (Alligator) or after

1 The depressor palpebre inferioris of Sphenodon is inserted into the

reflected angle of the conjunctival sac (text-fig. 29, p. 532), that of Chelone
and Alligator into both lids.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 531

“separation from it (Chelone), becomes a part of the skull,
and the quadrate becomes immovably fixed.

In Sphenodon the upper part of the myotome is at first an
elevator of the pterygoid process (text-fig. 24, p. 528) ; it sub-
sequently loses this function on fixation of the pterygo-quad-
rate, and ties its pterygoid and epipterygoid processes to the
side of the skull, with an insertion of a very few of its lowest

epipter. - retract oc
emp
otosph pi. - a
_- pty. exe
Tie
PI es
cr ;
Z — pty.ine
Me Sa © 4
Ea SEED >.
\o-= H S Ne x SK >
cer hya/ c. \

2 “Ayo gloss.
hb é yo gl

6 P gen Ayord

‘omohyoid

‘ant.my/o

trech
TExt-Fia. 28.—Transverse section through an embryo of Sphe-
nodon, in “stage R”’ of Howes and Swinnerton. (For explana-
tion of lettering see p. 555.)
fibres into the fixed palato-pterygoid bar (text-figs, 28 and
29, pp. 531, 532)—forming the spheno-pterygo-quadratus of
Firbringer.

In Agama the upper part of the myotome, after a temporary
insertion into the pterygoid process (text-fig. 30, p. 534),
becomes inserted into the pterygoid bone forming the ptery go-
parietalis and protractor pterygoidei! muscles. In some

1 Of Versluys.

5382 F. H. EDGEWORTH.

Lizards, e.g. Hemidactylus, Gephyra, Varanus, some of the
fibres are inserted into the quadrate (Fiirbringer).

In Chameleon there is no depressor palpebre inferioris
developed, and the whole of the upper part of the myotome,
after a temporary insertion into the pterygoid process, be-
comes divided into the pterygo-sphenoidalis anterior, pterygo-
parietalis,! and protractor pterygoidei;' of these the first and
third arise from the processus anterior inferior and adjacent
parts of the otic capsule; the second from the parietal bone.

In Tropidonotus there is also no depressor palpebre infe-

29.
retract oc “i is otosp/.pl.
j , hen.pterquad. /
alp ink \ Rexe. sp. yi }
ce as aus : / epiptor. V.
Ee Ne eS i

5 Z ,

cons sac. :
SS
Sw

ANI Wh)

FEZ. - omohyoid

Ya eee post. my/o.

/ ptyext. S g ~ :
cerhyalc. hyo.gloss.. Bri. epist.hyotd.
gen. hyord

Trxt-Fic. 29.—Sagittal section through an embryo of Sphen-
odon, in ‘stage S” of Howes and Swinnerton. (For explana-
tion of lettering see p. 555.)

rioris, and the upper part of the myotome, after a temporary
insertion into the pterygoid process (text-fig. 37, p. 543),
becomes divided into the vomero-sphenoideus, pterygo-
sphenoidalis anterior, pterygo-parietalis, and pterygo-
sphenoidalis posterior.

In the Lacertilia vera and Ophidia the quadrate—after
separation or atrophy of its pterygoid process—is movable ;

1 Of Versluys. Bradley states that these muscles are absent in the adult.
The condition in my embryos (one of which is fairly advanced in develop-
ment) supports the opinion of Versluys; they also show that a pterygo-
sphenoidalis anterior is present.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 533

in Chameleon it is fixed. It is probable that the movability
of the quadrate in the two former groups is a secondary
phenomenon and not a primitive Sauropsidan condition.
According to Broom it was initiated by loss of the quadrato-
jugal bone.

The formation of the processus ascendens (or epipterygoid)
from the pterygoid process in Crocodilia, Rhyncocephalia,
and Lacertilia was probably a development subsequent to
fixation of the pterygo-quadrate. Its development is inti-
mately associated in the Crocodilia with the upgrowth of the
pterygoid muscle; in the Rhyncocephalia and Lacertilia
vera with the upgrowth of the external pterygoid muscle. It
is not developed in Birds, Chelonia, or Ophidia. The ‘ epi-
pterygoid” of Chelonia is merely the anterior end of the
pterygoid process; on the other hand, in Rhiptoglossa the
anterior end of the pterygoid process, which also becomes a
part of the cranial wall, contains the basal part of a processus
ascendens (Broom).

Fiirbringer, from an analysis of the adult anatomy, homo-
logised the levator maxille superioris of Selachians with
the spheno-pterygo-quadratus of Lacertilia and the orbito-
quadratus of Birds, and deduced the theory that the monimo-
stylic (with fixed quadrate) condition of Crocodilia and
Chelonia is a secondary one, and that the streptostylic (with
movable quadrate) condition found in Lacertilia, Ophidia,
and Birds is the primary one. The theory sketched above—
based on comparison of developmental and adult features—
separates the streptostylic condition into two forms: a primi-
tive streptostylic pterygo-quadrate (Birds), and a secondary
streptostylic quadrate (Lacertilia vera and Ophidia). The
original Sauropsidan stock thus probably possessed a mov-
able pterygo-quadrate and a fixed (membranous) palato-
pterygoid bar; the former has been retained only by Birds,
the latter only by Chelonia, Crocodilia, and Rhyncocephalia.

The lower part of the mandibular myotome in Gallus passes
through three stages of development. At first, lke the
adductor mandibule of Scyllium, it forms a single muscle

534 F. H. EDGEWORTH.

passing from the pterygoid process to the lower jaw; it then
divides into an inner and outer part—pterygoid and temporal

A Bees Y | epipter. <>. cerhyalc
E. retract oc,’ ‘pty profgused. pty.imt ~ Ne.
mand my up

cer. Ayal.c
4
&r max

Brl
"4

a.
_. s6raoran

A yo gloss
an

jie” Me.
ae ant mylo.
gen gloss.

\
gen hyoid. \ sternohy.
post my/lo

Text-Fias. 30 and 31.—Sagittal sections through an embryo of
Agama. 30 is the more external. (For explanation of lettering
see p. 555.)

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 539

(text-fig. 15, p. 522). The outer part divides into the
quadrato-maxillaris which keeps its primitive origin from the
pterygoid process, and the temporal which grows up to gain
an origin from the skull. The inner part divides into the
external pterygoid which keeps its pterygoid-quadrate origin
(text-fig. 16, p. 523), and the internal pterygoid which arises
from the hind end of the palato-pterygoid bone (text-fig. 16,
p- 528).

The lower part of the mandibular myotome in Reptiles
similarly divides into inner and outer portions—pterygoid
and temporal (text-fig. 27, p. 530). The development of the
outer portion is much the same as in Birds. It is at first a
muscle passing from the pterygoid process to the lower jaw
(text-fig. 24, p. 528); subsequently, whilst retaining an
attachment to the quadrate, it also extends up to the side of
the skull. The part, however, which retains its original
upper attachment is not differentiated as a separate muscle
(quadrato-maxillaris) lying internal to the temporal as in
Birds. In Chameleon the development is a little excep-
tional: the outer portion separates into a temporal (which
becomes digastric in condition) and a quadrato-mandibularis
which arises from the front of the quadrate (text-fig. 35,
p. 539) ; the position of the latter muscle, below and not inter-
nal to the temporal, suggests that it is not homologous with
the quadrato-maxillaris of Birds, but is merely the lower part
of the temporal—that part which does not become digastric.

The development of the internal, pterygoid portion is very
diverse in the various groups of the Reptilia. The simplest
condition is present in Chelone, where there is during
development a single undivided pterygoid muscle with two
heads, one (inner) attached to the hind end of the palato-
pterygoid bar, and the other (outer) attached to the pterygoid
process of the quadrate. ‘These two heads extend upwards,
the former gaining an additional origin from the descending
plate of the parietal, the latter an additional one from the
prootic. The muscle does not become divided into two bellies.

In Sphenodon and Agama the pterygoid Anlage becomes

536 F. H. EDGEWORTH.

divided into an internal pterygoid muscle arising from the
palato-pterygoid bar (text-fig. 28, p. 531), and an external

mand my.

_- hyomand c.
-4---/9-s8.
Fores a = cerhyalc.

-}+ --post mylo.

Text-Fias. 32 and 33.—Sagittal sections through an embryo of
Chameleon. 32 isthe more external. (For explanation of lettering
see p. 555.)

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 537

pterygoid arising from the pterygoid process of the quadrate ;
subsequently, the upper end of the external pterygoid muscle
extends upwards outside the pterygoid process, and gains
attachments to the epipterygoid and side of the skull (text-
figs. 28 and 29, pp. 531, 532).

In Chameleon and Tropidonotus the pterygoid Anlage
remains single, it loses its attachment to the pterygoid pro-

3.

proc.ent.iof.
UX gea.gloss.

R\ Ayog/oss.

Ma cer hyalc.
y Srl.

lary.

-----443 b6r.eorar
Sses ~xu

ent.mylo. |”

gen hyoid ne
post mylo 166 gor ar.

Trxt-Fia. 34.—Sagittal section through an embryo of Chame-
leon. (For explanation of lettering see p. 555.)

cess and takes origin from the palato-pterygoid bar (text-
fies. 33 and 37, pp. 536 and 5498).

In Alligator the pterygoid Anlage also remains single, it
erows up outside the pterygoid process to the side of the skull
(text-figs. 21 and 22, p. 527).

It may be concluded that the fixation of the palato-quadrate
in the Crocodilia was intimately associated with the upgrowth
of the pterygoid muscle, and in the Rhyncocephalia and

538 Fr. H. EDGEWORTH.

Lacertilia vera with the upgrowth of the external ptery-
goid muscle, to the side of the skull. Investigation of the
process of development of the head-muscles did not throw
any light on the causation of the fixation of the pterygo-
quadrate in Chelonia, on the fixation of the quadrate in
Rhiptoglossa, or its movability in Ophidia.

The above differences, however, in the condition of the
upper part of the mandibular myotome and of the pterygoid,
suggest that fixation of the pterygo-quadrate was brought
about by different processes in, and so was independently
acquired by, ancestors of (1) Chelonia, (2) Crocodilia,
(3) Rhyncocephalia and Lacertilia vera, (4) Rhiptoglossa
and Ophidia.

Myotome of the Hyoid Segment.—The abducens
musculature of Gallus originates as a small clump of cells
which grows forward and then separates from the upper end
of the hyoid myotome. It passes forwards internal to the
Gasserian ganglion (text-fig. 4, p. 514), and divides into an
anterior and a posterior mass (text-figs. 10 and 14, pp. 518,
521); theanterior is the Anlage of the pyramidalis and quad-
ratus nictitantis muscles, the posterior that of the external
rectus. In Reptiles the abducens Anlage similarly divides into
an anterior and a posterior mass ; the former (except in Tropi-
donotus, where it atrophies) develops into the retractor oculi,}
the latter into the external rectus (text-figs. 22, i 30, pp.
527, 532, 534).

The myotome of the hyoid segment, after giving off the
abducens Anlage, develops into the depressor mandibule.
The primary insertion of this muscle is into the ceratohyal
(text-figs. 12, 18, 24, 30, and 32, pp. 519, 525, 528, 534,
536), but this is soon lost, and one into the hind end of the
lower jaw acquired. In Gallus (text-fig. 14, p. 521), Alligator,
and some Lizards (Parker), a stapedius muscle is also formed

1 The retractor oculiis greatly developed in Sphenodon, its origin extend-
ing backwards (text-fig. 28, p. 531). Its insertion varies, and accordingly

Huxley calls it “pyramidalis” in Chelonia and Crocodilia, “ bursalis’’ in
Lizards.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 539

from the upper part of the myotome; this may be regarded
as that portion of the hyoid myotome which retains its primi-
tive insertion, whereas the main mass has gained a secondary
one. The upper end of the depressor mandibule is attached
to the postero-lateral aspect of the skull; in Chameleon
and Tropidonotus (text-fig. 35, p. 539) it has additionally an
origin from the quadrate.

Myotome of the First Branchial Segment.—The
upper part of the first branchial myotome of Gallus dis-
appears, whilst the lower part develops into the branchio-
maxillaris muscle which extends forwards from the first
branchial bar to the lower jaw (text-fig. 16, p. 523).

¢ - 4 '
uo famx.an. |
RB exe. orb palp

Text-FiG. 35.—Sagittal section through an older embryo of
Chameleon. (For explanation of lettering see p. 555.)

The muscle is present in all the groups of the Sauropsida.
Its primary condition is that of a muscle—the branchio-hyoid
—which connects the first branchial bar with the ceratohyal
(text-figs. 25 and 31, pp. 529, 534). This condition is the per-
manent one in Sphenodon, and is found as a temporary one in
Gallus, Chelone, Agama, Chameeleon, and 'Tropidonotus. (‘The
embryo of the Alligator was too far advanced in development
for this stage to be seen.) In all these groups the muscle
subsequently extends forwards to the lower jaw. ‘This exten-
sion forwards is not dependent on loss of the continuity of the

540 F. H. EDGEWORTH.

ceratohyal, for it occurs in Chameleon before this takes place.
Probably it is related to the extension backwards of the jaw
into the hyoidsegment. In Chelonia the anterior attachment
of the branchio-maxillaris muscle is to the hind end of the
lower Jaw, in the other groups the muscle extends still further
forwards—in Rhiptoglossa even as far as the symphysis.

The branchio-hyoid or branchio-maxillaris muscle of
Sauropsida is homologous with the most anterior of the
coraco-branchiales muscles of Scyllium.

Myotome of the Second Branchial Segment.—A
myotome is developed in the second branchial segment in
Gallus (text-fig. 4, p. 514), but disappears leaving no trace.
In Lacerta, too, a second branchial myotome is described by
Corning, but there are no muscles present in the embryos of
Agama investigated or in adult Lacertilia which might have
developed from the myotome. And in the other Reptilian
embryos the stages were likewise too far advanced for any
rudiments of this myotome to be seen. In the Sauropsida,
then, a second branchial myotome, if formed, soon disappears .
and does not develop into any muscle.

Its existence, however, though only as a temporary structure
in Gallus and Lacerta, coupled with that of a second branchial
bar in Chelonia, Rhyncocephalia, and Lacertilia vera, sug-
gests that ancestors of Sauropsida may have possessed a
longitudinal muscle connecting the second to the first
branchial bar.

Cephalic Section of the Celom and Mylohyoid
Muscles.—There are certain similarities and differences
between Scyllium and Gallus in the relations of the ventral
cephalic ccelom to the heart and visceral muscles of the mandi-
bular and hyoidsegments. In early stages of Scyllium there
is, in the mandibular and succeeding segments of the head,
a ventral section of the ccelom which bears the same relation
to the ventral aorta as does the anterior portion of the body
section of the ccelom to the truncus arteriosus and the heart.
Subsequently, the developing heart bulges into the hinder
portion of the cephalic ccelom. Still later, the heart and

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 541

ccelom gradually retreat from the head. The mandibular and
hyoid mylohyoid muscles are formed from the obliterated
sections of the ventral cephalic ccelom in those segments.

In early stages of Gallus the cephalic section of the ccelom
similarly exists in the mandibular and succeeding segments
(text-fig. 3, p. 513), but its relations to the vascular system are
alittle different. The heart is situated more anteriorly, lying
in part in the cephalic and in part in the body portion of the
coelom; the truncus arteriosus is in the first branchial seg-
ment, and there is a very short ventral aorta between the
origins of the hyoid and mandibular aortic arches, the three

Trxt-ric. 36.—Sagittal section through an embryo of Tropido-
notus. (For explanation of lettering see p. 555.)

branchial aortic arches being subsequently given off from the
posterior end of the aorta. A little later the ventral aorta,
mandibular, and hyoid, aortic arches disappear, the ventral end
of the hyoid aortic arch being left as the ventral carotid
artery. The mylohyoids are formed, as in Scyllium, from
the walls of the obliterated portions of the ventral cephalic
ccelom in the mandibular and hyoid segments. The subsequent
retreat of the heart and ccelom from the head are also exactly
parallel events to those occurring in Scyllium. The condition
of the heart, truncus arteriosus, and ventral aorta in the early
embryo of Gallus is evidently a secondary one.

542 F. H. EDGEWORTH.

The anterior or mandibular mylohyoid of Sauropsida pre-
serves the form existing in Scyllium—a transverse sheet of
muscle passing from one Meckel’s cartilage to the other
(text-figs. 15 and 28, pp. 522, 531).

The posterior or hyoid mylohyoid in Scyllium forms a
transverse band passing from one ceratohyal to the other.
The primary attachment of the muscle in Sauropsida is also
to the ceratohyal, but this is lost in all groups with the possible
exception of Sphenodon.! It gains, m all Sauropsida, a
secondary attachment to the lower jaw, becoming more or
less continuous with the anterior mylohyoid, and spreads
down the neck forming the constrictor colli. This posterior
part, which is attached laterally to the fasciz of the neck (in
Chelonia also to the first branchial bar, in Rhiptoglossa also
to the crista occipitalis) is continuous with the anterior part.

In Gallus secondary changes take place in the posterior
mylohyoid. After a transitory stage of attachment to the
ceratohyal (text-fig. 12, p. 519) it becomes attached laterally
to the hind end of the lower jaw (text-fig. 13, p. 520) and first
branchial bar, and spreads down the neck. ‘lhe portion
attached to the first branchial bar becomes a separate muscle
—the anterior constrictor colli2—forming a muscular sling for
the second basibranchial (text-fig. 17, p. 524), whilst that in
front forms the serpihyoid and stylohyoid, that behind the
constrictor colli. ‘'he part attached to the lower jaw in the
Lamellicostren, however, forms a simple transverse band
(Gadow). ‘The differentiation of an anterior constrictor colli
is evidently related to the existence of a second basibranchial—
it is absent in Reptiles, and in those Birds, e.g. Rhea (Gadow),
where a second basibranchial is absent, and even when this
is present is not always developed.

Laryngeal and Syringeal Muscles.—The Anlage of
the laryngeal muscles in Gallus is developed in the splanch-

1 It was present up to stage S (of Howes and Swinnerton), but is not
mentioned by Osawa.

2'The “ceratohyoid” of Gadow, who included it among the lingual

muscles as a differentiated part of the ceratoglossus.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 543

nic mesoblast of the second branchial segment on the
sixth day. A day later each lateral mass has divided into
an apertor and its lateral half of the sphincter laryngis.

The laryngeal muscles of Sphenodon, Agama, Chelone,

gass.g.
»~ maend,my.u p
R sup HAS ~~ | V4 e> 3 oe
/ / / ve : ‘ 1 A “ > Rs 7 ;
| a ae y) oS yf ee

Rink - \
Oink i =

hyold.c.

SAM a cae iN

|

pty pr of quad. palpterbar pty.m. He

_ gen. gloss,

en _ Ayogloss.

|- Brel,

Fy gen.hyord ‘
ant mylo. post. mylo.

Trxt-Fias. 37 and 38.—Sagittal sections through an older embryo
of Tropidonotus. (For explanation of lettering see p. 555.)

Alligator, Chameleon, and Tropidonotus are formed simi-
larly from an Anlage in the second branchial segment.

The syringeal muscles of Gallus are formed from meso-
blast cells which have spread down the trachea, and their
Anlage is visible on the eighth day.

VoL. 51, pART 4.—NEW SERIES. A1

544, F. H. EDGEWORTH.

The Ventral Longitudinal Muscles are developed in
Scyllium from the five trunk-myotomes (4th to 8th), in Gallus
from the first five (text-fig. 9, p. 517), though the front part
of the first (cf. text-fig. 2, p. 513) has by then disappeared ; in
Lacerta (Corning) from the second to the fifth, the first having
previously atrophied. Ventral downgrowths take place from
each contributing myotome in the form of a curve, with
concavity forwards, round the branchial region of the head.
The ventral ends of these downgrowths fuse together, sepa-
rate from the rest of the myotomes, and form an elongated

#.sup. trab.

eri dy
'
{}

! 1 \ . . *
1 gengloss.\ mylo. es
Pe. genhyod trach

Trxt-Fic. 39.—Sagittal section through an older embryo of
Tropidonotus. (For explanation of lettering see p. 555.)

mass of cells in the ventral region of the branchial segments
dorsal to the cephalic portion of the ccelom. ‘The mass
extends forwards and backwards to form the Anlage of the
ventral longitudinal muscles of the head and neck.

In Scyllium it forms two parallel muscles—the coraco-
hyoideus and coraco-mandibularis. In the Sauropsida the
long column divides into an anterior and a posterior part—
the genio-hyoid and the sterno-hyoid. ‘The division takes
place opposite the first branchial bar, and the primary condi-
tion of the genio-hyoid is that of a muscle passing from the
anterior end of Meckel’s cartilage to the ventral end of the

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 945

first branchial bar, whilst the sterno-hyoid extends from this
down the neck to the sternum (text-figs. 19, 26, 31, 34, 38,
pp. 525, 529, 534, 537, 543).

This condition of the genio-hyoid persists in the Chelonia,
Sphenodon, Lacertilia vera, Rhiptoglossa, and Ophidia.
In the Alligator the muscle, after passing through this
stage, becomes connected by tendon with the anterior end of
one of the muscles into which the sterno-hyoid divides. In
Gallus the primary stage—of insertion of the genio-hyoid
into the first branchial bar—is rapidly passed through, and
the hind end becomes inserted into the under surface of the
first basibranchial (text-fig. 17, p. 524) ; later on the whole
muscle atrophies and disappears.

The sterno-hyoid divides in all Reptiles, except Ophidia,
into several parallel strips, some of which retain their original
attachment to the first branchial bar, whilst others become
inserted into the basihyobranchial. In the Alligator one of
the strips becomes connected with the genio-hyoid by tendon.

In Birds the sterno-hyoid, which does not become divided
into parallel strips, gains a new insertion into the dorsal sur-
face of the first basibranchial (text-fig. 17, p. 524)); and
in most Birds, though not in Apteryx and some others, it
divides into anterior and posterior portions, e. g.in Gallus into
sterno-thyroid and thyreo-hyoid (text-fig. 17, p. 524).

The lingual muscles of Reptiles are developed from the
genio-hyoid, and consist of a genio-glossus and hyo-glossus
(text-figs. 19, 20, 26, 31, 38, pp. 525, 526, 529, 534, 543).
The former is attached in front to the anterior end of
Meckel’s cartilage, and the latter behind to the posterior
branchial bar—just as is the muscle from which they
originate.

The genio-glossus is attached posteriorly to the front
end of the basihyobranchial in the embryos of Chelone (text-
fig. 20, p. 526), Alligator, and Sphenodon; this condition
persists in the two former, but in Sphenodon the muscle
subsequently also spreads into the tongue. ‘The muscle ends
free in the tongue in Agama, Chameleon, and Tropidonotus

546 F. H. EDGEWORTH.

(text-fig. 38, p. 543); in the last named a secondary protru-
sible tongue is subsequently formed (text-fig. 39, p, 544), and
the genio-glossus becomes its protractor, and also gives rise
to a genio-laryngeus.

The hyo-glossus is inserted into the side of the side of
the basihyobranchial in Chelone (text-fig. 19, p. 525), and
Alligator; it grows forward and ends free in the tongue in
Sphenodon (text-fig. 29, p. 532), Agama (text-fig. 31,
p- 534), and in early stages of Chameleon (text-fig. 34,
p- 537). In later stages of Chameleon the anterior part of
the muscle disappears (possibly developing into some of the
many intrinsic lingual fibres), whilst the hinder part forms
a muscle passing from the first branchial bar to the ventral
end of the ceratohyal cornu. In Tropidonotus the hyo-
elossus becomes a retractor of the protrusible tongue, and
also gives off a hyo-laryngeus.

The lingual muscles of Gallus are developed from an
Anlage which is homologous with the hyo-glossus of Reptiles
(text-figs. 13, 15, pp. 520, 522) ;? it at first extends from
the first branchial bar to the first basibranchial; it subse-
quently grows forwards and then divides into the cerato-
elossus, and hypoglossus rectus and obliquus. This dif-
ferentiation of the hyo-glossus is probably related to the
fact that the median hyobranchial cartilages in Birds, unlike
Reptiles, become jointed.

In some Birds, e. g. Procellaria,® though not in Gallus even
as a temporary structure, there is also a genioglossus passing
from the anterior end of the jaw to the os entoglossum.

The lingual muscles of the Sauropsida are thus somatic in
origin—arising from the anterior element of the ventral

1 The resultant muscle looks a little like the branchiohyoid of Sphenodon,
and the same name, “ Ceratohyoid,’”’ has been given to it, but it is of quite
different origin.

2 The Anlage of the lingual muscles of Gallus begins to be formed before
the genio-hyoid is quite separated from the sterno-hyoid.

3 The posterior insertion of this muscle—into the tip of the os ento-
glossum and not into the ventral surface of the first basibranchial—shows
that it is probably not a persisting genio-hyoid.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 547

longitudinal muscles; it would seem probable that their
original function was to connect the basihyobranchial with
the lower jaw and first branchial bar, and that their extension
into a tongue was a secondary matter. Only by this suppo-
sition do the phenomena occurring in Chelone, Alligator,
Sphenodon, Gallus, and Procellaria become explicable.

Some ‘THEORETICAL CONCLUSIONS.

The above observations suggest that some remote ancestor
of the Sauropsida possessed the following features :—A mov-
able pterygo-quadrate, fixed palate and pterygoid bones, a
ceratohyal which formed a continuous bar extending from
the hyomandibular above to a median unjointed basihyo-
branchial below, first and second branchial bars. The eye-
muscles consisted of four recti, two obliqui, and the retractor
oculi. The mandibular myotome had divided into an upper
and a lower part; the upper formed the depressor palpebrez
inferioris and an elevator of the pterygoid process of the
quadrate ; the lower consisted of an inner and an outer divi-
sion, the inner forming the pterygoid muscle which arose by
two heads—from the pterygoid bone and from the pterygoid
process of the quadrate, the outer forming the temporal
muscle, which arose partly from the quadrate and partly from
the skull wall above. ‘The hyoid myotome formed the
depressor mandibule. ‘he first branchial myotome formed
the branchiohyoid muscle passing from the first branchial
bar to the ceratohyal. The ventral longitudinal muscles
were formed by a genio-hyoid and a sterno-hyoid, whose
adjacent ends were attached to the first branchial bar. ‘he
lingual muscles—developed from the genio-hyoid—were the
genio-glossus and the hyo-glossus, the former passed from the
anterior end of the lower jaw to the anterior end of the basi-
hyobranchial, the latter from the first branchial bar to the
side of the basihyobranchial ; neither ended free in the tongue.
The anterior and posterior mylohyoids formed a continuous
sheet and extended down the neck.

548 . F. H. EDGEWORTH.

No one of the Sauropsida has preserved all these features,
but in each group they exist (though sometimes masked) in
embryonic stages, and the various changes which take place
and bring about the characteristics of each can be followed
in the subsequent development.

Birds differ from all living Reptiles in possessing the
following features. The pterygo-quadrate is movable; there
is a joint between the basihyal and the first basibranchial,
and a second basibranchial is generally present ; the retractor
oculi divides into pyramidalis and quadratus nictitantis; the
upper portion of the mandibular myotome, after giving off
the depressor palpebre inferioris, forms an elevator or eleva-
tors of the pterygoid process of the quadrate; a distinct
quadrato-maxillaris is formed; the genio-hyoid, after having
had a temporary insertion into the first basibranchial,
atrophies; the sterno-hyoid becomes, secondarily, inserted
into the dorsal surface of the first basibranchial, and in most
Birds divides into anterior and posterior portions ; the hyo-
glossus divides into several muscles ; a genio-glossus is seldom
developed ; a portion of the posterior mylohyoid generally
becomes a separate muscle—the anterior constrictor colli—
forming a muscular sling for the second basibranchial; in
most Carinate syringeal muscles are formed.

Birds resemble the Rhyncocephalia in possessing an upper
portion of the mandibular myotome inserted into the ptery-
goid process, but the adult condition in the latter group is
clearly a secondary modification correlated with a fixation of
the pterygo-quadrate.

The pterygoid process of the quadrate of Birds resembles
that of Chelonia in possessing no processus ascendens such
as exists in Crocodilia, Rhyncocephalia, Lacertilia vera, and
Rhiptoglossa, and such a structure (to which no muscles
are attached in early stages of development) does not seem
to be a part of a freely movable pterygo-quadrate, but rather
a formation occurring concurrently with or subsequently to its
fixation,

The condition of the pterygoid muscles in Birds might easily

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 549

be derived from that seen in the embryo of Chelone (i.e. a
single muscle with two heads, one from the pterygoid process
of the quadrate, and one from the hind end of the palato-
pterygoid bar), and which undergoes very little modification
in subsequent development, but not from that found in
Rhyncocephalia and Lacertilia vera (where the external
pterygoid muscle grows up outside the pterygoid process to
the side of the skull), nor from that found in Crocodilia
(where an undivided pterygoid muscle grows up outside the
pterygoid process to the side of the skull.)

The lingual muscles of Birds, in that they are only deriva-
ble from a genio-glossus and hyoglossus, which were attached
to the basihyobranchial and did not end free in the tongue,
are more like those of Chelonia and Crocodilia than any other
Reptilian group.

These features of resemblance suggest at first sight a very
distant Chelonian relationship for Birds, but are in reality
only ancestral traits, which are also present in embryonic
stages of other Sauropsidan groups. ‘The secondary fixation
of the pterygo-quadrate and atrophy of the elevator of the
pterygoid process, which occur in Chelonia, are strongly
marked differences from Birds.

The Crocodilia resemble the Chelonia in the atrophy of the
upper part of the mandibular myotome and in the fixation of
the pterygo-quadrate, but these features have come about
in different ways in the two groups. They also resemble one
another in the condition of the lingual muscles.

