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QUARTERLY JOURNAL

OF

MICROSCOPICAL SCIENCE.

Sir RAY LANKESTER, K.C.B., M.A., D.Sc., LL.D., P.R.S.,
VOLUME 62.— New Series.

IIONOIUUY FBI, LOW OK KXETKK COLLBGK AND HONORARY STUDENT OF CHRIST CHURCH, OXFORD;
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EDITED BY

WITH THE CO -OPERATION OF

SYDNEY J. HICKSON, M.A., F.R.S.,

BEYER PROFESSOR OK ZOOLOGY IN THE UNIVERSITY OF MANCHESTER;

REGIUS PROFESSOR OF ZOOLOGY IN THE UNIVERSITY OF GLASGOW;

AND

PROFESSOR OK ZOOLOGY AT THE IMPERIAL COLLEGE OF SCIENCE AND TECHNOLOGY.

plates awb fat-Jfigures,

LONDON:

GREAT MARLBOROUGH STREET.

1917.

J. & A. CHURCHILL

V

CONTENTS.

CONTENTS OF No. 245, N.S., NOVEMBER, 1916.

MEMOIRS : PAGE

On an Ichthyobdellid parasitic on the Australian Sand Whiting
(Sillago ciliata). By Charles Badham, B. Sc., Junior Demon-
strator in Zoology, University of Sydney. (With Plates 1 and 2,
and 6 Text-figures) . . . . . .1

The Development ofAlcyonium digitatum, with some notes on
the Early Colony Formation. By Annie Matthews, M.Sc.
(With Plates 3-5 and 53 Text-figures) . . .43

The So-called Labial Cartilages of Raia clavata. By Edward

Phelps Allis, junr., Menton, France. (With Plate 6) . 95

Index to Vols. 29 to 61 Inclusive (July, 1888 to July, 1916).

CONTENTS OF No. 24 6, N.S., FEBRUARY, 1917.

MEMOIRS :

On Phoronis ovalis, Strethill Wright. By Sidney F. Harmer,

Sc.D., F.R.S , Keeper of Zoology in the British Museum (Natural
History). (Published by permission of the Trustees of the British
Museum. (With Plates 7-9) .... 115

The Embryonic Development of Trichogramma evanescens,
Westw., a Monembryonic Egg Parasite of Donacia simpl.ex.

Fab. By J. Bronte Gatenby, Exhibitioner of Jesus College,
Oxford. (With Plates 10-12) .... 149

On the Development of the Cape Cephalodiscus (C. gilchristi,
Ridewood). By J. D. F. Gilchrist, M.A., D.Sc., Ph.D. (With
Plates 13 and 14). . . . . . 189

Note on the Sex of a Tadpole raised by Artificial Parthenogenesis.

By J. Bronte Gatenby, B.A., Exhibitioner of Jesus College,
Oxford. (With 5 Text-figures) .... 213

An Easy Way of Demonstrating the Nuclei of Nerve Fibres. By
Henry E. Reburn, Student of Medicine. (From the Physiological
Laboratory, King’s College, London) .... 217
On a Larval Actinian Parasitic in a Rhizostome. By C. Badham,

B.Sc., Demonstrator in Zoology, University of Sydney. (With 3
Text-figures) . . . . . .221

The Early Development of the Spleen of Lepidosiren and Protopterus.

By G. L. Purser, B.A. (Sometime Coutts-Trotter Student, Trinity
College, Cambridge. (With Plates 15-17) . . • 231

A Note concerning the Collar Cavities of the Larval Amphioxus.

By K. M. Smith, A.R.C.S., D.I.C., and H. G. Newth, Demonstrator
in Zoology, Imperial College of Science and Technology. (With
Plate 18) . . . . . . • 243

IV

CONTENTS.

PAGE

CONTENTS OF No. 247, N.S., AUGUST, 1917.

MEMOIRS :

On the So-called Pharyngeal Gland-cells of Earthworms. By
J. Stephenson, D.Sc., M.B , Lieutenant-Colonel Indian Medical
Service ; Professor of Zoology, Government College, Lahore. (With
Plate 19) . . . . . .253

The Chromosome Complex of Culex pipiens. Part II. — Fertilisa-
tion. By Monica Taylor, S.N.D., D.Sc. (With Plate 20 and 1 Text-
figure) ..... . 287

The Homologies of the Muscles related to the Visceral Arches of the
Gnathostome Fishes. By Edward Phelps Allis, jr., Menton,
France (With Plates 21 and 22 and 1 Text-figure) . . 303

The Cytoplasmic Inclusions of the Germ-cells. Part I. — Lepi-
doptera. By J. Bronte Gatenby, B.A., Exhibitioner of Jesus
College, Oxford. (With Plates 23-25, and 5 Text-figures) . 407

The Degenerate (Apyrene) Sperm-formation of Moths as an Index to
the Inter-relationship of the Various Bodies of the Spermatozoon.

By J. Bronte Gatenby, B.A., Exhibitioner of Jesus College,
Oxford. (With Plate 26) ..... 465

CONTENTS OF No. 248, N.S., DECEMBER, 1917.

MEMOIRS :

Notes on the Morphology of Bathynella and some Allied Crustacea.

By W. T. Calm an, D.Sc. (Published by permission of the Trustees
of the British Museum.) (With 14 Text-figures) . . 489

On Oxnerella maritima, nov. gen., nov. spec., a new Heliozoon,
and Its Method of Division ; with Some Remarks on the Centro-
plast of the Heliozoa. By Clifford Dobell, Imperial College of
Science, London, S.W. (With Plate 27) . . . 51S

“Proboscis pores” in Craniate Vertebrates, a Suggestion concerning
the Premandibular Somites and Hypophysis. By Edwin S.
Goodrich, F.R.S., Fellow of Merton College, Oxford. (With
Plate 28, and 3 Text-figures) ..... 539

The Cytoplasmic Inclusions of the Germ-Cells. Part II.— Helix
aspersa. By J. Bronte Gatenby, B.A., Exhibitioner of Jesus
College, Oxford (Dept, of Physiology). (With Plates 29-34 and 5
Text-figures) ...... 555

Note on the Development of Trichogramma evanesce ns.

By J. Bronte Gatenby, Exhibitioner of Jesus College, Oxford . 613

Title, Index, and Contents.

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NOVEMBER, 1916.

THE

QUARTERLY JOURNAL

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MICROSCOPICAL SCIENCE.

Sir RAY LANKESTER, K.C.B., M.A., D.Sc., LL.D., F.R.S.,

HONORARY FBI. LOW OF RXBTKK CO LI. KG K AND HONORARY STUDENT OF CHRIST CHURCH, OXFORD;
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GILBERT C. BOURNE, M.A., D.Sc., E.R.S.,

ACRE PROFESSOR OF COMPARATIVE ANATOMY, AND FELLOW OF MERTON COLLEGE, OXFOR

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WITH LITHOGRAPHIC PLATES AND TEXT-PIGURES.

J. & A. CHURCHILL

Adlard & Son and West Newman.,]

LONDON.

7 GREAT MARLBOROUGH STREET.
1916.

[London and Dorking.

CONTENTS OF No. 245-New Series.

MEMOIRS :

PAGE

On an Ichthyobdellid parasitic on the Australian Sand Whiting
(Sillago ciliata). By Charles Badham, B. Sc., Junior Demon-
strator in Zoology, University of Sydney. (With. Plates 1 and 2,
and 6 Text-figures) . . . . .1

The Development ofAlcyonium digitatum, with some notes on
the Early Colony Formation. By Annie Matthews, M.Sc.
(With Plates 3-5 and 53 Text-figures) . . .43

The So-called Labial Cartilages of Baia clavata. By Edward

Phelps Allis, junr., Menton, France. (With Plate 6) . 95

Index to Vols. 29 to 61 Inclusive (July, 1888 to July, 1916).

JAN 10 1917

AN ICHTHYOBELLID PARASITIC ON SAND WHITING.

: . | 1

On an Iehthyobdellid parasitic on the Australian
Sand Whiting (Sillago ciliata).

By

CBiarles Badtiani, B.Sc.,

Junior Demonstrator in Zoology, University of Sydney.

With Plates 1 and 2, and 6 Text-figures.

Introduction.

In August, 1912, during a visit to the Fish Hatcheries-
Institution, Port Hacking, an inlet south of Port Jackson,
Hew South Wa’es, my attention was called to a parasite
which, I was informed, had for some time past caused the
death of sand whiting kept in a large spawning pond. An
examination of this parasite showed it to be a marine leech.

The history of the infestation, so far as I have been able to
ascertain, is as follows.

It has been the custom for some years past to stock a large-
sea-water pond of the Institution with two or three dozen
sand whiting taken from the shoals in the vicinity. This
appears to have been done several times, and on each occasion
the fish were killed by the attacks of these leeches.

The fish, shortly after being placed in the pond, sickened,,
developed large ulcerated patches on their integument and
died. Within ten weeks of being introduced into the pond
most of the fish would be in a moribund condition.

It was easy to know when the fish were seriously affected,
for then they would swim very close to the surface* and on
their sides. An examination of a badly infested fish usually
VOL. 62, PART 1. NEW SERIES. 1

CHARLES BADHAM.

showed about a hundred leeches in various stages of develop-
ment, ranging in length from 1 to 13 mm.

The parasites were found on the tins — pectoral, pelvic, dorsal,
and caudal ; their presence was also noticed around ulcerated
patches on the sides of the fish, and a few were found in the
proximity of, but not on, the gills.

Owing to their transparent nature, and the mucous secre-
tions of the fish, they were not so readily visible as their
highly pigmented form seen when magnified would suggest.

On the occasion of the first visit in August I secured several
hundred specimens, and in March of the following year, having
determined to work out the anatomy and systematic position
•of this leech, I again visited the Fish Hatcheries, but found
that all the sand whiting had died. Dredging giving no
results, arrangements were made to again place a number of
the fish in the pond in order to obtain living specimens of
the leech.

Owing to an oversight, I was not informed of the state
of these fish until all save one had died. This remaining fish
was caught on June 8th, 1913, and was found but slightly
infested. Most of the leeches obtained died in the first few
days, but two hardy specimens were kept alive for three
weeks, and these served for an extended intra vitam
■examination, for taking photographs, and for making a
coloured drawing.

Owing to the transparent nature and small size of the
leeches, the details of the Blood-vascular, Nephridial, and
Doelomic systems could be followed, and this with only the
Might compression of the leech produced by the pressure of
u thin cover slip.

A number of photographs of the living form were secured
showing these systems in detail, and have been of value in
this work. The pond in which these fish were kept under
conditions which so favoured the increase of the leeches was
a large one, being about 50 x 100 ft. The water in it varied
in depth from 2 to 6 ft., and was changed by means of valves,
the water being run off at low and replaced at high tide.

AN ICHTHYOJBDELLID PARASITIC ON SAND WHITING. 3

About a dozen species of fish were kept in it: as well
as the sand whiting (Sillago ciliata), there were present
Pagrosomns auratus, Chrysophrys australis, and
Oaranx georgianus.

In no case were these leeches found on any fish other than
the sand whiting. That this pond formed a favourable place
for the development and increase of parasites was also shown
by the fact that most of the fish, except the sand whiting,
were infested by ectoparasitic Trematodes ; these will in du'e
course be described by Dr. S. J. Johnston, of the Sydney
University.

At the beginning of February, 1914,1 had the opportunity
of examining many hundreds of sand whiting netted by
fishermen or caught byline in the estuarine and ocean waters
of Wreck Bay, about 100 miles south of Port Jackson. Among
these fish it was rare to find an individual which did not have
from two or three to half a dozen specimens of this leech.
I examined a large number of other species of fish netted
along with the sand whiting, but never found them infested
with this or any other leech.

It was found necessary to create a new genus to contain
this leech, which will be described under the name of
Austrobdella translucens.

Austrobdella gen. nov.

Definition. — Small marine leeches with well-defined neck
and body regions. The body cylindrical in the young, but
much flattened in the adult. The lateral parts of the body
below the clitellum bulging out and forming a shoulder-like
appearance. Somite of six annuli. No pulsating vesicles
present, their place being taken by a continuous contractile
lacuna placed on either side outside the body musculature.
Dorsal and ventral median lacunae present, communicating by
segmental lacunae. Three ' pairs of pouches present in tho
thick- walled intestine, a fourth pair being represented by :i
flexure of the gut. Testes five pairs. Eyes one pair.

4:

CHARLES BADHAM.

Type species A. translucens, mihi. Type specimen' in
the Australian Museum, Sydney, No. W. 403.

External Form,

r The material on which this work is based consists of a large
number of specimens ranging from the young sexually
immature to the adult form. From a number of specimens it
has been possible to arrange a complete series of individuals
to show the change of form during growth. I would lay
stress on this very marked change in form, and my observa-
tions have convinced me of the likelihood of the younger
forms of similar marine leeches being described as new genera
or species in the absence of a series of individuals linking up
the young to the adult.

In Text-fig. 1 are shown the outlines of specimens which
measure 1*5, 3, 4, 55, and 7‘5 mm. long, to illustrate the
change in form which takes place during the growth of this
leech. Young forms possess a cylindrical body, and the chief
change in shape during growth is due to the great lateral
development of the body posterior to the cTitellum. The
appearance of a specimen 1*5 mm. long is shown in PI. 1,
fig. 3. When this leech is 4 mm. long the testes become
mature. At this stage the body is cylindrical (PL 1, fig. 4),
there is but a faint indication of the clitellum, and the ovaries
are quite undeveloped. In 6 mm. specimens maturation
divisions are seen in the ova. At this stage the clitellum has
become enlarged and is now well marked.

The young of Austrobdella have the anterior sucker of
the same diameter as the cylindrical body and the posterior
sucker nearly twice that size (Pl. 1, fig. 3). When a length
of 4 mm. is reached the anterior and posterior suckers
approach one another more closely as regards size and have a
diameter a little less than the greatest din meter of the body
(Pl. 1, fig. 4). When the leech has a length of 5*5 mm.
(Text-fig. 1) the typical form of the adult is foreshadowed.
At this stage the clitellum becomes evident, and owing to the
increased lateral development that part of the body posterior

AN IOHTHYOBDELLID PARASITIC ON SAND WHITING. 5

and lateral to it presents a shouldered appearance ; but the
body is only slightly flattened. When a length of 7*5 mm.
(Text-fig. 1) is reached the lateral development is quite pro-
nounced, causing a more flattened form, and the suckers have
assumed the ratio shown in the figure of an adult specimen,
9 mm. (PL 1, fig. 5).

Text-fig. 1.

Outlines of specimens of A ustrobdella translucens which
measure P5, 3, 4, 5’5 and 7‘5 mm. long. The figure showsthe
change in shape during the growth of this marine leech.

I give below a table to show the measurements at different
stages of development :

Length.

15 mm.
4*0 mm.
7‘5 mm.
9-0 mm.

Breadth below
Clitellum.

T3 mm.
‘52 mm.
-135 mm.

3 25 mm.

Height.

13 mm.
'5 mm.
‘7 mm.
10 111m.

Diameter
Oral Sucker.

13 mm.

‘30 mm.

•55 mm.

•8 X 5 mm. =
•65 mm.

Diameter
Posterior, Sucker.;

•20 mm.

•41 mm.

91 mm.
1-75 X 11-
142 mm. ,

The measurements given for the 9 mm. stage are from the
specimen which is shown in PI. 1, fig. 5. The following
additional .measurements are also from this specimen :

6

CHARLES BADHAM.

Distance from oral sucker to male opening 1’26 mm.

Oiameter of neck at base of oral sicker . *6 mm.

Breadth of clitellum ..... *95 mm.

All these measurements are taken from leeches killed by
means of boiling corrosive acetic solution while in a quiescent
condition.

In describing Platybdella micliaelseni, Johansson
(1911) states that the larger of two specimens secured was
6*9 mm. long. He found in this specimen that the testes were
ripe but the ovaries slightly developed, despite the presence
of spermatozoa. He therefore considers that this species
would not attain a much greater size. However, if my ob-
servations on the growth of Austrobdella are found to
be generally applicable to marine leeches, the species he
describes might grow to nearly twice the length given.

Coloration.

The transparent nature of the body of Austrobdella
allows the beautiful pigmentation to be clearly seen. The
drawing (PI. 1, fig. 1) is a careful representation in black
and white of a living specimen, viewed from the ventral
surface, as seen by transmitted light.

The specimen depicted is a young extended form in which
neither the clitellar region nor the lateral parts of the main
body region are as well developed as usual. This individual was
made use of owing to its having retained its colour in captivity
better than its fellows.

On the ventral surface most of the pigment cells are seen
to be wrapped round what appears to be a tube. These cells,
which are reddish-brown in colour, are in the walls of the
ventral lacuna ; their stellate nature is well represented.
Similar pigment cells are seen in relation to the ejaculatory
canals ; these are here, as in most specimens, rendered con-
spicuous by their dark colour.

As well as the reddish-brown pigment cells mentioned, a
smaller number of cells of varying shades of purple are

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 7

present ; some of these show through from the dorsal surface.
Large and more diffuse yellow cells are scattered about the
surface, and at the posterior region of the body certain of the
cocoon glands have a light brown colour.

Viewed from the dorsal surface the lighter coloured pigment
cells of the neck region are seen to be placed in a single
lateral and a medial double row.

The darker cells are found in a well-marked single row
along the mediad wall of the contractile lacuna and are
plentiful, but without any apparent regular arrangement in
the dorsal body wall.

The lighter cells appear as five irregular rows in each side
of the body.

In a living specimen the two eye-spots are very con-
spicuous. They are of a rich dark reddish-brown colour,
larger than any of the pigment cells and characterised by
their regular outline in place of the fringed appearance of
the pigment cells. In the drawing they are shown as seen
from the ventral surface through the body tissues.

A microscopic examination of the caudal fin of a living
sand whiting to which a leech was attached showed a remark-
able similarity in colour and arrangement of the pigment cells
of the two animals.

So far as my observations go the pigment cells lose their
fringed character in strong light and become more regular in
outline.

Movements.

During my observations of living specimens I never saw an
individual swimming, neither could I, by dropping the leeches
into salt water, cause any swimming motion. They would
fall straight to the bottom of the beaker and then move
slowly along the surface of the glass by leech-like movements.
These observations were made on leeches ranging in size from
2 to 12 mm. They have a bearing on the question of the
deposition of the cocoon and the manner of infestation of
sand whiting by young leeches. Johannson (1898), writing*

8 CHARLES BADHAM.

about Abranchus (a genus allied to Austrobdella), corre-
lates tlie fact that the members of fliis genus are unable tb
swim, with the fact that they infest fish which live in shallow
water among algal growths and so afford an opportunity for
leeches to creep on to them. He also supposes that when the
time comes for depositing the cocoon Abranchus drops to
the bottom and fixes its cocoon in seaweed. I have not
ascertained the method of depositing the cocoon in Austrob-
della, but would point out that the fish on which it lives
gets a great deal of its food by burrowing in the sand.

Annulation.

.

A typical somite consists of 'three annuli, and in the adult
these three annuli are divided so that the somite is of six
annuli. In young forms the somite consists of the three
primitive annuli ; this is followed by a stage in which there
are five annuli to the somite, due to the division of the annuli
anterior and posterior to the annulus in which the nerve
ganglion is situated. This stage of five annuli to the somite
“may persist in specimens which are quite large, but in the
largest specimens all the somites in the testicular region have
six annuli. Thus it happens that specimens 6 mm. in length
are seen, in which the primitive annulus containing the nerve
ganglion is still undivided. It is owing also to this subse-
quent division of primitive annuli that a difficuly arises in
regard to the number of annuli to be seen in the neck region,
for these increase in number with the length of the specimen.
In a leech of 4 mm. the male pore is between the 15th and
:16th primitive annuli and the female pore between the 17th
and 18th annuli. At this stage the somite in the testicular
region is still trimerous. In a leech of about 9 mm. there
are found twenty-three or twenty-four annuli in front of the
male pore. The primitive annuli which divide to form the
new annuli are the 4th, 5th, 7th, 10th, 11th, 13th, 14th, and
15th (Text-fig. 2). In regard to the total number of annuli,
my determinations have been varied owing to the fact that

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 9

the annulation of the 'posteriof part of the leech is very indis-
tinct,*so that in specimens in which the annulation of the neck

Text-fig. 2.

Diagram showing the character of the annulation of the
preclitellar and elitellar regions of Austrobdella trans-
lucens and the relations of the nerve ganglia, proboscis, and
sexual organs. S. gn. Subcesophageal nerve ganglion. N. gn.
Nerve ganglion. P. Proboscis. Ej. t. Terminal part of
ejaculatory canal. Sp. gl. Spermatophore glands. Male
opening. $ Female opening. Roman numerals indicate
primitive annuli, ordinary numerals the subdivision of the
primitive annuli.

and jlnid- body can be seen, that of this posterior part of the
body is pot evident.

10

CHARLES BADHAM.

The relations of the nerve ganglia, male and female
openings, and the male glands to the neck annuli are shown
in Text-fig. 2.

The Epidermis.

The cuticle is thin, 2 ju or less in thickness. The cells
of the epidermis are irregularly cubical in shape, measuring
about 8 fji. Towards the anterior and posterior suckers they
become more cylindrical and have a length of 14 fx by 5 /u
wide. On the ventral surface towards the median plane these
epidermal cells are not so numerous. But a few of them are
slightly enlarged and converted into gland cells with ducts
opening on the surface.

Hypodermal Glands.

There is developed on the lateral margin of the main body
region, partly surrounding the contractile lacuna, a remark-
able layer of cells, three or four deep and of great size
(PI. 2, fig. 9, L.gl.). Each is a unicellular gland and its
duct opens on the ventral surface. The largest of these cells
has a diameter of 63 fi and the size ranges from this to about
15 fj.. The shape varies considerably, but in all the secretion
space passes gradually into the duct, which has a very small
lumen.

The nucleus varies a good deal in appearance ; generally it
is more or less spherical, but frequently elongated and
twisted ; it has many chromatin particles staining heavily
with haematoxylin.

These glands appear to correspond to certain cells, not so
well developed, called the lateral glands in Branchellion
by Sukatsehoff (1912).

As is general in Ichthyobdellids, both mucous and albu-
minous unicellular glands are present in the two suckers.

In the oral sucker the albuminous glands are arranged
around the dorso-lateral three-fourths of the sucker. They
are placed inside the circular and longitudinal musculature of

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 11

the sucker wall, and, measuring about 20 /u in diameter, are
about half the size of the same glands in the posterior sucker.
Among them are placed the smaller mucous glands, and both
types of glands open by ducts on the concave surface of the
sucker. In the posterior sucker these glands are distributed
over the whole sucker. The mucous glands are found also
forming a pharyngeal group with ducts opening into the
pharynx, and they extend posteriorly and lie among the
salivary glands but nearer the body wall ; they are not
present below the glandular oesophageal pouches.

Clitellar Glands.

As has been remarked by various authors, the development
of the unicellular glands in the Ichthyobdellids is most
remarkable. To quote Bourne (1884): “They attain rela-
tively, and in Pontobdella and Branchellion actually,
huge dimensions.”

In this leech the largest among these cells have a diameter
of 170 /u, and, if they are situated far back in the body, a duct
7 or 8 mm. long. The clitellar glands extend in the body
region from the posterior sucker to the beginning of the
clitellum.

They are placed inside the body wall musculature and
occupy practically all the space between it and the alimentary,
reproductive, and lacuna systems. Text-fig. 3, drawn from a
transverse section through the region of the thick-walled
middle gut, shows the degree of development of these glands
(Cl. gl. 1, Cl. gl. 2).

Their ducts open all round the clitellum, that is from the
level of the 10th nerve ganglion to midway between the
12th-13th nerve ganglia. SukatschofPs (1912) excellent
work on these glands showed that in Branchellion there
were three types producing different secretions, and he gives
a description of extraordinary development of the branched
nucleus, which may come to measure 336 ju and which he
compares (p. 488) with other examples of nuclei of this type
in the animal kingdom.

12

CHARLES BAPHAM.

In Au strob della I shall content myself for the present
with the appearances presented upon staining with Ehrlich’s
hgematoxylin and eosin. By this means there are clearly
differentiated two types of clitellar glands. The larger of
these cells (Text-fig. 3, Cl. gl. 1), which are bowl-shaped or
more elongated, are characterised by their large secretion
space filled with a homogeneous substance showing a finely
granular nature when highly magnified, and staining a light
pink with eosin. These cells, which have an average diameter

Text-fig. 3.

A transverse section through the thick- walled, middle -gut region
of Austrobdella translucens (x 40), showing the arrange-
ment, development and character of the clitellar glands. The
relation of the intestinal sinus to the epithelium of the thick-
walled middle gut is shown. A portion of a segmental lacuna
of one side is present. The blind gut has been cut at its
narrowest part. Cl. gl. 1. Clitellar gland of the first type.

Cy. Cytoplasm. Nu. Nucleus. Sec. s. Secretion space.

Cl. gl. 2. Clitellar gland of the second type. Cl. gl. d. Groups
of ducts of clitellar glands. V.ne. Yentral nerve cord. B. g.
Blind gut. S. 1. Segmental lacuna. I. s. Intestinal sinus.

M. g. ep. Epithelium of thick-walled part of middle gut.

of 110 fi , have the bulk of their cytoplasm at the peripheral
end i;n a layer, which is 30 or 40 jjl thick, and which lines, as
it were, the secretion space, becoming finer towards the ducts.
This cytoplasm is of very coarse structure, and the nucleus,
wjiich is rich in large chromatin particles, has an irregular
shape, as described by Sukatschoff in Branchellion, and is

3

AN IOHTHYOBDELIAD PARASITIC ON SAND WHITING-. \ -

placed next to the secretion space (Text-fig. 3, Cy.} , riu.,
sec. s.).

The other type of clitellar gland cell (Text-fig. 3, Gl.gl.y 2)
is distinguished by the intense staining of the secretion by

Text-fig. 4.

Drawn from a whole mount of a specimen of Aust rob della
translucens 6 mm. long (x 100) which was killed when the
proboscis was protruding. The figure shows the character of
the salivary glands and their relations to the nerve ganglia.
P. Proboscis. S. gn. Suboesophageal nerve ganglion. N.gn. 7.
Seventh nerve ganglion. S. gl. Unicellular salivary gland.
S. gl. d. Unicellular salivary gland duct. Ej. t. Terminal part
of the ejaculatory canal. Sp.gl. Unicellular spermatopliore
glands.

14

CHARLES BADHAM.

eosin. This secretion is coarse and consists of particles of
2 fi in diameter. These cells, which are a good deal smaller
than those of the first type, have a diameter of about 60 /u, and
are placed in between the larger cells beneath the body wall
musculature, as well as internal to the larger cells, and a few
are found outside the salivary glands with their ducts passing

Text-fig, 5.

Transverse section through the preclitellar region of a specimen
of Austrobdella translucens 7 mm. long (X 186). The
drawing shows the character of two of the unicellular salivary
glands and their relatively large size. The position and
character of the nucleus which is next to the secretion space
is also shown. S. gl. Unicellular salivary gland. Cy. Cytoplasm.
.A lu. Nucleus. Sec. s. Secretion space. N. gn. A ganglion of
ventral nerve cord. P. Proboscis.

back to open on the clitellum. The nucleus, which is irregular
i ii shape, is either next to the secretion space or in it, and
separated from the cytoplasm. The ducts of these two types
of cells, filled with their characteristic secretion, are gathered
into four bundles, which, just before the clitellum, are placed
two in the dorsal and two in the ventral region.

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 15

The ducts of the larger glands have a diameter of 8 fi, the
smaller 6 ^ .

Salivary Glands.

The salivary glands, as shown in Text-fig. 4 (S. gl.) are
placed between the upper level of the eighth, and the lower
level of the tenth nerve ganglion. On either side of the pro-
boscis there are about five or six giant-cells and a number of
smaller ones. The large cells have a diameter of about 120 /u,
and other cells range in size from that down to 20^.
Their ducts curve at right angles to the body of the cell and
enter the base of the proboscis. The character of these cells
and their relatively huge size is shown in Text-fig. 5. The
position of the nucleus, which is next to the secretion space
is also shown.

The C(elomic System.

We owe to Johansson (1898) and to Selensky (1906) our
main knowledge of the coelomic system of the Ichthyobdellids
other than Pontobdella, Br anchellion, and Ozo-
branch us. The former in 1896 pointed out the great value
of the knowledge of this system in systematic work, giving
a number of examples of the chief features in different
Ichthyobdellids. This leech, while possessing the main
features of the coelomic system as described for Piscicola
and Callobdella, diverges widely in certain respects. This
divergence is most marked in that part which corresponds to
the contractile vesicles of Piscicola and Callobdella.

In Austrobdella, in place of a lateral row of such vesicles,
there is a continuous contractile lacuna. This lacuna occupies
the position of the contractile vesicles, as described for allied
genera, lying laterally just beneath the skin outside the
muscle layer. On either side it extends from the level of
the proboscis to the level of the anus, but is contractile only
in the region of the testes and the thick-walled intestine.
This genus differs also in wanting altogether the lateral

16

CHARLES BADHAM.

lacuna found in a numbed of Ichthyobdellid leeches, a fact
of considerable importance when considered in relation to the
direction of the flow of lymph in the body-cavity. Proceeding
to a detailed examination there are found the following
lacunae ; Dorsal, Yentral, Contractile, and the Segmental
Lacunae.

I shall follow Oka and Selensky in the use of the term
“lacuna” in place of “sinus” in dealing with the various
portions of the coelome, reserving the term “sinus” for the
blood-space found round the intestine called by Johansson the
“ intestinal lacuna.”

The Dorsal Lacuna may be considered in two parts, the-
testicular and the intestinal portions. It extends from the
beginning of the testes to the anal region. In the testicular
region it contains the dorsal blood-vessel (PI. 2, fig. 8, Dt.) ;
in the intestinal region it surrounds the thick-walled intestine
and the blood- stream in relation with it.

Posteriorly the intestinal portion communicates with the
ventral lacuna in the anal region. In each segment the dorsal
lacuna communicates by a pair of segmental lacunae with
similar extensions of the ventral lacuna (PI. 2, fig. 8, 8. 1.).
The wall of the testicular portion of the dorsal lacuna has a
thin well-defined membrane with small elongated nuclei,
which are scarce. Beneath this membrane are a few muscle-
fibres.

The musculature is increased where the dorsal blood-vessel
is fused with the wall of the dorsal lacuna.

Frequently the dorsal lacuna is divided into two parts by
the formation of septa placed dorsallv and ventrally to the
dorsal blood-vessel (PI. 2, fig. 10, Sep. d. Sep.v.). Such
septa generally begin with the origin of the valves of the
dorsal vessel, but do not extend to the preceding or succeeding
valve-origin ; so that at certain places the dorsal blood-vessel
lies free in the dorsal lacuna.

The Yentral Lacuna (PI. 2, fig. 8, v.l.) occurs as a
tube, which varies in size and runs from the anal region to
the proboscis, ventral to the alimentary canal. Throughout

AN ICHTHYOBDELLID PARASITIC ON SAND WHITINC. 17

its extension it contains the ventral nerve cord and the ventral
blood-vesssl.

Anteriorly it is considerably expanded and entirely sur-
rounds the proboscis and related organs.

A considerable expansion of the lacuna also contains the
ovaries, and again it is dilated in the region of the posterior
ganglionic mass. A pair of segmental communications is given
off at the level of each nerve ganglion, joining with those given
off more posteriorly in each segment by the dorsal lacuna.

Everywhere the ventral lacuna is lined by a membrane of
the same character as that of the dorsal lacuna, but the
muscle-fibres are few.

The Segmental Lacunae, as already mentioned, extend
from the dorsal and ventral lacunae. They unite towards the
lateral region of the body, just past the testes of each seg-
ment, and are also found in the thick-walled intestinal region.
Reference to the figure of the Ccelomic System (PI. 2,
fig. 8) will aid the explanation of the course and branching of
the segmental lacunae.

Immediately after the junction of the dorsal and ventral
extension, the lacuna divides and more laterally each division
again divides in two ; of the four ultimate branches, the two
inside ones unite and open into the contractile lacuna (PI. 2,

figs. 8, 9, s. q.

The two outside branches unite with the outside branches
of the preceding and succeeding segmental lacunae respectively.
Thus it follows that in each segment there are two openings
of the segmental lacunae into the contractile lacuna. These
openings into the contractile lacuna are furnished with muscle-
fibres of annular arrangement and sphincter action.

The lining of the segmental lacunae is a continuation of
that of the dorsal and ventral lacunae ; no muscle fibres are
found in their walls.

Contractile Lacunae. — These extend on either side from
the level of the base of the proboscis to the level of the
anus, but are only contractile from the beginning of the
testicular region. As already stated, their chief feature is their

VOL. 62, PART 1. NEW SERIES. 2

18

CHARLES BADHAM.

extension as tubes iti place of the row of vesicles found in
allied genera. They possess, as shown in PI. 2, fig. 9 ( C . 1),
the character of a series of pouches ; their walls are
furnished with delicate muscle fibres. Anteriorly the con-
tractile lacunae cease to be contractile at the level of the
neck but are continued forward as non-contractile parts to
the anterior sucker. I have not determined their course past
this point, but they certainly do not appear to break up into
capillaries.

Posteriorly their relations are more important. At the
level of the posterior sucker they curve sharply, and, passing
ventral to the two branches of the dorsal vessel, open into
the lacuna in the anal region. They receive on either side
of each segment the two openings of the segmental lacunae
(PL 2, fig. 8, C. 1.). Dorsally in each segment they give
off three or four pairs of capillaries (PI. 2, fig 8, Cap. 1.) ;
these run parallel to one another, just outside the muscle
layer. In one living specimen I observed these opening into
the dorsal lacuna, but was unable to demonstrate this in
other specimens.

In Callobdella Johansson has seen three or four pairs
of capillaries in each segment going from the dorsal lacuna
and stretching into the surrounding tissue.

The Circulation of the Lymph.

In the description of the circulation of blood in the vessels
mention will be made of the contraction of the pouches of
the thick-walled intestinal region. By these contractions a
space is produced between the wall of the intestinal sinus
and the wall of the intestinal lacuna, which is immediately
filled by the lymph flowing in from the lacuna formed in the
anal region by the fusion of the dorsal and ventral lacunas.
When the intestinal sinus is again in the condition of diastole,
the lymph is seen to be forced out of the dorsal lacuna and
to flow into the segmental lacunae. At the spot where the
dorsal and ventral extensions of the segmental lacunae join a
great deal of regurgitation takes place. The lymph corpuscles

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 19

are seen to be violently hurried in various directions. Some
are driven into the extensions of the segmental lacunae on
either side, some towards the ventral lacuna, but the majority
pass through the openings leading to the contractile lacuna.
'These are immediately closed by the annular muscle fibres
(PI. 2, fig. 9, S. m.f-), which function as a sphincter when
the contractile lacuna begins to contract. This contraction is
from before backward and the lymph is carried to the lacuna,
lying in the anal region, formed by the fusion of the dorsal
and ventral lacunae, whence it flows again into the dorsal
lacuna. The contraction of the contractile lacuna immediately
follows the diastole of the intestinal sinus, and the dorsal
blood-vessel in the testicular region; so that it is seen, in a
leech freshly taken from an ocean whiting, to take place
about thirty times per minute.

In this description of the cycle of the circulation of the
lymph, it will be observed that the events taking place in the
ventral lacuna are not mentioned. This is due to the relative
stagnation of the lymph in this lacuna. Despite repeated
attempts to find a definite direction of flow in the ventral
lacuna, I have been unable to observe anything more than
a great deal of regurgition both in this and the ventral parts
of the segmental lacunae. Frequently strong currents carry
lymph corpuscles from the dorsal segmental lacunae far into
these lacunae, and these, and other corpuscles therein, are
kept in constant movement by eddies. But there appears to
be no such definite direction of flow as is present in the dorsal
and contractile lacunae.

•Comparison with the Lymph Circulation of other

Leeches.

Salensky’s (1906) description of the valve arrangement in
the side vesicles of Piscicola makes evident the course of
the circulating lymph in this leech and affords an interesting
■comparison with the circulation above described.

In Austrobdella I consider the contractile lacunae, on
.account of their subcuticular position and pouched character.

20

CHAELES BADHAM.

to be homologous with the paired vesicles of other Ichthyob-
dellid leeches. They perform the functions of the side
vesicles and lateral lacunas of Piscicola-like forms. In place
of the valve described by Salensky, there are found openings
guarded by sphincter-muscle fibres.

The contraction of the dorsal vessel in the testicular region
is not caused by the lymph flowing back from the lateral
regions, as described by Johansson (1896 b) for Callob della,
for here in the segmental lacunas the flow is always to the
contractile lacunas. It is mainly caused by the flow of lymph
from behind and partly by the contractility possessed by the
blood-vessel itself.

Alimentary System.

Here I shall employ the terms used by Sukatschoff (1912)'
in his excellent description of this system in Branchellion.
The mouth opening is placed slightly towards the dorsal side
of the anterior sucker and leads into the short pharynx (Text-
fig*. 6, M.). Following the pharynx is the oesophagus, which
contains the proboscis characteristic of the Rhychobdellid
leeches and capable of being protruded by the eversion of the
pharynx and oesophagus. There follows then the entodermal
anterior gut.

The proboscis, when retracted, occupies somites 7, 8, and 9
(Text-figs. 2 and 6, P.). The proboscis sheath consists of a
thin ectodermal epithelium covering a number of longitudinal
muscle fibres. It corresponds very closely to the same
structure described in Branchellion by Sukatschoff — the

Text-fig. 6. — Diagram of the Nervous, Alimentary and
Reproductive Systems of Austrobdella translucens.
(X 20), A.g. Caeca of anterior entodermal gut. A.m.g. Caeca
of anterior thin-walled part of middle gut. A. s. Anterior
sucker. B.g. Blind gut. Ej.t. Terminal part of ejaculatory
canal. M. Mouth opening. M. g. Caeca of thick-walled part
of middle gut. M. g.f. Flexure of thick-walled part of middle
gut, representing a rudimentary pair of caeca. N. gn. 7. Nerve
ganglion 7, etc. (Fs. (Esophagus. (Fs. gl. (Esophageal glands.
Ov. Ovary. P. Proboscis. P. gn. Posterior ganglion of
ventral nerve cord. P. s. Posterior sucker. B. Rectum.
S. gl. Salivary glands. Sp. gl. Spermatopliore glands. S. gn .
Suboesopliageal ganglionic mass. T. Testis.

r AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 21
Text-fig. 6.

22

CHARLES BADHAM.

only difference being that the nuclei of the ectodermal
epithelium cells are more numerous, there being always one
and sometimes two between each bundle of muscle fibrils
(compare Sukatschoff, fig. 73).

The proboscis is covered by a thin epithelium, beneath
which are the longitudinal muscle fibres arranged in similar
fashion to those of other Ichthyobdellid leeches. A number of
radial muscle fibres more or less fan-shaped and 15 to 20 in
one plane (Text-fig. 5, P.) stretch from the periphery of the
proboscis to the epithelium lining the lumen. They frequently
surround the longitudinal muscle fibres at their expanded
ends. Midway between the periphery and the lumen of the
proboscis are a series of annular muscle fibres. The epithelium
lining the lumen of the proboscis is well developed. There is
little difference in the muscular structure of the proboscis
from that figured by Sukatschoff for Branchellion and by
Johansson (1896) for Callob della lophii and Abranchus
brunneus. In the spaces between the radial muscles are
placed the ducts of the salivary glands and the blood-vessels
of the proboscis.

When the proboscis is retracted it lies surrounded by its
sheath in the anterior lacuna, its apex lying close to the sub-
cesophageal ganglion.

The lumen of the proboscis opens posteriorly into a slight
expansion of the entodermal anterior gut, which has been
called the bulb in Branchellion.

The gut narrows immediately and gives off in somite 11a-
pair of oesophageal glands (Text-fig. 6, (Es . gl.). These lie
in somites 10, 11, and 12, and communicate with the oesophagus
by a very narrow lumen (5 to 10 p in diameter) in the eleventh
somite just above the nerve ganglion. These glands are
placed dorso-lateral to the accessory male glands and are
somewhat convoluted. They measure *3 mm. in a longitudinal
direction, and may, when distended, measure ’1 mm. trans-
versely. Glands of this nature were first described by
Johansson (1896) for the Ichthyobdellid leeches Piscicola,.
Callobdella, and Abranchus.

AN ICHTHY0BDELL1D PARASITIC ON SAND WHITING. 23

In 1913 Sukatschoff described similar glands in Brancliel-
lion, and compares them to the oesophageal glands of
Haementeria costata described by Ko wale vsky (1900), and
to similar glands of Clepsine plana figured by Whitman
(1891); Hemmingway (1912) found them also in the Grlos-
siphonid leeches Placobdella pediculata and P. para-
sitica.

Both Johansson and Sukatschoff state that they are
glandular organs and that fish blood-corpuscles are not found
in them, and I agree with them.

The nuclei of the epithelial cells which line these glands are
of the same character as those of the following pouches of the
thin-walled parts of the middle gut, but they are larger, some
being twice the size.

At the level of the opening of the female pore that is just
below the 12th nerve ganglion the second pair of pockets of
the anterior entodermal gut is given off (Text-fig. 6, A. g.).
They have wide openings into the gut and are somewhat con-
voluted at their blind ends. These pouches appear to corre-
spond to those figured in Branch ellion by Sukatschoff
(fig. 85, V. d. t. 2), and are homologous with the following
six pairs of the anterior thin-walled division of the middle
gut. Their difference in form is caused by their being pressed
forward by the cocoon gland ducts, which prevent lateral
distention. Behind this pair of pouches there is a sphincter
of annular muscle fibres.

Following the anterior entodermal gut is the middle gut,
which is divdded into —

(1) An anterior thin-walled part (Text-fig. 6, A.m.g.).

(2) The blind gut (Text-fig. 6, B.g.).

(3) A dorsal thick-walled part (Text-fig. 6, M. g.).

The anterior thin-walled division of the middle gut lies in
the testicular region, and consists of a small median tube
from which are given off six pairs of caeca opposite to nerve
ganglia 14 to 19 inclusive. The position and form of these
six pairs of caeca are shown in Text-fig. 6, A. m. g.

These pouches are lobed at their blind ends by the presence-

24

CHARLES BADHAM.

of dorso-ventral muscle fibres, which produce two or more
lobes in a distended pouch. A sphincter of annular muscle
fibres surrounds the tube just before and immediately behind
the first pair of pouches, and dorso-ventral muscle fibres are
also present about the neck of the following pouches and may
act as sphincters. A similar arrangement has been described
by Sukatschoff in Branchellion, but Johansson (1896)
states that all the pouches of this part of the gut of Callob-
della nod u lifer a have sphincters of annular muscle fibres.

The anterior thin-walled part of the middle gut is followed
ventrally by the blind gut (Text-fig. 6, B. g.), which in
Austrobdeila consists of a pair of elongated pouches which
open into the last pair of caeca of the thin-walled part of the
middle gut. They extend back to the anal region and are
fused with one another in five places. In fact the fusion is so
complete that the parts which are not fused form merely four
small apertures. When this blind gut region is filled with
fish corpuscles there is seen on either side a series of caeca
extending laterally, which decrease in size from before back-
wards. The extent of the development of this blind gut has
an important bearing on an interesting hypothesis put forth
by Johansson (1898). All the thin-walled parts of the middle
gut are lined by a single layer of flat 'epithelial cells, which
become considerably stretched when a pouch is distended, but
are well developed in the median parts. Weak muscle fibres
are occasionally present, and the pouches are generally filled
with undigested fish blood-corpuscles.

A comparison of the blind gut region of Austrobdeila
translucens with Johansson’s (1898) figures shows that here
the development of the blind gut is intermediate to that found
in Callobdella nodulifera and C. lophii. In this paper
Johansson compares the structure of this blind gut region in
the different Ichthyobdellids and divides them into three
types. The first, and in his opinion the most primitive, type
is represented exclusively by the genus Ab ranch us, which
has two completely, or almost completely, separated blind
pouches. The second type, represented exclusively by the

AN I0HTF1Y0BDELLJD PARASITLC ON SAND WRITING. 25

genus Pontobdella, has a single large undivided blind
pouch. The third type, to which all remaining genera belong,
shows transition forms between these two types. They have
blind pouches fused for a greater or less extent in five places.
In this group he placed the genera Platybdella, Piscicola,
Cy st obranchus, and Callob della, and by the researches
of Sukatschoff (1912) Branchellion must be included along
now with Austrobdella. The development of the structure
of the blind gut seen in the second and third types Johansson
correlated with the fact that the leeches possessing it are
enabled to exist for some time away from a host by reason of
the greater storage capacity produced by this partial or com-
plete fusion : and with the fact that it is found along with
well-develoded musculature indicating good swimming power
in such genera as are likely to experience difficulty in finding
a fresh host. To this likely hypothesis as to the cause of the
development of this fusion of the blind pouches Austrobdella
affords little support. In fact, the case presented here is almost
as hard to fit in with Johansson's hypothesis as that ofCallob-
della lophii, to explain which Johansson (1898) has to say
that ec it is hardly too bold to think that this leech never leaves
its host." He offers no explanation as to how the cocoons
are deposited, though elsewhere he states that all Ichthyob-
dellids, so far as is known, deposit their cocoons away from
their hosts. Here I may mention a note by Leigh- Sharpe
(1913) about the capture of a large angler (Lophius
piscatorius), only a few hundred yards from shore, with five
.specimens of C. lophii. Nowin Austrobdella the blind
gut is better developed than in C. lophii; the musculature
is weak (about the same as C. lophii), and the ability to
swim absent. The sand whiting, on which Austrobdella is
exclusively parasitic, so far as I can ascertain, is common, and
lives in very shallow water and feeds along shoals and beaches,
frequently burrowing in the sand. According to this mode of
life, following the reason used by Johansson for Ab ranch us,
there should be little need for extra storage of food, yet in
Austrobdella I find a development of the blind gut about

26

CHARLES BADHAM.

lialf that of Callobdella nodulifera, a species parasitic
on a deep-water fish ; however, the latter has better muscular
development. It seems that the explanation of these apparent
exceptions to what appears to be a well-reasoned hypothesis
can only be gained when the life history of these leeches is
discovered. The only record of Austrobdella trans-
lucens being found away from its host is a curious one, a
specimen being found by Prof. J. P. Hill some years ago in
the gastric pouch of a jelly fish (Cambessa mosaica).

The dorsal thick-walled part of the middle gut. —
In Austrobdella only three pairs of pouches are developed
in this division (PI. 1, fig. 1, M. g.). The fourth pair of thick-
walled pouches is present only in a rudimentary state. The
first pair of pouches lies between the 19th and 20th nerve
ganglia (Text-fig. 6, M. g.) ; the fourth, which in other
Ichthyobdellid leeches is placed between the 22nd and 23rd
nerve ganglia, is represented here by a flexure of the intestine
(Text -fig. 6, M.g.f.) The cells of the epithelium lining this
part of the gut have their free surfaces covered with a film of
densely-placed cilia, so that they present a fringed appearance
in section, similar to that described by Sukatsclioff as occur-
ring in Branchellion (1912, Fig. 78). There does not
appear to be present a section of the gut bearing a ciliated
epithelium of the usual type, such as occurs in Branch el lion
and in other Ichtliyobdellids (Johansson, 1896a). Following
the thick-walled part of the middle-gut there is the posterior
gut, formed chiefly by the rectum (Text-fig. 6, /?.), which
opens by the anus on the dorsaf surface of the 27th somite.
The walls of this portion of the gut are muscular and the
epithelium is similar to that lining the thick-walled part of
the middle gut, but is not ciliated.

The Blood- Vascular System.

Following the well-known work by Oka in 1894, dealing
with the blood-vascular system in Clepsine, there appeared
the investigations of Johansson (1896 b) and Selensky (1906).

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 27

relating to this system in Piscicola. In 1902 Oka published
a paper, in which he summarised his investigations concerning
the blood- vascular system in the various families of the
Hirudinea. In a lucid manner he showed that only in the
Grlossiphonidee and Ichthyobdellidas was a true blood-
vascular system present and that it had no communication
with the lacuna system. Again, in 1904, Oka described in
some detail the vascular system in Ozobranchus. My
investigations of Austrobdella have shown that a closed
blood-vascular system is also present here.

In general this system in Austrobdella resembles that
described in Ozobranchus and differs from the Piscicola
and Callobdella type.

There are, however, several important differences from
Ozobranchus. The lateral paired branches in the anterior
part of the body are three as compared with the four pairs
found in Clepsine and the Ichthyobdellid leeches so far
described. It is the second pair which are wanting. There
is also a ring vessel in the posterior sucker with which the
loops from the dorsal and ventral vessels connect; this is a
very different arrangement from any so far described.

Lastly, the division of the dorsal vessel into two parts takes
place in the 24th somite, which is much higher up than in
Clepsine and Piscicola.

I have been favoured in these observations on the blood-
vascular system by the transparent nature of the leech. The
diagram of the blood-vascular system (PI. 1, fig. 2), is a
careful representation of the course and relations of the
blood-vessels in the neck and anterior sucker.

The course of the blood-vessels is as follows :

The dorsal blood-vessel gives off, in the anterior part of
the body, three pairs of lateral branches, and an unpaired
proboscis branch. The first of these (PI. 1, fig. 2, L. v. 1.), is
formed by the forking of the dorsal vessel in the oral sucker.
The two branches given off run round the eye-spots and unite
in the region of the subcesophageal ganglion to form the
ventral blood-vessel. The course of this first pair of lateral

28

CHARLES BADHAM.

vessels is very similar to that found in Ozobranchus (Oka,
1904, Fig. 1). The second pair of lateral vessels (L.v.2),
is given off in somite 9 ; these branches run ventrally and
anteriorly, and unite with the ventral vessel just behind the
spot where the first pair join. The third pair of lateral
vessels (L.v. 3) are given off in somite 10 ; the two branches
run at first posteriorly to the end of somite 1 1 ; then they
curve sharply and, running forward, enter the ventral vessel,
just behind the point of entry of the second pair of lateral
vessels.

Immediately in front of the first valve the dorsal vessel
gives off the proboscis branch (P. v. 1.) which runs to the apex
of the proboscis and there bifurcates. The two vessels thus
formed unite almost at once to form the efferent proboscis
branch (P. v. 2), which runs to join the ventral vessel, just
behind the point where the second pair of lateral vessels enter
into it. After emerging from its intimate relations with the
•caeca of the thick-walled intestine posteriorly (J. s.), the dorsal
vessel divides in two at the beginning of the 24th somite.
These branches extend in such a way as to form a vessel
running round the periphery of the posterior sucker. This
part of the dorsal vessel gives off on each side four or five
.short-looped vessels, which communicate with a ring vessel
(P. v.) running right round the periphery of the posterior
sucker. This ring vessel receives on each side seven branches
of the ventral vessel. A certain degree of anastomosis is
.seen in these branches before they unite to form the ventral
vessel. I have followed the course of this ring vessel in
living leeches obtained from different places.

As stated in the account of the coelomic system, the dorsal
blood-vessel lies in the dorsal lacuna, or its extension the
intestinal lacuna, for almost the whole extent of these lacunae.
Anteriorly the dorsal vessel passes out of the lacuna at
somite 13. PI. 2, fig. 12, shows the vessels in the 11th
somite shortly after the dorsal blood-vessel has left the lacuna.
The ventral vessel is lying free in the dilated lacuna which
.surrounds the accessory male glands.

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 29'

Posteriorly the two branches of the dorsal blood-vessel in
the 26th somite leave the lacuna, formed by the union of the
intestinal and ventral lacunae, and enter the connective tissue
to course round the periphery of the posterior sucker.

The ventral vessel lies in the ventral lacuna from the 7tli
to the 26th somite. It is formed by the union of the first
pair of lateral vessels, just above the 7th somite. These
lateral vessels enter the lacuna opposite the spot where they
fuse. The ventral vessel lies quite free in the ventral lacuna
above or at the side of the nerve- cord and always above the
nerve ganglia. It leaves the ventral lacuna near the same
spot as the dorsal vessel in the 26th somite.

The histological features of the blood-vessel walls agree-
closely with those described for Callobdella by Johansson
(1896 6) and for Piscicola by Salensky (1906).

The dorsal vessel, immediately on passing out of the dorsal
lacuna, develops in the 13th somite a strong muscle layer of
annular nature internal to a layer of finer muscular fibres-
This structure continues until the diameter of the dorsal
vessel becomes smaller after the proboscis branch has been
given off. The anterior part of the dorsal vessel takes its
origin in a peculiar way from that part of the dorsal vessel
which has a much greater diameter. On the side of the-
dorsal vessel, opposite to the point of origin of the proboscis
vessel, this anterior part lies laterally to the dorsal vessel
and opens into it at two places, both of which are guarded
by valves. The lateral vessels, possessing this well-developed
muscle layer for but a little distance after they spring from the-
dorsal vessel, gradually come to resemble the ventral vessel
in the nature of their walls. The wall of the dorsal blood-
vessel in the dorsal lacuna possesses only a thin epithelium,
with scattered nuclei (PI. 2, fig. 10, Nu.epi.), save only at
those places where the valves are placed. Here there are
one or two annular muscle-fibres, such as are found in the
preclitellar region, and which, in contraction, form a sphincter, -
against which the valve is pressed by the backward pressure
of the fluid during the contraction of the dorsal vessel (PI. 2,.

30

CHARLES BADHAM.

fig. 11, S.m.f). In somite 19 the dorsal vessel enters into
intimate’connection with the caeca of the thick-walled intestine.
This remarkable arrangement was first described by Johansson
for the Ichthyobdellids inCallobdella, and in many respects
the relations of the blood-vessel with the gut-walls, as described
by him, hold good also for Austrobdella.

Text-fig. 3 shows the relations of this vessel with the
epithelium and muscular walls of the gut. At the beginning
of the thick-walled intestine, the dorsal vessel is seen connected
with the muscular layer of the gut, and almost immediately
it opens on either side into the intestinal sinus, and ceases to
be distinguishable from the walls of the sinus.

The intestinal sinus is formed by the separation of the
•epithelial and muscular walls of the gut. This separation is
not complete in Austrobdella, for here and there the
normal relations of the epithelial layer and the muscle layer
are seen (Text-fig. 3, M. g. ep.), but save at these places
of attachment, which are usually small, the blood-stream
surrounds the epithelial layer of the thick-walled intestine.
These relations are such as described for Callobdella. In
the intermediate portions of the thick-walled intestine, which
-connect the paired pouches, the dorsal blood-vessel separates
from the intestinal sinus and lies in the dorsal side of these
regions. Also, in the region of the fourth rudimentary pair
of caeca, the sinus developed from the dorsal vessel is very
small, and only for a short distance does the dorsal vessel
cease to be defined : following this part the dorsal vessel is
clearly defined and remains single until above the ganglion
of the 24th somite ; here it divides in two. The two branches
then run laterally to the gut closely connected with its
muscular wall. In the 27th somite these two vessels diverge
aud run round the periphery of the posterior sucker on either
.side and finally unite (PI. 1, fig. 2). The valves in the
•dorsal vessel are found from just before the giving off of the
.second pair of lateral vessels to the beginning of the intestinal
.sinus (PI. 1, fig. 2, VI.). They are placed somewhat irregu-
larly, one or two in each somite. They are generally shaped

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 31

like a fir cone, measuring 80 /u long, and are made up of
about eight cells. In some cases these cells become separated
and present the appearance in section shown in PI. 2, fig. 11.
The valve placed just before the intestinal sinus is twice the
length of the others.

Circulation of the Blood.

I have investigated the circulation of the blood and the
lymph in living specimens found on whiting which I caught
on the ocean beach. The chief mechanism for propelling the
blood is the peristaltic contraction of the muscular wall of
the intestinal sinus, first described by Johansson (1896).
This peristalsis occurs in the three pairs of pouches of the
thick-walled intestine and also in the rudimentary pouch. In
the living animal the whole of the thick-walled intestine is in
a state of active contractile movements. These begin where
the thick-walled intestine passes into the rectum. The general
movement is from behind forwards. The whole of the con-
tractile muscular wall appears to contract simultaneously
when the animal is very active, but in specimens in which the
rate of contraction is lowered it is seen that the peristalsis is
from behind forward. There is seen, however, a certain
amount of individual contraction of separate paired caeca.
The most active specimens I examined showed a rate of con-
traction of over thirty times per minute. The blood forced
forward by the contractions is prevented from flowing back
by the valves of the dorsal vessel.

In the dorsal vessel the backward pressure of the blood
causes the valve to press against the sphincter fibres of the
dorsal vessel just behind it. Very rapidly the blood passes
onward, and the next valve acting in the same manner, the
first valve is again forced forward by the incoming blood.
The constriction of the dorsal vessel is much greater at the
sphincter muscle fibres than elsewhere. So great indeed is
the contraction here that the dorsal vessel resembles a string
of sausages. The constrictions being at the points occupied

32

CHARLES BADHAM.

by the sphincters (PI. 1, fig. 2, VI.) , the dorsal vessel in the*
testicular region is caused to contract by the pressure of
lymph in the lacuna, so that its walls come into contact. In
this manner the blood is forced forward and ultimately enters
the paired branches ; these are not provided with valves.
The blood then passes along the non-contractile lateral vessels
into the non-contractile ventral vessel and so through the
complicated branches in the posterior sucker, until it again
reaches the intestinal siuus. The important relation of the
contractions of the muscular walls of the intestinal sinus to
the flow of the lymph and the contractions of the contractile
lacuna has been dealt with in the section describing the
circulation of the lymph.

Nephridial System.

There are eleven pairs of nepliridia arranged segment ally
in the 13th to the 23rd somite. These form what is practi-
cally a continuous network in this area. However, this is
so arranged that the segmental character of the nepliridia is
obvious. An inspection of PI. 2, fig. 7, will make this clear.
The best developed parts of the nepliridia are two tubes
( L.n.c .), which are placed ventral to that part of the lacuna
formed by the fusion of the dorsal and veutral segmental
lacunae. These tubes have a diameter of about 40 ju, while
the diameter of their lumen is 5 p. They pursue a tortuous
course and frequently branch, and the branches anastomose.
They give off in each somite branches which run to open at
the nepliridiopore (IVp.),and they receive the branches which
run from the dorsal and ventral networks.

The paired branches, which are given off in each somite ta
open at the nepliridiopore, are of the same size as the chief
branches of the lateral canals. They are given off from the
lateral canals near the level of the first annulus of the segment,
and run medially and posteriorly, curving sharply as they
approach the ventral lacuna at an angle of 45° (Np. b.). They
run laterally for about half the distance of their first course-

AN 1CHTHY0BDELLID PARASITIC ON SAND WHITING. 33

and open on the second annulus of each segment. The
diameter of the aperture of the nephridiopore is about 5
In each segment the lateral canals receive three or four main
branches, which are the outcome of the anastomotic canals.
These latter are best developed around the ventral lacuna, but
they also surround the dorsal lacuna. The arrangement and
relative size of these canals is shown in PL 2, fig. 7.

As is general in Hirudinea, the canals of this nephridial
system are intracellular. The cells, which are burrowed
through by these canals, have oval nuclei.

The nephridiopores open directly on the body surface and
not into pits, like those of Cystob ranch us.

Neither in the living animal nor in serial sections have I
been able to find internal openings, so that the ciliated
funnels, possessed by Bran chel lion and Pontobdella,
are here absent. In this respect then Austrobdella
resembles Piscicola, Callobdella, Cystobranchus,
Abranchus, and possibly Platybdella.

This nephridial system is most like that described by
Johansson for Callobdella. It differs in the much greater
development of the branch going to the nephridiopore, and
the greater degree of anastomosis of the smaller channels.

In Austrobdella there exists on either side a fine canal,
which may represent the dorso-lateral canal described for
Callobdella. The lateral tubes are well developed in
Austrobdella, being 40^ as compared with Abranchus
and Callobdella 20 jjl, Piscicola 30 p, but again am
smaller than Cystobranchus 50 /u (Johansson, 1896).

Reproductive System.