The Rhyncocephalia have preserved two features more
archaic than are found in any other Sauropsidan group—the
continuity of the ceratohyal, and the condition of the branchio-
hyoid muscle—but in the upgrowth of the external pterygoid
muscle and the condition of the lingual muscles are less
primitive than the Chelonia. Like the Chelonia and Croco-
dilia, they have preserved a fixed pterygoid bone.

The Lacertilia vera present great resemblances to the
Rhyncocephalia in the condition of the external pterygoid
muscle and the lingual muscles. The chief differences are

550 F. H. EDGEWORTH.

that in the former the greater portion of the pterygoid
process disappears, the quadrate becoming secondarily mov-
able, the continuity of the ceratohyal is lost, and the bran-
chio-hyoid muscle also extends forwards to the lower jaw.
Further, whereas in the Rhyncocephalia the upper portion
of the mandibular myotome is inserted into the pterygoid
process with a very few fibres to the (fixed) pterygoid bone,
in the Lacertilia it is inserted into the (movable) pterygoid
bone, with a few fibres in some Lizards to the quadrate.

The Rhiptoglossa and Ophidia present the Lacertilian
features of a movable pterygoid bone into which is inserted
the upper part of the mandibular myotome and (in embryonic
stages) lingual muscles ending free in the tongue. ‘Though
they differ markedly from one another in the adult form of
the tongue and in the associated condition of the hyobran-
chial visceral cartilages and lingual muscles, yet they possess
in common the following features which are not found in any
other groups—the non-development of a depressor palpebre
inferioris, an undivided pterygoid muscle arising from the
pterygoid bone, the partial origin of the depressor mandibule
from the quadrate, and the secondary position of the cerato-
hyal internal to the quadrate.

In conclusion, I have the pleasure of thanking Prof. W. N.
Parker for the loan of embryos of Chelone viridis, Prof.
F. S. Clarke for sections of Alligator mississipensis,
Prof. Moore for an embryo of Sphenodon, Mrs. Howes for
sections of Sphenodon, and Prof. Broom for embryos of
Agama and Chameleon. My thanks are also due to my
colleagues, Profs. Fawcett and Kent, for innumerable kind-
nesses.

REFERENCES.

BrapLEy.— The Muscles of Mastication and the Movements of the Skull
in Lacertilia,”’ ‘Zool. Jahrb., Anat. und Ontog.,’ Bd. xviii, 1903.
Broom.—“On the Development of the Pterygo-quadrate Arch in the

Lacertilia,” ‘Journ. of Anat. and Phys.,’ vol. xxxvii, 1903.
Cornina.— Ueber die Entwicklung der Kopf- und Extremititen-musku-
latur bei Reptilien,” ‘ Morph. Jahrb.,’ 1900.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 5051

EpcEwortu.—* The Development of the Head Muscles in Scyllium
canicula,” ‘Journ. of Anat. and Phys.,’ vol. xxxvii, 1903.

Fiirprineer, M.—‘* Zur vergl. Anat. Brustschulterapp. und Schultermusk,”
‘Jen. Zeitschr. Naturw.,’? Bd. xxxiv, 1900.

— “Zur Frage der Abstammung der Siugethiere,”” ‘ Haeckel’s Fest-
schrift.’

Frorrep.—“ Zur Entwicklungsgeschichte der Wirbelsaule insbesondere des

Atlas und Epistropheus und der Occipitalregion,”’ ‘ Archiv f. Anat.
and Phys.,’ 1883.

Gapow.—‘‘ Aves,” in ‘ Bronn’s Thierreich.’

Gaupp.—’ Die Entwicklung des Kopfskeletts,’ in ‘ Hertwig’s Handbuch
der vergl. und exper. Entwickelungslehre der Wirbelthiere.’

Huxtry.—‘‘ The Anatomy of Vertebrate Animals.”

Horrman.—‘ Reptilia,” in ‘ Bronn’s Thierreich.’

Howes AND SwINNERTON.—“ On the Development of the Skeleton of the
Tuatera, Sphenodon punctatus,’ ‘Trans. Zool. Soc.,’ vol. xvi,

1900.
KinestEy.—“ The Ossicula Auditus,” ‘Tuft’s College Studies,’ No. 6,
1900.

KaczanpDER.—“ Beitrage zur Entwicklungsgeschichte der Kaumuskulatur,”’
‘Mittheilungen aus dem embryol. Institut der k. k. Universitat
Wien.,’ 1883.

Osawa.— “ Beitraige zur Anatomie der Hatteria punctata,” ‘Archiv f.
mikroskop. Anat.,’ vol. li, 1898.

Parker, W. K.—“ On the Structure and Development of the Common Fowl
(Gallus domesticus),” ‘ Phil. Trans.,’ 1869.

—— “Report on the Development of the Green Turtle (Chelone
viridis),” ‘Challenger Reports,’ Zoology, vol. i.

—— “On the Structure and Development of the Skull in the Crocodilia,”’
‘Trans. Zool. Soc.,’ vol. xi, 1883.

— “On the Structure and Development of the Skull in the Lacertilia:
Pt. I. Lacerta agilis, L. viridis, and Zootoca vivipara,”
‘Phil. Trans.,’ 1879.

— “On the Structure and Development of the Skull in the Common
Snake (Tropidonotus natrix),” ‘ Phil. Trans.,’ 1878.

Rex.—‘‘ Ueber die Mesoderm des Vorderkopfes der Ente,” ‘Archiv f.
mikr. Anatomie,’ Bd. ], 1887.

SmirH, GEOFFREY.—‘ The Middle Ear and Columella of Birds,” ‘ Quart.
Journ. Micr. Sci.,’ vol. 48, pt. 1. 1904.

SuscHxin.—“ Zur Morphologie des Vogelskeletts. I. Schadel von Tinnun-

552 Ff. H. EDGEWORTH.

culus,” ‘Nouv. Mém. Soc. Impér. des Natural. de Moscou,’ T. xvi,
L. 2, 1899.
Verstuys.—‘ Mittlere und aussere Ohrsphiire der Lacertilia u. Rhyncoce-
phalia,” ‘Zool. Jahrb., Anat. u. Ontog.,’ Bd. xii, 1898.
“ Ueber die Kaumuskeln bei Lacertilia,’”’ ‘Anat. Anzeig.,’ 1904.
v. WisHE.—‘ Ueber die Mesodermsegmente und die Entwicklung der
Nerven des Selachierkopfes,” ‘ Verhand. der k. Acad. der Wissen. zu
Amsterdam,’ 1882.
“Ueber die Nerven und Somiten von Vogel- und Reptilien-
embryonen,” ‘ Zool. Anzeig.,’ Bd. ix, 1886.

On tHE NAMES EMPLOYED.

B=Bradley, F=Fiirbringer, G=Gadow, H= Hoffman, K= Kingsley,
O=Osawa, P= Parker, V = Versluys.

Orbicularis palpebrarum :

Birds (G), Sphenodon, Lacertilia vera, Rhiptoglossa.
Mandibular myotome—

Upper part divides :

Bods eae palpebre inferioris (G).
orbito-quadratus (G).

depressor palpebre inferioris et superioris (H).
atrophies.
depressor palpebre inferioris et superioris (H).
atrophies.

fee palpebre inferioris.

Chelonia

Crocodilia

Sphenodon spheno-pterygo-quadratus (F).

depressor palpebree inferioris.
Lacertilia veray pterygo-parietalis.
protractor ptery goidei (V) = pterygo-sphenoidalis poste-
rior (B).
[pees ree anterior,
Rhiptoglossa, pterygo-parietalis (V).

Ie ooicnots pterygoidei (V).
vomero-sphenoideus (H).
pterygo-sphenoidalis anterior (H).
pterygo-parietalis (1).
pterygo-sphenoidalis posterior (H),

Ophidia.

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 553

Lower portion, inner division—
Pterygoid (undivided) :
Chelonia—pterygo-maxillaris (H).
Crocodilia—pterygo-maxillaris (H).
Rhiptoglossa—pterygo-maxillaris (H).
Ophidia—trans verso-maxillo-pterygo-mandibularis (H).

Internal pterygoid :
Birds—pterygoideus internus (G).
Sphenodon—pterygoideus internus (OQ).
Lacertilia vera—pterygoideus internus (H)=pterygoideus (V)
= pterygo-mandibularis (B).

External pterygoid :
Birds—pterygoideus externus (G).
Sphenodon—pterygoideus externus (Q).
Lacertilia vera—pterygoideus externus (H) = pterygoideus (B).

Lower portion, outer division—
Temporal and quadrato-maxillaris: Birds (G).
Temporal :

Chelonia—occipito-squamoso-maxillaris (H).
Crocodilia—temporalo-maxillaris (H).
Sphenodon—capiti-mandibularis (O).

Lacertilia vera—temporalis (H) = capiti-mandibularis (B).
Rhiptoglossa—temporo-mandibularis et quadrato-mandibularis (H).
Ophidia—parieto-quadrato-mandibularis (H).

Hyoid myotome—
Stapedius: Birds (G), Alligator (K), Lacerta agilis, L. viridis, and
Zootoca vivipara (P).
Depressor mandibule :
Birds—digastricus (G).

Chelonia—squamoso-maxillaris (H).
Crocodilia—occipito-maxillaris (H).
Sphenodon—parieto-mandibularis (OQ).

Lacertilia vera—digastricus (H) = depressor mandibule, (V) = parieto-
mandibularis (B).
Rhiptoglossa—depressor mandibule (H).
Ophidia—occipito-quadrato-mandibularis (H).

First branchial myotome—
Branchio-hyoideus, branchio-maxillaris :
Sphenodon—cerato-hyoideus (QO).

554, F. H. EDGEWORTH.

Birds—genio-hyoideus (G).
Chelonia—cerato-maxillaris (H).
Crocodilia—maxillo-hyoideus (H).
Lacertilia vera—cerato-hyoideus (H).
Rhiptoglossa—genio-ceratoideus (H).
Ophidia—mylo-hyoideus (H).

Ventral longitudinal muscles—
Genio-hyoideus :
Birds—atrophies.
Chelonia—genio-hyoideus (H).
Crocodilia—anterior belly of maxillo-coracoideus (H).
Sphenodon—cerato-mandibularis (QO).
Lacertilia vera—cerato-mandibularis (H).
Rhiptoglossa—genio-hyoideus (H).
Ophidia—maxillo-hyoideus (H).

Sterno-hyoideus divides :
Birds—in Gallus into thyreo-hyoideus and sterno-thyroideus
(G).
Chelonia—coraco-hyoideus and coraco-cerato-hyoideus (H).
Crocodilia—coraco-ceratoideus, episterno-ceratoideus, and posterior
belly of maxillo-coracoideus (H).
Sphenodon—episterno-hyoideus and omo-hyoideus (0).
Lacertilia vera-—episterno-hyoideus profundus, sterno-hyoideus, and
omo-hyoideus (H).
Rhiptoglossa—sterno-hyoideus and omo-hyoideus (H).
Ophidia—cervico-hyoideus (H).

Genio-glossus :
Birds—in some, e. g. Procellaria (G).
Chelonia—maxilla-glossus (H).
Crocodilia—genio-glossus.

Sphenodon
Lacertilia vera | present, but not mentioned by (O) or (H).
Rhiptoglossa
Ophidia ) ......... it gives off the genio-laryngeus (H).

Hyo-glossus :
Birds—divides into cerato-glossus, hypoglossus rectus and

obliquus (G).
Chelonia—cerato-glossus.
Crocodilia—cerato-hyoideus (H).

HEAD-MUSCLES IN GALLUS AND OTHER SAUROPSIDA. 995

Splendor Ppresent, not mentioned by (QO) or (H).
Lacertilia vera

Rhiptoglossa—the posterior part forms cerato-hyoideus of (H).
Ophidia—hyo-glossus (H) ; it gives off the hyo-laryngeus (H).

Mylohyoids :
Birds—anterior mylohyoid = mylohyoideus (G).
posterior mylohyoid :
Anterior portion—serpihyoideus and stylohyoideus
(G); a simple band in Lamellicostren (G).
Anterior constrictor colli=ceratohyoideus (G); not
always developed (G).
Constrictor colli (G).
Chelonia—anterior mylohyoid =intermaxillaris (H).
posterior mylohyoid = constrictor colli (H).
Crocodilia—anterior mylohyoid = mylohyoideus (H).
posterior mylohyoid =sphincter colli (H).
Sphenodon—anterior and posterior mylohyoids form a continuous
sheet= subcutaneous colli (O).
hgeaa hire } mylohyoidens (H) and sphincter colli (H).
Rhiptoglossa
Ophidia—intermaxillaris (H) and transverso-hyoideus (H).

List or ABBREVIATIONS EMPLOYED IN THE ‘l'EXT-FIGURES.

ab.an. Abducens Anlage. ant.cons.collz, Anterior constrictor colli. ant.
card.v. Anterior cardinal vein. ant.mylo. Anterior mylohyoid muscle.
A.V. Auditory vesicle or involution. awr. Auricle of heart. 1B.B. 1st
basibranchial. 2.B.B. 2nd basibranchial. B.H. Basihyal. Bhbp. Basihyo-
branchial plate. 1 br.aov.ar. Ist branchial aortic arch. br.maa. Branchio-
maxillaris muscle. 1br.my. Ist branchial myotome. 2 b7.som. 2nd branchial
somite. buc.cav. Buccal cavity. cart.ento. Cartilago entoglossa. car.art.
Carotid artery. cer.br.1. Ceratobranchial of Ist branchial bar. cer.hyal.c.
Ceratohyal bar. cer.hyal.cornu. Ceratohyal cornu. cil.g. Ciliary ganglion.
conj.sac. Conjunctival sac. cons.colli. Constrictor colli muscle. ev, Coro-
noid bone. cost.proc. Costal process. d. Dentary bone. dep.mand.
Depressor mandibule. dep.palp.inf. Depressor palpebre inferioris.
dor.aor. Dorsal aorta. dor.ceph.ceel. Dorsal cephalic celom. EH. Eye.
enceph. Encephalomere. epipter. Epipterygoid or processus ascendens.
epist.hyoid. Episterno-hyoideus muscle. extrastap. Extrastapedial.
g.petr.iz. Ganglion petrosum of ix nerve. gass.g. Gasserian ganglion.

556 F. H. EDGEWORTH.

gang.Vi. That portion of gasserian ganglion from which the first por-
tion of v nerve proceeds. gen.gang.vii. Geniculate ganglion of vii nerve.
gen.gloss. Genio-glossus muscle. gen.hyoid. Genio-hyoideus muscle.
g.s. Gill pouch. ht. Heart. hy.aor.ar. Aortic arch of hyoid segment.
hyogloss. Hyo-glossus muscle. hyoid.my. Myotome of hyoid segment.
hyoid.som. Somite of hyoid segment. hyomand.c. Hyomandibular cartilage.
hypobl. Hypoblast. hypogloss.rect. Hypoglossus rectus muscle. int.orb.s.
Interorbital septum. j. Jugal bone. j.&ma.an. Anlage of jugal and
superior maxilla. lary. Larynx. lary.m.an. Anlage of laryngeal muscles.
lary.c. Anlage of laryngeal cartilages. ling.m.an. Anlage of lingual
muscles (in Gallus). mand.aor.ar. Aortic arch of mandibular segment.
mand.my. Myotome of mandibular segment. mand.my.u.p. Upper portion
of myotome of mandibular segment. Me. Meckel’s cartilage. med.pl. Me-
dullary plate. mid.nas.pas. Middle nasal passage. N. Nose. moto. Noto-
chord. O.inf. Obliquus inferior muscle. O.swp. Obliquus superior muscle.
O.sup.an. Anlage of obliquus superior. s. (Esophagus. ces.cons. Con-
strictor muscle of cesophagus. omohyoid. Omo-hyoideus muscle. orb.
palp. Orbicularis palpebrarum muscle. otosphen.pl. Otosphenoidal plate.
pal. Palate bone. pal.pter.bar. Palatopterygoid bar. per. Pericardium.
pg. Pterygoid bone. phar. Pharynx. pin. Pineal body. pit. Pituitary
body. p.o. Post-orbital bone. post.mylo. Posterior mylohoid muscle.
premand.seg.an. Anlage of premandibular segment. protr.pter. Protvactor
pterygoidei muscle. pty.an. Anlage of pterygoid muscles. pty.ext. Ptery-
goideus externus muscle. pty.int. Pterygoideus internus muscle. pty.m.
Pterygoideus muscle. pty.pr.of quad. Pterygoid process of quadrate.
pty.par. Pterygo-parietalis muscle. pyr. &qu.an. Anlage of pyramidalis
and quadratus nictitantis. quad.mand. Quadrato-mandibularis muscle.
quad. Quadrate. quad. & Me.an. Anlage of quadrate and Meckel’s carti-
lage. R.eat. Rectus externus muscle. R.int. Rectus internus muscle.
R.sup. Rectus superior muscle. retract.oc. Retractor oculi muscle. scler.
Sclerotic (cartilaginous). serpihy. Serpi-hyoideus muscle. som.meso. Somatic
mesoblast. sphen.p.n. Sphenopalatine nerve. sphen.pter.quad. Spheno-
pterygo-quadratus muscle. spl.meso. Splanchnic mesoblast. sq. Squamosal
bone. stap.m. Stapedius muscle. sé.cerv. Sterno-cervicalis muscle.
sternohy. Sterno-hyoideus muscle. sternothy. Sterno-thyroideus muscle.
suprastap. Suprastapedial. temp. Temporal muscle. temp.&ma.an. Anlage
of temporal and masseter muscles. ftrab. Trabecula. ér.art. Truncus arte-
riosus. trach. Trachea. 1 tr.som. 1st trunk somite. thyr. Thyroid.
ven.car.art. Ventral carotid artery. ven.ceph.ceel. Ventral cephalic celom.
vent. Ventricle of heart. vert.v. Vertebral vein. V.L.M. Anlage of
ventral longitudinal muscles. Roman numerals. Cranial nerves.

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. Ear

The Development of Ophiothrix fragilis.

By
E. W. MacBride, M.A., D.Sc., F.R.S.,
Professor of Zoology in McGill University, Montreal.

With Plates 31—36, and 4 Text-figures.

ContvENTs.

PAGE
Introduction E ; ; ; . 557
Historical Sketch : : : : . 558
Material and Methods : 5 ‘ - 567
Normal and Abnormal Development : ! 570
The Development of the Full-grown Larva : . 576
Metamorphosis of the Larva into the Brittle-star —. . 580

Comparison with Development in other Classes of Hchino-
dermata . : : ; : . 590
Summary and Conclusion : : : . 596

INTRODUCTION.

Tue present work, which has occupied my attention for the
last four years, was begun with the object of extending to
other classes of Echinoderms the researches which I had
already made on the development of Asteroidea (18) and
EKchinoidea (19). It has proved to be a task of extraordinary
difficulty owing to the minute and refractory character of the
larvee of Ophiuroidea. Nevertheless, the results obtained
will, I think, bear fair comparison with those which I have
already published concerning the development of Asteroidea
and Hchinoidea, whilst a number of new and unexpected
facts have disclosed themselves which possess interest for a

558 i. W. MACBRIDE.

wider range of students than specialists in the class Echino-
dermata. The most interesting result which was obtained
from the study of the development of Asteroidea and
Echinoidea was the discovery that the ccelom of the larva
showed distinct traces of metameric segmentation, a division
into three somites being clearly indicated. The pioneer in
this work is Bury, to whose stimulating papers (4, 5, and 6)
I wish on this occasion, as formerly, to express my deep
indebtedness. Bury, however, only recognised two somites,
whilst the main point of my previous researches was that
the hydroccele, or rudiment of the water-vascular system,
which Bury regarded as essentially a single organ, was
in reality paired, the left rudiment developing whilst the
right remained small and insignificant. This interpretation
of the facts has been challenged by other observers such as
Goto (11) and Masterman (20), who interpret what I regard
as the right hydroccele in a different manner. JI may summa-
rise the results of the present paper by saying that the study
of the development of Ophiuroidea has afforded me a com-
plete confirmation of my views. The state of affairs in the
Ophiuroidea is much clearer and simpler than in the other
classes of Echinodermata, and I hope to convince every
unprejudiced reader of this paper that the Echinoderm larva
really possesses three metameres.

HisroricAL SKETCH.

Our knowledge of the development of Ophiuroidea dates
back to Johannes Miiller. At the meeting of the Academy
of Sciences of Berlin, held on December 4th, 1845, this
celebrated naturalist described a number of new marine
animals which he had observed whilst at Heligoland in the
autumn of that year. Amongst these was one which Miiller
named Pluteus paradoxus, from its fancied resemblance
to a painter’s easel. This description was published in the
‘Archiv fiir Anatomie und Physiologie,’ in 1846 (24), and
on October 29th of that year Miller read before the Academy

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 559

of Sciences a paper entitled “ Uber die Larven und die Meta-
morphosen der Ophiuren und Seeigel” (25), in which he
describes the metamorphosis of Pluteus paradoxus and
shows that it is the larva of an Ophiurid. A somewhat
similar organism also referred to the genus Pluteus is
shown in this paper to be the larva of an Kchinoid, and
Miller adopted the regrettable custom of using the same
word “ Pluteus” to designate these two quite different larvze ;
this custom has persisted until quite recently and has been
the source of much confusion. In the case of Pluteus para-
doxus the change into an Ophiurid is traced step by step,
and Miller figures without describing it a specimen which has
a five-lobed rosette—the form assumed by the rudiment of
the water-vascular system—on the right as well as on the
left side of the cesophagus. In this figure also the metameric
division of the ccelom is beautifully shown, and, indeed,
Miiller’s drawing could have been used to illustrate this
paper. Of course, Miiller was unable to interpret his results
fully, and since the coelomic rudiments have extremely narrow
lumina it is not surprising that he did not recognise them as
the forerunners of the body-cavities of the adult. A second
form of Ophiurid pluteus with more slender arms is described
in the same paper, and in a later paper (27) read before the
Academy in 1851, Miiller describes two further forms of
Ophiurid larvee which he discovered during a visit to Trieste,
one of which he named Pluteus bimaculatus from the
brown pigment spots with which it was ornamented, the other
he recognised as the subject of the present paper—the larva of
Ophiothrix fragilis. In Pluteus bimaculatus Miller
discovered the larval anus of Ophiuroidea, thus showing that
the absence of the anus in this class of Hchinodermata is a
secondary, not a primary affair. The hydroccele or rudiment
of the water-vascular system is described as the palmate
organ, since it possesses five lobes like the five fingers of the
human hand. Miller saw that it was formed late in larval
life, and was at first round in shape. In its later stages it
extends ventral to the csophagus, and each lobe becomes
vot. 51, part 4.—NEW SERIES. 42

560 E. W. MACBRIDE.

five-lobed, so that each somewhat resembles the whole organ
in an earlier stage. Miiller was led astray by this resem-
blance and gave to each primary lobe the name “ palmate
organ,” and he imagined that the palmate organ of the
earlier larva was identical with the first of these, and that the
rest were formed de novo. He described also a semi-
circular wavy fold of skin, from which the rudiments of the
adult arms grew out, to which the “ palmate organs ” became
co-adapted later. These latter gave rise to the radial canals
and tentacles of the water-vascular system.

The larva of Ophiothrix fragilis was recognised by
Miiller owing to its possessing on the first tentacles of the
developing brittle-star the papillee characteristic of this species.
Miller only obtained late metamorphosing stages, and his
description concerns itself chiefly with the elaboration of the
details of the brittle-star. The last Ophiurid larva described by
Miiller is one which he called the “ worm-like larva,” which he
imagined to belong to the Asteroidea (28). It was re-dis-
covered by Krohn (15), who proved that it was the larva of
an Ophiurid. In 1900 Caswell Grave (12) found a similar
larva on the American coast, and showed that it was the
young stage of Ophiura brevispina. Owing to the
opacity of this larva neither Miiller nor Krohn made out
much of its structure.

The next great advance in our knowledge of Ophiurid de-
velopment was made by Metschnikoff in 1869 (21). Ina
paper on the development of Echinoderms and Nemertines
he records the results of investigations on all four classes of
Echinoderm larve, as well as on the embryos of the viviparous
species, Amphiurasquamata. Three kinds of Ophiurid
plutei are described, two of which are identified with larve
described by Johannes Miiller. Of only one form, however,
did he obtain sufficient stages to describe the development in
any detail, and this is the Pluteus bimaculatus of Miller.
In the youngest stage which he obtained he was able to make
out the right and left coelomic cavities lying at the sides of
the larval cesophagus. In the next stage he discovered a

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 561

pair of discs lying at the sides of the stomach, and concluded
that they had been segmented off from the cesophageal cavities,
which he could still make out. ‘This is the first assertion of
a trace of metameric segmentation in any Echinoderm larva.
In subsequent stages he discovered that the left anterior
cavity assumed the five-lobed form characteristic of the
hydroceele, whilst the right grew smaller and ultimately dis-
appeared. In the next stage he showed that the hydroccelic
rudiment had by growth changed its longitudinal position for
a transverse one, and was, in fact, beginning to surround the
larval cesophagus. Miiller had supposed that, asin Echinoidea,
the larval cesophagus disappeared, but Metschnikoff shows
‘that it persists. He further corrected Miiller’s error about
the “palmate organs,’ and showed that each of these corre-
sponded to one lobe of the original palmate organ or hydro-
cole. He showed also that the primary madreporic pore,
although formed on the left side, rotates during metamor-
phosis to the nght along with the left prz-oral arm of the
Pluteus. In aword, Metschnikoff made out almost everything
that it is possible to discover in a Pluteus without the use of
sections.

The viviparous species, Amphiura squamata, offered an
opportunity of studying the embryonic type of development
in an Ophiurid. Its viviparity was discovered by Quatrefages
in 1842. He communicated his results to Milne Edwards, by
whom they were published. Krohn published in 1851 a paper
on the subject (14), dealing with the relation of the embryo
to the maternal tissues. In 1852 Schultze (380) discovered a
transitory larval skeleton, which he compared to the skeleton
of the Pluteus.

In the paper referred to above Metschnikoff records most
valuable observations on the development of Amphiura
squamata, although the younger embryos are difficult to
obtain. He found a thick-walled blastula, and then a later
stage, in which the ccelom was represented by two thick-
walled bodies lying at the sides of the archenteron., In the
next stage each of these bodies had divided into anterior and

562 E. W. MACBRIDE.

posterior portions, and soon afterwards the left anterior por-
tion had assumed the rosette form characteristic of the hydro-
ccele. Metschnikoff expressly states that the right anterior
sac often likewise assumed the same form, and this is the first
clear statement that I have been able to find in the literature
that the hydroccele rudiment is paired. Miiller’s figure alluded
to above shows the same thing in Pluteus paradoxus, but,
as already mentioned, he gives no description of it.

Further researches on the development of Amphiura
squamata were made by Ludwig (16), Apostolides (2),
Fewkes (10), Carpenter (7), Russo (29), and myself (17).
Ludwig’s work has reference to the development of the
skeleton ; amongst other things he showed that the ossicle
termed a: “vertebra” is formed by the concrescence of two
calcareous plates, so that its homology with a pair of ambu-
lacral plates in an Asterid is proved. Apostolides dealt with
the whole development, and according to him the endoderm
is formed by delamination, not, as in other Echinoderms, by
invagination. This extraordinary statement is reaffirmed by
Russo, who also deals with the whole development. Russo
asserts further that the coelomic cavities arise as spaces in the
mesenchyme; but he utterly fails to confirm Metschnikoff’s
observations as to the division of the right ccelomic sac into
anterior and posterior portions, and the occasional assumption
of a five-lobed form by the anterior half. Russo’s results are
at variance with what is known of the early stages of
development of all other Echinoderms; and since he failed to
see what Metschnikoff was able to make out in the embryos
of Amphiura squamata his results have been received
with general scepticism, but cannot, of course, be formally
denied till some other investigator has the patience to re-
examine the early stages of development of this species. These
stages are opaque and difficult to obtain, hence, no doubt,
the unsatisfactory nature of our knowledge on the subject.
Fewkes, who had very little material of the early stages at
his disposal, deals chiefly with the development of the adult
skeleton, but made out also the ectodermal origin of the

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 563

cesophagus. Carpenter (7) was chiefly occupied in estab-
lishing a fanciful homology between the dorsal plates of
Amphiura squamata and the aboral plates of a Crinoid.
My own work (17) had reference to the first origin of the
genital cells, and dealt solely with post-larval stages. I dis-
criminated for the first time between various spaces which
had been confused under the name “axial sinus.” The
problematical organ lying along the stone-canal, which had
been regarded as a heart by some zoologists, was shown to be
of peritoneal origin, and to represent the first rudiment of the
genital cells. This paper contains one serious blunder which
I endeavoured to set right in my paper on Asterina gib-
bosa (18), viz. the vesicle representing the right fellow of the
hydroceele is confused with a space originating as a peri-
toneal invagination and labelled in my figures Sinus b.
Reviewing the narrative so far given, it will be seen that
the only two investigators who advanced our knowledge of
the early larval stages of Ophiuroidean development were
Miller and Metschnikoff. To these it is now necessary to add
a third name, viz. that of H. Bury. ‘This talented investi-
gator, who gave to zoology the first satisfactory account (4)
of the development of the Crinoid Antedon rosacea, was
led by his study of this species to revive the idea of a meta-
meric segmentation of the Echinoderm larva. ‘Taking up the
investigation of the larve of other classes of Echinodermata,
he found his ideas amply confirmed, and in 1889 he published
a paper (5) in which he showed that a transverse division of
the coelom took place in the larve of both Hchinoidea and
Ophiuroidea. Bury was the first to discriminate an anterior
ceelom from the hydroccele, which he regarded as an essen-
tially unpaired organ. ‘This hydroccele, as he showed, was
formed comparatively late in development, and arose as an
outgrowth from the left anterior ccelomic vesicle in EKchinoidea,
whilst in Ophiuroidea he felt himself compelled to assert that
it was an outgrowth from the left posterior vesicle. During
the interval between the appearance of the papers of
Metschnikoff and Bury quite a number of authors had made

564. E. W. MACBRIDE,

use of artificial fertilisation, and thus accumulated a certain
amount of information about the earliest stages of develop-
ment of Ophiurids other than Amphiura squamata.
Kowalevsky, in a paper devoted to Amphioxus (18), had even
before Metschnikoff’s time noted the fact that the eggs of an
undetermined Ophiurid formed a gastrula by invagination.
Balfour had observed the same in the case of Ophiothrix
fragilis (8), Selenka in Ophioglypha lacertosa (81),
and Fewkes in Ophiopholis aculeata (8). These results
naturally led to increased scepticism as to the supposed
formation of the endoderm by delamination in Amphiura
squamata. In 1895 Bury published another paper entitled
“The Metamorphosis of Echinoderms” (6). This paper,
although containing most valuable observations on the other
groups of Hchinoderms, as well as a general theory of the
phylogeny of the group, contains few new observations on
Ophiuroidea, the only notable one being a suggestion that
the left posterior ccelom encircles the stomach in the later
stages of development. This view Bury bases on a single
section, which showed a horizontal mesentery.