Thanks to the excellent work of Brumpt (1900), f Reproduc-
tion des Hirudinees/ it is possible to compare the repro-
ductive organs of Austrobdella with those of allied leeches.
This species in its male organs resembles most Callobdella,
(Trachelobdella) lophii as regards the structure of th&
ejaculatory canal, but it lacks the muscular organ of Johansson

VOL. 62, PART 1. — NEW SERIES. 3

34

CHARLES BADHAM.

and the conducting tissue of the bursa described by Brumpt.
Of the spermatophore glands, those described by Brumpt as
A and B glands are present, while I am doubtful as to the
presence of C glands. The glands A are well developed, and
are enclosed in the muscular tunic of the terminal parts of
the ejaculatory canals. The glands B, and perhaps C, surround
the terminal parts of the ejaculatory canals and open into the
•common part. Here the resemblance, owing to the develop-
ment of the A glands, is more to the Glossiphonid type than
to C. lopliii.

In the female organs, owing to the isolated ovaries and
the absence of both copulatory area and conducting tissue,
Austrobdella resembles Callobdella lubrica, Platy-
bdella solese, and Glossiphonia complanata.

Concerning the interesting fertilisation by means of hypo-
dermic injection of spermatophores, which Brumpt has shown
to be true in most marine leeches, I have not yet ascertained
if a similar phenomenon occurs in Austrobdella. I hope
later again to keep these leeches in captivity and to endeavour
to bring about copulation with a view to determining this
point.

There are five pairs of testes (PI, 1, fig. 1, T., Text-fig. 6, T .)
placed in somites 14-18 inclusive. The vasa deferentia
(PI. 2, fig. 12a, V. def.) on leaving the connective-tissue,
become more than doubled in their diameter and constitute
the ejaculatory canals, which lie in the expanded anterior
end of the ventral lacuna. These ejaculatory canals become
considerably coiled at the level of the 12tli nerve ganglion
(PI. 2, fig. 12a, Ej. c .), and the lumen of each increases
slightly and forms a seminal vesicle. They then come into
close relation with the dorsal blood-vessel (PI. 2, fig. 12,
Ej.c .) and shortly open dorso-laterally into the terminal parts
of either side (PI. 2, fig. 12a, Ej. t.). The terminal parts of
the ejaculatory canals are provided with a muscular tunic
(PI. 2, fig. 12, Ej. t.) which encloses the unicellular glands,
•called the A glands by Brumpt (1900). The two terminal
parts open into a common part, the spermatophore sac of

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 35

Kovalevsky (1900) (PL 2, fig. 12a, Sp.s.), into which open
the ducts of the unicellular spermatophore glands called the
B glands by Brumpb (PL 2, fig. 12, Sp.gl.). The sperma-
tophore sac leads into the bursa — an ectodermal invagination,
whose external opening is the male orifice (PI. 2, fig. 6).
The muscular tunic of the terminal parts consists of a single
layer of fibres imbricated at their ends, About four to six
fibres complete the circuit. At the anterior end the wall
becomes two or even three fibres thick. This circular
musculature is continued on around the spermatophore sac,
but here the fibres, placed two deep, are separated from those
on either side by the ducts of the spermatophore glands.
At the bursa the fibres decrease to a single layer, and placed
•external to them are several longitudinal fibres, apparently
the diverted fibres of the body-wall muscles round the male
genital opening, which may aid in the protrusion of the bursa
(PL 2, fig. 6). The terminal parts of the ejaculatory canals
are lined throughout with gland cells, the ducts of which
run radially and are directed towards the spermatophore sac
*(P1. 2, fig. 12, Ej. t.). These ducts almost obliterate the
lumen into which they pour their secretion. There are
found among these ducts the small cells of the supporting
tissue.

The cytoplasm of these glands stains more deeply with
haematoxylin than those of the accessory male glands outside
"the muscular tunic.

1'hese latter cells make up a well-developed glandular
mass, which lies in somites 11 and 12 (Text-fig. 6, Sp.gl.).
Each is a unicellular gland (PL 2, fig. 12, Sp. gl.) and opens
in o the spermatophore sac. On either side this mass presents
two lobes caused by dorso-ventrally placed muscle fibres.

'I he development of these glands is such that they wrap
round the terminal parts of the ejaculatory canals (PI. 2,
fig. 12, Sp. gl.).

The secretion space of each cell is generally filled with
numerous granular eosin-staining particles, as are also the
•ducts opening into the spermatophore sac.

36

CHARLES BADHAM.

I have not determined, by the staining methods of Brumpt,.
if there are two kinds of gland cells present ; but in sections
stained by haematoxylin and eosin they appear to be all of
one kind.

The bursa, which is shown in PJ. 2, fig. 6 (a medial
sagittal section) is lined by a continuation of the epidermis of
the body.

The ovaries, which are two simple sacs lying free in the
ventral lacuna, show considerable movement in the living'
animal — a fact which Moquin-Tandon (1846) says caused
Rondeau to take them for hearts in certain leeches. They
become united just above the 13tli nerve ganglion, and from
their junction the oviduct (PI. 2, figs. 6, 12a, Ovd.) runs
dorsally and curves to enter the glandular part of the bursa
(PI. 2, figs. 6, 12a, B. gl.). After their junction, the ovaries
are continued forward to form each an anterior horn. The
oviduct, which has a small lumen (PL 2, figs. 6, 12a, Ovd.),
has a wall of circular muscle fibres, and external to these-
a well-developed connective tissue layer with large nuclei,
such as Brumpt has described as general in Ichthyobdellids,.
and through which, he says, spermatozoa are frequently seen
working their way.

The epithelium lining the vagina is a continuation of the
epidermis of the body, which is thrown into folds in the
glandular part of the bursa.

The ovaries in adult specimens are filled with ova in
various stages of development. The development of the
ovum (PI. 2, fig. 6, Ov.) and the breaking-down of the yolk
cells appear to be similar to what occurs in Callobdella
lophii, as described by Brumpt. Near the oviduct the ova
are seen undergoing the first maturation division.

This work was begun while holding the John Coutts*
Scholarship. I wish to thank Prof. W. A. Haswell, in whose
laboratories I worked, for his advice and kindness. Mr.
F. W. Atkins, of the Sydney Technical College, re-drew the
figures, and Mr. W. Graham, of the Department of Zoology

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 37

here, gave me great assistance with photographs of the living
specimens.

Literature.

1. Moquin-Tandon, A. (1846). — * Monographie de la famille dea

Hirudinees.’ Paris.

2. Bourne, A. G. (1884). — “ Contribution to the Anatomy of the

Hirudinea,” ‘ Quart. Journ. Micr. Sci.,’ vol. 24.

3. Whitman, C. O. (1891). — “Description of Clepsine plana,”

‘Journal of Morph.,’ vol. iv.

4. Oka, Asajiro (1894). — “ Beitrage zur Anatomie der Clepsine,” ‘ Zeit,

Wiss. Zool.’ Bd. lviii.

5. Johansson, L. (1896a). — ‘ Bidrag till Kannedomen om Sveriges

Ichthyobdellider.’ Upsala.

6. (18966). — “ fiber den Blutumlauf bei Piscicola und Callob-

della,” 4 Festskrift Lilleborg.’ Upsala.

7. (1898). — “Einige systematisch wichtige Theile der inneren

Organisation der Ichthyobdelliden,” ‘ Zool. Anzeiger,’ Bd. xxi.

8. Kovalevsky, A. (1900). — “ I^tude biologique de l’Hsementeria

cos tat a Muller,” ‘Mem. Acad. St. Petersbourg (8) Cl.,’ phys.
math.,’ vol. xi.

9. (1900). — “ Plienomenes de la Fecondation chez l’Helob-

della algira (Moquin-Tandon),” ‘Memoires de la Societe
Zoologique de France,’ Tome xiii.

10. Brumpt, E. (1900). — “Reproductions des Hirudinees,” ibid.,

Tome xiii.

11. Oka, Asajiro (1902). — “ Uber das Blutgefasssystem der Hiru-

dineen,” ‘ Annot. Zool. Japon,’ vol. iv, part 2.

12. (1904). — Ueber den Bau von Ozobranchus,” ‘Annot. Zool.

Japon,’ vol. v.

13. Salensky, Yon. W. (1906). — “Zur Kenntnis des Gefasssystems der

Piscicola,” ‘ Zool. Anzeiger,’ Bd. xxxi.

14. Johansson, L. (1911). — “ Hirudinea, Die Fauna Sudwest-Australiens,

Hamburger Sudwest-Australischen Forschungsreise, 1905.”
Michaelsen und Hartmeyer, Bd. iii.

15. Sukatschoff, B. W. (1912). — “Beitrage zur Anatomie der Hiru-

dineen. 1. Uber den Bau von Branchellion torpedinis
Sav.,” ‘ Mittheil. Zool. Station, Neapel,’ Bd. xx.

16. Hemingway, E. E. (1912). — “Anatomy of Placobdella para-

sitica,” ‘Geol. and Nat. History Survey, Minnesota,’ Zool.,

series v.

38

CHARLES BADHAM.

17. Leigh- Sharpe, W. H. (1913). — “ Calliobdella lophii, Yan
Beneden & Hesse,” ‘Journal Marine Biological Assoc.,’ vol. x.

EXPLANATION OF PLATES 1 and 2,

Illustrating Mr. 0. Badham’s paper “ On an Ichthyobdellid
parasitic on the Australian Sand Whiting” (Sillago
ciliata).

[All figures refer to Austrobdella translucens.]

PLATE 3.

Fig. 1. — Drawing from life. Seen from the ventral aspect by
transmitted light. The specimen is somewhat extended so that the
shouldered appearance seen in large individuals (vide fig. 5) is absent.
The pigment cells (P. c.) are represented and the ejaculatory canals
( Ej . c.) show up on account of their pigmentation. The five pairs of
testes (T.) are a marked feature. Certain parts of the Blood- vascular
System appear, and the thick-walled part of the middle gut stands out
clearly (M. g.). A. m. g. Caeca of anterior thin-walled part of middle
gut. C. 1. Contractile lacuna. Cl. gl. Clitellar glands. E. Eye spots.
Ej. c. Ejaculatory canal. M. g. Caeca of thick-walled part of middle
gut. Ov. Ovary. P. Proboscis. P. c. Pigment cell. T. Testis.
V. v. Yentral vessel.

Fig. 2. — Drawing of the Blood-vascular System from life. The
dorsal vessel is shaded, the ventral vessel is outlined. The origin of
the dorsal vessel is seen in the posterior sucker ; just above the union
of the two branches from the posterior sucker this vessel enters into
intimate relation with the caeca of the thick-walled intestine to form
the intestinal sinus (1. s.). After leaving the sinus the dorsal vessel
(D. v.) has a series of valves (VI.), one being placed where a constriction
is seen on contraction. Anteriorly, the dorsal vessel gives off two
pairs of lateral vessels (L. v. 3, L. v. 2) and an impaired proboscis
branch (P. v. 1), and bifurcates just above the level of the eye spots
to form the first pair of lateral vessels. The paired lateral branches
are gathered together to form the ventral vessel (V. v.). This runs
posteriorly and gives off in the posterior sucker a series of anastomotic
branches which go to form the ring vessel (B. v.). D. v. Dorsal vessel.
E. Eye spots. L. v. Lateral vessel. P. v. 1. Afferent proboscis vessel.
P. v. 2. Efferent proboscis vessel. B.v. Ring vessel. VI. Yalve.

Figs. 3, 4, 5. — Drawings of alcoholic specimens (fixed in boiling
corrosive acetic), P5 mm., 4 mm. and 9 mm. long respectively. Fig. 3

AN ICHTHY0BDELL1D PARASITIC ON SAND WHITING. 39

is a side view, the other two figures are ventral views. This series
shows the change from the cylindrical form of the young specimen
(fig. 3) to the slightly flattened sexually mature specimen 4 mm.
long (fig. 4). The shouldered appearance of the body of the specimen
9 mm. long (fig. 5) and its greatly flattened form are in striking
contrast with the form of the younger specimens.

PLATE 2.

Fig. 6. — A medial sagittal section of a specimen 8 mm. long, showing
the genital openings, bursae, oviduct, ovaries, spermatophore sac, and
the relations of the ventral nerve cord and the ventral and dorsal
blood-vessels in this region. Compare with fig. 12a. The male
genital opening is seen at $ and the bursa leading into the
spermatophore sac ( Sp . s.). The female genital opening is seen at $
leading into the bursa, which has the walls of its dorsal part plicated
( B.gl .) to form the glandular part of the female bursa. The oviduct
( Ovd .) has been cut at two places, where it leaves the junction of the
ovaries and more dorsally where it is about to enter into the glandular
part of the bursa. The sections of the ovaries (Or.) show ova in
various stages of formation by the breaking down of yolk cells; the
two spindles represent maturation divisions. The relations of the
ventral nerve cord and ganglia 11, 12, 13 to the sexual organs are
shown, and may be compared with the model depicted in fig. 12a.
The ventral vessel ( V. v.) is seen lying free in the expansion of the
ventral lacuna which contains the sexual organs in this region. The
dorsal vessel ( D . v.) is seen as it is entering the dorsal lacuna.
The degree of development of the circular and longitudinal body-wall
musculature is displayed. B. gl. Glandular part of female bursa.
C. m. Circular body-wall musculature. D. v. Dorsal vessel. L. m.
Longitudinal body-wall musculature. N.gn. Nerve ganglion. Ovd.
Oviduct. Ov. Ovary. Sp. s. Spermatophore sac. V.v. Ventral vessel.

Fig. 7. — Diagram in relief of the Nephridial System in the testicular
region of the body viewed from the ventral surface. The posterior
end of the leech would be towards the observer. For the sake of
clearness the somite is shown as consisting of the three primitive
annuli. In order to show the segmental nature of the nepridial system
one whole somite and its portion of the nephridial system is drawn,
and parts of the preceding and succeeding somities with their portions
of this system. The ventral and dorsal lacuna are shown, but neither
the segmental nor the contractile lucunse. On either side the lateral
nephridial canal is seen receiving branches of the fine capillary network
of nephridial tubules which extend in the dorsal and ventral sides of
the body. The lateral nephridial canals are shown giving off in each

40

CHARLES BADHAM.

somite the pair of branches (Np. b.) leading to the nepliridiopores
( Np .). D. 1. Dorsal lacuna. D. v. Dorsal vessel. L. n. c. Lateral
nephridial canal. N. p. Nepliridiopore. Np. b. Duct leading to
nephridiopore. T. Testis. V. 1. Yentral lacuna. V. ne. Ventral nerve
cord. V.v. Yentral vessel.

Fig. 8. — Diagram in relief of the Lacuna System, viewed from the
dorsal surface, in a somite of the testicular region of the body. The
dorsal lacuna ( D.l .) is shown containing the dorsal vessel (D.v.). In
the ventral lacuna (V. 1.) are the ventral nerve cord (F ne.) and ventral
vessel (V.v.). The dorsal and ventral parts of the segmental lacuna
of either side of the somite are seen to junction laterally, and after
bifurcating to join up with branches from the preceding and succeeding
segmental lacuna) of the same side. The segmental lacuna opens into
the contractile lacuna in two places in each somite. The contractile
lacuna receives in each somite three capillaries dorsally on either side.
Cap. 1. Capillaries opening into contractile lacuna. C. 1. Contractile
lacuna. D. 1. Dorsal lacuna. I). v. Dorsal blood-vessel. S. 1. Segmental
lacuna. V.l. Yentral lacuna. V.ne. Yentral nerve cord. V.v. Yentral
vessel.

Fig. 9. — Drawing of horizontal section through the testicular region
of the body showing the character of the contractile lucana (C.l.) and
the branches of the segmental lacuna ( S.l .) leading to it. In one
place a branch opens into the contractile lacuna and the opening is
guarded by sphincter muscle fibres (S. m.f). The large unicellular
lateral glands ( L . gl.) are shown, and the clitellar glands medial to the
contractile lacuna. C. 1. Contractile lacuna. Cl. gl. Clitellar glands.
L. gl. Unicellular lateral glands. (S.m.f.) Sphincter muscle fibres.
S. 1. Segmental lacuna.

Fig. 10. — Drawing of a transverse section through the dorsal blood-
vessel and dorsal lacuna in the testicular region of the body, showing
the dorsal and ventral septa (Sep. d., Sep. v.). The nuclei of the
epithelial cells of the dorsal vessel bulge into the dorsal lacuna. D. v.
Dorsal vessel. D. 1. Dorsal lacuna. Epi. nu. Nucleus of epithelial
cell. Sep.d. Dorsal septum. Sep.v. Yentral septum.

Fig. 11. — Drawing of a section through a valve of the dorsal blood-
vessel in the testicular region of the body. Usually the valve (VI.)
is fir-cone shaped and when forced back rests against the sphincter
muscle fibre (S. m.f.), but sometimes, as shown here, the valve becomes
broken up into separate cells attached to a common stalk. On con-
traction of the dorsal vessel several cells of the valve may be forced
past the spincter, as is shown here. D.v. Dorsal vessel. VI. Yalve.
S. m.f. Sphincter muscle fibre.

Fig. 12. — Drawing of a transverse section through the neck region

AN ICHTHYOBDELLID PARASITIC ON SAND WHITING. 41

of a specimen 7 mm. long. The section passed through the eleventh
nerve ganglion and is cut obliquely so that the terminal part of the
ejaculatory canal ( Ej . t.) is shown on the right side and the main mass
of the spermatophore glands on the left (Sp. gl.). The terminal parts
of the ejaculatory canals are seen just before they have united to form
the spermatophore sac (compare with fig. 12a). The ventral vessel
is seen lying free in the ventral lacuna, here expanded to contain the
sexual organs. The relations of the ejaculatory canals, oesophagus
and dorsal vessel in this region are also shown. D. v. Dorsal vessel.
Ej. t. Terminal part of ejaculatory canal. Ej. c. Ejaculatory canal.
N.gn. 11. Nerve ganglion 11. (Es. (Esophagus. Sp.gl. Spermatophore
glands. V.v. Yentral vessel.

Fig. 12a. — Diagram in relief of the Reproductive System. The
terminal part of the right ejaculatory canal (Ej. t.) is shown as cut
away for the sake of clearness. The relations of the ventral nerve
cord and ganglia to the male and female organs are made evident.
B. gl. Glandular part of the female bursa. Ej. c. Ejaculatory canal.
Ej. t. Terminal part of the ejaculatory canal. Ovd. Oviduct. Ov.
Ovaries. Sp. s. Spermatophore sac. V. def. Yasa deferens. V. ne.
Yentral nerve cord. 11, 12, 13. Nerve ganglia. <$ Male opening.
^ Female opening.

THE DEVELOPMENT OF ALCYONIUM DIGITATUM.

43

The Development of Alcyonium digitatum,
with some notes on the Early Colony
Formation.

By

Annie Matttiews, JJI.Sc.

With Plates 3-5 and 51 Text-fignres.

Table of Contents.

1. Preface ....

PAGE

43

2. Introduction

44

3. Methods ....

44

4. General Account

45

5. Colony Formation .

55

6. Food ....

59

7. Segmentation

60

8. Pre-planula and Planula Stages, followed by Settling of
Larva .....

65

9. Mesogloea ....

70

10. Mesenteries

72

11. Tentacles, Mouth and Stomodaeum

78

12. Spicules ....

80

13. Mesenteric Filaments

81

14. Summary ....

86

15. Literature.

87

1. Preface.

I take this opportunity of thanking the Council of the
Marine Biological Association for the use of a table while
working out these results. Also I gladly acknowledge the
kindly and continued help given me by Dr. Allen and the
various members of his staff. This generous aid rendered

44

ANNIE MATTHEWS.

the work very much easier, and enabled me to get all the
necessary stages with a minimum amount of labour. My
warmest thanks are also due to Prof. Hickson, of the Man-
chester University, for many useful hints and for reading
through and criticising the completed paper.

2. Introduction to the Development of Alcyonium
Digitatum.

The broad outlines of the development of Alcyonium
digitatum were worked out by A. Uowalevsky in 1873 (8),
and amplified later by Hickson (2, 3, 4, and 4a), and it has
been the object of this paper to add further details to the
information given by these authors. On the whole the results
agree, except in some details concerning the sequence of
development of certain organs.

There is an interesting general resemblance between the
accompanying sketches of the segmenting egg, the planula
and the early fixed polyp, and those previously given by :

(1) de Lacaze-Duthiers, for Astroides calycularis (10).

(2) Wilson, for Renilla and Leptogorgia (16).

(3) Kowalevsky and Marion, for Sympodium and Clavel-
lina (9).

A comparison of the plates given by these authors with those
at the end of the present paper will demonstrate this. In
particular, PI. xiii, fig. 6, of de Lacaze-Duthiers, memoir (10)
would illustrate excellently the way in which Alcyonium larvae
settled in the finger-bowls in which they were reared, during
the experiments now described. Therefore, A. digitatum
bears out the collected evidence that the Anthozoa develop
roughly according to one and the same plan.

3. Methods used to Preserve and Stain the Alcyonium

Material.

(1) Preserving fluids.

(a) Schaudinn’s fluid (corrosive sublimate and absolute
alcohol).

THE DEVELOPMENT OF ALCYONTUM DIGITATUM.

45

(b) Corrosive acetic.

(c) Bouin^s picro-formol-acetic.

The above three reagents appeared equally good for
preserving all stages, except when the structure of the
spicules was required. Perhaps (c) was the best general
preserving fluid.

( d ) Osmic acid, for preparations showing spicules and
nematocysts.

(2) Staining reagents.

(a) Delafield’s hematoxylin. For morulas, and well-stained
segmentation spindles ; also for gland cells in the oesophagus
and ventral mesenteric filaments.

(b) EhrlicKs hematoxylin, as (a).

(c) Borax-carmine and picro-nigrosin. For planulas and
all subsequent stages ; for structure of mesogloea.

(d) RanviePs picro-carmine, followed by Kernschwarz,
after fixing with osmic acid. For spicule structure. The
picro-carmine stains the nuclei, while the Kernschwarz
stains the spicule and its surrounding protoplasm.

(e) Iron-brazilin. Good for all settled stages, gland cells,,
etc., in combination with some plasma stain, e. g. safranin.

4. General Account.

Ripe male and female colonies1 of Alcyonium digi-
tatum are brought in by the trawlers in the Plymouth
district from early December to early February, and fertilised
eggs were obtained :

(1) Between January 27th and February 13th, 1912.

(2) Between December 10th and February 10th, 191 2 —

1913.

(3) Between December 14th and February 10th, 1913—

1914.

1 Hermaphrodite colonies occasionally occur, male and female polyps
being present. Hermaphrodite individuals are also sometimes found
in these colonies, such exceptions finding a parallel in the case of
Corallium nobile (11).

46

ANNIE MATTHEWS.

These eggs were successfully reared in the Plymouth
laboratory. The above data shows that A. digitatum
spawns during two of the coldest and stormiest months of the
year, when the supply of material is necessarily uncertain,
and therefore the work of collecting the various segmentation
stages is, as a rule, unavoidably spread over the whole of the
spawning season. The colonies used for this paper came
from the trawling ground between the Dodman and the
E Idystone, i.e. outside and some miles west of Plymouth
Sound. They reached the laboratory in good condition in
buckets of sea-water, and after being well washed the ripest
colonies were selected, the choice being easily made, as ripe
ova are deep reddish-yellow in colour, and ripe sperm sacs a
very opaque white. The colonies were then placed by them-
selves in one of the laboratory tanks, through which a constant
stream of sea-water circulated, care being taken to avoid
overcrowding. As the colonies generally remain healthy for
only a few days in the laboratory tanks, even under the most
favourable conditions, rarely expanding fully and possibly
never feeding, those chosen must be in the best condition and
ready to spawn.

Some hours later the circulation was stopped and the female
colonies soon spaAvned in the still Avater. It seems essential
to have the Avater still, as this is favourable to the necessary
expansion of the very sensitive polyps. Simultaneously the
male polyps discharged from their mouths large quantities of
spermatozoa which SAvam freely and fertilised the floating
OAra. On microscopic examination the latter were then seen
to be completely covered by a delicate fringe of spermatozoa
Avliich gave them a pseudo-ciliate appearance, but the “ cilia,”
i.e. the tails, of the spermatozoa were non-motile.

It Avas impossible to distinguish the particular sperm which
fertilised the ovum, or to say when this occurred. Different
colonies began to extrude their ova at varying times of the
day or night (unlike Renilla, which spawns only between 6
and 7 a.m. (16)).

The eggs of any one colony were passed out con.

THE DEVELOPMENT OF ALCYONIUM DIGITATUM.

47

tinuously for several days, until the spawning was com-
plete.

On January 3rd, 1913, a spawning colony was placed in a
beaker of water and closely watched from 11 a.m. to 3 p.m.
The majority of the polyps were expanded while their ten-
tacles were half retracted ; others were only partly expanded
(c.f. Text-fig. 1). Five eggs were successively extruded
at regular intervals from the mouth of one polyp during a
period of fifteen minutes. They passed up the stomodaeum
one by one and after escaping from the mouth remained in
contact with the tentacles and oral surface until some slight

Text-fig. 1.

Solitary polyp, lateral view. Tentacles retracted, body slightly
contracted. This also illustrates the appearance of the colonial
polyp while spawning.

motion of the water finally dislodged them, when they floated
upwards. In some cases several eggs were seen in the stomo-
dseum simultaneously, one below the other, and in squeezing
upwards through this narrow tube they became temporarily
•oval but regained their round shape after extrusion. The trans-
parent membrane which surrounded them before spawning,
and which always envelops eggs taken forcibly from the mesen-
teries, was thrown off during the process of spawning, and the
empty membranes were ejected into the water after the ova.

Although artificially fertilised ova did in some cases seg-
ment satisfactorily, it was found more practicable for rearing
on a large scale to collect the fertilised ova from the tank
where they were naturally spawned and fertilised. To do
this the water was siphoned over into white dishes, from
which the eggs were removed with a pipette.

48

ANNIE MATTHEWS.

The eggs are opaque, very yolky, and reddisli-yellow in
colour. They are about 05 mm. in diameter, and float at

Text-figs. 1a-8.

Fig. 1a. Sixty-four celled stage, X 46. Fig. 2. Rather late
pre-planula, with prominent lobes, X 56. Fig. 3. Latest
stage pre-planula ; lobes softened into shallow wrinkles ;
seventy-two hours old, X 62. Fig. 4. Round ciliate planula
of sinuous outline, succeeding Fig. 3. The arrow indicates
the direction of progress, X 52. Fig. 5. Oval planula of
smooth outline, twenty-four hours older than last figure,
X 40. Figs. 6 and 7 show planarian-like movements of long
thin planula, X 40. Fig. 8. Planula showing lateral ridges
when contracted due to irritation, X 40.

various depths in the tank as though their specific gravity is
very near that of sea-water. After segmentation has begun

THE DEVELOPMENT OF ALCYONIUM DIGITATUM.

49'

they increase slightly in size, and become somewhat paler in
colour, and are thus distinct from newly-spawned eggs to the
naked eye.

They were pipetted into finger-bowls of “ outside 39 water
(water brought into the laboratory from outside Plymouth
Sound, and therefore in especially good condition), and
development took place at the ordinary and by no means
constant temperature of the laboratory. The developing
eggs appeared to do equally well in outside, Berkefeld-
filtered or ordinary tank water, and many larvae went through
all the stages of development, settled and produced tentacles
in the laboratory tank where the eggs were spawned.
Twenty-four hours after spawning the embryos were all
morulae, more or less advanced.

Fresh colonies were continually added to the tank, and
spent and unhealthy ones removed during the whole of the
spawning season. Judging by the proportion of ripe ones
brought in, this season reaches its height towards the end of
January, and soon declines after that date.

The segmenting egg (Text-fig. 1a) is in all stages typically
spherical, though during segmentation some examples may
become temporarily oval, regaining their globular shape later.
At the close of the morula stage the embryo undergoes a
curious change in shape. About the twenty to twenty-fourth
hour the whole surface of the sphere is slowly drawn out into
irregular blunt prominences with corresponding depressions
(Text-fig*. 2), the component cells meantime undergoing a
modification of structure and the segmentation cavity gradu-
ally disappearing. This condition lasts for some time, but
about the forty-fourth hour the knobs begin to withdraw
again. Wilson (16) mentions a similar stage for Renilla and
Leptogorgia, but apparently did not investigate it closely.
As this definite stage is followed by the swimming planula,.
it was convenient to call it the “ pre-planula ” stage. By the
end of the third day of development the pre-planula no longer
shows definite protuberances and depressions, these having
softened down into a gently wrinkled outline (Text-fig. 3)..

VOL. 62, PART 1. NEW SERIES. 4

ANNIE MATTHEWS.

-5 0

On the fourth day the cells near the centre of the solid pre-
planula begin to disintegrate, and so the larva again becomes
hollow and passes on into a free-swimming planula stage with
a definitely marked anterior pole. This planula developes
cilia, is at first roughly spherical and of sinuous outline (Text-
fig. 4), but lengthens somewhat in a few hours into a highly
contractile oval planula still of wavy outline. By the fifth
day it is a smooth oval planula swimming rather slowly at
various levels in the water, usually in a horizontal plane
(Text-fig. 5). Very soon the anterior end broadens and a
pear-shaped planula results, which rotates continuously on its
long axis while progressing in the water (Text-fig. 6). The
reddish -yellow colour of the ovum is still present, but gradu-
ally becomes paler as the yolk is absorbed, and the planula
increases in length. The larva continually changes its shape,
so that measurements of the ever varying length and breadth
are rendered difficult. While swimming it exhibits charac-
teristic planarian-like contractile movements, which are
represented in Text-figs. 6 and 7, and any irritation causes
strong contraction and lateral wrinkling (Text-fig. 8).1 By
the seventh day the planula is very long and slender, measuring
T3 mm. long and 03 mm. wide, but is not very often fully
extended. The anterior and aboral pole is deeper in colour
than the narrower posterior and oral pole where more yolk
has been absorbed (PI. 3, fig. 1). The surface at this time
is abundantly supplied with nematocysts and mucous cells
(PL 3, fig. 2), the latter being especially numerous at the
anterior pole. At first the planulae swim at varying levels in
the bowls, but towards the third free-swimming day they
become more sluggish, and most of them keep in a vertical
position with the thin aboral end hanging downwards (c.f.
<le Lacaze Duthiers (10), PI. xiii, fig. 6). Many then sink to
the bottom of the dishes in this position (possibly this is an

1 All the young stages are very sensitive to heat, and the microscope
lamp has to be used with caution while examining them or they quickly
die.

THE DEVELOPMENT OF ALOYONIUM DIGITATUM. 51

attempt to settle), but usually they get caught up here in
their own mucus and eventually degenerate.

Some larvae develop more quickly than others, but usually
on the fourth free-swimming day, i.e. the seventh day of
development, many larvae settle. After hovering motionless
for some time with the broad anterior pole apparently touching
the chosen place for settling, the planula becomes attached
(Text-fig. 9). In this they agree with Sympodium and

Clavellina (9). A thin disc of opaque white mucus (the
mucous plug) fastens them to the substratum (PI. 3, fig. 3,
M. P.), this mucus being secreted by the mucous cells in the
ectoderm of the anterior end. In bowls containing water
only, the circulation set up by the constantly varying
temperature of the laboratory carried many larvae to the top
of the water so that on becoming sluggish they were caught
up in the surface film and settled there, either on the film
itself or on the glass wall of the dish (cf.de Lacaze-
Duthiers (10), PI. xiii, fig. 6). In the former case the settled
polyps also developed perfectly, hanging upside down from
the film until this was disturbed, sending them down to the
bottom.

In a certain number of bowls, wherein small Pecten shells
were placed, the larvae settled in great numbers on both
surfaces of the shells and on the parts of the dish sheltered
by them, i.e. the base and lower part. In these cases the
circulation is modified by the presence of the shells, so the
larvae are less numerous in the surface film. In some dishes
the planulae did not settle until the fifth, sixth, to fourteenth
day of free-swimming life, and although these are results
obtained under laboratory conditions, a varying length of
free-swimming life would obviously help dispersal in the sea,
and give more larvae a chance to find Ascidians, Chaetopterus
tubes, Hydroids, Pecten shells or other suitable objects to
settle on. The planulae often settled so close to one another
as to render a group difficult to distinguish with the naked
.eye from a young colony. All the larvae did not settle, and

52

ANNIE MATTHEWS.

Text-figs. 9-14.

Fig. 9. Planula just settled (cilia not represented). Fig. 10. Same planula a
little later, length shortening; still ciliate. Fig. 11. Same, length considerably
shortened, cilia retracted. Fig. 12. Vertical section of young polyp arranged
to illustrate two stages in the growth of the mesentery. The vertical, dotted
line divides the two. The mesentery (c) is intermediate in development
between mesentery (b\) of Text-fig. 40, and mesentery ( d ) in this figure.
A broken line indicates the inner edge of the endoderm. Fig. 13. Similar
section on the third day when the stomodseum is developing. The tentacles
hive arisen, but are shown by dotted lines as they are alternate with the
mesenteries. Fig. 14. Lateral view of polyp about eighteen days fixed.
Tentacles bearing five to six pinnules and spicules showing well. Fig. 15.
Oral view of polyp with five to six pinnules. Tentacles well expanded.,
mouth almost closed (from bowl, x 25).

THE DEVELOPMENT OF ALCYONIUM DIGITATUM.

58

those which passed beyond the fifteenth to sixteenth free-
swimming day without fixing eventually degenerated.

The base of the newly fixed pear-shaped larva looks like a
round pinkish disc from below, the planula for a time retaining
its power of planarian-like contraction and expansion (Text-
fig. 10). Soon the cilia1 are retracted (Text-fig. 11), and the
fixed polyp shortens first to a stumpy oval shape, and then to
a flat mound shape by the end of the first twenty-four hours.
At the beginning of the second day the mesenteries show by
transmitted light as eight equidistant vertical ridges growing
up from the base of the lateral walls (PI. 3, fig. 4, P.M.).
These shallow ingrowths almost meet on the aboral and oral
surfaces by the end of the second day (Text-fig. 12, d.), while
eight small blunt conical outgrowths alternating with them
en the oral surface indicate the tentacles (Text-fig. 13, and
PI. 3, fig. 5,). On the third day the polyp has increased in
size, and the stomodmum is formed by an invagination of the
eral surface, within the circle of the tentacles (PI. 3, fig.
6, M.). This tube deepens rapidly, and by degeneration of
its base the coelenteron is put in communication with the
exterior on the fourth day. Meanwhile the tentacles and
body continue to lengthen (PI. 3, figs. 6 and 7) and soon the
former develop two rows of pinnules, seven being the maximum
number developed in the laboratory on any one row (Text-
figs. 14 and 15). The polyps had on an average produced in
each row :

One pinnule by the sixth day.

Two pinnules by the seventh day.

Two to three pinules by the eighth day.

Three to four pinnules by the tenth to twelfth day.

Five pinnules by the twelfth day.

Six pinnules by the eighteenth day.

Seven pinnules by the twenty-first day.

And while the older pinnules were carried along during
development towards the tips of the tentacles, the new ones

1 No cilia were seen on the ectoderm of the solitary polyp in any
.subsequent stage.

54

ANNIE MATTHEWS.

developed in succession below them, so that the youngest
pinnule was always at the base (Text-fig. 15, P1 and P2). The
numbers were variable in the two rows on any one tentacle,
and on the eight tentacles of any one polyp. The tentacles
were faint pink in colour at first, but this changed to pale
cream during development. Some polyps seemed to develop
rather quicker than others, as did the planulm. When fully
grown the measurements of the solitary polyp are only about
half the corresponding ones of the colonial polyp, i. e. about

Text-fig. 16.

Solitary polyp, settled fourteen to twenty days ; tentacles
bearing six pinnules, diameter of base T2 mm. Lateral view,
well expanded (from finger-bowl), X 10.

6 mm. in height, tentacles 3 mm. long, and diameter of base
T2 mm., while it is much more opaque, and consequently the
stomodseum shows less plainly and the mesenteric filaments
are obscured. As soon as the tentacles are long enough, they
curl gracefully about in the water, seeking food (Text-fig. 16).
Any vibration causes them to retract in part or wholly,
the polyp in a complete state of retraction appearing as a
small pink mound with an opaque centre. The spicules in
the tentacles and body nearly meet in the solitary polyp
(Text -fig. 14, T. Sp., and B. Sp.), whereas they are widely
separated in the individuals of older colonies. Some

THE DEVELOPMENT OF ALCYONIUM DIG1TATUM.

55

polyps lived in finger-bowls in the laboratory for about three
months, but eventually died without forming colonies. They
seemed healthiest when the bowls were immersed in much
larger cylinders of sea-water, with the growth of algse and
diatoms checked by limiting the amount of light, and with
frequent changes of water. In finger-bowls through which
air was constantly bubbled the polyps were nearly always
expanded, but in others they were nearly always closed until
dusk, although they usually opened if the bowls were removed
from their containing cylinders for examination. Hence the
polyps seem sensitive to light, currents in the water, and
temperature. No regular alternation of the expanded and
contracted stages was observed.

5. Colony Formation.

Some of the polyps produced one bud in the laboratory,
and one produced two daughter polyps. The finger-bowls
were difficult to keep clean, as the algae and diatoms in the
fine townetting, which was added from time to time as food,
settled down rapidly as a thick greenish coating. Therefore
at this stage, as it seemed unlikely that the colonies would
continue to develop in the laboratory, two bowls containing
numerous solitary polyps and young colonies were taken out
on March 17th, 1914, to Cawsand Bay, just outside Plymouth
Sound, to continue their growth. They were fastened in
wicker stands which were suspended inside a box-like raft
into which the sea penetrated, and were brought in tem-
porarily for examination on the following dates, after which
they were always returned :

(1) April 30th, after one and a half months on the raft.

(2) July 15th, after four months on the raft.

(3) September 16th, after six months on the raft.

This experiment was entirely successful, and the polyps
gave rise to disc-shaped colonies in the early stages of which
the new individuals arose according to a definite plan. As
the polyps increased in number the parent individuals seemed

56

ANNIE MATTHEWS.

to grow larger themselves, and the number of tentacular
pinnules increased gradually from seven to twelve or thirteen.
When last examined (September 16th, 1914), although the
best developed colony consisted of about thirty-two polyps,
they still all grew out of a common flat encrusting base.
The following account of the production of new polyps from
4he parent individual by stolonal gemmation is a summary of
observations made on the young stages in the laboratory and
the older ones from the raft. Young colonies trawled from
the Rame-Eddystone grounds, and growing on Chgetopterus
tabes, Polycarpa, etc., were found quite similar in their plan
of growth.

Detailed Account of Colony Formation.

About three weeks after fixation the circular base of the
polyp produced a blunt outgrowth opposite the two dorsal
mesenteries (Text-fig. 17, St.). This stolon increased in size,
became of circular outline and separated from the parent
polyp by a slight constriction. Soon it produced a bud which
rapidly grew into a second polyp (Text-figs. 18 and 19), with
its dorsal mesenteries adjacent to the dorsal mesenteries of
the parent.1 Then a second bud formed quite similarly from

1 Hickson (2) describes an Alcyonium polyp which bore one bud.
The figure he gives (PI. iii, fig. 24), indicates budding from the lateral
wall of the parent polyp, and not stolonal gemmation. It is difficult
to reconcile this example with the present account, and no explanation
can be offered.

Fig. 17. Aboral view of polyp, showing outgrowth of stolon
previous to formation of first bud. Fig. 18. Similar view of
well-expanded polyp with one bud. The two pairs of dorsal
mesenteries are seen opposite one another, and the long axes
of the mouths lie along one line. Fig. 19. Lateral view of
colony of two. Size of polyps very similar (from finger-bowl).
Fig. 20. Oral view of parent polyp and two buds (from
Clisetopterus tubes). [In Figs. 20 and 22-25 the common
stolon is ruled in with faint lines.] Fig. 21. Aboral view of
polyp with three buds (raft). Figs. 22-24. Oral view of
colonies showing four, six and eight buds respectively. Buds
numbered in order of appearance. Fig. 25. Colony with three
nows of buds — aboral view (raft).

Text-figs. 17—25-

d m .

-- A.B

20

22

23

24

25

58

ANNIE MATTHEWS.

a stolon-like outgrowth on the opposite side of the parent’s
base and with its dorsal mesenteries likewise turned towards
the original polyp (Text-fig. 20). By April 30th the parent
polyp bore ten pinnules on the tentacles, many parents
showing two buds, and one bearing three (Text-fig. 21). The
second bud showed no definite relation to any mesentery,
while the third one lay between the first and second (Text-
fig. 21, av bu, and Ci). By mid- July the colony, which consisted
on April 30th of four individuals, had altogether produced
eight buds by stolonal gemmation, and these lay in a circle
around, and all with their dorsal mesenteries turned toward
the original polyp (Text-fig. 22), and therefore with the long
axis of the mouth lying always along a radius. Possibly this
arrangement would facilitate a current of water and food
upwards through the young buds from the parent polyp, as
the dorsal filaments produce an upward current in Alcyonium
(Hickson, 3a, and Wilson, 17). Other polyps in the bowl had
also produced eight buds, and the rest from six buds down-
wards (Text-figs. 23 and 24). On September 16th it was
found that several colonies had produced about thirty-two
individuals, while others were not nearly so far advanced.
The younger colonies still retained their circular outline, but
the older ones had lost it. One had become quite oval
because barnacles had prevented its lateral expansion.

After the production of the first circle of eight buds a
second row forms outside these and alternate with them^
giving two concentric rows (Text-fig. 25). The second row
then increases greatly in number, and, later, young buds
appear between the parent of the colony and the first row
(Text-fig. 25). In still older colonies this regular system
becomes obscured, young and old polyps being irregularly
scattered throughout the colony. It was found that colonies
dredged in the Sound at this date and on the Rame-Eddystone
grounds contained a fair proportion of examples of similar
size to those reared on the raft.

THE DEVELOPMENT OE ALCYONIUM DIG1TATUM.

59

6. Food.

This appears to be almost wholly animal. In only one
instance was evidence found of any vegetable matter being
ingested — when a desmid was seen embedded in an endoderm
cell in one of the ventral mesenteric filaments. The polyps
reached quite an advanced stage of development while simply
using* up the embryonic yolk. Several pinnules had developed
before this was exhausted, in bowls to which no food had been
added. Cultures of Nitzschia, Pleurococcus, and other very
small green algae were tried as food with no success. Very
fine plankton was added regularly to some bowls and to the
water of the rearing tank, as adult colonies are known to
thrive on Nauplii and small Copepods (12), and on this food
the polyps developed six to seven pinnules, and small
colonies were produced. The reddish remains of a fairly
large copepod was one day found in a polyp, and on two
other occasions Temora longicornis was swallowed, while
Balanus nauplii were also accepted. However, those
polyps kept in Cawsand Bay flourished best, and while in the
laboratory for examination were frequently seen catching and
swallowing the larvae of Leptoclinum, which was also growing
on the dishes. They were successfully fed with these larvae
and with similar larvae of Botrylloides, from a pipette, and
would also take adult individuals removed from these colonies
when they were offered. The larvae were swallowed head
first, and the red Botrylloides larvae could be traced excel-
lently. Stages in the swallowing and disintegration of food
exactly like those figured by Miss Pratt (12) were obtained.
The young colonies showed no evidence of being preyed upon
on the raft, nor did the settled polyps in the laboratory tanks,
although shrimps ate the eggs and swimming larvae readily.
It was interesting to find a parasitic copepod in the ccelen-
teron of the polyps of the female colonies feeding on the
eggs.

-60

ANNIE MATTHEWS.

7. Segmentation.

The newly fertilised egg is of about 05 mm. diameter,
opaque, and fall of reddish-yellow yolk. It has accordingly
to be rolled round in a suitable vessel of sea-water in order
that the lobes and segments may be seen. Hence observa-
tions are less easy than in the case of the transparent eggs of
Echinus. The eccentric oosperm nucleus is somewhat oval in
shape, and resembles that figured by Hill (5). The problem
of the maturation and fertilisation of the egg together with
the first divisions of the oosperm nucleus was not attempted,
as it is rather outside the scope of this work.

The outstanding feature of the early segmentation in A.
digit at um is its great irregularity of procedure. It would
seem as though the many ways of reaching the morula stage
were highly variable and unimportant, the resulting embryos,
however, being apparently of one kind. This bears out
Wilson’s remarks on Renilla (16), and is the first of a series
-of similarities shown during the development of these two
Alcyonaria. Ho polar bodies are extruded and no outward
sign marks the time of actual fertilisation. Before any per-
manent alteration in appearance occurs the ovum seems to
make violent but unsuccessful attempts at division, i. e. the
spherical egg becomes temporarily polygonal, the surface
being drawn out into irregular high ridges with corresponding
depressions, possibly witnesses of internal activity (Text-
fig. 26) . These ridges disappear after some time and the egg
returns to its spherical condition. The whole process may be
repeated with no apparent result. In one case the ridges
softened down into eight vaguely defined, lighter coloured
areas which covered the whole surface of the egg, but these
•eventually disappeared, leaving a spherical egg as before. In
other cases the ovum became temporarily long and oval, but
regained its globular shape later. In artificial fertilisations
the time elapsing between the adding of sperm to the bowl of
ova and the above-described attempts at division varied from
half an hour to two and a half hours in different cases. The

THE DEVELOPMENT OF ALCYON1UM DIG! TATUM.

61-

interval between the end of this stage and the subsequent
formation of definite lobes, and again between the protrusion
of lobes and the actual production of blastomeres was also
very variable. Before segmentation begins the surface of
the egg usually becomes pushed out into eight equal or
unequal lobes (Text-fig. 27). However, sixteen lobes are
sometimes protruded instead of eight (Text-fig. 28). In one
such case the first lobe slowly formed about two hours after
fertilisation, and soon afterwards the second, third, and fourth
lobes followed, all at one pole (Text-fig. 29, l.). Next larger
lobes slowly formed towards the opposite pole (Text-fig. 29, L.),
and while these increased in number all the lobes became
more prominent. Two and a half hours after fertilisation
the lobes were still increasing in number, and half an hour
later still sixteen were counted embracing the whole surface
of the egg. Sections of this stage revealed one nucleus only,,
but in sections of other similar embryos the nucleus was
rapidly dividing into daughter nuclei. When only eight lobes
form they are necessarily larger in size than when sixteen
are protruded (cf. Text-figs. 27 and 28). Great variability,
however, exists in the protrusion of lobes ; many eggs seem
to throw them out in a very irregular manner, the lobes
themselves varying in number: thirty-two, sixty -four, and
other numbers have been counted.

In the majority of ova, while eight equal or unequal super-
ficial lobes are protruded, the centre yolk remains at first
undivided (PI. 3, fig. 8, C.'M.). Subsequently the grooves-
between the . lobes deepen, extending towards the centre of
the egg, and almost cutting it into eight segments. Finally
the segments become rounded off from one another at the
centre, leaving a little granular protoplasmic waste in the
newly formed segmentation cavity (Text-fig. .30, and PL 3,.
fig. 9, S. C.). This cavity persists until the end of the morula
stage. Usually the oosperm nucleus is found lying near the
region where the first lobes form, and, though it may divide
up into eight daughter nuclei while the lobes are being pro-
truded, the fission of the oosperm nucleus is sometimes-

02

ANNIE MATTHEWS.

•delayed until the lobing is complete. Sections of ova during
the continual subdivision of the parent nucleus show beautiful

Text-figs. 26-34.

32

33

34

Fig. 26. Egg making abortive attempts to divide, X 32. Fig. 27.
Egg with eight unequal lobes, the primary nucleus and
central protoplasmic mass being still undivided, X 48.
Fig. 28. Sixteen lobes protruded, eight large and eight small,
X 50. Fig. 29. Eai'ly lobed stage, two hours after fertilisa-
tion, X 54. Fig. 30. Egg segmented into four large and four
small cells, x 42. Fig. 31. Sixteen cells — eight large and
eight small, X 42. Fig. 32. Thirty-two celled stage, X 42.
Fig. 33. Segmenting egg with many small cells at one pole,
and fewer large ones at the other, X 45. Fig. 34. Sagittal
section of late morula, just passing into pre-planula stage,
X 78.

THE DEVELOPMENT OE ALCVONIUM DIGITATUM.

63

karyokinetic figures, one daughter nucleus passing to each of
the eight lobes (PI. 3, fig. 10). After the first segmentation
the blastomeres may be uniform or may fall into groups of
four large and four small cells (Text-fig. 30), or again may
be quite irregular in size. The nucleus in each segment now-
halves, exhibiting meanwhile well-formed spindles, some with
equatorial chromosomes and others with the halved chromo-
somes at each pole. Then the eight cells divide into sixteen,
generally eight smaller at one pole and eight larger at the
other. All the blastomeres do not divide simultaneously, and
hence stages are found with ten, twelve, or fourteen cells
only, but an examination of the nuclei shows that the undivided
cells will also shortly segment, giving the typical sixteen cell
stage. In some fourteen-celled embryos the nuclei of many
of the segments had again halved ready for the thirty-two
celled stage before the two slowest of the original eight cells
had completed their first division. When sixteen lobes are
protruded the egg divides immediately into sixteen cells
instead of eight (PI. 3, fig. 11, and Text-fig. 31). These
segments again may be equal or unequal, and are, as in the
case of the eight cell stage, separated by a segmentation
cavity containing a little waste protoplasm. In all cases the
sixteen cells halve, giving thirty-two blastomeres, and here
again some segments divide very slowly, and so twenty,
twenty-four, and twenty-eight celled embryos occur. The
thirty-two blastomeres (Text-fig. 32) may or may not be
uniform, and the irregularity in all the previously mentioned
stages prepares one for and helps to explain other embryos
where the segmentation seems quite irregular, or where
numerous very small cells form a cap at one pole over a few
large cells at the other (Text-fig. 33). Possibly the large
amount of yolk in the ovum causes the unequal segmentation
as well as the initial futile attempts at division and the
retardation of fission in some blastomeres. The thirty-two
celled embryo divides repeatedly, giving sixty-four, one
hundred and twenty-eight, etc., cells. By this time the
embryo has become two-layered, the segments at each division

64

ANNIE MATTHEWS.

becoming' smaller and more numerous. Between the periods
of division the segments are flattened, but just after segmen-
tation they are very prominent, so that the contour alters
considerably. The late morula is approximately spherical,
and though during late segmentation it may become tem-
porarily oval it soon regains its round shape. The first
delamination cleavage occurs when the nuclei of the sixteen
cell stage divide (PI. 3, fig. 11, D. N.). Some of the spindles
lie along* a radius of the sphere, and hence the resulting*
daughter nuclei lie similarly, so that a cell is split off towards
the centre of the embryo in all such cases, and thus an inner
endodermic layer arises (PI. 3, fig. 12, End.). Other spindles
lie in a plane at right angles to the radius, and hence these'
daughter cells lie side by side with the parent cell in the
outer ectodermic row (PI. 3, fig. 12, N.). It is thus evident
that all of the sixteen cells do not simultaneously contribute-
to the endoderm layer, and while both ectoderm and endoderm
cells continue to divide, later radial spindles in the ectoderm
afford evidence that the early endoderm cells are continually
reinforced from the ectoderm (PI. 3, fig. 12) } Hence from
the thirty-two celled stage onwards the larva is two-layered
(Text-fig. 34). The outer layer is somewhat irregular at first
(PI. 3, fig. 12), and, as Wilson says of Renilla (16), the
ectoderm cells dovetail into those forming the inner mass.
The endodermic yolk globules are much larger than those of
the ectoderm at this stage (PL 3, fig. 21). As the number
of cells increases the ectoderm becomes a more regular row
of cuboid cells, staining much more deeply than the inner
layer of larger polygonal endoderm cells (PI. 3, fig. 21).
Towards the end of the morula stage about thirty-two small
cells can be counted round the circumference of the sphere,
the ectoderm being now columnar (Text-fig. 34). The yolk
in the endoderm has been partly used up, the vesicles having
become smaller and similar to those in the ectoderm (PI. 3,
fig. 20).

1 The writer hopes to discuss the question of the origin of th£
endoderm in greater detail in a subsequent paper.

THE DEVELOPMENT OF ALCYON1UM DIGITATUM.

65

Early Embryos in Section.

The granular protoplasm of the unsegmented egg is full of
yolk globules. These are largest towards the periphery, while-
a shallow surface layer of the egg is finely granular and
devoid of yolk. These areas are still visible in the eight and
sixteen cell stages (PI. 3, fig. 13), the finely granular surface
layer being confined to the outer edge of the blastomeres.
The yolk distribution in the later morula stages has been
already described, the large round nuclei being surrounded
by a deeply staining finely granular area (PL 3, fig. 20).

Other Types of Segmentation.

One embryo was sectioned in which an irregular outer layer
of blastomeres had been cut off from an inner undivided mass.1
This example agrees with lvowalevsky’s description of the
segmentation of the egg of Alcyonium (8). In this he relates
how a complete covering layer of nucleate ectoderm cells of
various sizes was cut off from an undivided central yolk mass,
which itself split later into a few large cells, while further
divisions of all the cells resulted in a morula similar to those
now described. Hickson (4) records a four-celled embryo,
and during the present investigations one embryo was followed
to the late morula stage from two unequal blastomeres.

8. The Pre-planula.

After the twentieth hour the spherical morula becomes very
gradually distorted, slowly protruding blunt lobes separated
by corresponding depressions (Text-fig. 2). It is found that
at this time the numerous yolk globules in the columnar
ectoderm and the polygonal endoderm are still small and
quite similar in the two layers. About the fiftieth hour, when,
the prominent lobes very slowly begin to soften down again,

1 Very possibly this egg was unhealthy; it is quoted because
Kowalevsky's account of the early development of the egg of Alcyo-
nium differs so greatly from what is described above.

VOL. 62, PARI’ 1. N'EW SERIES.

5

66

ANNIE MATTHEWS.

the yolk globules become fewer but larger in size, and
continue this decrease in number and increase in bulk for
some little time, apparently by fusion of the smaller globules
(PL 3, fig. 18, and Pl. 3, fig. 14, Y. V.). Meanwhile the
segmentation cavity, which has persisted until now (Text-fig.
34), disappears. The columnar ectoderm cells increase greatly
in length and number, becoming very slender, while smaller
rounded ones appear among them (PL 3, fig. 15, Ect. and
R. C.). The outer halves of the columnar cells have become
finely granular, the inner portions still containing yolk
globules (Pl. 3, fig. 15, Gr. E. and Y. E), while the round
cells each contain about four large yolk globules.1 The
innermost endoderm cells, which are also the largest, now
begin to degenerate (PL 3, fig. 16, c.), and by the continued
absorption of these cells a series of cavities forms which
presently fuse into one — the coelenteron — the wall of which
consists of a layer of endoderm about three cells deep, with
the columnar ectoderm outside. As development continues
the eudodermic layer becomes progressively thinner by the
degeneration and ultimate absorption of the inner cells, until
it consists of only one row of columnar cells about half as
long and twice as broad as the columnar ectoderm cells.
Round cells are at this time found lying between the tall
•ectoderm cells at both their inner and outer ends (PL 4,
fig. 27, a drawing of the next subsequent stage, which would
also serve as a figure of this stage). At times the columnar
ectoderm cells proliferate very rapidly, and so the ectoderm
becomes temporarily multilayered, soon, however, regaining its
normal columnar character (Text-fig. 35). In the final stages
(Text-fig. 3), the ectoderm cells of the pre-planula are very
deep indeed, and become separated from tlie endoderm by a
thin structureless membrane (Text-fig. 35, S.M.). There is
no direct evidence to show which layer secretes this, but it
seems identical with and indeed an early part of, the meso-
1 Hickson (4a) describes the endoderm in the planula of Alcyonarians
as a plasmodium, but from the present account it is clear that in
Alcyonium digitatum tlie endoderm is always a definite cellular
layer.

THE DEVELOPMENT OF ALOYONIUM DIGITATDM.

67

gloea of the adult polyp. This being so, the endoderm must
be regarded as its source of origin (see p. 72 seq.).

The Planula.

On the fourth day the pre-planula merges into a spherical
swimming planula of wavy outline (Text-fig. 4). Cilia are
developed by the ectoderm cells, and a definite anterior pole
is marked by being held foremost while swimming. The
coelenteron is still partly filled by groups and strings of
loose endoderm cells, empty cells, protoplasmic cell contents
and yolk. The definite endoderm consists of a row of
columnar cells full of yolk vesicles, and roughly pyriform,
i. e. rather wider at the free nuclear end than at the base.
To the inner edge of the permanent endoderm one or more
rows of rounded cells still adhere as in Text-fig. 36. The
yolk has all been absorbed in the columnar ectoderm, the
cell contents being now granular, while the separating
membrane is much more distinct. These ectoderm cells have
slightly expanded and flattened bases where they rest on the
membrane, and are about as tall as the permanent endoderm
cells, and half as wide. Rapid proliferation will again
temporarily obscure the columnar character of this layer so
that it seems to consist of several rows of round cells, but, as
in the previous and subsequent stages, they soon return to
their columnar state. In a few hours the round planula has
become oval, but at first its outline remains sinuous. The
microscopic structure is little altered, save that the degen-
erating endoderm cells have been absorbed in increasing
numbers, while the permanent row of endoderm cells stand
out clearly (PI. 3, fig. 17, c., End.). By the fifth day the
outline of the planula is smooth, and soon the anterior pole
broadens, so that the larva assumes its characteristic pear-
sliape (Text-fig. 6). It rapidly lengthens, while the permanent
row of endoderm cells still shows several rows of rounded
or polygonal cells clinging to it in places, and often forming-
club-shaped projections into the coelenteron (PL 3, fig. 1,
.and PI. 3, fig. 17, C. P.j. Increase in the number of endoderm

68

ANNIE MATTHEWS.

Text-figs. 35-37.

THE DEVELOPMENT OF ALCYONIUM DIGITATUM.

69

and ectoderm cells is brought about by the multiplication of
very small interstitial cells found at their bases. When very
young these are even smaller than the nuclei of the adult
cells. Nematocysts are now formed by the rounded cells
lying at the outer edge of the ectoderm (Text-fig. 36), these
cells being also recruited from the interstitial cells (PI. 3^
fig. 19, We.). Previous to this many ectodermic interstitial
-cells give rise to broad columnar mucous cells, i . e . refringent
cells with a protoplasmic network surrounding the mucus
which stains deeply with haematoxylin (PI. 3, fig. 2, M. C.).
At the anterior pole the endodermic tissue forms a deeper
layer than elsewhere (PI. 3, fig. 1). The ectoderm cells of
the anterior pole become specially long in the well-grown
planula, and the mucous cells are particularly numerous.
Now the larva settles by the broad anterior and aboral pole
(see general account), and for some hours after fixation the
still ciliate planula hangs freely in the water, retaining its
characteristic contractile power. If forcibly detached from
the mucus plug which fastens the broad anterior end to the
substratum, it will swim freely again for some time and then
resettle. The round flat disc formed by the anterior pole
becomes the base of the new polyp, and hence the nucleus of
-attachment of a fresh colony. For some time after settling
the coelenteron still contains much yolky detritus, which is
gradually used up. The endoderm is similar to that described
for the late planula, i. e. many cells deep at the fixed aboral
pole and one layer elsewhere, clumps of non-permanent cells
clinging to it in places (PI. 4, fig. 24, End. M., and End.).
Very many round cells arise next to the supporting membrane
in the ectoderm of the fixed base of the polyp, soon giving it
a multilayered character (PI. 4, fig. 23, R. G.). Mucous cells
are still abundant in the ectoderm, but gradually disappear,

Fig. 35. Ectoderm of stage shown in PI. 3, fig. 16, at time of
rapid proliferation of cells (temporarily multilayered). Fig. 36.
Ectoderm and endoderm cells from late planula, showing
nematocysts. Fig. 37. Sagittal section of newly settled polyp,
now shrinking in length. The pointed posterior and oral pole
of the planula is still visible ( X 133).