Ziegler in 1896 published a short paper on the early stages
of development of Ophiothrix fragilis (88). His account
only reaches as far as the gastrulation, but he describes a
vacuolated crest which is very characteristic of these larve.

In 1900 Caswell Grave published an interesting and valuable
paper (12) on the development of Ophiura brevispina.
This species lays comparatively large eggs and has ashortened
development. It transpires, indeed, that the eggs develop
into the “worm-like” larva described by Johannes Miiller
and Krohn, which is thus shown to be the larva of Ophiura
brevispina, or of a closely allied species. Grave’s material
was, as he himself explains, decidedly scanty, a circumstance
which renders it possible that some of his conclusions may
require revision should more abundant material become avyail-
able. ‘The first stage which he describes is an oval larva, 14
days old, uniformly ciliated and swimming freely. In this
stage an anterior, bilobed, coelomic vesicle is being constricted

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 565

off from the apex of the archenteron. A large cellular plug
projects from the apex of the ccelomic vesicle and extends
into the cavity of the archenteron. From this circumstance
Grave draws the somewhat rash conclusion that the archen-
teron has been formed by the hollowing out of the inner mass
of a planula-like larva. In the next stage, which is only a
few hours older, a remarkable state of affairs has come about.
The first vesicle is completely constricted from the archenteron
and divided into right and left halves. A second vesicle,
however, has been budded from the archenteron and is still in
open communication with it. This vesicle is already partially
divided into two by a constriction, and its anterior portion
shows the five lobes which proclaim it to be the hydroceele,
whilst its posterior portion gives rise to the hypogastric
coelom, i.e. the left posterior ccelom. This state of affairs
is widely different from that which has been described in the
case of any other Hchinoderm. The very early appearance
of a lobed hydroccele shows that the first stages of develop-
ment have been very much hurried over, and hence we must
regard differences from the normal type of Echinoderm
development as secondary modifications, not as indications of
a primary state of affairs. In the next stage the ectodermal
stomodxum has met the cesophagus. It passes under the
arch of the hydroccele, so that this forms a bridge half-way
around it. The stone-canal is formed as a secondary connec-
tion between the left anterior ccelom and the hydrocele. <A
so-called epigastric ccelom—corresponding to the right pos-
terior coelom of the free-swimming pluteus—appears in this
stage, and after some discussion of the point, Grave concludes
that it must have been formed from the right anterior ccelom.
In the next stage the cilia are restricted to four transverse
rings, which produce an appearance of metameric segmenta-
tion and give rise to the “worm-like ” appearance of the
larva. The posterior eud of the larva is broadened, and
contains practically all the organs of the Ophiurid; the
anterior portion contains mesenchyme and forms a large
pre-oral lobe. ‘The hydroccele has rotated to such an extent

566 E. W. MACBRIDE.

that its most anterior lobe, numbered 1 by Grave, has not
only passed over the cesophagus to the right, but has con-
tinued its revolution underneath it to the left again, so that
it has been rotated through an angle of 180°, whilst the most
posterior lobe, No. 5, has rotated through 90° only, the whole
hydroceele having grown longer. The left anterior ccelom,
together with the madreporic pore which has now appeared,
follow the rotation of the hydroccele and pass over the
cesophagus on to the right side. The lobes of the hydrcele
become trifid, the lateral lobes being the rudiments of the
first paired tube-feet.

From the left posterior coelom, termed by Grave the
“hypogastric,” there arise four out-growths, which, insinua-
ting themselves between the lobes of the hydroccele, form the
rudiments of the radial perihemal canals and of the perihemal
ring in the same manner as I have described in Asterina
vibbosa. A fifth is given off from the left anterior ccelom.
In later stages the nervous system makes its appearance as
thickenings of the neutral ectoderm overlying the ring and
radial canals. These thickenings are then invaginated into
grooves and so the epineural canals of the adult are formed.
The larval organ gradually disappears, but the young
Ophiurids retain for a considerable period some of the larval
transverse rings of cilia.

I have described Grave’s work at some length, since it is
the latest work of any importance on the formation of organs in
an Ophiurid which has been published. Making allowances for
differences in nomenclature, it will be seen that Grave confirms
Bury in the opinion that the hydrocele springs from the left
posterior celom. ‘The independent origin of this portion of
the coelom as a separate evagination of the gut from that
which gives rise to the two anterior ccelomic vesicles is
almost unique in the published accounts of Hchinoderm
development. It is true that Masterman (20) has described
a somewhat similar state of affairs in a star-fish, Cribrella
oculata, in which, according to him, the hypogastric coelom
(left posterior coelom) does not arise from the common rudiment

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 567

of the anterior and epigastric (right posterior) cceloms, but has
an independent origin from the gut ; in this case, however, the
hydroccele is derived from the anterior ccelom. In both cases we
have to do with yolky eggs anda shortened development, and
in both cases a re-examination of the facts is desirable, as Grave
would be the first to admit, since throughout his paper he
laments the scantiness of his material and the gaps between
the stages. It is to be hoped that he will be able at no
distant date to give the whole subject a thorough revision.
Mortensen in 1898 published a systematic review of all the
forms of Echinoderm larve known up till that date (22). In
this valuable paper he discusses the homology of the various
larval appendages, and introduces a nomenclature which will
be employed in this paper. Another service which he has
rendered is to introduce the terms Ophiopluteus and
Echinopluteus for the larve of Ophiuroidea and Kehino-
idea respectively, thus doing away with the confusion imphed
in the use of the term “ Pluteus.”’ In a second paper (28) he
makes an addition to the number of varieties of Echinoderm
larvee known, and with this second paper our review of the
knowledge so far gained may be said to be complete.

MatERIAL AND MeEtTHops.

In the summer of 1898, whilst I was staying at Plymouth
investigating the development of Echinus esculentus, I
received a large number of specimens of Ophiothrix
fragilis, which appeared to be sexually ripe. I therefore
attempted artificial fertilisation, but as the developing eggs
remained at the bottom I concluded that they were abnormal.
About eight days afterwards, to my delight, I found a certain
number of perfectly formed, free-swimming larve in the jar.
These larv continued to develop, and at the end of twenty-
six days had completed their metamorphosis. ‘The jar in
which the larvee were placed was shaded from the light and
fitted with a Browne plunger. During the summer of 1898
the Phytoplankton was particularly abundant, and so the de-

568 E. W. MACBRIDE.

veloping larvee obtained plenty of food. Returning to com-
plete my work on Echinus esculentus in 1899, I again
tried to rear the larve of Ophiothrix fragilis, but although
I obtained ripe adults and fertilised the eggs I was unable to
keep the larve living for longer than five days. Towards the
end of my stay the larve became very abundant in the Plankton,
and from July 14th to 19th, 1899, it might be fairly said to
swarm with them. As I observed that the Echinoderm larve
were in a somewhat pathological condition when the bottles
containing the Plankton were brought into the laboratory, I
went out in the steamer belonging to the station and pre-
served the Plankton immediately on its being brought to the
surface. For this purpose I used 1 per cent. osmic acid
followed by Miiller’s fluid—a method which has the advantage
of giving an excellent preservation of the tissues, but it
labours under two disadvantages: it makes the specimens
brittle and it completely dissolves the calcareous matter, and
hence no attention is paid in this paper to the development of
the adult skeleton. The Plankton contained in all four varieties
of Ophiurid larve, but the larve of Ophiothrix fragilis
were in an enormous majority, and in all stages of develop-
ment up to the completed metamorphosis. They were easily
distinguishable by the greatly developed postero-lateral arms,
which in this species are extraordinarily long. In this way
a large amount of material was obtained, but I was unable to
commence work on it till 1908, when I had completed the
work on Echinus esculentus. In 1905 I paid another
visit to Plymouth in order to fill the gap in the series of
stages which I had obtained from the Plankton. It happened
that the season was rather later, so far as the breeding habits
of this species were concerned, than it had been in 1898 and
1899, and though I examined hundreds of adults I only
succeeded in obtaining two females which were capable of
fertilisation. Of these the first gave larvee which pursued a
development similar to that undergone by the larve which I
obtained by artificial fertilisation in 1899, and which I believed
to be the normal course of events, but I was unable to keep

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 569

them alive more than eight or nine days. The second female
was placed in a half-gallon jar along with two males and
spawned in a natural manner. The larve were exceedingly
vigorous and developed with great rapidity; they differed in
a marked manner from the result of previous cultures so far
as the earliest stages were concerned. I was able to rear
them for sixteen days, by which time the hydroccele had made
its appearance. ‘heir eventual death was clearly due to starva-
tion, as the Phytoplankton was extraordinarily scanty in that
year. Reviewing the account just given, it will be seen that
in 1898 a culture was obtained as the result of artificial
fertilisation in which some larve completed the entire de-
velopment, until the end of metamorphosis. In 1899 and 1905
similar cultures gave larvee which were only able to carry on
the development for about a week; whilst in 1905 a culture
obtained by what may be termed natural fertilisation gave
larvee which grew and developed vigorously as long as food
was available, and which in their earlier stages were different
from those which resulted from artificial fertilisation. The
main mass of the material was obtained from Plankton. If
we take the successful culture of 1898 as giving an indication
of the time required to effect complete development, it will
follow that metamorphosis is finished in about a month, and
the approximate time for the appearance of other organs is
given in the following table:

Free-swimming blastula 24 hours.
Gastrula . 30s
Formation of ccelom 2 days.
Stomodeeum joins gut Dae ahs
First transverse division of ccelom . Sa S
Formation of hydroccles 10.
Left hydrocele five-lobed ‘ 20
Encircling of cesophagus by hydrocele . 23) gs

Absorption of larval arms complete ‘ 26,
‘hese times, however, are only approximate, and the rate
of development varies very much with the amount of food
present, and possibly from other causes too. In particular

570 E. W. MACBRIDE.

it will be noticed that the culture obtained by natural
fertilisation in 1905 developed at a quicker rate than that
indicated by these figures.

The larvee were observed and drawn living. Those pre-
served in osmic acid and Miiller’s fluid were sectioned. For
this purpose the celloidin paraffin method was used, and the
procedure adopted was similar to that employed in the case
of Echinus esculentus and described in my paper on the
subject (19). One additional caution, which it was found
necessary to adopt, may be mentioned here. It transpired
that if the sections, after being spread out on hot water in
order to flatten them, were left to dry for longer than forty
minutes on the top of the thermostat, great cracks developed
in them owing to the shrinkage of the celloidin.

When it was necessary to supplement the information
obtained from views of the living larve by whole mounts of
preserved ones, these were cleared from osmic acid. This
was done by placing them in water or weak alcohol. The
vessel containing them was then placed (open) inside a larger
vessel on the bottom of which was a layer of crystals of
chlorate of potash on which strong hydrochloric acid was
poured. The larger vessel was closed. The euchlorine gas
evolved became dissolved in the fluid of the smaller vessel
and oxidised the black deposit of metallic osmium in the tissues.

In the orientation of sections the postero-lateral arms of
the larva were of the greatest assistance, for they persist
uutil the metamorphosis is quite complete, so that they mark a
constant plane amidst the varying position of the other organs.
This plane will be called the frontal plane, and most of the
sections were cut parallel to it. Sections parallel to the
median sagittal plane of the larva were also employed as
were transverse sections when they became necessary in
order to elucidate special points.

Norma AND ABNORMAL DevELOPMENT.
It has already been mentioned that eggs fertilised under
different conditions give rise to larvae which in their early

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 571

stages differ considerably from one another. From eggs
artificially fertilised larvee are obtained which are shown in
Pls. 52 and 33, figs. 18-23, whilst when a male and female
are brought together and allowed to spawn naturally the
fertilised eggs develop into larve, the corresponding stages
of which are shown in PI. 31, figs. 1-5, and Pl. 383, figs, 24-28.

Considering first the normal development due to natural
fertilisation we find that the eggs, which are small (about
‘1 mm. in diameter) and opaque, owing to the presence of
a yellow yolk, develop in seven or eight hours into small thick-
walled blastule (PI. 51, figs. 1 and 2), from one side of which
mesenchyme cells are budded off (Pl. 33, fig. 24, mes.). At
the end of twelve to eighteen hours the embryo has escaped
from the egg membrane and become ciliated and free-swim-
ming, and is now henceforth to be designated a larva. The
larva is egg-shaped (PI. 51, fig. 3), the more pointed end, which
is directed forwards, being formed of a great crest of vacuolated
cells, which possibly acts as a hydrostatic apparatus (Pl. 33,
fig. 25), whilst at the opposite pole the formation of mesen-
chyme is going on, and the invagination which is to form
the primary gut or archenteron is beginning (Pl. 33,
figs. 25-27). During the next fifteen or twenty hours the
form of the larva changes from an egg-shape to a lozenge-
shape, owing to the appearance of two lateral outgrowths,
which are the earliest rudiments of the postero-lateral arms
of the larva (Pl. 33, fig. 28, pl.). Into these outgrowths
the most of the mesenchyme cells wander, where they form
the skeletogenous basis of the calcareous rods which support
these arms. The space between the archenteron and the
ectoderm is of course identical with the blastocele or cavity of
the blastula; it has been termed the archicele or primary
body-cavity by German authors; both these names will be
employed in this paper. The secondary body-cavity or celom
(Pl. 33, figs. 28 and 29, cw.), arises as a thin-walled bilobed
outgrowth from the apex of the archenteron when the larva
is from thirty to forty hours old. The postero-lateral arms
grow rapidly and the vacuolated crest dwindles in similar

572 E. W. MACBRIDE.

proportion. ‘The characteristic longitudinal ciliated band by
means of which locomotion is effected has already appeared
as a transverse ridge, which rapidly becomes a horizontal
loop (PI. 31, figs. 4 and 5) ; as the postero-lateral arms grow,
they receive extensions of this loop, and so the swimming
powers of the larva are increased. It may be partly owing
to this circumstance that the vacuolated crest disappears,
but it is also partly replaced by a conversion of the ecto-
derm of the posterior end of the larva into an appendage of
vacuolated cells, which persists until metamorphosis is com-
plete (figs. 29 and 44, vac.).

Leaving the consideration of eggs which have received
natural fertilisation, and turning to those which have been
artificially fertilised, we find that they are distinguished by
such an early and abundant formation of mesenchyme that
sections through earlier stages (fig. 18) show a practically
solid mass of blastomeres, and when segmentation is complete
there is an outer covering of cylindrical ectodermal cells, and
an inner mass of rounded cells, so that an appearance is
presented which strongly recalls that of a Cceelenterate
planula (Pl. 32, fig. 19). A flattening and thickening of
one pole now follows, together with a renewed production of
mesenchyme (fig. 20, mes.), and then invagination takes
place in such a way as to leave a wedge of cells projecting
into the cavity of thearchenteron (fig.21). This results from
the fact that the endodermic plate of cells becomes in one
meridian several cells deep before it is invaginated. In the
next stage the postero-lateral arms make their appearance
(fig. 22) and receive most of the primary mesenchyme, whilst
soon after the ccelom appears as a vesicle at the apex of the
archenteron. Part of the projecting tongue of cells is
included within it and part within the lower portion of the
archenteron, both parts being eventually absorbed. At no
time is there the slightest trace of the vacuolated crest of cells
so characteristic of normal development. Caswell Grave, in
his paper on the development of Ophiura brevis (12),
describes a tongue of cells projecting into the archenteron as

THE EVELOPMENT OF OPHIOTHRIX FRAGILIS. 573

a normal feature of the development of that species, and he
suggests (as he did not have the earlier stages) that it is an
indication that the archenteron is formed, not by invagination,
but by the hollowing out of an originally solid endoderm. It
is but just to record that when I gave an account of the
abnormal development of Ophiothrix fragilis before the
American Society of Zoologists in Philadelphia in 1903, Dr.
Grave, in commenting on my paper, stated that he had

TEXT-FIG, I.

os =
oS

F

=F

NE
=

L

“HIN

“A,

if.

la.le Three stages in development of eggs artificially fertilised.
ld.-lf. Three stages in development of eggs naturally fertilised.
Mes. Mesenchyme. Ca. Coelomic rudiment. (From the ‘ Proceed-
ings of the Royal Society,’ B, vol. 79, 1907.)

obtained further material of Ophiura brevis, and that he
had become convinced that the course of events in that
species was identical with what I had described for Ophio-
thrix fragilis, in that form of the development which I at
that period took to be the normal one.

The contrast between the normal and abnormal methods of
development is well shown in text-fig. 1.

Turning now to the question of the cause of the difference
between normal and abnormal development, it will hardly be

574 E. W. MACBRIDE.

contended that the mere act of cutting out the ovary is
competent to produce such an effect. This is negatived by
the fact that in one culture raised from artificial fertilisation
some larvee were found showing a well-developed vacuolated
crest. The much more probable assumption is that abnormal
development results from the fertilisation of eggs which are
not quite ripe.

Up till now development following on fertilisation has been
taken as adequate proof of the ripeness of an egg, as anyone
who reads the numerous papers of Driesch, Herbst, Loeb, and
other workers on what is called “ Developmental Mechanics ”
may readily see. But when working with Echinus escu-
lentus I obtained some evidence that this is not necessarily
the case. I was, however, so much astonished by the facts
which came to lght that I have not until now dared to
publish them, and only do so now that a similar state of
affairs has been discovered with reference to the development
of Ophiothrix fragilis. I found that in order to obtain
good cultures of the larve of Echinus esculentus it was
necessary to choose for artificial fertilisation specimens which
were thoroughly ripe. This thorough ripeness was evidenced
by the condition of the ovary. When this organ at a touch
dissolved into eggs, then the resulting culture was highly
successful. When, however, I tried to fertilise eggs from
half-grown specimens, I obtained indeed some larve, but
these were small and had imperfectly developed arms, and
lived only a few days.

Of course, as all know, an egg is incapable of fertilisation
until it has formed its two polar bodies, but this process, in
some animals at least, is hurried on by the mere presence of
the spermatozoa. In the American oyster it is impossible by
inspection under the microscope to discriminate between ripe
and unripe eggs, for in this species the membrane of the
nucleus of the odcyte remains undissolved until it comes into
contact with the spermatozoa. It seems, therefore, allowable
to conclude that although in Echinoderms polar bodies are
normally formed independently of the presence of sperma-

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 9795
tozoa, yet they may be prematurely produced by the presence
of these, or possibly by the mechanical irritation of shaking
the ovary, and that in these cases the resulting development
is abnormal. This abnormality must be due therefore to a
difference in the chemical constitution of the egg, and the
fact that one abnormal feature appears to be normal in
another species seems to be an indication that very slight
chemical alterations in the constitution of the fertilised egg
may produce marked structural alterations in the organism
which results from it, and that these alterations may be
inheritable. Here, perhaps, is to be found the origin of those
mutations which, according to recent speculation, constitute
the raw material with which evolution works. But for the
solution of this problem animals must be selected, which are
easier to breed and in which the infant mortality is less than
in Echinoderms.

THE DEVELOPMENT OF THE FULL-GROWN LARVA.

We left the consideration of the normal development at
the point where the ccelom had arisen from the apex of the
archenteron as a bilobed vesicle, and the vacuolated crest had
diminished in size, whilst the postero-lateral arms were making
their appearance. By the end of the second day the crest has
entirely disappeared, whilst the postero-lateral arms have
grown so much that the organism takes on the form of a V
with a thickened point. The ccelom separates as a single
vesicle from the archenteron, while the rest of the archenteron
constitutes the definite eut (Pl. 33, fig. 29). Soon afterwards
the single coelomic rudiment divides into right and left halves,
and the stomodeum makes its appearance as a wide, shallow
pit just ventral to the place where the crest disappeared (PI. 31,
fig. 6). In fig. 30 the stomodzum is shown in the act of open-
ing into the gut, and from a comparison of this specimen with
older specimens, sections of which are shown in figs. 81 and
32, the conclusion is arrived at that, as in Echinus escu-
lentus, the V-shaped adoral ciliated band which projects

volt. D1, PART 4,—NEW SERIES. 45

576 EK. W. MACBRIDE.

into the cesophagus is formed partly from ectoderm and partly
from endoderm cells (fig. 30, Cil. ad.). As in other Echino-
derm larve the blastopore forms the anus, whilst the definitive
gut becomes divided by constrictions into intestine, stomach,
and esophagus, the last-named of which joins with the
stomodeum. On the third day the antero-lateral arms make
their appearance (fig. 31, a./.), and these are supported from
their first inception by branches given off from the primary
skeletal rods which support the postero-lateral arms. ‘The
posterior ends of these primary rods undergo a branching just
inside the vacuolated posterior end of the larva, which is
characteristic of Ophiothrix fragilis. Hach rod branches
into anterior and posterior spikes, and each of these divides
into a dorsal and ventral branch, so that by the juxtaposition
of right and left halves of the skeleton a kind of basket is
produced. In none of the figures is the forking into dorsal
and ventral spikes shown, but the division into anterior and
posterior branches is shown in many of the figures (cf. figs.
6-12).

When once right and left coelomic sacs have been established
from their inner walls muscular fibres grow out and surround
the cesophagus (fig. 31, musc.). Thus constrictor muscles are
provided by means of which swallowing movements can be
carried on and the larva becomes able to feed. At the same
time the madreporic pore is formed. As shown in the trans-
verse sections represented in figs. 35 and 36, an ectodermic
ingrowth (m.p.) consisting apparently of two large clear cells,
meets a short outgrowth (p.c.) from the left coelomic sac, and
by the fusion of the two a tube is established which leads
from the left coelomic sac to the exterior. This tube may be
termed the pore-canal, whilst its opening may be called the
primary madreporic pore. ‘This pore, as in Hchinus
esculentus, is at first markedly on the left side of the larva,
but subsequently shifts so as to reach the mid-dorsal line ;
indeed, in Ophiothrix it even passes beyond the mid-dorsal
line. The left coelomic sac has a wider lumen than the right,
thus foreshadowing its eventual conversion into the so-called

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 577

“ampulla” of the stone-canal, the equivalent of the axial sinus
in Asterina gibbosa. About the fourth day the post-oral!
arms make their appearance and the ventral side of the larva
becomes markedly concave. ‘The new arms are supported by
outgrowths from the primary skeletal rods, which arise at the
same point as those which support the antero-lateral arms.
The left coelomic sac now grows out into a posterior tongue of
cells at first solid (fig. 32, l.p.c.), which soon hollows out to
form a vesicle (fig. 33). This vesicle becomes separated from
the left coelomic sac as the left posterior ccelom, and moves
back so as to be at the side of the stomach (fig. 8, /.p.c.), whilst
the original sac now termed the left anterior ccelom (fig. 8,
l.a.c.) remains at the side of the cesophagus. About the fifth
day the right coelomic sac undergoes a similar series of changes.
The solid posterior outgrowth is shown in fig. 34, whilst in
fig.8 is represented a specimen in which this outgrowth (7.p.c.)
having become a hollow vesicle is in the act of separating
from the original sac, now termed the right anterior coelom
(r.a.c.), which remains at the side of the cesophagus. In fig. 9,
a slightly older specimen is shown in which the division is
complete on both sides. Thus in Ophiothrix fragilis as
in Asterina gibbosa and in Hchinus esculentus the
transverse division of the coelom of the left side precedes that
of the right, thus foreshadowing that predominance of the
left side which plays such an important part in the meta-
morphosis.

The larva now continues to grow in size without under-
going any important changes for a day or two, but at a
period varying from seven to ten days from fertilisation,
according to the amount of food-supply present in the water,
the last pair of arms, the postero-dorsal * (figs. 10 and 11, p.d.)

1 Owing to an unfortunate misunderstanding of Mortensen’s paper (22) the
arms of the larva of Echinus esculentus are wrongly named in my paper
on the subject (19). What I have called in that paper postero-dorsal arms
are really post-oral and vice-versa.

2 These arms correspond to those arms of the larva of Echinus
esculentus, wrongly named post-oral by me in my paper on the subject.

578 EK. W. MACBRIDE.

make their appearance. They are supported by branches
from the rods which support the antero-lateral arms. At
the same time the posterior ends of both the right and the left
anterior coelomic sacs become thickened and the thickenings
are hollowed out so as to form vesicles (fig. 37a). These
vesicles are the rudiments of the right and left hydroceles
respectively. On the left side the vesicle becomes constricted
from the left anterior coelom (figs. 10 and 11), so that it is
connected therewith by a narrow neck only, which is the
rudiment of the stone-canal. On the right side no such con-
striction takes place, but the cavity of the right hydroceele
becomes entirely shut off from that of the right anterior
coelom (figs. 38a and )). Bury supposed (5) that the
left hydroccele was formed by an outgrowth from the left
posterior colom. This supposition is an _ exceedingly
natural one, for it corresponds with the appearances presented
by slightly older larve than those which we are at present
discussing. The left posterior ccelom grows forward so as to
become closely apposed to the rudiment of the future hydro-
cole, and an observer who had not an unbroken series of
stages before him might easily imagine that the hydroccele
was being nipped off from the posterior coelom. With
regard to the right hydroccele, this is larger relatively to the
left rudiment than it is in the case of either Asterina
gibbosa or Echinus esculentus. In both the last-named
forms it occupies from the beginning a position near the mid-
dorsal line, for which reason Masterman (20) calls it the central
ceelom and identifies it with the pericardial cavity of
Balanoglossus. Goto (11) also on the same ground denies
that it is the fellow of the left hydrocceele. Butin Ophiothrix
fragilis there can be no doubt on the subject ; the structure
in question is on the right side from the beginning and
remains on the right side, and occasionally, as Miiller (25) has
figured and as is shown in fig. 53, takes on a five-lobed form.
Shortly after this period when all the larval arms have
attained their full length, the left hydroccele begins to take
on a five-lobed form (figs. 12 and 39). The five lobes soon

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 579

increase in length and become finger-shaped; they are at
first arranged in a straight line antero-posteriorly, and will
be numbered 1 to 5, commencing with the most anterior. It
is a question of considerable interest where the stone-canal
enters the hydroccele. The latter organ, as it grows, extends
forward beneath the anterior coelom and eventually reaches
beyond it, although the anterior celom was originally
situated entirely in front of the hydroccele.

The stone-canal was thought by Bury (6) to open into the
hydroccele between lobes 4 and 5. Bury regards the opening
of the stone-canal as a fixed point when comparing one class
of Echinoderms with another, and imagines that ‘ the inter-
radius in which the ring-canal closes” is the point which
varies. I confess that I cannot follow this reasoning. In
dealing with an organ like the hydroccele, which at one
period of development has an identical form in all groups
of Echinoderms, viz. an open hoop with five projecting lobes, it
seems to me impossible to evade the conclusion that the
front and hind ends of the structure correspond throughout
the phylum, and hence the point where they meet so as to
“close the ring”’ must be identical throughout the group.
The stone-canal opens on the inner surface of the hoop or
ring, whilst the lobes project from its outer surface, and
hence it is quite conceivable that the position of the opening
of the stone-canal could shift without involving any such
morphological impossibility as jumping a lobe. But before
we have recourse to this modest hypothesis it would be well
to satisfy ourselves that as a matter of fact the point where
the stone-canal enters the hydroccele is different in the
different groups. In Asterina gibbosa this point is situated
between lobes 1 and 2, and I have found that this is also true
of Ophiothrix fragilis, as the sagittal sections shown in
figs. 42a and 42 clearly demonstrate. The first figure shows
the opening of the stone-canal and its oblique course back-
wards to join the anterior ccelom ; the second shows the pore-
canal opening to the exterior. This second figure shows how
Bury’s error arose, for he did not use sections, but based his

580 E. W. MACBRIDE.

statement on views of living larvee. Now, fig. 42) shows that
the pore is opposite the interval between lobes 4 and 5, and
if one were to imagine that the stone-canal led vertically
downwards beneath the pore one would conclude that it must
open between lobes 4 and 5. With regard to the other
groups of Echinoderms, the evidence at present to hand
certainly does suggest that the opening is placed in a different
inter-radius from that in which it opens in Asteroidea and
Ophiuroidea, but until this point has been thoroughtly investi-
gated by means of sections it would be premature to give up
the hope that the rest of Echinoderms may be shown to
agree in this respect with these two groups. The intestine,
as fig. 41 shows, is a loose, baggy structure in the Ophio-
pluteus larva. The anus is kept closed, except during the
emission of faces; the cells lining the intestine differ in tone
and size from those lining the stomach, these latter being
more cylindrical and staining more deeply than those lining
the intestine.

Tae METAMORPHOSIS.