70

ANNIE MATTHEWS.

while nematocysts are very abundant over the whole surface.
The long planula-shaped polyp now shrinks in length (Text-
figs. 10, 11, and 37), the ectoderm and supporting lamella
each becoming crumpled up on itself so that it is of wavy
outline in section. The endoderm cells are indeed heaped up
into a series of blunt processes separated by deep hollows, and
projecting into the ccelenteron (Text-fig. 37, End. C.). Soon
the pointed aboral end flattens down, the larva in vertical
section now appearing like PI. 4, fig. 23. The endoderm
is a single row, except at the base, where it forms a deep
multilayered mass of cells each containing one huge vacuole
(in stained preparations). Thus a mound-like stage is reached
at the end of the first day of sedentary life. A little later
the mesenteries arise, and simultaneously a great and rapid
increase in the number of rounded cells at the base of the
columnar ectoderm occurs all over the surface. Next these
become surrounded by mesogloea, and from now onwards an
intermediate layer separates the endoderm and ectoderm of
the polyp. The rounded cells are young scleroblasts and
nematocysts.

The next section of the paper deals with the secretion of
mesogloea, as the further development of the polyp may then
be more easily understood.

9. Mesoglcea.

In this paper the word mesogloea1 will be applied to the
structureless, deep-staining, jelly-like substance which lies
between the ectoderm and endoderm of the polyp, and, aided
by the spicules, gives rigidity to the body wall. The “ endo-
mesogloeal ” cells are the cells which become embedded in it.
The thin supporting lamella which first appears between the
endoderm and the ectoderm of the late pre-planula, and can
be traced in the planula (PI. 4, fig. 27, S. M.) and earliest
settled stages, stains quite similarly to the mesogloea of later

1 This word was introduced with the significance given above by
Prof. Gilbert Bourne, F.B.S., in 1887 (see ‘ Quart. Journ. Micr. Sci./
vol. 27, p. 303).

THE DEVELOPMENT OF ALCYONIUM DIGIT ATTJM. 71

stages, and seems to be simply an earlier secreted part of it. At
the time of the early formation of the mesenteries the lamella
of the body wall is -first thickened by further secretion of meso-
gloea which is deposited on its outer side and separates it from
the ectoderm. This process continues during later stages, and
it is found that the most recently secreted part of the meso-
glcea, i. e. that part lying nearest the ectoderm (PI. 5, fig. 39,
R.Mes.), always stains more feebly than the rest (E.Mes.).
The mesogloea, after thickening the supporting lamella of the
attached base of the polyp (PI. 5, fig. 40, Mg.), streams
between the round cells of the multilayered ectoderm (Ect.)
and unites with the original disc of adhesive mucus ( M. P. ),.
thereby strengthening the attachment of the polyp to the
substratum. The mesogloea is thickest in the body wall, much
thinner in the bases of the tentacles, and very little thicker
than the original supporting lamella in the distal ends of the
tentacles, the pinnules, the oral disc, and stomodseum. No
cells have been found embedded in the mesogloea which could
be shown responsible for its secretion. On the other hand,
streams of newly secreted mesogloea (PI. 5, fig. 35, S. Mg.),
which would seem in this stage to be a very viscous fluid,
are found running outwards, firstly from the very slightly
thickened supporting lamella (PI. 5, fig. 35, Mg.), and later
from the gradually thickening mesogloea of the body wall
(PI. 5, fig. 36, Mg.) towards the ectoderm. They encroach
on and finally surround the round cells at its base (PJ. 5,
fig. 36, Sc. and Ne.), isolating them singly or in small groups
in the mesogloea (S. cell and G-r. cell). These mesogloeal cells
arise as interstitial cells (PI. 5, fig. 37, Sc.), and give rise
either to spicules or nematocysts. The formation of the latter
has not as yet been followed, but it was supposed during
these investigations that the small rounded cells which stain
very blue with picro-nigrosin aud are only about half the size
of the young scleroblasts when engulfed are young nemato-
cysts. Perfectly formed nematocysts may also be thus sur-
rounded, and occasionally a scleroblast secretes a young
spicule while still lying at the base of the ectoderm, i. e.

72

ANNIE MATTHEWS.

before it becomes surrounded by mesogloea. Evidence seems
to indicate that the mesogloea flows out from the endoderm
(Pl. 5, figs. 36 and 39). It is certainly not secreted by any
of the ectoderm cells, and the direction of flow is always out-
ward from the endoderm to the ectoderm, and then, as
previously stated, in among and around the basal cells of this
layer. During the later growth of the mesenteries they con-
sist almost wholly of a thin sheet of structureless mesoglea,
covered on both sides by endoderm (PI. 5, fig. 38, Mgl and
End.), so that this layer is probably capable of providing for
all further secretion of mesogloea required by the growth of
the mesenteries (see paragraph on mesenteries).

Nothing has been found to correspond with the irregular
nells described by Bourne (1), full of minute highly refringent
granules, which concealed the nucleus. He suggested that
these might be the mesogloea-secreting cells, while Woodland
(18) described some small rounded cells which he also noticed
as full of refringent granules, without deciding whether these
corresponded to Bourne’s. As both these papers were founded
on work done on Alcyonium colonies, cells may be present in
the mesogloea which are not found in the solitary polyp. Still
it seems very possible that Woodland’s cells were merely
young scleroblasts, judging from his figures. The “oval
bodies ” described by Hickson (3) and Woodland as occurring
in the mesogloea are nematocysts which may be in process of
migration. The coiled thread in the cell was clearly stained
with picro-nigrosin after preservation with osmic acid (PI. 3,
fig. 22, N. T .)

10. Mesenteries.

The eight characteristic mesenteries of Alcyonium are thin
vertical sheets stretching radially into the coelenteron from
the body wall and dividing it into eight incomplete inter-
mesenteric compartments, without meeting one another in the
centre. They consist of thin sheets of mesoglceal substance
arising from the supporting lamella of the body wall (PI. 3,
fig. 19, Mes.), and covered on both sides by endoderm cells

THE DEVELOPMENT OE ALCYONIUM DIGITATUM.

73

{End.), which in the early stages are long and columnar, but
become low and broad later when the mouth has opened. The
mesenteries arise simultaneously early on the second day of
fixation, and usually do not appear until the flattening of the
larva is complete. They are visible before the original
supporting lamella of the body wall is thickened by the
addition of fresh layers of mesogloeal substance, and therefore
before the development of the spicules, tentacles, and mouth,
these organs following them in the order stated. Between
the normal eight as many as eighteen rudimentary mesenteries
are often developed (Text-fig. 38, Sub.M.), but these either
remain very small or eventually disappear, and are possibly
vestiges of a more primitive condition when the mesenteries
were very numerous (cf. Clavellina (9)). In colonial forms
the new polyps formed by budding never show these rudi-
ments. In the early stages the mesenteries are indicated
from outside by eight equidistant shallow vertical grooves,
stretching up a little way from the base of the lateral wall
(PL 3, fig. 3), while sections of rather older stages indicate
that the mesenteries are already arranged in four pairs (Text-
fig. 39). At this stage the mesenteries in vertical radial
section resemble Text-fig. 12, c. During the earliest stage
observed (Text-fig. 40, a.) the supporting lamella of each
mesentery, consisting of a very thin sheet of mesoglcea, can
be easily traced. Sections show it growing radially inwards
from the supporting lamella of the body wall (Text-fig. 41,
Mes.) between the endoderm cells lining the latter. At this
time it is structureless, stains slightly with picro-nigrosin, and
encloses no cells. Each mesentery grows rapidly upwards
along the body wall and along the base of the polyp towards
the centre (Text-figs. 40, bi, and 12, c. and d.). Meanwhile it
increases in radial depth (Text-fig. 40, Zq), and as the supporting
lamella deepens, the endoderm cells surrounding it grow
inwards with it, so that each mesentery soon projects into the
coelenteron as a shallow ridge formed by an infolding of the
endoderm supported by a central ridge, the lamella (Text-
fig. 42, Mes.). Figures of subsequent stages show that the

ANNIE MATTHEWS.

Text-figs. 38-45.

45

THE DEVELOPMENT OF ALCYONIUM BIG L TATUM.

75-

mesentery is free along its inner edge but attached elsewhere
to the body wall and base of the polyp. By the end of the
second day the mesenteries nearly meet in the centre of the
oral and aboral surfaces of the polyp, but they are still fairly
shallow (PL 3, fig. 5, and Text-fig. 12, d.). After the forma-
tion of the tentacles the stomodaeum develops as an invagina-
tion of the oral surface, in the space encircled by the upper
edges of the mesenteries, and as it grows inwards the upper
edges of the mesenteries are carried down with it, so that all
eight become attached along the entire length of the stomo-
daeum (Text-fig. 13,/. and S.). There has been some discussion
as to whether a mesentery is purely endodermic in origin (the
view taken by Wilson and Kowalevsky), or whether both the
endoderm and ectoderm contribute to its formation (the view
held by de Lacaze-Duthiers), and the following account may.

Fig. 38. Aboral view of polyp, some hours older than PI. 3, fig. 4.
The eight permanent mesenteries are distinguished from the
subsidiary ones by their greater development. Spicules just
appearing (x 27). Fig. 39. Transverse section of polyp about
stage drawn in Text-fig. 12, showing paired arrangement of
mesenteries. Fig. 40. Vertical section of polyp with very
young mesenteries, early on second day of fixation. The
vertical dotted line divides the diagram into halves, the right
showing a more advanced stage than the left. At a. a very
young mesentery is drawn, which does not yet project beyond
the endoderm. At bx a rather older mesentery is shown (at
a stage similar to the mesenteries in Text-fig. 42). Fig. 41.
Diagram of a transverse section of a young polyp near its
base. The mesentery is similar to that shown in Text-fig. 40
(a). Hence section cuts through a solid layer of endoderm
(the multilayered endoderm at the base of the young settled
polyp), and through the supporting lamella of eight young
mesenteries ( Mes .). Fig. 42. Transverse section of a rather
older polyp, at stage drawn in Text-fig. 40 (&/ some distance
higher up than the level of Text-fig. 41. The mesenteric
ridges have deepened radially, and are covered by columnar
endoderm cells. Fig. 43. Transverse section of polyp some-
what older than Text-fig. 42, showing how the lamella of the
lateral wall becomes pulled in during the inward growth
of the mesenteries. Fig. 43a. Lamella of young mesentery,
reconstructed from sections. Fig. 44. Here the mesoglcea is
being rapidly thickened. It is seen penetrating between the
ectodermic cells which have been drawn into the root of the
mesentery, and isolating them (Mg. and I. C. S.). Fig. 45.
Transverse section of polyp, showing mesenteries cut across-
near base, i.e. at level M. B. in Text-fig. 43a.

76

ANNIE MATTHEWS.

serve to show how support for both views could be obtained
in the case of Alcyonium according to the part of the
•mesentery studied :

(1) Sections show that by the time the mesenteries reach
up to the oral surface of the polyp, the supporting lamella
contains a great many ectoderm cells which lie very near the
body wall. Text-fig. 43a is reconstructed from a series of
sections of a polyp with mesenteries at this stage. It is a
diagram of the supporting lamella of one mesentery, while
the covering endoderm is not represented. Many small
round cells are seen embedded in it towards the outer edge
and at the base (Ect. C.). These are nematocysts and young
scleroblasts which have entered from the ectoderm in a
manner to be discussed later. Therefore both ectoderm and
endoderm elements occur in the mesentery at this stage.
However, it is seen that the greater part of the lamella con-
tains no ectoderm cells, while as the mesentery grows larger
the proportion of the lamella which contains ectoderm elements
grow necessarily less and less, so that in the adult the
mesentery is almost wholly endodermic in structure. This
.seems to support the view that the function of the ectoderm
cells in the mesentery may be unimportant, and that the
endodermic covering can provide for all further increase in
the size of the lamella.1

(2) When the mesentery first develops, its supporting
lamella grows radially inwards from the lamella of the body
wall and is directly continuous with it (Text-figs. 41 and 42,
Mes.). A little later, as the mesentery continues to grow
inwards, the lamella of the lateral wall often appears slightly
pulled in where the mesentery joins (Text-fig. 43, L. /.), and
these two cases may appear in one section. Interstitial cells

1 The fate of the nematocysts is uncertain: possibly they migrate
to some definite place for future use. It is known that they occur
in the six ventral mesenteric filaments, but probably all of these are
derived from the ectoderm of the stomodseum, as described later.
The spicules secreted by the scleroblasts found in the lamella would
•certainly help to stiffen it at the base and near the body wall.

THE DEVELOPMENT OF ALCYON1UM DIGITATUM.

77-

are constantly arising in the ectoderm of the lateral wall, and
some are often seen lying near the inner edge of the mesentery,
and are therefore frequently found in the groove formed at
this point. As the mesenteries grow these grooves deepen,
and so more interstitial cells canenter. While the mesogloea
of the body wall is being laid down outside the original
supporting lamella (Text-fig. 44, Mg .), it appears to flow
between these interstitial cells, cutting them off from the
ectoderm either singly or in small groups, just as it envelops
and isolates interstitial cells in other parts of the body wall
(see chapter on Mesogloea) . It is not impossible then that any
ectoderm cells found in the mesentery are introduced by
mechanical means, i.e. the ingrowing of the mesentery
draws a few interstitial cells after it, which become enclosed
in the supporting lamella, this process continuing in some
regions until a large number are enclosed.

(3) Transverse sections of the mesentery in Text-fig. 43a
at level T. (indicated by a dotted line) would show a few
cells in the supporting lamella near the body wall. At level V. .
a similar condition would obtain. Lower still, the lamella in
cross-section would show a very deep groove full of cells
near the lateral wall, and a very short single sheet or none at
all (Text-fig. 45, V. or Z. ). Towards the base of the mesen-
tery the lamella is double and full of ectoderm cells, or in
some cases it contains a group of cells near its inner edge
(Text-fig. 45, IF.), cut off from the outer deep groove T. by a
region E.} containing no cells. A vertical section of Text-
fig. 43, A., at line R. would obviously show a double lamella
fall of ectoderm cells and surrounded by endoderm, and this
would appear to be the kind of section figured by de Lacaze-
Duthiers (10).

Considered apart from the early history of the mesentery,.
Text-fig. 45 seems to indicate that the mesenteries are true
invaginations .of the body wall. A solid cord of ectoderm
cells .seems to grow in, surrounded by a tubular ingrowth of
the supporting lamella, .and a covering of endoderm cells, but
whether the former cells are to be regarded as drawn in.

78

ANNIE MATTHEWS.

mechanically during the ingrowth of the mesentery, or
whether they have any embryological significance imply-
ing a true invagination of both layers is not altogether
certain. If the first, then the mesentery is purely endo-
dermic, as Wilson believes ; if the second, then de Lacaze-
Duthiers is correct. The total evidence afforded by these
investigations seems, perhaps, to support the latter view. It
mav be added that the rudimentary mesenteries contain many
ectoderm cells embedded in the supporting lamella.

11. Tentacles, Mouth and Stomoda]um.

Eight short broad tentacles develop on the third day
(PI. 3, fig. 5). They are simple hollow outgrowths of the
upper surface of the young polyp, consisting, therefore, of
ectoderm and endoderm separated by a supporting lamella
(Text-figs. 46 and 47, T .), and arise between the mesenteries
in a circle round the oral disc. A little later a shallow
invagination of the latter forms the beginning of the stonio-
daeum. This grows considerably in size and dept i, the
exterior and only opening being the mouth (PI. 3, fig. 7, and
PI. 4, fig. 28). The lumen of the invagination soon widens
considerably at the base, and the floor of the cavity is
correspondingly enlarged (Text-fig. 46). During the fourth
day the endoderm and ectoderm of this floor degenerate,
either at the centre or near the side or in both regions simul-
taneously (Text-fig. 47). By this means communication is
established between the ccelenteron and the exterior, and
food may enter from outside. Until now all requisite nourish-
ment has been provided by the embryonic yolk. By the fifth
day the disintegration is complete, and the formation of the
mouth and stomodaeum is accomplished. The ectoderm
covering: the latter consists of taller columnar cells than else-
where. Like the tentacles it is plentifully supplied with
nematocysts, which, indeed, at this stage are abundant all
over the surface of the polyp. Mucous cells and granular
^land cells are soon formed in the stomodaeum and pour their

THE DEVELOPMENT OF ALCYONIUM DIGITATUM.

79

Text-fig. 46.

Text-fig. 47.

;Same, when communication is established between the coeienteron
and the exterior, by the degeneration of the endoderm and
ectoderm at the centre of the base.

80

ANNIE MATTHEWS.

secretion upon the food while it is being passed down. The-
supporting lamella of the stomodaeum always remains a thin
sheet, while the endodermic lining is similar to that found
throughout the coelenteric cavity, as the endoderm covering
the attached base of the polyp is by now reduced to one row
of columnar cells (PI. 3, fig. 19, End.). In section the top
of the newly-opened stomodaeum is round, but lower down it
is keyhole shaped (PI. 4, fig. 34), the narrow ventral end
of the keyhole being the siphonoglyph. Hence in vertical
sections which cut the stomodaeum dorso-ventrally, the latter
seems much wider than when cut in a plane at right angles
to this.

12. Spicules.

Investigation into the origin of the spicule in the young
solitary polyp confirms Woodland’s work on colonial forms
(18), adding thereto one or two minor points of interest-
Each spicule, as he states, is the product of a single cell, and
during1 its elaboration the nucleus halves, each daughter
nucleus apparently controlling one pole of the spicule (PI. 4,
fig. 31, Nos. 1 and 5). It was found, however, during the
present investigations that some large spiculoblasts from
Alcyonium colonies contained three or four nuclei. It was
also seen that the young scleroblasts are of ectodermic origin
(PI. 4, fig. 32), and arise as rouud interstitial cells at the-
base of the ectoderm of the body wall and tentacles (Pl. 5,.
fig. 35, Sc.). These scleroblasts become stellate, spindle-
shaped, oval or rounded, as they increase in size (PI. 4,.
fig. 32), and, as explained previously, are encroached on and
eventually surrounded by mesoglea, either singly or in small
groups. In later stages the spicules become entirely isolated
from one another by mesogloea. As Woodland states, the
cytoplasm of the scleroblasts eventually becomes reduced to a
mere thin granular covering-layer over the greatly enlarged
spicules (PI. 4, fig. 31, Nos. 4 and 5). PI. 4, fig* 30, shows
that after dissolving away the spicules by an acid stain
(picro-nigrosin), the mesogloea is left full of corresponding;

THE DEVELOPMENT OF ALCYONIUM DIGIT ATUM.

81

cavities lined with the cytoplasm of the scleroblast, each
cavity shaped exactly like the spicule which occupied it.
The earliest spicules arise on the second day of fixation,
rather later than the mesenteries and before the tentacles,
and may be examined with low powers of the microscope.
They appear as small refringent nodules in the upper surface
of the mound-shaped polyp, and show through by transmitted
light. One row of small unbranched spicules is present in
the body wall by the time the mouth opens (PI. 5, figs. 36
and 37), and in older polyps they are found in the upper and
lower regions of the body wall, the bases of the tentacles,
and in the outer edge of the mesenteries very near the base
(Text-fig. 14). According to Woodland, after the two
daughter nuclei have formed in the scleroblast, the steadily
growing round spicule lengthens and assumes a simple
dumb-bell shape (PI. 4, fig. 31, No. 3). The ends of this
dumb-bell become gradually elaborated into processes, so that
the spicule then resembles a caudal vertebra in shape
(Nos. 4 and 5). The colonies he utilised in following out the
development of the spicule were small — about half an inch
across — and the drawings he made have all been verified on
the solitary polyp, with the exception of his figures illustrating
the cavities which the spicules occupied.

13. Mesenteric Filaments.

Each mesentery bears a filament on its free inner edge
running down for some distance from the lower opening of
the stomodaeum (Text-fig. 48). While the two dorsal fila-
ments create an upward current of water in the coelenteron
by the active lashing of the cilia borne on the cells which
cover them (Text-fig. 49), the six ventral filaments are
secretory and absorptive. The ferment poured upon the food
by these filaments continues and probably completes the
disintegration begun in the stomodaeum. In the oldest
solitary polyp examined (fixed from thirteen to fifteen days),
the ventral filaments reached almost to the bottom of the free
VOL. 62, PART 1. NEW SERIES. 6

82

ANNIE MATTHEWS.

edge of their respective mesenteries, and the dorsal not quite-
so far (Text-fig. 48). The first signs of each ventral filament

Text-figs. 48-51.

Fig. 48. Vertical section of polyp on thirteenth day of fixation,
showing relative length of dorsal and ventral mesenteric
filaments. Fig. 49. Transverse section of young dorsal
mesenteric filament. Fig. 50. Longitudinal and radial section
of very young ventral mesenteric filament, showing its relation
to the thickened edge of the mesentery. Fig. 51. Transverse
section of young ventral mesenteric filament.

are found on the fourth day of sedentary life, shortly before
the mouth is open. Immediately below the base of the

THE DEVELOPMENT OF ALCYONIUM DIG [TATUM. 83-

stomodoeal invagination the free edge of the mesentery
becomes thickened by a proliferation of the endoderm cells in
that region. These cells are longer than they are wide,,
whereas those on the rest of the mesentery are short and
broad. After the mouth and stomodseum have completed
their formation, on the fifth day, a narrow strap-like process-
of the ectoderm of the stomodseum grows out over the endo-
dermic part of each filament (Text-fig. 50, Ect. M. F., and
PI. 4, fig. 33, E. S. 0.; cf. Wilson's fig. 21 (15)). In
this ectodermic band, which is only 6 ju (or one section) broad
at first, nematocysts, mucous and granular gland cells
develop. The band, lying over the endodermic part of the
filament, soon widens and forms an ectodermic layer covering
the outer surface of the latter (Text-fig. 51). [In this figure
the two layers in the filament are marked Ect. M. F. and End..
M.F., and are shown in transverse section, while Text-fig. 50
represents a much earlier stage in longitudinal section (about
stage of PI. 4, fig. 33), the dotted line marking the edge of
the mesentery before the endodermic thickening began
( Mes . E.). The letter W. marks the lower limit of the stomo-
dseal wall ($.), before the ectodermic downgrowth developed.]
By the sixth day these filaments extend half way down the
free edge of the mesentery (PI. 4, fig. 25, S. 0.). During
subsequent growth the filament becomes somewhat twisted
laterally and gathered up into puckers on the edge of the
mesentery as a result of growing faster than the latter..
Consequently, sometimes the ectoderm and sometimes the
endoderm cells lie uppermost, and this can be realised easily
by imagining the filament shown in transverse section in
Text-fig. 51 twisted from side to side. Longitudinal sections
through the convoluted filaments are therefore somewhat diffi-
cult to understand at first sight, as groups of ectoderm and
endoderm cells lie side by side.1

While the ectodermic portion is secretive, the endodermia

1 A model in red and white clay of the endodermic and ectodermic-
parts of the filament was similarly twisted from side to side and then
sectioned to check this result.

84

ANNIE MATTHEWS.

part is absorptive. Food is engulfed by the amoeboid processes
of the latter cells in common with the endoderm cells lining
the general body cavity (12). The above observations
probably explain why in Miss Pratt’s paper ((12) PI. 21,
fig. 4) the carmined food is absorbed, and has reddened the
filament in certain definite areas and not in others. The
colourless parts are the ectodermic and the reddened parts
the en do dermic areas twisted uppermost. It also explains
the observation made early in the same paper, that histo-
logical study of the “stomodEemn and ventral mesenterial
filaments in several members of the family reveals many
points of similarity, if not identity, in their elemental consti-
tution. Both granular and mucous gland cells, as well as
nematocysts, occur in all these structures.” Briefly, this is
because the secretory part of these filaments is ectodermic,
the requisite gland cells and nematocysts being supplied by
the downgrowth from the stomodseum. The dorsal filaments
are very much narrower and straighter than the ventral in
the solitary polyp. A transverse section of these dorsal
filaments two days after they first appear is only two-tliirds
the size of a similar section of the ventral filaments on
the same date. No indication of these dorsal filaments is
found until the sixth day, i.e. they arise later than the
others. Narrow processes of the stomodseal ectoderm are
then seen growing down over the uppermost part of the
free edge of the two dorsal mesenteries, thus giving rise
to the filaments (PL 4, fig. 26, D. 0.). No appreciable
thickening of the endoderm forms, however, as a support
for this ectoderm, whereas it was visible before the ecto-
dermic part in the ventral filaments. A transverse section
reveals that in rough outline the dorsal filament is quite
comparable to the ventral, the difference being one of
degree of development only (Text-figs. 49 and 51). In the
former the ectodermic band, consisting of tall ciliate cells
with deep staining nuclei, rests on a slender endodermic
support, which is smaller than in the ventral filament.
Therefore both kinds of filament consist of ectodermic and

THE DEVELOPMENT OF ALCYON1UM DIGITATUM.

85

endodermic portions, the endodermic being well developed in
the ventral, and much less so in the dorsal. Growth seems
slower in the dorsal than the ventral filaments, so that by the
end of the seventh day they are still much shorter than the
latter (PI. 4, fig. 26).

Sections made of polyps for the examination of the fila-
ments at this stage, also show that the retractor muscles of
the mesenteries are now developing. By the thirteenth to
fifteenth day of sedentary life the ventral filaments are
twice as long as the dorsal, although they extend very little
below them, because of their convoluted condition. By this
date, also, the filaments approximate more nearly to the
adult in transverse section, i.e. the ciliated surface of the
dorsal filament has become more concave, while the glandular
ectoderm of the ventral has become more convex, and extends
further round (cf. Text-figs. 49 and 51 with PI. 4, fig. 29,.
and (3) PI. 38, figs. 18 and 19).

Summary of other Writers’ Yiews on the Derivation of
the Mesenteric Filaments, and Remarks on these.

H. Y. Wilson (15) considers that the ventral mesenteric
filaments in the coral Manicina are wholly ectodermic in
origin, and gives a very similar figure to the present PI. 4,
fig. 33, showing the down growth from the stomodgeum of the
ectodermic bands which give rise to the filaments. E. B.
Wilson (17) states that the dorsal filaments in colonial polyps
of Alcyonium are of ectodermic origin, but while he shows
(16) that endoderm certainly enters into the ventral filaments
of Renilla, he did not find any ectodermic outgrowth con-
tributing to them. It is not impossible that he passed over
this stage in development. J. Stanley-Gardiner (13) con-
siders that the ventral mesenteric filaments of Coenopsammia
are purely ectodermic, basing this view on histological
grounds. Also he mentions the same fact for Flabellum (14).

It is possible that investigations into the early development
of other Anthozoa would confirm the fact that all the fila-
ments throughout the group consist of ectodermic and endo-

86

ANNIE MATTHEWS.

dermic elements, both being well developed in the ventral,
while the ectodermic part is alone elaborated in the dorsal,

14. Summary.

(1) The fertilised eggs segment in various ways, but typical
morulae always result.

(2) When the sixteen cell stage again divides to produce
the thirty-two celled embryo, delamination occurs, and from
now onwards the larva is two-layered.

(3) The morula at the twentieth hour begins to undergo a
series of contortions which last from the first to the third day.
This solid contorted stage is here termed the pre-planula, as
it passes on into the hollow planula stage.

(4) The pear-shaped planula, while swimming, exhibits
-characteristic “ planarian-like ” movements, and on the fourth
free-swimming day (the seventh day of development) it
settles down by the broad anterior and aboral pole.

(5) The settled larva soon flattens, assuming a mound-like
$hape, and on the second day of fixation eight mesenteries
grow out simultaneously into the ccelenteron from the base of
the lateral wall. The ccelenteron is identical with the hollow
central space in the endoderm of the planula.

(6) The mesenteries, arranged in four distinct pairs, grow
simultaneously and rapidly along the lateral walls and attached
base, and soon nearly meet on the basal and oral surfaces of
the polyp.

(7) Many round cells now appear at the base of the columnar
ectoderm.

(8) Mesoglcea is at this time secreted by the endoderm, and
flows round the above-mentioned round ectoderm cells, cutting
them off either singly or in groups. These isolated ectoderm
cells produce either nematocysts or spicules, the spicules
appearing soon after the mesenteries.

(9) Early on the third day eight simple hollow tentacles
grow out, alternating with the mesenteries, and encircling the
oral surface.

THE DEVELOPMENT OF ALCYONIUM DKHTATUM. 87

(10) Later on tlie third day the stomodasum and month
arise by the appearance of a rapidly deepening invagination
of the oral surface, in the centre of the circle of tentacles.
Yolky detritus is still present in the coelenteron.

(11) On the fourth day the base of this invagination de-
generates, and so the coelenteron communicates with the
exterior.

(12) While the mouth invagination is still in process of
formation, the endodermic portion of the mesenteric filaments
arises on the six ventral mesenteries by a proliferation of
cells on the upper part of the free edge.

(13) On the fifth day the ventral mesenteric filaments are
completed by strap-like ectodermic downgrowths from the
stomodasum over the endodermic thickening on each mesen-
tery.

(14) On the sixth day the dorsal mesenteric filaments arise.

(15) The dorsal and ventral mesenteric filaments appear
homogeneous in origin, though of diverse function.

(16) On the seventh day the eight filaments reach about
half way down the free edges of their respective mesen-
teries.

(17) Further development consists of elaboration of the
organs already present.

(18) At the end of the third week the first bud is formed,
and the solitary polyp becomes a young colony by stolonal
gemmation.

(19) The young colonies were successfully fed in the labor-
atory on larvae and adult individuals from colonies of Lepto-
clinum and Botryllus.

15. Literature.

1. Bourne, G-. C. — “ The Calcareous Skeleton of the Antliozoa,”

‘ Quart. Journ. Micr. Sci.,’ vol. 41, 1898-1899.

2. Hickson, S. J. — “ Alcyonium,” ‘ L.M.B.C. Memoirs,’ No. v, 1901.

3. “The Anatomy of Alcyonium digitatum,” ‘Quart.

Journ. Micr. Sci.,’ vol. 37, new series, 1894-5.

88 ANNIE MATTHEWS.

Sa. Hicksou, S. J. — “On the Ciliated Groove (Siphonoglyphe) in the
Stomodseum of the Alcyonarians,” ‘Phil. Trans, of the Royal
Society,’ part iii, 1883.

4. “Report on Mr. Wadsworth’s Collection of Material for

the Study of the Embryology of Alcyonium,” Section D,

‘ Report British Association, Bristol,’ 1898, p. 585.

4a. — “ Coelenterata and Ctenopbora,” ‘Cambridge Natural

History,’ vol. i.

5, Hill, M. D. — “Maturation of the Ovum of Alcyonium

digitatum,” ‘Quart. Journ. Micr. Sci.,’ vol. 49, 1905-6.

0. Hoffman und Schwalbe. — ‘ Jahresbericht Anat. u. Physiol.,’ Bd. ii,
1875.

7. Korschelt and Heider. — ‘ Text-book of the Embryology of the

Invertebrates,’ part i, 1895.

8. Kowalevsky, A. — “ Untersuchungen liber die Entwickelung der

Coelenteraten,” ‘ Nachrichten der Kaiserl. Gesellschaft. der
Freunde der Naturkentniss der Anthropologie und Ethnographie,’
Moscow, 1873-4. [In Russian ; the figs, can best be followed by
using them together with paper (6).]

9. et Marion, A. F. — “ Documents pour l’histoire embryogenique

des Alcyonaires,” Memoire iv, 1883 ; ‘ Annales du Musee

d’Histoire Naturelle de Marseille. — Zoologie,’ tome i, part ii.

10. de Lacaze-Duthiers, H. — “ Developpement des Coralliaires,” Deux-

ieme Memoire, ‘Archives de Zool. Exp. et Gen.,’ tome ii, 1873.

11. ‘ Histoire Naturelle du Corail,’ Paris, 1864.

12. Pratt, Edith M. — “ The Digestive Organs of the Alcyonaria, and

their Relation to the Mesogleal Cell Plexus,” ‘Quart. Journ.
Micr. Sci.,’ vol. 49, 1905-6.

13. Stanley- Gardiner, J. — “ The Anatomy of a supposed New Species

of Coenopsammia from Lifu,” ‘Willey’s Zool. Results,’ part iv,
Camb. Univ. Press, 1899.

14. “Flabellum, Anatomy and Development,” ‘Marine Invest.

S. Africa,’ Cape Town, 1902.

15. Wilson, H. Y. — “On the Development of Manicina areolata.”

‘ Journ. Morph.’ vol. ii, 1888-9.

10. Wilson, E. B. — “The Development of Renilla,” ‘Phil. Trans, of

the Royal Society,’ part iii, 1883.

17. “ The Mesenterial Filaments of the Alcyonaria,” ‘ Mitteilun-

gen aus der Zoologischen Station zu Neapel,’ Leipzig, Bd. v,
1884.

THE DEVELOPMENT OF ALOYONIUM DIGITATUM. 89

18. Woodlapd, W.— “ Studies in Spicule Formation, II,” ‘Quart. Journ.
Micr. Sci.,’ vol. 49, 1905-6.

EXPLANATION OF PLATES 3-5,

Illustrating Mrs. Annie Matthews’s paper on “The Develop-
ment of Alcyoniuin cligitatum, with some notes on
the Early Colony Formation.”

Reference Letters of Plate Figures.

a. Radial vertical section of very young mesentery, before it projects
into the coelenteron. av First developed hud of colony. A. B. Fixed
base of polyp. An. Anterior aboral pole. b. Small blastomere. bY.
Radial vertical section of mesentery, rather older than stage (a.) of
same text-fig., and stretching higher up lateral wall of polyp. bn. Second
bud. B. Large blastomere. B. Sp. Spicules of body wall. c. Mesentery:
somewhat older than stage (b x.) of Text-fig. 40. cv Third bud. C. Ccelen-
teron. C. End. Columnar endoderm. C. M. Undivided central part of egg.
C. P. Club-shaped processes of endoderm. Col. E. Columnar ectoderm.
Col. E. C. Columnar ectoderm cell. Con. Ect. Contracted and wrinkled
ectoderm. Ci. Cilia, d. Mesentery at a stage rather older than (c.)
of the same figure, d. Ect. Deep columnar ectoderm in stomodseum.
d. m. Dorsal mesenteries. D. A. Deeply staining, finely granular area
round nucleus. D. M. Dorsal mesentery. D. M. F. Dorsal mesenteric
filament. I). N. Delamination spindle. D. 0. Dorsal filament just
arising as downgrowth of ectoderm of stomodseum over free edge of
mesentery. E. Mes. Earlier secreted mesoglea. E. S. 0. Early stage
of formation of ventral mesenteric filament, showing ectodermic band
growing from stomodseum over edge of mesentery (Mes.). Ect. Ecto-
derm. Ect. M. F. Ectodermic part of mesenteric filament. Ect. d.
Deep columnar ectoderm at base of lateral wall. Ect. C. Ectoderm
cells. End. Endoderm. Endv Endoderm cells, one row deep in this
region. End. C. Contracted endoderm. End. M. Endoderm, several
rows deep, lining attached base of polyp. End. M. F. Endodermic part
of mesenteric filament. End. S. Endoderm cells in region where they are
several rows deep. /. Radial vertical section of mesentery, at time when
stomodseal invagination is forming. F. First row of buds. G. Glass.
G. P. Granular protoplasm. Gr. Granular, yolk free area. Gr. cell
Group of cells becoming embedded in mesogloea. Gr. E. Granular ecto-
derm. Gr. Gld. C. Granular gland cell. I. C. Interstitial cells. I. C. S.

•90

ANNIE MATTHEWS.

Interstitial cells cut off singly from octoderm by mesogloea. 1. Small lobe.

L. Large lobe. L. J. Junction of supporting lamella of mesentery with
that of body wall. Lx Wv Somewhat contracted and therefore wrinkled
lateral wall of polyp. L. W. Lateral wall of polyp. mv Supporting
lamella of mesentery growing radially inward from lamella of body
wall. M. Mouth. M. B. Base of mesentery. M. C. Mucous cell.

M. F. Mesenteric filament. M. P. Adhesive plug of white mucus,
attaching base of polyp to substratum. Mes. Mesentery. Mes3, Mess,
Two lateral mesenteries. Mes. E. Edge of mesentery before ventral
filament developed. Mg. Mesogloea. Mg±. Mesogloea of mesentery.
Mgn. Mesogloea flowing between interstitial ectoderm cells at root of
mesentery. Mg. Tli. Thickened ridge of mesogloea stiffening the line
of attachment of mesentery to base of polyp. N. Nucleus halved
by karyokinesis. N«. One of daughter nuclei, formed before first
segmentation. N3. Nucleus. N. T. Nematocyst thread. Ne. Nematocyst.
Nev Nematocyst, in mesogloea of mesentery. Ne4. Nematocyst, with
thread stained. 0. P. Oral pole. 0. S. Oral surface, surrounded by
tentacles. P. Pinnule. Pr Earliest developed pinnule on the tentacle.
P2. Latest and youngest pinnule on the tentacle. P. C. Pre-oral
cavity. P. M. One of eight permanent mesenteries. Pa. Parent
polyp. PI. Remains of degenerate base of stomodseal invagination.

R. C. Rounded cells in ectoderm. R. E. Rapidly proliferating and
therefore temporarily multilayered ectoderm. R. Mes. Recently
secreted mesogloea. R. Sc. Remains of scleroblast. S. Stomodseum.
Sc. Scleroblast. Se. Second row of buds. Su. Substratum. St.
Stolon. Sp. Spicule. Spv Spicule embedded in mesogloea. Spb>
Spiculoblast with spicule dissolved out. Spbv Spicule left in scleroblast,
embedded in mesogloea. S. cell. Cell embedded singly in mesogloea.
Sub. M. Subsidiary mesentery. S. B. Termination of wall of stomodseum.

S. C. Segmentation cavity. £?. L. Supporting lamella. S. M. Separating
membrane. S4 Mv Position occupied by supporting membrane
in mesogloea of body wall. S. Mg. Mesogloea streaming out from
endoderm among rounded cells at base of ectoderm. S. 0. S. Sloping
oral surface. S. 0. Early stage in appearance of strap-like outgrowth
of ectoderm of stomodseum, over endodermic part of ventral mesenteric
filament. T. Tentacle. T. B. Swollen base of tentacle. Tl Bv Very
young bud of the third row. T. R. Tentacles retracted. T. Sp.
Tentacular spicules. V. M. Yentral mesentery. V. M. F. One of the
six ventral mesenteric filaments. V. M. F1. Lower limit of ventral
mesenteric filament. Wr. Wrinkled outline of latest pre-planula stage.
Y. Yolk globules. Yv Small yolk globules. Y2. Large yolk globules.
Y. D. Yolky detritus. Y. E. Inner half of columnar ectoderm cells,
.still containing yolk globules. Y. V. Large yolk-containing vacuoles.

THE DEVELOPMENT OF ALCYON1UM DIGITATUM.

91

PLATE 3.

Fig. 1. — Sagittal section of long highly contractile planula, towards
end of free- swimming life. Yolky detritus still present in coelenteron.
Ectoderm very full of mucous cells and nematocysts. X 78.

Fig. 2. — Portion of ectoderm of full-grown planula, showing mucous
cells and nematocysts among the ordinary cells. Granular reticulate
protoplasm in mucous cells very deeply stained, x 507.

Fig. 3. — Lateral view of polyp with older mesenteries than fig. 4.
Second day of fixation. Adhesive plug shows well. X 16.

Fig. 4. — Lateral view of polyp during early part of second day of
attachment. Eight mesenteries appearing, x 16.

Fig. 5. — Lateral view of polyp on the fifth day of attachment.
Tentacles partly contracted, body expanded. X 37.

Fig. 6. — Oral view of young polyp, with tentacles well expanded and
mouth widely opened. The tentacles were still reddish-yellow and the
body wall cream ; spicules abundant (about five days fixed). X 35.

Fig. 7. — Oral view of polyp about the sixth day. Tentacles partly
contracted, but mouth still exposed, x 39.

Fig. 8. — Section through the eight-lobed egg of Text-fig. 30. X 78.

Fig. 9. — Sagittal section through eight cell stage. One of the several
nuclei that have again divided ready for the next segmentation is
shown at N. X 78.

Fig. 10. — Section through an egg protruding sixteen lobes ; two
daughter nuclei are drawn, x 78.

Fig. 11. — Sagittal section through sixteen cell stage, showing de-
lamination spindle. X 78.

Fig. 12. — Transverse section of a young morula showing delamination
and ordinary spindles. X 78.

Fig. 13. — Enlarged drawing of section of two blastomeres from the
sixteen cell stage. The finely granular outer area, and large and small
yolk globules are shown, x 133.

Fig. 14. — Five endoderm cells from a late pre-planula, in which the
lobes are almost withdrawn again. The yolk globules are few and very
large and the segmentation cavity has disappeared. X 760.

Fig. 15. — Ectoderm and endoderm cells from transverse section of a
late pre-planula, when the ectoderm cells have become much longer
nd narrower, while large vacuoles are still present. X 78.

Fig. 16. Transverse section of a very late pre-planula, with rapidly
proliferating ectoderm (stage of Text-fig. 3), just before it passes into

92

ANNIE MATTHEWS.

the early round planula stage. A definite membrane is now present
between the ectoderm and the endoderm. X 78.

Fig. 17. — Sagittal section of smooth oval planula, with broadened
anterior end. Endoderm cells in places only one row deep, but in
others form club-like processes. (Second free-swimming day.) x 133.

Fig. 18. — Ectoderm and endoderm cells from a somewhat younger
pre-planula than PI. 3, fig. 14, and at a later stage than Text-fig. 2.
Yolk globules fewer than in early pre-planula and increasing in size.
X 760.

Fig. 19. — Part of a vertical section of a young polyp with mesenteries
and spicules. One mesentery is cut through vertically near its attach-
ment to the lateral wall. Many scleroblasts and nematocysts are
thus cut across, which have been drawn into the mesoglcea of the
mesentery here. At the lower right-hand corner young scleroblasts
and nematocyst cells are shown in situ at the base of the columnar
ectoderm, x 380.

Fig. 20. — Ectoderm and endoderm cells from a late morula stage,
x 133.

Fig. 21. — Transverse section of an earlier morula stage than fig. 20,
showing large yolk globules in the endoderm and smaller ones in the
cuboid ectoderm. X 167.

Fig. 22. — Nematocysts (oval bodies of Hickson), from mesoglcea of
solitary and colonial polyps.

PLATE 4.

Fig. 23. — Sagittal section of a polyp about stage shown in fig. 3.
This section does not cut through the mesenteries. X 133.

Fig. 24. — Sagittal section of newly-fixed larva, still planula-shaped
and ciliate. At the base the outer edge of the ectoderm has been
diagrammatically thickened in the drawing, to indicate clearly the
extent of the fixed area. X 133.

Fig. 25. — Vertical section of polyp with only one pinnule on tentacles,
i.e. about six days fixed. At Mes2 and Mes4 the basal attachments
of the two lateral mesenteries Mes3 and Mesd are seen. At V. 31. Fx
is shown the termination of the ectodermic outgrowth forming the
mesenteric filament of mesentery 31es3, i.e. about half way down the
free edge of the mesentery. At S. 0. the continuity of the stomodaBum
and this outgrowth is shown. X 133.

Fig. 26. — Vertical section of a polyp fixed from six to seven days,
showing the strap-like ectodermic process on the right, which forms
the ventral mesenteric filament, and on the left the shorter ectodermic

THE DEVELOPMENT OF ALCYONIUM DIGIT ATUM.

93

outgrowth over the free edge of the dorsal mesentery, which forms
the dorsal mesenteric filament, D. 0. The dorsal mesentery is only
partly shown at D. M. x 133.

Fig. 27. — Part of ectoderm and endoderm of swimming planula.
Multilayered and adjacent one-layered endoderm both shown. Granular
ectoderm cells expanded at base rest on the separating membrane.
Round interstitial ectoderm cells seen. Endoderm still full of yolk
globules. X 760. (Ectoderm and endoderm of the pre-planula in the
latest stage are very similar in detail.)

Fig. 28. — Vertical section of polyp showing stomodseal invagination.
There is as yet no communication with the exterior. Yolky detritus
is still present, and mesoglea is flowing between the ectoderm cells of
the attached base, and round the inner ectoderm cells of the lateral
walls. X 133. (The stomodseal invagination is cut across laterally,
and so is very narrow.)

Fig. 29. — Transverse section of polyp settled from thirteen to fifteen
days, cut below stomodseum ; the six ventral and two dorsal mesenteric
filaments are shown. X 133.

Fig. 30. — Section through the mesogloea of a fairly old polyp after
dissolving the calcareous part of the spicules by staining with picro-
nigrosin. The nuclei and organic remains of the spicules are shown,
and it can be seen that the cavities occupied in the mesoglea by the
spicules are replicas of the latter, x 245.

Fig. 31. — Young spicules in scleroblasts, from solitary polyp. X 760.

Fig. 32. — Young scleroblasts before secretion of spicule. X 760.

Fig. 33. — Vertical section through the stomodseum of a polyp five
days fixed. The remains of the degenerate base of the stomodseum
are still visible, and the ectoderm of the stomodseum is growing down
over a mesentery as a strap-like process (E. S. 0 .) ; the section is
beyond the actual mouth opening. The rest of the mesentery ( Mes .)
was seen in subsequent sections. X 450.

Fig. 34. — Transverse section of a polyp settled from thirteen to
fifteen days, cutting through the siplionoglyph. x 133.

PLATE 5.

Figs. 35, 36, 37 and 39. — Vertical radial sections of part of wall of
young settled polyp, showing ectoderm, endoderm and origin of
mesoglcea :

Fig. 35. — Mesogloea secreted by endoderm, beginning to stream
between the interstitial cells at the base of the ectoderm, at the time
of the early formation of the mesenteries. X 380.

94

ANNIE MATTHEWS.

Fig. 36. — Streams of mesogloea flowing between ectodermic interstitial
cells, and cutting them off singly ( Sjob .), or in groups ( Gr . cell), x 760.

Fig. 37. — Scleroblasts surrounded by mesogloea. (The spicules have
been dissolved during preservation.) The mesogloea is seen streaming
round the interstitial cells at the left hand of the diagram, x 760.

Fig. 38. — Part of a transverse section of a young polyp, cutting
through stomodseum and mesenteries, and showing the thin sheets of
mesogloea, devoid of cells which together with the surrounding endo-
derm form the mesenteries, x 380.

Fig. 39. — Faintly staining streams of mesogloea, flowing in between
the newly formed interstitial cells, from the endoderm. X 760.

Fig. 40. — Vertical section of part of attached base of young polyp.
The mesogloea is shown streaming in between the ectoderm cells, and
then strengthening the mucous plug, x 608.

LABIAL CARTILAGES OF RATA CLAVATA.

95-

The So-called Labial Cartilages of Raia
clavata.

Bj

Edward Pholps Allis, junr.,

Menton, France.

With Plate 6.

Gegenbaur (1872), in his classical work on “ Das Kopfskelet
der Selachier,” describes (1. c., p. 216), in Raia (species not
given) and Raia vomer, two cartilages which he considers
to be the homologues of the labial cartilages of Selachii. In
Raia (species not given), which is the one first described, one
of these two cartilages is shown lying definitely nearer the
anterior end of the ventral surface of the snout than the
other cartilage, and, doubtless because of this, the former
cartilage is called the anterior upper labial and the other
the posterior upper labial. The so-called anterior labial lies,'
however, farther from the symphysis of the upper jaw and
farther from the upper edge of the mouth than the so-called
posterior one, and if the mouth were terminal it would be
the posterior instead of the anterior cartilage. Doubtless
because of this, Gegenbaur says (l.c., p. 218) that it is
evident that the so-called anterior upper labial of the Batoidei
corresponds to the posterior upper labial of the Selachii, and
the posterior upper labial of the Batoidei to the anterior
upper labial of the Selachii. The posterior (oral) edge of the
posterior labial is shown (1. c., PI. 17, fig. 1) in contact, its
full length, with the palatoquadrate; its anterior (aboral) edge
is said to be bound to the posterior (oral) edge of the anterior

96

EDWARD PHELPS ALLTS.

labial; and the lateral (absymphysial) end of the latter cartilage
is said to be in contact with the palatoquadrate. There is
accordingly no space either between the adjoining edges of
the two cartilages, or between their posterior (oral) edges and
the palatoquadrate, and the nasal groove (Nasenrinne) must
accordingly lie either wholly lateral (absymphysial) to both
labials or external to them.

In Raia vomer the posterior labial is said to be overlapped
externally, in its middle portion, by the broad lateral (absym-
physial) portion of the anterior labial, the latter labial thus
not here having the markedly anterior and aboral relations to
the other labial that it has in Raia (spe'cies not given). It
however has the same absymphysial relations to that cartilage.
The nasal g'roove (Nasenrinne) is apparently shown (l.c.,
PI. 16, fig. 7) lying between the lateral (absymphysial) ends
of the two labials, but it is said that both labials lie, in part,
in the nasal flap, and hence necessarily external to the nasal
groove, as will be later fully explained. The anterior labial
is referred to, both in the figure and in the text, by the index
letter L , while in Raia (species not given) that letter refers
to the posterior labial.

In Rhinoptera, Gegenbaur says (1. c., p. 219) that there are,
in addition to a cartilage that corresponds strictly to the so-
called anterior upper labial of Raia, two small cartilages
found near the angle of the gape of the mouth which together
form a rudimentary labial arch similar to the arch formed
by the posterior upper and the single lower labials of the
Selachii. The presence of this posterior pair of labials in
Rhinoptera is said by Gegenbaur to definitely confirm his
already expressed conclusion that the other labial of Rhino-
ptera must be the anterior upper one. But it also evidently
proves, if correct, that the so-called anterior upper labial of
Rhinoptera, and hence also the corresponding labial of Raia,
must be the homologue of the similarly named labial of the
Selachii and not of the posterior one; which is in direct con-
tradiction to the positive statement made on p. 218 of his
work and already above referred to.

97

LABIAL CARTILAGES OF RAIA CLAVATA.

On a still later page of his work (l.c., p. 222) Gegenbaur
says that the first (anterior) labial of selachians corresponds
to the premaxillary bone of teleosts and the second (posterior)
labial to the maxillary bone of those fishes, and as he includes
both the Selachii and the Batoidei in the term selachians
(Selachier), and as he makes no qualification whatever of the
statement, it evidently implies that the so-called anterior and
posterior labials of both these sub-orders of the Plagiostomi
are homologous, which is again in direct contradiction to his
positive statement made on p. 218.

T. J. Parker (1884) gives a figure of these labials in Raia
nasuta which somewhat resembles Gegenbaur’ s figure of
them in Raia vomer, but the so-called first labial of Parker’s
descriptions, which is Gegenbaur’s anterior labial, is so long
that it crosses the opening of the mouth and overlaps ex-
ternally the mandible. This labial is said to support the
•corresponding flap of the fronto-nasal process, while the
second labial, Gegenbaur’s posterior one, is said to lie in a
fold of skin external to the naso-buccal groove. But as there
is no fold of skin external to the naso-buccal groove excepting
only the flap of the fronto-nasal process, this labial is thus
here said to also lie in that flap.

W. K. Parker (1878) also gives figures and descriptions of
these labials in Raia maculat a and Raia clavata, but they
differ so radically from Gegenbaur’s and T. J. Parker’s figures
and descriptions that there is no possibility of comparison.

These several descriptions of the labials of the Batoidei are
accordingly not clear, and I have, in connection with my
-present work on the cranial anatomy of Chlamydoselachus,
-examined these cartilages in such specimens of these fishes as
I happened to have at my disposal. These specimens con-
sisted of a single head of Raia clavata, two small speci-
mens of Raia, r a di ata, two small specimens of Myliobatis,
and two partly dissected specimens of Torpedo ocellata.
The head of Raia clavata was a fresh one, while all the
other specimens had been long preserved in alcohol, and were
-not in good condition for this particular dissection.

VOL. 62, PART 1 . NEW SERIES.

7

98

EDWARD PHELPS ALLIS.

In Rai a clay at a (3?]. 6, figs. 1-3) tlie posterior (oral) edge
of the nasal flap of either side occupies about two-fifths of the
distance from the angle of the gape to the symphysis of
the upper jaw, and it covers a depressed region that will be
referred to, in its entirety, as the nasal-flap furrow. The term
nasal groove is avoided, because that term (Nasenrinne), as
employed by Gegenbaur, would seem to refer to a lateral and
deeper portion, only, of the entire furrow, as will be later
fully explained. Between the nasal flaps of opposite sides
the edge of the upper lip of the fish is deeply re-entrant,
exposing the teeth and a considerable portion of the upper jaw.
A well-marked furrow separates this part of the upper lip
from the underlying upper jaw, and may be called the upper
labial sulcus. Laterally, on either side, this sulcus runs into
the mesial (symphysial) edge of the corresponding nasal-flap
furrow, and the posterior (oral) edge of the nasal flap of
either side accordingly appears as a direct continuation of the
upper lip. It is, however, not a continuation of that lip, the
lip and its related sulcus being prolonged a certain distance
along the floor of the nasal-flap furrow, internal to the nasal
flap, and there gradually vanishing, as shown in PI. 6, fig. 2.

In the nasal flap, occupying approximately its entire-
posterior (oral) half and extending mesially (symphysially)
somewhat beyond the base of the flap into the subdermal
tissues between the upper lip and the nasal capsule, lies the
cartilage called by Gegenbaur the anterior upper labial.
This so-called anterior upper labial of my specimen, like the
corresponding one in Gegenbaur’s figure of Rai a vomer,
lies external to the so-called posterior upper labial and farther
from the symphysis of the upper jaw than that labial, but
not definitely anterior to it. It is, however, not a labial
cartilage, as will be later shown, but a cartilage developed
strictly in supporting relations to the nasal flap. It can
accordingly be called the nasal-flap cartilage, which will
sufficiently distinguish it from the Nasenfliigelknorpel of
Gegenbaur’s descriptions, which latter cartilage also in part
supports the nasal flap and is the ala nasalis of certain.

LABIAL CARTILAGES OF RAIA CLAVATA.

99'

English authors. J. Muller (1834) also did not consider this
nasal-flap cartilage to be a labial cartilage, and he called it
the inner Nasenflugelknorpel.

This nasal-flap cartilage of Eaia clavata has the shape
shown in the accompanying figures, and it is connected with
what Gegenbaur calls the anterior process of the correspond-
ing ala nasalis by the dermal and connective tissues of tho
nasal flap. At its anteror (aboral) corner there is, on
one side of the head of my one specimen, a small and
independent bit of cartilage. In about the middle of the
postero-mesial edge of the cartilage there is a curved
incisure, and slightly lateral to the bottom of this incisure,,
and parallel to the edge of the cartilage, there is a ridge on
the internal surface of the cartilage. The mesial surface of
this ridge is flat and slopes gradually to the edge of tlie-
cartilage, and, on either side of the incisure, it rests upon and
is firmly bound to the posterior upper labial of GegenbauEs
descriptions, this contact with the latter labial being
particularly large and strong anterior (aboral) to the incisure.
The lateral (absymphysial) surface of the ridge is abrupt
and curved, and forms the mesial (symphysial) boundary
of the nasal-flap furrow, thus marking the base of the nasal
flap. The nasal-flap cartilage thus extends mesially beyond
the base of the nasal flap into the general tissues on the
ventral surface of the head, but it in no place reaches or
touches the palato-quadrate, being everywhere separated
from it either by the so-called posterior labial cartilage or
by the nasal-flap furrow. The incisure in the postero-mesial
edge of the nasal-flap cartilage arches over the posterior
(oral) end of a short section of the nasal latero-sensory canal
(Garman, 1888) that is directed antero-posteriorly.

The posterior upper labial of Gegenbaur's descriptions
above referred to, is the only labial found in either Eaia
clavata or Eaia radiata, and it will accordingly be called,,
in the following descriptions, the upper labial, or simply the
labial. It consists of two broad and approximately parallel
portions connected by a narrow neck of cartilage which

100

EDWARD PHELPS ALLIS.

extends from the middle of the mesial portion to the mesial
end of the lateral portion. The mesial portion lies quite
closely upon the palatoquadrate, but is separated from it by
Tranches of the nervus trigeminus and other tissues. Its
mesial end projects antero-mesially beyond the palatoquadrate.
The antero-mesial end of the lateral portion of the labial lies
against the posterior surface of the nasal capsule, the
remaining portion lying external to the muscles of the
region, but separated from them by tough connective tissues,
the cartilage being bent in conformity with the shape of the
underlying structures. The postero-lateral portion of this
lateral portion of the labial lies immediately beneath the
external dermis and parallel with it, and its posterior end
lies at a considerable distance from the angle of the gape,
separated from that angle by the bulging muscles of the
region. The connective tissues in which it lies are attached
to it, but it cannot be said that the cartilage runs gradually
into ligamentous tissues that are continued into the mandible,
as Gegenbaur says is the case in Raia vomer.

The narrow neck of cartilage that connects the mesial and
lateral portions of the labial lies in the hollow between the
aboral edge of the palatoquadrate and the posterior wall of
the nasal capsule, and it is always somewhat bent or twisted.
In my specimens of Raia radiata this twist is so pronounced
that the primarily posterior (oral) edge of the cartilage is
presented ventrally, the cartilage thus here lying, as Gegen-
Tauer has said for Raia vomer, in a vertical position. The
primarily external surface of the neck was thus, in these
specimens, presented anteriorly instead of ventrally, and the
nasal-flap furrow, having crossed the primarily posterior
(oral) edge of the labial, had, anterior (aboral) to that edge,
somewhat the appearance of lying on the internal rather
than the external surface of the labial.

There were, in my specimen of Raia clavata, no special
ligamejitous attachments of the mesial (sympliysial) end of
either the labial cartilage or the nasal-flap cartilage to the
ventral surface of the rostrum, such as Gegenbaur describes

LABIAL CARTILAGES OF RAIA CLAYATA.

101

in Raia vomer; this end of the labial of Raia clavata
simply lying in dense connective tissues of the region, and
the corresponding end of the nasal-flap cartilage lying upon
and being firmly bound to it. On each side of the head the
ventral edge of the posterior wall of the nasal capsule was
partly membranous, and in this membrane there was a narrow
and independent strip of cartilage. In the mesial wall of the
capsule, near its ventral edge, there was a hiatus closed by
membrane. One or more branches of the nervus trigeminus
perforated the cartilage between the hiatus and the edge of
the capsule.

The nasal latero-sensory canal was so named by G-armaii
(1888) in his descriptions of these fishes, and is the antorbital
portion of the main infraorbital canal of my descriptions of
Mustelus (Allis, 1901). Running mesial ly across the external
surface of the musculi adductor mandibulae and levator labii
superioris, this canal reaches the lateral edge of the lateral
portion of the labial cartilage immediately anterior to the
point where that cartilage assumes a position parallel to the
external surface of the head. The canal then crosses the
external surface of this portion of the labial and reaches its
mesial edge, where it continues onward and reaches and
traverses the narrow neck of cartilage that connects this
lateral portion of the labial with its mesial portion. Having
reached the mesial portion of the labial the canal turns
abruptly posteriorly (orally) and crosses the external, surface
of this part of the labial, lying close to its lateral edge.
When the canal reaches the postero-mesial edge of the labial
it traverses the incisure in the mesial edge of the nasal-flap
cartilage and then turns abruptly antero-mesially along the
postero-mesial edge of the labial ; and continuing in that
direction it joins its fellow of the opposite side in the median
line to form the median canal of Harman’s descriptions.
The canal lies internal to the nasal-flap furrow, and internal
also to the nasal-flap cartilage, and in no part of its course
does it enter any part of the nasal flap. It lies everywhere
external to the labial cartilage and is firmly attached to

102

EDWARD PHELrS ALLIS.

that cartilage, but, excepting where it crosses the mesial
portion of the labial, there is no noticeable groove to mark
its course. In my specimens of Raia radiata, because
of the marked twist in the neck of the labial, the canal
there has markedly the appearance of lying on the internal
rather than on the external surface of the labial. In Raia
cl a vat a some of those branches of the nervus buccalis
lateralis that innervate the organs of the canal perforate
the labial, but most of them pass over the anterior (aboral)
edge of the labial and then turn posteriorly (orally) across
its external surface. They always lie internal to the nasal-
flap cartilage.