In the case of the larva of Asterina gibbosa the meta-
morphosis may be said to begin from the moment that the
larva fixes itself. At this period the hydroceele is an open
hoop with five lobes, which are just beginning to show them-
selves externally by causing protrusions of the ectoderm. In
the case of the larva of Echinus esculentus and that of
Ophiothrix fragilis, which remain swimming until the
metamorphosis is complete, it is impossible to fix in such a
way a definite point which indicates the beginning of the
change. In the larva of Asterina gibbosa, when it has
just fixed itself, the hydroccele is an open hoop; one may,
therefore, regard the beginning of metamorphosis in the larva
of Echinus esculentus as marked by the acquisition of
lobes by the hydroccele, which is at first a hoop but almost
immediately passes into the ring form. In Ophiothrix
fragilis, where the lobes appear whilst the hydroccele is
still straight, metamorphosis may be regarded as begun, by

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 581

the commencement of the encircling of the cesophagus of the
larva by the hydroceele ; in a word, by the assumption of a
hoop-like form by this organ. This stage is shown in dorsal
and ventral views in figs. 13@ and 136, and in section in
figs. 43 a and 43 b.

As yet the external symmetry, as shown by the larval arms,
is unaffected, but the first lobe of the hydroccele has extended
towards the mid-dorsal line above the stomodzal portion of
the cesophagus, whilst the fifth lobe has begun to pass beneath
the cesophagus towards the right. Corresponding to this
extension of the dorsal portion of the hydroccele goes a cor-
responding extension of the thickened side of the cesophagus,
which constitutes the left half of the adoral ciliated band.
This pushes the dorsal, thin-walled, scoop-like portion of the
cesophagus, which is part of the stomodzum, over to the right.
The beginning of this shifting is shown in fig. 13). The
outer wall of the left posterior ccelom becomes thickened and
produced into five elevations, which are the rudiments of the
arms of the adult brittle-star. Three of these are shown in
fig. 15a; besides these there are two ventral ones. These arm
rudiments consist at first of mere conical thickenings, but
each soon acquires a lumen which is an evagination of the
coelomic cavity (fig. 43a, arm 1). This lumen forms the
dorsal coelomic canal of the adult arm. The greater proportion
of the cells of the thickening seem to be destined to form the
plates which ensheath the adult arm, but as I have already
intimated, all trace of the adult calcareous structures is lost
owing to the method of preservation.

As the metamorphosis proceeds the dorsal and ventral
ends of the hydroccele extend over to the right.

Soon the external features are involved, as shown in fig. 14
and in figs. 44a, b, and ¢ (which are sections of a specimen of
the same age); the left autero-lateral arm is carried over to the
right. In fig. 44a it is seen that the madreporic pore and
left anterior coelom have shared in the movement, and have
reached the mid-dorsal line; in fig. 446 the left half of the
adoral ciliated band is seen to have increased tremendously,

582 E. W. MACBRIDE.

so that the thin-walled, scoop-like portion of the cesophagus
has been pushed completely over to the right and very much
diminished in size. In fig. 44 ¢ the lobes of the left hydroccele
are seen to project as the primary tentacles into the outer
and ventral part of the stomodeum. The left posterior
coelon has meanwhile developed two extensions which may
be termed the dorsal and ventral horns (fig. 44a, 1’. p”. ¢”,
and figs. 44b and c, I’. p’. c’). Of these the first extends
forward along the left side of the cesophagus outside the

AID pa ae f*

eames ee
Cecile C@ ) 2)

A series of diagrams to show the changes undergone by the celom. a.
Single ccelomic rudiment. 4. Right and left ccelomic sacs. c. Left
coelomic sac preparing to divide. d. Left sac divided, right pre-
paring to divide. e¢. Both sacs divided. jf. Formation of hydro-
ceeles (indicated by heavy lines). g. Further development of hydro-
ccele, and of left posterior ccelom.

lobes of the hydroccele, but only reaches as far forward as
the second lobe. The second reaches across to the right side
of the larva. Eventually the two meet and complete the
ring form of the left posterior ccelom, outside and parallel to
the ring of the hydroccele. <A precisely similar develop-
ment takes place in Asterina gibbosa, and in Echinus
esculentus the front end of the left posterior ccelom
grows in a ring-form round the stone-canal and axial sinus.
The intestine becomes diminished in size and pushed over to
the right; meanwhile its cells commence to develop large

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 583

vacuoles and thus show obvious signs of degeneration.
When matters have reached this point the animal may be
said to be in the first stage of metamorphosis. The changes

TEXT RIGS ox

R.HY.

RPC. Ni sk

Diagram showing relations of organs in a_ full-grown larva of
Ophiothrix fragilis. ap. Adoral ciliated band. a.t. Antero-
lateral arms. nv. Intestine. w.a.c. Left anterior coelom. Ly,
Left hydroceele. 1.p.c. Left posterior ccelom. M.p, Madreporic
pore. P.D. Postero-dorsal arm. p.L. Postero-lateral arms. — P.0o,
Post-oral arm. R.a.c. Right anterior celom. R.uy. Right hydro-
cele. R.p.c. Right posterior cclom. st. Stomach. stom.
Stomodeal portion of the cesophagus.

TEXT-Fic. 4.

‘SL

iNT.
Diagram showing the changes which supervene during the first period
of metamorphosis. Lettering as in preceding diagram ; in addition:
u.p’.c’, Ventral horn of the left posterior ecelom. 1.2.0”. Dorsal
horn of the left posterior ccelom.
which initiate metamorphosis are easily understood if the
two following text-figures be compared with one another.
Text-figure 3 shows the arrangement of organs in a larva

584. EK. W. MACBRIDE.

just before the metamorphosis has commenced, whilst text-
fivure 4 shows the changes which have supervened during
the first period of metamorphosis.

Whilst these changes in the relative growth of the internal
organs and in the external symmetry have been taking place,
new structures have been making their appearance. From
the inner wall of the left posterior ccelom four outgrowths are
given off which alternate with the lobes of the left hydroccele.
These outgrowths have thick walls and a narrow lumen. At
first their cavities are in open communication with the left
posterior coelom (cf. fig. 49), but this communication is rapidly
narrowed (fig. 45), and soon entirely cut off. A similar out-
erowth is given off from the anterior ccelom (fig. 44 a, p.h. 1
and 2), which insinuates itself between lobes 1 and 2 of the
hydroceele, whilst those originating from the left posterior
coelom intervene between lobes 2 and 3, 5 and 4, 4 and 5,
and (when the hydroccele ring is completed) lobes 5 and 1
respectively. These outgrowths are the rudiments of the
perihemal system of cavities and of the muscles and nerves
which originate from their walls. As already mentioned, the
tips of the hydroccele lobes now appear as tentacles freely
projecting into the outer part of the stomodeeum. Alternating
with these tentacles there appear inter-radial ridges of ecto-
derm (fig. 46, ep.), from which flaps grow out to the right and
left. The flaps of adjacent inter-radii meet one another over
the bases of the tentacles and in this way the epineural canals
or closed ambulacral grooves characteristic of the Ophiuroidea
are formed. I own that I was disappointed to find that in
this respect Ophiuroidea so closely resemble Echinoidea, for
the development which I have just described is identical with
the process of formation of the epineural ridges in Echinus
esculentus (19). We possess a fairly full geological history of
the evolution of Ophiuroidea out of Asteroidea, and of the
gradual conversion of the open ambulacral groove into a
canal by the meeting of the edges, and I expected to find
that the star-fish form would be definitely attained by the
larva before the groove became closed. Such is, however, not

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 585

the case ; the process as repeated in ontogeny is pushed back
to an earlier stage in development than that at which (to
judge from paleontology) it actually occurred in the history
of the race.

When by the meeting of the epineural flaps the epineural
canals are formed, and when at the same time the hydroccele
ring is completed by the meeting of its dorsal and ventral
ends, the second stage in the metamorphosis may be said to
be reached. Its external appearance is shown in fig. 15, which
represents a larva twenty-three days old raised from artificial
fertilisation in 1898. It will be observed that the left antero-
lateral arm has been carried over the cesophagus until it has
come into close contact with its right fellow, by the double
process of the preponderant growth of the organs of the left
side, and the shrinkage of the thin-walled forehead of the
larva which intervenes between the two antero-lateral arms.
This forehead is the very much reduced representative of the
pree-oral lobe of the Asterina gibbosa larva, and its shrink-
age corresponds to the atrophy of the pre-oral lobe or stalk
of Asteroidea. At the same time, all the larval arms except
the postero-lateral become reduced in size. The two horns of
the left posterior ccelom meet one another, and thus the arm
rudiments are brought into juxtaposition with the hydroccele
lobes. This is, however, effected in such a way that arm
rudiment 1 comes to rest on hydrocele lobe 2, whilst
hydroccele lobe No. 1, which has been carried over the
oesophagus, is supported by arm rudiment 5, which is an out-
growth from that horn of the left posterior ccelom, which has
passed under the cesophagus. Here, again, the larva of
Ophiothrix fragilis agrees with the larva of Asterina
gibbosa.

Kach hydroccele lobe has given rise to two or three pairs
of lateral lobes which are the rudiments of the first paired
tentacles, the original lobe giving rise to the terminal or
azygous tentacle of each arm. New tentacles are formed by
acropetal buds from the median tentacle. The perihemal
cavities shut off from the left posterior ccelom have expanded

586 E. W. MACBRIDE.

into V-shaped spaces, one limb of each V extending up each
arm adjacent to the cavity beneath the lobe of the hydroccele.
From the wall of this extension of the cavity are derived the
motor ganglion cells of the coelomic nervous system (fig. 50,
nc’), whilst the cells of the ectodermic nervous system
(fig. 50, m. c.) are derived from the ectoderm covering the
tentacles themselves. The first nerve-fibres are seen in fig.
50 as fine wavy lines connecting the two sets of nuclei.
Other outgrowths from the wall of this cavity, consisting of
larger cells with sparser nuclei, are in all probability to be
regarded as the rudiments of the ventral intervertebral
muscles, though in the latest stages examined by me histo-
logical differentiation had not progressed far enough to be
absolutely decisive on this point. Similarly, cellular out-
growths from the dorsal coelomic canal (fig. 48, muse.) are
in all probability the rudiments of the dorsal intervertebral
muscles.

The intestine is reduced to a small solid vestige consisting
of vacuolated cells. The dorsal thin-walled portion of the
stomodseum has completely disappeared, and the whole
cesophagus is now surrounded by a thick wall derived from
the adoral ciliated band, chiefly from its left half. In
Asteroidea and Hchinoidea a new adult mouth is formed by a
meeting of ectoderm and endoderm on the left side of the
larva; in Holothuroidea, where the larval mouth persists, it
moves (as Bury has shown [6] ) at the decisive moment of
metamorphosis to the left side; the outer thin-walled portion
of the cesophagus becomes evaginated at a later period to
form the buccal membrane or peristome, whilst the inner
thick-walled portion persists as the cesophagus. Ophiothrix
fragilis resembles in this respect the larva of Holothuroidea
in that the permanent cesophagus is formed from the inner
portion of the larval one, whilst the outer portion of the
larval cesophagus by the shrinkage of the forehead is, so to
speak, uncovered, and forms the peristomial membrane of the
adult.

The right hydroccele has, during this stage, a very variable

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 587

development. In one specimen (fig. 53), it had actually
assumed a five-lobed form, presenting a resemblance to the
shape assumed by its left fellow at an earlier period of
development. Its normal form may be regarded as that
shown in fig. 47, viz. a small vesicle with a comparatively
thin wall. But quite often it is large, thin-walled, and
conspicuous, sometimes, indeed, forming quite a projection on
the external surface of the larva. Sometimes a projection of
its inner wall is noticeable similar to that which gives it a
crescentic form in the larva of Asterina gibbosa. The
right anterior coelom has disappeared with the alteration in
form of the cesophagus whilst the left anterior coelom forms,
as it does in both Asterina gibbosa and Hchinus
esculentus, the axial sinus or “ampulla” of the stone-
canal.

As is well shown in fig. 47, the madreporic pore has been
brought close to the right hydroceele, although it was at first
widely separated from it. ‘his is due to the rotation of the
madreporic pore from its original position on the left side
over the csophagus and so on to the right side. Thus is
eventually brought about a juxtaposition of organs which in
the larve of Asterina gibbosa and Hchinus esculentus
exists from the first. The position of the right hydroccele in
these two species close to the mid-dorsal line of the larva, has
led both Goto (11) and Masterman (20) to combat my view of
its homology. But Ophiothrix fragilis is quite decisive
on the point, and a little consideration will show why the
Ophiurid in this respect retains a much more primitive
arrangement than the other two forms. In it the larval
mouth is gradually transformed into the adult mouth, and
instead of the mouth moving to the left the stomach is
displaced towards the right (fig. 47, st.) ; in the Asterid and
Kehinid, on the other hand, the permanent mouth is formed
on the left, whilst the stomach retains its original position.
The right hydroccele in these two groups is orientated with
respect to the eventual position of the permanent mouth, and
“right”? with respect to a mouth which is going to be

588 E. W. MACBRIDE.

formed on the left side of the larva is practically ‘ mid-
dorsal” with respect to the median sagittal plane of the
larva and to the Jarval mouth.

The madreporic pore in these two groups attains its adult
position before the right hydroceele is formed, so that when
the latter appears it is close to the madreporic pore.

With the disappearance of all the larval arms except the
two postero-lateral, the third and last stage of the meta-
morphosis is begun. Its external appearance is shown in
fig. 16, and a frontal section is given in fig. 48. The adult
arms have become longer and are folded ventrally over the
disc—this is not general among Ophiuroidea, but is character-
istic of Ophiothrix fragilis. ‘he oral spines which even-
tually give rise to the so-called “teeth” have already
appeared (fig. 16, sp.). In the internal anatomy the great
point to be noted is the appearance of the peri-oral coelom
(fig. 48, peri.), as an outgrowth from the left posterior
celom. ‘The peri-oral ccelom closely surrounds the ceso-
phagus (figs. 51 and 52, peri.), and corresponds to the
similarly-named space in the larva of Asterina gibbosa.
But whereas in Asterina gibbosa the wall of this space
gives rise to the ten retractor muscles which restore the
everted stomach to its place on the conclusion of a meal, in
Ophiothrix the space remains as an apparently functionless
vestige throughout life, as the stomach of an Ophiurid is not
eversible.

The formation of the primitive germ cells commences at this
period. As shown in the sagittal section represented in
fig. 51, they make their appearance as a series of large nuclei
in the wall of the left posterior coelom overlying the stone-
canal (fig. 51, gen.). The further history of these cells has
been worked out by me in my paper on Amphiura
squamata (17), to which the reader is referred. A section,
however, of one of the embryos of Amphiura squamata is
given in fig. 52, The single layer of cells shown in fig. 51 is
here seen to have become a projecting nodule (fig. 52, gen.),
the first rudiment, in fact, of the string called the genital

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 589

rachis which eventually surrounds the disc and from which
the genital organs arise as buds. The ampulla of the stone-
canal (/.a.c.) is seen in fig. 52 to be still in continuity with
the perihemal space to which it gave rise. This ampulla—
the original left anterior coelom—into which both pore-canal
and stone-canal open (cf. fig. 51) is the equivalent of the
axial sinus of Asteroidea and Kchinoidea, but has nothing to
do with the space which originally received that name in
Ophiuroidea. ‘This latter space, lettered sinus b in my paper
on Amphiura squamata, is not formed till the post-larval
period, and hence is not described in this paper. Its outer
wall separating it from the general hypogastric (i. e. left
posterior) coelom is a thin membrane, and the space itself
appears to be due (as far as I can make out from a re-
examination of my sections) to an invagination of the ccelomic
cavity into the tissue lying above the stone-canal. Now in
Asterina gibbosa the cells which give rise to the genital
rudiment are likewise invaginated, but the cavity of the
invagination is occluded. When we reflect also that the true
axial sinus of Ophiuroidea is of limited extent, the confusion
between the true axial sinus of Asteroidea and the wrongly
named axial sinus of Ophiuroidea becomes explicable. The
right hydroceele is seeu in fig. 51 as a closed space (r. hy.).
When I commenced my work on the late development of
Amphiura squamata I had made no researches on the
early development of Echinodermata, and hence I was much
puzzled by the appearance of this space, whose origin I could
not trace. After long wavering I persuaded myself on the
evidence of one or two sections that it, too, was derived from
an evagination of the general ccelom, and so it is lettered as
if it were part of sinus b, the wrongly denominated axial
sinus. Of course, this is a mistake which I endeavoured to
correct in my paper on Asterina gibbosa, and which
receives renewed correction here.

As the metamorphosis nears its conclusion the ciliated epi-
thelium covering the long postero-lateral arms begins to de-
generate, and the animal sinks lower and lower in the water;

590 Kk. W. MACBRIDE.

such larve are only captured when the tow-net is dragged
along the bottom. Soon the animal rests on the bottom
altogether, and then the ciliated epithelium is rapidly
absorbed, leaving sometimes, as fig. 17 shows, a portion of
one of the calcareous rods, which it originally covered, as a
bare, projecting spine. The arms of the young Ophiurid
are armed at the sides with hooks. ‘These hooks strongly
resemble the hooks which form the sole armature of the arms
in such primitive genera as Ophiohelus, and it is interesting
to find them recurring in the brephic stage of Ophiothrix. As
a matter of fact these hooks persist in this genus throughout
life, but as growth proceeds there is added to each one of
them a vertical row of those delicate, thorny, lateral spines
from which the genus takes its name.

COMPARISON OF THE DEVELOPMENT OF OPHIOTHRIX WITH THAT
OF OTHER ECHINODERMS.

Before discussing the bearing of the facts which have just
been laid before the reader, it is proper that a word or two
should be spoken as to the difference between my results and
those of Dr. Caswell Grave on Ophiura brevis (12). Some
of these differences, such as the rapid development, are attri-
butable to the fact that Ophiura brevis has yolky eggs
and never develops a typical Ophiopluteus larva; but others
are of greater import. ‘The origin of the ccelom, as two dis-
tinct evaginations from the archenteron, of which the hinder
forms the hydroccele and the left posterior ccelom, is un-
paralleled among Echinoderms. We can only venture to
suggest that, as Dr. Grave distinctly states that he had only
an exceedingly limited supply of material, that his interpreta-
tion of the appearances presented to him is mistaken, owing
to stages being missed out.

It is true Masterman has announced somewhat similar results
in the case of Cribrella oculata (20). Here, according to
him, the coelom originates as two evaginations, of which one
gives rise to the single anterior ccelom, characteristic of

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS, 591

Asteroidea, from which the right posterior ccelom, called by
him the epigastric ccelom, arises as does the true right hydro-
coele, termed by him the central ccelom. This tallies fairly
well with Ophiura brevis, where the anterior evagination
divides into right and left anterior cceloms, and from the right
anterior coelom Grave infers that the right posterior ccelom (the
epigastric) is subsequently derived. In Cribrella oculata
the posterior evagination, as in Ophiura brevis, gives
rise solely to the left posterior ccelom (hypogastric coelom).
The difference between the two isthatin Cribrellaoculata
the left hydroccele originates from the anterior rudiment,
whereas in Ophiura brevis it originates from the posterior
vesicle. This irreconcilable difference between these two
authors prevents us placing as much stress on the points of
agreement between them as we should otherwise feel bound
to do. When we recollect that Masterman’s results would
fall at once into line with what is known of the development
of other Asteroidea, if we make the assumption that the
supposed independent origin of the left posterior ccelom from
the gut is founded on a mistaken observation, we shall, I
think, be justified in provisionally assuming that this is the
case. All that is known of the development of the ccelom in
the classes of Hchinoderms, apart from these two papers, can
be subsumed under the formula that, in all cases, the ccelom
arises as a single bilobed evagination from the apex of the
archenteron. In the single Crinoid whose development is
known (viz. Antedon rosacea) the whole archenteron is
bodily transformed into the ccelom, which then divides into
anterior and posterior portions, whilst from the anterior
portion the definitive gut subsequently arises as a pair of
out-growths. This late appearance of the gut is rendered
possible by the fact that it is functionless in the embryo.
In Holothuroidea the single out-growth also divides first
into anterior and posterior portions, and whilst the anterior
subsequently divides into anterior ccelom and left hydroccele,
the posterior portion divides into right and left halves. The
right hydroceele is not developed. In Asteroidea (Bipinnaria),
vou. 51, PART 4,—NEW SERIES, 44,

592 E. W. MACBRIDE.

Ophiuroidea and Echinoidea, the ccelomic rudiment first
divides into right and left halves, and then these sub-
sequently divide into anterior and posterior portions. ‘This
has been doubted in the case of Asteroidea. From Agassiz’s
figures (1) some have drawn the conclusion that in the
Bipinnaria larva the right and left coelomic sacs originate as
independent evaginations of the gut. But an inspection of
these figures shows that Agassiz did not observe enough
stages to warrant such a conclusion, and Driesch (8), who
has used the larve both of Asterias and Astropecten for
experimental embryology in all cases shows a single vesicle
as the first rudiment of the ccelom. The course of develop-
ment in Asterina gibbosa (and apparently in Cribrella
oculata also) differs from that of the Bipinnaria larva in
that the anterior portion of the ccelom never divides into
right and left halves; whereas in the Bipinnaria it first
divides into right and left coelomic sacs, and then these
reunite in the pre-oral lobe to form the single anterior
coelom. It is obvious that in Asterina and Cribrella the stage
when the anterior coelom becomes divided into right and left
halves is missed out owing to the shortening of development.

If we regard the free-swimming larva as representing a
bilaterally symmetrical ancestor we may then with confidence
consider that the development of the ccelom, according to the
scheme which I have now shown to obtain in Asteroidea,
Ophiuroidea and Kchinoidea, gives us the best idea of the
condition of the ccelom in this ancestor. We must credit this
ancestor with a long pre-oral lobe, which is not found in
the Ophiurid because in Crinoidea and Asteroidea this lobe
is used as a fixing organ or stalk, and a fixed stage in the
development supplies us with the best idea of a transitional
condition between a bilaterally symmetrical animal swimming
freely and a radially symmetrical creeping animal. At the apex
of this pree-oral lobe there was a brain in the form of a neuro-
epithelial plate ; such an organ I have not been able to find
in the Ophiopluteus, but it exists in the Crinoid larva and in
the larva of Echinus esculentus and as a thickened plate

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS, 593

of cells in the Bipinnaria. The ccelom on each side was three-
lobed, and the central division in all probability was produced
into tentacles, whilst locomotion was effected by means of a
ciliated band. Now, if we survey the existing animal
kingdom in order to find some type still living which will
approximately answer to this description we are led to the
group of the Ctenophora. Here we find a group of animals
with an anterior neuro-epithelial plate, swimming by means
of cilia. Connected with the endodermic stomach or archen-
teron, as we may term it, are right and left outgrowths.
From each of these a canal runs forward into the anterior
portion of the body to end near the brain (so-called excretory
canal) whilst another runs backwards to near the mouth
(paragastric canal). The middle division of the outgrowth
leads into a tentacular canal which also gives rise to canals
which open into the meridional canals lying beneath the
ciliated ribs. Now Iam very far from suggesting that the
ancestor of KHchinoderms resembled in details a living
Ctenophore, but if Ctenophora are the remnants of a great
group of pelagic animals with considerable variation in
structure, the Echinoderm ancestor may well have belonged
to that group. The Ctenophore represents a stage before
the ccoelom had been separated from the gut or the primi-
tive mouth divided into mouth and anus. It is to me a
matter of extreme interest to find that not only have the
researches of Lang and the discoveries of Willey and others
with regard to Ctenoplana led to the conclusion that 'l'urbellaria,
and therefore all the group of Platylelminthes, are descended
from a Ctenophore-like ancestor, but that Woltereck (82),
working on the development of Polygordius, is led to the same
conclusion with regard to this animal also. This conclusion,
if justified, carries with it the consequence that the whole
of the Annelida and Mollusca, which are bound together
by the common possession of the trochophore larva, are also
derivable from this root. When such diverse lines of research
seem to converge upon one point, it really does seem as if we
were within measurable distance of gaining some idea of what

594 K. W. MACBRIDE.

the common ancestor of Coelomate animals was like. I think
it will be admitted that to call this ancestor a Ctenophore
would be extremely ill advised, as this would suggest that
in details of the arrangement of cilia, etc., it agreed with
the Ctenophora. Better, I think, to adopt the name already
suggested by me in my paper on Asterina gibbosa (18),
viz. Protoccelomata, for the ancestral group from which
Ctenophora, Platyhelminthes, Annelida, Mollusca, and Hchi-
nodermata have sprung. ‘To this group Vertebrata are also
to be traced back, since the Tornaria larva of Balanoglossus
exhibits the three-fold division of the ccelom and the apical
nervous plate, which we have determined as characteristic of
the ancestor of Echinodermata, whilst the middle division of
the coelom in the allied form Cephalodiscus is produced into
long tentacles, just as is the case with the hydroccele in
Kchinodermata.

Turning now to the later development of Ophiothrix
fragilis, the principal point to be noticed is that the ontogeny
bears out the conclusion of comparative anatomy that the
Asteroidea and Ophiuroidea are closely allied. ‘Thus in the
formation of one perihemal rudiment from the axial sinus, in
the apposition of arm-rudiment 1 and hydroccele-lobe 2, in
the opening of the stone-canal between lobes 1 and 2, in the
development of dorsal and ventral horns from the left posterior
coelom, and finally in the development of the peri-oral ccelom,
the development of Ophiothrix recalls that of the Asterid.
The points in which it differs are, first, that the fixed stage is
entirely missed out; second, that the cavities are in general
smaller and their walls thicker; third, in the development of
epineural folds; and fourth, in the persistence of the larval
mouth. Taking these points in order, the missing out of the
fixed stage is in accordance with the rule everywhere exempli-
fied in the animal kingdom, that there is a tendency to reduce
the number of larval stages. In each stage the organism is
exposed to a special set of dangers; if, therefore, enough food
can be accumulated during one stage to enable the necessary
changes in structure to be made, so that the next stage can

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 595

be missed out, this will be done. So we explain the missing
out of the Mysis stage in the embryology of the crab, for
instance, and the passing over numerous larval stages within
the egg-shell in other cases. The smallness of cavities and
thickness of the tissues derived from their walls is a general
feature of Ophiuroidea which runs throughout the entire class,
and is an adult feature retrospectively affecting development.
The epineural folds require, of course, no comment, as
they are one of the features in which Ophiuroidea have
progressed beyond Asteroidea. ‘They made their appearance
in ontogeny rather earlier than they should, and in so
far are another example of adult features affecting the
ontogeny. The retention of the larval mouth, however,
cannot be viewed otherwise than as a primitive feature.
We cannot imagine that in the history of the race a period
intervened when the previously existing mouth disappeared
and a totally new one made its appearance, but why the
mouth should persist in Ophiuroidea and Holothuroidea and
be lost in Asteroidea and Mchinoidea is a mystery. We may
notice, however, that the mouth is retained in Crinoidea also,
for though the larval stomodzum, as Bury calls it (4), never
opens into the yolky, functionless gut, yet there is no doubt
from its position of its homology with the stomodweum of
other Echinoderm larve. Now this stomodeum is shifted
posteriorly and opens out to form the vestibule of the fixed
larva, out of which the tentacles project. Asimilar fate befalls
the stomodeum in Ophiuroidea. Owing to the shrinkage
of the forehead it opens out, and the primary tentacles
of the hydroccele project through it. The same thing happens
in the development of Holothuroidea. <A difficulty may here
occur in the minds of some that, in the development of
Synapta digitata, the only Holothurid of which the whole
ontogeny is known, the first tentacles to make their appear-
ance are the buccal tentacles, not those terminating the radial
canals. But from Metschnikoff’s paper (21) it appears that as
“atrium ” (as
the stomodzeum is called), accompany them, and hence it may

the radial canals grow back, extensions of the

596 E. W. MACBRIDE.

be inferred that here, too, these terminal tentacles when they
appear, project at first into the atrium. ‘To the stomodzxum
or atrium, containing the primary tentacles, found in Ophiu-
roidea, Holothuroidea, and Crinoidea may be compared the
amniotic cavity of Echinoidea and we must: suppose that in
the last-named group the original stomodzeum is divided into
two parts—one developed in the middle line in order to
function for the larva, the other in the adult position to
protect the first tentacles.

As to Asteroidea, there occurs in the text-books a statement,
attributed to Agassiz, that in some Bipinnarie the larval mouth
persists and moves to the left. Later researches on the de-
velopment of Bipinnaria by Bury and Goto have failed to
confirm this statement, but considering of how few types the
development is known, it is quite possible that in some form
the larval mouth does persist, and the detailed study of the
development of such a form would be of rare interest. In the
meantime we can only conclude that as the groups of Asteroidea
and Ophiuroidea diverged from one another, the Asteroid
larva, whilst retaining the fixed stage, lost the primitive
features of the mouth, whereas the Ophiuroid larva retained
these features whilst losing the fixed stage, just as the lower
insects have lost the caterpillar larva although the higher
insects have retained it.

SUMMARY.

The principal points which have been brought out in this
paper may be summarised as follows:

(1) The early development of Ophiothrix fragilis varies
with the condition of the egg at the moment of fertilisation,
and the development of the unripe egg resembles in certain
features that of Ophiura brevis.

(2) The coelom originates as a single vesicle from the apex
of the archenteron, and this appears to be true for all classes
of Echinoderms.

(3) The segmentation of the ccelom proceeds along the
same lines as those already elucidated for Asteroidea and

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 597

Kchinoidea, viz. into three somites on each side, but the
middle somite on the right side is not shifted dorsally as it
is in Asteroidea and Echinoidea. This somite occasionally
assumes a five-lobed form, proving beyond doubt that it is a
right antimere of the water-vascular system.

(4) The left hydroccele is budded from the anterior division
of the left ccelom, not from the posterior division as Bury
supposed (5), and its persistent connection with the left
anterior coelom constitutes the stone-canal; this opens into
the hydroccele between lobes 1 and 2, as in Asteroidea, not
between lobes 4 and 5 as Bury asserted.