In Raia batis Ewart (1892) shows two loops in the nasal
latero-sensory canal. No such loops were found in Raia
clavata, and it is probable that they are exaggerated in
Ewart’s figure, the loops simply representing points where
the canal follows bends or twists in the labial such as I find
in Raia radiata.

In my specimens of Myliobatis the nasal-flap furrows are
so wide (deep) that they nearly meet in the median line, a
narrow “frenulum” (Gegenbaur) there alone separating them.
In correlation with this extension of the nasal-flap furrows
the nasal-flap cartilages have been carried toward the median
]ine, and are there separated from each other by only a
narrow space in which lies the small median bit of cartilage
that Muller (1834) describes as the “ Trager der Nasen-
fliigelknorpel.” The nasal-flap cartilage, called by Muller the
inner Nasenfliigelknorpel, has the triangular shape shown by
that author in his figure of Myliobatis aquila (1. c. PI. 9,
fig. 13), bufc it is more deeply fimbricated in my specimens
than shown by Muller. The ala nasalis is as shown in Muller’s
figure. The nasal-flap furrow lies internal to both these
cartilages. The nasal latero-sensory canal runs internal to
the nasal- flap furrow, and then outward and forward (aborally)
in the frenulum to meet and fuse, in the median line, with its
fellow of the opposite side. In one of my specimens the canal
is enclosed in the ventral edge of a strip of cartilage that has

LABIAL CARTILAGES OF RAIA CLAVATA.

103

somewhat the position of the narrow strip found along the
ventral edge of the posterior wall of the nasal capsule in
Raia cl a vat a, and already described. In the other speci-
men the canal is enclosed in an independent tubule of tissue
that has a semicartilaginous appearance. In Trygon
tuberculata Gregenbaur describes (l.c., p. 220) and figures
what would seem to be a strictly similar tubule, but it is said
by him to be a rod; and although he says that this so-called
rod is of fibro-cartilage, he nevertheless considers it to be the
homologue of the anterior upper labial of his own descriptions
of Raia and Myliobatis, which latter labial is said to be of
hyaline cartilage. The lateral portion of this tubular or rod-
like cartilage of Trygon is shown, in Gfegenbaur’s figure,
lying definitely internal to the ala nasalis, and it seems as if it
must accordingly lie, as does the latero-sensory tubule in my
specimen of Myliobatis, internal also to the nasal-flap furrow.
If such be the case it cannot be a nasal-flap cartilage, or
so-called anterior labial, of this fish. It probably contains,
in both Trygon and Myliobatis, a remnant of the upper
labial of the present descriptions. If not, then that upper
labial is entirely wanting in my specimens of Myliobatis, as it
was in those examined by Gregenbaur. The cartilage described
by Gregenbaur, in Myliobatis, as the posterior upper labial I
could not find in my specimens.

In Torpedo ocellata I find the nasal flap much less long,
antero-posteriorly, than the flap of Myliobatis, this being due,
as Gregenbaur has said, to the nasal capsules lying nearer the
anterior edge of the mouth. The nasal flap is supported by
a marked prolongation and development of the ala nasalis,
similar to the prolongation of that cartilage in Myliobatis,
but there is no indication of a separate nasal-flap cartilage.
The frenulum is supported by a small median Trager der
Nasenfliigelknorpel, as in Myliobatis, and the posterior (oral)
end of this little cartilage is strongly attached by connective
tissues to the adjoining mesial (symphysial) ends of the
palatoquadrates of opposite sides. No upper labial cartilage
was found, and a cord of connective tissue lying internal

104

EDWARD PHELPS ALLIS.

to the nasal-flap furrow alone represented the aborted nasal
latero-sensory canal.

Certain of Gegenbaur* s statements regarding the labials of
the Raiidge and their relations to the nasal flap may now be
considered. In Raia (species not given), Gegenbaur says, as
already stated, that the oral edge of the anterior labial is in
contact with and bound to the aboral edge of the posterior
labial, and that the lateral (absymphysial) end of the anterior
labial is in contact with the palatoquadrate. Of Raia vomer
he says (l.c., p. 216), that the posterior labial, where it
bends posteriorly along the external surface of the musculi
adductor mandibulge and levator labii superioris, “ gelangt
dadurcli mit seiner Flaclie an die hintere resp. obere Flaclie
des vorderen Knorpels, den er mit seinem Endabschnitte
seitlich iiberragt.” This statement certainly implies that the
lateral ends of the two so-called labials of Raia vomer are
in contact, as they had previously been said to be in Raia
(species not given), but the figure given of Raia vomer
apparently shows the nasal-flap furrow (Nasenrinne) lying'
between them; and it certainly shows the lateral end
of the posterior labial lying internal to the nasal furrow.
On p. 219 Gegenbaur says: “Wenn wir bei Raja erfahren,
dass Labialknorpel in die mit der Bildung der Nasenfurche
zusammenhangende Nasenklappe gelangen, in deren nicht
bedeutende seitlich e Zipfel sie einragen, so folgt daraus,
dass bei einer medialen Yerbreiterung des labialen Endes
der Furche die Labialknorpel von ihrer Lagerung vor
dem Oberkieferknorpel gelost werdenniiissen. Indem die
lateral en Zipfel der Klappe auf eine grossere Strecke hin
von der Unterflache des Kopfes sich trennen, kommen die
Labialknorpel mehr oder minder vollstandig in die Klappe
zu liegen. . . . Je mehr die beiderseitigen Nasenklappen

gegen die Medianlinie zu frei werden, um so mehr werden
die Labialknorpel in sie eintreten.” And on p. 226 he
says: “Die beiden oberen Labialknorpel kommen ins Yelum
zu liegen. Der zweite obere Labialknorpel wird aber nicht
immer vollstandig vom Yelum umschlossen. Ein Theil davon

LABIAL CARTILAGES OF BAIA CLAVATA.

105

tritt manchmal lateral iiber das Velum hinaus in den Boden
der Nasenrinne, die er dann noch lateral mit begranzen hilft.
Dem Nasenvelum gehort somit streng genommen nur der eine
vordere, obere Lippenknorpel an.’5

These several statements of Gegenbaur’s certainly
definitely affirm that both the anterior and the posterior
labials of either side of Raia enter into some part of the
corresponding nasal flap, and they are apparently both
said to extend into the tip of the flap. This is, however,
evidently impossible, in so far as the so-called posterior
labial is concerned, for in both Raia clavata and Raia
radiata, which cannot differ markedly in this respect
from Raia (species not given) and Raia vomer, the
nasal-flap furrow lies definitely between the lateral portions
of the two so-called labials, and it would necessarily
continue so to lie however much the furrow might be
reduced, or be extended inesially. The mesial edge, or
bottom, of the furrow marks, or rather determines, the
base of the corresponding* nasal flap, and the so-called
posterior labial could not possibly enter any part of that
flap, as the flap is found in my specimens, nor could it
enter into a velum formed by the fusion, in the median
line, of two such flaps. Gegenbaur’s several statements,
above referred to, are accordingly certainly incorrect.

The nasal flap of Raia and the other non-electric rays is
said by Gegenbaur to be derived from the much smaller and
quite different nasal flap found in most of the Selachii, the
Scylliidae being said to present several intermediate stages in
the process of development. A nasal velum is said to be
formed in Myliobatis, in certain others of the non-electric
rays, and also in certain of the Selachii, by the fusion, in the
median line, of the nasal flaps of opposite sides. In the
electric rays the method of development (Genese) of the
velum is said (1. c., p. 221), to be totally different from
that in the non-electric rays, the inference accordingly being
that the vela in these two groups of fishes are equivalent but
not homologous structures. This will be further discussed

106

EDWARD PHELPS ALLIS.

after considering the intimately associated nasal-flap furrow
and naso-buccal groove.

The nasal-flap furrow, as I have used that term, is the entire
space that lies in the angle between the nasal flap and the
underlying external surface of the dermis of the ventral
surface of the head. The lateral (absymphysial) portion of
this space is in Paia cl a vat a deepened, and this depressed
portion, beginning immediately mesial (sympliysial) to the
angle of the gape, runs at first anteriorly (aborally) and then
turns antero-mesially to fall into the postero-mesial portion
of the nasal pit. This deepened portion of the entire furrow
thus forms a marked groove in the dorsal (internal) wall of
the furrow, and it will be referred to hereafter as the naso-
buccal groove, this term being taken from T. J. Parker’s
(1884) descriptions of this fish. Parker, however, used this
term to designate, not the naso-buccal groove alone of my
descriptions, but the entire nasal -flap furrow.

Gegenbaur says of the nasal groove of his descriptions
(l.c., p. 224): “ Durch den geschilderten Yorgang der

Yelumbildung werden nicht bloss die Nasenklappen dem
Munde genahert, sondern die von der Klappe bedeckte
Paumlichkeit dehnt sich dabei von der Nasengrube her gegen
den Mundrand zu aus und bildet eine flache oder tiefere
Pi line, die von der anderseitigen durch ein verschieden
breites Frenulum getrennt ist, oder auch dei bedeutender
Kiirze jenes Frenulums mit derselben zusammenfliesst. Eine
solche Einrichtung kann als eine Weiterbildung des bei den
zuletz aufgefuhrten Scyllien bestehenden Yerhaltens gelten.
Sie findet sich bei Chiloscyllium, ahnlich auch bei Stegostoma,
bei denen die ziemlich tiefe Rinne zum Mundwinkel lierab-
fiilirt. Entfernter vom Mundwinkel fuhrt sie bei Crossorliinus
zum Munde, indem sie den Rand der Oberlippe durchbricht.”

The “ Paumlichkeit 99 or “Rinne” here described by
Gegenbaur is evidently the entire nasal-flap furrow of ray-
descriptions, but several others of Gegenbaur’ s statements
seem quite definitely to make the term apply only to the
naso-buccal groove. In a footnote on p. 218 he says :

LABIAL CARTILAGES OF RAIA CLAVATA.

107

R Scy Ilium besitz keine Nasenfurche ” ; and this notwith-
standing that there is a .well-marked nasal-flap furrow in
•certain of the Scylliidas, and that in his own figure of
Scyllium canicula a so-called “Nasenrinne” is indicated
by index letters. On p. 217 he says: “Denkt man sich an der
Yorderflache des Oberkiefers einen sich bedeutend verbreit-
ernden Labialknorpel gelagerfc, so wird derselbe, da die
Flachenvergrosserung nicht gegen den Mundrand zu statt-
finden kann, nach vorn zu sich ausdehnen miissen und wird
mit der Bildung einer von der Nasengrube zum Mundwinkel
fuhrenden Nasenfurche median von derselbeu zu liegen
kommen.” On p. 218 he says : “ Auf die Nasenfurche lege
ich hiebei grosseres Gewicht als auf die Nasenklappe, denn
dnrch den Yerlauf der ersteren zum Mundwinkel ist die
Zutheilung der bezuglichen Knorpel zu dem zwischen beiden
Nasenfurchen gelegenen zur Nasenklappe sich differenzir-
enden Abschnitte des Integumentes zu erklaren.” And on
p. 224 he says : “ Diese Nasenrinne oder Nasenfurche erscheint
unter den Roclien allgemein verbreitet. Sehr ausgepragt ist
^ie bei den liajae,meist gerade zum Mundwinkel lierabziehend.
Durch eine mediale Yerbreiternng* erfahrt die Binne eine
Abflachung, und beiderseitige Rinnen konneu vor der Mun-
doffnung zusammenfliessen, was bei einer geringeren Ausbil-
dung des Yelums, wie z. B. bei manchen Rhinobatiden,
fast zu einem Yerschwinden der ganzen Einrichtung fiihrt.
Aus demselben Grunde ist auch bei Trygon die Rinnenbildung
schwer zu erkennen und eben so bei Myliobatis. Die ganze
Erscheinung erlangt bei diesen den liochsten Grad ihrer
Ausbildung und zwar in einem das Verlmltniss bei den Rajae
weit uberschreitenden und es damit unkentlich machenden
Masse.^

In all these several quotations the so-called nasal groove
(Nasenrinne) is evidently considered to be a groove that runs
primarily from the nasal pit to the angle of the gape of the
mouth, and the appearance of this groove is apparently
conceived to precede the differentiation of the nasal flap.
There is, however, no slightest indication in any of the many

108

EDWARD PHELPS ALLIS.

fishes described by Gegenbaur of such a groove existing
independently of the nasal flap and its related nasal-flap
furrow, and it is quite certain that the groove is simply a
secondary differentiation of the furrow. Such being the case
the nasal-flap furrows of all the Plagiostomi are strictly
homologous structures, and this is the conclusion that
Luther (1909) arrives at from physiological considerations.
A continuous nasal velum would then be formed if the
furrows of opposite sides were to coalesce in the median
line by the complete or partial breaking through of the
intervening frenulum. There is, however, no indication
whatever, in any of my specimens, that this frenulum is
ever broken through, for even in Myliobatis the oral edge
of the frenulum forms a part of the upper lip of the fish
and not a part of the nasal flap of either side. The
nasal-flap furrows of opposite sides are here certainly in
communication with each other beneath the velum, but it
is through the intermediation of the small persisting median
section of the upper labial sulcus and not because of the
coalescence of the furrows. I am accordingly convinced that
a complete velum, extending across the median line, must, if
ever found, be formed by the coalescence of the opposing-
mesial edges of the nasal flaps of opposite sides in fishes
where those flaps have been prolonged beyond the oral edge
of the upper lip ; and this would seem to be confirmed by
the conditions that I find in a small specimen of Scyllium.

In this small specimen of Scyllium, which I am quite
certain is Scyllium canicula, I find the nasal flaps of
opposite sides so much more developed than those shown in
Gegenbaur’s figure of this fish that it would seem as if the
two fishes could not be of the same species. The flaps of
opposite sides are separated by a small median incisure which
extends to the oral edge of the frenulum, that edge certainly
representing a small persisting median portion of the upper
lip. There is accordingly no complete velum in my specimen
of this fish. Such a velum would, however, be formed if the
adjoining edges of the incisure were to fuse, and this is

LABIAL CARTILAGES OP RA1 A CLAVATA.

109

apparently wliat does take place in older specimens, for
'Gunther (1870) says of this fish: “ The nasal valves confluent,
without cirrus, forming together a simple broad flap in front
of the month, the posterior edge of the flap being nearly free,
not interrupted in the middle.”

The nasal flaps of all of the Plagiostomi, whether Selachii
-or Batoidei, are accordingly simply folds of the dermal tissues
of the internasal portion of the snout, this internasal portion
of the snout being presented more or less ventrally according
to the greater or less development of the rostrum and the corre-
lated configuration of the head. If the mouth were terminal
and the nasal apertures disposed as in Amia and most of the
Teleostei, this internasal region would lie on the dorsal surface
of the snout, and the relations, anterior and posterior, would
be the reverse of what they are in Raia. In the Batoidei the
nasal flap always lies external to the nasal section of the
latero-sensory canals, and the nasal-flap cartilage, which lies
in large part in the flap, also always lies external to that
canal, and external also to the nervus buccalis lateralis. In
most Selachii the nasal flap lies wholly aboral to the nasal
latero-sensory canal, that is, on the opposite side of the canal
to the labial cartilages; but in my specimen of Scyllium it
lies external to the canal, #as it does in the Batoidei. In
Chlamydoselachus both labials lie oral to the suborbital
latero-sensory canal but internal both to the third group of
ampullae of Merritt Hawkes* (1906) descriptions and to those
branches of the buccalis that supply those ampullae; the
labials thus lying morphologically internal to the suborbital
canal. In Mustelus (Allis, 1901) the labials have similar
relations to the latero-sensory canals, ampullae, and related
nerves. In my specimen of Scyllium the anterior end of the
single upper labial (Gegenbaur, 1872) lies directly internal to
the latero-sensory canals at the point where the nasal canal
joins the suborbital canal ; and, in Stegostoma tigrinum,
Luther (1909) says that the rostral (external) surface of the
-anterior labial is grooved to lodge the nasal canal.

The so-called anterior upper labial of Gegenbaur's descrip-

110

EDWARD PHELPS ALLIS.

tions of the Batoidei, the nasal-flap cartilage of the present
descriptions, can not accordingly be the homologue of either
of the labial cartilages of that author’s descriptions of the
Selachii, and it is apparently a fibro-cartilage developed
wholly in supporting relation to the nasal flap. Sections of
it show the interior of the cartilage a mass of fibrous strings
running gradually, toward the exterior on either side, into
hyaline cartilage.

The nasal-flap cartilage of Raia thus not being the liomo-
logue of either of the labials of the Selachii, the single upper
labial of the former fish might represent either one of the
labials of the latter fishes. I am, however, strongly inclined
to believe that it represents both the labials of the latter
fishes, here secondarily connected by a narrow neck of
cartilage ; the mesial and lateral portions of the labial of
Raia representing', respectively, the anterior and posterior
labials of the Selachii. The general shape and disposition of
the cartilage favours this view, and this composition of the
labial would offer a possible explanation of the peculiar course
of the nasal latero-sensory canal. The labials, in the Selachii,.
lie either oral or internal to the nasal latero-sensory canal, as
just above explained. In Raia the labial lies in large part
aboral to the canal, and, in acquiring this position, the two
parts of which I consider the labial to be composed have
necessarily pushed against and carried with themselves those
branches of the nervus buccalis lateralis that supply the
organs of the related portion of the caual. This push on the
nerves would naturally tend to displace the caual, but
the mesial section of the canal was held in place by the
attachment of the nasal-flap cartilage to the lateral end
of the anterior labial. The lateral portion of the canal was
not so held in place, and would in consequence be carried
aborally by the pull of the nerves, and these nerves, becoming
more or less enveloped in the pushing edge of the labial,,
would be found perforating the cartilage in the adult. The
sharp bend actually found in the canal would thus be
accounted for. The relations of the nerves to the mesial

LABIAL CARTILAGES OF RAIA CLAVATA.

m

portion of the labial, in Raia, and the relations of the canal
itself to the lateral portion of the labial are both against the
view that these cartilages are developed in direct relation
to the canal, but the cartilage of Raia is nevertheless
evidently of fibro-cartilaginous origin, for sections of it
show certain fibrous strings in the interior of the cartilage.
They are, however, much less numerous than in the nasal-
flap cartilage.

Gegenbaur considered the anterior and posterior upper
labials of his descriptions of the Selachii, and their assumed
liomologues in the Batoidei, to be cartilages that served as
groundwork (Grundwerk) on which the premaxillary and
maxillary bones, respectively, of the Teleostei were developed.
My work has as yet offered nothing decisive either in favour
of or against this view, in so far as it applies to the two
labials of the Selachii and the one upper labial of the present
descriptions of Raia, but the relations of the labial of Raia to
the branches of the nervi buccalis and trigeminus favour the
view that its two portions may represent the two bones of the
Teleostei. There is, however, doubt as to which part of the
labial represents the maxillary and which the premaxillary.
The nasal-flap cartilage, Gegenbaur’s anterior labial, can not,,
however, represent either of the two bones of the Teleostei.
Its general position, in Raia, and its relations, in Myliobatis,.
to the so-called Trager der Nasenfliigelknorpel, strongly
suggest that it may represent the ascending process of the
premaxillary bone of the Teleostei, and that the Trager der
Nasenfliigelknorpel may represent the rostral cartilage of
certain of those fishes.

In two of my earlier works (1898, 1909) I came to the
conclusion that the ascending process of the premaxillary
bone of the Teleostei was primarily an independent bone,,
the so-called dermal ethmoid, which later fused with the
premaxillary. This primarily independent bone was said to
have been developed in protective relation to a line of latero-
sensory organs, and to be found as such a protective bone
not only in certain ganoids (Amia, Polypterus), but also in

112

EDWARD PHELPS ALLtS.

Elops and probably also in Belone. In certain other Teleostei
the corresponding bone, the supra-ethmoid of current descrip-
tions, was said to underlie a line of surface pit organs that
corresponded to the canal line in Aniia. This supra-ethmoid
bone was accordingly considered to be a bone of membranous
origin that represented a deeper component of the canal-
bearing bone of Amia, just as, in certain others of the canal-
bearing bones of Amia and other fishes, there is au uuderlying
membrane component apparently developed somewhat inde-
pendently of the canal-bearing component. The conditions
now found in Raia suggest that this supra-ethmoid bone
of the Teleostei is represented in the nasal-flap cartilage of
Raia. If this be so, the supra-ethmoid bone can not represent
an underlying component of a canal-bearing bone, for the
cartilage of Raia lies definitely external to the related canal.
This origin of the supra-ethmoid bone from the nasal-flap
cartilage might then account for the absence, in those fishes
in which that bone is found, of the canal line found in Amia;
for this cartilage, or bone, in sinking from the position which
it has in Raia to that which it has in the Teleostei, would
necessarily smother the uuderlying canal and ultimately lead
to its complete abortion. The line of pit organs that over-
lies the supra-ethmoid bone in certain Teleostei would then
be a secondary outgrowth from the end of the infraorbital
canal line, and hence not the homologue of the cross-commis-
sural canal line of Amia and the other fishes in which it is
found. In Amia, Polypterus, and Elops, it is to be especially
noted that the presence of a canal-bearing ethmoid bone is
associated with the relation of the maxillary bone to the pre-
maxillary that Sagemehl (1884) described as lateral, and that
is said by that author to be found in only a few of the
Teleostei. These two conditions may accordingly be related,
but the want of proper material does not at present permit me
to farther investigate it.

O

Palais de Carnoles, Menton;

January 20th, 1916.

LABIAL CARTILAGES OF RAIA CLAYATA.

113

Literature.

Allis, Edw. P., jr. (1898). — “On the Morphology of Certain of the
Bones of the Cheek and Snont of Amia calva,” ‘Journ.
Morphol.,’ vol. xiv, No. 3, Boston.

(1901). — “The Lateral Sensory Canals, the Eye-Muscles, and

the Peripheral Distribution of the Cranial Nerves of Must el us
lsevis,” ‘ Quart. Journ. Micr. Sci.,’ vol. 45, part 2.

(1909). — “ The Cranial Anatomy of the Mail-Cheeked Fishes.”

‘ Zoologica,’ Hft. 57, Bd. xxii, Lief. 3/5, Stuttgart.

Ewart, J. C., and Mitchell, J. C. (1892). — “ The Lateral Sense Organs
of Elasmobranclis. II. The Sensory Canals of the Common
Skate (Raia bat is),” ‘Trans. Boy. Soc. of Edinburgh,
vol. xxxvii, part 1 (Nos. 5 and 6), Edinburgh.

Garman, S. (1888). — “On the Lateral Canal System of the Selachia
and Holocephala,” ‘Bull. Mus. Comp. Zool. Harvard College,”
vol. xviii, No. 2.

Gegenbaur, Carl (1872). — Untersuchungen zur vergleichenden Anatomie
der Wirbelthiere,” Hft. 3, ‘Das Kopfskelet der Selachier, ein
Beitrag zur Erkenntnis der Genese des Kopfskelets der Wir-
belthiere,’ Leipzig.

Gunther, Albert (1870). — “ Catalogue of the Fishes in the British
Museum,” vol. viii, London.

Hawkes, O. A. M. (1906). — ■“ The Cranial and Spinal Nerves of
Chlamy doselachus anguineus” (Gar.), ‘Proc. Zoolog. Soc,
London for 1906,’ part 2.

Luther, Alex. (1909). — “Beitrage zur Kenntnis von Muskulatur und
Skelett des Kopfes des Haies Stegostoma tigrinum Gm.
und der Holocephalen, mit einem Anhanguber die Nasenrinne,”
‘ Acta Soc. Sc. Fennicae,’ Helsingfors.

Muller, Johannes (1834). — “ Yergleichende Anatomie der Myxinoiden
der Cyclostomen mit durchbohrtem Gaumen, Th. 1, Osteologie
und Myologie,” ‘Abh. d. Berliner Akad. d. Wiss. (Phys-math.
Abt.), Jahrg. 1834,’ Berlin, 1835.

Parker, T. J. (1884). — ‘A Course of Instruction in Zootomy,’ London.

Parker, W. K. (1878). — “ On the Structure and the Development of the
Skull in Sharks and Skates,” ‘ Trans. Zool. Soc. London,’ vol. x,
part 4, No. 1, London.

Sagemehl, M. (1884). — “Beitrage zur vergleichenden Anatomie der
Fische. II. Das Cranium der Characiniden, etc.,” * Morph.
Jahrb.’ Bd. x, Hft. 1, Leipzig.

VOL. 62, PART 1. — NEW SERIES.

8

214

EDWARD PHELPS ALLIS.

EXPLANATION OF PLATE 6,

Illustrating Mr. Edward Phelps Allis’s paper on “ The
So-called Labial Cartilages of Raia clavata.”

Index Letters.

a. n. Ala nasalis. a. n. a. Anterior nasal aperture, ant. Antorbital
cartilage (Parker), Schadelflossen-Knorpel (Gegenbaur). f.n. Fenestra
nasalis. m. mouth, md. Mandibula. n. Nasal section of latero-
sensory canal, n. b. g. Naso-buccal groove, n. c. Nasal capsule. n.f.
Nasal flap, n.f.c. Nasal-flap cartilage, n.f.f. Nasal-flap furrow.
orb. Orbital section of latero-sensory canal, pn. Pre-nasal section of
latero- sensory canal, p. n. a. Posterior nasal aperture, pq. Palato-
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CONTENTS OF No. 246-New Series.

MEMOIRS :

PAGE

On Phoronis oval is, Strethill Wright. By Sidney F. Hakmer,
Sc.D., F.B.S., Keeper of Zoology in the British Museum (Natural
History). (Published by permission of the Trustees of the British
Museum. (With Plates 7-9) . . . .115

The Embryonic Development of Trichogramma evanescens,

W estw., a Monembryonic Egg Parasite of Donacia simplex,

Fab. By J. Bronte Gatenby, Exhibitioner of Jesus College,
Oxford. (With Plates 10-12) .... 149

On the Development of the Cape Cephalodiscus (C. gilchristi.
Bidewood). By J. D. F. Gilchrist, M.A., D.Sc., Ph.D. (With
Plates 13 and 14) ..... 189

Note on the Sex of a Tadpole raised by Artificial Parthenogenesis.

By J. Bronte Gatenby, B.A., Exhibitioner of Jesus College,
Oxford. (With 5 Text-figures) .... 213

An Easy Way of Demonstrating the Nuclei of Nerve Fibres. By
Henry E. Beburn, Student of Medicine. (From the Physiological
Laboratory, King’s College, London) .... 217

On a Larval Actinian Parasitic in a Bhizostome. By C. Badham,

B.Sc., Demonstrator in, Zoology, University of Sydney. (With 3
Text-figures) . . . . . . 221

The Early Development of the Spleen of Lepidosiren and Protopterus.

By G. L. Purser, B.A. (Sometime Coutts -Trotter Student, Trinity
College, Cambridge. (With Plates 15-17) . . .231

A Note concerning the Collar Cavities of the Larval Amphioxus.

By K. M. Smith, A.B.C.S., D.I.C., and H. G. Newth, Demonstrator
in Zoology, Imperial College of Science and Technology. (With
Plate 18) . . . . . . .243

ON PHORONIS OVAL IS, STRETHILL WRIGHT.

115

On Phoronis ovalis, Strethill Wright.

By

Sidney F. Haimci’, Sc.D., I.K.S.,

Keeper of Zoology in tlie British Museum (Natural History).

{Published by permission of the Trustees of the British Museum.)

With Plates 7, 8, and 9.

Introduction.

In 1913 Miss R. E. Roper, who was working with Professor
A. Meek at the Polvzoa of the Northumberland coast, was
kind enough to send to the British Museum an empty shell
of Neptunea antiqua bearing specimens of Alcyo-
nidium mammil latum, Alder. On examining the surface
of the shell on which this Polyzoon was growing, a curiously
eroded appearance was noticed. In order to ascertain the
meaning of this appearance, a fragment of the shell was
decalcified ; and it was at once obvious that the substance of
the shell was traversed by the burrows of numerous boring
animals. A few of these belonged either to the Sponge,
Cl ion a, or to a small Poly chaste, probably Polydora
ciliata. The great majority of them belonged, however, to a
minute species of Phoronis, which has proved to correspond
closely with the description of P. ovalis given by Strethill
Wright (18561, 18562) in the papers in which the genus
Phoronis was established. The Neptunea which is here
considered was obtained to the south-east of St. Mary’s Island,
off the Northumberland coast, in 16 fathoms ; and it has

VOL. 62, PART 2. — NEW SERIES. 9

116

SIDNEY F. HARM Eli.

been registered in the British Museum collection as
13. 7. 10. 1-2.

Since the publication of the original description, P. oval is
appears never to have been rediscovered (cf. Selys-
Longchamps, 1907, p. 188) ; and it has been supposed that
the species had been founded on the immature condition of
some other species. I am happy to be able to confirm the
accuracy of Strethill Wright’s account; and to show, by
the occurrence of well-developed ovaries and testes, that it
must be regarded as an adult form, in spite of its minute size-
and the small number of its tentacles. The examination of
the Northumberland material has furnished some explanation
of the fact that this interesting species has so long escaped
notice. Although present in very large numbers in the
material under consideration, it is so completely concealed in
the substance of the shell that its presence would not have
been suspected unless the shell had been decalcified. Although
I have not obtained other specimens, there seems every reason
to think that the species will be discovered in equal abundance
when shells of Neptunea or other Molluscs from the north-
east coast of England and the east coast of Scotland are
examined by the method of decalcification.

A further result of the present investigation has been to
demonstrate the occurrence of a remarkably active process
of reproduction by fission, in confirmation of the results of
certain other observers, for other species ; though taking
place with far greater frequency than is indicated by anything
that has previously been published.

The genus Ph or on is was established by T. Strethill
Wright in a paper communicated to the Royal Physical
Society of Edinburgh on April 23rd, 1856, and published in-
two Edinburgh journals (18561, 18562). Two species were
distinguished — P. hippocrepia, the tubes of which wer&
embedded in a stone obtained at Ilfracombe; and P. ovalis,.
found in a decayed oyster-shell, inhabited also by Cliona
celata, dredged near Inchkeith in the Firth of Forth.
Of P. hippocrepia an excellent description is given, so far

ON RHORONIS OVALIS, STRETHILL WRIGHT.

117

as the structure could be made out in the part of the animal
protruded from the membranous tube. This account includes
an accurate description of the hippocrepian lophopliore, the
number of tentacles being given as about sixty ; of the
descending oesophagus and the ascending rectum, the position
of the mouth, epistome, and anus being well described ; of
the blood, containing red corpuscles ; and of the principal
vessels, including the afferent and efferent trunks, the
tentacular vessels, and some of the lophophoral vessels.
The structure of P. oval is is described in less detail, but
stress is laid on the form of its lophophore, which is oval but
slightly flattened on one side. The tentacles were eighteen
in number, and the blood-corpuscles were noticed. -The
entire animal was about half an inch in length, and the gullet
terminated in a globular gizzard, which communicated with a
thick-walled stomach. Good figures are given of the oral
ends of both species, the body of P. ovalis being figured as
protruding from a delicate tube, embedded in the substance
of the oyster-shell. The examination of Strethill W right's
figures and description leaves no doubt that the specimens
described in the present paper belong to P. ovalis.

Although the eroded appearance of the outer surface of
the Nept unea-shell furnished the clue which led to the
discovery of the Phoronis, it does not appear to have been
caused by the presence of this animal. The outer layers of
the shell, both on the outer and on the inner side, are traversed
by a number of branching’ hypha-like threads, which reach a
diameter of as much as 24 /u ; and it appears probable that
these are the principal cause of the erosion noticed on the
outer surface. This is in accordance with the statements of
Bornet and Flahault (1889), who give an account of various
Algae and Fungi which bore in the shells of Molluscs.
According to these authors the organisms in cpiestion
commence their work by extending horizontally in the
epidermic layer of the shell, subsequently sending branches
vertically into the shell-substance and others parallel with
the first set. These become so numerous and their branches

118

SIDNEY F. HARMER.

so close together that the interposed calcareous substance
finishes by disappearing, and the plant thus conies into
contact with the external water, and is able to discharge its
reproductive cells. The surface of the shell is thus rendered
rugose and uneven. This process is supposed to be the
principal cause of the disappearance of empty shells in quiet
bays.

I have not succeeded in determining the vegetable organisms
found with the Ph or on is oval is, though they appear to have
some resemblance to the Alga described by Bornet and
Flahault as G-omontia polyrhiza. The hyphae of this
plant are said to have a maximum diameter of 12 /u — a size
which is considerably exceeded in the largest filaments found
in the Northumberland material.

The thickness of the N eptunea-shell varies between about
2 and 4’5 mm. In the neighbourhood of the columella it
reaches its greatest thickness, while it is much thinner in the
middle of the whorls. The diameter of the tubes of the
majority of the Phoronis individuals is from *250 to
*275 mm. Even in the thinnest part of the shell the
diameter of the burrow of the Phoronis is thus not more
than about one-eighth of the thickness of the shell, and there
is accordingly plenty of room in the substance of the shell
to accommodate a large number of these burrows.

The general arrangement of the cavities inhabited by the
Phoronis maybe indicated by comparing the shell with a
mass of wood excavated by the burrows of Teredo. The
Phoronis is present in very large numbers, its burrows
passing in all directions through the shell, and opening to the
exterior either on the outer side or on the inner side.
The distal end of the burrow is commonly placed at right
angles to the surface, but in some cases part of the tube lies
in a superficial groove of the shell. In addition to the
Phoronis and the Alga already mentioned, the substance
of the shell is inhabited by other boring organisms, and
particularly by the. Sponge C lion a and a Polychaete which

ON PHORONIS OVALIS, STRE I'HILL WEIGHT.

119

is probably Poly cl ora. The Sponge forms much larger
cavities than those produced by the Phoronis, and these
naturally have a form corresponding with the lobes of the
Sponge, being quite different in shape from the cylindrical
Phoron i s-tubes, which remain of approximately the same
diameter throughout their course. The Polychsete tubes are
larger than those of the Phoronis; and, instead of having
the hyaline character of the tubes of this organism, their
transparency is affected by the presence in them of numerous
granular particles.

The tubes of the Phoronis are represented in several of
the figures (e.g. PI. 8, fig. 15; PI. 9, fig. 37). It will be
seen that they are by no means uniform in shape, but that
they are generally curved in various ways. The thin mem-
branous tube is closely applied to the inner surface of the
excavation in the shell, and the burrows are accordingly
curved in correspondence with the form of the tubes seen in
a decalcified preparation.

The most superficial examination of a number of the tubes
set free by decalcifying the shell shows that there is an
extraordinary amount of variation in the included organisms.
It is hardly going too far to say that it is difficult to find two
individuals alike on a slide containing a large number of
individuals. The length of the animal varies within wide
limits, while differences in the transverse diameter of the
specimens are also marked. The most striking differences
are seen, however, in the extent of the development of the
lophophore. While some of the individuals are provided
with a lophophore bearing well-developed tentacles (PI. 7,
figs. 1—3), the lophophore is completely absent in others
(PI. 9, figs. 29, 30). In others again a lophophore in an
early stage of development can be made out at the distal
end (PI. 8, fig. 13) ; while all stages between this and the
fully-developed lophophore can be found without difficulty
among the other individuals on the slide (PI. 7, figs. 5, 4, 8).
It is impossible to interpret these appearances on any other
supposition than the assumption that regeneration of the

120

SIDNEY F. HARMER.

lopliopliore takes place with great readiness in this species of
Phoronis. Although it is probable that this regeneration
may be no more than the replacement of the lopliophore pre-
viously present, there is reason to suppose that in other cases
it indicates the occurrence of a process of asexual repro-
duction, the regenerating lopliophore being formed, in such
cases, at the distal end of a proximal part of the body
separated off from the remainder by a zone of transverse
fission. Before considering the evidence in favour of this
view it will be convenient to notice previous observations
bearing on this subject.

The power of regeneration possessed by Phoronis early
attracted the attention of observers of this animal. Dyster
(1858, p. 251) states that “an abstracted head [of P. hip po-
or epia] is renewed within forty-eight hours, not completely
developed, but with a serviceable mouth and its covering-
valve and stumpy tentacles which do their work of pro-
viding food.” In the same year Van Beneden (18581, p. 460,
Plate, figs. 4-6, and 18582, p. 18, PI. v, figs. 4-6) describes
and figures the spontaneous loss of the lopliophore and its
subsequent regeneration in P. gracilis. Cori (1890,
p. 502), in describing P. psammophila, mentions the same
phenomenon, which occurs spontaneously, although he refers
to the belief of the fishermen of Messina that the “ heads ”
are bitten off by small fishes. In the course of a paper
dealing with Ccelenterates, Cerfontaine (1902, p. 262) records
some interesting observations on P. kowalevsky i,1 which
occurs at Naples in a very restricted situation under a bridge
in the “ arriere-port de Naples.” The animal forms large
colonies in this locality, and these are found in a flourishing
condition during a certain part of the year, namely from
May to November. They are provided, at this period, with
lophophores, among the tentacles of which occur numerous

1 De Selys-Longchamps (1907, p. 173) points out that this form is
indistinguishable on anatomical grounds from P. liippocrepia, but
that its tubes are encrusting, while P. liippocrepia is a boring
species.

ON PHORONIS OVA MS, STRKTHILL WRIGHT.

121

•eggs and developing embryos, a great number of larvas being
set free from time to time. At the end of this season the
lophophores are lost and the colonies then consist of blackish
u cakes” of matted tubes, from 1 to 2J cm. in thickness.
At the recommencement of the favourable season these
eakes become covered by aune riche vegetation de Phoronis.”
The tubes, examined during the “ mauvaise saison,” were
found to contain remains of the body of the Phoronis, with
lophophores in all stages of regeneration. Actinotrocha is
said to occur rarely in the plankton at Naples at any time in
the year, and Cerfontaine points out that it is difficult to
suppose that the innumerable Phoronis which appear on
the surface of the old cakes in a few days, at the commence-
ment of the favourable season, can have been derived from
larvas. He concludes, therefore, that P. kowalevskyi
possesses a mode of spontaneous annual regeneration.

A more detailed account of the process of regeneration is
given by Schultz (19031), who describes the spontaneous loss
of the lophophore in P. m tiller i at Heligoland, and compares
it with the loss of the calyces in the Polyzoa Pedicellina
and Urnatella, or of certain parts in Compound Ascidians
(Diplosomidae) and Hydroids. He states that the process
occurs, in Phoronis, whenever the conditions become
unfavourable ; and that it is followed by the regeneration of
the lophophore as soon as better conditions return. The loss
of parts of the body under unfavourable conditions is
regarded as a physiological necessity which has become a
normal process in various animals and plants (as in the loss of
leaves by deciduous trees) ; this iC reduction ” being explained
as the loss of parts which can be dispensed with temporarily
during a period of hunger, thus leaving fewer structures to
be nourished during times when nutriment is not abundant.
Schultz points out that a reduction-process of this nature, in
an animal which has a high capacity for regeneration, may
lead to transverse fission, “ and so indirectly to budding,”
although he does not prove that an asexual method of repro-
duction occurs in Phoronis.

122

SIDNEY E. HARMED.

On dividing a Phoronis transversely with a pair of
scissors Schultz found that regeneration took place readily in
both the pieces thus separated. Although he did not deter-
mine the minimal size of the fragments which were capable of
regeneration , he states that this process occurred, in both the
proximal and the distal portions, wherever the cut was made.
The distal portion regenerates a proximal end, for instance,
even if the part separated consists only of the lopliophore and
a part of the body containing the commencement of the
oesophagus and the extreme end of the rectum. The proximal
portion regenerates a new distal end whether the cut be made
through the commencement of the oesophagus, or at practi-
cally any lower level, while the distal end regenerates a new
proximal end with a similar disregard of the region where the
section has been made. The details of the process of
regeneration are described.

In a later paper (19032) Schultz shows that regeneration
takes place in the Actinotrocha larva of Phoronis, similarly
divided by transverse cuts, although it proceeds at a much
slower rate than in the adult animal.

None of the papers so far quoted contain any suggestion
that the regeneration in Phoronis may be associated with a
process of reproduction by fission.

In his monograph of the Phoronidea of the Gulf of Naples
(1907, pp. 161—) de Selys-Longcliamps gives further infor-
mation with regard to the regeneration of species of this
group. No evidence was obtained that the lophophoral end
spontaneously thrown off was capable of regeneration. The
lophopliore may be' thrown off several times in succession by
the same individual, which regenerates this portion after each
reduction. The proximal end of the body, or “ ampulla,” is
incapable of regeneration, a process which appears to be con-
fined to the muscular region. On cutting this part, in P.
psammophila, into six pieces of approximately equal size,,
each of these pieces regenerated so as to become a complete
individual, while the lophopliore and the ampulla did not
regenerate. Fragments hardly longer than wide in which tho

123

ON PHOEONIS OVALIS, STEETHILL WEIGHT.

lophophore was being regenerated were found in certain
colonies.

On p. 164 of his monograph, de Selys-Longchamps makes
the significant remark that it is difficult to believe that the
numerous individuals which compose a colony of P. ko wa-
le v sky i can have been derived from as many Actino-
trocha larvae. It seems most unlikely that these larvae, which
lead a pelagic existence, can assemble at the time of their
metamorphosis in sufficient numbers to build up a colony of the
kind characteristic of this and other species. Allusion is made
to the observations of Cerfontaine, who found numerous re-
generating fragments in colonies ofPhoronis which appeared
to be dead and decomposing ; and toothers by Ikeda (1901,
p. 580), who had found young animals which he supposed —
probably wrongly — to have been derived from larvae, in the
debris of old colonies. De Selys-Longchamps believes that
fragmentation of the individuals is a normal process, and, as
this is followed by regeneration of the pieces, that it is a
method of asexual reproduction. The correctness of this con-
clusion is borne out by my own observations on P. oval is.

Specific Characters of Phoronis ovalis.

It has already been pointed out that P. ovalis has not
hitherto been recognised since its original description by
Strethill Wright in 1856. As doubts have been cast on its
claim to be regarded as an adult form (cf . de Selys-Long-
champs, 1907, p. 188), it is important to notice that the
Northumberland material includes specimens possessing fully
developed ovaries (PI. 7, fig. 2) or testes. The following*
diagnosis of the species may be given :

Size very small compared with that cf other species of the
genus. 'Total length reaching at least 6 mm., the diameter of
the body being about 250 p, and of the tube, in large
specimens, about 250-350 ju. Tube delicate, hyaline, em-
bedded in the substance of shells of molluscs. Lophophore
oval, not much broader than long, one of the longer sides of

124

SIDNEY F. HAltMER.

the oval indented, thus indicating the hippocrepian form of
the lophophore in other species. Number of tentacles very
small, about twenty-two. Metasome (body) sharply divided
into two distinct regions, the distal portion with strong longi-
tudinal bundles of muscles, the proximal portion with an
extremely thin body-wall in which muscles are absent or at
most very slightly developed. The proximal end of the
muscular region is slightly invaginable, so that in contracted
specimens this portion forms a shallow cup surrounding the
more distal part of the muscular region. About fourteen
bundles of longitudinal muscles occur on each side in the
distal portion of the body. Regeneration of the lophophore
occurs with great facility, and this regeneration is frequently
the result of transverse fission.

Phoronis oval is differs from other species in its rela-
tively minute size, in the remarkably simple character of its
lophophore, which is, however, hippocrepiform, in the very
small number of its tentacles, and in the sharp differentiation
of its body into two regions, the proximal end of the muscular
region being slightly invaginable. As the occurrence of
functional gonads in some of the specimens indicates that it
is really an adult form, the claims of the species here
described to be regarded as a distinct species seem to be
incontrovertible.

P. oval is has often been referred to in literature, but as
no subsequent observer has hitherto succeeded in obtaining
it, these references are all based on Strethill Wright’s original
account. De Selys-Longchamps (1903, p. 32) has stated that
he considers the claims of P. ovalis to specific rank not
improbable, and that Actinotrocha pallida, Schneider,
may be its larva.

The only description which might refer to the same species
is Van Beneden’s account of C rep in a gracilis (18581,
18582). This was described as having from twenty -four to
forty tentacles, and as reaching a length of 8-10 mm. The
epidermis is provided with numerous stiff hairs, the points of
which project to the exterior. The lophophore, as shown in

ON PHORONIS OVALIS, STRETHILL WRIGHT.

125

tlie original figures, has a simple structure, its ends not being
in-rolled. In this respect it agrees with P. ovalis; but
Van Beneden’s figures represent animals with about forty
tentacles, a number which is considerably in excess of that
given by Stretliill Wright for his species, and of that found
by myself in the Northumberland material. De Selys-
Longchamps (1903, p. 25) has found a form at Heligoland,
which he refers to Van Beneden’s species; and in support of
this conclusion he emphasises the occurrence, in the Heligo-
land specimens, of very numerous epidermic structures (see
his Plate ii, figs. 22-26), which he identifies with Van
Beneden’s. “ hairs.” This resemblance is certainly a striking
one, especially as the author points out that he has not found
these structures in any other species. The number of
tentacles found by de Selys-Longchamps was, however,
greater than that given by Van Beneden, being commonly
fifty to sixty, but sometimes as much as eighty. The length
of the tube of the Heligoland species is said to be 10-20 mm.

It may be remarked that P. h i ppocrepia, P. gracilis,
and P. ovalis are all found in burrows in the shells of
Molluscs, or in other calcareous substances. They appear to
differ from one another in size and in the number of their
tentacles; P. hippocrepia having the largest dimensions
and the greatest number of tentacles, P. ovalis occupying
the other end of the series in both respects, and P.
gracilis taking an intermediate position.

P. millleri, also described by de Selys-Longchamps
(1903, p. 6) from Heligoland, does not form colonies. It
reaches a length of 40-80 mm. and has fifty to sixty
tentacles, of which those on the oral side of the lophophore
are specially short.

Structure of P. ovalis.

The general structure of the members of this genus is so
well known1 that it will not be necessary to describe that of

1 See especially the elaborate monograph of de Selys-Longchamps
(1907), who gives full references to the literature of the subject.

126

SIDNEY E. HARMER.

P. ovalis in great detail. I confine myself, therefore, to
a description which is sufficient to show that the subject of
this paper is rightly referred to Ph or on is, and also brings
out some of the more noteworthy features of P. ovalis.

Tube.

The characters of the tube can be readily examined after
decalcification of a fragment of the shell containing the
animals. The single shell which furnished the whole of
the material must have contained hundreds of individuals,
whose tubes penetrated the substance of the shell in all
directions. Although accompanied by other boring animals
(C lion a, Polychasta) there is not the slightest reason to
suppose that the Phoronis inhabits burrows excavated by
other organisms. The diameter of its tubes is distinctly
smaller than those of its associates; and each Plioronis-
tube closely lines the burrow in which it lies, along the
whole of its course. De Selys-Longchamps (1907, p. 28)
thinks that the tube is secreted by the proximal end of the
ampulla, and that its growth takes place at this end. I have
no observations to indicate how the boring is effected, but I
am inclined to think that the main increase in length takes
place as suggested by that author. The set of tubes shown
in PI. 8, fig. 15, seems to prove, however, that this explanation
is not sufficient, and that the faculty of boring and secreting
a tube is not restricted to the region of the ampulla. The
figure shows that secondary deposits of tube-material may
be formed inside the original tube. Some of these are more
or less curved transverse septa (E, B, E), occurring on the
proximal side of the ampullar region. Others may be formed
in an irregularly longitudinal direction, as at L. In three
places (C, G, J) a lateral opening has been formed on the
proximal side of a transverse septum, and a new tube has
grown out at an angle with the original tube. These lateral
tubes, which will be considered below in the section dealing
with regeneration, can hardly have been formed by the

ON PHORONIS OVALIS, STRETB1LL WRIGHT.

127

proximal end of an individual ; and it seems necessary to
assume that the faculty of producing a tube and of boring
in the shell is possessed by a considerable part of the body-
wall.

Structure of the Animal.

Owing to its small size many of the principal points in the
structure of this species can be made out in stained prepara-
tions of the entire animal mounted in Canada balsam. The
frequent occurrence of regenerating lophophores gives rise
to an extraordinary want of uniformity in the appearance of
the individuals. The even more striking variation in size, as
exemplied, for instance, by PI. 7, figs. 2 and 5, appears to
be due to the reduction in length produced by transverse
fission.

A. specimen with expanded lophophore is represented in
PI. 7, fig. 1. The small number of the tentacles is at once
apparent, and it constitutes one of the most characteristic
features of the species. How striking is the difference
between P. oval is and some other species of the genus
may be illustrated by the comparison with P. buskii, the
number of whose tentacles is estimated by de Selys-Long-
champs (1907, p. 33) at about one thousand.

In the great majority of the specimens the tentacles lie in
their retracted condition inside the tube. This condition of
the tentacles is shown in PL 7, fig. 3, and other figures. In
favourably prepared specimens (PL 9, fig. 40) the epistome
(ep.) can be seen as a distinct lip overhanging the mouth and
surrounded by the bundle of tentacles.

The distal part of the body-wall is thick (PL 7, fig. 3), a
condition which is largely due to the presence of strong
bundles of longitudinal muscles. These end abruptly at
about the middle of the length of the body in this particular
individual, although the proportion which the muscular part
of the body-wall bears to the non-muscular part is highly
variable. In PL 7, fig. 2, for instance, the muscular region

128

SIDNEY E. HA EM EE.

is not more than a quarter of the entire length, although
absolutely of about the same length as in PI. 7, fig. 3. In
many of the specimens the proximal region of the muscular
part of the body is slightly invaginated (PI. 7, figs. 4, 8),
thus forming a sort of shallow cup surrounding the base of
the remainder of the muscular portion. The cavity of the
cup faces distally, towards the lophophore.

The remainder of the body-wall is extremely thin and
transparent. In some individuals (PI. 7, fig. 8) the extreme
proximal end has the ampulla-like form usually found in
Phoronis. The absence of a typical ampulla in other
specimens is doubtless due to the loss of this region when
transverse fission takes place ; but the ampulla is probably
regenerated in due course by the distal individual formed by
fission. Muscles have not been detected in the “non-
muscular part ” ; and if they occur they must be excessively
thin.

The alimentary canal has the form usual in the genus.
The first part of the descending limb is formed by an
oesophagus, sharply marked oft from the succeeding part,
and occupying from half to a quarter of the length of the
muscular part of the body (PL 7, figs. 3, 8). The remainder
of the descending limb, constituting the proventriculus ( pr .),
is relatively narrow throughout the muscular region, but it
gradually ddates in the non-muscular part, reaching its
maximum size in the ampullar region, but before the extreme
proximal end is reached. From the dilated stomach ( st .)
thus formed (PL 7, fig. 8) a short section of the descending
limb, of distinctly smaller size than the stomach, continues
to the proximal end of the body, where it curves round into
the ascending limb ( int This portion is for the most part
of small diameter, though its size depends partly on the
amount of the remains of food (commonly Diatoms) or the
faeces which it contains. The last part of the intestine
is of small size, and opens by the anus (Pl. 9, fig. 40, an.)
close to the lophophore, and on the side corresponding with
the base of the epistome.

ON PHOEON1S OVALIS, STRETH1LL WEIGHT.

129

In a few individuals (PI. 7, fig. 2) a number of large eggs
may be seen lying in the body-cavity of the noil-muscular
region. These no doubt constitute the ovary, and the
occurrence of this organ is of importance as evidence that
animals in this condition are mature.

In most of the specimens a considerable amount of granular
tissue is visible, lying in the body-cavity, principally of the
non-muscular region, between the alimentary canal and
the body-wall (PI. 9, figs. 29, 33, ad.). This is the “ adipose
body ” or “ vaso-peritoneal tissue ” of other authors ; and,
as in other species of Phoronis, a part of this tissue
commonly has the histological characters of a testis. I have
not convinced myself that ovary and testis may occur in the
same individual, and it is possible that P. ovalis is dioecious.
If this difference really occurs between P. ovalis and other
species (which are usually hermaphrodite) it is perhaps the
result of the small size of the animal.

Some of the anatomical features have been examined in
sections ; but the material, contained as it was in the burrows
in the shell, is not sufficiently well preserved to show the
finer details.

PI. 8, fig. 14, an approximately sagittal section of the
distal end of the animal, shows the muscular region of
the body-wall and the cup-like invagination (inv.) at its
base. The strong muscular bands ( l . m.) are clearly seen, as
well as the origin of the bundles from the body- wall in
the region of the invaginated part. The epistome (ep.) is
visible, surrounded by the tentacles, while the oesophagus ( aes .)
is cut along the whole of its length, and is separated from
the proventriculus (pr.) by a circular valve. The terminal
portion of the intestine (int.) is seen by the side of the
oesophagus; and the position of the anus (an.), which opens
into a depression of the body-wall close to the lophopliore
and the base of the epistome, is indicated.

A few sections from a series cut transversely to the long*
axis of the body have also been figured. In the first of these
(PI. 8, fig. 16) the tentacles are seen to be arranged in the

130

SIDNEY F. HARM KB.

form of a horse-shoe, though the ends of the lophophore
are not drawn out to the extent found in species with
numerous tentacles. Twenty-two tentacles can be counted?
and this was the full number present in this individual. The
tube (t.) is seen to consist of several superposed cuticular
layers.

In the next section shown (PI. 8, fig. 17) the bases of the
tentacles have become confluent on the anal side, and the lopho-
phore is now clearly seen to be liippocrepian in form. Some
indication of the tentacle-vessel can be seen in several of the
tentacles, in addition to the cavity of the tentacle. The tip
of the epistome is cut at ep.

In PI. 8, fig. 18, the union of the tentacle-bases is more
complete, and a considerable part of the epistome ( ep .) is
visible. The next figure (PI. 8, fig. 19) shows the epistome
at its largest part. In PI. 8, fig. 20, the tentacle-bases
have all united, so that the mouth (m.) is completely out-
lined. The anus {an.) opens into a depression between a
lobe of the metasome and the lophophore, and a part of the
nerve-ring ( n.r .) is visible between it and the mouth. The
two nephridia are seen in one or two of the sections which
come next in the series ; but they have not been drawn, as the
preparations are not very favourable for showing their details.
In PI. 8, fig. 21, the oesophagus (ass.) and the intestine (int.)
are seen, as well as the oral part of the nerve-ring ( n . r.).
The afferent blood-vessel (a. v.) occurs between the oesophagus
and the intestine, and some of the longitudinal muscles of the
body-wall are visible.

PL 8, fig. 22, shows a complete median mesentery ( mes .)
supporting the two limbs of the alimentary canal ; and both
the afferent {a.v.) and the efferent (e. v.) blood-vessel. The
longitudinal muscles of the body-wall are now well developed.
In PL 8, fig. 23, the longitudinal muscles (Z. m.) are still
stronger, and about fourteen bundles can be seen on each side
of the median mesentery. Both the longitudinal blood-vessels
are still visible. Pl. 8, fig. 24, is through the proximal end
of the muscular part of the body-wall, and shows part of the

ON RHORONIS OVALIS, STRETHIIjL WRIGHT.

131

shallow invagination ( inv .) above described. PL 8, fig. 25,
represents a section passing through the non-muscular part
of the body, and shows the thin character of the body-wall
in this region.

PI. 8, fig. 26, is from another series of sections, and it
represents a section through the distal end of the body, not
far from the lophopliore. A considerable part of the nerve-
ring [n. r.) is visible, as well as both nephridia ( neph .). The
small lobes projecting into the body-cavity, near the tubes of
the nephridia, are probably parts of the funnels of these
•organs.

Regeneration and Fission.

In the material under consideration there is no need to
make a careful search for evidence of regeneration. It is
more difficult to find a lophopliore provided with tentacles of
the full length than to find one with immature tentacles.
It is, moreover, a striking and most obvious fact that the
dimensions of the individuals vary to such an extent as to be
unintelligible on any hypothesis of orderly growth from the
immature to the mature condition. It may further be noted
that there is no relation between the condition of the lopho-
phore and the size of the specimen. It will be convenient to
analyse the facts under the following heads :

(a) Regeneration of the lophophore.

(b) Direct evidence of transverse fission.

(c) Method by which the tube of the proximal segment is
-completed.

(d) Size of regenerating individuals as indirect evidence of
fission.

(e) Position of die zones of fission.

(a) Regeneration of the Lophophore.

Assuming provisionally that transverse fission is a process
of normal occurrence, it is obvious that the conditions under
which a new lophophore is formed is not quite the same in

VOL. 62, PART 2. NEW SERIES. 10

132

SIDNEY F. HARMED

the two individuals formed by the fission. In the case of the
proximal individual, the entire lophophore has to be formed
de novo from a region which is far removed from the
original lophophore. In the distal individual, regeneration
if it takes place, presupposes the loss of the original lopho-
phore. This latter process is of the same nature as that
which has been described in other species, where the lopho-
phore is thrown off, the wound closes, and a new crown of
tentacles is formed from the extreme distal end of the animal*

Many of the specimens figured illustrate the regeneration
of the lophophore at the distal end ; and where the relations-
of the tube give no indication of the previous occurrence of
fission in this region (cf . section (c)) the regenerating lopho-
phore appears to be a replacement of the original lophophore..
But as none of the regenerating specimens figured are as
long as the fully adult specimen shown in PI. 7, fig. 2, it
may be considered probable that they are all fractional
portions of individuals produced by the metamorphosis of
larvae. Neglecting for the moment the differences in the
length of the individuals, the regeneration of the distal end
may be illustrated by the following cases :

In the distal individual shown in PI. 9, fig. 32, the thick-
walled muscular region of the body is clearly indicated, with
the collar-like partial invagination ( inv .) characteristic of the
proximal end of this part. The new lophophore is repre-
sented merely by the thickened body-wall at the extreme-
distal end.

In PI. 9, fig. 36, the distal thickening indicating the new
lophophore is more distinct, and is separated by a slight
annular constriction from the beginning of the muscular part
of the body-wall.

In PI. 8, fig. 13, the lophophore is still more distinct and
shows distal lobulations which will become the new
tentacles.

Further stages in the growth of the tentacles are shown in
PI. 8, fig. 11, and PI. 7, figs. 5, 4, and 8 ; and in the last of these-
the formation of the new lophophore is practically complete.

ON PHORON1S 0 VALIS, STRETHILL WRIGHT. 133

For a more detailed description of the growth of the
regenerating lophophore reference should be made to the
memoir of Schultz (19031).

(6) Direct Evidence of Transverse Fission.

It has not been very easy to obtain unmistakable evidence
of the occurrence of this process, but several specimens have
been found which appear to be demonstrative in this respect.

PI. 9, fig. 31, represents what may be regarded as the
commencement of this process. At the extreme distal end of
the non -muscular part of the body an annular layer of tube-
substance has been formed, projecting into the cavity of the
tube, and slightly constricting the body-wall, which does not
as yet show any indication of transverse division.

In PI. 9, fig. 29, a similar process of constriction is
taking place in the non-muscular region, at some distance
from its distal end. The tube lies in very close contact
with the body of the animal, but a constricting lamina
can be seen on the left side of the figure. The body- wall
now shows evidence of being constricted, and it may be
noticed in particular that the mass of adipose tissue (ad.)
which fills up most of the proximal region of the body-cavity
is being divided into two parts by the constriction. This
specimen furnishes the most direct evidence which has been
obtained of the occurrence of the process in question.

In PI. 9, fig. 32, two individuals lie in the same tube, the
cavity of which has been divided by a transverse septum.
The proximal end of the distal individual has a bilobed
character, differing from the evenly rounded surface which
characterises the normal ampulla. This lobed appearance of
the proximal end has been noticed in many of the individuals,
and may be taken as evidence of the occurrence of fission,
the rounded form of the ampulla not being yet reconstituted.
The figure shows that the mesentery of the alimentary canal
is attached to the emargination between the two lobes. The
distal end of the individual on the proximal side of the

134

SIDNEY F. HARMKR.

septum is also lobed, and this region shows indications of
regeneration, particularly in the commencing differentiation
of an oesophageal portion of the descending limb of the
alimentary canal. It appears to be practically certain that
this specimen represents a stage not long after the occurrence
•of transverse fission.

In PI. 9, fig. 37, there are also two individuals in what
may be considered one original tube. The individual on the
proximal side of the septum already shows a recognisable
lophophore and oesophagus, but it is constituting a new distal
end to its tube by growing out laterally from the original
tube.

(c) Method by which the Tube of the Proximal
Segment is completed.

The specimen last described furnishes the evidence
required, and the explanation it suggests is fully confirmed
by a number of other cases which have been noticed. In
most of the specimens referred to, a tube makes a sudden
bend outwards, immediately on the proximal side of a trans-
verse septum ; and tliis outwardly bent portion contains the
distal end of an individual. This is represented, for instance,
in PI. 8, fig. 9, and PI. 9, fig. 34 ; and the natural interpretation
of the conditions shown is that the portion of the tube contain-
ing the proximal end of the individual in question is part of
an original tube, from the rest of which it is separated by the
transverse septum ; and that at the formation of this septum
the proximal part of the tube, being cut off from the exterior
by the septum, has grown out laterally so as to form a new
opening for itself. It may be noted that the formation of
these laterally growing tubes makes it almost impossible to
accept the view of de Selys-Longchamps, alluded to on p. 12,
that the tube is secreted only by the ampullar end of the
animal.

The system of empty tubes represented in PI. 8, fig. 15,
may be taken as distinct evidence, in the light of the facts

ON PHORONIS OVAL1S, STRETHILL WRIGHT.

135

already recorded, that the process of fission may be repeated
several times in one original individual and its products.
The tube A-K appears to be part of a tube originally
inhabited by a single individual, and added to from time to
time as the result of successive transverse fissions of its
inhabitants. A is the proximal end, and K the distal end
of the portion represented. The transverse septa at B ,
F, and I, may be taken as indications of as many transverse
fissions The segment of the tube between B and F has
been occupied by an individual which has formed a new
distal end to its tube at G-, and has restricted the size of the
rest of its tube by the formation of the irregular, longitudinal,
secondary deposit of tube-substance seen at L. The septum
H may indicate merely a part of this process of reducing the
size of the tube, but fission may have occurred at this point,
in which case it must be assumed that the segment of the
animal which occupied the portion B-H had not succeeded
in forming a new distal end to its tube. The portion of tube
situated proxitnally to the septum I has grown out into the
irregular tube J. On the proximal side of the septum B a
considerable length of the distal part of a tube has been
formed at C. This individual occupied only a short portion
of the original tube, a septum having been formed at M;
and it then appears to have grown out proximally, in the
direction P, a further fission of the inhabitant of the tube
being indicated by the septa E. It is not impossible that the
fragment of the individual left in P may have turned com-
pletely round in its tube, so that E became the proximal end
and P the distal end of its tube, but of this there is no
evidence.

The appearances presented by this system of tubes,
together with the evidence brought forward in the next
section ( d ) suggest that fission occurs repeatedly in this
species, and it seems not improbable that all the numerous
individuals found in a given area of the shell may have been
derived by fission from a single metamorphosed larva, or
from a small number of individual larvae which succeeded in

136

SIDNEY E. HAIiMER.

effecting* their metamorphosis in the neighbourhood of the
shell.

Further evidence of the correctness of the interpretation of
PI. 8, fig. 15, suggested above, is furnished by PL 8, figs. 9,
28, and PL 9, figs. 34, 37, which show individuals, in varying
conditions of regeneration, in which the distal part of the tube
arises laterally from another tube, and, usually, immediately
on the proximal side of a tube-septum which appears to
indicate the position of the zone of fission.

( d ) Size of regenerating individuals as indirect
evidence of fission.

The variation in length of the regenerating individuals is a
very striking fact. The individual shown in PL 7, fig. 2, is
about 6 mm. long, while that represented in PL 7, fig. 7, is
only ’3 mm. long. If what is here described as regeneration
were really explainable as the various stages by which a
metamorphosed larva reaches its adult condition, there would
be some definite relation between the size of the specimen
and the stage of development of the body and lophophore.
Nothing of the kind can be made out. The earliest stages in
the development of the lophophore may be found in very long
individuals, as in PL 9, fig. 36 ; and, conversely, very small
specimens (PL 7, fig. 5) may have a well- grown lophophore.
The only legitimate explanation of the facts seems to be that
regeneration of the lophophore may occur indifferently in
large and in small specimens; and from the evidence which
has already been brought forward it appears to be fair to
conclude that this process is commonly the result of fission.
In cases where no tube-septum can be discovered on the
distal side of a regenerating specimen, the process appears
to be the consequence of the spontaneous loss of the lopho-
phore, as has been described in other species of Phoronis.
But in cases like PL 9, figs. 32, 33, the new lophophore is
clearly being developed as the direct result of the formation
of a fission-zone. PL 9, fig. 35, represents what appears to

ON PHOBONIS OVALIS, STBE THILL WRIGHT.

137

be an unusual condition. The position of the fission-zone
which cut off the small individual shown is clearly indicated
by the annular tube-septum. But in this case the regenerating
distal end is growing into the part of the original tube situated
distally to the septum, instead of growing out laterally to
form a completely new opening. Perhaps the septum was not
a complete one ; but if not it must be assumed that the central
part of the septum has been absorbed by the regenerating
fragment. It is not more difficult to make this assumption than
to assume that in other cases a lateral part of the tube can be
absorbed, in order to allow the proximal fission-segment to
form a new orifice to its tube.

The great capacity for transverse fission possessed by
P. oval is is indicated by the very small size of the re-
generating fragments. The smallest specimen shown (PI. 7,
fig. 7) is only *3 mm. long, but it shows clear signs of re-
generation in the differentiation of a new muscular region of
the body-wall, indicated by a greater thickness of this part
distally, and by the formation of a distinct line of separation
between it and the future non-muscular portion. The appear-
ances here shown give reason to suppose that a fragment no
more than *3 mm. long can regenerate a complete individual.
The complicated arrangement of the tube-septa in this case
implies that the cavity of the original tube has been reduced
in size several times, probably in correlation with the small
size of the living fragment left in this section of the tube.

(e) Position of the zones of fission.

The direct evidence obtained on this subject points to the
non-muscular part of the body as the region where fission
may occur. This is illustrated by PI. 9, figs. 29-31. It may
be noted that this is not in agreement with the statement of
de Selys-Longcliamps (1907, p. 163), according to whom it is
the muscular region that is specially capable of regeneration.
The observation by this author that, having cut the muscular
region of a Phoronis psammophila into six fragments,

138

SIDNEY F. HARMER.

each of these regenerated a complete individual, is too
precise to be disputed. But in P. oval is I have found no
certain evidence that the muscular region shares the power
of division which is undoubtedly possessed by the non-
muscular region. It is possible that PL 8, fig. 10, indicates
that fission may occur in the muscular region, since in this
case the longitudinal muscles are well differentiated in a frag-
ment which is only just beginning to develop a new lopho-
phore. PL 9, fig. 35, may also imply that the fission-zone
was formed just distally to the junction of the two regions of
the body-wall. But in most of the specimens drawn, the mus-
cular part is at first indicated merely hy a thickening of the
body-wall, and no distinct muscle-fibres can be recognised
in the early stages. The regeneration of the distal end in
fact commences, as has already been pointed out, with the
regeneration of a muscular region, and the lophophore ap-
pears subsequently at the distal end of the muscular region.

The general result of these observations is that fission may
occurin P. ovalis at practically any point of the non-muscular
body-wall, and that very small fragments separated off in
this way are capable of complete regeneration. No certain
evidence has been obtained that the muscular part can form
fission-zones, though this possibility is not excluded. A lobed
condition of the proximal end of the body, as shown in Pl. 9,
figs. 32, 36, 39, appears to indicate that the ampullar region has
not been completely reconstituted since the last fission took place.

Many of the individuals, whether regenerating or not, show
a great development of the adipose tissue which accompanies
the two longitudinal blood-vessels. In many cases, as in Pl. 7,
fig. 6, PL 9, fig. 29, the body-cavity of a regenerating fragment
contains a large quantity of this tissue, which may probably
be regarded as a reserve of nutrient material, at the expense
of which the fragment can continue to survive until it has
reconstituted its alimentary canal and has formed a new
orifice to its tube. Larger specimens which have developed
a considerable amount of this tissue are probably in a favour-
able condition for undertaking fission ; and it may be noticed

OX PHOKON1S OVA MS, ST RETHILL WRIGHT.

139

that the specimen shown in process of dividing in PL 9,
fig. 29, is well provided with adipose tissue. The regenera-
tion of the lophopliore without fission may also be facilitated
by the previous deposition of a sufficient reserve which can
be drawn on for the nourishment of the other tissues during
the temporary closure of the alimentary canal. It may be
noted that the intestine of a regenerating fragment without
a functional lophopliore frequently contains the remains of
Diatoms, which must have been taken in during a period
when well-developed tentacles occurred.

The presence of a large amount of adipose tissue is not,
however, a necessary prelude to fission, even though it may
favour this process. Some of the specimens of full length
are remarkable for being of smaller diameter than usual,
their tissues being more transparent than in other cases, and
the adipose tissue being deficient in amount. These seem to
be ill-nourished individuals, and their occurrence probably
accounts for certain abnormally slender regenerating frag-
ments, of the kind shown in PI. 9, fig. 38, which are some-
times found. The muscular part of the body-wall has com-
menced to differentiate in PI. 9, fig. 38, and although it&
small diameter points to a want of vigour, this individual, and
others like it, may have been in a condition to complete tlio
regeneration.

The Occasional Complete Invagination of the Muscular
Part of the Body-Wall.

In several cases individuals have been found in the peculiar
condition shown in PI. 9, figs. 41 and 39. In PI. 9, tig. 41,
the partial invagination which normally occurs at the proximal
end of the muscular body-wall has become so complete a&
to result in the invagination of the whole of the lopho-
phore and of the tentacles. The invaginated muscular wall
is now turned entirely inside out, forming a sheath
opening distally (or.), containing the tentacles ( tent .), and
having its epidermal portion lining the cavity of the intro-
vert and its longitudinal muscles on the outer side of the

140

SIDNEY F. HARMER.

epidermis. The junction between the muscular and non-
muscular parts of the body-wall now lies at the distal end of
the introvert, and the outermost layer in this region is the
part of the non-muscular wall into which the more distal part
has been invaginated. The invagination has resulted in the
formation of a loop of the alimentary canal which passes
distally along one side of the invagination.

PI. 9, fig. 39, is in a similar condition, except that the
introvert contains no tentacles. In their place may be seen
a projection which obviously consists of a regenerating mus-
cular part of the body-wall, terminated by a commencing
lophophore.

I am unable to give a satisfactory explanation of these
appearances, although the fact that three or four specimens
have been found in this condition shows that the complete
invagination of the muscular body-wall happens not infre-
quently. It is perhaps one of the methods by which the
lophophore may be regenerated, as it seems probable, from a
comparison of the two specimens figured, that PI. 9, fig. 41, is
the earlier stage in the process, and that the invaginated ten-
tacles would have been thrown off somewhat later, the wound
closing, and the body-wall in that neighbourhood then growing
out into the part seen inside the introvert in PI. 9, fig. 39.
It is not obvious what the later course of the regeneration
would have been, though it is possible that the introvert
Avould have been evaginated and the muscular wall recon-
stituted, partly from the old wall and partly from the portion
which is being regenerated in PI. 9, fig. 39. It does not
seem probable that the loss of the original lophophore always
takes place in this way. It is not easy to explain the mechan-
ism by which the complete invagination of the muscular part
of the body-wall takes place in these cases.

The condition shown in PI. 9, fig. 41, resembles that found
in Ectoproct Polyzoa during the retraction of the tentacles,
though the Phoronis has no retractor muscles comparable
with those of the Polyzoa. The resemblance does not appear
to me, however, to lend any support to the view maintained

ON PHORONIS OVALIS, STRETH1LL WRIGHT.

141

by some authors of a relationship between these two groups.
The Plioroniclea differ from the Polyzoa in their embryonic
development, as well as by striking morphological characters,
and in view of these differences the resemblance of the
invaginated body-wall to the tentacle-sheath of the Polyzoa
seems to be merely a fortuitous one. The formation of an
introvert containing the retracted lophophore might perhaps
be compared with more reason with the similar introvert in
Sipunculoid Gephyrea, though the affinity of the Plioronidea
to that group has not been established with any certainty.
The introvert of these specimens of P. oval is is not unlike
that which occurs in certain Gasteropoda (e.g. Buccinum),
but it would hardly be maintained that this resemblance is
any indication of affinity.

The Larval Form of Phoronis ovalis.

Although the observations here recorded do not throw any
direct light on this question, one or two remarks on the sub-
ject may not be out of place. P. ovalis is known from
Strethill Wright's original account to occur in the Firth of
Forth, while the large number of individuals found by me in
a single shell from the Northumberland coast suggests that
the species is common along the eastern coast of the northern
part of Eugland, although it has hitherto been overlooked
owing to its retiring habits. Actinotrocha has more
commonly been found in these regions than the adult Pho-
ronis; and it is, for instance, of frequent occurrence at St.
Andrews.

The adult characters of P. ovalis seem to be so distinc-
tive, particularly the small number of the tentacles and the
restriction of the bundles of longitudinal muscles to a small
part of the metasome, that a recently metamorphosed Actino-
trocha belonging to this species might well be recognisable.
It should, however, be pointed out that the characters of the
individual produced by the metamorphosis of a larva may
differ from those of any of the specimens examined from the

142

SIDNEY F. HARMER.

Northumberland material. Reproduction by fission appears
to take place so frequently in these specimens that all the
observed individuals may well have been produced in this
way. It would thus be unsafe to assume that primary indi-
viduals metamorphosed from larvae have so restricted a
muscular region as that of their fission-products. This may,,
however, be the case ; and it would be desirable to bear in
mind the short muscular region and the tendency for its
proximal end to be slightly invaginated, should the oppor-
tunity occur of examining recently metamorphosed specimens
from this part of the British coast.

Observations which I have attempted to make on this
subject have led to no definite result. By the kindness of
Prof. W. C. McIntosh, F.R.S., I have been able to examine
specimens of Ac tin otr ocli a branchiata from St. An-
drews ; and amongst them I have found one or two specimens
which have recently completed their metamorphosis. There
appear to be no sufficient reasons for referring these speci-
mens to P. oval is. I have also examined three recently
metamorphosed Phoronis kindly lent to me by Prof. J.
Graham Kerr, F.R.S., who obtained them on the West
Coast of Scotland, off the Island of Arran. The number of
tentacles in these specimens seems to be not less than twenty-
eight to thirty, and there is no obvious differentiation of
muscular and non-muscular portions in the metasome. The
evidence thus appears to indicate that the specimens in
question do not belong to P. ovalis.

De Selys-Longchamps (1903, p. 43) has convinced himself
that Actinotrocha branch iata is the larva of P. mulleri,
a species described by him from Heligoland, but not, so far
as I am aware, at present recognised as a member of the
British fauna. In the same memoir (p. 47) he has advanced
reasons for believing that A. pallida, Schneider, is not the
larva of either P. hippocrepia or P. gracilis; and he
suggests that it may belong to P. ovalis, if that form is
really a distinct species. His statement (p. 47) that the
worm produced by the metamorphosed larva of A. pallida

ON PHORONIS OVALIS, SI RUT HILL WRIGHT.

143

has eighteen handles of longitudinal muscles appears to be
significant in this connection, though it should be remarked
that the number of muscle-bundles which I have found in
P. oval is (cf . PI. 8, figs. 22, 23) appears to exceed eighteen.

Actinotrocha pallida was described by Schneider
(1862, p. 64, PI. ii, fig. 12) from Heligoland, where it is said
to be as common as A. branchiata. It is stated to have
not more than ten tentacles, which are broader and shorter
than those of A. branchiata. It possesses only a single
mass of larval blood-corpuscles, while A. branchiata has a
pair of these masses, one in connection with each of the
nephridia. De Selys-Longcliamps (1907, p. 190) has found
A. pallida at Wimereux (Pas-de-Calais) as well as at Heli-
goland, and he represents two young stages in PI. xi, figs. 21,
22. He states that there are never more than six pairs of
larval tentacles, and that the length of the larva does not
exceed *6 mm., while that of A. branchiata (p. 189) is as
much as 2 mm.

The evidence at present available thus seems to point to
A. pallida as being the larva of P. ovalis, and the small
dimensions of this larva are in accordance with the small size
of the adult form to which it is supposed to belong.

Summary.

Phoronis ovalis, which has usually been regarded as the
immature form of some other species, is shown to be a well-
characterised adult form. It inhabits burrows which it
excavates in the shells of molluscs. It possesses in a high
degree the faculty of regenerating the distal end, which is of
common occurrence in the genus. Its gregarious habit is
probably the result of its power of reproducing by transverse
fission, a process which takes place repeatedly and profusely.
There is reason to believe that a similar process occurs in
certain other species which are found as colonies consisting
of numerous individuals, though it is uncertain whether other
species have the power of reproducing by fission.

144

SIDNEY F. HARMER.

Literature.

18581. Beneden, P. J. Van. — “Notice sur un annelide cephalobranche
sans soies, designe sous le nom de Crepina,” ‘Bull. Acad. Roy.
Belgique ’ (2), v, p. 450.

18582. — “ Notice sur un annelide cephalobranche sans soies, designe

sous le nom de Crepina/' ‘Ann. Sci. Nat.’ (4), Zool. x, p. 11.

1889. Bornet, E., and Flahault, C. — “ Sur quelques Plantes vivant dans
le Test Calcaire des Mollusques,” ‘ Bull. Soc. Botan. France/
xxxvi (Congres de Botanique, Paris, 1889), pp. 1-31 (sep.).

1902. Cerfontaine, P. — “ Reclierclies experimentales sur la Regeneration
et FHeteromorphose cliez Astroides Calycularis et Pen-
naria Cavolinii,” ‘Arch, de Biol./ xix, p. 245 (p. 262 : Account
of Phoronis kowale vskyi).

1890. Cori, C. J. — “ Untersuchungen iiber die Anatomie und Histologie
der Gattung Phoronis,” ‘ Zeitschr. f. wiss. Zool./ li, p. 480.

1858. Dyster, F. D. — “Notes on Phoronis liippocrepia,” ‘Trans.
Linn. Soc./ xxii, p. 251.

1901. Ikeda, I. — “Observations on the Development, Structure and
Metamorphosis of Actinotrocha,” ‘ J. Coll. Sci. Tokyo/ xiii,
p. 507.

1862. Schneider, A. — “ Ueber die Metamorphose der Actinotrocha
branchiata,” Arch. f. Anat. u. Physiol./ 1862, p. 47.

19031. Schultz, E. — “Aus d. Gebiete d. Regeneration,” iii, “ Ub.
Regenerations - erscheinungen b. Phoronis Mulleri Sel.
Long.,” ‘ Zeitschr. f. wiss. Zool./ lxxv, p. 391.

19035. lb., iv, “ Ub. Regen. b. Actinotrocha branchiata

Muller,” ‘ Zeitschr. f. wiss. Zool./ lxxv, p. 473.

1903. Selys-Longcliamps, M. de. — “ Beitrage zur Meeresfauna von
Helgoland,” “Ueber Phoronis und Actinotrocha bei
Helgoland." ‘Wiss. Meeresunt. Kiel u. Helgoland,' n.f., vi Bd.,
Abt. Helgoland, Heft 1.

1907. “Phoronis,” ‘ Fauna u. Flora G. v. Neapel/ 30 Monogr.

18561. Wright, T. Strethill. — “Description of two Tubicolar Animals,”
‘Proc. Roy. Phys. Soc. Edinburgh/ i, 1858, p. 165.

18565. “ Description of two Tubicolar Animals,” ‘ Edinb. New

Phil. Journ/ iii, n.s., p. 313.

ON PHORONIS OVALIS, STRETHILL WRIGHT.

145

EXPLANATION OF PLATES 1, S, and 9,
Illustrating Mr. Sidney F. Harmer’s paper “ On Phoronis
ovalis, Strethill Wright.”

Reference Letters.

ad. Adipose tissue, or vaso-peritoneal tissue, amp. Ampulla, an .
Anus. a. v. Afferent blood-vessel, ep. Epistome. e. v. Efferent blood-
vessel. /. Fission-zone. int. Intestine, or ascending limb of the
alimentary canal, inv. Invagination of the proximal end of the
muscular part of the body-wall. Z. Lophophore. l.m. Longitudinal
muscles, m. Mouth, mes. Median mesentery, muse. Muscular part
of the body-wall. neph. Nephridium. n.r. Nerve-ring. ces. (Esophagus.
or. Orifice of invaginated body-wall. ov. Ovary, pr. Pro ventri cuius,
constituting the greater part of the descending limb of the alimentary
canal, s.s1., Septum of tube. st. Stomach, t. Tube. tent. Tentacles.

[All the figures refer to Phoronis ovalis. The sections, PI. 8,.
figs. 14 and 16-26, were drawn with a Zeiss C Obj.; the remaining
figures with a Zeiss A Obj. All the figures have been reduced two-
thirds.]

PLATE 7.

Fig. 1. — The expanded lophophore of an adult specimen. Eighteen
tentacles can be counted. Slide L.

Fig. 2. — A fully adult specimen with expanded tentacles. The ovary
(ov.) is developed. The lobed character of the proximal end of the body
probably indicates, as in other similar cases, that the ampulla (amp.)
has not been completely reconstituted after transverse fission. Slide O.

Fig. 3. — A. smaller specimen with retracted tentacles. Slide M.

Fig. 4. — A small regenerating fragment. The marked angle between
the axes of the proximal and distal parts of the body probably
indicates, as in fig. 3 and other specimens drawn, the lateral outgrowth
of the new distal part of the tube necessitated by the closure of
the original tube by the septum formed during the process of transverse
fission. Slide O.

Fig. 5. — A smaller regenerating fragment. Slide N.

Fig. 6. — A small regenerating fragment in which the lophophore
is not yet developed. The muscular part of the body-wall (muse.) is
already indicated. The adipose tissue (ad.) fills most of the body-
cavity. Slide M.

Fig. 7. — An extremely small fragment in about the same stage of
regeneration as the preceding figure. The cavity of the tube is
restricted by a complicated system of septa (s.). Slide Q.

146

SIDNEY F. HARM KR.

Fig. 8. — A regenerating specimen resembling tliose shown in figs. 4
and 5, bnt more completely developed. Slide M.

PLATE 8.

Fig. 9. — A completely regenerated individual. The original tube
has been subdivided by septa (s.), and the new distal end of the tube
has been developed as a lateral outgrowth starting immediately on the
proximal side of the septa. Slide Q.

Fig. 10. — A small regenerating fragment in which the muscular part of
the body-wall is unusually long; l., the commencing lophopliore ; l.m.,
longitudinal muscles. Slide M.

Fig. 11. — The appearances of this specimen suggest that a small
fragment produced by fission on the distal side of the septum (s.) has
formed a new distal end to its tube by lateral outgrowth instead of
making use of the original distal end of the tube. Slide P.

Fig. 12. — Another small fragment. The muscular part of the body-
wall (muse.) is already in vagina ted at its base (inv.) ; l., the commencing
lopliophore. Slide N.

Fig. 13. — A specimen with strongly marked invagination (inv.) of
the proximal end of the muscular region (muse.) and a commencing
lopliophore (l.). Slide M.

Fig. 14. — A sagittal section passing medianly through the distal end
of the body, inv., invaginated part of body-wall ; ep., epistome ; ces.,
oesophagus, separated by a circular valve from the proventriculus (pr.) ;
an., position of anus. (Zeiss C Obj.) Slide F.

Fig. 15. — A system of empty tubes from which the course of the
transverse fissions of the animal can be inferred. For explanation see
text, pp. 12, 20. Slide K

Figs. 16-25. — From a series of sections transverse to the principal
axis of the body of an adult specimen. (Zeiss C Obj.)

Fig. 16. — Through the retracted tentacles, of which 22 are present,
arranged in the form of a horse-shoe. Slide A1.

Fig. 17. — The tentacles have united at their bases on the anal side
(l.) ; ep., the tip of the epistome. Slide A1.

Fig. 18. — A more proximal section; ep., epistome. Slide A1.

Fig. 19. — The epistome (ep.) is cut at the level where it reaches its
greatest size. Slide A1.

Fig. 20. — The tentacles have completely united at their base, and
the mouth (m.) is thus outlined. The anus (an.) lies between the
lopliophore (l.) and a lobe of the metasome; n.r., part of the nerve-
ring. Slide A1.

ON PHOItONIS OVA US, STRE THILL WRIGHT.

147

Fig. 21. — Through the commencement of the metasome. The
afferent blood-vessel (a. v.) is visible ; n. r., the part of the nerve-ring
on the oral side. Slide A1.

Fig. 22. — A more proximal section. Both limbs of the alimentary
canal are supported by the median mesentery ( mes .). Both longitudinal
blood-vessels (a.v., e.v.) are visible. Slide A1.

Fig. 23. — A more proximal section. The longitudinal muscles (l. m.)
form strong bundles. Slide A1.

Fig. 24. — Showing part of the invagination (inv.) at the proximal
end of the muscular part of the body. Slide A2.

Fig. 25. — Through the distal part of the non-muscular region of the
body. Slide A2.

Fig. 26. — From another series of “ transverse ” sections, through
the region close to the base of the lophophore ; n. r., part of the
nerve-ring; neph., nepliridia. (Zeiss C Obj.) Slide B1.

Fig. 27. — A small regenerating fragment which is not yet provided
with an orifice to its tube. Slide M.

Fig. 28. — Regeneration practically complete, the distal end of the
tube having been formed as a lateral oiitgrowth developed on the
proximal side of the septum (s.). Slide Q.

PLATE 9.

Fig. 29. — A regenerating specimen which is commencing to divide.
At the distal end, the muscular part (muse.) is developing, while the
lophophore ( l .) is recognisable owing to the presence of a septum
dividing its body-cavity from that of the metasome; /., zone of
transverse fission, accompanied by the formation of a tube-septum.
The adipose tissue (ad.) is present in large quantity. The proximal
end is lobed, indicating a previous fission, further evidence of which is
afforded by the tube-septum (s.). Slide L.

Fig. 30. — A larger specimen in the same condition. Slide O.

Fig. 31. — The first indication of fission is afforded by the formation
of the annular tube-septum (s1.), which in this case occurs at the
commencement of the non-muscular part of the body. Slide Q.

Fig. 32. — A later stage in the fission-process. The two products
of fission are completely separated, the distal one showing a lobed
proximal end, and the proximal one showing signs of regeneration
distally. Slide N.

Fig. 33. — The proximal member of the result of the occurrence of
fission, the evidence of which is the tube-septum ( s .) and the lateral
outgrowth of the tube (t.) on its proximal side. Slide Q.

VOL. 62, PART 2. NEW SERIES.

11

148

SIDNEY F. HAEM Ell.

Fig. 34. — A similar specimen of much smaller size. Slide N.

Fig. 35. — A very small regenerating fragment, the distal end of which
is growing through the annular tube-septum (s.) which presumably
indicates the previous occurrence of fission. Slide Q.

Fig. 36. — A large specimen, of a kind frequently observed, in which
the alimentary canal is thin and occupies only a small part of the
body-cavity : a condition which is probably due to deficient nutrition.
Regeneration of the distal end is taking place. Slide O.

Fig. 37. — The two specimens here shown have probably been
separated from one another by fission, as indicated by the septum
(s.) and the lateral outgrowth of the tube on its proximal side. The
lopliophore (Z.) of the proximal individual is in an early stage of
development. Slide N.

Fig. 38. — A very slender and presumably ill-nourished regenerating
specimen. Slide N.

Fig. 39. — The significance of the condition here shown has not been
ascertained. The entire muscular region has been invaginated, forming
an introvert opening to the exterior at or. The introvert contains
a regenerating distal end, in which the new muscular region (muse.)
and lopliophore ( l .) can be distinguished. By the formation of the
introvert the alimentary canal has been thrown into a loop, the portion
of which belonging to the descending limb (pr.) is seen to the right
of the introvert. The ascending limb of the alimentary canal probably
has a similar course, but it was not observed in this specimen. The
outgrowth of the proximal end of the body in a direction at right
angles to the axis of the original tube probably indicates a lateral
extension of the proximal end of the tube in order to provide room
for the elongation of the corresponding region of the body, the growth
of which, in this direction, would otherwise be prevented by the
septum s. Slide N.

Fig. 40.— Lateral view of the distal end of a mature specimen,
showing the epistome (ep.) inside the group of retracted tentacles.
Slide M.

Fig. 41. — Another stage of the condition shown in fig. 39. The
introvert contains a bundle of fully developed tentacles. This may be
either an earlier stage than fig. 39, in which case the original lophophore
has been completely retracted into the introvert, and was destined
to be thrown off later ; or a later stage, in which the new lophophore
has been completely regenerated. In this specimen there is evidence
that the ascending limb of the alimentary canal, as well as its
descending limb, forms a loop passing up one side (left in the figure)-
of the proximal end of the introvert. Slide M.

DEVELOPMENT OE TRICHOGRAMMA EVANESOENS. 149

The Embryonic Development of Tricho gramma
evanescens, Westw., Monembryonic Egg
Parasite of Donacia simplex, Fab.

By

J. Bronte Oatenby,

Exhibitioner of Jesus College, Oxford.

With Plates 10, 11, and 12.

Introduction.

Since the description of polyembryony in some parasitic
Hymenoptera by Marchal (1) the attention of a few zoologists1
has been turned to the interesting problems these forms
offer.

1 Sir Ray Lankester has kindly drawn my attention to the writings
of the Polish Embryologist, Ganin, whose work on Platygaster, carried
out forty years ago, is of great interest. Platygaster is a parasite on
the larvae of some Diptera (Cecidomyia), and its larval form is curiously
modified in early stages. According to Ganin the larva has neither
nervous, vascular; nor respiratory systems (Compare p. 20 of this paper),
and its last abdominal segment terminates in a curious caudal organ of
a tree-like nature, almost certainly concerned in nutrition. The larva
undergoes a number of moults, looses its caudal organ, and gradually
becomes vermiform. I have lately noticed that the larva of an Apanteles
parasitic on Porthesia has a remarkably modified ultimate abdominal
segment, which is very large, vesicular, and formed of hypertrophied
cells. The gut of this larva is in all the early stages completely blind,
and the animal depends ou the swollen abdominal segment for its
nutrition. Like Ganin’s larvae this form looses the vesicular segment
just before pupation. It is a very remarkable fact that the ultimate
abdominal segment should be modified for this purpose (Zeit. f. Zool.,
Bd. xv.).

150

j. bront£ gatenby.

Silvestri (2) has contributed some important papers on the
subject, but these have appeared in an Italian agricultural
journal only taken by a small number of the scientific
libraries of this country. Considering the vast number of
parasitic Hymenoptera which exist, and their diversity and
remarkable instincts, a rich field, only now being explored,
is opened to zoologists. But it is a field full of difficulties,
for the Trichogrammids, to mention one group alone, are, as
Perkins (4) , has said, among the smallest of known insects.
In several other groups of parasitic Hymenoptera there are
to be found numbers of forms whose life history and habits
are of absorbing interest. The pure observer finds problems
and instincts of wonderful diversity, and the embryologist is
impressed with the remarkable adaptations for the modus
vivendi which these forms follow.

The remarkable oogenesis of some of these parasites has
been the subject of some interesting papers by R. Hegner (3).

The parasite, a part of whose embryology I have described
in this paper, is a member of that important family the
Chalcididae, a numerous and highly interesting assemblage of
minute Hymenoptera. These insects are of great importance
to the economic entomologist, because among them one finds
forms which aid the agriculturist, and which often injure.
Trichogramma might be said to aid.

It is a pleasant duty to express my thanks to Mr. Goodrich
for his kind interest in this work, and for advice and criticism,
which has been of great value.

The Host1 (Donacia simplex, Fab.).

Donacia simplex is quite common around Oxford in the
early summer. Commander Walker informs me that he has
occasionally taken this species at Oxford in winter. In the
early summer one can always find the beetles on the water-

1 Kindly identified by Mr. H. Britten, Assistant of the Hope Depart-
ment, Oxford Museum.

DEVELOPMENT OF TRICHOGRAMMA EVANESOENS. 151

reeds which grow from shallow ponds and the sides of
streams ; they may be observed copulating and laying their
eggs. The latter are laid in masses in a regular manner, the
whole group forming a rectangular mass containing a varying
number of eggs. In one mass eggs of several shades of
brown may occur in patches, as if a number of beetles had
oviposited in the same place. Whether this is so I do not
know. The egg groups do not adhere very closely to the
surface of the reed, and they are easily removed by bending
the surface upon which they are laid. From the number of
parasitized eggs which one can find there is no doubt that
this Trichogrammid must cause a great deal of destruction
among the broods of beetles, and were the Donacia a pest on
valuable plants it would be quite easy and worth while to
rear batches of parasites. This has been done in the case
of parasites of injurious insects, particularly in America, and
such methods of attacking pests have so far met with a good
deal of success. In the case of Donacia almost the entire
number of eggs laid in a locality where the parasites are
common will be found parasitised. In PI. 10, fig. 3, is drawn
an enlarged figure of Donacia simplex; in A the egg
mass (OF.) viewed in profile upon the reed (P.) is shown,
and resting on the lower eggs a Trichogramma (P.P.) is seen,
drawn to about the same scale as the beetle.

The Parasite (Trichogramma evanescens, Westw.)

I have to thank Commander Walker for drawing my
attention to some literature on Trichogramma. The Rev. J.
Waterston, B.D., of the Imperial Bureau of Entomology, in
kindly identifying this insect, writes that Trichogramma
evanesce ns is generally found as a parasite upon the eggs
of insects whose habits and place of oviposition are similar to
that of Donacia.

As is common with many of the parasitic Hymenoptera,
Trichogramma evanescens has very gaudy colouring.
The wings, which are a shiny blue, at once attract attention

152

J. BR0NT6 gatenby.

to the insect as it walks over the Donacia egg mass. In
collecting my material I found it most convenient to examine
the rushes for Donacia egg-masses from a boat, and those
upon which parasites were seen were removed from the
water-plant and placed in a box. A most unfortunate circum-
stance, unknown to me then, was the fact that the time1 taken
to walk to the laboratory with the material was just long
enough to allow the newly-laid eggs to form polar bodies,
segment, and enter upon the blastoderm stage. Except in
the case of a small number of eggs laid in the laboratory, all
my sections begin from the blastoderm stage onwards, and
some important stages are missing. If the insect is taken
into the laboratory and placed with an egg mass of Donacia,
it is possible to watch oviposition taking place. The little
parasite may be observed to walk somewhat rapidly over the
eggs, continually tapping them with its geniculate antennae.
When is satisfied with the egg it has chosen it stops,
unsheaths its ovipositor, and moves its abdomen backwards
and forwards with a sawing motion about eight times, until
the chorion of the Donacia egg is pierced. When this
happens the parasite may be seen to depress its abdomen,
thrusting home the ovipositor. It pauses about five seconds
while the egg passes down the evipositor into the Donacia
egg, withdraws its ovipositor, and generally begins on the
next egg in the row. Though the parasite does not seem
to work systematically along the rows, in many cases all the
eggs in a mass are parasitised, though more often a few are
left untouched.

In cases where all the eggs have been parasitised several
parasites may have laid in one mass. It is quite common to
observe two or three Trichogrammids on one Donacia egg-
mass. In very rare cases there are two eggs laid in the
same Donacia egg ; one so seldom finds this that it is
probable that a parasite is able to tell whether one of its
fellows has previously given attention to an egg. What

1 Added to the fact that the fixative I used does not penetrate the
chorion of the beetles’ eggs as quickly as desirable.

DEVELOPMENT OF TRICHOGRAMMA EVANESCENS. 153

happens in development when two eggs are laid in the same
Donacia egg I do not know, but one generally finds the two
eggs in different stages of development. Probably the older
embryo succeeds in the end in killing the other, for I have not
yet found more than one insect imerging from one egg.

I am unable to say whether there is more than one brood
of parasites during the summer, but it is possible to collect
at the same time eggs containing parasites ready to emerge,
and some containing newly laid eggs. This points to there
being more than one brood. There are two or three species
of Donacia fairly common at Oxford, and they appear one
after the other, so that this strengthens the view that several
broods occur in one season. During the winter months I
have not found the empty egg-cases of Donacia on the stems
of the reeds, and I have not been able to satisfy myself as to
whether the parasite hibernate in the eg’g-cases or whether
they emerge in summer and creep into crevices with a view to
wintering there. Nearly every year the reeds upon which the
egg-masses are laid are submerged in the floods, and become
withered and torn, and thoroughly soaked. For this reason
it is unlikely that the parasites would remain in the eggs
which they have destroyed.

In remarking on the parasite and its host, I do not over-
look the possibility of T. evanescens being found on the
eggs of other insects.1

Technique.

The egg of Donacia is covered by a thick chorion which,
added to the yolk, makes sectioning a very difficult business.
The parasitised egg-masses were generally preserved in
Petrunchekewitsch, with a little more nitric acid than usual.
This often gave splendid results. A mixture of Petrunche-
kewitsch2 and Bouin2 was also tried with about equal results.

1 Prof. Poulton informs me that this Chalcid parasitises the eggs of
Dragon flies. I have since been able to observe this interesting fact
myself.

2 For these fixatives see Bolls Lee's Microtomists’ Vade-Mecum.

154 J. BRONTE GATENBY.

Ill some cases the eggs were pricked and the whole thrown
into picro-nitric.

After some trials Petrunchekewitsch was almost exclusively
used, and in most cases it gave a fine fixation, but not always.
In using this fixative it is not necessary to prick the eggs.
Ordinary preservatives like Bouin, corrosive acetic, or
Flemming will not penetrate the chorion. This at once
causes difficulties, for alcoholic fixatives are not always
reliable. The eggs were left over night in the Petrunche-
kewitsch and washed out in 70 per cent, alcohol.

When in xylol the eggs were pricked with a fine needle
and placed in the paraffin bath. It was not always possible
to successfully prick the eggs, but unless this was done it
was necessary to leave the masses longer in the bath. This
hardens the eggs and makes sectioning a dreadfully difficult
task. The eggs were cut in their groups, 5 jjl in thickness,
on a Yung microtome, each section being painted with
celloidin and ether. One could not be sure that the eggs
were not parasitised until after staining, and three or four
batches would often be cut without finding any stages. It
was only by staining overnight in Iron Haematoxylin that a
a suitable differentiation could be got. Ehrlich and the
carmines were useless. In some cases alternate slides were
counterstained in orange G. or dilute acid fuchsin.

General Facts Concerning the Appearance of the
Material in Sections and in Whole Mounts.

In PL 11, fig. 8, there is drawn a part of the section of a
parasitised egg-mass. The larval parasite (D.P.) lies in the
yolk of the Donacia egg, and a little to the right and lower
edge of the larva is the remains of the embryonic gut of the
host {Gr.). At N.S. are the remains of the Donacia larva’s
nervous system, and below at I is a still recognisable de-
generate leg. The parasite has reached the stage just before
it begins to swallow the yolk in which it lies. Abutting
against the chorion of the egg in the middle of the field are

DEVELOPMENT OF TR1CHOGRAMMA EVANESCENS. 155

the chorions of the neighbouring eggs, all of which were
parasitised.

It will be seen that when these eggs were attacked the
contained embryos had become far advanced and were almost
ready to hatch. Though one can observe a Donacia ovi-
positing, and a parasite on the same mass piercing and
depositing its eggs in the newly laid beetle's eggs, it is
possible to find eggs parasitised at any stage. If one removes
Donacia embryos from their chorion by means of fine needles
and stains them in paracarmine, one can often find the
developing parasites as in PI. 10. fig. 2, at _D.P. Now this
embryo lies at the posterior pole of the embryo beetle, and is
too far down to have been oviposited there. It may be that
this egg was an outside one of the mass and that the parasite
bored it from the side ; but such cases occur too frequently
in sections of eggs in the middle of the mass, and I am
inclined to think that in those Donacia eggs laid in a
horizontal position the developing Trichogramma embryo
may sink downwards. I cannot otherwise explain how
parasites* eggs are found in this position, because the beetle's
eggs seem too closely applied to one another to allow the
parasite to get its ovipositor between them, and reference
to PI. 10, fig. 1, will show how short the little insect's
ovipositor is. (Both fig. 1 and 2 are drawn to the same
scale: x 75.)

The Effect of the Deposition of the Egg in the
Developing Embryo's Body.

Primarily the effect is to arrest further development of the
host, but all life is not killed immediately, for living nuclei
are to be found much later on as the parasite develops. As
is well known, the nuclei in the yolk of an insect's egg are
very large, and such vitellophags become larger than the
other cells almost from the time they are established. I
believe that it is the vitellophags which manage to live longest
after the parasite has oviposited in the beetle's egg, and in

156

J. BRONTE GATENBY.

some cases degenerate, but evidently still living yolk cells
can be found in the gut of the young larval parasite.

In degeneration the nuclei become hy perch romatic, large
stainable masses collecting in both nucleus and cytoplasm,
the cell finally becoming a black shapeless mass. I am
inclined to believe that the large cells forming the serosa
also live longer than the ordinary embryonic cells, after the
Trichogramma embryo lias been developing some time.

The Ovarian Egg when Ready to be Laid.

In PI. 11, fig. 9, a longitudinal section of the nearly mature
ovarian egg is drawn. The egg is of an elou gated oval
shape, the anterior pole (J..) being somewhat broader than
the posterior, and the cytoplasm appears homogeneous except
for the occurrence at the posterior pole of a large dark
mass ( G.G.D. ), the so-called germ-cell, or germ line, deter-
minant. The probable nature, mode of appearance, and the
fate of this protoplasmic inclusion will be dealt with under a
separate heading. The follicle cells are much drawn out in
PI. 11. fig. 9, and it is very difficult to distinguish between
the wall of the ovary and the follicular layer. The nucleus
lies slightly towards the anterior yolk of the egg in the mid
line. It consists of a large condensed mass of chromatin
surrounded by a clear nucleoplasmic zone. In the latter
minute stainable granules may be found. The manner in
which this condensed form of nucleus is produced is, as far
as I am able to judge from my material of adult insects, the
same as that described by Hegner for Copidosoma (3).

The Newly Laid Egg.

In the eggs at this period I have found a small body near
the surface, which, I think, is the spermatozoon. In PI. 11,
fig. 10, this darkly staining body is seen to be surrounded by
a number of small granules. I have been unable to find any
signs of activity around this body as one would expect if it

DEVELOPMENT OF THICHOGRAMMA EVANESOENS. 157

were a spermatozoon, though in some insects no clearing of
the cytoplasm around the male pronucleus, or other event,
takes place at this period. In the stage later, during the
formation of the polar bodies, the granules which were
present in PL 11, fig. 10, around the male pronucleus ( M.P.N .)
cannot be seen, but the latter has penetrated further into the
egg. In all the sections of newly laid eggs that I have found,
the cytoplasm towards the central region of the egg has
become partially vacuolated and thinner, while the germ cell
determinant has become much more faintly staining. My
collection of newly laid eggs is not complete enough to show
whether this thinning out of the central region of the egg is
the rule, and it should be observed that in PI. 11, fig. 11,
which shows the formation of the polar bodies, this vacuolisa-
tion was quite absent. In PI. 11. fig. 7, I have drawn a
transverse section of an egg which shows the nucleus lying
in a central clear region, and quite close a denser part
of the cytoplasm containing a cloud of granules ( Q.G. ).

The egg, when laid, lies almost always towards the top of
the Donacia ovum, and it never has a definite orientation, for
in a section of a group of the host's eggs one cuts across eggs
in all directions. In a brood of parasites which I caught
emerging, some had their heads downwards in the Donacia
egg, some their abdomens. At the stage when the larva
begins to feed, it is forced to lie lengthwise in the host’s egg;
because it has by then become too long to lie in any other
way. It is obvious that the orientation of the pupating
Trichogramma larva in relation to the Donacia egg is not
governed by any special circumstance. Nevertheless it is
possible, though to my mind unlikely, that the larva may be
able to turn around at will witbin the Donacia egg.

The newly laid egg is provided with a vitelline membrane
and a thin chorion ( G.H. ).

Formation of the Polar Bodies.

In the one egg I found at this stage there were two polar
bodies (PI. 11, fig. 11). One polar body ( P.B b) has been

158

J. BRONTE GATKNBY.

extruded and lies on the surface of the egg. The spindle of
the second polar body is in the telophase and the chromo-
somes seem fused. No aster or centrosomes could be seen.
Around the neighbourhood of the forming polar body is a
clear zone, and a little above the dumb-bell shaped figure are
two large granules. I do not know exactly how these
granules arise, but I think that they are possibly extrusions
of the polar figures, for expulsion of granules from the nuclei
can be observed in later stages. The fate of the polar bodies
is not known. In Oophthora and Encyrtus they eventually
degenerate (Hegner, 3a).

The Stages between Formation of Polar Bodies and the

Blastoderm.

These are not described in the present paper ; through lack
of material last spring I have been unable to get the stages.
This spring I was able to procure a great deal more material,
with which I hope to describe the early segregation of the
germ-cells and the accompanying phenomena.

The Blastoderm Stage.

The material from the blastoderm stage onwards to the
formation of the young larva is very complete. The germ-cell
determinant at the posterior pole of the egg has, by the time
of formation of the polar bodies, become more faintly staining,
and considerably broken up (PI. 11, fig. 20, broken pieces
P.P.). Such broken pieces come apart, and the whole deter-
minant loses almost all affinity for stains of any kind. The
exact time at which the determinant usually disappears is at
present unknown, but very rarely one can find rather darkly
staining patches in the germ-cells of the blastoderm stage,
which may be the remains of the germ-cell determinant

(PI. 12, fig. 36 G.)

In PI. 11, fig. 12, the earliest blastoderm stage is seen in
longitudinal section. The number of germ-cells is very diffi-
cult to determine, for at about this stage the latter lose almost

DEVELOPMENT OF TRICHOGRAMMA EVANESCENS. 159

all affinity for stains. I feel quite sure that there are at least
six germ-cells in the blastoderm stage, but often one counts
more during later stages, in some cases as many as nine.
Mitotic division of the germ-cells between the blastoderm
stage and the adult larva I have not yet found, and I feel the
more certain of this at the period of germ layer formation,
because one never finds the germ-cells assuming a greater
affinity for chromatin dyes as they would do if they were in
the prophases of mitosis. From the time of their segregation
onwards to the formation of the larva the germ-cells are
resting. At the time when the larva has swallowed almost
all the yolk (see PL 12, fig. 38 and p. 25) the germ-cells seem
to become active again, but though I believe they begin to
divide by amitosis, I have not enough material of this stage
to feel quite certain of this. The germ-cells have the ap-
pearance of fig. 36 of PI. 12, and the granules (6r.), which
one can in rare cases discover, may be the remains of the
germ-cell determinant. In PL 11, fig. 21, the structure of
the germ-cells and the blastoderm nuclei is shown. The
arrangement of the latter is very peculiar and characteristic.
The nucleus consists of an oval nucleoplasmic zone, PL 11,
fig. 21 A. ( N.P .) in which is placed excentrically, and always
towards the periphery of the blastoderm, a large chromatin
nucleolus ( M.G. ). This large granule is rounded on the side
touching the edge of the nucleus and generally more irre-
gular on its inner surface. Placed on the periphery of the
nucleus, and always pointing towards the central region of
the egg, is a granule, or two, much smaller, and quite sphe-
rical ( G.R.C .) (P. 11, fig. 21). This remarkable arrangement
and the peculiar orientation of the nucleus and its granules
is quite clear. Observe also the transverse section in PL 11,
fig. 12, and in fig. 17.

This arrangement is quite constant and typical, but in one
blastoderm alone did I find a difference, and this lay in the
presence of other granules (0. G.) near the large main
nucleolar granule (M. G.)y PL 11, fig. 16. It may be possible
that the blastoderm was younger than its fellow drawn in

160 J. BRONT/2 GATENBY.

PI. 11, fig. 12, and that the exceptional nucleus is an inter-
mediate form.

The nuclei at the anterior end of the egg are orientated in
relation to the centre, as are those of the posterior. In the
central region of the egg are found a number of black masses
(Iron Hsematoxylin staining) of approximately the same size
and shape as the excentric nucleolar mass of the blastoderm
nuclei. That these masses are extrusions from the latter is
proved by the fact that all stages in their expulsion can be
found. In PI. 11, fig. 12, there were twenty- three in the
egg. When the nucleolar mass is shot out towards the centre
of the egg the nucleoplasm and the other granules break
apart. The former disappears, the latter may be found in
the egg (PI. 11, fig. 12, G. R. C.). In PI. 11, fig. 17, at X,
there is a space left in the row of nuclei ; exactly on the
same level, and quite near, are two nucleoli labelled Y. I
believe that the empty space was occupied by the chromatic
masses, both of which have lost their nucleoplasmic zone and
their small granule. Additional proof that my conclusion
concerning the character of these masses is correct will be
mentioned below. In PI. 11, figs. 12, 13, 16, and 17, the
central part of the egg is seen to contain extruded nucleoli.
I have been able to count the number extruded in various
eggs.

In PI. 11, fig. 12, there were twenty- three ; in PI. 11, fig. 13,
there were fifty-three; in PI. 11, fig. 16, there were twenty-
four; in PI. 11, fig. 17, there were thirty; and so on, the
number usually varying from twenty to fifty. In PI. 1 1, figs. 12,
16, and 17, are younger than Pi. 11, fig. 13, so fewer nucleoli
have been expelled. It is generally true that the younger
the blastoderm, the fewer the extruded nucleoli. Examina-
tion of the figures of blastoderm stages will fail to reveal
any dividing nuclei, and none are ever found in the sections.
It is quite obvious .that if it is true that nuclei are extruded
and no division takes place, one should find a decrease in the
number of nuclei in the growing blastoderm. Up to a certain
point this is so. In PI. 11, figs. 12 and 14 are both longitudinal

DEVELOPMENT OF TRICHOGRAMMA EVANESCENS. 161

sections through the egg, the former at a time when most
nuclei are present, the latter when fewest are present and
just before multiplication begins again. PI. 11, fig. 12, ha&
thirty-eight nuclei in the section; PI. 11, fig. 14, has thirty.
Counts of a large number of sections yield similar results,
though the total number of nuclei in a number of blastoderm
stages varies a good deal. From PI. 11, fig. 12 to fig. 14, it
will be noticed that the egg has broadened and contracted in
length a good deal. Measured roughly from the camera
lucida drawing, PI. 11, fig. 12, is a centimetre longer than
the much older stage PI. 11, fig. 14. We then realise that
two curious processes take place at this time ; one, the expul-
sion of as many as fifty nuclei, the other, an obvious
shortening and broadening of the egg. Explanations for
both occurrences are difficult to formulate. In cases where
no shortening can be shown to have occurred it is equally
true that no lengthening has taken place, so that it remains
correct that the developing egg departs from the proportions
which it had when laid. A relative shortening always occurs,.,
i. e., in comparison of lengths and breadths of the eggs at
different stages, for PI. 11, fig. 8, is one and three-quarter
times as broad again as PI. 11, fig. 12, and a little shorter.
PI. 11, fig. 13, is the later stage of the blastoderm. The egg
has become relatively broader and shorter, and important
changes have been taking place in the nuclei. It has already
been remarked that in this egg fifty-three of these have been
extruded. The germ-cells now stain quite faintly, but their
arrangement is still unaltered. Most of the blastoderm
nuclei in PL 11, fig. 13, are the same as those in PI. 11,
figs. 12 or 21, but others show differences. Many of them
have lost their small spherical granule, which was directed
centrally, and in these the large nucleolar mass has shifted
from its position in the periphery of the nucleoplasmic wall
(PI. 11, fig. 21a) to the middle of the nucleoplasmic zone
(PI. 11, fig. 13a, 2 and 3). The latter fig*ure is much
enlarged and shows three stages in the alteration of the
nuclear arrangement. At a later stage these changes become

162

J. BRONTE GATENBY.

widespread, and by the stage in PI. 11, fig. 14, no granules
are left and all nucleolar masses are found in the mid-region
of the nucleoplasmic mass. I have not discovered the early
blastoderm form of nucleus in any other stages.

PI. 11, fig. 18, is a transverse section of an interesting-
stage. It shows that the blastoderm nuclei have grown and
that changes have taken place in their disposition, while the
mass of extruded nuclei which, in PL 11, figs. 12, 13, 16, and
17, was situated in the centre of the egg, appears to be
shifting outwards. Now, an examination of all later stages
after the blastoderm will reveal the fact that the extruded
nuclei leave their central position in the egg, and pass to the
periphery (see PI. 11, figs. 14, 15, 18, 19, 24, 25, and 27,
E. X.N.).

It is just after the stage drawn in PI. 11, fig. 12, that this
occurrence takes place, and PI. 11, fig. 18, shows what
happens. The central mass containing the nuclei, as is seen
in PI. 11, fig. 13, is somewhat vacuolated. Almost the whole
of this central region streams out to the periphery, carrying
the extruded nuclei with it, and breaking through and
disarranging the layer of blastoderm nuclei on one side; in
the process several healthy nuclei are carried out as well
(PI. 11, figs. 18 and 19, L. E. N.). The space left by the out-
streaming mass is soon closed up, and the disarranged nuclei
resume their places ; the new membrane appears between the
re-formed blastoderm and the extruded mass (M. B., in PL 11,
fig. 19).

Regarding the position in which this final expulsion of
extruded nuclei takes place, though no absolute regularity
exists, it is a fact that the outbreak appears generally towards
the middle at any place, but more often than not on the future
dorsal side of the embryo. In PL 11, figs. 14, 18, and 19, it
was ventral; in PL 11, figs. 15 and 25, it was dorsal. In
PL 11, fig. 14, it wiis near the posterior pole; in the others
about median.

As will be seen in PL 11, figs. 15, 18, 19, and 27, at
E. X. N. this extruded mass is quite large and consists of the

DEVELOPMENT OF TRICHOGRAMMA EYANESCENS. 163

wider reticulate central part of the egg. After its expulsion
the widely reticulate central part of the egg (PI. 11, fig. 13)
disappears (observe PI. 11, figs. 14, 18, and 19). A partial
vacuolisation may reappear secondarily, as in PI. 11, fig. 15,
but this is rare. Further description of the fate of this
extruded mass will be postponed, but it remains for a good
while lying between the chorion (PL 11, fig. 19, V.M.)
and the re-formed blastoderm, often becoming much
flattened.

The Appearance of the Germ Layers.

The expulsion of the inner waste mass is a preliminary to
the incipient formation of the germ layers. On what is later
the dorsal surface of the embryo a longitudinal groove
appears, and beneath this groove the regularity of the
arrangement of the blastoderm nuclei becomes disturbed. On
a space oocupied by about five or six nuclei broad and seven
or eight nuclei long a gradual sinking-in begins. In PL 11,
fig. 18 and 19, the groove is marked I. N. V. and the sinking
nuclei N. 8. 1. In PL 11, fig. 14, the egg at this period is seen
in longitudinal section.

This process is undoubtedly gastrulation, though in view
of the fact that the representative of the blastula is solid the
event is somewhat disguised. PL 11, fig*. 19, has a striking
resemblance to a gastrulating blastula, though there is no
segmentation cavity or blastoccele. That the groove repre-
sents the early blastopore ( I.N.V .) I have no doubt, and were
the depression to become deeper it would form the mesenteron.
As it happens, this never takes place, the cavity of the gut
being formed in a different way.

It has already been shown that about the stage in PL 11,
figs. 13, 14, the nuclei loose their granule, and the large
nucleolus becomes placed in the centre of the nucleoplasmic
zone. By the stage in Pl. 11, fig. 14, this has taken place in
every nucleus. In this figure the blastopore (I.N.V.) appears
on the dorsal surface of the anterior end of the egg, but its

VOL. 62, PART 2. NEW SERIFS. 12

164

J. BRONTE GATENBY.

position varies little. The row of nuclei which will form
most of the gut, and which are now sinking in ( N.S.I .) are,
on the average, a little bigger than the other nuclei. At thb
anterior pole of the egg, near the letters E.X.N., is seen an
extruded nucleus. It is a fact that though the main expul-
sion of nuclei occurs between the stages in PI. 11, fig. 12 and
fig. 13, even after the throwing out of the central part of the
egg which contains these large granules, sporadic extrusion
may take place. That these later extrusions do really occur
is shown by comparing the size of expelled granules. In
PL 11, fig. 14, the granule in the anterior end of the egg is
twice as large as those extruded earlier at the posterior region.
(Compare also PI. 12, fig. 32.)

The germ-cells in PI. 11, fig. 14, have changed their posi-
tion somewhat, becoming arranged towards the ventral edge
of the posterior pole. In this figure the germ-cells are drawn
a little darker than they should be. PL 11, fig. 19, is drawn
from such a transverse section as that through K. in fig. 14.
The insinking nuclei (N.S.I.) are shown.

Such an arrangement does not last long, for as the nuclei
sink inwards they lose their order. This is caused by
the fact that some lag behind while others penetrate more
quickly towards the centre of the egg. This is shown in
PL 11, fig. 15, at N.S.I. By this time these nuclei have
become very large. The relationship of the various nuclear
elements in the egg now becomes more complicated, because
at intervals around the periphery other nuclei grow larger
and sink inwards (PL 11, fig, 15, at X.Y.). All these nuclei
are quite distinct from those which were the first to begin
sinking inwards, and I feel sure that some of them at least
contribute to the formation of the gut. Others form loose
cells lying in the cavity between the gut and the ectoderm.
Often just before and at this stage amitotic division of nuclei
is found taking place. Moreover, the chromatic arrangement
of some of the nuclei changes curiously. In these the large
centrally placed nucleolus becomes ragged at the edges and
pieces break off and become arranged around the periphery

DEVELOPMENT OF TH1CHOGRAMMA EVANESCENS. 165

of the nucleoplasmie zone. This process may go on till the
nucleus becomes normal, that is, until a rough reticulum is
produced, and sometimes the chromatin becomes very sparse.
These changes are shown in PI. 12, figs. 40-43. In PL 11,
fig. 15 such nuclei are marked N., and in PL 11, fig. 25, there
is a large group of them towards the centre of the embryo.

I do not know the reason for this reversion to the usual
chromatic arrangement, but at a much later stage during
pupation the early abnormal form of nucleus gives place to
the normal one. The nervous system of the larva, for instance*,
is formed of nuclei having quite a different chromatic
arrangement from that of the adult. I feel convinced that the
curious form of nucleus found in the larva is connected with
the unusual metabolic conditions to which the developing egg
is exposed. Inspection of PL 11, fig. 15, will show that a
large number of nuclei are sinking inwards, but among them
the nuclei marked N.S.I., which are the original endoderm,
are remarkable for their size. In this figure the extruded
nuclei and the inner mass of the egg have been thrown out on
the mid-dorsal side, and lie in the space formed by the gas-
trulating periphery of the ovum ( INV .). The germ cells lie
towards the ventral edge of the posterior pole of the egg
and have sunk inwards ; at Z. the edge of the blastodernl
tends to embrace the pocket in which the germ cells lie.
The latter stain very faintly, and form a light area on the
posterior pole of the egg. Fig. 23 of PL 11 is a transverse
section through this part of the egg, near the letters A -A ill
PL 11, fig. 15. The latter figure is a little earlier than the
former. In PI. 11, fig. 22, drawn at twice the magnification
of either PL 11, figs. 9 or 23, is an oblique longitudinal sec-
tion of the posterior pole of the egg to show both the manner
in which the germ cells sink into the egg in the form of a
pocket (G-CP.), the neighbouring blastoderm nuclei ( X.X .)
surrounding and protecting the pocket, and the relative
staining power of the egg cytoplasm and the germ cell cyto-
plasm. Up to the stage drawn in Fig. 15 of PL 11, the
somatic nuclei of the egg are scattered in a syncytium ; in

166

J. BRONTE GATENBY.

Fig. 24 of PL 11a transverse section of the embryo is drawn
at a stage when the cell outlines begin to appear. At the
places where the body cavity is formed, the syncytium becomes
thin and vacuolated, and between the future cell elements,
cell walls are deposited. In PI. 11, fig. 24, the large endo-
derm cells have become arranged in a definite manner
(END.N.) , and the beginning of the lumen of the future gut
is seen at GL. Beneath the ring of endoderm nuclei (END.N.)
a large cavity ( CAV .) has already appeared, but otherwise
the separation into regions is still slight. On what is the
ventral side of the embryo, at the letters NCN, will be noticed
three rows of nuclei. The upper row ( MCN .) just beneath
the embryonic body cavity (CAV.) becomes detached by
further vacuolisations in the region marked X, X, and in the
larva becomes loose in the body cavity (PL 12, fig. 37, MCN.).