(5) The metamorphosis is initiated by a preponderant
growth of the organs of the left side, which affects the
larval arms and the sides of the oesophagus, and which not
only carries the hydroccele round the cesophagus but also the
madreporic pore and the left anterior ccelom, so that these
come to be near the right hydroceele.

(6) The perihemal system of canals originates as a series
of five hollow evaginations, the cavities being small and the
walls thick. The first originates from the left anterior
ccelom, the other four from the left posterior celom. From
their walls originate the motor ganglion cells and in all
probability the ventral inter-vertebral muscles.

(7) The left posterior ccelom gives rise to a dorsal and a
ventral horn, which eventually meet, causing it to assume a
ring-shape. From it five evaginations give rise to the arm
rudiments and the first of these comes to overlie hydroccele
lobe No. 2.

(8) A peri-oral coelom closely surrounding the adult
cesophagus originates from the left posterior ccelom. |

(9) A series of epineural ridges, the tops of which grow
out so as to form arch-like folds, are formed inter-radially
alternating with the primary tentacles. By the union of
adjacent arches the basal portions of the tentacles are
covered, exactly as occurs in the Hchinoid larva.

(10) The adult cesophagus is formed from the left inner
portion of the larval one: its covering is made of the adoral

- 598 E. W. MACBRIDE.

ciliated band, chiefly of the left half thereof, which grows
part passu with the left hydroceele. The outer part of the
larval oesophagus opens out in consequence of the shrinkage
of the forehead to form an atrium into which the primary
hydroceele lobes project.

(11) The larval intestine slowly diminishes in size and
degenerates, this process being accompanied by a vacuo-
lisation of its cells. The larval stomach becomes pushed
over to the right—a process which leads to the same result
as the moving of the larval mouth to the left in Holothu-
roidea, or the formation of the adult mouth on the left
in Asteroidea and Kchinoidea.

(12) The primitive germ-cells originate from the left
posterior coelom covering the stone-canal.

In conclusion, I have to express my thanks, first to the
University of Cambridge and to the British Association, who
granted me the use of their tables at Plymouth; next to
Dr. E. J. Allen, Director of the Marine Biological Laboratory
at Plymouth, who in the most whole-hearted and generous
manner placed the resources of the laboratory at my dis-
posal; then to my friend and colleague, Mr. J. Simpson,
B.Sc., Demonstrator of Zoology in McGill University, to
whose skill I owe a large proportion of the drawings which
illustrate this paper; and lastly to my wife, who also assisted
with the more complicated drawings, with the text-figures,
and with the general revision of the text.

List OF WoRKS REFERRED TO IN THIS Paper.

N.B.—It has not seemed to me desirable to encumber this list with
references to works dealing with groups of animals other than the
Echinoderms, such as that of Lang on the Turbellaria, or Willey on
Ctenoplana, ete., since the facts referred to have been embodied in
text-books and are the common property of all zoologists. Woltereck’s
recent paper forms the sole exception.

1. Acassiz, L.—‘Selections of Embryological Monographs, I, Echino-
dermata,” ‘Mem. Mus. Comp. Zool. Harvard.,’ vol. ix, pt. 2, 1883.

2. Arostotipes, N. C.—‘“‘ Anatomie et développement des Ophiures,”’
‘Arch. Zool. Exp.,’ vol. x, 1882.

10.

11.

12.

13.

14

15.

16.

17.

18.

19.

20.

21.

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 599

. Batrour, F. M.—‘ Text-book of Comparative Embryology,’ vol. i, 1881.
. Bury, H.—“The Early Stages in the Development of Antedon

rosacea,” ‘Phil. Trans. Roy. Soc.,’ vol. elxxix, 1888.

. Bury, H.— Studies in the Embryology of Echinoderms,” ‘ Quart, Journ.

Mier. Sci.,’ vol. 29, 1889.

. Bury, H.— “The Metamorphosis of Echinoderms,” ‘Quart. Journ.

Mier. Sci.,’ vol. 38, 1895.

. Carpenter, P. H.—‘‘On the Development of the Apical Plates in

Amphiura squamata,” ¢ Quart. Journ. Micr. Sci.,’ vol. 27, 1887.

. Driescu, H.—“ Zur Analysis der Potenzen embryonaler Organzellen,”

© Arch. Entwicklungsmechanik,’ vol. 11, 1895.

. Fewxes, J. W.—“ Preliminary Observations on the Development of

Ophiopholis and Echinarachnius,” ‘ Bull. Mus. Comp. Zool. Harvard,’
vol. xii, 1886.

Fewxes, J. W.—“‘On the Development of the Calcareous Plates of
Amphiura squamata,” ‘Bull. Mus. Comp. Zool. Harvard,’ vol. xii,
1887.

Goro, S.—* The Metamorphosis of Echinoderms, with Special Reference
to the Fate of the Body Cavities,” ‘ Journ. Coll. Sc. Imp. Univ. Tokyo,’
vol. x, 1898.

Grave, C.—“ Ophiurabrevispina,” ‘Mem. Biol. Lab. Johus Hopkins
Univ.,’ vol. iv, No. 5, 1900.

Kowatrvsky, A.— Entwicklungsgeschichte des Amphioxus lance eo-
latus,”’ ‘Mém. Acad. Imp. des Sc. St. Petersburg,’ vol. xi, series 7,
1867.

Kroun.— Ueber die Enutwickelung eines lebendig-gebarenden Ophiura,”’
‘Arch. Anat. u. Physiol.,’ 1851.

Kroun.—* Ucber cinen neuen Entwicklungsmodus der Ophiuren,”
‘Arch. Anat. u. Physiol.,’ 1857.

Lupwie, H.—‘‘ Zur Eutwicklungsgeschichte des Ophiurenskelettes,”
‘Zeit. fiir Wiss. Zool.,’ vol. xxxvi, 1881.

MacBripr, E. W.—‘“ The Development of the Genital Organs, Ovoid
Gland, Axial and Aboral Sinuses in Amphiura squamata,”
‘Quart. Journ. Micr. Sci.,’ vol. 34, 1892.

MacBripg, FE. W.—*The Development of Asterina gibbosa,”
* Quart. Journ. Micr. Sci.,’ vol. 38, 1896.

MacBripr, KE. W.—‘“‘ The Development of Echinus esculentus,”
‘Phil. Trans. Roy. Soc.,’ vol. excv, 1903.

Masterman, A. T.—“ The Early Development of Cribrella oculata
(Forbes), with Remarks on Echinoderm Development,” ‘Trans. Roy.
Soc. Edin.,’ vol. xi, pt. 2, 1902.

Metscunikorr, .—‘‘ Studien tiber die Entwickelung der Echinodermen
und Nemertinen,” ‘ Mém. Acad. Imp. des Sc. St. Petersburg,” vol. xiv,
series 7, 1869.

600 E. W. MACBRIDE.

22. Morrensen.— “Die Echinodermenlarven der Plankton Expedition,”
‘ Ereeb. der Plankton Exp.,’ Bd, 2, J.

23. Mortensen.— Die Echinodermenlarven,” ‘ Nordisches Plankton,’ ix,
1900.

24. Miier, J.—“ Ueber einige neue Thierformen der Nordsee,” ‘ Archi.
Anat. u. Physiol.,’ 1846.

25. Miuirr, J.—‘ Ueber die Larven und die Metamorphose der Ophiuren
und Seeigel,” ‘ Konig. Akad. Wiss. zu Berlin,’ 1846.

26. Miter, J.—‘ Ueber die Larven und die Metamorphose der Holothurien
und Asterien,” ‘ Konig. Akad. Wiss, zu Berlin,’ 1850.

27. Miuuer, J.—‘ Ueber die Ophiurenlarven des Adriatischen Meeres,”’
Konig. Akad. Wiss. zu Berlin,’ 1851.

28. Miuirr, J.—‘‘ Ueber den allgemeinen Plan in der Entwicklung der
Echinodermen,” ‘ Konig. Akad. Wiss. zu Berlin,’ 1852.

29. Russo, A.—‘‘ Embriologia dell’ Amphiura squamata (Sars),” ‘Atti.
dell R. Academia dell Sc. fis. e mat. di Napoli,’ vol. v, series 2, 1891.

30. Scuutzn, M.—‘ Ueber die Entwicklung von Ophiolepis squamata,”
‘Arch. Anat. u. Physiol.’ 1851.

31. Sevenka, .—“ Keimblatter der Echinodermen,” ‘Studien tiber Entwick-
lung der Thieren,’ Wiesbaden, 1883.

32. Wo.rereck, R.— Wurm-‘kopf,’ Wurmrumpf und Trochophora,” ‘ Zool.
Anzeiger,’ Bd. 28, Nos. 8 and 9, 1904.

33. Zizcuer, H. E.—* Einige Beobachtungen zur Entwicklungeschichte der
FEchinodermen,” ‘ Verh. der deutschen Zool. Ges.,’ 1896.

EXPLANATION OF PLATES 31-36,

Illustrating Professor EK. W. MacBride’s paper ‘On the
Development of Ophiothrix fragilis.”

List or ABBREVIATIONS KMPLOYED.

The Arabic numerals 1—5 are employed to denote the lobes of the left
hydroceele. When a dash is added to the numeral, thus—l’, 2’, ete.—a lobe of
the right hydroceele is indicated. The Roman numerals I—V are employed to
denote the developing arms of the adult. In all cases the numbers commence
with the lobe or arm which is situated in the larva farthest forwards and the
enumeration proceeds backwards.

al, Antero-lateral arm of larva. az. ¢. Azygous tentacle of the young
Ophiurid. 4. ¢. Buccal tentacle of the young Ophiurid. céi/. Longitudinal

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 601

ciliated band of the larva. eé/. ad. Adoral ciliated band of the larva. coe.
Undivided ccelomic rudiment. ep. Mpineural fold. ep. s. Epineural sinus or
canal. gen. Primitive germ-cells. iz¢. Intestine. /.a@.c. Left anterior
ceelom. 7. coe. Left coelomic vesicle as yet undivided. /. Ay. Left hydroceele.
l. p.c. Left posterior ceelom. @’, p’.c’. Ventral horn of left posterior ccelom.
W’. py”. ce”, Dorsal horn of left posterior ceelom. mes. Mesenchyme. mes!.
Primary mesenchyme. mes?. Secondary mesenchyme. mp. Primary madre-
poric pore. musc. Muscle-fibres. x. c. Nerve-cells of ectodermic origin.
x’, c’. Nerve-cells of ccelomic origin. 2.7. Nerve-fibres. @s. Gsophagus.
p.e. Pore-canal. pd. Postero-dorsal arm of larva. ph. Perihemal space.
ph. 1,2. Periheral rudiment originating between hydroccele lobes 1 and 2.
ph. 2, 3, ph. 3, 4, ph. 4,5, ph. 5,1. Rudiments originating between lobes
2 and 3, 3 and 4, 4. and 5, and 5 and 1 respectively. pl. Postero-lateral arm
of larva. p.o. Post-oral arm of larva. 7. ac. Right anterior celom. 7” ca.
Right coelomic vesicle as yet undivided. +r. dy. Right hydrocele. 7. p. e.
Right posterior ceelom, s&, Larval skeletal rod. sk’. Rudimeut of adult
skeleton. sp. Buccal spines, or ‘‘teeth” of adult. s¢/. Stomach. — st. e.
Stone-canal. séom. Stomodseum. vac. Vacuolated ectoderm at posterior end
of larva.

PLATE 31.

Fic. 1.—Optical section of blastula, seven hours old, reared from natural
fertilisation. x 260.

Fic. 2.—Optical section of blastula, seven hours old, reared from natural
fertilisation, but more advanced in development than that shown in fig. 1.
mes, Primary mesenchyme. x 260.

Fig.3.—Optical section of gastrula, twenty-one hours old, reared from natural
fertilisation, ‘The vacuolated crest is already strongly developed. x 260.

Fig. 4.—Ventral view of larva, thirty hours old, reared from natural
fertilisation. cd/. longitudinal ciliated band. x 260.

Fic. 5.—Ventral view of larva of same age and origin as that shown in fix. 4,
but of more advanced development. x 260.

Fic. 6.—Ventral view of larva, fifty-five hours old, reared from natural
fertilisation, 7. c@., 4. ce. Right and left undivided coelomic vesicles. p. /.
Postero-lateral larval arms. sk. Calceified rods supporting the larval arms.
x 260.

Vic. 7.—Ventral view of larva, tlree days old, reared from natural fertilisa-
tion. a.é, Antero-lateral larval arms. p.o. Post-oral larval arms. x 260.

Fic. 8.—Dorsal view of rather older larva picked out of Plankton. ‘The
larval skeleton has been dissolved out by the preservative. /.a.c., d.p.c. Anterior
and posterior halves of the left celom. r.a.¢., 7.p.c. Auterior and posterior
halves of the right ccelom, still connected. x 260.

602 EK. W. MACBRIDE.

Fic. 9.—Dorsal view of a larva, five days old, reared from natural fertilisation.
The ccelomic vesicle is completely divided on both sides into anterior and
posterior halves. m.p. Primary madreporic pore. xX 260.

Fic. 10.—Dorsal view of a larva, six days old, reared from natural fertilisa-
lion. p.d. Rudiments of postero-dorsal arms. 1. Ay., /. Ay. Rudiments of right
and left hydrocceles respectively. x 260.

Fic. 11.—Dorsal view of a larva, eight days old, reared from natural
fertilisation. The postero-dorsal arms have acquired a fair length. x 150.

PLATE 32.
Fic. 12.—Ventral view of a larva, sixteen days old, reared from artificial
fertilisation, All the arms have attained their full length. 1—5. Lobes of the

left hydrocele, which has grown forward beyond the left anterior ccelom.
x 150.

Vics. 13 @ and 6.—Dorsal and ventral views respectively of a larva picked
out of Plankton, in which the metamorphosis is just beginning. ‘The pre-
servative has dissolved out the larval skeleton. 1—5. Lobes of the left hydro-
cele, which is beginning to extend ventrally under the cesophagus ; I—LV.
Rudiments of three of the arms of the adult brittle-star. cz¢. Lutestine.
st. Stomach. sfom. Stomodeum. x about 100.

Vic. 14.—Ventral view of a larva picked out of Plankton, in which the
metamorphosis is more advanced than in the preceding figures. The prepara-
tion was somewhat opaque, and the larval skeleton has been dissolved by the
preservative. Observe that the left antero-lateral arms and lobes 1 and 2 of
the left hydroccele have passed over the esophagus. x 100.

Fic. 15.—Ventral view of a larva twenty-three days old, reared from arti-
ficial fertilisation, in which the metamorphosis is very far advanced. All the
larval arms except the postero-lateral are reduced in size. az. ¢. The terminal
tentacle of one of the radial water-vascular canals. 4. ¢. Buccal tentacle.
x 100.

Fic. 16.—Ventral view of a larva of the same age as foregoing, but of more
advanced development, reared from artificial fertilisation. All the larval arms
except the postero-lateral arms have disappeared ; the developing adult arms
are bent over the dise. ‘The spines of the mouth-angles (sp.) have made their
appearance. X 100.

Vic. 17.—Ventral view of a young brittle-star which has just completed its
metamorphosis. Artificial fertilisation, twenty-six days. p/. The calcareous
rod of one of the two postero-lateral arms which have now disappeared. The
hooks characteristic of the brephic stage of Ophiothrix fragilis are seen
attached to the sides of the arms, x 100.

Fig. 18.—Section of a segmenting egg. Artificial fertilisation. x 500.

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 603

Fre. 19.—Longitudinal section of a blastula. Artificial fertilisation. mes.
Primary mesenchyme filling the interior. x 500,

Fie. 20.—Longitudinal section of a blastula in which gastrulation is about
to commence. Artificial fertilisation. mes’, Primary mesenchyme filling the
interior. mes®. Secondary mesencliyme budded from the endodermic areca.
x 500.

Fig. 21.—Longitudinal sagittal section of a gastrula. Artificial fertilisa-
tion. Notice the tongue of endodermic cells projecting into the archenteron.
x 500.

PLATE 38.

Fic, 22.—Longitudinal frontal section of a larva two days old. Artificial
fertilisation. p.é. First rudiment of one of the postero-lateral arms. x 500.

lic, 23.—Longitudinal frontal section of a larva two and a half days old,
Artificial fertilisation. ca@. Colomic rudiment still opening into the archen-
teron. xX 500.

Fic. 24.—Longitudinal section of blastula seven hours old. Natural fertilisa-
tion. mes. Beginning mesenchyme. xX 500.

Fie. 25.—Longitudinal frontal section of incipient gastrula, one day old.
Natural fertilisation. x 500,

Fic. 26.—Longitudinal frontal section of more advanced gastrula, one day
old. Natural fertilisation. x 500.

Fic. 27.—Longitudinal frontal section of gastrula of one and a half days.
Natural fertilisation. x 500.

Fie. 28.—Longitudinal frontal section of larva, two days old. Natural
fertilisation. Notice the origin of the ceelom. ca@. Coelomic rudiment. p. l.
Rudiment of the postero-lateral arm. x 500.

Fie, 29.—Longitudinal frontal section of larva, two days old. Natural
fertilisation. Notice that the ccelom is separated from the archenteron as a
single vesicle. x 600.

Fie, 30.—Longitudinal frontal section of a larva two and a half days old.
Natural fertilisation, The stomodzum is in the act of opening into the ceso-
phagus. @s. Gsophagus. stom. Stomodeum. x 500.

Fie. 31.—Longitudinal frontal section of a larva picked out of Plankton.
Estimated age three and a half days. mase. Muscular fibres encircling ceso-
phagus developed from coelomic sacs. x 260.

604 E. W. MACBRIDE.

PLATE 34.

Fic. 32.—Longitudinal frontal section of a larva picked out of Plankton
Estimated age four and a half days. 2. p.c. Outgrowth from the left ecelomic
sac, which will be cut off as the left posterior celom. al. Antero-lateral arm,
x 260.

Fie. 33.—Longitudinal frontal section of a larva four and a half days old.
Natural fertilisation. U.p.c. The rudiment of the left postericr celom. It
has now assumed a rounded shape. X 260.

Fic. 34.—Longitudinal frontal section of a larva picked out of Plankton.
Estimated age five days. 7.p.c. Outgrowth from right ecelomic sac, which will
be cut off as the right posterior celom. x 260.

Fic, 35.—Transverse section of a larva three days old. Natural fertilisation.
mp. Hetodermic invagination to form the madreporic pore. x 500.

Fic. 36.—Transverse section of a larva three days old, more advanced in

development than that shown in the preceding figure. Natural fertilisation.

mp. Ketodermic invagination to form madreporic pore; this has now fused
with an outgrowth from the left coelomic sac. s¢. Stomach. x 500.

Fics. 37 a and 6.—Two longitudinal frontal sections through a larva picked
out of Plankton of estimated age fourteen to sixteen days. Fig. 37a is the
more dorsal section passing through the cesophagus. Fig. 374 is a more
ventral section passing through the stomach alone. 7. Ay., 7. hy. Rudiments of
the right and left hydroceles respectively. pd. Postero-dorsal arms: the
last to appear. x 260.

Fries. 88 a and 6.—Two longitudinal frontal sections through a larva some-
what older than that represented in fig. 37, picked out of Plankton. Fig. 38a
passes through the left hydroccele, fig. 38 4 through the right hydroceele. The
latter is the more dorsal section. x 260.

Fie. 39.—Longitudinal frontal section through a larva picked out of
Plankton, still older than that shown in fig. 38. The left hydroccele is just
beginning to be lobed; 1—5. Its lobes. x 260.

Vic. 40.—Longitudinal frontal section through a larva picked out of
Plankton older than that shown in fig. 39. The lobes of the left hydroccele
are finger-like. r.a.¢c., d.a.c. Right and left anterior cceloms, just grazed.
cil. Longitudinal ciliated band. cil. ad. Adoral ciliated band. x 260.

Fic. 41.—Longitudinal median sagittal section through a larva picked out
of Plankton, about the same age as that shown in fig. 40. x 260.

PLATE 35.
Fies. 42 a and 4.—Two longitudinal sagittal sections through a larva

picked out of Plankton, of same age as that shown in the preceding figure,
Fig. 42 a is more to the left of the middle line than fig. 42 6. Fig. 42 @ shows

THE DEVELOPMENT OF OPHIOTHRIX FRAGILIS. 605

the opening of the stone-canal into the left hydroccele between lobes 1 and 2.
Fig. 42 6 shows the opening of the pore-canal to the exterior. m.p. Madre-
poric pore. sé. c. Stone-canal. xX 260.

Fics. 43 a and 6.—Two longitudinal frontal sections through a larva picked
out of Plankton, in which there is the first discoverable trace of metamorphosis.
Fig. 43 @ is the more dorsal section, and passes through lobes 1 and 5 of the
left hydroccele, whilst fig. 43 6 passes through lobes 2, 3, and 4, thus showing
that the hydroceele is commencing to encircle the esophagus. In fig. 43 @
the right hydroccele (7. hy.) is grazed, and the thickening of the outer wall of
the left posterior ccelom, which is the first rudiment of an adult arm, has made
its appearance. The prolongation of the lumen of the ceelom into the thicken-
ing is the origin of the dorsal ccelomic canal of the arm. Pari passu with the
extension of the left hydroccele goes the extension of the left half of the adoral
ciliated band (cil. ad.). x 260.

Fics. 44 a, 6, and c.—Three longitudinal frontal sections through a larva
in the first stage of metamorphosis. Fig. 44a@ is the most dorsal section,
fig. 44c the most ventral. Fig. 44a@ shows the pore-canal (p.c.) opening
into the left anterior ccelom (/. a. ¢.) from which is given off the perihzmal
rudiment (p4.1,2). Both left dorsal (2. py”. c”.) and right ventral (/’. yp’. c’).
horns of the left posterior coslom are seen. From the former is given off the
periheemal rudiment (pA. 2, 3), from the latter the perihemal rudiment
(ph. 3,4). The right hydroccele is seen, as are also the adult arms I and
IT, which now show as external protuberances. In fig. 44 8 all five lobes of
the left hydroccle are shown. ‘The stomodum, or thin-walled portion of the
cesophagus, is pushed over to the right, owing to the enormous growth of the
adoral ciliated band. p. d. Left postero-dorsal larval arm carried forward by
the preponderant growth of the left side. In fig. 44 ¢ the tips of the lobes of
the left hydroceele are seen projecting into the stomodeum. Each lobe is
covered with an ectodermal thickening. ‘The left antero-lateral arm has passed
over towards the right, so as to assume a mid-dorsal position. The opening
of the stomach into the intestine is shown, the different histological characters
of these two portions of the alimentary canal being strongly marked. x 250.

PLATE 36.

Fie. 45.—Longitudinal frontal section through a larva of about the same
age as that shown in fig. 44. Picked out of Plankton. ‘The rotation of the
left antero-lateral larval arm has not proceeded so far as in fig. 44. All five
lobes of the left hydroccele are shown, ‘Three perihemal rudiments are seen ;
one of them (pA. 3, 4) appears as a hollow outgrowth of the left posterior
celom. The stomach (s¢.) is seen opening into the intestine. External to
the right posterior coelom a mass of mesenchyme cells is seen—probably the
rudiment of one of the adult plates. x 250.

606 E. W. MACBRIDE.

Fic. 46.—Longitudinal frontal section through a larva of about the same
age as that shown in figs. 44 and 45. Picked out of Plankton. The epineural
folds (ep.) are shown; these roof over the ambulacral grooves (i. e. the sur-
faces of the primary lobes of the hydrocele) and give rise to the epineural
canals. X 250.

Tic. 47.—Longitudinal frontal section of a larva picked out of Plankton in
the second stage of metamorphosis. The opening of the stone-canal into the
water-vascular ring is shown; the pore-canal is indicated by dotted lines, as it
does not lie in the plane of the section. The evagination of the left posterior
ceelom to form the dorsal canal of the fourth arm is shown (arm IV). x 250.

Fic. 48.—Longitudinal frontal section of a larva picked out of Plankton in
the third and final stage of metamorphosis. a./. Disappearing remnants of the
antero-lateral arms. p.d. Remnant of right postero-dorsal arms. peri. Peri-oral
coclom originating as an outgrowth from the left posterior ccelom. muse.
Muscles originating from dorsal canal (arm-ccelom) of arms IV and V.
x 250.

Fre. 49.—Portion of a longitudinal frontal section of a larva picked out -of
Plankton in the first stage of metamorphosis. ps. 4.5. A perihemal rudiment
showing its origin as an evagination of the left posterior celom. sé, Stomach.
x 466. .

Fie. 50.—Portion of a longitudinal frontal section of a larva picked out of
Plankton in the second stage of metamorphosis. ep.s. Epineural canals. muse.
Muscular cells originating from the walls of the perihamal space (pA. 3. 4.).
n.c. Ganglion cells originating from the ectodermic wall of the epineural canal.
n.'c' Ganglion cells originating from the wall of the periheemal space. 2./. First
nerve-fibres. p./. Postero-lateral arm cut obliquely. x 466.

Fic. 51.—Sagittal longitudinal section of a larva picked out of Plankton
in the third stage of metamorphosis. gez. Primitive germ-cells originating
from the wall of the left posterior celom. x 233.

Fie. 52.— Sagittal section through a young Amphiura squamata ‘25 mm.
in diameter taken from the maternal brood-pouch. muse. Muscular fibres
developing from the cells lining a tentacle. gez. Primitive germ-cells forming
a swelling, which is the beginning of the genital rachis. x 233.

Fic. 53.—Longitudinal frontal section through an abnormal. larva of
Ophiothrix fragilis in the second stuge of metamorphosis picked out of
Plankton. 1’, 2’, 8’, 4’, 5’. Lobes of abnormally developed right hydroceele.
x 178,

SEGMENTATION OF THE HEAD OF DIPLOPODA, 607

On the Segmentation of the Head of Diplopoda.

By

Margaret Robinson,
University College, London.

With Plate 37 and 6 Text-figures.

INTRODUCTION.

Taroucn the kindness of Professor Minchin and Mr.
Pocock I have been able to examine embryos and larvee of a
South African species of Archispirostreptus from Port
Elizabeth. The adult specimens were obtained by Mr.
Pocock for the Zoological Society, and the eggs were laid in
the insect house at the Society’s gardens.

The work was undertaken with a view to finding out, from
embryological evidence, the number of segments and
appendages concerned in the formation of the Diplopod
enathochilarium. Unfortunately none of the larve from
the two batches of eggs that were laid lived long enough to
allow the development of the gnathochilarium to be observed.
The examination of some of the embryos has, however, led
to one or two interesting results, and incidentally to the
solution of the gnathochilarium problem itself.

Many thanks are due to Mr. Pocock, who spent much of
his valuable time in preserving the earlier stages daily.

HISTORICAL SKETCH.

In an account of the developmental stages of several
Chilognatha, Metschnikoff (1874) states that in them the
VoL, 51, PART 4,—NEW SERIES, 45

608 MARGARET ROBINSON.

mouth parts are only two in number; namely, mandibles and
one pair of maxille. Heathcote’s (1888) observations con-
firm this statement as also do those of vom Rath (1886),
whose dissertation unfortunately I have been unable to see.
While Heathcote merely mentions the fact of there being
but one pair of maxille, Metschnikoff insists upon it as being
important, and includes it among other points of resemblance
between the Poduridz and the Chilognatha.

More lately, zoologists have been quite awake to the
significance of there being only two pairs of mouth parts in
the Chilognatha, though but few observations have been
made on the development of these myriapods. Folsom (1900)
quotes Packard (1883) as saying that there can only be two pairs
of oral appendages ; but adds that he (Folsom) would suggest
that further embryological studies on Diplopods might show
more. In his summary he homologises the Diplopod gnatho-
chilarium with three Hexapod somites, namely, those of the
superlingue, maxille, and labium.

Korschelt and Heider (1898) also, in their text-book quote
Metschnikoff and vom Rath, and allude to the necessity for
further investigation into this matter.

Heymons (1897), in an account of the embryo of Glomeris,
states very decidedly that its gnathochilarium is formed of
only one pair of appendages and the hypopharynx (hypo-
stome), but ventures no surmises as to the origin of the
hypostome.

Silvestri (1898), in describing the larva of Pachyulus
communis says that though there is only one pair of
appendages in the gnathochilarium yet two sterna are also
concerned in its formation; namely, the sternum of the
maxillary segment and that of the post-maxillary segment,
and that it is this latter sternum which corresponds with the
hypostome of Latzel. his implies the existence of two
maxillary segments, though one of them is without appendages,

The fact that only two jaw-bearing segments were known
in Diplopods was one of several reasons given by Professor
Lankester (1903), for placing the Diplopods (Dignatha

SEGMENTATION OF THE HEAD OF DIPLOPODA. 609

monoprosthomera) far away from the Chilopods, Hexapods,
and Crustacea (Pantagnatha triprosthomera), in his
recent classification of the Arthropoda.

The latest writer on the subject, Carpenter (1905), has
found what he interprets as two pairs of maxille in the
gnathochilarium of Polyxenus (adult). He also expresses
the confident anticipation that the median parts of the
enathochilarium will be found to belong to the post-maxillary
segment. This would imply the existence of three maxillary
segments in Diplopoda.

Among the recent writings on the segmentation of the
Diplopod head it seems only necessary to mention those of
Professor Lankester (1903), Heymons (1901), and. Carpenter
(1905).

Professor Lankester, considering the head as monopros-
thomerous, places the Diplopoda far away from the Chilopoda,
Hexapoda, and Crustacea.

Heymons considers the differences between the Diplopod
head and those of the other groups to be not radical, for he
admits the existence of a tritocerebrum in the adult Diplopod
(St. Rémy, 1890), and also because he would include the
post-maxillary segment in the head, considering it as a
rudimentary maxillary segment, thus giving the head six
segments as in the other tracheates.