Of the two lower rows, the bottom one, and at least some
of the upper row nuclei, form the nerve chain of the adult.
Fig. 39 of PL 12 should be compared with this figure.

In PI. 12, fig. 39, the body cavity is better formed (CAV.).
It will be noticed in PL 11, fig. 18, that there are four large
nuclei marked Z which do not seem to be included in the
forming gut. The upper two may form such large glandular
cells as those marked Z in PL 12, fig. 39, for in this figure
it will be noticed that in places the wall of the gut (GL.) is
formed of two rows of cells. One can often find very large
unattached cells in the newly-formed body-cavity, and these
may break up later on (PL 12, fig. 39, XX.). Immediately
after the final sorting up of the cell elements, and after each
nucleus has taken its place, there is an expulsion of super-
fluous cells, which degenerate either in the hsemocoel, or are
cast from the surface of the ectoderm (PL 12, fig. 39, X, X.).

The Formation of Stomodeum, Mesenteron, and
Proctodeum.

As far as one can tell in a case where such wide variation
occurs, the large dorsal mass of nuclei which sinks inwards

DEVELOPMKNT OF TRIUHOGRAMMA EVANESCENS. 167

takes part in the formation of no organ except the mid-gut,
but, as I have already pointed out, some of the cells forming
the mesenteron may conceivably be of another origin,
namely from nuclei which sporadically wander in from the
periphery on other parts of the surface of the embryo
(PI. 11, fig. 15, XT.). From the first the nuclei destined to
form the mid-gut are conspicuous by their large size and
rapid growth. The lumen of the mesenteron appears just
after the stage drawn in PI. 11, figs. 27 and 28. It seems to
be formed by an internal delamination of the solid endo-
dermal cell mass in some cases, but in others it looks as if,
during growth, the ring of cells, gradually enlarging, left a
lumen in their centre, just as the lumen is known to appear
in an ordinary duct. In any case there is always a residuum
left in the developing lumen (PI. 12, figs. 27 and 28). After
the endodermal cells have grouped themselves as shown in
PL 11, fig. 24, the proctodaeum aiid stomodaeum begin to be
quite recognisable ; and there is no doubt that the latter is
formed by a regular invagination (PI. 12, fig. 31, ST.). The
manner in which the proctodaeum is formed is a little more
doubtful. In the case of the stomodaeum the invagination is
normal (PI. 11, fig. 27). The iupushing cells meet the
roughly disposed endoderm cells, and when the final dis-
solving out and disintegration of that part of the embryo
which forms the body-cavity takes place the connection
between the stomodasal and mesenteron cells remains unbroken.
The same thing applies to a region where the proctodaeum is
formed, but it is difficult to be sure of a true invagination
such as occurs with the stomodaeum. The latter is formed of
much smaller cells than the proctodaeum, and is longer, while
the demarcation between mesenteron and proctodaeum is
quite indistinct. In PI. 12, fig. 30, which is a horizontal
section of the front region of a larva of the same age as that
drawn in PI. 12, fig. 38; the stomodaeum, mouth, and
mesenteron are shown. In PI. 12, fig. 34, there is a longi-
tudinal section of the proctodaeum of a somewhat younger
larva, but it serves to show how short the hind gut is. This

168

J. BRONTE GATENBY.

seems to be the rule in many Hymenopterous larvae. In the
oldest larvae I have found there is no oesophageal valve
formed, nor is there any differentiation in the proctodaeal end
of the gut.

As the larva grows it swallows all the host’s yolk in the
egg, and no defecation takes place until every yolk disci et
has been swallowed ; by this time the animal is enormously
stretched, and the body-wall and gut-wall are so thin as to be
overlooked unless care is taken. PI. 12, fig. 38, is drawn
when the swallowing is well advanced, PI. 12, fig. 33, when
the first food has reached the mesenteron. When the larva
has finished swallowing the yolk, it occupies almost the whole
extent of the egg.

The Head Region of the Larva of Trichogramma.

In PI. 12, fig. 30, I have drawn the horizontal section of
the head region. The mouth {MTU.) is a simple opening;
but pointing forwards and outwards are two extraordinary
horn-like processes {PRC.). These are seen to protrude
from a pair of lateral thickenings — one on each side of the
head. These thickenings arise quite early, and are closely
associated with the inner side of the epidermis. In PI. 12,
fig. 29, {TH.) 1 have drawn a transverse section of a younger
head to show the thickenings before the horn is secreted from
them. Beyond this curious organ I have been unable to
discover any other mouth parts whatsoever.

The Late Larva.

In the stage when the larva has swallowed all the yolk
several facts may be noticed.

The first is absence of tracheae ; the second, absence of any
external sign of segmentation ; and the third, the absence of
completely differentiated muscles or heart.

The larva is merely an ovoid sac, provided in front with
two horn-like processes, and with an opening at either end,
for taking in food and casting out waste matter ; internally
there is a gut divided as usual into three regions ; and finally

DEVELOPMENT OF TRICHOG-RAMMA EVANESCENS. 169

there is the single median ventral germ-cell pocket, beneath
the proctodaeum.

In PI. 12, fig. 30 ( CU .), a distinct cuticle could be seen. It
dipped into the pockets from which the horn-like jaw-pro-
cesses protruded, and the latter are probably cuticular in
nature. The thickening ( TH .) is ectodermal. Cuticle (chitin)
was found in the stomodaeum, but I am not quite sure
of its presence in the proctodaeum. It is possible that the
processes are used for scooping up the yolk of the host as the
larva feeds, and they are probably much modified mandibles.

When the larva has swallowed all the yolk, very often not
the smallest particle can be found outside its gut, and exactly
how the yolk at the posterior end of the host's egg is worked
to its mouth is impossible to say ; but it is probably by means
of movements of the body that the unswallowed parts are
brought forward.

The Fate op the Extruded Matter.

In PL 11, fig. 15, the extruded mass still lies within the
vitelline membrane of the egg. As the larva grows the
membrane becomes stretched and the waste mass flattened;
but, though it remains intact for a good time, it eventually
bursts. The extruded mass then floats free in the yolk of
the Donacia egg. In PI. 11, fig. 27, EM., it is shown to the
right of the ventral side of the posterior pole of the embryo.
In PI. 12, fig. 35, it is seen quite close to the embiyo at EM.

Curiously enough these fragments seem to live a good
while, and nuclear changes, such as those undergone in the
blastoderm, take place in some cases.1 The mass may becom
spherical, as in PI. 12, fig. 32, and may resemble the egg
itself. Eventually the mass either degenerates outside the

1 One is tempted to entertain the view that this peculiarity may he in
some way or other connected with a faculty that culminates in the
establishment of polyembryony. Were the extruded mass to contain
enough live nuclei it might partially follow the development of the
embryo.

370

J. BRONTE GAT UN BY.

embryo or is swallowed by the latter. The live nuclei, to
which the temporary persistence of the extruded mass is due,
may develop the microsome granule ( GEC .) drawn in PL 11,
fig. 21 a. This is the case with the nuclei marked LEN. in
Pi. 12, fig. 82. (See addendum, p. 30.)

The Nervous System.

The nervous system can be recognised very early ; it arises
from the multiplication of ectodermal cells in the usual
manner found in insect larvae, but it never becomes properly
separated off from the ectoderm. Even in late larval life the
nervous system seems l< coarsely” made; that is to say, it is
formed of comparatively few cell elements which are not
differentiated in the characteristic manner, and there are no
such things as nerves in the sense of offshoots or twigs to
organs, such as exist in other larvae, such as Yespa. The
nerve-cells do not differ in any way from other cells in the body,
always excepting germ-cells. In PI. 11, fig. 18, the nerve-
chord is seen in a rudimentary condition, and consists of the
bottom row of nuclei marked N. C. N., and an unknown
number of the row above. In PL 11, fig. 21, the brain (BE.)
and nerve-chord ( N.C .) are cut longitudinally. In PL 11,
fig. 22, PL 12, figs. 33 and 38, a better view of the chord in
transverse section is seen, and in PL , fig. 29, the brain (BE.)
is cut transversely, to illustrate its close connection with the
epidermis (E/P.) and oesophagus (S TD.). No such things as
ganglia exist, and the chain ends a little before the germ-
pocket; it does not reach the proctodaeum. In late stages
(PL 12, fig. 88, N. C.) it becomes an increasingly difficult
matter to recognise the chain, so stretched does it become,
and by the time the larva has swallowed all the yolk in the
Donacia egg, the nervous chain is for most of the hinder
part of its length quite unrecognisable. The oesophageal
connectives seem to consist of single cells applied to one
another (PL 12, fig. 30, CES. CON.), and are extremely
rough.

DEVELOPMENT OF TK1CHOGKAMMA E-VANESCENS. 171

The Amitotic Division in the Developing Embryo.

It has been shown that the number of nuclei in the
blastoderm stage becomes subsequently reduced, but that soon
afterwards, at about the stage in PI. 11, fig. 15, amitosis can
be found. Mitosis never occurs in the stages I have examined,,
and I suspect that it never occurs at any stage of develop-
ment ; but between polar bodies and blastoderm, and larva and
pupa, I have no stages. I cannot find mitosis in the ovary
of the imago, but my series is not satisfactory, and subse-
quent work may cause me to alter my views. In the dividing
nucleus the large median chromatic body may be seen to
elongate (PI. 10, figs. 6a and 6b), while the nucleoplasmic
zone ( NP . Z.) is unaltered in shape. The nucleoplasmic
zone soon constricts and becomes elongate. The chromatin
mass becomes roughly dumb bell-shaped, and the nucleus
divides into two by a constriction (PL 10, figs. 15 a and b).

From the scanty evidence afforded by PL 11, fig. 11, it
seems probable there is no proper mitotic figure in the polar
bodies. The figure draw in PI. 11, fig. 11a, closely resembles
the stages of amitosis in the embryonic nuclei, except for the
absence of the nucleoplasmic zone. It is probable that mitotic
figures will be found during and after the formation of the
pupa. . The probable reason for the absence of mitosis during
early development is evidently connected with the explana-
tion of the form of the nucleus. (See the discussion, p. 26.)

Mesoderm.

In the section of the young larva one always finds loose
cells in the body cavity. These I believe to be mesoderm ;
such cells are shown in PI. 11, fig. 27, MC. ; PI. 12,
fig. 33, MC. ; fig. 34, X. The formation of mesoderm is quite
unaccompanied by the appearance of mesoblastic somites;
these cells which form the mesoderm are derived from nuclei
which sink inwards from the periphery in the stage of fig. 15,
PI. 11, but as the disposition of such nuclei varies I find it impos-
sible to state exactly where they arise. It will be clear, after

172

J. BRONTE OATEN BY.

an examination of PI. 12, fig. 39, that the cells marked MCX.,
which form the mesoderm, appear in a scattered manner,
being set free by vacuolisations which rise around them as
the body-cavity is formed. It has already been noticed that
at this stage many such cells degenerate completely ( X , X.,
PI. 12, fig. 39), and the number which persists in the young
larva is never constant.

It is the body cavity cells which most usually exhibit that
curious resumption of the reticulum of the nucleus shewn in
PI. 11. figs. 27 and 28, X , and in PL* 12, fig. 34, X. The fate
of these cells, and the part they play, if any, in histolysis, I
do not know at present, but at the stage when the larva has
swallowed up all the yolk in the Donacia egg, they seem few
in number and much compressed, while their nuclei never
show the reticulate structure. Some of the loose cells in
the body-cavity also form muscles, becoming slightly flattened
under the ectoderm.

The Germ Cells.

Trichogramma evanescens is one of those remarkable
animals where a definite difference can be seen very early to
exist between germ cells and soma cells; the difference
between the two lies in the presence of a germ cell determi-
nant in the former. At the time of segregation we know,
from the cases of such insects as Chiron omus or Calligraplia,
the germ cell determinant becomes included in the pole cells
which later form the gonads, a'nd in some special examples
pieces of the broken-up determinant can be found in fairly
late stages of development. The germ cells at the blasto-
derm stage PL 11, fig. 12, fig. 14, and fig. 15) have been
described. They have already lost a great deal of affinity
for any stains, and in bad preparations the nuclei can hardly
be found. Xot long after the extruded centrally-placed
nuclei are finally thrown out to the periphery of the egg, the
germ cells begin to sink inwards. Exactly what causes them
to move in this manner I am quite at a loss to say, but it is
easy to watch the eveut taking place. In the fully ferified

DEVELOPMENT OK TRICHOGRAMMA EVANESCENS. 173

larva the germ cells lie in a pocket beneath the proctodeum,
that is, on the ventral edge of the body-cavity. In the
earliest stages the germ cells may be seen moving in this
direction (fig. 14 of PL 11, in the direction of the arrow).
One germ cell (M.) has begun its migration. By the stage in
PL 11, fig. 15, the germ cells have sunk right into the ventral
edge of the posterior pole, pushing aside the blastoderm
nuclei. In Pl. 11, fig* 22, which is a somewhat oblique longi-
tudinal section, this inpushing is finished, and the germ
pocket is formed by the nuclei ( X ., X.X.). The latter are
quite early set aside for this work, and continue in that
position in late larval life. During the time the other organs
are being differentiated the germ cells remain closely embraced
by these cells ; and just when the lumen of the gut is appearing
(Pl. 11, figs. 27 aud 28) the germ pocket has the appearance
drawn in PL 12, fig. 37, in transverse section, and in PL 12,
fig. 34, in longitudinal. The germ cell socket is enclosed by
about four cells, and contains the germ nuclei in what appears
to be a syncytium, though faint cell outlines and slight
vacuolisations can sometimes be noticed. The germinal
cytoplasm stains very faintly in plasma dyes. In PL 12,
fig. 34a, I have drawn an enlarged view of the pocket in
order to show the staining reactions. In the case of nearly
every nucleus the nucleolus alone can be made to stain.
Regarding the number of nuclei in the pocket I could count
seven in one, and in another six, but there were always
doubtful nuclei at or on the edge of the syncytum, which
may or may not have been germ nuclei ; it is probable that
the number of germ cells is subject to variation, though I
have never found less than six.

After the stage drawn in PL 12, fig. 34, my material is not
very good, but at a stage a little after the time the larva has
distended itself with the yolk of the host, the germ cells
seem to become almost similar to the somatic cells, and
•amitotic division begins. The exact details and further
confirmation of the facts cannot be given at present.

It will be noticed in PL 11, figs. 21, 22, and Pl. 12, figs.‘34

174

J. BRONTE GATENBY.

and 35, that the germ nuclei gradually lose all staining
power, except that of the chromatic nucleolus, the reticulum
disappearing. In later stages, when the germ cells begin to
stain more heavily, only the nucleolus can be made to take
up chromatin dyes.

With regard to the migration of the germ-cells from out-
side the embryo inwards (PI. 11, figs. 21 and 22, no pole
canal could be recognised. The germ cells seem to sink in
passively, and never become amoeboid as in Calligrapha

(3).

The (Term C ell Determinant .

The origin of the germ cell determinant, even in those
insects where the eggs are larger and technique easier, is
still in doubt. I have examined several Hvmenopterous
insects parasitic upon Aphids, and find that the determi-
nants appear as a cloud of granules towards the posterior
pole of the egg.

In Trichogramma the determinant is densest and most
darkly staining during the period in which it still lies in the
ovarian tubule, but is just about ready to lay (PI. 11, fig. 9).
Py the time the egg has been laid and the polar bodies are in
process of formation the determinant loses a great deal of
its affinity for stains, and begins to break into pieces (PL 1 J,
fig. 26, P, P.) At the blastoderm stage the determinant has
completely disappeared, and with the exception of the rarest
cases nothing of it remains (PL 12, fig. 36, G.). Indeed at
at this stage the cytoplasm of the germ-cells, instead of
staining more heavily than that of the somatic syncytium,
as one would expect, has lost a great deal of staining power,
both in nucleus and cytoplasm. This soon becomes very
accentuated (PI. 11, fig. 22). In the newly laid eggs in
PL 11, figs. 10 and 20, the germ cell determinant has become
rather shrunken and faintly staining*, though in the case of
Pi. 11, fig. 11, the determinant has a good deal more affinity
for stains.

DEVELOPMENT OF TKICHOGRAMMA EVANESCENS. 175

Discussion.

The Significance of the Nuclear Changes during
Blastoderm Stage. — Many nuclei (from twenty-five to
fifty-five) are cast out altogether. Others, as far as I can tell
all of them, extrude the microsome or small chromatin
granule, marked GRC. in PI. 11, figs. 16 and 21. In some
cases there are two granules of the same size, both of which
are expelled into the cytoplasm. No granule can be found
to be extruded from the germ cells, and it might follow
therefore that the latter, at this period at least, contain
more chromatin than the ordinary blastoderm nucleus. In
Miastor, Kahle (5) and Hegner (3) have described a definite
chromatin diminution process whereby the somatic nuclei are
deprived of a part of their chromatin during certain divisions.
Though I do not overlook the possibility of a homologous
occurrence taking place in Tricliogramma evanescens,
I am more inclined to believe that another explanation
should be attached to the remarkable chromatin diminution
in the parasite. In the first place the chromatin diminution
in Miastor takes place quite early, before the blastoderm is
formed completely, and, moreover, the process is brought about
in a different manner, not by extrusion of a granule, but by the
discarding of the larger part of the chromosomes during the
mitotic division, only the extreme ends of the chromosomes
going to the opposite spindles at the telophase. The residual
mass in the middle of the spindle undergoes degeneration.

No satisfactory explanation of the occurrence in Miastor
has been advanced, bat in Trichogramma evanescens I
would suggest that the process is connected with the curious
metabolic influences which must affect the nuclei. It must be
remembered that all nourishment which is necessary for the
development of the egg, and which is ordinarily provided
by the central mass of yolk of the insect-egg, is, in the case
of this parasite, derived from the yolk of another insect’s
egg and without the aid of vitellophags. Such nourishment

J. BliONTE GATENBY.

176

must be received over the surface of the ovum, and it follows
that the surface nuclei must be partly engaged in the taking
up of the food matter. A glance at PL 11, fig. 7, and fig. 28,
will show how enormously the egg has grown during develop-
ment. Both figures are drawn to the same scale, and the
embryo in PI. 11, fig. 28, had not yet begun to swallow food.
All the food necessary for this growth has been derived
through the surface of the embryo and of the developing egg,
and without the help of yolk cells, which are so characteristic in'
hexapod embryology. The form of nucleus in the blastoderm
must be the one suited to the requirements of the developing
embryo, and the occasional expulsion of whole nuclei, and
the constant extrusion of the granule, is probably due to the
fact that the nuclei become liyperchromatic. That this
nuclear arrangement is artificial and temporary is shown, in
the first place, because it is not found in the adult insect
(follicle cells of ovary excepted) ; and secondly, because
there is always a tendency for the nuclei to regain the
normal reticulate arrangement. It is as if the forces which
suppressed the usual chromatic arrangement were overcome
now and again, but soon recovered their power. To illustrate
this suggestion it may be mentioned that the changes shown
in PI. 12, figs. 40-48 take place sporadically. Nuclei like
that figured in PL 12, fig. 43, occurred in the embryos in
PL 11, figs. 27 and 28 (A.), were absent in Pl. 12, fig. 33,
but were common in PL 11, fig. 15 (N.N.), and were found
to occur in a scattered manner right up to the formation^of
the larva, when they became suppressed. It was particularly
in the loose cells in the body cavity that such nuclei were
found, and it seems fair to conclude that these are the cells
which would be least affected by the metabolic influences
surrounding the embryo.

The occurrence of the modified nucleus in the follicle cells
of the adult iu sect's ovary is due to the fact that such cells
are exposed to somewhat the same conditions as the nuclei in
the embryo, and are engaged in passing on food to the ovum
(PL 11, fig. 9, FN.).

DEVELOPMENT OF TKICHOGUAMMA EVANESCENS 177

Hypercliromatic nuclei are known to occur in nurse cells
of insects, in various cells of vertebrate foetal membranes,
and in many tissues concerned in nourishment, and where
these nuclei do not become noticeably hypercliromatic, they
generally hypertrophy.

The extruded granules are, therefore, to be regarded as
superfluous chromatin, which has arisen through the peculiar
conditions to which the blastoderm nuclei are exposed.

Formation of the Gferm Layers. — In view of the-
fact that the egg of Trichogramma is not provided with yolk
the formation of the germ layers is of great interest, for the
yolk profoundly alters the organogeny in the usual hexapod
development. That one would receive a faithful representa-
tion of the ancestral mode of development of the insect,
from the case of Trichgramma is too much to expect, because
the method of development, though primitive in some
respects, is overshadowed by the effects of the parasitic mode
of life. The blastoderm stage is without doubt quite
normal, and except for minor nuclear phenomena differs not
at all from that of the host or of Miastor (3), but the events
leading to the formation of the endoderm are interesting.
That the progress figured in PI. 11, figs. 14 and 19, is one of
gastrulation one hardly doubts. In the case of Polygnotus
minutus Marchal (7) describes how the embryo is formed
by a complete invagination of one side of the hollow blastula,
to form a two-layered gastrula. The method of gastrulation.
in the parasite treated in this paper is somewhat less distinct
than in the case of Polygnotus, and before the process is
far advanced a secondary insinking of other peripheral
nuclei almost completely obscures it (compare PI. 11, figs. 14
and 15.)

The manner in which the endoderm is formed in Tricho-
gramma is of very considerable interest in view of the dis-
cussions which have been caused by the different opinions
expressed by several authors (Dohrn, Kowalevsky, and
Granin (7) ), but it is not intended here to review their widely
different suggestions in the light shed by Trichogramma.

178

J. BRONTE GATENBY.

Germ Cell and Determinant.

In the ordinary Hymenopterous larva (e.g. Vespa) the
germ cells lie about two-thirds way in the length of the body
and above and resting upon the mid-gut.

In the Tricliogramma larva the germ cells are situated at
the posterior pole and ventral to the proctodasum. In the adult
insect the ovaries occupy the same position as they do in the
Vespa imago. Migration of germ cells is very small in the
developing embryo. In most insect embryos the germ cells
.are carried into the tail fold, and may be said to either
migrate or be passively carried a good distance, but except
for the early insinking of the germ cells and the formation of
the germ pocket in Tricliogramma the position of these cells
is hardly altered.

I have looked carefully at my sections of the adult ovary,
and find that the germ cell determinant appears as a cloud of
granules, which become more and more heavily staining, and
denser and denser, until the determinant resembles a dark
spherical ball at the posterior pole of the egg. The whole
history of the germ cell determinant, in so far as the
ovary is concerned, has been exhaustively treated by
Hegner (3) in more suitable insects. I have examined a
number of sections of the Hymenopterous parasites common
on Aphids, and I am able to substantiate most of his remarks ;
but in the nurse cells, as well as in the developing oocyte, 1
have found curious large spherical granules which have not
hitherto been mentioned. These seem to appear after
synezesis in the oocyte, and whether they have anything to
do with the germ cell determinant I cannot at present say.
If suitable material is procured I hope to examine this
point.

Addendum.

When this work had been finished I had not had the
opportunity of acquainting myself with Prof. Silvestri’s
writings, only knowing of them through short reviews in

DEVELOPMENT OP TRICHOGRAMMA EVANESOENS. 179

•other papers more accessible to me. Since then I have
been enabled, through Mr. Goodrich's kindness, to read
Silvestri’s valuable articles. I have been impressed by the
similarity between all stages in the development of Oophthora
and of Trichogramma. To my eye, untrained in the apprecia-
tion of small systematic differences in Chalcids, the adult
insects in these species are closely similar, and the peculiar
larvae of both species are structurally identical.

Apart from differences due to different interpretation there
is no doubt that the course of organogeny in these parasites
is parallel.

Silvestri (‘ Bolletino del Laboratorio de Zoologia Generale
•e Agraria/ vol. i and iii) identifies the darkly staining masses
of the inner region of the blastoderm stage (PI. 11, figs. 12
and 13 in my drawings) as a “ piccolo numero di nuclei, die
in seguito degenereranno,” but lias overlooked the small
granule ( G-RC .) (if really present in Oophthora) which is so
characteristic of stages such as that of PI. 11, figs. 12, 13,
and 21. In Oophthora the germ cells have sunk into the egg
before any marked differentiation of the primary germ
layers has taken place (vide Silvestri, vol. iii, p. 78, fig. xxx,
vii, 5), for it will be remembered that in the stage drawn in my
fig. 15, PI. 11, the germ layers are distinctly forming and
the germ cells still situated at the pole of the egg.

Regarding Silvestri's statement that the extruded masses
are nuclei, it might be well to mention that these darkly
staining masses are but a part (i.e. the nucleolus) of the
•original nuclei (see p. 11, and the figs. 13a and 21 of
PI. 11).

In Trichogramma I have not described the formation of an
■embryonic membrane, nor do I believe that such exists. In
Encyrtus apliidivorus and in Oophthora Slivestri des-
cribes the formation of a “ pseudoserosa ” from a delamination
of the surface cells of the embryo. He states : “ L'involucro
embrionale dell* Oophthora h in parte omologo a quello dell’
Encyrtus, perche in questo sembra che derivi completamente
per delaminazione delle cellule embrionali, mentre nell'
VOL. 62, PART 2. NEW SERIES. 13

180

J. BRONTlS GATENBY.

Oophthora la parte di esso, che prima si forma, deriva dalla
parte spugnosa del protoplasma clie occupava, a blastoderma
completo, il centro dell ovo. Intorno a tale differenza io
pero non voglio insistere troppo perch e potrebbe essermi
sfnggito il primo vero periodo di formazione dell' involucro
embrionale nell' Encyrtus, mentre lio potuto seguirlo con
ogni precisione nell' Oophthora.”

In Encyrtus Silvestri gives several figures (vol. iii, 1908,
p. 67) of the “inizio della psedoserosa,” which I find not
unconvincing, but I cannot see any delamination taking place
in fig. xxvi, 2, except at P., which I think has little in
common with the “pseudoserosa” drawn in fig. xxv, 3. I
will leave my comment at this point because Encyrtus is in
some ways different from Trichogramma, and will consider
Oophthora (vol. iii, pp. 71, 79). Whether Prof. Silvestri's or
my views concerning these forms are correct, I am convinced
that we have to deal with two species whose development is
closely similar. I find stages such as those drawn by Silvestri
in figs, xxxvii and xxxviii, and in almost all others of his
figures. Not only this, but the modified larvae of both
Trichogramma and Oophthora are similar.

He believes that one part of the pseudoserosa is formed
by the extruded inner mass (protoplasma superficial spug-
noso), while the other is formed like that of Encyrtus, and is
homologous with this membrane in the latter.

In my figs. 15, 18, 19, 24, and 25 of PI. 11, I have drawn at
EXN. what Silvestri calls the “ pseudoserosa.” Since read-
ing the Professor's papers I have very carefully re-examined
my sections, and find nothing to alter in my interpretations;
but I have drawn PL 10, fig. 4, with a view to the clearer
explanation of my view of the “pseudoserosa” of Silvestri.

The egg when laid is surrounded by a vitelline membrane
and a thin chorion, which, however, is quite distinct (PI. 10,
fig. 6, CH.) As development goes on the waste nucleoli
collect in the centre of the egg, and are soon extruded
(Iff. 11, figs. 18 and 19). They come to the surface of the
egg, and at first form a slight cavity in the ovum. But as

DEVELOPMENT OF TRIOHOGRAMMA EVANESCENS. 181

the egg grows rapidly the chorion becomes slightly stretched,
and the lump of “ protoplasma spugnoso ” becomes pressed
flat, and mechanically spreads around the egg (PL 10, fig. 4,
X, X, X.). Now should the chorion by any chance burst,
as it sometimes does, the extruded mass is released and lies
near the egg and embryo (PL 11, fig. 27; PL 12, fig. 35,
JSJXN.).

In Pl. 10, fig. 4, the extruded mass ( EXN .) lies inside the
chorion, and has been flattened out between the points X,X,X.x
on the dorsal surface of the embryo, but on the ventral
surface (F) the chorion, though somewhat stretched and
thinner, is still recognisable, and cannot be confused with
any other structure. The “ protoplasma spugnoso ” of
Silvestri is an extruded dead mass, and is in no way com-
parable or homologous with either the ammon or serosa of
other insects, and since, as Silvestri shows, there is really a
living embryonic membrane around the egg of Bncyrtus, it is
incorrect, in my humble opinion, to say that “ LTnvolucro
embrionale delL Oophthora e in parte omologo a quello delL
Encyrtus.” In his figure on p. 67 of vol. iii, he depicts a
membrane (P.) which has nuclei evenly distributed, and the
tout ensemble is far more convincing than his fig. xxxvii, 6,
of Oophthora. In the latter figure there are no nuclei in the
“ pseudoserosa ” except those on one side, which he had
already declared were “ in seguito degenereranno.”

I feel convinced that in Trichogramma and Oophthora the
“ pseudoserosa ” of Silvestri is merely an artefact produced
by the mechanical flattening out of a waste mass of proto-
plasm and chromatin. If the chorion bursts early no
“ pseudoserosa ” can be formed.

I agree with Silvestri’s description of the larva except that
his fig. XL., p. 81, which, he says, is a sagittal section, he
marks what I consider to be the longitudinal nerve-chord,
as “ cellule muscolari M.” It is true that no properly
differentiated muscles seem to exist in the larva of either
species, and the movements of the animal are brought about
by flattened mesoderm cells lying here and there under the

182

J. BRONTE GATENBY.

ectoderm. These cells only differ from the other somatic
cells in that they are more elongate, their nuclei and cyto-
plasmic structure being normal.

It is a curious fact that Silvestri, though not paying much
attention to the formation of the germ layers, has not figured
the invagination of the endoderm (PI. 11, figs. 14 and 19).
I cannot but believe that this happens in Oophthora, where all
our other stages are almost identical.

In Oophthora that remarkable nuclear arrangement of
early stages (PI. 11, figs. 16 and 21) has not been described,
and it possibly is absent ; however, Prof. Silvestri does not
appear to have paid great attention to the nuclei of early
stages, and it may have been overlooked. I mention this
because the early changes in the nuclei of Trichogramma are
so striking.

As Silvestri has pointed out, Encyrtus aphidivorus is
not a parasite on aphids, but a hyperparasite on one or two
other true aphid parasites. With regard to the fate of the
embryonic membrane which he figures enveloping the larva
(on p. 69, fig. xxix) he says: “E in tale stato di sviluppo
che la larva allungandosi rompe nella parte anteriore e nella
posterior e la serosa e libera comincia a nutrirsi dei tessuti
dell’ ospitatore.”

It will be seen that, with the exception of those parts of
organogeny which Silvestri has not treated at length, his
admirable work agrees fairly well with the few remarks I
have been able to pass on the embryology of Trichogramma,
and I have no doubt that when the Professor examines his
stages in greater detail, his results will fall into line with
my own.

Summary.

(1) Trichogramma evanesce ns lays its eggs on the
egg mass of a beetle, Donacia simplex, a single parasite
emerging from one host’s egg.

(2) The ovum has a large germ cell determinant at its
posterior pole, and in segmentation the determinant is

DEVELOPMENT OP TRICHOGRAMMA EVANESUENS. 183

divided among the large cells at the posterior pole, which are
the germ cells.

(3) In the single case found there were two polar bodies.

(4) The blastula is fairly normal except for the curious
arrangement of the chromatin in the somatic nuclei.

(5) Many nucleoli are cast out into the centre of the egg,
where they collect till from twenty-five to fifty are present;
the mass is then extruded on the periphery of the egg.

(6) As the blastoderm grows it broadens without lengthen-
ing up to the stage where the germ layers begin to form.

(7) About thirty-five nuclei sink inwards from the dorsal
surface of the embryo to form endoderm.

(8) From the blastoderm stage to that of the gastrula no
nuclear division appears to take place.

(9) Shortly after the formation of the endoderm amitosis
may be found, and from this onwards the number of nuclei
increases.

(10) The mesoderm seems to be formed from peripheral
nuclei, which sink in sporadically; no somites can be made
out, nor does any segmental method of formation of the
mesoderm occur.

(11) The nervous system, stomodaeum, and probably procto-
dasum, are normally formed.

(12) The germ cells lie in a pocket formedjby several somatic
cells, which embrace them.

(13) Ordinary mouth parts, tracheae, heart, and oesophageal
valve are wanting ; the head has two horn-like mandibular
processes, which may assist in scooping forwards the food.

(14) The larva does not feed on the food little by little,
defecating as it eats ; instead, it begins by swallowing all the
yolk at once, so that its body becomes enormously distended
and stretched.

(15) Metameric external segmentation is absent, the body
and head being continuous and sac-like.

184

J. BRONTfi GrATENBY.

Bibliography.

1. Paul Marchal. — “ Reclierches sur la biologie et le developpement

des Hymenopteres parasites,” ‘Arch, de Zool. Exp.,’ 4e Serie, 2.

2. Silvestri, F., 1906-08. — “ Contribuzioni alia conoscenza biologica

degli Imenotteri parasitici,’ i-iv, * Bollet. Scuola sup. Agric.,
Portici, T. 1 and 3.

3. Robert W. Hegner.* — “ Studies on Germ Cells : IY, Protoplasmic

differentiation in the oocytes of certain Hymenoptera,” ‘Journal
of Morphology,’ vol. xxvi, No. 3.

3a. ‘ The Germ Cell Cycle in Animals,’ New York, 1914.

4. Perkins. — “ Tricliogramma evanescens and Pentarthron,” ‘ Trans.

Ent. Soc.,’ 1913.

5. Kahle, W. — “ Die Paedogense der Csecidomyiden,” ‘ Zoologica,’

1908, Bd. xxi.

6. Henneguy. — ‘ Les Insectes,’ Paris, 1904.

7. ‘ The Embryology of the Honey Bee,’ J. A. Nelson, Princeton

Univ. Press, 1915, p. 72. -

* After Hegner’s paper (3) a useful bibliography is found.

EXPLANATION OF PLATES 10, 11, and 12,
Illustrating Mr. J. Bronte Gatenby's paper on “Triclio-
gramma evanescens (W.) : a Monembryonic Egg
Parasite of Donacia Simplex.”

Lettering.

ANT. Anterior pole. B. 0. Body cavity. BB. Brain. CAV,
Developing body cavity. CH. Chorion. CU. Cuticle. D. Dorsal
surface. D.P. Developing parasite. E. Ectoderm. ECB.N. Ecto-
dermal nuclei. E. M. Extended mass of cytoplasm and chromatin.
END.N. Endodermal nuclei. F. Food. F.N. Follicle nuclei; G.
Stainable granule. G. C. Germ cell. G. C. D. Germ cell determinant.
G. C. N. Germ cell nucleus. G. C. P. Germ cell pocket. G. L. Gut
lumen. G. B. C. Minor granule of nucleus. GT. Gut. INV. In-
vagination. L. Leg of host. L. E. N. Healthy nucleus extruded.

M. Muscle cells. M. C. Cells of body cavity. M. T. H. Mouth.

N. Nucleus. N. C. Nerve chord. N. P. Z. Nucleoplasmic zone of
nucleus. N. S. I. Nuclei sinking inwards (endoderm). (ES. (Esophagus.
(ES. COM. (Esophageal commissure. OV. Eggs of Donacia. P. Broken
pieces of germ cell determinant. P. P. Parasite. P. B. 1st polar body.

DEVELOPMENT OF TRLOHOGRAMMA EVANESOENS. 185

PD. Proctodseum. POST. Posterior pole. P. B. C. Frontal process
of larva. B. Reed. S. L. N. Mostly vitellopliags of host. ST.
Stomodseum. TH. Glandular thickening secreting frontal process.
V. Yentral. V. C. Yacuoles in cells. V. M. Yitelline membrane.
Y. Yolk.

[In reproduction all figures reduced by one-half.]

Figs. 7, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 23, 24, 25, 28, 29,
30, 32, 33, 34, 35, 37, drawn with a Zeiss oil immersion and compen.
eye-piece 8. A camera lucida was used, with drawing-board slightly
inclined towards the microscope, and at table level. Magnification
about 1,760 diameters.

Figs. 27, 31, and 38 from Zeiss ^ an(^ comP- eye-piece 4.

Figs 10a, 11a, 13a, 16, 21, 22, 26 enlarged about twice from camera
drawings with O. ^ E. 8.

Figs. 36, 40, 41, 42, and 43 enlarged in the same way about four times.

Fig. 8 drawn with O. £ E. 2, drawing-board at table level. (Camera
lucida).

Fig. 39 was drawn with Zeiss O. F. E. 4.

Fig. 4. — x 2400 (Koristka T\tli X Hug. oc. 5.

PLATE 10,

Fig. 1. — Tricliogramma evanescens (Westwood), adult female
(now x 75.)

Fig. 2. — Donacia embryo X 75 containing at its posterior pole a
parasite ( D.P. ). Whole preparation. The parasite was at the stage
drawn in PI. 11, fig. 24.

Fig. 3. — Donacia simplex ( F .) X 4.

Fig. 3a. — Egg mass of Donacia, viewed from side with parasite (P.).
All to same scale as beetle.

Fig. 4. — Transverse section in mid region of an embryo when the gut
lumen has formed, Shows the flattening out of the extruded mass
(EX. N.) under the chorion ( CH .).

Fig. 5. — Stages in amitosis of somatic cells.

Fig. 6. — Part of early blastoderm stage to show chorion (CH.) and
extruded nucleolus (EX. N.).

PLATE 11.

Fig. 7. — Part of Donacia egg showing the newly-laid egg of the
^parasite in transverse section.

186

J. BRONTE GATENBY.

Fig. 8. — Part of egg mass of Donacia in transverse section showing a
parasite at the stage drawn in fig. 28.

Fig. 9. — Nearly mature ovarian egg of parasite to show nucleus (N.y
and germ cell determinant (G. C. D.).

Fig. 10. — Newly-laid egg, with spermatozoon (M. P. N.).

Fig. 11. — Formation of second polar body.

Fig. 12. — Typical blastoderm stage showing extruded nuclei (EX. N .)
and germ cells ( G . C.).

Fig. 13. — Later blastoderm to show stages in nuclei and shortening
of egg.

Fig. 14. — Late blastoderm stage showing beginning of formation of
endoderm (N. S. I.).

Fig. 15. — Stage after fig. 14 to show beginning of insinking of
peripheral nuclei (x Y.), and penetration and change of position
of germ cells.

Fig. 16. — Part of transverse section of blastoderm stage to show
structure of nuclei.

Fig. 17. — Transverse section of a blastoderm stage to show expulsion
of nuclei (Y.).

Fig. 18. — Transverse section showing final extrusion of nuclei
(EX. N.) and beginning of gastrulation.

Fig. 19. — Gastrula stage in transverse section after expulsion of
nuclei (EX. N.). Nuclei in this specimen a little larger than usual.

Fig. 20. — Transverse section of posterior pole of the same egg as
that in fig. 7, to show germ cell determinant.

Fig. 21. — Posterior pole of blastoderm stage to show germ cells
(G. C.) and structure of nuclei.

Fig. 22. — Posterior pole of egg just after sinking inwards of germ
pocket (G. C. P .) and when the nuclei (X. X.) form a covering for
the pocket.

Fig. 23. — Transverse section of same embryo as that in figs. 24, 25,
and PL 12, fig. 31, to show germ cells. Such a section as this is
through the points A ... A in PI. , fig. 15.

Fig. 24. — Transverse section of embryo during the formation of gut
(END. N.) and nervous system (N. C.), etc.

Fig. 25. — Section through anterior region near stomodseum.

Fig. 26. — Enlarged view of posterior pole of the egg drawn in fig. 11,
to show breaking up germ cell determinant (P. P.).

Fig. 27. — Obliquely sagittal section through embryo to show forma-
tion of gut (G. T.), stomodseum (STD.), proctodseum, brain, and nerve
chord.

DEVELOPMENT OF TIUCHOGRAMMA EVANESCENS. 187

Fig. 28. — Section such as that through points X y X y in fig. 27.
X is a cell whose nucleus has temporarily resumed the usual reticulate
arrangement. Compare PI. 12, figs. 40-43.

PLATE 12.

Fig. 29. — Transverse section through brain and thickening
which secretes the horn-like process. Same age as embryo in PI. 11,
figs. 27 and 28.

Fig. 30. — Horizontal section through head region of a young larva
to show liorn-like processes (P. R. C.), mesenteron ( MES .), and mouth
(M. T. H.).

Fig. 31 — Transverse section through developing stomodseum. Same
embryo as that in PI. 11, figs. 23, 24, and 25.

Fig. 32. — Cast-out mass of cytoplasm with the extruded nuclei. Has
become round, and the nuclei still live (Zb E. N.).

Fig. 33. — Transverse section of larva near midgut when it begins to
take in food ( F .).

Fig. 34. — Longitudinal section through germ pocket ( G . C. P.) of
same embryo as that in PI. 11, fig. 27.

Fig. 35. — Part of embryo and the extruded mass ( E . HP.) with some
nuclei still living ( L . E. N.).

Fig. 36. — A germ cell of blastoderm stage containing the faint
remains ( G .) of the germ cell determinant.

Fig. 37. — Transverse section through germ cell pocket ( G.C.P .) in
same embryo as that in Pi. 11, fig. 28.

Fig. 38. — Larva in transverse section after it has began to swallow
yolk ( F .), and when the body becomes stretched thereby.

Fig. 39. — Transverse section through embryo after the stage drawn
in PI. 11, fig. 24, to show formation of body cavity (C. A. V.).

Fig. 40-43. — Stages in the resumption by mesoderm nuclei of the
typical reticulate arrangement. Compare fig. 34 at X.

THE DEVELOPMENT OF THE CAPE CEPHALOD1SCUS. 189

On the Development of the Cape Cephalodiscus
(C. gilchristi, Ridewood).

By

.1. I>. F. Gilchrist, M.4., l>.Sc., Pli.D.

With Plates 13 and 14.

In October, 1915, I recorded some observations on living
specimens of the Cape Cephalodiscus, its eggs and
larvae (4). I had hoped in the following summer to be able
to procure additional specimens, more especially of advanced
larvae, showing the process of metamorphosis. Contrary
however, to all expectations, not a single living specimen
was procured by the trawlers during the summer months.
One colony, very much damaged, and with the zooid cavities
filled with sand grains, was found on a sandy bottom, some
six or eight miles from the usual habitat of the animal, of
value only as indicating that the animal may be carried some
distance by currents. The reason for this scarcity probably
was, as suggested by the captains of the trawlers, that there
had been no heavy seas, and no great “drawback ” or strong
currents to detach the colonies from the rocky ground
which, there is reason to believe, is their natural habitat.

The material, procured during the previous summer, how-
ever, has proved on examination sufficient to indicate some
new facts regarding the development of the animal, which it
may be desirable to put on record, without waiting an
indefinite time for the uncertain possibility of procuring and
rearing the larvse to later stages.

This material was preserved in a variety of ways. Subli-

190

J. D. F. GILCHRIST.

mate, sublimate-acetic, Gilson’s fluid, formalin-alcohol, alcohol,
Fleming’s fluid, and formalin in sea-water were employed.
Sublimate preparations seemed to be unfavourably affected,
more especially in the yolk-laden parts; osmic acid caused
great contraction, though the fixing was good, at least in the
larva; the best results were obtained from 10 per cent,
formalin in sea-water, provided care was taken to pass the
tissue slowly through gradations of absolute alcohol and
xylol. The passage from absolute alcohol to xylol was best
effected by using half a dozen gradations of these up to pure
xylol, though good results were obtained by passing the
material from absolute, through to one-tliird and two-tliirds
xylol to pure xylol. By the use of this method some very
distinct preparations were procured, showing cellular struc-
ture, of the early stages within the egg capsule, though, for
some reason, the method was not successful in larvae hatched
out from the egg.

The history of the investigation into the development of
Cep halo disc us need not be here gone into, further than
to indicate certain points on which further information is
desirable, or which are not beyond dispute. Masterman
(1900) observed some segmenting eggs in the material pro-
cured by the “ Challenger ” Expedition. Andersson (1903)
noted the planula-like larva for the first time. Harmer (1905)
described the eggs, heavily laden with yolk, their liolo-
blastic and nearly equal segmentation, and suggested that
they may give rise to solid embryos, in which the endoderm
arises by delamination. He showed that the five body
cavities of the adult arise at an early stage in the embryo.
Andersson (1907) described his material more fully, and
recorded the occurrence of a gastrula-like stage, in which
there is a centrally placed mass of yolk with a narrow lumen ;
he believes that this is the endoderm formed by a process of
invagination. He also confirmed the early appearance of the
body cavities. Scliepotieff (1909), though adding nothing-
further towards the elucidation of the gastrula stage, con-
firms the existence of the planula-like larva, and adds a

THE DEVELOPMENT OF THE CAPE CEPHALOD1SCUS. 191

further stage, which is free-swimming, and in which the
five body cavities may be recognised. He regards the central
mass of yolk in the embryo and larva as representing an
endoderm formed by involution. Braem (1911) draws a
comparison between this free-swimming larva and that of
certain Polyzoa. Harmer (1915) draws attention to the
different disposition of the body cavities, and gives a summary
of the points in the development of Cep halo discus on
which there appears to be unanimity, drawing attention to
the unexplained origin of the gastrula-like stage.

There are, as will be seen from this review, several
important gaps in the development of Cep halo discus
which have not yet been filled up, and on which further
information is very desirable. Thus, nothing is known of
what occurs between the first segmentation stages and the
gastrula — this alleged gastrula requires further investigation ;
the exact mode of origin of the body cavities has not been
explained ; and finally, the metamorphosis of the larva into
the adult still remains to be elucidated.

The chief points on which the present work seems to throw
further light with regard to these questions are : Segmenta-
tion stages leading to the formation of a blastula, which does
not become invaginated to form a gastrula, but develops into
a solid body, the outer parts of which become differentiated
into an ectoderm and endoderm, the main inner yolk mass
not representing the endoderm, nor its cavity the arch enter on ;
the origin of the body cavities, the anterior as a part parti-
tioned off from the archenteron, and the posterior as a lumen
internal to the developing endoderm.

Ovary and Ovarian Egg.

The structure of the ovary of this species has been briefly
described by Ridewood (7). It consists of a very narrow
oviduct, leading into a mass of developing eggs in which no
distinct lumen can be made out. The great majority of the
eggs are comparatively small, one or two, at the point furthest

192

J. D. F. GILCHRIST.

from the oviduct, being greatly developed, and constituting
the main part of the whole ovary. Each of these eggs is lodged
in a follicle of small cells. Scliepotieff (8, p. 81, fig. 63) has
described the large ova of certain species as lying between a
central epithelial lumen and the wall of the oviduct, where
they are surrounded by a relatively large quantity of blood.
This has not been observed in the present species, in which
the ovum in its follicle is always in close contact with the
surrounding ova. The ova appear to arise in the walls of
the oviduct.

Two questions have been raised with regard to the ovary —
the function of the pigment of the oviduct, and the method
of discharge of the very large ova. With regard to the
first, there is nothing new to add, except that the pigmented
oviduct does not seem to be a luminous organ as has been
suggested. None of the living animals examined, with a
special view to ascertaining this, showed any trace of
luminosity. With regard to the second question, Masterman's
suggestion, with which Andersson (2, p. 86) does not agree,
that the ova are set free on the death of the animal, seems to
have some partial confirmation, from the fact that, in the
fresh material, detached ovaries were frequently found.
These may, of course, have been forcibly detached in the
trawl, but living zooids were also observed in which the
ovary, loosely attached to the animal, was seen to be quite
exposed, suggesting that the whole ovary, or part of it, may
break away from the body, without, however, necessarily
involving the death of the animal — a condition which may
also, perhaps, have been brought about by pressure in the
trawl-net.

Certain histological features of the ovarian egg, which do
not seem to have been noted, may be worthy of mention, as
they seem to indicate that the subject is worthy of further
study. The nucleus (PI. 13, fig. 1) is a prominent feature of
the developing egg. It is of a clear, almost homogeneous
appearance, with only indistinct indications of chromatin
elements. It has a distinctly demarcated border, which may,

THE DEVELOPMENT OF THE CAPE CEPHALODISCUS. 193

however, assume various irregular shapes, as if it were of an
amoeboid nature, though there were no prolongations into the
surrounding yolk mass. It is never of the elongate or semi-
lunar form figured by Schepotieff for his species. A con-
spicuous, deeply staining nucleolus is always present, and, in-
some preparations, was observed to have a series of rounded
vacuole-like spots, arranged around its border ; in other case&
there appeared to be a single large vacuole, seemingly con-
firming the view that the vacuoles are of a changing nature.
That the nucleolus takes a part in the functional activity of
the egg at this stage is indicated by these different appear-
ances, and also by the fact that in some cases it was observed
drawn out in a tapering manner quite to the periphery of the
germinal vesicle, and, iu one case, a small detached part of it
was observed lying in the germinal vesicle not far from it.

Stages in the formation of the yolk granules are well illus-
trated. These granules are very numerous, of an oval or
rounded shape, with well-defined borders, and stain deeply
with eosin. Scattered throughout them appeared a number
of minute bodies (PL 13, fig. 1, y. ?^.), which readily stained
with hematoxylin. The transformation of the homogeneous
substance of the ovum into yolk granules does not appear to
begin in the immediate neighbourhood of the nucleus, as in
some other cases, for, in several instances, the nucleus with
its nucleolus was observed in a homogeneous matrix in the
form of a crescent at the periphery of a large mass of yolk
granules. In others, the homogeneous part assumed the form
of a portion slightly constricted off from the main mass of
the egg. The boundary between the homogeneous and the
granular part of the egg in these cases was well defined, and
in it occurred a layer of the deeply staining bodies above
mentioned, which may be termed yolk nuclei, though a
variety of objects seem to be included under this term. So
definite was the demarcation that it was at first supposed
that there were here two cells, the semilunar homogeneous
cell with its nucleus being a nourishing cell, assisting in
building up the relatively enormous yolk mass. The fact that

194

J. D. F. GILCHRIST.

both were enveloped in a common follicle was not sufficient in
itself to disprove this, but, as no nucleus could be found in the
larger mass, it must be concluded that we are dealing* here
with one ovum. More advanced ova, completely transformed
into yolk granules and yolk nuclei, possessed a large nucleus
and nucleolus, located now in the centre.

The origin and nature of the yolk nuclei in general is still
an obscure question. It has been suggested that they arise
independently in the cytoplasm, that they are derived from
the nucleus, and that they are derived from the nucleolus.
The evidence in this case seems to be in favour of the last
suggestion, in view of the appearances in the nucleolus noted
above. The nuclei did not appear to originate from a single
large yolk nucleus as is the case in other instances of such
structures. The further study of the change in the ovarian
egg seems to be worthy of attention.

The Fertilised Ovum.

Two or three specimens only of the unsegmented ovum,
enclosed in its clear capsule and presumably fertilised, were
procured. Such eggs (PI. 13, fig. 2) were quite spherical, in
contrast to later stages. In sections, among the numerous
eosin-stained yolk granules, were seen some small bodies,
stained, though not conspicuously, with haematoxylin, pre-
sumably the yolk nuclei.

The eggs of Cephalodiscus have been described as oval
and of a varying diameter. These are probably late stages
of the ovum, in which the embryo is fairly advanced, and the
egg proper may not vary much in diameter, though sufficient
material at this stage was not available to give any certainty
on this point.

Segmentation .

The first division of the ovum, from the beginning of the
constriction to the complete separation of the blastomeres,
was observed. More examples of this stage were found than

THE DEVELOPMENT OF THE OAPE CEPHALODI8CU8. 195

of the undivided egg’, but still comparatively few (about ten
out of several hundreds examined). This probably indicates
that this stage is passed through at a comparatively rapid
rate. Segmentation was in all cases total and usually about
•equal. A typical case is shown in PI. 13, fig. 3, in which the
blastomeres are about equal. Cases of decidedly unequal
division, however, occurred as shown in PI. 13, fig. 4, and in
one case the smaller blastomere was *21 x *16 mm., the
larger *29 x ’37 mm. A large nucleus with nucleolus was
conspicuous in some cases in each segment.

Stages of four blastomeres were about as numerous as
those of two. In some the second division was of the typical
form, equal and at right angles to the first (PL 13, fig. 5).
In others there were decided departures from this type.
Thus a stage was found (PI. 13, fig. 6), in which the blasto-
meres did not lie in one plane, each of them being so placed
that it was in contact with the other three, as if a relative
change in position had taken place subsequently to the second
division, or the division spindles of the second division had
been at right angles to each other. A second aberrant type
(PI. 13, fig. 7) was found in two cases, in which two segments
were widely separated, the other two being in close contact
with each other. Both of these types may be connected with
the fact, shown in another case, in which the division in one
segment has been more rapid than in the other, resulting in
the formation of three blastomeres, one large and two small
(PI. 13, fig. 8).

This segmentation may therefore be unequal, not only in
quantity, but in point of time and method of division,
probably connected with the great amount of yolk in the egg,
a fact which also, as will be seen later, has a striking effect
in further development. It apparently indicates a very inde-
terminate type of segmentation which is seen also in the next
stage observed. This consisted of six cells (PL 13, fig- 9)-

Blastula.

The earliest appearance of the segmentation cavity was at

VOL. 62, PART 2. NEW SERIES. 14

196

J. D. F. GILCHRIST.

a stage showing six segments in section (PL 13, fig. 10) ; here-
it was very small, and was occupied by a homogeneous sub-
stance stained faintly with haematoxylin. A well-marked
blastula is soon developed after this stage, for a section
showing nine cells has a relatively large blastocoele (PL 13,
fig. 11). Here the cells at one side appear larger than at the
other. Other sections, however, show that the grouping of
large cells at one point does not appear to be constant.

In a blastula of fourteen cells in section (Pl. 13, fig. 12)
one cell was observed entirely within the hitherto complete
circle of cells. It seems from subsequent events that this
arises by proliferation of an outer cell rather than by ingrowth
of a cell. It marks, as will be seen in later stages, the
posterior end of the developing embryo.

The beginning of a further change is seen in a blastula
of about twenty-nine cells in section (Pl. 13, fig. 13), in which
a more marked polar disposition of parts becomes evident.
At one end, which, as subsequent development shows, becomes
the anterior end of the animal, the cells are decidedly elongate,
while more posteriorly they are still rounded. The nucleus
in the elongate cells appears at the distal end, while, in the
rounded posterior cells, it appears in the centre.

Formation of the Yolk Columns.

The elongate outer cells begin to assume a columnar form,,
whose main body consists of an elongate mass of yolk cells
with a peripherally placed nucleus (PL 13, figs. 14 and 15,
e.r. y. e.). This is probably due to their increase in number,
and consequent mutual pressure. The elongate character is
gradually assumed by the other cells in a more posterior
position, and ultimately all the outer cells assume the form
of columns with peripheral nuclei. The last of these outer
cells to assume the columnar form is a group of rounded
cells at the extreme posterior end, from which the internal
cells are proliferating. The internal cells are still some-
what rounded, and ultimately completely fill the blasto-
coele, so as to form a solid mass of cells.

THE DEVELOPMENT OF THE CAPE CEPHALODISCUS. 197

An interesting result of the rapid increase of these yolk
columns is that an invagination is formed near the point
(PL 13, fig. 15, p. inv.) where they are attached to the inner
mass, apparently due merely to mechanical causes associated
Avith the increase of the outer layer. This gives the
appearance of a gastrula-like structure, which, however, can
only be fully discussed when the changes in the inner yolk
mass are considered.

Origin of Ectoderm.

After the formation of the external yolk columns, their
cells divide rapidly, and the dermarcation between them, so
clearly marked before, disappears. In view of their origin,
Ave must regard each of the yolk columns as representing a
single cell, and the breaking down of the cellular structure
as due probably to the great abundance of yolk, the cell
having lost control of the elongate and attenuated column.
What Avas observed to occur at this stage was that the
multiplying nuclei, with a certain amount of protoplasm,
became each closely applied to a yolk granule, the two
forming an ovoid body, in which the yolk granule, deeply
stained Avith eosin, could be clearly distinguished from the
nucleus, which Avas as distinctly stained with hsematoxylin.
Presumably the yolk granules serve as nourishment for the
rapidly multiplying cells, for, ultimately, the yolk granules dis-
appear, first from the peripheral parts, and later from the
deeper parts of the ectoderm, till finally only a network of
protoplasm, or rather a vacuolated protoplasmic mass, with
numerous nuclei embedded in its substance, is left.

Two other points may be noted in connection with the
origin of the ectoderm, viz. the formation of a basemeut
membrane and the occurrence of excretory matter. With
regard to the first, the outer cells were always distinguishable
from the inner, except posteriorly, and an intervening space
is seen in preparations of the more advanced stages. This
demarcation becomes more distinct by the appearance of a

198

J. D. F. GILCHRIST.

fine basement membrane at the base of the ectoderm cells,
apparently secreted by these cells (PL 13, fig. 17, b. m.).
This basement membrane was not, however, usually so dis-
tinct as in the case figured, and it may be formed by the
endoderm cells described later.

With reference to the second fact, there are to be seen in
the developing ectoderm, after the cellular structure has
been lost, a number of small bodies about the size of nuclei,
but readily distinguished from them by their black colour.
These become larger, and are frequently fused together to
form elongate black masses. That they are ultimately passed
out to the exterior was evident from some which were
observed partly protruding beyond the surface of the develop-
ing embryo. This accounts for the presence in the living
state of dark particles floating in the space between the
embryo and the egg capsule, the rotation of the ciliated
embryo causing them to move about rapidly, so that their
presence is readily detected. It also accounts for the charac-
teristic pigment spots of the embryo, which sometimes
assumed an elongate shape, and formed a ring round the
anteriorly situated sense organ.

Certain areas of the ectoderm seem to retain their distinctly
cellular structure throughout the changes which take place
in the ectoderm. The most prominent of these is the part
which appears as a ventral thickening in the embryo
(PI. 13, fig. 18, v.th.), and which may, as Harmer suggests,
represent the disc-like face of the proboscis. Its early
appearance is noteworthy. The sense organ also consists of
independent cells, as also the glandular cells of the ectoderm,
but these are not seen at this early stage, and their cellular
condition may be of much later origin.

Origin of Endoderm.

The origin and mode of formation of the endoderm is,
as already indicated, one of the outstanding problems of the
development of Cephalodiscus.

THE DEVELOPMENT OF THE CAPE CEPHALODISCUS. 199

The cells of the ectoderm before fusion are arranged
radially, and are at this stage clearly marked off from the
inner cells ; a little later they are further marked off from
them by the basement membrane. Soon after the fusion of
these ectodermal cells a few cells appear below the basement
membrane. These are closely applied to yolk granules, and
form with them a distinct layer round the anterior end of
the yolk mass, but clearly marked off from it (PI. 13, figs. 17,
18 and 19, end.). As the yolk granules in this layer are
used up, each of the cells sends out a long protoplasmic process
towards the other, so that, ultimately, they form a chain of
attenuated cells devoid of yolk granules. These cells appear
first at the anterior end and later more posteriorly, so that
the chain of cells gradually extends backwards, over the
internal yolk mass, as an uninterrupted series, to the posterior
end, at the point where the internal cells remain in connection
with the ectoderm.

Formation of the Yolk Cavity and Andersson's
‘ ‘ Gastrula.”

Meanwhile a change has taken place in the inner cells,
associated perhaps with the appearance of the endoderm.
Unlike the ectodermal cells, they do not assume a columnar
form, but remain more or less rounded, each, however, with a
nucleus and a distinct cell boundary, and as heavily laden
with yolk granules as the ectodermal cells. On the appearance
of the endodermal cells their cellular structure can no longer
be distinguished. It appears as if the nuclei, with their
associated protoplasm, no longer controlled these cells, and
had wandered to the periphery, as in the case of the ecto-
dermal cells, leaving a central non-cellular mass of yolk
granules.

All the cells of the inner mass do not pass to the periphery,
but some find their way to the centre, where they form a
small but distinct group, embedded in a substance nearly
devoid of yolk granules. A small cavity then appears in

200

J. D. F.- GILCHRIST.

this substance. The cavity as seen in sections is usually
rounded or oval in shape, but that it is in reality of an elongate
nature is evident from the fact that it can be followed
through a series of consecutive sections. It ends abruptly
when traced in one direction, but may be followed in the
Other direction to the periphery of the embryo, where it
suddenly ends in a shallow pit in the ectoderm, apparently
the involution or invagination of the ectoderm already noted.
This was most clearly seen in sections which passed through
the axis of this part of the embryo (PI. 13, fig. 16). The
whole assumed the form of a structure, which, without this
explanation of its origin, might be taken to be a typical
gastrula, in which the central yolk-laden mass represents an
endoderm, formed by invagination, and a central cavity, the
archenteron ; the only suspicious feature being the very
narrow lumen and the absence of cellular structure of the
endoderm.

Andersson (2, p. 87) was the first to notice and figure this
gastrula, and he has apparently no doubt as to how it has
arisen. He notes HarmePs suggestion (5, pp. 109, 110) that
the gastrula is probably formed by a process of delamina-
tion, and considers that for his species at least this is not the
case, but that “ die Gastrula durch eine typisclie Invagina-
tion sicli bildet 99 (p. 89). He was unfortunately unable, lie
adds, to carry out any study of the cellular structure, as
owing, he believes, to imperfect preservation, the ectoderm and
endoderm appeared uniformly filled with yolk granules, which
he notes, however, were absent in the immediate vicinity of
the central lumen. His figure, however, indicates the existence
of the external yolk columns. Schepotieff (8, p. 437) states
that he found gastrula stages in C. indicus, but was unable
to follow out their formation. That, however, he accepts the
view that the central yolk mass represents an endoderm
formed by invagination, is apparent from his description and
figures of larval stages of the species. Harmer also
(4, p. 245) accepts the view that the central yolk represents
the wall of the archenteron.

THE DEVELOPMENT OF THE CAPE CEPHALODISCUS. 201

It appears from what lias been observed that, in this species,
the central yolk-laden cells arise solely by unipolar prolifera-
tion of cells into the cavity of the blast ula, that the cellular
structure breaks down, and some of the nuclei, with their
associated protoplasm, goto form the endoderm, while others
pass towards the centre and become vitellopliags. Owing
to the activity of these latter the yolk granules become
used up, leaving a homogeneous detritus in which a cavity
subsequently appears. This cavity extends at first to an
•ectodermal involution, and it may be that the excretory
matter passes out in this way to the exterior, just as the
•excretory products of the growing ectoderm are given off in
another manner already indicated. The subsequent changes
•in this cavity and its relation to the posterior involution, as
well as to the cavity immediately enclosed by the endoderm,
will be described in later stages.

Formation of Internal Yolk Columns.

After the inner yolk-laden cells become a homogeneous
mass of yolk with scattered cells, and soon after these
reduced cells begiu to migrate, some towards the periphery,
some to the centre, a change takes place in the uniform
distribution of the yolk granules, and they assume the form
of a number of yolk columns, or rather pyramids, whose
apices meet round the central cavity, and whose broader
distal extremities are in the proximity of the cells which form
the endoderm (PI. 13, figs. 18 and 19, i. y. c.). The result
bears some resemblance to what has occurred in the ectoderm,
but it has apparently been attained in a different way, for
the large yolk-laden cells do not individually become yolk
columns ; at least there was no appearance of this, and it
can hardly be imagined how they could do so, unless perhaps
the cells, 'migrating outwards to form the endoderm, retained
for a time some control over their original yolk masses, and
similarly the cells migrating inwards draw out their associated
yolk into lenticular masses.

202

J. D. F. GILCHRIST.

The functional significance of the whole process seems very
evident. A very little of the yolk is necessarily used up in
the formation of the thin endodermal layer, and the main
mass is reserved to be transformed by vitellopliags into a
form which can be absorbed by the archenteron to feed the
rapidly developing ectoderm, which has now used up it&
original supply of yolk.

The inner yolk pyramids persist as such for a considerable
time, but later, when their yolk granules have been much
reduced, they seem to disappear.

Origin of Body Cavities.

As development proceeds and the anterior part of the
endoderm increases in size, a space (the archenteron) appears
between it and the central yolk mass (PI. 13, fig. 19, * arch .).
Posteriorly the endoderm is still in close contact with the
yolk mass, but later a few cells, evidently arising from the
yolk mass, as the endodermal cells did, appear below it on
the yolk. These increase in numbers and ultimately form a
distinct layer, so that the endoderm here seems double. The
cavity between these two layers is very evidently the
beginning of the first pair of posterior body cavities (PI. 13,
fig. 19, and PI. 14, fig. 20, b. c.2). The second pair of body
cavities is subsequently formed in a similar manner (PI. 13,
fig. 18, and PI. 14, fig. 20, b. c.3). The body cavities may
therefore be regarded as of endodermal origin, which, though
not typically enteroccelic, is a modified form of such a method
of development. At later stages both pairs of body cavities
may be seen with a complete epithelial lining (PI. 14, fig. 21,
b. c.2 and b. c.3).

The definite origin of the single anterior body cavity was
not observed until later, but it may be mentioned here that it
is developed from the anterior part of the archenteron.

The Laeva.

The structure of the larva soon after hatching is not very
different from that of the late embryo, but certain points.

THE DEVELOPMENT OE THE GAPE CEPHALODISCUS. 203

obscured by the compression of the embryo in a small space,
become clearer or assume a different aspect in the early larva.
Thus the ectoderm, very much folded in the embryo, now
expands, and the body cavities can more readily be made out.
Certain more definite changes, however, were observed in
older larvae.

The general structure of the larva has already been
described by Harmer (5), Andersson (2), and Schepotieff (8).
The chief new points to be added are in connection with (1)
the fate of the internal yolk mass, (2) the arrangement of the
body cavities and the mode of origin of the anterior body
cavity, (3) the origin of the anus, (4) the involution of the
sense organ and its nervous tissue, and (5) a postero-ventral
thickening and involution.

(1) Fate of yolk mass. — Neither Harmer nor Andersson
found any trace of cellular structure in the yolk. Schepotieff,
however, indicates clearly (8, PL 8 fig. 16) that this part
is divided up into large columnar cells, which, but for their
distinct demarcation and single nuclei, might pass for the
internal yolk columns, already described for the species
under consideration. His fig. 7 also shows the walls of what
he regards as the proboscis-ccelom, ending abruptly at the
anterior part of the yolk, instead of passing round it to the
posterior extremity, as here described. The cells of this
body cavity are obviously very diagrammatically drawn, and
it may be that those of the “Urdarm” in fig. 16 are of
the same nature, in which case it would not be so difficult to
interpret them as internal yolk columns. It is not, however,
absolutely necessary to reconcile other accounts of the for-
mation of the endoderm with that given here, as both may
be correct; the mode of development, even in closely related
forms of animals, having been proved in some cases to be
very different.

The further changes observed in the central yolk mass were
as follows : The anterior space (archenteron) between the
endoderm and the yolk becomes very large, and at the same
time the yolk lumen increases in size (PI. 13, fig. 19, y. L).

204

J. D. F. GILCHRIST.

This lumen appears somewhat triangular in longitudinal
section ; in sagittal sections of some advanced embryos it is
seen that the part of the yolk forming the upper portion or
roof of the central cavity has disappeared, and it is now in
connection dorsally with the archenteron. The consumption
of these yolk granules in this particular region is apparently
associated with the active growth in the tissues immediately
adjacent to it. More posteriorly, a part of the roof of the
lumen is still present, and a transverse section of this part
would show a circular cavity in the yolk. This is very well
illustrated in Andersson’s figs. 73-78, and in transverse
sections of younger larvae of this species. He still regards
this diminishing cavity as the archenteron, and its walls,
including the homogeneous substance which is interpreted
here as the detritus of yolk granules attacked by vitellophags,
as the endoderm formed by invagination. The very large
cavity in front of and above the yolk can in this case only be
considered, as he does, to represent the cavity of the pro-
boscis. Later embryos, however, show that the roof of the
yolk lumen disappears even from the posterior part, just as it
did in the anterior, so that no yolk cavity is left at all. This
is seen in transverse sections (PL 14, figs. 23-30) of an
advanced larva the exact age of which cannot be determined;
it was found crawling over a ccenoecium and kept alive for
about two days afterwards. Here the roof of the yolk lumen
has entirely disappeared, though the floor, or ventral part,
still persists as a fairly large mass, with the cavity of the
archenteron above it. The ventral part of the yolk is
probably used up in the next step in the metamorphosis of
the larva, as yet unknown, but probably most marked on the
postero-ventral aspect of the larva, where this yolk mass
lies.

(2) The body cavities of the larva. — The four
posterior body cavities can usually be seen distinctly in suit-
ably prepared material, provided there is not too great con-
traction of the tissues. Their epithelial lining can also be
sufficiently distinguished. As they are of importance in the

THE DEVELOPMENT OF THE CAPE CEPHALODISCUS. 205

organisation of Cephalodiscus, and as tlieir relative
position and extent may have a bearing on the subsequent
processes of metamorphosis, some details may be added to
what is already known for other species.

Only a limited number of larvae were available, and several
of these were, for various reasons, unsuitable for detailed
examination of the cavities, some being too contracted or
distorted, others were somewhat broken up, owing' to the
difficulty of getting whole sections through the yolk mass.
One or two series of transverse sections were, however, satis-
factory, and showed the body cavities clearly. In one of
these, cut into a series of 140 sections, the sixth from the
posterior end (PI. 14, fig. 22) showed that the two posterior
body cavities extended backwards beyond the yolk and archen-
teron. At the 17th section (PI. 14, fig. 23) the body cavities
are very large, extending almost completely round the yolk,
but are -separated from each other dorsally by a mesentery -
and ventrally by the posterior thickening already mentioned.
That there is a here a ventral mesentery obscured by the
pressure of the yolk is shown in other series, and it is evident
in the next or 24th section (PI. 14, fig. 24). Here the upper
wall of the archenteron has become broadly attached to the
ectoderm, and the body cavities are beginning to disappear
from the dorsal side. A few sections further on, at the 25th,
the beginning of the second body cavity appears on the right
side at its dorsp-lateral coiner, and, at the 27th section
(PI. 14, fig. 25), it is of considerable size. At the 29th section
(PI. 14, fig. 26) it has extended to the right side of the
archenteron, and in this section the second body cavity of the
left side appears at the upper angle formed by the ectoderm
and the wall of the archenteron. That the point at which
the second body cavities begin on each side is therefore not
similar is evident from this, and in other series of sections it
also shows a variation, as, for instance, in one in which it
begins quite at the lateral wall of the ectoderm. The 36th
section (PI. 14, fig. 27) shows further advance, and, at the
45th (PI. 14, fig. 29), the third body cavity has disappeared

206

J. I). F. GILCHRIST.

on the right side. In the 67th section (PI. 14, fig. 30) both
pairs of body cavities have disappeared. At the 95th section
(PI. 14, fig. 31) the yolk mass is much smaller, and the ecto-
derm has the clear spaces and the elongate cells characteristic
of the dorsal and ventral parts of this region of the body
respectively.

The formation of the definite body cavity of the proboscis
was not seen in these transverse sections, but in some longi-
tudinal sections a division appeared running obliquely across
the cavity of the archenteron anteriorly, and cutting off a
portion of this cavity, the portion cut off being about one-
fourth of the whole archenteron. This division was observed
in two longitudinal sections only, and in these the thin wall
of the archenteron was incomplete in places (PI. 14, fig. 37).
How this division arose is not quite clear, and the
question is perhaps better left open till further confirmation
is possible.

(3) Appearance of the anus in the larva. — In a
sagittal section (PI. 14, fig. 38) of a larva the cavity of the
archenteron extends to the posterior end, and comes in contact
with the ectoderm. At the point of contact there is a slight
involution and indication of a pore, though there is no well-
marked opening. There seems little reason to doubt that this
is the point of origin of the anus. In the section it is situated
towards the dorsal aspect of the body. It doubtless, there-
fore, represents the point at which the yolk mass remains in
contact with the ectoderm, but has no connection with
what is described later as a postero-ventral thickening und
involution of the ectoderm. The section, however, was not
entirely convincing, and later stages are desirable to clear
up and confirm this point. There is no indication of the anal
opening in PI. 14, fig. 22, a transverse section posterior to
yolk and archenteron.

(4) Changes in the sense organ appear in the larva.
In the earlier stages it consisted of a group of elongate
ciliated cells, at the base of which appeared a small patch of
nervous tissue, as described for other species. In the more

THE DEVELOPMENT OF THE CAPE CEPHALODISCUS. 207

advanced larvae the nervous tissue is seen to extend in a
posterior direction under the general tissue of the ectoderm
(PI. 14, fig. 32), and the cells of the sense organ now assume
the same character as this nervous tissue. They lose their
cilia, and become sunk in an ectodermal pit (PL 14, fig. 33),
which may be drawn out posteriorly into a tubular structure
(PL 14, fig. 35, inv.s. o.).

(5) A postero- ven tral thickening and involution
of the ectoderm was observed in some sections under the
hinder end of the yolk mass. This thickening is seen in
Pl. 14, fig. 23, and, a few -sections posterior to it, it is seen to
lead to an involution. This involution is, however, more clearly
seen in another series (PL 14, fig. 36). It may not prove to
be of any particular significance, but may be noted, as it is in
this region that the greatest change will probably take place
in the metamorphosis of the larva.

Summary of Results.

(1) Certain facts are noted with regard to the formation of
yolk granules, presence of yolk nuclei, character of nucleus
and nucleolus.

(2) The segmentation is holoblastic, equal, or markedly
unequal, and apparently indeterminate.

(3) A blastula stage occurs.

(4) The blastula becomes solid by proliferation of cells at
one end ; there is no invagination at this stage.

(5) The point of proliferation marks the posterior end,
and the anterior end is distinguished by the elongation of its
cells.

(6) All the outer cells become elongate, and assume the
character of columnar cells full of yolk. As these increase in
number a small posterior invagination appears.

(7) The cellular character of the yolk columns disappears;
the yolk granules are used up, and an ectoderm consisting of
many nuclei in a protoplasmic network, with a basement
membrane, is formed.

208

J. D. F. GILCHRIST.

(8) Excretory matter in the form of dark specks and
elongate rods is formed during this process and constitutes
the characteristic pigment of the late embryos and larvae.

(9) The ventral thickening of the ectoderm is found at an
early stage.

(10) The endoderm appears under the ectoderm, first as a
number of cells at the anterior end, and ultimately as a com-
plete chain of cells extending over the inner yolk, except at
the point of proliferation at the posterior end.

(11) The cells occupying the blastocoele break down like
the outer cells, and become a mass of yolk granules, in
which are scattered a number of nuclei with associated proto-
plasm.

(12) Some of these pass outward to form the endoderm,
others pass inwards to form vitellophags.

(13) A lumen is formed in the yolk mass and it becomes
connected to the posterior involution, giving rise to a gastrula-
like structure.

(14) The internal mass of yolk assumes the form of a
number of yolk columns or pyramids.

(15) The posterior body cavities arise by a number of cells
from the yolk mass forming a second layer under the endo-
dermal layer.

(16) The yolk lumen increases in size, the yolk granules
becoming converted into a homogeneous substance. This
takes place chiefly on the dorsal side, where the yolk lumen
becomes connected with the archenteron.

(17) The position and extent of the five body cavities in
the larva are shown.

(18) The yolk in the larva is in the form of an elongate
mass of granules and homogeneous matter, lyiug on the floor
of the archenteron.

(19) Changes are described in the larval nervous system,
and the appearance of a posterior thickening and involution
of the ectoderm below the yolk mass is noted.

TH 15 DEVELOPMENT OF THE- CAPE CEPHALODISCUS. 209'

References.

1. Andersson, K. A. — “ Eine Wiederentdeckung von Cephalo-

discus,” ‘Zool. Anz.,’ xxvi, 1903, p. 368.

2. “ Die Pterobranchier der schwedischen Siidpolar-expedi-

tion, 1901-1903,” * Wise. Ergebn. schwed. Siidpolar-Exped.’ v,
1907.

3. Braem, E. — “Pterobranchier mid Bryozoen,” ‘Zool. Anz.’ xxxviii,

1911, p. 546.

4. Gilchrist, J. D. F. — “Observations on the Cape Cephalodiscus

and some of its Early Stages,” with an Appendix by Sidney F.
Harmer, ‘Ann. Mag. Nat. Hist.,’ ser. 8, vol. xvi, 1915, p. 233.

5. Harmer, S. F. — “ The Pterobranchia of the ‘ Siboga ’ Expedition,.

with an Account of other Species,” ‘ Resultats des Explorations
a bord du “ Siboga,” Monogr.’ xxvi bis, 1905.

6. Masterman, A. T. — “ On the further Anatomy and the Budding

Processes of Cephalodiscus dodecalophus,” ‘Trans. Roy.
Soc. Edinb.,’ xxxiv, 1900, p. 507.

7. Ridewood, W. G.— “A new species of Cephalodiscus (C.

gilcliristi) from the Cape Seas,” ‘Mar. Inv. S. Africa,^
vol. iv, 1908.

8. Schepotieff, A. — “Die Pterobranchier des Indischen Ozeans,” ‘Zool.

Jalirb. Abt. Syst.,’ xxviii, 1909, p. 429.

EXPLANATION OF PLATES 13 and 14,

Illustrating Dr. J. D. F. Gilchrist's paper “On the Develop-
ment of the Cape Cephalodiscus (C. gilchristi,
Ridewood)."

Explanation of Figures.

The following reference letters are used in the figures : an. Anus.
arch. Archenteron. b. m. Basement membrane. d. Yolk detritus.
end. Endoderm. ex. y. c. External yolk columns, i. y. c. Internal yolk
columns, inv. s. o. Involution in nervous tissue of sense organ. N..
Nucleus, n. Nucleolus, p. inv. Posterior invagination. p. v. inv.
Postero-ventral involution, p. v. th. Postero-ventral thickening, s. o.
Sense organ, vit. Vitellophag. v. th. Ventral thickening, y. Yolk.
y.gr. Yolk granules, y.l. Yolk lumen, y.n. Yolk nucleus.

[All the figures have been drawn by camera lucida except figs. 5.

210

J. D. F. GILCHRIST.

und 8; figs. 1, 17, and 20 with a Zeiss F objective; figs. 2-13 with
a Zeiss A, and the remainder with a Zeiss C. The ectoderm i-j
represented diagramatically by a light shading, the yolk grannies by
a stippled shading, where details are unnecessary. The scale of
magnification is shown by a line representing 50 /*.] -

PLATE 13.

Fig. 1. — Section of ovarian egg. N. Nucleus, n. Nucleolus, y.gr
Yolk granules, y.n. Yolk nucleus.

Fig. 2. — Fertilised ovum.

Fig. 3. — Two-celled stage with nearly equal division.

Fig. 4. — Two-celled stage with unequal division.

Figs. 5-7. — Four-celled stage showing various methods of division.

Fig. 8. — Three-celled stage.

Fig. 9. — Six-celled stage.

Fig. 10. — Section of egg showing 6 blastomeres and segmentation
-cavity.

Fig. 11. — Section o£ blastula showing blastoccele and contents.

Fig. 12. — Section of blastula showing beginning of internal prolifera-
tion at posterior end.

Fig. 13. — Section of blastula showing elongation of cells at anterior
end of embryo.

Fig. 14. — Section showing a solid embryo, the blastocoele being filled
with cells from the posterior proliferation. The external cells assume
the form of external yolk columns (ex. y. c.).

Fig. 15. — Longitudinal section of an embryo showing the external
yolk columns in increased numbers, and an invagination at the
posterior end.

Fig. 16. — Section of gastrula-like structure showing vitellophags
(vit), homogeneous detritus ( d .), yolk lumen (y. L), posterior invagina-
tion (p. inv.), and traces of external yolk columns (ex. y. c.), now
disappearing at the anterior end.

Fig. 17. — Section of part of anterior end of embryo showing the
formation of the endoderm (end.), and the appearance of a basement
membrane ( b . m.).

Fig. 18. — Longitudinal section of an embryo showing the formation
of inner yolk columns ( i . y. c.), the further development of the endoderm
(end.), and the early appearance of the ventral thickening (v. th.).

Fig. 19. — Longitudinal section showing further development of
endoderm, and formation of posterior body cavities (b. c.2 and b. c.3),

THE DEVELOPMENT OF THE CAPE OEPHALODISCUS. 211

Increase in yolk lumen ( y . 1.), and disappearance of inner yolk columns
from dorsal region of yolk lumen.

PLATE 14.

Fig. 20. — Longitudinal section showing details of formation of
posterior body cavities, a second layer of cells forming the inner wall
of b. c.2 but not yet in b. c.3.

Fig. 21. — Horizontal section showing the completed epithelial lining
of b. c.2 and b. c.3.

Figs. 22-34. — Transverse sections selected from a series of 140 of
a larva to show the positions and relations of the posterior body cavities
(b. c.2 and b.c.3), the archenteron (arch.), the yolk (y), and the sense
organ (s. o.).

Fig. 22 is the 6th from the posterior end.

23

17th

24 .,

24th

25 .

27th

26 ..

29th

27

36th

28

. 40th

29

, 45 th

30 .

, 67th

31

95th

32 ,

. 123rd

33 .

, 132nd

34 ,

, 138th

Fig. 35. — Transverse section from another series showing involution
(inv. s. o.) in nervous tissue of sense organ below ectoderm.

Fig. 36. — Transverse section showing postero-ventral thickening
(p. v. th.), and involution ( p.v.inv .).

Fig. 37. — Longitudinal vertical section of larva showing the anterior
body cavity (b. c.x).

Fig. 38. — Longitudinal vertical section of posterior end of larva
showing origin of anus (an.).

VOL. 62, PART 2. NEW SERIES.

15

TADPOLE RAISED BY ARTIFICIAL PARTHENOGENESIS. 21&

Note on the Sex of a Tadpole raised by
Artificial Parthenogenesis.

By

J. Bronte Gatenby, B.A.,

Exhibitioner of Jesus College, Oxford.

With 5 Text-figures.

With the object of ascertaining what is the sex of tadpoles
of It. temporaria raised by artificial parthenogenesis [ I
undertook some experiments last April. As my intention was
to procure as many tadpoles as possible, I adhered to the
method of smearing the eggs with a mixture of blood and
lymph and then pricking each one with a very fine glass
needle. The usual precautions were taken in this work, even
the water in which the eggs were raised being drawn from a
tank where it had remained for several days; the frogs were
carefully washed in alcohol before opening, and the eggs were
not allowed to touch the skin while being withdrawn from
the swollen uterus.

Two sorts of glass needles were used ; one was drawn from
glass tubing and the other from solid glass rod; the former
gave a higher percentage of burst and spoilt eggs, but while
the latter sort of needle gave fewer irretrievably ruptured
eggs, the percentage of successful segmentations was lower.
There is little doubt that the minute lumen left in the glass-
tube needle served to introduce more of the blood and lymph
into the egg, and hence to promote segmentation. In some
experiments carried out by the late Dr. Jenkinson different

214

J. BRONTE GATENBY.

Text-fig, 1. — Partlienogenetic tadpole three months old. X 1.

Text-fig. 2. — Control fertilized tadpole at same age raised under
same conditions, x 1.

Text-fig. 3. — Gonad and surrounding organs of the parthenogenetic
tadpole. F. B. Fat body. K. Kidney. B. Rectum. T. Testis, x 10.

Text-fig. 4. — Gonad and surrounding organs of control male. X 10.

Text-fig. 5. — Obliquely longitudinal section of part of the gonad
and fat body ( F . B.) of the parthenogenetic tadpole, A. T. Attachment
of gonad to roof of peritoneal cavity. G. E. Germinal epithelium.
L. A. C. Lacunee in gonad. S. P. T. Spermatic tubules. X 270.

TADPOLE RAISED BY ARTIFICIAL PARTHENOGENESIS. 215

fluids were injected into tlie egg, but though very large
numbers were treated, only one abnormal tadpole was procured.
The data got from these experiments and from those since
carried out by myself seem to show that there are almost
certainly other factors in the problem, as, for instance, in one
batch of eggs pierced by a solid needle a very good percentage
of tadpoles was got, while in another lot pierced by a hollow
needle, not one ev.en segmented. Nevertheless the whole
series of experiments clearly showed in my case that the
hollow needle was the better. Individual frogs differed
markedly in the number of tadpoles raised from their eggs.

I pricked five thousand eggs of R. temp or aria and raised
about fifty tadpoles to the closure of the neural folds. There
were, as is usual, many abnormal specimens, and the death-
rate of those which hatched was high. Without going into
details, it may be mentioned that fifteen tadpoles were raised
to a stage when the external gills become covered by the
epidermal overgrowth. Two of these were scarcely able to
swim, and they soon died. Of the remainder all died except
two, just before their hind limbs broke through. Those which
died at this time did so, I believe, because the weather was
most inclement, for the tadpoles born under natural con-
ditions in the ponds were extremely backward for the season
of the year. One of the survivors died at the critical period
when the germ cells were beginning to become grouped in
the manner which shows their sex. I believe this one would
have been a male, but there was still undifferentiated material
in the gonad. The sole survivor grew at a great pace and
quickly outstripped the controls, so that it was nearly two
and a half times normal size. In Text-fig. 1 and 2 are natural
size drawings of this tadpole and a normal control raised

n the same way ; the parthenogenetic tadpole is normally
proportioned, its hind limbs, tail, faeces, and its general
outward morphology being proportionately large. The rectum,
as was shown by the size of its faeces, and as subsequent
dissection showed, was also very large.

At the age of three months the tadpole was placed in an

210

J. BRONTE GATENBY.

aquarium from which it was known normal tadpoles could
not escape. To my regret I found that just as the front
limbs had broken through, the tadpole jumped out on to the
floor, where it died before I discovered its plight. In figs. 3
and 4 are drawn the gonads (T), kidneys (K), and rectum ( R )
of the parthenogenetic and normal tadpole respectively.
When I sectioned the gonads of the former, I found that it
was a well developed male, as the external appearance seemed
to show, for the gonads were distinctly testiculiform.

In section the germ cells are clearly marked into numerous
incipient spermatic tubules ; though the section drawn in
fig. 5 was across the least well-differentiated region, the
spermatogonial nests are well marked. Undoubtedly the
gonad had passed beyond the indifferent stage during which
it is impossible to speak with certainty as to the sex. The
part marked X in fig. 5 contains germ cells which have just
begun to form spermatogonial groups, while that marked
Y is apparently nothing more than a non-germinal core, the
cells and their nuclei staining like the tissue forming the
mesorchium (A.T.).

I feel quite sure that this tadpole was a male. Mr. Goodrich,
whom I have to thank for his usual kind interest and sugges-
tions, lately drew my attention to an abridged account of a
paper by J. Loeb read before an American philosophical
society on the same question as that dealt with in this note.
Loeb found that the sex of an American species of partheno-
genetic frog a year old was male. I have not yet seen
LoeVs paper, but his results agree with mine as to the sex.

DEMONSTRATING THE NUCLEI OF NERVE FIBRES. 217

An Easy Way of Demonstrating the Nuclei of
Nerve Fibres.

By

Henry E. Relmrn,

Student of Medicine. (From the Physiological Laboratory, King’s
College, London.)

While attending the histology class this summer, I, in
common with my neighbours, found great difficulty in render-
ing visible the nuclei of teased, fresh nerve fibres. Using
gentian violet as recommended in Sir Edward Schafer’s
* Essentials of Histology/ the nerve fibres were almost
uniformly stained, and the nuclei did not stand out convinc-
ingly. I tried other stains with equally disappointing results,
and at last, in desperation, I used a mixture of nearly all the
stains on the table (Ehrlich’s haematoxylin, methylene blue,
and alcoholic solution of eosin) and thus obtained a prepara-
tion in which blue nuclei stood out prominently on a reddish
background. The preparation was shown to Dr. de Souza
and Prof. Halliburton, who suggested to me that I should
proceed to investigate the matter and ascertain what was the
cause of success in this “ blunderbuss ” experiment.

Without going into all the details of the numerous prepara-
tions I made subsequently, I may state at the outset that the
principal factor is the alcohol, in which, in my first successful
experiment, the eosin had been dissolved. Aqueous solutions
of eosin are quite as ineffective as the other stains. Fresh
nerve fibres (and especially their nuclei), teased on the slide
in the usual way, stain with great difficulty. But the nuclei
stain readily with practically any dye (methylene blue, haema-

218

HENRY E. REBURN.

toxylin, picro-carmine, gentian violet) after preliminary treat-
ment with alcohol. Or if the dye is added first and the
nuclei remain unstained, the stain in the nuclei becomes
evident on subsequent addition of alcohol to the preparation.

The difficulty of staining these nuclei in fresh preparations
appears to have been noticed by others. Thus in Foster and
Langley's ‘ Practical Physiology and Histology' (7tli edition,
p. 125) I find this statement: “ The nuclei of the sheath
may be stained by placing a piece of nerve after brief treat-
ment with osmic acid in picro-carmine or haematoxylin for
an hour." In Stirling's f Practical Histology' (2nd
edition, p. 206) the directions for staining the nuclei include
the statement that after osmic acid picro-carmine may be
used, but it is best to leave them for several days in the
stain.

In consequence of these statements 1 made numerous
preparations in order to see whether preliminary treatment
with osmic acid will take the place of the alcohol, but with
very indifferent results. Haematoxylin after osmic acid gave
a brownish appearance to the whole nerve fibre, but the nuclei
did not stand out ; and in the case of gentian violet after
osmic acid the nuclei were apparent because they were
stained less darkly than the rest of the fibre. Picro-carmine
after osmic acid did stain the nuclei red, but this took a con-
siderable time, and the preparations were not nearly so good
as after treatment with alcohol. In A. B. Lee's ‘ Microtomists*
Vade-mecum ' (7th edition, p. 136) I find the following,
which seems germane to the present question : “ Living tissue
elements in general do not stain at all, but resist the action of
colouring reagents till they are killed by them.

Objects which have been passed through alcohol generally
stain better than those which have only been in watery fluids.
But long preservation of tissues in alcohol is generally
unfavourable to staining."

It is well known that the nuclei of nerve fibres are usually
quite well stained in sections, and in this case alcohol is
usually employed in the stages of preservation or embedding.

DEMONSTRATING THE NUCLEI OE NERVE FIBRES. 219'

I can, however, confirm Lee’s statement that long preservation
in alcohol is not beneficial to the staining of nerve nuclei;
after a nerve has been kept for some days in alcohol it is
almost as difficult to stain its nuclei as when it is in the fresh
condition.

The following is the method I would recommend for general
routine work when a rapid result is wanted. The fresh nerve
is teased on a dry slide in the usual way, the preparation
being kept moist with the breath. A drop of absolute
alcohol is added and then a drop of Ehrlich’s hsematoxylin,
followed by a drop of methylene blue. Either dye may be
used alone, but the nuclei are most deeply stained when both
are employed. An alcoholic solution of eosin may be substi-
tuted for the absolute alcohol ; the alcohol here is the
essential reagent, but the eosin provides a red counter-stain.
The preparation is then washed, cleared, and mounted in the
usual way, and the whole operation is completed within a few
minutes.

ON A LARVAL ACT1NIAN PARASITIC IN A RHIZOSTOME. 221

On a Larval Actinian Parasitic in a Rliizo-

stome.

By

C. Badlmm, 15. Sc.,

Demonstrator in Zoology, University of Sydney.

With 3 Text-figures.

Our knowledge of the medusophilous larval Actinians lias
been summarised by Haddon (1) and by McMurrich (2).
The last-named author says : “ The available evidence seems
accordingly to point strongly in favour of the various medu-
sophilous forms being the young stages in the development
of Peachia rather than Halcampa; but a direct linking-up
of the immature examples of Bicidium1 with their respective
adults is necessary to settle the question.”

In this paper I will show that I have found the larvae of
Peachia hilli parasitic in a Rhizostome up to the stage in
which they have been found free-living — thus linking up this
medusophilous form with the adult. There is described, for
the first time, the function of certain structures (the conchula
and pores of the physa) found in Peachia larvae, and
their importance in connection with the parasitism of the
widespread genus Peachia is emphasised. So far as I am
aware, this is the first time that a larval Actinian has been
•described as parasitic in a Rhizostome. The parasitic larvae
hitherto described differ from the larvae of P. hilli in this

1 A genus in which he places these medusophilous forms.

222

C. BAPHAM,

respect — that they live either on the exterior of their hostr
or in the gut which opens freely by a mouth. The larvae of
P. hilli, however, live for a considerable period in the radial
canals of a Rhizostome, from which they can only escape by
perforating the body-wall of their host. This host is a large
form, Crambessa mosaica, which is found in the land-
locked harbours along the coast of New South Wales. This
medusa is frequently found in large numbers, possibly brought
together by currents and tidal action. At other times it is
widely scattered. At all times it forms a very characteristic
faunal element of the various inlets. So far as I can ascer-
tain, it passes through its life-history in these waters.

Peach ia hi lli, the adult form of these larvae, is found
in Broken Bay, and was described by Miss Wilsmore (3) in
1911. She also described a free-living larval form, the
internal anatomy of which showed that it was the larva of
P. hilli.

The larvae which are found parasitic correspond in their
older stages with the larva found free-living, and so link up
the medusopliilous forms with the adult.

Character and Occurrence of Larvae.

The larvae are found in various parts of the large radial
canals adhering to the ex-umbrella wall of the gut, excepting
when they are making their way out of their host. I found
them at various stages of development from 5 mm. to 40 mm-
long. They occur in about every tenth medusa examined
during the months of September and October, but by January
it is rare to find them.

In October they were noticed in the act of escaping from
their host, going through a hole, regular in outline, made in
the sub-umbrella wall of the gut, near its periphery. I have
found larvae lying free in the gut, But near a hole, others
actually filling up such a hole, with their oesophageal end
protruding, and yet others, having effected their escape,,
adhering to the tentacles of their host. This latter condition

ON A LARVAL ACTINIAN PARASITIC IN A RHIZOSTOME. 223

recalls the discovery in a similar situation of another and
possibly closely related Actinian found by the Astrolabe
Expedition (4) off the east coast of Australia in 1833, and
recently investigated by Pax (5). The escaping larvae varied
in length from 20 mm, to 40 mm.

Description of Larvae.

Both the adult and the larval form of P. hilli found free-
living have been described by Miss L. J. Wilsmore from
specimens obtained from Broken Bay by Prof. J. P. Hill.

In regard to the colour of the larva, the body is light
amber, the oesophageal folds somewhat flesh-coloured or
tawny, and the twelve tentacles have purplish-brown mark-
ings. There is a spot at the apex of each tentacle, and next
to this is a line encircling the tentacle. The five markings
which follow are V-shaped, and are on the oesophageal surface
of the tentacle only. The colour of the apex of the V is weak
or absent. There are no processes on the body resembling
the suckers described by Haddon and Dixon (6) in the adult
P. hastata; and neither while the larva is in its host nor
“in vitro,” have I seen any attempt on the part of the larva
to attach itself except by its oesophageal folds.

There are two points which call for a further description,
and which are, moreover, of considerable interest in con-
nection with the parasitic life of the larva : I refer to the
conchula and the pores present in the physa. Both of these
structures are best studied in the living animal.

The Conchula and Pores of the Physa.

In the genus Peachia there is a single deep siphonoglyph.
When the lips of the siphonoglyph come together, there is
formed a tube which runs from the enteron to the exterior.
In some species the external opening is surrounded by a
complicated series of lobes, forming a conchula. In others
the conchula is of a simpler nature.

224

C. BADHAM.

McMurricli (2) gives a useful account of the conchula in
the various species of Peachia. In the larva of P. hi lli
the peripheral ends of the lips of the siplionoglyph project
as a pair of small processes (Text-fig. 2, S. 1.), while from the
base of the external opening there projects a median lobe.
In this manner the conchula presents a simple three-lobed

Text-fig 1.

Drawing of a fully-extended, living larval specimen of Peachia
liilli (x 1|). The subject has been kept detached from its
host for a few days and the conchula (C.) is somewhat con-
tracted. The oesophageal folds and the character of the
tentacles are seen. The grooves on the surface of the body
are clearly shown and some of the pores of the physa. The
trilobed character of the conchula is evident.

structure, such as appears to be the basis of the conchula of
all species of the genus Peachia. Haddon (6), writing*
of P. hastata, says that the conchula varies greatly in
complexity, but that “ one basal, and two lateral lobes may

ON A LARVAL ACTINTAN PARASITIC IN A RHIZOSTOME. 225

always be detected, which are larger and carry more secon-
dary lobes than the remainder. The basal lobe forms a
kind of lid or operculum to the siplionoglyph.” An original
observation which I have made is that, when the larva is
attached by the oesophageal folds, it is through the conchula
that a constant stream of fluid bearing food particles goes to
the enteron. This fact I have made out by studying living
larvm which were still attached to the gut-wall of a Rhizo-
stome. Correlated with the function of the conchula is the

Text-fig. 2.

Drawing of the oesophageal surface of a living larval form of
Peachia liilli (x 2). The nature and markings of the
tentacles are shown and the character of the oesophageal
lobes. The conchula (C.) is seen to consist of a basal and two
lateral lobes, the latter being borne by the lips of the siplio-
noglyph (S.I.), which are in contact near the periphery but
slightly separated near the centre.

presence of a large number of pores in the physa. These
pores were mentioned by Haddon (6) as occurring in
P. hastata and included in his definition of the genus
Peachia (7). They were noted by Miss Wilsmore (3) in
sections and described by her so far as her material per-
mitted. They are placed in the twelve external grooves of
the physa, which, however, is not marked off from the scapus
except in extreme contraction. There are generally twenty
pores in each row arranged somewhat irregularly (Text-fig. 3).
There is no central pore.

226

0. BADHAM.

These pores are a conspicuous feature when the anemone
is extended. The intake of fluid by the conchula is con-
tinuous, and as these pores, which lead from the interior of
the animal, are widely opened at such times, I consider that
they serve to carry away the fluid taken in by the conchula.
So that, in the attached larva, there is a constant stream of
water, bearing food particles, going into the interior of the
anemone through the conchula, and a stream of water passing
out through the pores of the physa. It would appear as if the
-conchula has been developed as a larval organ, correlated with

Text-pig. 3.

Drawing of the physa of a living larval Peachia hilli, showing
the character and arrangement of the pores as seen when the
anemone is extended and the pores open.

the parasitic existence of the larval forms of the genus
Peachia; and, associated with it, is the development of the
pores of the physa.

The manner in which the medusophilous larvae of Actinians
take in their food has not been elsewhere described. Haddon ( 1 ) ,
however, suggests that the form he described as the larva of
Hal cam pel la chry san thellum detaches itself and floats
in the water. Other authors assign this form to the genus
Peachia. The deep siphonoglypli present has an external

ON A LARVAL ACTINIAN PARASITIC IN A RHIZOSTOME. 227

opening (Hacldon (8) ), and when the anemone is attached,
would, I take it, function as does the conchula of P. hilli.

McIntosh (9), writing of the parasitic larva of P. hastata,
says :

“ They appear to adhere to the medusa by the sucker-
like action of the mouth, which is widely open, though
the tentacles are closely applied to the surface. The
free-swimming larval forms are thus, at a subsequent
stage, carried about, without effort, by the medusae, and
as there is abundance of nourishment of a suitable kind
around, it is not necessary to limit the view only to the
possibility of their feeding on Thaumantias, for by
the use of their tentacles as organs of attachment the
mouth may, at any time, bes et free.”

My observations show that the larva of P. hilli adheres
h>y the sucker-like action of the closely-applied oesophageal
folds — the closed lips of the siphonoglyph completing the
sucker — and that the tentacles are not brought into use.
The larva is always found adhering in this way, save when
escaping from its host, and it is through the conchula that
the food is taken in.

I am tempted to put forward the hypothesis that the
larvae of the widespread genus Pea chi a are all meduso-
philous, and that the single deep siphonoglyph, possessing
as it does an opening below the oesophageal folds, is a larval
organ, correlated with such parasitism. The development of
a conchula, as a series of processes round the external open-
ing, is seen in the older larvae.

In support of this hypothesis I would draw attention to
the distribution of the larval forms — of Peachia hilli para-
sitic in Crambessa mosaica on the east coast op Aus-
tralia; of Peachia parasitica on Cyanea arctica in
the North Atlantic; of Peachia hastata on various
medusae in the North Sea; and of Bicidium aequoreae,
the probable larval form of P. quinquecapitata, off the
-coast of British Columbia.

In regard to the second and third forms, McMurrich (2)

VOL. 62, PART 2. NEW SERIES. ] 6

228

0. BADHAM.

prefers to place these larvae in the genus Bicidium, until
they are proved to develop into the adult Pea cilia. He
regards the occurrence of P. quinquecapitata in the
same locality as Bicidium aequoreae as suggestive, and
considers it not improbable that their differences are due to
age.

It is to be noted that there are so far described, according
to McMurrich (2), seven species of Peach i a, and these,
together with an eighth species described by Miss Wils-
more (3), are as widely distributed as the medusophilous
Actinian larvas.

In conclusion, it would seem that the parasitism of these
larvae is only compatible with the presence of a deep siphono-
glyph having an external opening or conchula, and such a
structure is possessed only by the genus Peachia, if we
except the little-known genus Actinopsis, which is said to
possess a double conchula.

I wish to thank Prof. Haswell, in whose laboratory this
work was done, for his kind help and advice.

The figures were re-drawn by Mr. F. W. Atkins, of the
Sydney Technical High School.

Literature.

1. Had don, A. C.— “Note on the Arrangement of the Mesenteries in

the Parasitic Larva of Halcampa chry santhellum
(Peach),” ‘ Proc. Hoy. Dublin Soc.,’ vol. v, 1886-7.

2. McMurricli, J. Playfair. — “ On Two New Actinians from the Coast

of British Columbia,” ‘ Proc. Zool. Soc. London,’ 1913, p. 963.

3. Wilsmore, Leonora J. — “ On some Hexactinise from New South

Wales,” ‘ Journ. Linnean Soc., Zoology," vol. xxxii, 1911.

4. Quoy et Gaimard. — “ Zoologie du Yoyage de la corvette ‘Astro-

labe,’ ” Paris, 1833.

5. Pax, F. — “ Revision des types des Actinies decrites par Quoy et

Gaimard,” ‘ Ann. Sci. Nat.,’ ser. 9, xvi, 1912.

6. Haddon, A. C. and Dixon, G. Y. — “ The Structure and Habits of

Peachia liastata (Gosse),” £ Sci. Proc. Roy. Dublin Soc.,’ new
series, vol. iv, 1885.

ON A LARVAL ACTIN1AN PARASITIC IN A RHIZOSTOME. 229

7. Haddon, A. C. — Revision of the British Actiniae,” ‘Trans. Roy.

Dublin Soc.,’ vol. iv, ser. 2, 1888-92.

8. “ Researches at St. Andrew’s Marine Laboratory, on Larval

Actiniae parasitic on Hydromedusae at St. Andrew’s,” ‘Ann. Mag.
Nat. History,’ vol. ii, ser. 6, p. 256.

9. McIntosh, W. C. — “ On the Commensalistic Habits of Larval Forma

of Peachia,” ‘ Ann. Mag. Nat. History, ser. 5, vol. xx.

SPLEEN OF LEPIDOSIREN AND PROTOPTERUS. 231

The Early Development of the Spleen of Lepido-
siren and Protopterus

By

Cl. L. Purser, B.A.,

(Sometime Coutts-Trotter Student, Trinity College, Cambridge.)

With Plates 15, 16, and 17.

My first words must be those of thanks to Professor Graham
Kerr for providing me with every facility for carrying out
the research, the results of which are embodied in this short
paper. He invited me to work in his laboratory, provided me
with material already stained and mounted, and was especially
helpful in suggestion and criticism.

Owing to the uncertainty as to the time available for this
work, I have done no fresh section-cutting, nor have I re-
stained any of the sections already made. This being so, I
found that the earlier stages of Protopterus were more suit-
able for the present investigation than those of Lepido siren,
and so I will first describe its development in detail, and then
contrast and compare that of Lepido siren, adding further
details where I can.

The Spleen of Protopterus.

In the earlier stages than N.T.XXXII.1 the foregut is short,
thick, and solid, its enveloping mesoderm being yolky, and
with difficulty distinguishable from the subjacent endoderm.

1 The stages are designated by the numbers used in Keibel’s
Normentafeln.’

232

O. L. PURSER.

Just about this stage, however, the foregut elongates and
narrows, and the mesoderm loses its granules, but remains
fairly compact.

On the right-hand side of the foregut, where it overlies the
intestine, there is a distinct thickening of its layer of meso-
derm, which is particularly vascular. This is where the
spleen will arise (fig. 1 and la). It is in front of the dorsal
rudiment of the pancreas, the only one which has developed
at all at this stage, and, except for the posterior lobe of the
liver, is entirely posterior to that organ. This posterior lobe
is ventro-lateral on the right side of the position of the spleen,
but more or less in the mesial plane of the embryo itself, the
foregut being to the left of the middle line.

The vascularisation is entirely venous, being part of the
gut circulation.

There is nothing to suggest that the endoderm has anything
to do with the formation of this thickening, but it is impos-
sible to be dogmatic on the point, because at stages just prior
to the one under discussion the two layers seem completely
fused, and it is impossible to decide in many instances whether
a nucleus belongs to a cell of the endoderm or of the meso-
derm. Since, however, all the organs known certainly to be
of endodermal origin, e.g. liver, pancreas, or thyroid, arise
as lieavily-yolked rudiments, therefore the development of
the spleen from tissue entirely free from yolk granules and in
all other respects resembling the mesenchyme of other parts
of the embryo seem to lend support to the view upheld by
Laguesse against that by Maurer and Kupffer.

At Stage XXXIII (fig. 2 and 2a), the spleen rudiment is
distinctly visible as a flat structure on the right side of the
foregut extending as far forward as the point where the latter
begins to bend to the mesial plane of the embryo over the
lung outgrowth. The anterior ventral portion of the intestine
extends for a considerable distance in front of the developing
pyloric valve, and so the spleen rudiment is entirely dorsal to
it. With regard to the glands of the alimentary canal, it lies
behind the origin of the liver and the ventral pancreatic

SPLEEN OF LEPIDOSIREN AND PROTOPTERUS.

233

buds but anterior to the dorsal bud, a position it keeps
throughout life.

As yet the cells of the organ are quite undifferentiated
from those of the rest of the mesenchyme, but the rudiment
is apparent owing to its vascularisation. The vein from the
intestine here breaks up into a number of branches which
run into the rudiment in a more or less forward and dorsal
direction. This is the afferent system. The efferent is formed
by a number of small tributaries which unite and enter the
liver as the Hepatic Portal Vein. The organ itself contains
large sinuses, chiefly peripheral, with which these two
systems communicate. They have no visible endothelial
lining.

About this time in the development of the embryo the
foregut greatly increases in length, and since the spleen lies
embedded in its sheath it is to be expected that it would
grow in that direction too. Examination of embryos at
Stage XXXIV proves this to be the case, its length increasing
from about; *2 mm. at Stage XXXIII to about *5 mm., while its
greatest diameter remains at about T5 mm. Anteriorly it is
slightly twisted towards the dorsal side of the foregut, where
the latter begins to arch over the lung rudiment : which seems
to point to this portion of the foregut being at this stage
included in the twisting which occurs chiefly in the intestine.
The position of the spleen with regard to the gut appendages
is the same as earlier, i.e., the bile-duct opening and the
ventral pancreatic buds lie roughly in tho same transverse
plane as its anterior end, and the dorsal pancreas lies at the
posterior end. The yenous circulation of this portion of
the embryo has undergone no change, except, perhaps, that
the peripheral sinuses are better marked, and I have been
unable to trace any arterial supply.

There is active cell-division going on throughout the organ,
but particularly in the venous spaces, where the erythroblasts
are multiplying freely.

At this stage (fig. 3 and 3a) the differentiation between the
cells of the spleen and those of the rest of the mesenchyme

234

Gr. L. PURSER

commences. The nuclei at the periphery begin to take up a
position with their long axes tangential to the organ ; the
cells containing these will form the capsule. External to
these the cells form a rather compact connective tissue, and
within them the splenic tissue is but little more differentiated,
but its spaces are, of course, venous sinuses.

The alimentary canal between Stages XXXI Y and XXXY
undergoes a considerable amount of remodelling (I use the
word in the same sense as Professor Graham Kerr, ‘ Quart.
Journ. Micr. Sci./ vol. liv, p. 484). Whereas the first turn
of the intestine has up to now been very prominent and has
caused the embryo to be rather tadpole-like in shape, at this
time there is considerable shrinkage in this region so that
there is but little bulging exteriorly (vide Keibel’s f Nor-
mentafeln/ vol. x). The result of this is that the relative
positions of the organs in connection with the foregut and
the anterior portion of the intestine are altered. The foregut
itself is rotated so that the spleen lies laterally anteriorly and
ventro-laterally posteriorly on its right side : the dorsally
placed wall of that part of the intestine projecting in front
of the pyloric valve shortens, so that the three pancreatic
buds become fused together and the bile-duct opening comes
to lie just behind the posterior end of the spleen, a very
marked difference from its earlier position : the pancreas
extends forwards under the posterior half of the spleen, but
gradually retreats further back as development proceeds.

The histology also has advanced, although the tissue is still
very condensed. The trabeculae are developing, as will be
seen by examining fig. 4, but the cells, except for their arrange-
ment, are indistinguishable from the rest of the mesenchyme.

By Stage XXXYI, the last one examined, the organ has
become very well-marked and obvious in sections, but it
never becomes so in dissection, because it remains embedded
in the sheath of the foregut. It is just over 1 mm. long, and
its greatest diameter, towards its posterior end, is about a
third of this.

The first turn of the intestine has shrunk still more, so

SPLEEN OF LEPIDOSIREN AND PROTOPTERUS.

235

that the spleen projects for most of its length, but the
posterior end, where it continues to overlap the pancreas, is
contained within it.

The histology is as follows : The cells are arranged to form
closely-packed trabeculae, surrounded by blood spaces. These
have no endothelial lining, and are the venous sinuses pre-
viously mentioned. The peripheral sinuses, which were so
well marked in the earlier stages, are now completely broken
up by trabeculae to form the channels of the sponge-work.

The blood supply is rather doubtful. There appears to be
a small branch of the coeliac artery actiug as an afferent
vessel in addition to the branches of the intestinal vein ; but,
I think, some of the blood from the intestinal goes direct to
the liver. The factors of the Hepatic Portal Vein compose,
as before, the efferent system.

The Spleen of Lepidosiren.

Turning now to the development of the spleen in Lepi-
dosiren, the differences observable are not of much morpho-
logical significance.

It apnears at just about the same stage as in Protopterus,
but develops rather more quickly, so that by Stage 34,
(figs. 5 and 5a) it is aleady about 1 mm. long, and shows the
beginnings of a trabecular arrangement among its cells.

Its position, too, differs from that in Protopterus; it is
dorsal to the foregut anteriorly, turning over to the right side
posteriorly, and lies almost entirely in front of the intestine.
This last point of contrast is due to the difference of distribu-
tion of yolk in the two species.

I have been unable to discover a branch of the coeliac
artery supplying it, but the intestinal vein, besides supplying
the spleen, does continue directly to the liver.

Already, however, at Stage 35, a branch of the coeliac
artery can be made out going to the spleen, which is as far
developed as the latest stage of Protopterus that I have
examined (figs. 6 and 6a).

236

G. L. PURSER.

At Stage 36 (figs. 7 and 7a) the organ is clearly defined
from the rest of the mesenchyme dorsal to the endodermal
wall of the gut. It has sharply-marked boundaries, and is
compact, while the neighbouring mesenchyme has developed
the alveolar structure typical of ordinary connective-tissue.
The capsule-forming cells appear as a single layer of
nuclei bounding the organ. These cells have formed a
definite connective-tissue sheath over a large portion of the
surface by Stage 37, the most advanced stage I have
examined.

For the histogenesis of the spleen in Lepi do siren I
cannot do better than refer to Dr. Bryce’s paper on the
“ Histology of the Blood of the Larva of Lepidosiren.”
One point only will I mention. The cellular elements show a
marked reduction in size between Stages 35 and 37. This
change seems to affect the whole of the mesoderm cells of
the foregut, as will be seen by examining figs. 6, 7 and 8.

I thought at first that it was due to variation in the amount
of contraction which the larvae had undergone during the
preparation of the sections, but that this is not the case is
shown by two facts : (1) the blood corpuscles do not show
this change, and (2) the effect is observable in all the series
examined.

To return to the question of the blood circulation. It is
quite clear that this is, to begin with, entirely venous, so that
there is a sort of splenic portal system. This is in connection
with the veins draining the intestine. The development of
these had not been fully worked out in either form, nor have
I had the time to do it properly myself, so the following
remarks must be accepted with all reserve.

Apparently, in both species the main intestinal vein which
drains the intestine breaks up in the spleen. (It will have
been noticed that I have referred to the vein which supplies
the spleen as the intestinal. There is, of course, no vein
ordinarily called by that name : I use it simply as a matter of
convenience, because I do not wish to make any definite
statement on the point.) The blood from the spleen runs to the

SPLEEN OF LEPIDOSIREN AND PROTOPTERUS.

237

liver via the hepatic portal vein. There is, at first, no direct
communication between the intestine and the liver, and no
arterial supply to the spleen. This latter point seems to be
general throughout the phylum, for Dr. Bryce states, in
QuaiiTs f Elements of Anatomy/ vol. i, p. 237, that in the
human embryo the artery develops late.

What the chief factor of this intestinal vein is, is uncertain.
In Pro top ter us it appears to be the intra-intestinal, while
in Lepidosiren it is the subintestinal. This, however, is
most likely only a question of which is most developed at the
stage under consideration.

At about Stage XXY in Pro top ter us, and earlier in
Lepidosiren, there is visible a small vein which communi-
cates directly with the liver. This is the Hepatic Portal
Vein proper, which, in the adult, becomes a well-marked
vessel.

In Lepidosiren at the latest available stage I carefully
examined the veins of this region (omitting the smaller
tributaries from the intestine) . The arrangement is as follows,
from before backwards. There are three veins which com-
municate between the spleen and the hepatic portal vein, and
behind the last of these the latter forks ; the right branch is
confined to the liver, and the left, passing straight through
the tissue of the pancreas and turning over the left side of
the gut, gradually fades away in the lattePs ventral
mesenchyme.

I interpret this in this way. The subintestinal vein1 runs
round the left side of the intestine to the dorsal surface, and
then gives off a branch running backwards into the right
lobe of the liver. It then continues forwards and gives off
one branch to the splenic spongework, receives two from it,
and then disappears. After reaching the side of the liver the
portal vein seems to give off small branches into the liver
tissue along the entire length.

1 Dr. Jane Robertson (‘ Quart. Journ. Micr. Sci.,’ vol. lix, p. 121)
describes this vein as the posterior part of the original subintestinal and
the proximal part of the left vitelline vein.

238

G. L. PURSER.

W. N. Parker, describing the adult condition in Protop-
terus, states that the main factor of the hepatic portal is a
large mesenteric which runs close to the intra-intestinal artery-
in the axis of the spiral valve and comes to the surface at the
pylorus (this is the intra-intestinal vein of Laguesse). He
mentions a subintestinal vein, the connections of which he has
not made out, and then says that just anterior to the pylorus
the mesenteric can be traced into an anterior and posterior
branch, the latter supplying the posterior lobe of the liver
behind the gall-bladder. (This is the first branch I have
mentioned above.) He continues: “The former receiving a
large lieno-gastric vein (the factors of which form a dense
meshwork in the spleen) and a pancreatic vein, and then
dividing into branches which supply the anterior lobe of the
liver.” According to him, therefore, there is but one
efferent vessel, and the afferent supply is wholly arterial.
This is in marked contrast with what obtains iu other fish.

(a) T. J. Parker on Must el us, for instance, describes two
large veins connnected with the spleen, an anterior and
posterior lieno-gastric. The former runs with the lieno-
gastric artery, and is most likely . efferent ; the latter lies
between the pyloric division of the stomach and the right lobe
(morphological posterior portion) of the spleen and “ receives
feeders from both.”

(b) Laguesse on Acanthias says the hepatic portal is
composed of two trunks : the supra-intestinal, running the
length of the intestine, and, after passing the liilum of
the spleen, receives from that organ the splenic vein ; and the
subintestinal, which receives blood from the pancreas, at the
edge of which it receives the accessory splenic. These two
veins correspond to the anterior and posterior lieno-gastric
veins of T. J. Parker respectively. He states that in the
adult they are anastomosed, and in the embryo it is on this
loop that the spleen appears. He also states that there is a
double anastomosing arterial supply, one directly from the
aorta and one from the coeliac, a condition which, he says, is
found in the Trout as well.

SPLEEN OF LEP1DOSIREN AND PKOTOPTERUS.

239

Judging, therefore, from these three descriptions, taken
with the facts of embryology already known, one comes to the
conclusion that the original circulation of the spleen must
have been entirely venous, being a portal system between the
intestine and the liver. Later it was “ shunted ” oft the main
vessel so as to lie on a loop alongside. Later still, the delivery
of arterial blood removed the necessity of an afferent venous
supply, so that in all forms above the Pisces there are present
a single splenic artery and a single splenic vein only. In the
class mentioned, however, both the veins persist, and there
may be a second artery as well. The direction of the blood-
flow in the veins is of some importance, a point on which
authors are not very clear. Judging from T. J. Parker's
description of Mustelus, it seems as if both the veins are
efferent in function. This entails a reversal of the current, in
the one serving the right lobe, during development. This is
not a serious difficulty (it would be by no means an isolated
case), but it makes the efferent system extraordinarily large
compared with the afferent, both the veins being so much
larger than the artery.

It seems, therefore, as if detailed investigation of the blood-
vessels of this part of the body in Lepidosiren and Pro-
topterus would be of much morphological value, and would
most likely help to bring into line the various descriptions
which have been published for the different fish investigated.

Summary.

(1) The spleen arises in a thickening of the mesenchyme
of the foregut, just after that mesenchyme has become free
from yolk granules.

(2) It is, at first, a mass of mesenchyme cells, round about
which are comparatively large venous sinuses without any
endothelial walls; later the cells become arranged to form
trabeculae across these sinuses, which thus get broken up
into the channels of a spongework.

(3) The afferent and efferent veins are in very close con-

240

G. L. PURSER.

nection with the veins from the intestine and to the liver
respectively. The arterial supply of blood develops from the
coeliac artery rather later.

(4) The organ remains throughout ontogeny embedded in
the sheath of the foregut, and is therefore inconspicuous.

Bibliography.

Brvce, T. H. — “ The Histology of the Blood of the Larva of Lepido-
siren paradoxa,” ‘ Trans. Roy. Soc. Edin.,’ vol. xli, p. 454,
1904.

Kerr, J. Graham. — “Notes on Certain Features in the Alimentary
Canal of Lepidosiren and Protopterus,” ‘ Quart. Journ. Micr. Sci.r
vol. liv, p. 484, 1909.

Lagtjesse, E. — “ Recherches sur le developpement de la Rate chez les
poissons,” ‘Journ. de l’Anat.,’ vol. xxvi, pp. 345-406 and 425-495,
1890.

Parker, T. J. — “ The Blood-vessels of Mustelus,” ‘ Phil. Trans. Roy.
Soc. Lond.,' vol. clxxvii, pt. 2, 1886.

Parker, W. N. — “The Anatomy and Physiology of Protopterus
annectens,’* ‘ Trans. Roy. Irish Acad.,’ vol. xxx, pt. 3, 1892.

EXPLANATION OF PLATES 15, 16, and 17,

Illustrating Mr. G. L. Purser's paper on u The Early Develop-
ment of the Spleen of Lepidosiren and Protopterus."

All these figures have been drawn with the aid of a Zeiss Abbe
drawing apparatus. I have to thank my sister, Miss Dorothy Purser,
for making the diagrammatic drawings forming Plate 1 5.