Carpenter too is unable to consider the Diplopod head as
monoprosthomerous, partly because he believes in the
existence of an ocular segment, and also because he is in
hopes of a tritocerebral segment being demonstrated in an
embryo millipede.

MetHops.

The eges were fixed for the most part in Perenyr’s fluid,
but for some corrosive sublimate and acetic acid were used.
Perenyi is excellent for hardening the yolk, but the second
method gives far better histological results.

T can fully endorse Folsom’s (1900) statement that the

610 MARGARET ROBINSON.

best and easiest way of observing the minute embryonic
appendages is to study the embryos whole with simply their
shells removed. When this has been done sections can be
cut, and they are of great use in giving complementary and
explanatory evidence. The whole embryos were stained with
Delafield’s hematoxylin, the sections with Kleinenberg’s
hematoxylin and orange.

DESCRIPTION OF THE STAGES.

My observations were made chiefly on the two embryonic
stages which are described below.

Stage A (fig. 1).

At this stage one can see on the ventral surface of the
blastoderm :

(1) The mouth which has the appearance of a semicircular
pore.

(2) The clypeus lying in front of the mouth, and already
showing division into two lobes. |

(3) A rudimentary brain consisting of three lobes on either
side.

(4) An incomplete nerve ring on which lies one pair of
post-oral ganglia.

(5) Two other pairs of post-oral ganglia.

(6) A large pair of procephalic lobes.

(7) Four pairs of rudimentary post-oral appendages.

(8) Markings or grooves showing divisions between the
segments in which the appendages lie.

The nerve-ring, with its thickenings, is here very minute.
I have represented it (fig. 1) and them as being slightly
raised above the rest of the blastoderm. This is done to
emphasise their appearance.

HWeymons (1901), in his account of the development of

SEGMENTATION OF THE HEAD OF DIPLOPODA. 611

Scolopendra, describes the primitive brain as consisting
of three lobes on either side. In his drawing, which, like
mine, is somewhat diagrammatic, the lobes lie more in series
than do those shown here (PI. 37, fig. 1). Nevertheless, there
is a great similarity between the two brains.

It seems that we have in the Archispirostreptus embryo
an archicerebrum, in Professor Lankester’s (1881) sense of
the word (Heymons uses the words “ archicerebrum” and
“syncerebrum” with a different meaning), and one cannot help
being reminded of the fact that in the Chetopod brain, also
an archicerebrum, there are three areas (Pruvot, 1885, and
Racovitza, 1896).

The first post-oral ganglion, which is here very small, con-
sisting of a just visible expansion of the thickening, is that
belonging to the first post-oral segment which bears the
antenna. I can find no trace of a pre-antennary segment in
this embryo.

The segment which lies immediately behind the imperfectly
closed nerve-ring is apparently the tritocerebral segment
(intercalary segment of Heymons and others). It has no
appendages, but shows distinct rudiments of a pair of ganglia,
and is definitely cut off by grooves from the antennary
segment in front and the mandibular segment behind.

The “intersegmental furrows” (Heymons) seem here to
appear first in the median region of the blastoderm, while in
Scolopendra, according to Heymons (1901) they make
their first appearance laterally.

The rudimentary nervous system is here extremely small and
difficult to make out. From the deep way in which it stains
with Delafield’s hematoxylin, I believe it to be as yet merely
a thickening of the ectoderm.

Behind the mouth, a very little way in front of the first
“‘intersegmental furrow,” there is a very slight expansion of
the thickening on both sides of the nerve-ring. This expansion
I take to be the first indication of the antennary ganglion.
The ring is not completely closed, but the two cords lie very
near each other behind the imperfect closure. These cords

612 MARGARET ROBINSON.

have a rather wavy outline all through their length, but after
much careful observation I was able to make out in each cord
a dip or incurving, surrounded by a semicircular thickening,
of the cord. This incurving lies about half-way between the
imperfect closing of the nerve-ring and the second “ inter-
segmental furrow ”—i. e., in the middle of the second post-
oral segment. This appearance is repeated in the mandibular
segment. These semicircular, thickened incurvings of the
cords must be the rudimentary tritocerebral and mandibular
ganglia.

One cannot help being struck by the well-marked double-
ness of the nervous system shown here. In Scolopendra,
according to Heymons, the median unpaired ectodermal
thickening (which he calls the archicerebrum) is the part of
the nervous system which appears first. Later on it forms a
junction between the two halves of his syncerebrum, ultimately
becoming fused with them. These two halves, therefore, are
never separated from each other by non-nervous tissue. But
Heathcote (1886), if I understand him rightly, records a
double origin for the nervous system of Julus, and
Metschnikoff (1874), with more clearness, speaks of the first
rudiment of the brain in Strongylosoma as consisting of
two “ Scheitelplatten.”

I do not believe that the tritocerebral segment has, up till
now, been observed in a Diplopod embryo. It is, however,
not surprising to find it, since St. Rémy (1890) has described
a well-marked tritocerebrum in the brains of Julus and
Glomeris (adult).

Before leaving the future head, a word or two must be said
about the procephalic lobes. They resemble those found in
embryonic insects, arachnids, etc., and those figured by
Heymons (1897) in the embryo of Glomeris. Their size
would seem to me to almost preclude the possibility of find-
ing a separate pra-antennary segment here.

Behind the tritocerebral segment lies that of the mandibles,
and following that two segments bearing maxille. I have not
been able to demonstrate the ganglia belonging to these last

SEGMENTATION OF THE HEAD OF DIPLOPODA. 613

two segments. They have probably not yet made their
appearance.

The antennz and mandibles are slightly raised above the
rest of the blastoderm, and each of these appendages shows a
slight depression in its centre. The anterior pair of maxille
are less rounded and smaller than the second pair, and they
also lie rather nearer to the middle line. They are most likely
homologous with the maxillule or superlingue described by
Hansen (1893) and Folsom (1900) in some of the Thysanura
aud Orthoptera. They are also the homologues of the first
maxillz in Chilopoda and the first maxille in Crustacea.

I am guided to the making of this homology mainly by
Hansen’s (1893) observation that the structure of the first
maxille in Machilis agrees with that of the second maxilla
in the Eumalacostraca, though I am well aware that serial
position is of more importance than structure in such a case
as this.

Folsom’s (1900) account of the development of the maxillule
or superlinguz in Anurida is somewhat unsatisfactory, for he
says that they do not make their appearance until some little
time after the maxillee have become evident. This statement
may, however, be due to an error in observation, and the
ultimate position of these small appendages is between the
mandibles and the first maxille.

Another piece of evidence which, though structural, seems to
me to count fora good deal is this: —My observations on the de-
veloping Archispirostreptus have enabled me to giveample
confirmation to Heathcote’s (1888) statement as to the meso-
dermal origin of the salivary gland in Diplopoda. This gland,
then, must be a ccelomoduct, and is most probably homologous
not only with the salivary gland in Peripatus, but also with
maxillary gland (in the second maxilla) in Crustacea.

Hansen has recently (1903) found maxillule in Scolo-
pendrella, and this gives another reason in favour of the
above-mentioned homology ; for if the Symphyla be not true
Diplopods they are, at any rate, very nearly related to them,
as they are also to the Thysanura.

614 MARGARET ROBINSON.

I would, therefore, arrange the mouth parts in the four
groups thus:

Hexapoda. Crustacea. Diplopoda. Chilopoda.
Mandibles. Mandibles. Mandibles. Mandibles.
Maxillule (Hansen First maxillx. First maxille. First maxille,

and Folsom).

First maxille. Second maxille. | Gnathochilarium. Second maxille.
Labium. First maxillipede. Post-maxillary seg- Maxillipede.
ment.

In stage B the size of the first maxillze as compared with
that of the other appendages is much reduced, and no trace
of them can be found in the mouth parts of older larva. My
material was very scanty, and only three series of longitudinal
sections through stage B were cut. In each of these series,
however, one could see the first maxillee apparently in process
of fusion with the mandible. Perhaps it would be better to
say that one could see it being absorbed by the mandible
(fig. 4). And just as one could see the apparent absorption
of this maxillula by the mandible, so also could one see the
absorption of its ganglion by the mandibular ganglion (fig. 5).
Provided that any fusion of ganglia or appendages takes place
the above is the manner in which one would expect it to do
so. For the tritocerebral rudiments undoubtedly join the
deuterocerebrum which lies in front of them, and not the
mandibular ganglion which lies behind them, and the
general tendency of fusion in the Arthropoda is from
behind forwards.

If, as I believe, these maxillule fuse with or become part
of the mandibles, they can hardly be homologous with
the maxillula found by Carpenter (1905) im the adult
Polyxenus, for the inferences which he draws from his
observations on the mouth parts of that Diplopod would lead
one to suppose that the maxillule there had fused with the
maxille during the growth of the gnathochilarium.

This matter certainly requires further investigation, though
it seems, for reasons which are given below, extremely

SEGMENTATION OF THE HEAD OF DIPLOPODA. 615

improbable that more than one pair of appendages are con-
cerned in the formation of the gnathochilarium.

Stage B (fig. 2).

This is the stage which immediately precedes the breaking
and final casting off of the egg-shell. In an external view of
it there can be seen:

(1) The three-lobed archicerebrum.

(2) The antennary ganglia, to which the tritocerebral rudi-
ments are now joined, and which still le behind the mouth.

(3) The ganglia of the mandibles.

(4) The ganglia of the maxillule, much reduced in compara-
tive size.

(5) The ganglia of the maxillary segment.

(6) The ganglia of a post-maxillary segment which is
without appendages.

(7) The ganglia of the three segments bearing rudi-
mentary legs.

There are also to be observed the mouth and the clypeus,
as well as a pair of appendages for each pair of ganglia,
excepting that in the post-maxillary segment.

In each of the three lobes of the archicerebrum and in
every other ganglion there can now be seen a distinct
depression, a little pit in fact, the opening of which lies in
about the middle of the ventral surface of the ganglion.
Heathcote (1888) mentions these depressions as occurring in
the ganglia of the embryonic Julus, but states that they
do not appear until the ganglia have left the ectoderm. He
also says that he does not consider them to have anything to
do with the “cerebral grooves.” These ‘cerebral grooves ”
are similar cavities which he found in the ganglia of the
embryonic brain.

In my specimens I can find no difference between the
depressions in the ganglia forming the archicerebrum and
those in the ganglia of the cords; and further, these depres-
sions seem to me to appear before the ganglia have com-

616 MARGARET ROBINSON.

pletely left the ectoderm (see fig. 5), which represents a
longitudinal section through part of one of the nerve cords in
stage B). In this section there can also be seen the small
tritocerebral rudiment as well as the rudimentary ganglion
of the maxillule. This Jast is being absorbed by the mandi-
bular ganglion.

The tritocerebral rudiment here shows no depression. It
is simply a mass of nervous tissue, lying between the anten-
nary ganglion and that of the mandible. The ganglion of
the maxillula on the other hand, does show traces of a
depression, although it is fusing with, or rather becoming
part of, the mandibular ganglion.

It can be noticed that all the appendages (first maxille or
maxillule excepted) have grown in size, as also has the
clypeus. ‘he mouth has moved a little further back, but
still lies in front of the antenna and its ganglion. The first
maxillee appear very much flattened between the mandibles
and the second maxillee.

Behind the second maxillary segment there is a segment
definitely marked off from those in front of and behind it,
and showing distinctly a pair of ganglia. This is the post-
maxillary segment. Behind it there follow three segments,
each of which bears a pair of rudimentary legs. These
appendages spring from the hindmost region of the segments
in which they occur, so that they have the appearance of
belonging to the segment behind their own. Heymons (1897)
has observed a similar appearance in the embryo of
Glomeris, and, what is far more important, he has noticed in
the same embryo a post-maxillary segment which bears no
appendages. While being firmly of the opinion that there
go to the making of the gnathochilarium only one pair of
appendages and the hypopharynx, he homologises the post-
maxillary segment with the labium-bearing segment in
Hexapods, and the second maxillary segments in Chilopods.
The knowledge of the existence of a pair of maxillz in front
of the gnathochilarium compels one now to homologise these
segments differently. (See Table given above.)

SEGMENTATION OF THE HEAD OF DIPLOPODA. 617

The Gnathochilarium.

Silvestri (1898) found a post-maxillary, legless segment in
the larva of Pachyulus communis. He expresses the
opinion that the sternum of this (post-maxillary) segment corre-
sponds with the hypostome of Latzel, and that therefore the
gnathochilarium consists of the sternum and appendages of
the maxillary segment together with the sternum of the labial
(post-maxillary) segment.

Further, he says that the dorsal part of this labial (post-
maxillary) segment does not join the head at all, but forms a
neck joined to the first segment of the body. This last state-
ment is fully confirmed by my observations in Spiro-
streptus. My reasons for thinking that the sternum of this
segment does not form the hypostome are given further on.

Unfortunately none of the larvee hatched in the Zoological
Gardens lived long enough to enable the actual formation of
the gnathochilarium to be worked out. But that is now a
matter of comparatively small importance since the real
question at issue was not the formation of the lower lip, but
the number of maxillary segments present in the Diplopoda.

When the larval Archispirostreptus leaves the egg
the mandibles and the second pair of maxillee are clearly laid
down, as indeed can be seen from PI. 37, fig. 2, which represents
an embryo still within the egg-shell. The first maxillz have
disappeared, being by this time completely absorbed by the
mandibles. But the gnathochilarium is far from being fully
formed, and in this my observations do not agree with those
of vom Rath (1891), who found that the young larve of the
Julide and Glomeride left the egg with mouth parts like
those of the adult.

As the larvee grew in age and size the maxille gradually
approached each other, and in that which lived longest they
touched each other; but as yet they were not fused together,
nor could I see that they had fused with any other part of
the head, or body (see text-figures 1—4).

618 MAKGARET ROBINSON.

There were no adult Archispirostreptus at my disposal,
I have therefore made use of some adult Spirostreptus
collected by Professor Minchin in Uganda.

In the adult Spirostreptus there is behind the hypo-

Trext-Fic. l.

rn am

Mex. =

Diagrammatic plan of the mouth-parts of the larva of Archispirostreptus
five days after it leaves the egg-shell.

stome a piece of chitin, which, though larger than the hypo-
stome, resembles it in shape (text-fig. 5). The simplest way
of accounting for this would be to consider it as representing
the sternum of the post-maxillary segment. The tergum of

TextT-FIG. 2.

Mouth-parts of larva ten days after leaving the shell.

this segment is exceptionally large, and seems to represent
two terga, namely, that of the post-maxillary segment and
that of the first leg-bearing segment. ‘The segment follow-
ing that which bears the third pair of legs (fifth body segment)
is apodous in Spirostreptus.

Heathcote (1888) in his account of the development of

SEGMENTATION OF THE HEAD OF DIPLOPODA. 619

Julus says that he believes the first body segment, i.e. the
post-maxillary segment, to be the apodous one. He states
that he believes it to be equivalent to the second maxillary

TEXT-FIG. 3.

- Ant,

Mouth-parts of larva fifteen days after leaving the shell.

seoment in the Insecta, and also asserts that its ganglion
fuses early with that of the segment in front of it. I can
find no trace of such a fusion in either the embryos or larvee
of Archispirostreptus. Moreover, I have cut longitudinal

Text-Fic. 4.

== ely:

— ——Ant.

mxto7
Mouth-parts of larva twenty days after leaving the shell. dz¢, Antenna.
Cly. Clypeus. Md. Mandible. Mz.” Second maxilla.

sections through the subcesophageal ganglion in several adult
Spirostreptus, and have found it to consist of only two
pairs of ganglia (PI. 37, fig. 6), namely, those of the mandibles
and maxille.

I am well aware that the evidence is incomplete and that

620 MARGARET ROBINSON.

what is really needed is an examination of larve intermediate
in age between the oldest which I have had and the adult.
But such evidence as we have at present seems to show that

TEXT-FIG. 5.

IN

.

Cn.-
2 iMy:
fe 8 ( eesercearas Ja)
sate
Gnathochilarium from adult Spirostreptus, with hypostome as usually
present.

only one pair of appendages (the second pair of maxille) are
concerned in the making of the gnathochilarium, though
the sternum of the segment which bears them most probably

Trext-ric. 6.

Nl

~

Se

‘
ee

alee yet eet
GaSe - 58.

Gnathochilarium from adult Spirostreptus, showing double hypostome.
Gu. Gnathochilarium. Hy. Hypostome. S¢. Sternum of post-

maxillary segment.
also takes part in it. The fact that the subcsophageal
ganglion consists of only two pairs of ganglia seems to point
strongly to this conclusion,

SEGMENTATION OF THE HEAD OF DIPLOPODA. 621

In taking out the subcsophageal ganglia from several
adult Spirostreptus I naturally looked at their gnatho-
chilaria. In two of these I found the hypostome to be dis-
tinctly double (text-fig. 6). This would seem to show that it
had its origin rather in two appendages than in one sternum.

If, as above suggested, the gnathochilarium consists of but
one pair of appendages we have another reason in favour of
the homology proposed in the first part of this paper.

Of making many homologies for the segments in different
arthropods there is no end, but most authorities are agreed
in assigning six segments to the head in Crustaceans, Hexa-
pods and Chilopods. Now the head of the adult Diplopod is
very distinctly marked off from the body, being separated
from it by a narrow neck, which forms, as it were,a joint. This
is more apparent in the recently hatched larva before the
chitin has developed than in the adult (fig. 3). Even in so
young an embryo as that shown in fig. 2 the ganglia of the
head have a different appearance from those of the body.
The Diplopod head can now be shown to consist of six
segments thus:

(1) A segment which, together with the acron (Heymons),
forms the procephalic lobes.

(2) The antennary segment.

(3) The tritocerebral segment, representing the second
antennary segment of Crustacea, and tritocerebral rudiments
in Hexapoda and Chilopoda.

(4) Mandibular.

(5) First maxillary (rudimentary).

(6) Second maxillary segment, the appendages of which
are fused to form the gnathochilarium.

As the head of Spirostreptus is distinct from the body
so also, notwithstanding the forward movement of the
ganglia, is the subcesophageal ganglion distinct from that of
the post-maxillary segment. I mention this as further
evidence to show that the post-maxillary segment belongs to
the body and not to the head.

622 MARGARET ROBINSON.

SumMMARY OF RESULTS.

There are, in the embryo of Archispirostreptus, two
segments, the possession of which would seem to give
the Diplopoda a place in the Arthropod system nearer to the
Chilopoda and Hexapoda than that which has of late been
assigned to them.

These additional segments are :

(1) A tritocerebral segment representing the tritocerebral
rudiments found by Wheeler (1893) and others in Hexapoda,
and by Heymons in Scolopendra, and also the tritocerebral
segment in Crustacea.

(2) A pair of maxille (rudimentary) lying in front of the
pair which forms the gnathochilarium in the adult. These
are most likely homologous with the first maxillz in Chilo-
_ poda and Crustacea, and with the superlinguze (Folsom) of
Hexapoda.

With regard to the development of the gnathochilarium I
have unfortunately not been able to add much to our previous
knowledge. But the importance of this matter has now
become comparatively small, since the existence of a first
pair of maxilla has been demonstrated.

The evidence that I have is incomplete, but it certainly goes
to show that the gnathochilarium is part of the head, being
formed by the second pair of maxille which are the only
appendages in it; and that the post-maxillary segment of
Heymons and Silvestri takes no part in the formation of this
enathochilarium, but is purely a body segment.

May 27th, 1907.

REFERENCES TO LITERATURE.

Carpenter, G. H.—‘‘Notes on the Segmentation and Philogeny of the
Arthropoda, with an Account of the Maxille in Polyxenus lagurus,”
‘Quart. Journ. Micr. Sci.,’ vol. xlix, 1905.

Forsom, J. W.— The Development of the Mouth-parts of Anurida mari-
tima,” ‘Bull, Mus. Comp. Zool, Harvard,’ vol. xxxvi, No, 5, 1900.

SEGMENTATION OF THE HEAD OF DIPLOPODA. 623

Hansun, H. J—‘‘ Zur Morphologie der Gliedmassen und Mundtheile bei
Crustacen und Insekten,”’ ‘Zool. Anzeiger.,’ Jhg. xvi, 1898.

Hansen, H. J.—“ The Genera and Species of the Order Symphyla,” ‘ Quart.
Journ. Mier. Sci.,’ vol. xlvii, 1903.

Heatucotr, F. G.—‘The Early Development of Julus terrestris,”
op. cit., vol. xxvi, 1886.

Heatucote, F. G.—‘ The Post-embryonic Development of Julus terres-
tris,” ‘Phil. Trans. Roy. Soc.,’ vol. clxxix B, 1888.

Heymons, R.—“ Mittheilungen tiber die Segmentirung und den Korperbau
der Myriapoden,” ‘ Sitz. k. Preuss. Akad. Wiss.,’ 1897.

Herymons, R.—“ Die Entwicklungsgeschichte der Scolopendra,” ‘Zoologica,’
Bd. xiii, 1901.

KorscHELT AND Hetper.—‘ Text-book of Embryology,’ 1899.

Lankester, WN. R.—‘‘ Observations and Reflections on the Appendages and
on the Nervous System of Apus cancriformis,” ‘Quart. Journ. Micr.
Sci.,’ vol. xxi, 1881.

Lankester, 2. R.—“On the Structure and Classification of the Arthropoda,”
op. cit., vol. xlvii, 1903.

Metscanikorr, }.—‘ Embryologie der Doppeltfiissigen Myriapoden (Chilo-
gnatha),” ‘ Zeitschr. f. wiss. Zool.,’ Bd. xxiv, 1874.

Pruvor, G.—‘‘ Systéme nerveux des Annélides polychétes,” ‘Arch. de Zool.
expér. et générale,’ tom. iil, 1885,

Racovirza, E. G.—‘ Lobe céphalique et encephale des Polychétes,” ‘ Arch.
de Zool.,’ 1896.

vom Ratu, O.—* Beitrage zur Kenntniss der Chilognathen,” ‘Inaug. Diss.
Bonn.,’ 1886.

vom Ratu, O.—‘ Ueber die Fortpflanzung der Diplopoden,” ‘ Berichte
Naturforsch. Gesellsch. Freiburg-im-Breisgau,’ Bd. v, 1891.

Saint Rémy, G.—‘ Arch. de Zool.,’ Suppl. Théses, 1887-1890.

SitvesTrI, F.— Sulla Morfologia dei Diplopida III. Sviluppo del Pachyu-
lus communis,” ‘Atti R. Accad. dei Lincei.,’ vol. vii, 1898.

WueeLter, W. M.—“A Contribution to Insect Embryology,” ‘Journ. of
Morph.,’ vol. viii, 1893.

VoL. 51, PART 4,—NEW SERIES. 46

624. MARGARET ROBINSON.

EXPLANATION OF PLATE 37,

Illustrating Miss M. Robinson’s paper “On the Segmenta-
tion of the Head of Diplopoda.”

REFERENCE Lerrers.

4g. Antennary ganglion. dAné, Antenna. Are. Archicerebrum. Cly.
Clypeus. He. Ectoderm. M. Mouth. Jd. Mandible. Mdg. Ganglion of
mandibular segment. Jes. Mesoderm. Mz’. First maxilla. Mz'y. Ganglion
of first maxilla segment. Ma”. Second maxilla. Mz’g. Ganglion of second
maxilla segment. Pmx. Post-maxillary segment. Pmarg. Ganglion of post-
maxillary segment. Pmt. Post-maxillary tergum. Pre. Procephalic lobe.
Isf. Intersegmental furrow. Zire. Trito-cerebral segment. 'reg. ‘Trito-
cerebral ganglion.

Fie. 1.—Embryo of Archispirostreptus (sixteen days old) about four
days before leaving the shell.

Fic. 2.—Embryo of Archispirostreptus one day before leaving the shell

Fic, 3.—Larva of Archispirostreptus five days after hatching.

Fic. 4.—Longitudinal section through appendages of embryo shown in fig. 2.

Fie. 5.—Longitudinal sectiou through ganglia of embryo shown in fig, 2.

Fic. 6.—Longitudinal section through the subeesophageal and post-maxil-
lary ganglia in the adult Spirostreptus.

CYPRIS LARVA OF SACCULINA CARCINI. 625

The Fixation of the Cypris Larva of Sacculina
carcini (Thompson) upon its Host,
Carcinus menas.

By

Geotirey Smith, M.A.,
New College, Oxford.

With 6 Text-figures.

‘ue fixation of the Cypris larva of Sacculina has hitherto
been successfully observed only by Delage (1), and since a
considerable degree of doubt has been thrown on his account
it may be of interest to record that I have been able this
autumn, at Plymouth, to rear the Jarve and observe all
the stages of fixation upon their host. My inability to obtain
the Cypris larvee of any of the Rhizocephala at Naples had
left a gap in my observations, which I am now in a position
to fill, and thus Iam able to present a complete account of the
life-history of Sacculina from my own observations (2).

It is extremely easy to obtain large batches of the Naupli
of Sacculina or Peltogaster by selecting a crab whose para-
site is nearly ready to emit its Nauplii from the brood pouch,
and keeping it for a few days in a vessel of sea water. The
readiness of the parasite to emit the Nauplii can be judged
in Sacculina by the purple colour of the mantle, and in Pelto-
easter by the mantle becoming a paler pink than usual.

The Nauplii of Peltogaster are not actively heliotropic, but
they are negatively geotropic, and in a short time all of them
reach the surface film of the water and perish, and I have
been unable to obviate this difficulty in rearing the larve.
The Nauplii of Sacculina are actively heliotropic, but there is
no marked negative geotropism; nevertheless, with the

626 GEOFFREY SMITH.

Neapolitan races of Sacculina which infest Inachus mauri-
tanicus! (Lucas) and Pachygrapsus marmoratus
(Fabr.), I did not succeed in keeping them alive for more
than four days, or in observing the transformation into
the Cypris stage.

The Naupli of the Sacculina on P. marmoratus appeared
to be hardier than those of the parasites on J. mauri-
tanicus, and I suggested (2, p. 44) that this might be due
to the former crab being a distinctively littoral species, and
thus subjected to more varying conditions than the Spider-
crabs ; this suggestion is probably correct since the Nauplii
of the Sacculina of Carcinus menas are evidently the
hardiest of all, and this crab is more decidedly littoral in
habit than P. marmoratus.

The successful rearing of the Nauplii of Sacculina
carcini was effected by selecting “purple” Sacculine and
obtaining a batch of healthy Nauplii; they were then trans-
ferred to sea water which had been purified by mixing with
powdered charcoal and filtering in the manner used by the
Director of the Plymouth Laboratory, Dr. EH. J. Allen, in his
preparations of pure cultures.

It is of extreme importance that water of great purity
should be used, since the larve are to live in this water for
nearly a fortnight, and the multiplication of Infusoria and
Algee in the water is highly injurious to them. The larve, of
course, require no food, since they possess no gut and subsist
entirely on the yolk reserves which they contain.

The Naupli, actively swimming the whole time, undergo
four successive moults in four days; they are then transformed
by a single moult into the Cypris larve. Before these larve
fix upon their host, Delage observed that they had to spend
at least two more days in a free-swimming state; fixation
also only takes place in the dark. These two points I am
able to confirm from my own observations.

The fixation of the Cypris larvae, as followed by myself,

1 By an error in nomenclature I. mauritanicus (Lucas) was called I.
scorpio (Fabr.) throughout in my monograph (2).

CYPRIS LARVA OF SACCULINA CARCINI. 627

~takes place exactly in the manner described and figured by
Delage (1). In every case the larva was fixed by one of its
antennule to the base of a hair, most frequently on the legs of
the crab, and preference was shown for the sparsely scattered,
plumose hairs upon the flat surfaces of the proximal joints of
the legs. The larve appeared to avoid fixing on the dense
hairs which fringe the edges of the appendages. This fact
is fortunate, since the Cypris when fixed upon an isolated
hair on the smooth surface of an appendage is a most con-
spicuous and unmistakable object.

~~ The crabs which I placed with the Cypris to be infected
were about 7—15 mm. in breadth, and I selected specimens
which had recently undergone a moult, since the skin of
these individuals is clean and easy to manipulate.

Soon after fixation the Cypris larva casts away bodily all
its thoracic appendages with their attached muscles; I was
fortunate to obtain this stage, the particular specimen exactly
resembling Delage’s Plate 238, fig. 21 (2). During the next
two days a process goes on inside the Cypris shell which
leads to the formation of the Kentrogon larva. According to
Delage, the ectoderm of the larva draws away from the
Cypris shell and comes to surround a mass of mesodermal
cells lying in the anterior region of the body; the ectoderm
secretes a layer of chitin externally to the whole, the Cypris
shell falls off, and with it the degenerated remains of the
larval muscles, pigment, sense-organs, and broken-down food
material, and the so-called Kentrogon larva, a little oblong
sac encased in chitin, is left attached to the hair of the crab

__by means of the antennule of the Cypris.

Delage’s figures representing these changes were so com-
pletely reproduced in the larve observed that I have nothing
to add to his description.

With regard to the contents of the Kentrogon larva a word
is necessary. Delage considered that a layer of ectoderm is
present surrounding a mass of cells which he calls the ovary.
This conception I disputed (2, p. 43) after finding the earliest
internal stages of the parasite in which no visible differentia-

628 GEOFFREY SMITH.

tion of the cells into distinct layers or organs was present. I
therefore suggested the term “embryonic cells” to designate
the cellular contents of the Kentrogon. After observing in
several instances the formation of the Kentrogon larva, it
appears to me that the ectoderm of the Cypris is undoubtedly
included in the Kentrogon together with the mesodermal
cells, as Delage originally maintained. At the same time,
his designation of the mesodermal mass of cells as the ovary
is Obviously a misnomer, since this mesodermal mass gives
rise to all the mesodermal organs and tissues of the adult,
and, at the early stages of the endoparasitic development, is
quite undifferentiated (see 2, pp. 47 and 55).

We may, therefore, retain the term “ embryonic cells” for
the cellular contents of the Kentrogon, with the admission
that these embryonic cells are composed of embryonic ecto-
derm and embryonic mesoderm. It may be remarked that,
in this manner, the Kentrogon of the Rhizocephala almost
exactly corresponds to the post-nauplius stage of Monstrilla,
as described by Malaquin (8), which, on penetrating the
ectoderm of its host, loses all the larval organs, and consists
of a little chitinous sac containing embryonic ectoderm and
meso-endoderm, from which the adult organs are regenerated.
In Monstrilla, of course, the endoderm is not entirely sup-
pressed, as is the case in the Rhizocephala, though it is on
the way to becoming so.

This interesting parallelism between the Rhizocephala and
the Monstrillide has not been sufficiently insisted on.

In the extreme anterior region of the Kentrogon, where it
is attached to the base of the crab’s hair, I have been able to
observe the formation of the dart which gives the name to
this larval stage (kévrpov, dart; ydvoc, larva). ‘This hollow
dart, as Delage described, elongates and pierces the base of
the hair, thus opening a passage through which the embry-
onic cells of the Kentrogon can pass into the hemoceel of
the host. The dart is continuous with the inner cuticle of
the Kentrogon larva, and is presumably a product of the
ectoderm,

CYPRIS LARVA OF SACCULINA CARCINI. 629

After the formation of the Kentrogon larva with its dart,
a process obviously preparatory to the infection of the host,
the next stage in the life-history which is known is that
which I have described (2, p.47) under the name Sacculina
interna migrans.

The parasite at this stage consists of a mass of embryonic
cells undergoing rapid division by mitosis; it has an irre-
gular shape, a few small roots beginning to grow out from a
central tumour, and the whole is enclosed in a thin chitinous
cuticle. There is no visible differentiation of any of the
adult organs. It is clear that the gap between this stage
and the Kentrogon larva is very slight, since the morpho-

Text-Fies. 1 and 2.—1. Nauplius of Sacculina.
2. Cypris of Sacculina,

logical structure is practically identical. These small migrant
Sacculine were found in the hzemoccel of the crabs applied
to the upper part of the intestine just below the stomach,
that is to say, far away from the point where the body of the
adult Sacculina externais situated. This position of the
earliest known internal stage of Sacculina is completely in
accord with the indefinite position of the fixation of the
Cypris larvee upon the crab, and is irreconcilable with the
theory that the Cypris fixes upon the under surface of the
crab’s abdomen, and is transformed into the adult Sacculina
in situ.

We are still confronted with the problem how the cells of
the Kentrogon that enter the hemoccel of the crab at any

630 GEOFFREY SMITH.

point are transported to the position of the Sacculina
interna migrans. Delage, who did not observe the latter
stage, believed that the cells of the Kentrogon, on entering
the crab, at once began to send out roots, and to grow always
in a determinate direction until the central tumour reached
the point where the adult Sacculina was to be evaginated.
But, since the youngest Sacculine of the migrant stages
which I observed were not provided with elongated roots
stretching to the skin at any point where the Cypris might
have fixed, I incline to the opinion that the embryonic cells
of the Kentrogon, after entering the hzmoccel, are carried
passively about in the blood stream until they, sooner or
later, reach the large blood spaces surrounding the intestine,
and that, arrived there, they begin to throw out the root
system, while the central tumour grows down toward the
junction of thorax and abdomen where the body of the
Sacculina externa is differentiated. The method of this
differentiation, the formation of the adult organs, and the
evagination of the Sacculina to the exterior have been fully
described by Delage (1) and myself (2).

The life-history of Peltogaster, the only other of the
Rhizocephala that has been at all worked out (2), is very
similar to that of Sacculina, save that it seems probable that
the Cypris fixes upon the abdomen of the hermit-crab, and
that the migrant phase of the internal development is not so
marked.

The life-history of Sacculina may be shortly summarised as
follows :

The eggs undergo maturation in the brood pouch, and are
self-fertilised.

Development up to the Nauplius stage proceeds in the
brood pouch.

The Nauplii are expelled to the exterior, and lead a
free-swimming existence for four days, undergoing four
moults.

The Cypris stage is attained at the fifth day, and, after two
or three days of free existence, the Cypris lary attach

CYPRIS LARVA OF SACCULINA CARCINI.

Tpxt-Fies. 3—6.—Diagrams illustrating the life cycle of Saccu-
lina, 3. Cypris fixed on hair; degeneration of larval organs, and
formation of Kentrogon (K). 4. Kentrogon, formation of dart
(d.); Em.ec. Embryonic ectoderm; Hm.mes. Embryonic mesoderm.
5. Sacculina interna migrans (S.) on gut of host. 6. Saccu-
lina interna, later stage, with central tumour (e¢.) passing to
position of evagination (X.).

632 GEOFFREY SMITH.

themselves by their antennulz to a hair upon any portion of
a young individual of the host, preferably upon the append-
ages.

The Cypris casts off its thoracic appendages, the ectoderm
draws away from the shell, and comes to surround a mass of
mesodermal cells; it secretes a chitinous coat, and in this
manner the Kentrogon larva is formed. The Cypris shell,
together with all the larval organs, are thrown off.

The ectoderm of the Kentrogon secretes a hollow dart in
the anterior region which projects up into the antennule by
which the larva is fixed to the base of the hair on the crab,
and gradually penetrates the base of the hair so as to open a
means for the cells contained in the Kentrogon to enter the
heemoccel of the crab.

The embryonic cells of the Kentrogon, consisting of ecto-
derm and mesoderm, enter the hemoccel of the crab, and are
carried about in the blood-stream until they reach the large
blood-spaces surrounding the intestine. They are enclosed
in a thin chitinous cuticle. The Sacculina interna
migrans now proceeds to grow rapidly, to throw out roots
in all directions, while the central tumour grows down the
intestine towards the junction of thorax and abdomen of the
crab. As it grows in this manner, the adult organs are
differentiated in the most posterior portion of the central
tumour, which soon arrives at the position of evagination of
the adult Sacculina. Here differentiation proceeds, and the
pressure of the growing tumour upon the epithelium of the
crab causes it to degenerate, and thus, when the crab next
moults, a hole is left in the new chitin, through which the
Sacculina protrudes, and so gains the exterior.

LITERATURE.

” ser. 2,

1. Deracr, Y.—“ Evolution de la Sacculine,” ‘Arch. Zool. Expér.,
t. li, 1884.

2. Smiru, G.—‘ Fauna des Golfes von Neapel,’ Monograph, No. 29, “The
Rhizocephala,” 1906.

3. Mataquin, A.—‘‘Le Parasitisme Evolutif des Monstrillides,” ‘Arch.

Zool. Expér.,’ ser. 3, t. ix, 1901.

PHYSIOLOGICAL DEGENERATION IN OPALINA. 633

Physiological Degeneration in Opalina.

By
Cc. Clifford Dobell, B.A.,

Scholar of Trinity College, Cambridge.
With Plate 38 and 2 Text-figures.

INTRODUCTION.

In many respects Opalina ranarum, Purk. and Val., is
one of the most interesting of the ciliated Protozoa. Although
its adult appearance has been familiar to all zoologists for a
long time, it is only in the course of the last year that its
interesting life-cycle has become fully known. We owe this
knowledge to Neresheimer, who has, however, only given us
a preliminary account of his important researches. His full
paper will be awaited with much interest.

It will be unnecessary for me to give a detailed description
of this very common species. But I would remind the reader
that it is a large, multinucleate, holotrichous form found in
the large intestine of our common frog and toad (Rana
temporaria, L., and Bufo vulgaris, L.). The nuclei may
be regarded as consisting of meganucleus and micronucleus
fused toform asynkaryon. For an account of the life-history
the reader is referred to accounts already published, but
more especially to the memoirs of Zeller and Neresheimer.
It will be necessary for me to give a brief account of the
earlier part of the life-cycle, however; that is to say, of the
part prior to encystment, which takes place in the adult host.

634 G. CLIFFORD DOBELL.

Neresheimer has accurately, though briefly, described this
phase, and I have been able to confirm fully his observations.

In the spring, at the frog’s breeding season, Opalina
encysts, and is cast out into the water in the excreta. The
changes which take place before encystation are as follows :—
The adult animal divides in an oblique manner, giving rise to
two daughter individuals. Each of these then divides into
two, and these again divide until small Opaline containing
several nuclei are formed. During these divisions important
changes occur in the nuclear apparatus. The nuclei are seen
to become less distinct, this being due to the fact that the
chromatin is cast out into the cytoplasm in the form of small
strands and particles, or chromidia. Sometimes I have
observed these united to form a network through the
creature. Finally, the original nuclei vanish, and we are left
with only distributed chromatic material. The chromidia
soon become aggregated at certain centres, and thus synthe-
sise new nuclei, which are from two to ten in number.
These nuclei are seen to be composed of large chromatin
granules, arranged irregularly. They change, however, with
the approaching encystment of the animal, which soon takes
place. The chromatin travels to the periphery of the nucleus
where it becomes arranged in a thin layer with two to four
large cap-like thickenings.' In optical section these nuclei
appear as rings. ‘The cap-like projections are soon cast off
into the cytoplasm, where they degenerate, this constituting
the first nuclear reduction. Encystment follows, and in the
normal course of events the cyst is cast into the water, where
a second reduction of chromatin occurs. I will leave the
description of the life-cycle at this point, merely noting that
each nucleus is now a reduced gamete nucleus, and takes part
in the formation of a single ciliated gamete whose destiny is
to conjugate in the tadpole’s gut. The essential points in the
process just described are (1) formation of chromidia,
(2) synthesis of fresh nuclei from these chromidia, (3) reduc-
tion of chromatin, and (4) encystment.

1 Tirst described by Loewenthal as ‘‘ micronucleus-like structures,”

(Se)
Or

PHYSIOLOGICAL DEGENERATION IN OPALINA. 6

DESCRIPTION OF DEGENERATION IN OPALINA.

Having cleared the ground by describing the ordinary
course of events preceding gamete formation, I will now
pass on to a description of the physiological degeneration of
the forms whose usual destiny is to encyst.

Two different kinds of degenerative changes may be distin-
guished—the one caused by removal from the host, the other
within the host. The former is much the more rapid, and is
easily induced at any time. Degeneration results from drying,
from increase in the number of bacteria, and also doubtless
from lack of food, and increase of metabolites. The entire
organism simply decomposes, often after first throwing out
its nuclei in fragments. It is the second kind of degene-
ration—that within the host—with which I am here con-
cerned.

In nature, as we have seen already, the encystation of
Opalina is contemporary with the sexual activity of its host.
The set of degenerative changes which I am about to describe
took place when the ordinary activities of the host animals
were modified by captivity and starvation. Frogs, as is well
known, can endure starvation for many weeks. But the con-
tained Opaline, apparently, cannot do so—at all events
at their encystation period. Starvation is, I believe, the
determining factor in their degeneration. Other causes,
which materially influence degeneration in the organisms
when removed from their host—such as change in reaction of
the medium, drying, increase in the number of bacteria, etc.
—do not appear to come into play. For after lengthy starva-
tion the rectal contents of the frogs and toads examined con-
sisted of only a small quantity of a clear, mucous fluid,
alkaline in reaction, and containing but few bacteria. It was
in cases such as this—where starvation of the host had some-
times lasted for at least two months—that the most advanced
stages in the degeneration of Opalina were encountered.
My observations extend over a period from the middle of

636 O. CLIFFORD DOBELL.

January to the beginning of April, and are based upon the
careful examination of the rectal contents of over fifty frogs
and toads. The original object of the research was to study
the life-histories of the small Protozoa (flagellates and
amoebee) which occur in this situation. My attention was
attracted by the curious degeneration forms, although their
true nature was not, for some time, understood. However,
after a careful study of fresh material, the complete series of
degeneration changes here described became apparent.

TEXT-FIGURE 1.

One of the earliest changes which the Opalinex undergo
is a change of shape. Instead of remaining of a flattened,
ovate form, they become modified into all sorts of indefinite
shapes. Some of these are shown in text-fig. 1, but a great
number of other forms may be seen. The drawings are
schematic, and merely indicate the kind of thing which one
encounters. These forms do not divide in the normal manner,
but simply constrict off pieces, apparently at random, of all
shapes and sizes. These again divide until small, irregularly
discoidal, or ovoid forms are produced which have a length
of about 10u to 304. These usually contain from one to

PHYSIOLOGICAL DEGENERATION IN OPALINA. 637

four nuclei, but much larger individuals—up to 50, with
nine or ten nuclei—are also found in the same condition.
Such forms undergo two remarkable changes—(1) they com-
pletely lose all cilia, and (2) they give rise to globules of a
substance of high refractivity in their cytoplasm. The nature
of these globules I am unable to determine. I may mention,
however, that they have the following properties :—

In the fresh state they are somewhat greenish, and very
highly refringent. They are coloured a bright pink with
eosin, and a bright greenish-yellow with picric acid. With
iodine they appear to become slightly more greenish, but the
reaction is not well marked. They are insoluble in water,
alcohol, and weak acids and alkalies. Heidenhain’s iron
hematoxylin colours them a dark greyish- or brownish-black
—not so dark, however, as the chromatin. Delafield’s hama-
toxylin does not colour them—neither does borax-carmine.

From their remarkably vivid coloration with eosin I have
termed these globules ‘‘ eosinophile ” bodies, in ignorance of
their chemical constitution. Although they first appear as
separate globules of small size, they ultimately run together,
forming large masses lying inthe cells. ‘hey do not appear
to have any connection with the nucleus.

If these degenerate forms be obtained at the right stage,
the loss of cilia may be observed in the living animals. It
takes some days for all to be completely lost, and all do not
seem to disappear in the same way. Apparently some of
them actually dissolve, for they become gradually fainter
and fainter, and finally disappear. Others are thrown off
entirely, and after moving spontaneously for a short time
after detachment, they become motionless and fade away.
Still others undergo fusion with one another, and ultimately
with the cytoplasm. In this manner many individuals arise
which are completely divested of ciliary covering, and con-
tain refringent eosinophile bodies. The nucleus also under-
goes remarkable modifications. I term these forms the
atrichous forms.

The nuclear changes which the atrichous forms undergo are

638 C. GLIFFORD DOBELL.

as follows, and may be easily seen in the living animal :—
‘he chromatin which was at first evenly distributed through
the nucleus becomes massed in granules at the periphery,
whilst the nucleus itself increases in size until it becomes
sometimes double its original diameter (text-fig. 2, a, b).

a. é. c. d. e

TeXT-FIGURE 2 (from permanent preparations, stained with Heidenhain’s
iron-hematoxylin).

The chromatin becomes evenly disposed in a single layer,
so that in optical section the nucleus has a very character-
istic annular appearance (c), the ring being thickened at
various points (see also Plate, figs. 8, 12). A typical atri-
chous form is therefore distinguished by having no cilia, by
possessing ‘eosinophile globules” and a large ring-like
nucleus (or nuclei). When first seen they have a remarkable
appearance, and their connection with the ordinary Opalina
would hardly be suspected. These forms are quite motion-
less.

In many of the larger atrichous forms division of the
nucleus takes place, followed frequently by division of the
cytoplasm. Division may be equal, by a constriction appear-
ing in the middle (text-fig. 2, d), or unequal. In this latter
process a blister-like elevation of the chromatin appears, and
is finally constricted off. This is shown in text-fig. 2, e.
Above is a cap-like outgrowth of the chromatin, whilst below
a later stage is seen in optical section (cf. also Plate, figs. 10,
Lt).

When cytoplasmic division follows the result is either
equal bipartition or budding (cf. Plate, figs. 9, 10). The
buds so produced are sometimes very small, not reaching a
greater diameter than 4—5y. Occasionally buds are pro-
duced in which no nuclear material whatsoever can be

PHYSIOLOGICAL DEGENERATION IN OPALINA. 639

detected, and very commonly small buds are given off which
contain an eosinophile globule, but no nucleus. All these
enucleate buds appear to die and disintegrate. Finally, a
number of uninucleate atrichous forms result, which are of
an average diameter of about 20 u. At this stage they showa
marked tendency to attach themselves to one another, thus
forming small colonies (cf. Plate, fig. 12). No fusion, as a
rule, appears to take place.

The chromatin of the nucleus, which is of a very variable
size, but on an average about 8—10y in diameter, is seen to
be arranged in lumps peripherally. It soon leaves the
nucleus, however, and fills the cytoplasm, where it takes the
form of irregular granules and masses of different shapes
and sizes. These chromidia, as they may be called, appear
to be sometimes in the form of minute hollow spheres, ring-
like in optical section. By their formation the original
nucleus dwindles away, and finally disappears (see Plate,
figs. 1, 2, 7). As arule most of this chromatin is cast out of
the organism, which then dies and breaks up. But occa-
sionally a remarkable thing happens. Only a part of the
chromatin is cast out and perishes. The remaining granules
run together again, very much as drops of oil might run
together in a watery medium. All the irregular chromidial
masses may become aggregated at a single centre, but at
other times two such centres are formed, so that finally two
nuclei, consisting of solid chromatin, are synthesised. These
two solid lumps then approach one another and fuse (see
Plate, figs. 3—6). During the chromidial stages a soft cyst-
wall is sometimes formed.

I have been unable to obtain any further stages after this,
except such as are disintegrative. Kept under a waxed
coverslip or in a hanging drop they always perish by
discharging their nuclei in fragments and then breaking up.
This also appears to happen in the frog’s gut. It would be
exceedingly interesting to know whether a recovery could be
made under suitable conditions or not. I have endeavoured
to restore some of these degenerate fragments by transfer-

VoL. 51, PART 4,—NEW SERIES. A7

640 CO. CLIFFORD DOBELL.

ring them into the boiled rectal contents of a normal frog,
but without success. I have not been able to obtain sufficient
material for more extended experiments in this direction.

The final result then is death; and with this I finish my
description of degenerative changes in Opalina ranarum
so far as I have observed them. Before leaving the subject,
however, I must draw attention to the extraordinary parallel
which exists between these changes and certain so-called
“sexual” processes. In many Protozoa gamete nuclei are
formed from the origimal compound nuclei by resynthesis
from chromidia, very much in the same way as the solid
chromatin nuclei which I have just described in Opalina.
In certain autogamic processes the nuclei are formed and
fuse in the same cell. Compare, for example, the autogamy
of Bodo lacerte, Grassi, as described by Prowazek. ‘The
animal encysts, and inside the cyst the nucleus gives off
chromidia into the cytoplasm. From these chromidia a new
nucleus is built up, and this divides intotwo. Hach daughter
nucleus forms two “ polar bodies,’ and the reduced nuclei
(the gamete nuclei) approach one another and fuse. In
Bodo lacertz heterogamy also occurs, but in Tricho-
mastix lacertw, Blochmann, and in Entameba coli,
Losch, only autogamy is known—no other “ sexual ” act.

It is possible that the process which I have just described
in Opalina is a kind of autogamic attempt on the part of
the organism to reconstitute itself. But 1 believe that the
sole explanation of this curious set of changes is to be sought
in the alteration in chemical and physical properties which
living protoplasm undergoes in dying.

In conclusion, mention may be made of certain other
observations which have been made on degeneration in other
Protozoa. But little attention has been bestowed upon the
matter, although in a few species degenerative changes have
been studied in considerable detail. Among these I may
name Actinospherium (Hertwig), Amceba (Prandtl),
Paramoecium (Maupas, Calkins, etc.), 'Trichospherium
(Schaudinn), and the sporont of Cyclospora caryolytica

PHYSIOLOGICAL DEGENERATION IN OPALINA. 641

(Schaudinn). In the first three of these increase in the size
of the nucleus has frequently been observed as a preliminary
occurrence. In Actinospherium giant nuclei are formed
when the animal degenerates owing to overfeeding. Break-
ing up and discharge of the nucleus usually follows—that is
to say, chromidia play a part in the degenerative phenomena.
In Trichospherium, when degeneration is induced by
starving the organism, the nuclei clump themselves together
at certain points. This agglomeration is not followed by
fusion. Stole has observed a similar condition in starved
Pelomyxe. And I may here recall the observation of
Maupas on Paramcecium, that the fragments of the old
meganucleus sometimes fuse with the new meganucleus of an
exconjugant if it be starved.

In the degenerating sporont of the coccidian Cyclospora
the polar bodies divide until eight are formed, and microga-
metes then attempt to fertilise each of these. In later stages
of degeneration pigment is formed; and Prandtl has shown
that pigment appears in a degenerating Amceba proteus,
and is formed from the chromatin of the nucleus. In this
particular form there is also a curious tendency for the
nucleus to surround food masses in the cytoplasm.

Nuclear fusion sometimes takes place—independently of
any sexual process—in multinucleate Protozoa during, or
following, encystment: e.g. in Dileptus (Prowazek).

Hyper-regeneration occurs in Stylonychia if mutilated
when in a degenerate condition (Prowazek). Loss of ap-
pendages has been frequently observed in many different
Protozoa undergoing degenerative changes. It is unneces-
sary to give a number of examples, but Trichospherium
and Paramcecium may be cited as good instances.

Chromidia of the type I have described in Opalina (i.e.
bladder-like, or blaschenformig) have only been noticed, so
far as I am aware, in one other Protozoon, Bodo lacerta,
Grassi. And here they are formed as a preliminary to
oeamete formation and autogamy (Prowazek).

Very curious in many other ways is the parallel which

642 CG. CLIFFORD DOBELL.

exists between degenerative and “sexual” processes. Be-
sides the fact that chromidia are formed in both, we have
the observation that an amoeboid condition may occur in
degenerating Protozoa, and also sometimes just before
conjugation. Fusion also occurs in degenerating forms of
various kinds. I have observed it especially in flagellates,
e.g. Trichomastix, Trichomonas, etc. Senile Para-
moecia enter upon what Calkins calls the “ miscible state,”
when they tend to adhere to one another. Similarly, Roux
has observed that isolated, living blastomeres of frog’s eggs
become amoeboid and run together; though, according to
Driesch, this is merely due to the capillary forces between
the cells. These facts, and many others of a similar nature,
are not without interest, both from a pathological and from a
zoological point of view. I may mention merely their pos-
sible bearing upon the remarkable fusion which appears to
take place between leucocytes and cancer cells, and its un-
known significance. And since the work of Calkins seems to
indicate that chemical change in protoplasmic composition is
the chief beneficial effect of conjugation, and there is some
proof that Protozoon individuals have chemical compositions
differing from one another (cf. Jensen), is it not at least
possible that the physico-chemical changes which cause
fusion in degenerating cells are of a similar nature to those
which gave rise to the first cell-couplings, and which still
determine the fusion of one gamete with another?

ZOOLOGICAL LABORATORY,
CAMBRIDGE.

ADDENDUM.

Neresheimer’s full account of the life-cycle of Opalina
has appeared since this paper was submitted for publication.
It is a very complete description, with full references to the
literature, and is to be found in ‘ Arch. f. Protistenk ;’ Supple-
ment i (Festband fiir R. Hertwig), 1907, p. 1.

15.

PHYSIOLOGICAL DEGENERATION IN OPALINA. 643

LITERATURE REFERENCES.

. Carxins, G. N.—“ Studies on the Life-history of Protozoa.” —I, in ‘Arch.

Entwickmech.,’ Bd. xv, 1902, p. 189; IL (with C. C. Lies), in ‘ Arch.
f. Protistenk.,’ Bd. i, 1902, p. 355; ILI, in ‘ Biol. Bull.,’ vol. iii, 1902,
p. 192; LV, in ‘Journ. Exp. Zool.,’ vol. i, 1904, p. 423.

. Gotpscumipt, R.—“ Der Chromidialapparat lebhaft funktionierender

Gewebszellen,” in ‘ Zool. Jahrb.’ (Abth. f. Anat.), xxi, 1904, p. 41.

. Gotpscumipt, R.— Die Chromidien der Protozoen,” in ‘ Arch. f. Protis-

tenk.,’ Bd. v, 1905, p. 126.

. Herrwic, R.—< Ueber physiologische Degeneration bei Actinosphe-

rium eichhorni,” in ‘ Festschr. f. E. Haeckel,’ Jena, 1904, p. 301.

. JENSEN, P.—‘ Ueber individuelle physiologische unterschiede zwischen

Zellen der gleichen Art,’ in ‘Pfliiger’s Arch. f. Physiol.,’ Bd. lxii,
1895, p. 172.

. Lopwentuat, W.— Das Auftreten eines Mikronukleusartigen Gebildes

bei Opalina ranarum,” in ‘Arch. f. Protistenk.,’ Bd. ili, 1904,
p. 387.

. Maupas, E.—‘‘La rajeunissement karyogamique chez les ciliés,” in

‘Arch. Zool. Exp. et Gén.,’ vii, 1889, p. 149.

. NeresHEImer, B.—‘ Der Zeugungskreis von Opalina,” in ‘Sitz. Ber.

Ges. Morph. Physiol. Miinchen,’ 1906.

. Pritzner, W.—“ Zur Kenntnis der Kerntheilung bei den Protozoen,”

in ‘Morph. Jahrb.,’ Bd. xi, 1886, p. 454.

. Pranptt, H.—‘ Die physiologische Degeneration der Ameba pro-

teus,” in ‘Arch. f. Protistenk.,’ Bd. vili, 1907, p. 281.

. Prowazex, S.—‘“‘ Der Encystierungsvorgang bei Dileptus,” in ‘Arch.

f, Protistenk.,’ Bd. iii, 1904, p. 64.

«Degenerative Hyperregeneration bei den Protozoen,” in ‘ Arch.
f. Protistenk.,’ Bd. iii, 1904, p. 60.

“ Untersuchungen ueber einige parasitische Flagellaten,”’ in
‘Arb. Kaiserl. Gesundheitsamte,’ xxi, 1904, p. 1.

. Roux, W.—“ Ueber den ‘ Cytotropismus’ der Furchungszellen des Gras-

frosches (Rana fusca),” in ‘Arch. Entwickmech.,’ Bd. i, 1894, pp.
43 and 161.

Scuaupinn, F.—“ Untersuchungen iiber den Generationswechsel von
Trichospherium sieboldi, Schn.,” in ‘ Anh, Abh, Akad. Berlin,’
1899.

644 C. CLIFFORD DOBELL.

16. Scnaupiny, F.—‘“Studien iiber krankheitserregende Protozoen: I.
Cyclospora caryolytica, Schaud., der Erreger der pernicidsen
Enteritis des Maulwurfs,” in ‘Arb. Kaiserl. Gesundheitsamte,’ Bd.
xviii, 1902, p. 378.

17. Srotc, A.—‘Beobachtungen und Versuche iiber die Verdauung und
Bildung der Kohlenhydrate bei einem amdbenartigen Organismus,
Pelomyxa palustris, Greeff,” in ‘ Zeitschr. f. wiss. Zool.,’ Ixviii,
1900, p. 625.

18. Zevier, E.—“< Untersuchung wber die Fortpflanzung und die Entwick-
lung der in unseren Batrachiern schmarotzenden Opalinen,” in ‘ Zeitschr,
f. wiss. Zool.,’ xxix, 1877, p. 352.

EXPLANATION OF PLATE 38,

Illustrating Mr. C. Clifford Dobell’s paper on “ Physiological
Degeneration in Opalina.”

[Figs. 7 and 8 are drawn from living specimens, under a 2°5 mm. (apert.
1:25) apochromatic water immersion objective by Zeiss ; compensating ocular
12. The remainder are from permanent preparations: fixed sublimate-alcohol
(2:1). Figs. 1—6, 9 and 12 from specimens stained with Heidenhain’s iron-
hematoxylin. Fig. 10, Heidenhain and eosin. Fig. 11, Weigert’s iron-
hematoxylin and eosin. Drawn under a 2 mm. (apert. 1°40) apochromatie
oil immersion (Zeiss), with compensating ocular 12.

All figures are drawn in monochrome for sake of uniformity. In all cases
the darker masses are chromatin; the paler masses surrounded by a light area
are the eosinophile bodies. ]

Fie. 1.—Atrichous form in which the nucleus has largely broken up into
chromidia. The remains of the nucleus are seen near the middle, with a
single large cap-like mass of chromatin, Beneath is seen one large eosino-
phile body. Note that many of the chromidia are in the form of hollow
spheres—annular in optical section.

Fic. 2.—Smaller specimen, completely filled with chromidia.

Fic. 3.—Specimen in which the chromidia are running together to form two
new nuclei. Some indication of the formation of a cyst can be seen.

Fic. 4.—The chromidia have fused to form two new nuclei, which are
approaching one another,

~

PHYSIOLOGICAL DEGENERATION IN OPALINA. 64.5

Fic. 5.—A cyst-wall has been formed, and the two solid chromatin nuclei
are applied to one another.

Fic. 6.—Specimen showing a still later stage in the fusion of the nuclei.
A thick, soft cyst has been formed.

Fic. 7.—Fresh preparation in which a cyst has been formed and the nucleus
has broken up into chromidia.

Fie. 8.—Atrichous form in fresh condition, showing nucleus with peri-
pherally placed chromatin masses (annular in optical section).

Fic. 9.—Large multinucleate atrichous form breaking up to form smaller
ones. At least one nucleus is dividing.

Fie. 10.—An individual in the act of forming buds. Small blister-like
nuclei are budded off, and the cytoplasm has become constricted round one of
these, forming a complete bud.

Fie. 11.—Similar form, in which nucleus is seen in optical section. One
nucleus has been completely separated off, and another is almost so.

Fie. 12.—Association of eleven typical atrichous forms. None of these
have as yet formed chromidia, though one appears to be enucleate.

isfap «tamer ay or

LATER DEVELOPMENT OF THE FROG. 64.7

Some Facts in the Later Development of the
Frog, Rana temporaria.

Part I.—The Segments of the Occipital Region of the Skull.

By
Asnes I. M. Elliot, B.Sc.,
Associate of Newnham College, Cambridge.

With Plates 39 and 40.

THE question of the segmentation of the vertebrate head is
one which for a long time has attracted the attention of many
investigators. With regard to the segmentation of the
anterior and middle parts of the head the facts are conflict-
ing, and opinions differ; but, with regard to the post-otic
portion, there is a converging mass of evidence, drawn from
the neural, muscular, and skeletal tissues, that a varying
number of segments serially homologous with the trunk
segments have been drawn into the head. Besides this there
is also evidence that in this region, in some groups at all
events, a certain number of segments have altogether dropped
out, and are not now represented by any rudiments, even in
the embryo.

In the Cyclostoma the cranium terminates with the laby-
rinth region—there is no occipital portion of the skull.

The post-otic region of the Selachian head has been inves-
tigated by Neal, Fiirbringer, Van Wijhe, Rabl, Beard,
Sedgwick, C. K. Hoffman, Sewertzoff, Braus, Froriep, and
Dohrn. The chief results of their inquiries about this region
may be summarised as follows.

There is present an occipital region of the cartilaginous
skull, lying behind the labyrinth region. ‘This is penetrated
by a number of nerves, serially homologous with the spinal
nerves, and known as the spino-occipital nerves. These are
often three in number in the adult, but may vary from none

648 AGNES I. M. ELLIOT.

(Torpedo) to five (Hexanchus), and only retain their ventral
roots; but in the embryo as many as seven may be indicated,
and all provided with the rudiment of a dorsal ganglion
(embryo Torpedo). More usually, however, there are only
five or six (Spinax) of these nerves in the embryo, and only
the last two or three show dorsal ganglia. But even in these
cases we may still find seven pest-otic somites (Spinax).

The skull itself is not usually segmented, but the myo-
comata are attached to the parachordal masses in segmental
fashion. Rosenberg has shown in some sharks (Carcharias)
distinct signs of extra vertebree joined to the post-otic region
of the skull, but leaves it uncertain whether these represent
the spino-occipital segments of other sharks or whether they
are additional trunk vertebree which are being drawn into the
skull.

In the Selachians we may therefore conclude that at least
seven trunk segments have been fused with the head in the
post-otic region, though in some forms (Scyllium and Pris-
tiurus) traces of no more than five are still visible during
development.

In other fishes there has been a further pushing up of
trunk segments into the head. In Accipenser as many as six
more segments have been fused, and in the Teleostei probably
only three more.

In Amphibia the line of division between head and trunk
is supposed to lie in the same place as in Selachians, but if so
a number of the occipital segments have entirely disappeared,
as at most three spino-occipital segments can be demonstrated
in the embryo, and in the adult there are no spino-occipital
nerves. In the Anura, indeed, the first spinal nerve and its
ganglion also disappear.

In the Urodeles, Miss Platt has shown for Necturus that
there are three post-otic segments in the head of the embryo,
the first not giving rise to any muscles, the second partly
uniting with the third, and the muscles from the second and
third segments being hard to distinguish in the adult. She
has also shown that there are two rudimentary dorsal arches

LATER DEVELOPMENT OF THE FROG. 649

connected with these segments: a pre-occipital, which fuses
with the otic capsule, and an occipital, which forms the
occipital region of the skull.

Dr. Gadow suggested to me that, in view of the absence of
the first spinal nerve in the Anura, I should cut a series of
sections in the neck region of the developing frog to see what
is the embryonic condition in this region—whether the first
spinal nerve is present in the embryo, and what other traces
there are of the disappearance of segments in this region.

Fig. 1 is a horizontal section of this region in a 9-milli-
metre tadpole.

The vagus is seen arising by a number of roots—five in
this section with traces of a sixth—of which the first is larger
than the rest and ganglionated, and there is a distinct gap
between the first and the succeeding roots.

The latter have no ganglion-cells, and this fact, together
with their more posterior position, suggests that they are not
homologous with the first, and that they may be ventral roots
of spinal nerves drawn into the head, and running forward to
join the original cranial vagus,

In another section there are as many as seven roots to the
vagus.

The third myotome is the first visible in this section, and in
front of it les a string of mesodermic tissue with elongated
nuclei running from the anterior myotome to the otic sac.

If we follow the series of sections we can trace two myo-
tomes in front of this one. In fig. 2 the second is shown, and
in fig. 3 we have the complete series. The first is smaller
than those posterior to it, and extends forward almost to the
otic sac. There is, however, some mesoderm anterior to the
first myotome, but it does not give rise to a muscle plate,
though in a similar section of an animal of about the same
age I saw signs of a muscle fibre in this region. We may, I
think, fairly look on this mesoderm as representing at least
one or more somites in front of that from which the first
myotome is developed.

The second myotome has a trace of a rudimentary ganglion

650 AGNES I. M. ELLIO!

(see fig. 4) associated with it, and the third myotome is
accompanied by a well-developed ganglion.

In fig. 2 the distinction between the first ganglionated
root of the vagus and the succeeding group of roots is well
marked. The more posterior roots have formed a common
trunk which is running forward to join the first root. There
is a rudimentary ganglion to the second myotome, but it is
not cut in either fig. 2 or fig. 3, and the ganglion of the
third myotome is only just shaved in the section shown in
fig. 3. Neither in this nor in any other section is there any
trace of a ganglion in connection with the first myotome,
nor with the mesoblast lying in front of the first myotome.
If the segments are counted back to the cloaca (the only
approximately fixed point, as the limbs have not begun to
develop) they are found to be eleven in number.

Fig. 4 is a horizontal section through another tadpole of
about the same age, showing all the anterior myotomes and
associated ganglia. ‘The first myotome is small, and has no
trace of a ganglion, the second has on the left side a rudi-
mentary ganglion associated with it; each of the other myo-
tomes corresponds with a well-developed ganglion. The vagus
roots on the right side are seen crossing outwards over the
first myotome. In fig. 4a we have the rudimentary ganglion
of myotome 2 enlarged. In fig. 4b we have a typical spinal
ganglion from the same tadpole enlarged to show the contrast.

Fig. 5 is a horizontal section through a much older tadpole,
about 13 millimetres long, in which the cartilaginous dorsal
neural arches are developed. ‘The first neural arch lies at a,
on the myoseptum between the first and second myotomes
now present. In front of this, however, are two small carti-
lages b and c, which look serially homologous with the neural
arches, and lie just behind the ganglion of the vagus. In
more ventral sections they may be seen to fuse with cartilage
continuous with the otic capsule. These, I believe, represent
two neural arches—are, in fact, homologous with the pre-
occipital and occipital arches which Miss Platt found in
Necturus. The first myotome now present has a distinct

LATER DEVELOPMENT OF THE FROG. 651

though degenerating ganglion, and the myotome is small.
This ganglion is lost in the adult, and belongs to the missing
first spinal nerve. This is obvious from its position just in
front of the first neural arch. The number of myotomes now
present as far as the cloaca is only 9.

I therefore conclude that the first two myotomes and the
rudimentary ganglion associated with the second, present in
a 9-millimetre tadpole, have now disappeared—that these
segments are represented by the two small cartilages b and c
—and that the third myotome and its ganglion are much
reduced in size, and are disappearing.

In fig. 6 we have a figure drawn combining two sagittal
sections of a somewhat larger tadpole, about 20 millimetres
long, in which the tissues of the hinder limb are beginning
to show differentiation. The limb is drawn in from a section
some distance farther on in the series, but the myotomes have
been counted so as to correspond. The section shows the
neural cord, cut a little on one side, the myotomes of that side,
and the neural arches and gangha almost all the way from the
anterior end to the cloaca. The letter a points to the first
myotome still remaining at this stage, and is, as in fig. 5, the
third of the complete series. Anterior to it we see a blood-
vessel b, and just to the right of this blood-vessel, at f, there
are some cells which are evidently degenerated muscle-cells
of the second myotome. ‘Two small nodules of cartilage,
ec and d, are united to the parachordal bar by connective
tissue, which already shows signs of chondrification. ‘These
are the same as the cartilages b and c in fig. 5. The first
myotome present in this section—a—has a well-developed
nerve and ganglion, and as this ganglion is in the space
between the posterior end of the skull and the first neural
arch, it is the first spinal nerve, which disappears in the adult.

Behind this we see ganglia, neural arches, and myotomes
in orderly series, and counting myotome a as the first, we find
that the ninth myotome gives off muscles to the cloaca.

The limb which is drawn from another section of the same
animal is provided with three nerves, which we can see are

652 AGNES I. M. ELLIOT.

probably those of the eighth, ninth, and tenth myotomes—
the connection of the middle one with the ninth myotome is
quite clear. It is now possible to make a table showing the
connection of the segments found in the tadpole with the
nerves and vertebre of the adult frog.

: allen oli aes eee | Distribu-
Skel. a raced Gang. Nerve. ane Remarks,
| BE: | Post. eat
ALE WA Nonell None if aa Ik ) Mesoblast in front of 1st myo-
‘Lu: lof een Comes
Post.
coo ‘| Pres. | None eaetlet | Disa rs by time li
A jeceip-| | g. ppears by time limbs appear.
ee te of “sere |
2 Os ema | |
ove | 9] Pres. | Rudy. Disappears by time cartilaginous}
arches appear. May be de-) —
generating cells. (See Fig. 6.) |
3} Pres. | Pres. Nerve and ganglion disappear!
Meret al | in adult—still” present when| —
ert. Hypo.
| | A eee ieee ee Muscles, cart. arches are formed.
ae. iaar | ie Fo tongue ee ee Spinal nerve.
el | | | Really Sp. II.
5] Pres. | Pres. Ill Brachial
Vert. 3 | | | plexus
a 6) Pres. | Pres. IV Brachial
Vert. 4 Bae pieaue
w “| Pres. | Pres. Vv Body wall
Vert. 5 & | |
el
=a a 8} Pres. | Pres. VI Body wall
Vert. 6
= 9} Pres.) Pres.| VII |Bodywall
Vert. 7 | | :
rar Pres. | Pres. VII Sciatic j
lexus
Vert. 8 | | ie P
ll) Pres. | Pres. IX | Sciatic || muscles to legs.
Vert. 9 Pay | | plexus | / muscles to cloaca. :
. 19] Pres. | Pres. x | Sciatic | :
Dorsal arch | | | plexus [
f urostyle
mses Noe ve teres lia) aeen | epeee | ee pale |

Pose

LATER DEVELOPMENT OF THE FROG. 653

Rana therefore entirely agrees with Necturus in the seg-
mentation of the head. ‘There are two post-otic segments
giving rise to the first and second myotomes, and in front of
this a mass of mesoblast, which may represent one or an
indefinite number of disappearing segments. ‘There is no
ganglion connected with the first myotome, and an extremely
rudimentary one with the second.

The neural arches between the first and second and second
and third somites persist, and form the occipital region of
the skull. ‘They are homologous with the pre-occipital
and occipital arches which Miss Platt found in Necturus.

In the 9-millimetre tadpole the third myotome is of full
size, and so is the corresponding ganglion. But they have
already begun to show signs of diminution in the tadpole, in
which the dorsal cartilaginous arches are present (see fig. 5).
The nerve of this segment is the missing first spinal nerve of
the adult.

If the division between head and trunk is homologous in
Selachians and Amphibia, as is generally assumed, a number
of post-otic somites must have entirely vanished in the
Amphibia, leaving no obvious trace at any stage of develop-
ment. or in the Selachians Braus has demonstrated seven
spino-occipital segments, and in the Amphibia we have traces
at most of three, as shown for both Urodela and Anura.

The curious origin of the vagus may point to a possible
reminiscence during development of these otherwise totally
missing segments. The difference between the first and the
succeeding roots, the presence of ganglion cells on the
former and their absence on the latter, and the union of all
the posterior roots into a common trunk, which passes
forward to join the first root, are, | think, indications of a
difference of origin of the posterior group. ‘The position of
the first myotome opposite to these roots, together with the
absence of any trace of nerve connected with it, suggests at
once that its nerve may have run forward to join this group ;
and it is easy to carry the suggestion further, and see in the
more anterior roots of the posterior set a rudiment of the

654. AGNES I. M. ELLIOT.

nerves of segments now lost; segments whose myotomes are
represented by the undifferentiated mesoblast in front of the
first existing myotome.

To sum up the results obtained—

1. The first spinal nerve with its ganglion and associated
myotome is well developed in the young tadpole, and still
present, though somewhat reduced, in the tadpole which has
developed dorsal cartilaginous arches, and whose limbs have
begun to develop.

2. Two myotomes are present in the 9-millimetre tadpole
in front of this segment. Cartilaginous arches appear in
connection with these, and fuse with the parachordals, from
which they are still distinct in a 20-millimetre tadpole.
There is no ganglion in the segment of the first myotome,
but there is a rudimentary one in the segment of the second
myotome. Both these myotomes and the rudimentary gan-
glion disappear by the time that the cartilaginous dorsal
arches are present, while the cartilaginous arches corre-
sponding to them form the occipital region of the skull.

3. The vagus arises by numerous roots. ‘The first root
has a ganglion, and lies well forward and distinctly separated
from the remaining roots. ‘The latter may possibly repre-
sent ventral roots of the nerves of the missing post-otic
segments, and also of the segment in which the first myotome
is developed.

4, There is a string of true mesoblast lying between the
otic sac and the first myotome. ‘This may also be the
remains of the tissue of segments anterior to that of the first
myotome.

5. The segmentation of the post-otic region of the skull in
Rana agrees completely with that in Necturus.

My sincere thanks are due to Dr. Gadow, both for his
suggestion of the subject of this investigation, and for his
kind interest and advice during its progress.

LATER DEVELOPMENT OF THE FROG. 655

BIBLIOGRAPHY.

Batrour.—* Monograph on the Development of Elasmobranch Fishes,” 1875.

Bearp.—*System of Branchial Sense Organs and their Ganglia in Ichthy-
opsida,” ‘ Quart. Journ. Mier. Sci.,’ xxvi, 1886.

Braus.—“ Beitriige zur Entwickelung der Muskulatur und des peripheren
Nervensystems der Selachier; I Theil. Der metotischen Urwirbel und
spino-occipitalen Nerven,”’ ‘ Morph. Jahr.,’ Bd. xxvii, 1899.

Dourn.— Studien zur Urgeschichte des Wirbelthierkérpers,” 1$—22,
‘Mitt. z. Stat. Neapel,’ Bd. xv.

Frorter.—“ Uber ein Ganglion des Hypoglossus und Wirbelanlagen in der
Occipitalregion,” ‘Arch. Anat. u. Phys.,’ Anat. Abt., 1882.

“Uber Anlagen von Sinnesorganen,” etc., etc., ‘Arch. Anat. u.
Phys.,’ Anat. Abth., 1885.

“Uber die Ganglienleisten des Kopfes und des Rumpfes,” etc.,
‘Arch. Anat. u. Phys.,’ Anat. Abth., 1901.

“Zur Entwickelung und Geschichte des Wirbelthierkopfes,” ‘ Verh.
Anat. Ges.,’ 16 Vers., Halle, 1902.

FURBRINGER.—‘“ Spino-occipitalen Nerven der Selachier und Holocephalen,”
‘Festschrift Gegenbauer,’ Bd. ii, Leipzig, 1897.

GEGENBAUER.—“ Kopfnerven von Hexanchus,” ‘Jen. Zeits. fiir Naturwissen-
schaft,’ Bd. vi, 1871.

‘“‘Vergleichende Anatomie der Wirbelthiere,”’ Leipzig, 1898.

“Die Metamerie des Kopfes und die Wirbeltheorie des Kopfske-
lettes,” ‘ Morph. Jahrb.,’ Bd. xiii, 1888.

Hertwic.—* Vergleich u. Exper. Entwickelungslehre,” Leipzig, 1898.

Horrman.—“ Zur Entwickelungsgeschichte der Selachii,” ‘Morph. Jahrb.,’
Bd. xxiv, 1897; Bd. xxvii, 1899.

Pratr.— Development of the Cartilaginous Skull and Branchial and Hypo-
glossal Musculature in Necturus,” ‘ Morph. Jahrb.,’ Bd. xxiv, xxv, 1897.

Rasu.—‘‘ Uber die Metamerie des Wirbelthierkopfes,” ‘ Verh. Anat. Ges.,’
5 Vers., Wien, 1892.

Sepewick.—“ Notes on Elasmobranch Development,” ‘Quart. Journ. Micr.
Sci.,’ xxxiii, 1892.

SewErtzorr.— Entwickelung der Occipitalregion der niederen Vertebraten
in Zusammenhang mit der Frage iiber die Metamerie des Kopfes,” ‘ Bull.
de la Société Imp. des Naturalistes de Moscou,’ 1895.

“Beitrag zur Entwickelungsgeschichte des Wirbelthierschadels,”
‘Anat. Anz.,” Bd. xiii, 1897.

voL. 51, part 4.—NEW SERIES. — 48

656 AGNES I. M. ELLIOT.

Sewertzorr.— Die Metamerie des Kopfes von Torpedo,” ‘Anat. Anz.,’
Bd. xiv, 1898.

Van Wisun.—‘ Uber die Mesodermsegmente und die Entwickelung der
Nerven des Selachierkopfes,” Amsterdam, 1883.

EXPLANATION OF PLATES 39 AND 40,

Illustrating Miss Agnes I. M. Elliot’s paper on Some Facts in
the later Development of the Frog, Rana temporaria.” .

ABBREVIATIONS.

Br. Brain. C.c.sp.c. Central canal of spinal cord. C.#.C. Cartilaginous ear
capsule. C/. Cloaca. Cl.mus. Cloacal muscles. C.N S. Central nervous
system. C.¢. Connective tissue. Deg.cs. Degenerating cells. 2. Hye.
Gan.V. Ganglion of V cranial nerve. Gan.VIII. Ganglion of VIII cranial
nerve. Gan.X. Ganglion of X cranial nerve. Gr.m.sp.c. Grey matter of
spinal cord. H.K. Head kidney. H.Z. Hind limb. JZ.S. Lymph space.
Ig. Lung. Mes.Ant. Mesoblast cells stretching forward from anterior
myotomes to otic capsule. Mes”. Mesenchyme. My. 1—13. Myotomes
numbered from anterior end. In figures 5 and 6 myotome 1 is the first
myotome still remaining, and corresponds to myotome 3 in figures 1, 2, 3,
and 4. N.e. Nerve cell. Meh. Notochord. Pch. Parachordal cartilage.
Pr.O.A. Preoccipital arch. R.sp.gan. Rudimentary spinal ganglion. Sp.ed.
Spinal cord. Sp.gan.1—1ll. Spinal ganglia 1—1l. V.as.dr. Nerve trunk
running forward, formed from posterior roots of vagus. Vagus R.1. First
root of vagus. Vagus R.6. Sixth root of vagus. V.1—7. Dorsal cartila-
ginous arches corresponding to vertebra 1—7. Wh.m.sp.c. White matter of
spinal cord.

PLATE 39.

Fic. 1.—Horizontal section of 9-millimetre tadpole, showing roots of vagus.
Magnification 260.

K ie. 2.—Horizontal section of same tadpole, more ventral; showing second
myotome and ascending roots of vagus. Magnification 100.

LATER DEVELOPMENT OF THE FROG. 657

Fic. 3.—Horizontal section of same tadpole, still more ventral; showing
first and succeeding myotomes and spinal ganglia corresponding to myotomes
3, 4,5, 6, and 7. Magnification 83.

Fic. 4.—Horizontal section of 10-millimetre tadpole ; showing myotomes
and spinal ganglia, including the rudimentary spinal ganglion corresponding
to the second myotome. Magnification 50.

Fie. 4a.—Rudimentary spinal ganglion corresponding to second myotome,
enlarged from Fig. 4. Magnification 800.

Fig. 4b.—Spinal ganglion corresponding to fourth myotome, enlarged from
Fig. 4. Magnification 800.

PLATE 40.
Fie. 5.—Horizontal section of 13-millimetre tadpole; showing cartila-
ginous arches, myotomes, and spinal ganglia at anterior end. (Combined

from three sections.) For explanation of a, 6 and ¢ see page 650. Magnifi-
eation 100.

Fig. 6.—Sagittal section of 20-millimetre tadpole; showing cartilaginous

arches, myotomes, and spinal ganglia of anterior region, and developing hind

‘limb. (Combined from two sections.) For explanation of a, , c, d and / see
page 651. Magnification 36.

INDEX TO” VOR:

ole

NEW SERIES.

Ajnigma exnigmatica, by G. C.
Bourne, 253

Anatomy of Notoryctes, by G. Sweet,
325

Anomiacea, structure of Alnigma, by
G. C. Bourne, 253

Antedon and Synapta, spicules of, by
W. Woodland, 31

Ascidians, spicules of, by W. Wood-
land, 45

Auricularia, wheel-spicules of, by W.
Woodland, 483

Babesia bovis (see Piroplasma
bigeminum, 297)

Bourne, G.C., structure of Ainigma
enigmatica, 253

Buds of Cephalodiscus, plumes of, by
W. G. Ridewood, 221

Carcinus, fixation of Sacculina upon,
by G. Smith, 625

Cattle-fever, parasite of Texas, by
H. B. Fantham, 297

Cephalodiscus, development of plumes
in, by W. G. Ridewood, 221

Cephalodiscus, Neurospori-
dium, a sporozodn in, by W. G.
Ridewood and H. B. Fantham, 81

Chetognatha, or primitive Mollusca,
by R. T. Gtinther, 357

Chemical composition of Radula, by
[ENB SollassukD

Chromatin masses of Piroplasma
bigeminum, by H. Bb. Fantham,
297

Convoluta, green cells of, by F.
Keeble and F. W. Gamble, 167

Cypris larva of Sacculina, fixation of,
by G. Smith, 625

Degeneration in Opalina, by C. C.
Dobell, 633

Dendy, A., parietal sense-organs of
Geotria, 1

Development of Frog, occipital re-
gion, by A. I. M. Elliot, 647

Development of head-muscles in
Gallus, by F. H. Edgeworth, 511

Development of Ophiothrix fra-
cilis, by E. W. MacBride, 557

| Development of plumes in Cephalo-

discus, by W. G. Ridewood, 221

_ Development of Radula, by I. B. J.

Sollas, 115

_ Development of teeth in Ornitho-

rhynchus, by J.T. Wilson and J.
Py Hill, 137

660

Diplopoda, segmentation of head of,
by M. Robinson, 607

Dobell, C.C., physiological degenera-
tion in Opalina, 633

Dobell, C. C., Trichomastix ser-
pentis, 449

Doncaster, L.,
Nematus ribesii, 101

Edgeworth, F. H., head-muscles of
Gallus and otlier Sauropsida, 511
Elliot, A. I. M., later development of

Frog, 647

Factors in production of various forms
of spicules, by W. Woodland, 55
Fantham, H. B., chromatin masses of
Piroplasma bigeminum, 297
Fantham, H. B., and W.G. Ridewood,
on Neurosporidium cephalo-
disci, $1

Fertilisation in Nematus ribesil,
by L. Doncaster, 101

Fixation of Cypris larva of Sacculina,
by G. Smith, 625

Fleure, H. J., and M. M. Gettings,
notes on Trochus, 459

Formation of skeleton in Madrepo-
raria, by M. M. O. Gordon, 473

Forms of spicules, factors concerned,
by W. Woodland, 55

Frog, later development of, by A. I.
M. Elliot, 647

Gallus, development of head-muscles,
by F. H. Edgeworth, 511

Gamble, F. W., and F. Keeble, green
cells of Convoluta, 167

Gametogenesis in Nematus ribesil,
by L. Doncaster, 101

Geotria, parietal sense-organs of, by
A. Dendy, 1

Gettings, M. M., and H. J. Fleure,
notes on T'rochus, 459

gametogenesis in |

INDEX.

Gordon, M. M. O., formation of ske-
leton in Madreporaria, 473

Green cells of Convoluta, by F.
Keeble and F. W. Gamble, 167

Giinther, R. T., the Chetognatha, 357

Hair, skin, and reproductive organs
of Notoryctes, by G. Sweet, 825
Head-muscles in Gallus and other
Sauropsida, by F. H. Edgeworth,
511

Head of Diplopoda, segmentation of,
by M. Robinson, 607

Hewitt, C. G., structure of House-
fly, 395

Hill, J. P., and J. T. Wilson, tooth-
development in Ornithorhynchus,
137

House-fly, structure of, by C. G.
Hewitt, 395

Keeble, F., and F. W. Gamble, green
cells of Convoluta, 167

Lamprey, New Zealand, (Geotria),
parietal sense-organs of, by A.
Dendy, 1

Later development of Frog, by A. I.
M. Elliot, 647

MacBride, E. W., development of
Ophiothrix, 557

Madreporaria, formation of skeleton
in, by M. M. O. Gordon, 473

Mollusca, Cheetognatha or primitive,
by R. 'T. Giinther, 357

Mollusca, spicules of, by W. Wood-
land, 45

Molluscan Radula, by I. B. J. Sollas,
115

Musca domestica, structure of, by
C. G. Hewitt, 395

Nematus ribesii, gametogenesis
in, by L. Doncaster, 101

INDEX.

Neurosporidium cephalodisci,
by W. G. Ridewood and H. B.
Fantham, 81

New Zealand Lamprey, parietal sense-
organs of, by A. Dendy, 1

Nicoll, W., Parorchis acanthus, |

anew Trematode, 345
Notes on skeleton of Madreporaria,
by M. M. O. Gordon, 473

and M. M. Gettings, 459
Notoryctes, skin, hair, and reproduc-
tive organs of, by G. Sweet, 325

Observations on tooth-development
in Ornithorhynchus, by J. T.
Wilson and J. P. Hill, 137

Occipital region of Frog, develop- |

ment, by A. I. M. Elliot, 647

Ogilvie Gordon, M. M., formation of
skeleton in Madreporaria, 473

Opalina, degeneration in, by C. C.
Dobell, 633

Ophiothrix fragilis, development
of, by E. W. MacBride, 557

Ophiuroidea and Kchinoidea, spicules
of, by W. Woodland, 31

Origin and nature of green cells in
Convoluta, by F. Keeble and F. W.
Gamble, 167

Ornithorhynchus, tooth-development
in, by J. ‘I. Wilson and J. P. Hill,
137

Parasite of Texas cattle-fever, by H.
B. Fantham, 297

Parietal sense-organs of Geotria, by
A. Dendy, 1

Parorchis acanthus, a new Tre-
matode, by W. Nicoll, 345

Physiological degeneration in Opalina,
by C. C. Dobell, 633

Piroplasma bigeminum, chro-
matin masses of, by H. B. Fant-
ham, 297

661

Plate-and-anchor spicules of Synapta,
by W. Woodland, 483

Plumes of buds of Cephalodiscus, by
W. G. Ridewood, 221

Preliminary consideration, forms of
spicules, by W. Woodland, 55

Primitive Mollusca, the Chetognatha
or, by R. T. Gtinther, 357

| Production of various forms of spi-
Notes on 'Trochus, by H. J. Fleure —

cules, by W. Woodland, 55

Radula, composition and development,
by I. B. J. Sollas, 115

Reproductive organs, skin, and hair
of Notoryctes, by G. Sweet, 325

Ridewood, W. G., development of
plumes in Cephalodiscus, 221

Ridewood, W. G., and H. B. Fantham,
on Neurosporidium cephalo-
disci, 81

Robinson, M., segmentation of head
of Diplopoda, 607

Sacculina, fixation of Cypris larva of,
by G. Smith, 625

Sauropsida, head-muscles in, by F.
H. Edgeworth, 511

Scleroblastic development of spicules
in Mollusea and Ascidians, by W.
Woodland, 45

Scleroblastic development of spicules
in Ophiuroidea, ete., by W. Wood-
land, 31

Segmentation of head of Diplopoda,
by M. Robinson, 607

Segments of occipital region of Frog,
by A. I. M. Elliot, 647

Sense-organs, parietal, in Geotria,
by A. Dendy, 1

Skeleton of Madreporaria, by M. M.
O. Gordon, 473

Skin, hair, and reproductive organs of
Notoryctes, by G. Sweet, 325

662

Smith, G., fixation of Cypris larva of
Sacculina, 625

Sollas, I. B. J., the Molluscan Radula,
115

Spicule formation, studies in, Part V,
by W. Woodland, 31; Part VI, 45;
Part VII, 483

Spicules, various forms of, by W.
Woodland, 55

Sporozoén from Cephalodiscus, by
W. G. Ridewood and H. B. Fant-
ham, 81

Structure of Hnigma exnigmatica,
by G. C. Bourne, 253

Structure of House-fly, by C. G.
Hewitt, 395

Studies in spicule formation, Part V,
by W. Woodland, 31; Part VI, 45 ;
Part VII, 483

Sweet, G., skin, hair, and reproduc-
tive organs of Notoryctes, 325

Synapta, plate-and-anchor spicules,
by W. Woodland, 483

INDEX.

Texas cattle-fever, parasite of, by H.
B. Fantham, 297

Tooth-development in Ornithorhyn-
chus, by J. T. Wilson and J. P.
Hill, 137

Trematodes, new genus, Parorchis, by
W. Nicoll, 345

Trichomastix serpentis, by C.
C. Dobell, 449

Trochus, notes on, by H. J. Fleure
and M. M. Gettings, 459

Various forms of spicules, factors
concerned, by W. Woodland, 55

Wheel-spicules of Auricularia, by W.
Woodland, 483

Wilson, J. T., and J. P. Hill, tooth-de-
velopment in Ornithorhynchus, 137

Woodland, W., forms of spicules,
factors concerned, 55

Woodland, W., studies in Spicule For-
mation, Part V, 31; Part VI, 45;
Part VII, 483

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