List of Abbreviations.

ao. Dorsal aorta. er. Erythroblasts. /. g. Foregut. g. b. Gall-
bladder. g.b.d. Bile-duct. h.p. v. Hepatic portal vein. int. Intestine.
int.v. Intestinal vein, i.v.c. Inferior vena cava. k. Nepliridial tubes.
li. Liver, lu. Lung. pa. Pancreas, sp. Spleen, sp. a. Splenic artery.
tr. Trabeculae, v.s. Yenous sinus.

SPLEEN OE LEPIDOSIREN AND PROTOPTERUS.

241

PLATE 15.

Series of diagrammatic figures of transverse sections through the
embryos of Pro top ter us and Lepido siren, to show the position of
the spleen with regard to the neighbouring organs. Their numbers
correspond to those of the lithographic figures, which are drawings of
the same sections at a higher magnification.

Pigs. la-4a. — Protop terus. X 20 (circa).

Figs. 5a-8a. — Lepidosiren. X 10 (exc. 5a X 20).

PLATE 16.

Transverse sections through the spleen of Protopterus.

Fig.

1.-

— N. T. xxxii.

X

180.

Fig.

2 _

— N. T. xxxiii.

X

180.

Fig.

3.-

— N. T. xxxiv.

X

220.

Fig.

4.-

— N. T. xxxv.

X

220.

PLATE 17.

Transverse sections through the spleen of Lepidosiren.

Fig. 5. — N.T. 34.

X

180.

Fig. 6. — N.T. 35.

X

180.

Fig. 7. — N.T. 36.

X

180.

Fig. 8.— N.T. 37.

X

180.

COLLAR CAVITIES OF THE LARVAL AMPHIOXUS. 243

A Note concerning the Collar Cavities of the
Larval Amphioxus.

By

K. M. Smith, A.R.C.S.,

And

II. Q. Newth,

Demonstrator in Zoology, Imperial College of Science and Technology.

With Plate 18.

Introduction.

Notwithstanding the great volume of literature dealing
with the development of Amphioxus, there are many points
still outstanding on which the light of further investigation
must he thrown. The present short communication attempts
to dispose of one of these.

It is well known that in Amphioxus the cavities of the
typical somites give rise, on the one hand, to all the myocoels
except the first pair, and, on the other hand — by the fusion
of their ventral moieties on each side — to longitudinal spaces
which are the splanchnoccels. But the fate of the so-called
“ collar cavities has not hitherto been satisfactorily estab-
lished. It was known that from the walls of their dorsal
parts were formed the first pair of myotomes ; but there has
been difference of opinion about the behaviour of their ventral
parts.

MacBride (1) at first maintained that "the metapleural
‘ lymph canals ’ found in the atrial folds are the persistent

VOL. 62, PART 2. NEW SERIES. 17

244

K. M. SMITH AND H. G. NEWTH.

ventro-lateral extensions of the collar pouches ” — having
observed this relation to hold in the case of the right collar
cavity, and inferring that it was true of the left side also.

Lankester and Willey (4) have described a pseudocoelic
origin of the metapleural spaces, and MacBride, in answer to
criticism by Lankester and others, returned to this subject in
his paper of 1900 (2). Some of the conclusions arrived at,
as a result of this re-investigation, were as follows :

“The ectoderm on the external side of these ridges ”
(i. e. the atrial ridges) “becomes thickened, the cells
composing the thickening become clear and glassy and
eventually are hollowed out to form a f lymph canal/
My former statement as to the coelomic nature of this
lymph canal is therefore incorrect.

“The extensions of the collar cavities into the atrial
ridges become first separated off as the metapleural
coelom on each side ; later this coelomic space becomes
converted into a solid mass of cells from which arise
muscular fibres in the neighbourhood of the gill openings,
and almost certainly, later, the sub-atrial muscle.”

In the following year van Wijhe (5) affirms: “Nach den
Verlialtnissen beim ausgebildeten Thiere halte ich die Angabe
von MacBride aus dem Jahre 1898, nach welcher die Seiten-
flossenhohlen in Continuitat mit den f collar pouches *
entstehen wurden, fur richtiger als die spatere Behauptung
des selbstandigen Auftretens der Seitencanale.”

Finally, MacBride, in his latest contribution to this subject,
reiterates his former assertion — made in his second paper —
that the collar cavities form spaces in the atrial ridges,
longitudinally co-extensive with the pharynx, and distinct
from the splanchnoccel (3).

The present investigation was begun at the suggestion of
Prof. MacBride, and was carried out in his laboratory. We
wish here to make grateful acknowledgment of our indebted-
ness to him for the assistance he has given throughout the
progress of the work, and for the generous permission to make
use of his preparations for purposes of comparison.

COLLAR CAVITIES OF THE LARVAL AMPHIOXUS. 245

Material and Method.

Four larval stages were examined. Larvae which had been
fixed in Hermann's fluid were obtained from the Naples
Zoological Station. They were cut into series of transverse
sections by the method of double embedding in celloidin and
wax, and the sections, 4 /u or 5 u thick, were stained variously
with Delafield’s liasmatoxylin, thionin, alcoholic haematein ;
or with an aqueous solution of picro-nigrosin for the special
purpose of making plain the relations of the myosepta.

It is, as other workers have found, difficult to obtain
perfectly preserved material ; larvae of the same batch
apparently vary in their reaction to the fixative. To obtain
reliable results it was found necessary to section a large
number of animals, discarding those which showed distension
or contraction, and basing conclusions on those alone in which
the histological detail was convincing.

Our drawings are, to the best of our ability, faithful
reproductions of the appearance of the sections, except that
in some of them the irrelevant cytological detail is omitted.
They were all made at the level of the microscope stage with
the aid of a camera lucida, the magnification being, in each
case, that obtained with a 2 mm. apochromatic oil-immersion
objective and No. 6 compensating ocular of Leitz.

Description.

The earliest stage examined was one in which the larvae
show as yet no indication of the formation of a mouth. The
left head cavity is a vesicle unconnected with the ectoderm —
though in contact with it — the club-shaped gland opens
widely into the floor of the enteron, and the endostyle
appears as a slightly thickened area of the right side of the
swollen pharyngeal region. The formation of somites from
the archenteron is still occurring at the extreme posterior
end of the animal, and the tail has not begun to grow.

In such a larva the cavities of the collar somites have no

246

K. M. SMITH AND H. G. NEWTH.

ventral extension round the sides of the fore-gut, and, indeed,
in sections in front of the club-shaped gland the mesoblast
does not appear at all between the ectoderm and the gut-
wall (PI. 18, fig*. 1), save for an occasional isolated cell.
In sections a little further back, in the region where the
mouth will be formed, the collar somite on either side sends
a ventral horn downwards round the gut as a thin plate of
cells ; but since there are no cavities in these extensions and
the myosepta have not yet assumed their characteristic
appearance, it is impossible to make out the relations of the
somites in this stage.

The right collar cavity is completely separated from the
gut, but on the left side there is a virtual communication,
marked by the peculiar orientation of the cells of the gut-

Avall.

Our second (and critical) stage is one in which the mouth
has just become established, but is still a mere pore. PI. 18,
fig. 2, shows the appearance of a section about 5 /u behind the
blind anterior end of the gut. The collar somites have con-
siderable cavities which extend ventrally on either side of
the pharynx. We will first deal with that of the right side.
Into its dorsal part projects the mass of the first myotome,
the cells of which are already differentiated as muscle ; its
ventral horn can be traced, with diminishing lumen, to the
mid-ventral line. The next section of the series (PI. 18,
fig. 3) shows the ventral horn as before ; but in the muscle
a crescentic septum has appeared, dividing its mass into an
inner and an outer portion. This septum is the first myo-
septum, and the inner muscle mass (in . contact with the
notochord) is the anterior end of the second myotome (i.e ,
first trunk somite). Succeeding sections show the gradual
increase in size of the trunk somite at the expense of the
collar somite, as evidenced by the outward and downward
migration of the septum (PI. 18, figs. 4 and 5). Two sections
further on (PI. 18, Jig. 6) the cavity of the trunk somite is
seen to be well established above the dorsal edge of the
septum, and four sections beyond this the septum has just

COLLAR CAVITIES OF THE LARVAL AMPHIOXUS. 247

lost its apical attachment to the gut, so that the cavities
above and below now communicate (PL 18, fig. 7). The
remains of the septum have disappeared in the next section.

It will be plain from this description that the collar cavity
of the right side is continuous with the splanchnocoel. It
has a more extensive (longitudinally) communication with
that space than have the succeding myocoels, but essentially
its relations are the same. We have not found anything
comparable to the septum described by MacBride as separating
a postero-ventral extension of the collar cavity from the
splanchnocoel.

Turning now to the left side of the larva, we see that the
first myoseptum is well in advance of that of the right side.
In PI. 18, fig. 2, the ventral horn of the collar cavity is
already pushed down to the level of the middle of the
notochord, and in tracing sections back the first trunk
myocoel is found to be well established dorsally when the
section passing through the mouth is reached — the septum
between it and the collar myocoel having passed obliquely
downwards and backwards to meet the upper lip. The cavity
of the upper lip (virtual at this stage) is that of the first
trunk somite.

What happens in the lower lip is more difficult to make
out. A thickened layer of ectoderm, applied to the left head
cavity and to the antero-ventral wall of the gut, suppresses
altogether the ventral extension of the somite (PL 18, fig. 2) ;
just behind this thickening the somite passes down, its lumen
often occluded, to become continuous with the mesoblast of
the lower lip, which, in its turn, is continuous with the
splanchnocoel (PL 18, figs. 8, 4, 5). Again, no septum
is observable between collar cavity and splanchnocoel ;
but, the spaces being for the greater part merely virtual,
it is impossible to state with certainty whether there is actual
continuity.

The great dilatation of the collar cavities, which MacBride
took to be the first appearance of the atrial folds, is, in our
opinion, largely a fixation effect. It depends, no doubt, as he

248

K. M. SMITH AND H. GL NEWTH.

suggests, upon tlie amount of fluid contained in the cavities,
but the actual ectodermal profile we take to be an artifact.
On the left side, where the ectoderm is anchored, so to speak
— by its fusion with the gut, to form the mouth, and with the
head cavity to form the pree-oral pit — the swelling occurs to
a lesser degree than on the right side, where no such attach-
ments exist; and in the trunk region, where the cavities of
the myotonies are small or virtual, and the close apposition of
the walls of the splanchnocoel would preclude osmotic disrup-
tion, it occurs not at all. The artificial nature of the disten-
sion in question is frequently made apparent by a separation
of the extremely thin outer wall of the somite from the
ectoderm. This is seen even in so young a larva as that
shown in PI. 18, fig. 1 (q. v.) ; in later larvse the action of the
fixative is often to rupture the ectoderm on the right side.

The communication between the left collar cavity and the
gut, described by MacBride, is indicated in larvge of this
stage (PI. 18, figs. 2 and 3). It still appears in the majority
as a funnel-shaped depression in the dorso-lateral wall of the
gut, with sometimes a plug-like fascicle of columnar cells
filling it (PI. 18, fig. 2). It is never, in our sections, a very
definite structure, but it gives a characteristic shape to the
lumen of the gut, which is generally found to persist through
several sections. In the preparations we have examined of a
slightly later stage (mouth and one gill established) we have
failed to demonstrate its presence ; but the swollen, vacuo-
lated condition of the gut cells in this later stage makes it
easily possible that the connection, though not recognisable,
is present.

In later larvae still (seven or eight primary gills present)
the nephridium of Hatschek appears, as described by
Goodrich (6), lying in a space which is apparently a backward
prolongation of the left collar myocoel. But whether this
space is the original portal of communication, drawn out into
a tube by the growth of the larva, and, if so, how the
nephridium (an organ presumably of ectodermal origin)
comes to lie naked within it, are questions that our materials

COLLAR CAVITIES OF THK LARVAL AMPHIOXUS. 249

do not enable ns to answer. Never theless, attention should
perhaps be called to a constant but inconspicuous feature of
our second stage larvas — the mass of cells marked with a
point of interrogation in PI. 18, fig. 4. They occur just
behind the communication above mentioned, and it is tempting
to regard them as a proliferation of ectoderm into the first
myoseptum and as the rudiment of the nephridium. We
cannot, however, assert that this is so.

In conclusion, our acquaintance, limited as it is, with the
early stages of development of Amphioxus has convinced us
of the need of careful experiment with the object of dis-
covering better methods of fixation of the larvae. This can
only be done by rearing them in large numbers in the
laboratory — an undertaking never yet* achieved. Only so
can material be obtained the study of which will give satis-
factory answers to the many morphological questions which
still remain doubtful.

Note by Prof. E. W. MacBride.

The investigation carried out by Messrs. Smith and Newth
in my laboratory on the development of body cavities in the
larva of Amphioxus has led them to results which in some
respects are different from those which I have published on
the same subject.

In particular they find that the space into which the right
collar cavity opens as it sweeps downwards towards the mid-
ventral line is the splanchnocoel, and not, as I supposed, a
distinct cavity lying external to the splanchnocoel, which
later became the cavity of the atrial fold, or, as van Wijlie
terms it, the pterygocoel.

After a careful examination of the preparations made by
Messrs. Smith and Newth, which were based on better
preserved material than was available to me, I have come to
the conclusion that these authors are right, and I am prepared
to accept their view. A re-examination of my own prepara-
tions leads me to believe that the septum which I believed to

250

K. M. SMITH AND H. G. NEWTH.

divide the splanclmocoel from another cavity external to it
is the parietal wall of the coelom, which in the process of
preservation has become separated from the ectoderm.

The facts elucidated by Messrs. Smith and Newth enable
us to compare the collar cavity of Ampliioxus directly with
the mandibular cavity of the embryos of Petromyzon and the
Elasmobranch embryo. This cavity has been observed by
Hatta to originate in Petromyzon from the wall of the gut
independently of the outgrowth which gives rise to the
myotomes behind, but in both Petromyzon and the Elasmo-
branch embryo the mandibular cavity becomes subsequently
connected by a long tongue-like ventral extension with the
splanclmocoel.

List of Woeks Referred to in this Paper.

1. MacBride, E. W. — ‘’ The Early Development of Ampliioxus,” ‘ Quart*

Journ. Micr. Sci.,’ vol. xl, 1898.

2. “ Further Remarks on the Development of Ampliioxus.”

‘ Quart. Journ. Micr. Sci.,’ vol. xliii, 1900.

3. “ The Formation of the Layers in Ampliioxus and its Bearing

on the Interpretation of the Early Ontogenetic Processes in
Other Vertebrates,” ‘Quart. Journ. Micr. Sci.,’ vol. liv, 1909.

4. Lankester, E. Ray, and Willey, A. — “ The Development of the Atrial

Chamber of Amphioxus,” ‘ Quart. Journ. Micr. Sci.,’ vol. xxxi,
1890.

5. Wijhe, J. W. van. — “ Beitr. z. Anat. des Kopfregion des Amphioxus

lanceolatus,” ‘Petrus Camper,’ vol. i. 1901.

6. Goodrich, Edwin S. — “ On the Structure of the Excretory Organs of

Amphioxus,” ‘ Quart.. Journ. Micr. Sci.,’ vol. liv, 1909.

EXPLANATION OF PLATE 18,

Illustrating Messrs. K. M. Smith and H. G. NewtlTs paper,
“ A Note concerning the Collar Cavities of the Larval
Amphioxus.”

Abbreviations.

The asterisk marks the communication between the left collar somite
and the gut.

COLLAR, CAVITIES OE THE LARVAL AMPHIOXUS.

25 L

ch. Notochord, c. g. Club-shaped gland, esty. Endostyle. 1. c. s.
Left collar somite. 1. h. c. Left head cavity. 1. m. 2. Second myotome
of the left side. 1. me. 2. Second myocoel of the left side. m. Mouth.
n. c. Nerve cord, r.c.s. Right collar somite, r.h.c. Right head cavity
r. m. 1. First myotome of the right side. r. m. 2. Second myotome of the
right side. r. me. 2. Second myocoel of the right side. s. Septum.
spl. Splanchnocoel. t. me. Trunk myocoel.

Fig. 1. — Transverse section through the pharyngeal region of a very
young larva, showing the relations of the collar somites to the gut.
The section is 4 y thick and was stained with tliionin. Note the
separation of the outer wall of the right collar somite from the
ectoderm.

Figs. 2-7. — Transverse sections through the pharynx of a larva in
which the mouth is just formed. Figs. 2-5 are of consecutive sections ;
between 5 and 6 one section is missed, between 6 and 7 three sections
are missed. The preparation from which the drawings were made was
overstained, in picro-nigrosin to make plain the septa, and this has
obscured the cytological detail. For further description see text.

Fig. 8. — Diagrammatical representation of the relations of the coelomic
spaces of the left side of a larva in which the mouth is just formed ,
The dotted lines show the condition on the right side where the wide
opening of the collar somite to the splanchnocoel is not interfered with
by mouth or head cavity.

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CONTENTS OF No. 247-New Series.

MEMOIRS :

On the So-called Pharyngeal Gland-cells of Earthworms. By
J. Stephenson, D.Sc., M.B., Lieutenant-Colonel Indian Medical
Service ; Professor of Zoology, Government College, Lahore. (With
Plate 19) .......

The Chromosome Complex of Culex pipiens. Part II. Fertilisa-
tion. By Monica Taylor, S.N.D., B.Sc. (With Plate 20 and 1 Text-
figure) .......

The Homologies of the Muscles related to the Visceral Arches of the
Gnathostome Fishes. By Edward Phelps Allis, jr., Menton,
France (With Plates 21 and 22 and 1 Text-figure)

The Cytoplasmic Inclusions of the Germ-cells. Part I. — Lepi-
doptera. By J. Bronte Gatenby, B.A., Exhibitioner of Jesus
College, Oxford. (With Plates 23, 24, and 25, and 5 Text-figures)

The Degenerate (Apyrene) Sperm-formation of Moths as an Index to
the Inter-relationship of the Various Bodies of the Spermatozoon.
By J. Bront£ Gatenby, B.A., Exhibitioner of Jesus College,
Oxford. (With Plate 26) .....

PAGE

253

287

303

407

463

PHARYNGEAL GLAND-CELLS OF EARTHWORMS. 253

On the So-called Pharyngeal Gland-cells
of Earthworms.

By

J. Stephenson, D.Se., M.B.,

Lieutenant-Colonel, Indian Medical Service ; Professor of Zoology,
Government College, Lahore.

With Plate 19.

Contents.

PAGE

1. Historical ...... 253

2. Material and Methods .... 260

3. Pheretima posthuma .... 261 ‘

4. Pheretima heteroch^ta , . . . 265

5. Pheretima hawayana .... 267

6. Lumbricid,e . . ... 269

7. The Appearances in Young Specimens . . 274

8. The Cells in the Lumbricid Embryo . , 279

9. Function of the Cells .... 281

10. Summary ...... 283

11. References to Literature .... 284

12. Explanation of Figures .... 285

Historical.

Succeeding the buccal cavity in all earthworms is a swollen
portion of the alimentary tube, the pharynx. The usual
description of this portion of the tube in Lumbricus may
be given in the words of Parker and Haswell (7) : the

“buccal cavity ... is followed by a much larger
thick-walled, rounded chamber, the pharynx. From the wall
vol. 62, part 3. — new series. 19

254

J. STEPHENSON.

of the pharynx there run outwards to the body-wall a number
of radially arranged bundles of muscular fibres which, when
they contract, draw the pharynx backwards, and at the same
time dilate it.”

One of the constituents of this pharyngeal thickening, not
mentioned in the ordinary descriptions of the earthworm, is
nevertheless a prominent feature, easily visible under the
lens in the ordinary dissection, and immediately obvious,
owing to its staining properties, in sections through the
region where it occurs. This constituent is a cellular mass
which forms soft white projecting lobules on the dorsal and
lateral aspects of the pharynx ; the lobules surround the
muscular strands which issue from the pharynx, and in
addition, the cells of the mass penetrate inwards between
the interlacing muscular bundles of the thick dorsal pharyn-
geal wall in the direction of the lumen of the canal.

Though these cells have received some attention from
previous writers, an adequate account of their nature and
origin has not yet, I believe, been given.

References to previous authors are given by Vejdovsky
(9, 1884), from whose account of them I quote, since the
older literature is inaccessible to me. The earlier investi-
gators— Leo, Clarke, Lankester — who saw these masses of
pharyngeal cells in Lumbricus, interpreted them as
glandular. Perrier described pharyngeal glands in several
genera; in Pontodrilus they are said to be variously coiled
tubes whose walls are composed of large cells with granular
contents; in Moniligaster they pour their secretion into
the pharynx by a multitude of small canals visible with the
lens; Perichseta houlleti has several layers of glands
which open into the interior by three pairs of orifices.
Claparede refers to those cells of the pharyngeal mass which
penetrate inwards between the muscular bundles as “ . . .
numerous polygonal cells with large round nuclei 6 fi% in
diameter. The import of these cells is at present not clear
to me. Their similarity to ganglion-cells is not to be denied,
though a connection with nerves could not be recognised.

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

255

The matter is best left undecided at present.” The projecting
lobules on the dorsum of the pharynx Claparede called
“ ganglia of the previously described pharyngeal plexus.”

Vejdovsky’s own account of the pharyngeal cells is not
very clear, and is interpolated here and there amongst
descriptions of the muscular and vascular apparatus of the
pharynx, and of the occurrence and mechanism of its extrusion.
Unlike Claparede, who recognised the identity of the cells of
the lobules with those which penetrate inwards between the
muscular strands (interpreting both as nervous), Vejdovsky
considers them as distinct. Those which penetrate inwards
he looks on simply as cellular elements of the coelomic fluid,
which become attached to the pharyngeal muscles as to other
organs ; and he makes the rather surprising statement that
“ had Claparede compared these cells with those suspended
in the coelomic fluid, he would certainly have recognised them
as the latter.” The projecting lobules, on the other hand,
are interpreted as mucous glands (Schleimdrusen) ; in vertical
sections the glandular masses, contracting anteriorly to form
long ducts, wind between the muscular bundles of the
pharynx, and most probably empty their secretion into the
pharyngeal cavity ; these glands extend backwards far into
oesophageal segments, and correspond to the septal or mucous
glands of other Oligochseta. Vejdovsky also describes the
ducts of the “septal glands” of Criodrilus as winding
through the layer of muscular and vascular tissue on the
dorsum of the pharynx, and the exceptionally large and
numerous mucous glands of Dendrobaena rubida are said
to consist each of a pear-shaped mass of cells with large
round nuclei and containing a substance which stains deeply
in picrocarmine.

Vogt and Yung (10, 1888) describe irregularly dispersed
cells between the muscular fibres on the dorsum of the
pharynx (in Lumbricus agricola). These cells have ill-
defined outlines, a granular protoplasm, and a clear spherical
nucleus containing a nucleolus. The authors refer to
Claparede’s interpretation of them as nerve cells ; they

256

J. STEPHENSON.

resemble, however, the unicellular glands found in a corre-
sponding position in other animals; and though the authors
had not succeeded in discovering their ducts, they thought it
not impossible that they secrete the viscid substance which
the worm mixes with its food.

Hesse (6, 1894) considers the pharyngeal cells of Oligo-
chseta in general as belonging fo the epithelial layer; in the
Naididac and Tubificidas the ventral end of each cell is pro-
longed into a duct, which debouches between the lining
epithelial cells of the pharyngeal cavity ; the ducts of these
cells are more drawn out in Lumbricus.

Beddard (2, 1895) does not treat of the pharyngeal gland-
cells of earthworms apart from the well-known “ septal
glands ” of EnchytraBidae, etc. The septal glands in general,
and so by implication the cells under consideration, appear
to him to be simply epidermic glands which have been
invaginated along with the stomodaeum, though their position
causes him some doubt.

The author who has examined these cellular aggregates in
detail in the largest number of species, and who has given
the most precise accounts of their supposed ductules and
manner of discharge is Eisen (4, 5, 1895, 1896). In
Phoenicodrilus taste the masses (called “salivary
glands ”) discharge through ducts which follow the muscle
strands into the pharyngeal cavity ; and it is probable that
all the suprapharyngeal glands in Lumbricids open similarly
and without any great variation as to detail ; narrow ducts
penetrate the pharyngeal epithelium, forming near the free
surface small ovoid pockets for temporarily storing a small
amount of the salivary secretion. These (suprapharyngeal)
glands are connected posteriorly with the septal glands, —
four pairs, superposed on several main longitudinal muscular
bands which connect the pharyngeal glands with the body-
wall in segment IX ; their ducts, both wide and narrow, follow
these muscles, so that the secretion of the septal glands also
is emptied into the pharynx. InPontodrilus michaelseni
the pharyngeal or salivary glands have a similar position,.

PHARYNGEAL GLAND-CELLS OF EARTHWORMS. 257

and are directly connected by means of ducts with the
epithelium of the pharynx ; arrived at the pharyngeal
epithelium the ducts branch out, sending numerous discharge
tubes between the epithelial cells ; these ductules are fre-
quently, though not generally, branched while in the
epithelial layer, and each ductule is furnished at the distal
end with a small storage chamber of oblong form and con-
siderably smaller than the nucleus of the epithelial cells.
There are also in this species five pairs of septal glands,
ventral to the oesophagus, and principally attached to blood-
vessels, in segments V-IX, of similar structure ; a very thin
duct runs backwards and upwards from the upper end of
each towards the alimentary canal at its junction with the
septum, “ but I have some doubt about it emptying into the
intestine, and it is much more probable that . . . these

septal glands empty into the pharynx. None of my sections
however show this to be the case.” The distribution of the
septal glands in this species may be compared with what
is found in Helodrilus (Bimastus) parvus (v. p. 23
post.).

In Benhamia nan a Eisen states that the glands are
evidently unicellular, and the fine ducts penetrate between
the epithelial cells of the pharynx, the discharge pockets
being almost globular ; here and there the duct of a single
glandular cell may be followed clear to the discharge pocket.
“ But to draw the conclusion . . . that all the pharyngeal

and septal glands are unicellular is, I think, premature. In
Fon tod rilus, at least, there may be seen plainly numerous
nuclei on the gland ducts, which of course indicates that we
have here a fusion of several cells. . . . In Pontod rilus

the majority, and all the large glands, consisted of several
cells, the respective ducts of which finally united into one.
In Benhamia I could see no such union, and the single ducts
could be followed with great facility to the outlets.” In
Benhamia liana the septal glands, in segments IX, X, and
XI, are very narrow and only one cell thick in the row. In
B. palmicola the small septal glands are in IX and X, but

258

J. STEPHENSON.

the author could not find that they were in any way connected
with the pharyngeal system of glands.

The pharyngeal and septal glands ofAleodrilus keyesi
are also described. Here it will be sufficient to call attention
to the author’s statements regarding the discharge of the
gland-cells. The pharyngeal glands have discharge pockets
which are much thicker than those seen in any other species;
the septal glands are of the same nature as the pharyngeal,
“ but I have good reasons to believe that the glands in this
species discharge into the tubular intestine. I have been
able to follow fclie discharge duct as far as the muscular
layers of the intestine, which would hardly have been the
case if the ducts had continued forwards into the pharynx,
as do those of the forward septal glands in many genera.”
In some other small aggregations of similar cells the author
was unable to follow the ducts.

In Sparganophilus (which, though aquatic, belongs to
the Grlossoscolecidse, and so may be considered along with
the earthworms), it is noted that in one species the ducts of
the septal glands with precipitated secretions can be followed
along the septum down towards the intestine, but the con-
nection with the latter, if any, was not ascertained; in
another species the discharge tubes and chambers are very
large, the chambers occupying more than half the width of
the pharyngeal wall (the meaning is more than half the
height of the pharyngeal epithelium).

De Ribaucourt (8, 1901), describes in a few words the
deeply staining mass of cells in the Lumbricidas : “ On
staining with methyl blue and iodine green one can easily
establish the fact that these cells are continued as far as the
epithelial layer by a fine prolongation ; thus the cells may
quite possibly have a secretory function.” Miss Raff (7a,
1910), recognises the cells in the Australian Megascolecidae,
but finds no trace of a duct in connection with the “ glandular
mass.”

I omit the literature which deals with the septal glands of
the specially aquatic groups — the Microdrili — as 1 hope to

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

259

return to these at a later date. Nor need I refer to a
number of observations on the occurrence, form, and position
of the pharyngeal and septal glands of earthworms by
systematic writers, since these do not deal with their intimate
structure.

The cells in question, therefore, are usually considered
as gland-cells belonging to the epithelial layer of the
alimentary tube, and they are supposed to pour their secre-
tion into the lumen of the tube by means of long, fine
ductules prolonged from the cell-body. Clapar&de saw no
ductules, and believed the cells on the pharynx to be nervous
in nature ; Yogt and Yung, who nevertheless believed the
cells to be secretory, could not discover the ductules ; Raff
also saw no duct; Vejdovsky saw “long ducts,” but not,
apparently, their connection with the pharyngeal cavity ;
de Ribaucourt saw the continuation of the cells as far as the
pharyngeal epithelium ; Eisen has given detailed accounts of
the ductules, of their branching in the pharyngeal epithelium,
and of their discharge pockets ; Perrier (according to
Vejdovsky) saw the ducts with a lens, and observed their
paired orifices.

According to my observations the cells in question are not
of epithelial origin, and have no connection with the pharyn-
geal epithelium. They originate at the peripheral limit of
the pharyngeal mass, and are congeneric with the peritoneum ;
in the adult they extend deeply into the pharyngeal mass,
and there become largely transformed into connective tissue ;
but what their primary function is I am unable to say. It
will save repetition to state here that in none of my sections,
which were taken in all three planes, have I seen structures
that could be interpreted as ductules.

Claparede’s view of the nervous nature of the cells prob-
ably originated in their superficial resemblance to the spinal
ganglion cells of higher Vertebrates; there is no resemblance
to the ganglion cells of the Oligochaeta. Vejdovsky’s state-
ment as to the similarity to the coelomic corpuscles of those

260

J. STEPHENSON.

of the cells which lie deep among the muscular bundles of
the pharynx is frankly unintelligible to me. The authors
who have seen ductules and their endings in the pharyngeal
epithelium have, I believe, been misled by preconceived ideas
on the nature of the cells, and by the appearances due to the
transformation of the deeper cells into connective tissue.

Material and Methods.

I have investigated in detail the five common species of
earthworms found in Lahore; three of these, Pheretima
posthuma (L. Vaill.), P. heterochseta (Mchlsn.), and
P. hawayana (Rosa), belong to a genus of Megascolecidse ;
two, Helodrilus (Allolobophora) caliginosus subsp.
trapezoides (Ant. Dug.), and Helodrilus (Bimastus)
parvus (Eisen), to the Lumbricidae. In addition to adult
specimens, I have examined a number of younger worms of
both families, and also several Lumbricid embryos in various
stages, taken from the cocoons ; but only one of these latter
gave me additional information. I am also familiar in a
general way with the cell masses as they occur in a large
number of other worms, which I have sectioned from time
to time in the course of systematic work on Indian Oligo-
chaeta ; though as I cannot answer for the histological con-
dition of this material (which mostly formed part of the
Indian Museum collections) I have not made use of it in the
present account.

The methods of fixation employed were Zenker’s fluid and
sublimate-acetic for the embryos and smaller worms, including
the adults of Helodrilus parvus; some specimens of
Pheretima were also treated by one or other of these
methods. Narcotisation with chloretone and fixation by
10 per cent, formalin were employed for most of the adult
specimens of Pheretima and Helodrilus caliginosus.

For staining, the most generally useful method is some
degree of overstaining with Delafield’s hsematoxylin, differen-
tiation with acid alcohol, and counterstaining with alcoholic

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

261

eosin. Dobell’s modification of Heidenhain’s iron-hsematoxylin
method (3) has also given me excellent results, and I should
like to confirm what its author says regarding its value and
convenience. One or other of the above methods was em-
ployed for all specimens used in descriptions of the cells. In
addition, I have used Heidenhain’s original chromhaematoxylin
method, which gives unsurpassed differentiation of epithelial
cells (skin, pharynx, oesophagus), but in my hands has been
useless for the cells of the pharyngeal mass. Van Gieson’s
stain, and borax-carmine followed by picroindigo-carmine,
were useful in differentiating the connective tissue and in
distinguishing it from the muscular fibres.

I have to thank my friend and former pupil, L. Baini
Prashad, M.Sc., Alfred-Patiala Research Student of the
Punjab University, for kindly giving me the embryos and
some of the youngest specimens used in the investigation.

Pheretima Posthuma.

General description. — In this species, in front of
septum 4/5, a soft mass extends forwards almost to the
anterior end of the body, filling up the available space, and
hence narrower in front where the cerebral ganglion lies
across it. The posterior end, or base of the somewhat conical
mass, can be separated only with difficulty from septum 4/5,
against which it lies, on account of the numerous- strands of
muscle which issue from the mass and pass backwards
through the septum. When the separation has been accom-
plished, the posterior part of the mass is, seen to be composed
of numerous micronephridial tubules the pharynx with its
associated aggregations of “ gland-cells 39 lies in front of
this.

Emerging from the dorsal and lateral surfaces of the
pharyngeal mass are numerous strands and sheets of muscle
which take in general an obliquely backward direction ; the
obliquity is less in front, where the strands are more nearly
transverse in direction, and greater behind, where they are

262

J. STEPHENSON.

more longitudinal. Around the bases of these strands are a
number of soft whitish lobular masses ; these are either one
to each strand, or the lobules are fused at their bases to
form a transversely extended mass enveloping the origin of
several strands. The whitish lobular masses are arranged in
about four transverse series, and the muscle strands emerge
in a corresponding number of transverse rows. The most
anterior portion of the mass is smooth, and represents the
thick muscular and connective tissue wall of the pharynx
itself. The condition is similar to that shown in PI. 19,
fig. 1, for P. heterochae ta, omitting the masses in seg-
ment Y.

In segment Y, concealing the oesophagus, there is on each
side posteriorly a considerable tuft of micronephridia, and
anteriorly a mass of follicles of the so-called blood glands
(cf. Beddard, 1) ; these latter rest against and are connected
with the posterior face of septum 4-5 ; they interest us here
because some are found more anteriorly, embedded in the
cells of the posterior portion of the pharyngeal mass.

On examining longitudinal sections through the anterior
end of the worm the lobules previously mentioned are found
to consist of the “ pharyngeal gland-cells ” of earlier authors ;
these cells also penetrate in for some distance between the
muscular fibres, which, crossing and interlacing, form the
main portion of the pharyngeal mass. The pharyngeal
lumen is lined by a columnar epithelium ; the ventral wall
of the pharynx is thin, in contrast to the massive dorsal wall ;
the muscular coat is here no thicker than the layer of
epithelium, and the tc gland-cells ” are absent.

Since these cells are certainly not glandular in the sense
intended by previous writers, and since their function is not
fully known, it is advisable to drop the earlier name. I
propose to call them chromophil cells, because of their
peculiar staining properties ; which, in sections stained by
hsematoxylin, for example, render the masses immediately
obvious even on a naked-eye inspection.

The Chromophil cells (PI. 19, fig. 2). — The individual

PHARYNGEAL GLAND-CELLS OF EARTHWORMS. 263

cells are of various shapes — more or less polygonal, triangular,
crescent-shaped, or altogether irregular — according to the
disposition of the adjacent cells. They do not however as a
rule fit closely together, and are mostly well separated by
clefts from their neighbours. They are usually longer in
one direction than the other; the longer diameter may
measure, on an average, 17 ; the shorter, perhaps, 10 /.i.

Their outlines are not definite, and they are frequently
continuous at their periphery with an amorphous or fibrillar
coagulum-like substance, which partly fills up the intercellular
spaces, and by the intermediation of which the cells may be
continuous with each other.

The nucleus is often obscured by the deeply-staining portion
of the cell-body to be described. It is subspherical or
shortly oval, 4*5-6 g in its long diameter. The nucleolus
is large and distinct, evenly staining, and often somewhat
excentrically situated ; granules of chromatin occupy the
more peripheral region of the nucleus (obscured in the
figure).

The cell-body may be distinguished into deeply and more
lightly staining portions. The deeper staining portion is
always considerable in amount, and may form almost the
whole of the cell-body ; no further structure can be made out
in this portion ; it is seldom well defined in its extent, and
merges into the more lightly staining portion at its periphery.
The outer portion of the cells stains more lightly, and has a
granular, or sometimes apparently a reticular constitution ;
it has often no definite peripheral boundary, the cell having
a ragged edge as if its outer portion were disintegrating ; or
it merges into the loose substance between and sometimes
connecting the cells.

Transformation of the Cells. — These cells are typi-
cally seen, and in large numbers, dorsally and posteriorly on
the pharyngeal mass ; where, as a compact aggregate, they
form the lobules previously described, which are penetrated by
the emerging muscular bundles ; near the posterior limit of the
mass there is in addition an admixture of follicles of “ blood-

264

J. STEPHENSON.

glands.” Further forwards in the pharyngeal mass, dorsal
to the - cavity of the pharynx, in what may be called the
transition zone, the cells become sparser, and interlacing
muscular fibres form the bulk of the mass. In this zone the
cells are seen to change their characters as they are traced
gradually forwards and inwards. They become rather smaller
in size ; the deeply staining matter becomes less in amount,
and is aggregated in smaller masses ; and the cell-body
becomes continued into the now abundant fibrillar strands
between the muscle fibres. Numbers of such cells can be
seen, which, with still a considerable amount of deeply-
staining matter, dissolve at their periphery into the fibrillar
or reticular packing tissue (“ Fullgewebe”) between the
muscle fibres (compare PL 19, fig. 5, from P. hawayana).

Still further inwards and nearer the pharyngeal epi-
thelium the deeply staining matter disappears altogether,
and the tissue passes into the abundant connective tissue of
the deeper portion of the pharyngeal mass, which is absent
from the more superficial region where the typical chromophil
cells are aggregated. The nuclei, no longer obscured,
become conspicuous ; the nucleolus diminishes in size, and
ultimately disappears ; the chromatin grains are distributed
more evenly through the otherwise clear nucleus. But even
quite near the pharyngeal epithelium occasional cells are
still met with which retain the characters of those in the
more superficial parts of the mass.

In this deeper region the nuclei appear to undergo a final
change by becoming smaller; maintaining the above cha-
racters, they can be traced down to a size measuring 4 g in
greatest diameter. Along with these, in the connective tissue,
another type of nucleus is abundantly represented ; these,
about 3 /x by 2 fx, are often irregular in shape ; the smallest ones
stain darkly, and are almost homogeneous ; some appear
clearer, with a few grains of chromatin. These I believe
to represent the nuclei of the original connective tissue
element of the muscular dorsal wall of the pharynx. They
are similar to connective tissue nuclei elsewhere, and, as will

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

265

be seen, are found numerously in young specimens, where the
chromophil cells have undergone little change.

I am doubtful if it is always possible to distinguish between
these smaller nuclei and the last stage of transformation of
the nuclei of the chromophil cells. But, in spite of the fact
that discrimination of the separate elements may be impossible
in the adult, it seems necessary to attribute a double origin to
the connective tissue of this region.

The Capsule. — In view of what will be said later, the
relation of the cells to the peritoneum is of interest. The
lobular masses are surrounded by a thin capsule, — a membrane-
like expansion, with fairly numerous ovoid or flattened nuclei,
which show scattered chromatin granules but no nucleolus.
The membrane bridges over the clefts between adjacent cells
at the surface of the mass ; it is in many places distinctly
differentiated from the underlying cells, staining pink with
eosin, and hence sharply marked off from the chromophil
cells beneath. In places the membrane may contain numbers
of brown chloragogen grains ; in this condition it may be
still a moderately thin (3-4 n) membrane, or it may be swollen
so as to be fairly described as being composed of somewhat
flattened chloragogen cells ; but there are no chloragogen
cells of the usual elongated type. In places the capsule is
absent, and the — sometimes indefinite — limits of the chromo-
phil cells themselves form the boundary of the mass.

PHERETIMA HETER0CH2ETA.

G-eneral description (PI. 19, fig. 1).— In this species the
cells, as in P. posthuma, form lobular masses on the
pharynx (c1) ; but in addition lobules composed of chromo-
phil cells extend backwards, dorsal to the oesophagus, into
segment Y (c2, c3), where they are altogether behind the
pharyngeal region of the alimentary tube. Crossing seg-
ment Y in a more or less longitudinal direction are a
number of muscular bands which pass backwards from the
pharyngeal mass in front; the more superficial of these (m2)

266

J. STEPHENSON.

are partly, the deeper are wholly, surrounded by the soft
white masses of the cells (c3, c3). The “ blood-glands ”
appear as masses of grape-like follicles in segment YI,
clustering round the backward prolongations of the muscle
bands ; follicles also occur, as seen in sections, within the
lobular aggregations of the chromopliil cells, both in seg-
ment Y and on the pharynx.

The Chromopliil Cells (PL 19, figs. 3, 4). The cells
resemble, on the whole, those described for P. posthuma;
but those of the posterior portion of the mass are in general
more definite in outline than in the previous species, and do
not here dissolve into the intercellular and connecting
substance to the same extent. The nucleus is again cha-
racteristic,— a spherical or shortly ovoid vesicle with large
nucleolus and scattered chromatin.

Transformation of the Cells. — In the backwardly
projecting lobular masses of pharyngeal cells are strands
of connective tissue, — a lightly-staining substance, scarcely
definitely fibrillar in structure, though with an obvious longi-
tudinal differentiation which is manifested by the deeper
staining of small streaks in the direction of the length of
the strand. In these strands are contained numerous cells,
of the general nature of those already described ; many of
these dissolve at their extremities into the substance of the
strand without any demarcation ; some however are dis-
tinctly outlined; the nuclei may still be perfectly distinct
when most of the cytoplasm has dissolved away. Indefinite
masses of deeper staining material, continuous with the
substance of the strands, and without nuclei, are also seen
(possibly nuclei are not present merely because of the plane
in which the section happens to be taken). (Compare PI. 19,
fig. 5, from P. liawayana).

Similarly amongst the muscular fibres on the dorsum of
the pharynx are strands of connective-tissue of the above
type with small islets of cells. The cells are in part indi-
vidually distinct, in part continuous with the connective-
tissue. As the transformation of the cells proceeds, the

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

267

nuclei become smaller; the nucleoli also diminish in size ;
and when the deeply staining substance has altogether dis-
appeared the nuclei (or some of them) seem to disappear
also, becoming fainter and less easily distinguishable ; so that
ultimately tracts of connective-tissue of some little size — at
least as large as several of the original cells — show no nuclei
at all.

The Capsule. — The lobes are surrounded by a capsule,
which consists of a thin membranous sheet with not infre-
quent oval nuclei. This constitutes a very definite peritoneal
covering over the posteriorly projecting lobules ; over the
more anterior masses it is less evident. But even there it
can be made out in places by means of the somewhat
flattened nuclei contained within a lightly staining material,
which fills up little inequalities in the surface or forms small
projections. In other parts however no capsule is discover-
able; the limit of the mass is the limit of the chromophil
cells themselves ; and, as owing to the disintegration of the
periphery of the cells this is not always sharply defined, it
would be easy in such places to distinguish a limiting
membrane if one were present (PI. 19, fig. 4). A definite
peritoneal investment covers the muscular strands which
issue from the pharyngeal mass.

Pheretima hawayana.

General description. — The condition is not unlike that
of the last species (PJ. 19, fig. 1). The pharynx is covered
by a soft white mass, from which muscle bands emerge.
Projecting behind the pharynx, and therefore in segment V,
there are on each side two lobes, one above the other. The
upper lobe has a smooth surface, and three muscular bands
emerge from its posterior border; the lower is larger,
triangular in shape with its apex backwards, smooth for the
greater part, but the posterior tapering portion consists of
follicles of the “ blood-glands ” clustering round a muscle
strand. Other strands also emerge from this lobe ; and on

268

J. STEPHENSON.

sectioning, follicles of the “ blood-glands ” are found numer-
ously within the cellular masses, even deep amongst the
chromophil cells of the dorsum of the pharynx.

The Chromophil Cells and their Transformation.
— The cells which compose the main portion of the white
masses on and behind the pharynx are polygonal or irregular
in shape, 20-25 /n in longest measurement, sometimes separated
from each other by linear spaces ; such have therefore a
definite outline. The nucleus, up to 6 fx in greatest diameter,
is conspicuous, vesicular, with large nucleolus and numerous
granules of chromatin. The cytoplasm as a whole stains
deeply but not homogeneously, and the lighter staining or
non-staining portions of the cells appear sometimes as rela-
tively large areas which may resemble vacuoles. (A similar
condition is shown in PL 19, fig. 5, which, however, is from
P. heterochasta.)

Besides the cells with definite outline, a number are
also visible in which the central deeply staining cytoplasm
shades off into a peripheral region, less deeply staining and
with a fibrillar structure ; this peripheral region again in
places is indistinguishable from an intercellular substance.

Passing inwards towards the pharyngeal epithelium the
continuity of the cells with the connective tissue, now con-
siderable in amount, is very evident. The connective tissue
accompanies the muscular fibres in close association, its
fibrillas often running parallel with the fibres. The cells still
retain some of the darkly staining substance (PI. 19, fig. 5).

Still deeper in the pharyngeal mass there may be no
stainable cytoplasm in association with the nuclei ; these
then lie in the connective-tissue. Such nuclei are smaller,
more irregular in shape, sometimes appearing shrivelled ;
the nucleolus decreases in size, and may become indistinguish-
able from the chromatin grains. Appearances suggest that
some at least of these nuclei break up and disintegrate,
sometimes by dividing into two small vesicles each with a
staining granule in its interior, sometimes by becoming as a
whole progressively more indistinct.

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

269

The Capsule. — A peritoneal covering limits the lobes
in some places. The nuclei of this membrane are rounded or
slig*htly flattened; the membrane itself is in places a distinct
pink-staining (in hsematoxylin and eosin preparations) mod-
erately thick expansion. In some regions, while it is still
possible to speak of an investing membrane, the cells com-
posing this latter are seen to be continuous with the chromophil
cells and to have the same cytoplasmic constitution. In
other places no investing membrane is present.

In the adult Pheretima therefore, the chromophil cells
form lobular aggregations covering the muscular mass of
the dorsum of the pharynx ; and in some species they also
extend backwards behind the pharynx as lobe-like masses.
The cells also extend deeply inwards amongst the muscular
fibres in the direction of the pharyngeal epithelium; but
here they become modified, the cytoplasm being progressively
converted into connective tissue. The connective tissue of
this region has therefore probably a double origin. The
descriptions of the peritoneal capsule suggest that it and the
chromophil cells are modifications of the same tissue ; where
the capsule is absent, the cells lining the coelomic cavity have
become chromophil cells; where present, the cells in immediate
relation to the cavity have become flattened, while those
underneath have taken on the chromophil character.

Lumbricid^;.

As an example of what is seen in the dissection of one of
the Lumbricidse, it will be sufficient to describe Helodrilus
caliginosus, perhaps the commonest of all earthworms;
the histological appearances in this species are similar in all
main features to those of Pheretima (except that there are
no “ blood-glands ” among the chromophil cells), and they
therefore need not be detailed. Instead, an account of the
microscopical structure of the chromophil tissue in Helo-
drilus parvus will be given; this species is too small to

VOL. 62, PART 3. NEW SERI US. 20

270

J. STEPHENSON.

allow of much being seen in dissection, but examined micro-
scopically it presents a number of interesting features which
go some distance towards elucidating the origin of the cells.

The Disposition of the Cell-masses in H. cali-
ginosus (PL 19, fig. 6). — The combined mass of chromophil
cells is situated dorsally on the pharynx, and extends back-
wards as far as septum 5/6. The cellular aggregate appears as
a number of white lobes amongst the muscular strands ; the
general arrangement is one of four transverse bands. The
posterior of these transverse elevations is divided into two
by a cleft in the mid-dorsal line, and forms a single rounded
pillow-like mass on each side (c4). The next is not divided,
and forms a single transverse elevation across the dorsum of
the pharynx (c3). The second is divided up into a number of
separate lobules, (c2), and appears therefore as a transverse
row of rounded projections. The first is similar to the
second (c1).

The cellular masses extend downwards on the sides of the
pharynx about as far as the lateral line or a little further ;
the first transverse row may be shorter.

Each lobule of the two anterior rows is associated with a
muscular strand (m), the base of which it surrounds. The
third, undivided elevation, has a number of muscular bands
emerging in a transverse series from its posterior face. The
fourth is not associated with muscular strands.

The General Relations of the Cell-masses in H.
parvus (PI. 19, fig. 7). — As seen in sections, the much
lobulated pharyngeal cell-mass (c1, c2, c3), situated dorsal to
the pharynx, extends also behind this region, and partially
surrounds the first part of the oesophagus. It thus occupies
segments IY, Y, and YI ; the portion in segment YI is to
some extent separate, being divided from the rest by septum
5/6, through which it communicates with the anterior portion
by a constricted neck. In segments IV and Y the mass is
penetrated by a number of muscular strands.

But in this species the characteristic cells have a con-
siderably greater extent of distribution than in the forms

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

271

previously described. Thus in segments V and VI the main
mass extends downwards on each side to within a short distance
of the mid- ventral line ( cv ). An aggregate ot cells is present
in segment VII, ventrolateral to the oesophagus on each side,
in close association with the lateral oesophageal (“ intestino-
tegumentary ”) blood-vessel. Similar small aggregates occur
in segments VIII and IX. Small masses of cells are present
dorsally in VIII, between the wall of the oesophagus and the
dorsal vessel ; and, at least in one specimen more minutely
examined in this connection, also dorsally on the oesophageal
wall in IX, in the angle between the alimentary tube and
septum 8/9; on both anterior and posterior faces of septum 9/10
below the oesophagus ; ventrally in segment X in association
with a blood-vessel ; and on the wall of the oesophagus below
the dorsal vessel at the level of septum 10/11.

The Chromophil Cells. — (PI. 19, fig. 8). — The cells are
oval or irregular in shape, a small one measuring 9 jjl, a
large one 18 fi in greatest length. They do not fit closely
together ; the interspaces are empty or contain an inter-
cellular matter.

The nucleus is large and conspicuous, vesicular, spherical
or ovoid, 4 /ul — 6 /u in longest diameter, often peripher-
ally situated, and clearer than the stained cytoplasm around
it. Besides small grains of chromatin there is a large
nucleolus, of different material from the chromatin grains, the
central portion of a bluish tinge in alcoholic iron-hsema-
toxylin preparations, the periphery darker and more opaque.
This large nucleolus may be absent; and then the deeply
staining chromatic granules are alone visible, of which one
may be larger than the rest.

The cell-body contains masses of deeply-staining material,
the remainder of the cytoplasm being more slightly coloured.
The less deeply staining areas are more peripherally situated ;
the more densely coloured portion usually encloses the
nucleus, and on the whole is more central in position; it may
be prolonged in one or other direction as fibril -like strands.

The intercellular substance is not as a rule sharply

272

J. STEPHENSON.

marked off from the cells ; the periphery of the cell fades
away into the intercellular substance, and in the measurements
of the cells as given above, the reference is to the deeply
staining portion only, on account of the impossibility of
determining the limits of cell and intercellular matter. In
amount this latter may be very considerable, and the staining
portions of the cells are then comparatively widely isolated
from each other. It has the character of a granular amor-
phous matrix, into which the bodies of the cells merge, and
through which some of the fibrillar processes of the deeper
staining matter are continued.

Transformation of the Cells. — The chroinophil cells
in this species are more completely aggregated together on
and behind the pharnyx than, for example, in Pheretima
posthum a; the number of the cells which penetrate inwards
amongst the interlacing muscular fibres on the dorsum of the
pharynx is much smaller. The chromopliil cells which occur
between the muscular fibres are mostly isolated, or in twos and
threes; in them the densely staining matter becomes less in
amount, the periphery of the cell may show a reticular
structure, and the cell processes are distinctly fibrillar. At a
further stage the deeply staining matter disappears ; the
cell elongates to form a strand, the nucleus is at one.side, the
pale-staining fibrillse form a reticulum. Longer strands
appear, composed apparently of several cells, since they may
contain one, two, or more nuclei. The nuclear changes are
similar to those previously described; the nucleolus becomes
smaller, and disappears or becomes indistinguishable from
the chromatin grains ; the nucleus itself decreases in size, and
becomes faint and difficult to distinguish ; appearances here
again suggest that at this stage it sometimes divides ; ulti-
mately it seems to disappear.

The Capsule. — In the adult, a peritoneal capsule is
present in parts over the main mass of the cells, especially
posteriorly ; in other species also the posterior surface
appears to be the region where a recognisable capsule is
best developed. But it is absent in other parts, — perhaps in

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

273

most parts ; and then the chromophil cells themselves form the
limit of the mass.

The smaller masses of chromophil cells, which occur in
some abundance in this species in several segments behind
the main mass, show interesting relations and give consider-
able help in elucidating the origin of the tissue.

Relation of Chromophil Cells to Septa. — The small
masses of cells on septum 9/10 are directly in contact with
the muscular fibres of the septum, taking the place of the
peritoneum at the spots where they occur. At one point a
still smaller aggregation appears to be essentially a slight
swelling of the peritoneal covering of the septum. Again
at another point a single cell of the chromophil type takes its
place in the series of peritoneal cells with flattened nuclei on
the septum. One of the larger aggregates is continuous
through the septum with a smaller mass on the other side.
The aggregates, of all sizes, are continuous with the peri-
toneum.

Relation to the Alimentary Canal . — The cells which
lie on the alimentary wall in segment IX are situated imme-
diately outside the muscular layer. Others are in close
contact with the blood-vessels which occur external to the
muscular- layer on the surface of the oesophagus, and not
only on the outer side of the vessels, but also between the
vessels and muscular fibres of the alimentary wall. In
places where the muscular layer of the wall is not visible
(probably because of gaps in the arrangement of the fibres),
the cells are in actual contact with the epithelium of the
oesophagus. Occasional cells are found singly here and
there internal to the muscular layer, in the irregular space
between the muscle fibres and the base of the epithelial
layer.

Relations to Blood-vessels. — The cells which are
situated on the lateral oesophageal trunks are in direct
apposition with the muscular or connective tissue coat of the
vessels, which they surround on all sides. There is no
separate peritoneal coat surrounding the vessel apart from

274

J. STEPHENSON.

the chromophil cells; nor any peritoneal membrane outside
the cell mass.

The appearances in Helodrilus are therefore con-
firmatory, in general, of the results obtained from a study of
Pheretima; but in addition, the facts relating to the small
masses of chromophil cells on the septa, on the blood-vessels,
and on the alimentary canal allow us, more decidedly than in
Pheretima, to derive them from the peritoneum, — to consider
them as modifications of the peritoneal layer, with which
they are continuous, or the place of which they take. The
occurrence of a few cells or cell aggregates in close relation
to the alimentary canal is interesting in connection with
former views on the nature of the cells. But here also they
are to be regarded as modified peritoneal cells, which in
places come in contact with the base of the epithelial layer
through a hiatus in the muscular coat, or perhaps here and
there make their way inwards between the muscle fibres.

The Appearances in Young Specimens.

1 turn now to the results obtained from the examination
of young worms, of various ages, of both genera. In the
case of the Pheretimas it is impossible to be certain of the
species to which young examples belong, since the discrimina-
tion of the three species which are found in Lahore is made
by means of the genital system (including especially the
external sexual marks). 'The young Lumbricids examined
belonged to the smaller species, Helodrilus parvus.

Non -sexual Pheretima. — In a Pheretima which is
approaching its full size but is still without sexual marks,
the condition is not markedly different from that previously
described. The cells of the lobular mass are irregular in
shape but definite in outline ; they do not dissolve at their
margins into an intercellular substance. In size, 20 g would
be the greatest length of a moderately large one. The
nucleus has the same general characters as in fully-grown

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

275

specimens ; the large nucleolus, always present, may measure
a third to two-fifths of the long, and even a half of the short
diameter of the usually ovoid nucleus. Here too the cyto-
plasm is not uniform, but shows darker and lighter patches,
the latter sometimes almost clear and vacuole-like ; the darker
patches are homogeneous, more or less central, and contiguous
to the nucleus.

Deeper in the pharyngeal mass (PL 19, fig. 9) the ad-
mixture of cells is not great. The nucleus enlarges; the
cell-body, smaller, dissolves at its periphery into a reticulum
of fibrillar connective tissue ; or the cell-body may be absent
as such, having wholly broken up into fibrils, so that one
side of the nucleus is bare. Still nearer the pharyngeal
epithelium the nucleolus decreases in size.

The chief features of this stage are, therefore, the integrity
of the cells in the lobular masses, where they have not begun
to disintegrate ; and, apparently, the larger size of the
nucleolus. The connective tissue change is proceeding in
the cells which have penetrated inwards amongst the muscle
fibres.

Pheretima of diameter 1*5 mm. — The cells in the
posterior and superficial portion of the mass measure 15-25 p,
are of various shapes, and well defined in outline. The cell-
body consists as before of two portions, a more lightly and a
more deeply staining ; the latter occurring as amorphous
masses, the former having a granular structure. The granules
of the lighter portion appear to be the same in substance as
the deeper staining masses, only not so closely aggregated;
and the deeper staining portion merges into the other by
becoming looser in texture.

The passage forwards towards the pharyngeal epithelium
is interesting. The cells, which posteriorly are in a compact
mass with only an admixture of muscle strands, become
much more scattered ; the muscle fibres, now arranged in a
variously interwoven felt, contain within their meshwork
isolated cells ; the whole texture is loose. In a few cells here
and there the beginning of a connective tissue change is to

276

J. STEPH ENSON.

be recognised ; but in general even the deepest cells retain
their original characters.

The cells cease altogether some distance from the pharyn-
geal epithelium ; in other words, they have not yet distributed
themselves throughout the whole pharyngeal mass. Near the
pharyngeal epithelium and between the interlacing muscle
fibres are scattered nuclei belonging to the sparse con-
nective tissue of this region. These nuclei are of various
and sometimes of irregular shape, and scarcely any structure is
to be made out in them; the connective tissue, reticular or
amorphous, is non-staining ; and there is no transition between
this tissue and the chromophil cells. It represents the
ordinary connective tissue of the muscle, and is comparable
to the connective substance between the muscle fibres in the
body-wall, or in other regions of the alimentary tube. The
adult connective tissue of this region has, therefore, as pre-
viously surmised, a double origin.

For the greater part of the surface of the mass there is
nothing of the nature of a capsule; the margin of the mass
is the distinctive cytoplasm, coarsely granular in character, of
the chromophil cells, and there is an entire absence of any
superficial differentiation, or of any special covering. In
places however a little pinkish-staining (in hsematoxylin and
eosin preparations) matter, of a membranous or connective
tissue-like appearance, is seen on the surface ; sometimes the
membrane is of linear tenuity, sometimes more bulky.

Where the muscle strands leave the mass a few chromophil
cells appear sometimes to have travelled a little way along
the strand, and hence are seen adhering to the strand just
after it has emerged from the main aggregate of the cells.
While some such cells appear to be underneath the peritoneal
investment of the strand, others are absolutely continuous
with it ; in other words, some of the peritoneal cells, instead
of retaining the usual flattened form, are swollen, and contain
the chromophil substance.

Pheretima of diameter 1 mm. — In a still younger
stage the cells, which already have a very marked chromophil

PHARYNGEAL GLAND-OELLS OF EARTHWORMS. 277

character, are still more definitely confined to the posterior
and dorsal portions of the mass. They are entirely absent
from half of the thickness of the mass, — that half which is
nearest to the pharyngeal epithelium.

The shape of the cells is, as before, various ; the outlines
are well-defined, and there is for the most part no shading off
into an intercellular substance. An average measurement in
the longest diameter would be 15 fi; 20 would be excep-
tional. The nuclei are to a considerable degree obscured ;
they measure 3 ’5 — 4^u in greatest diameter, are vesicular,
shortly ovoid, with large equably staining nucleolus and
scattered, sometimes mainly peripheral, chromatin grains.
The nuclear characters are thus already remarkably like those
of the adult. The cells are rather loosely arranged, with
considerable intervals.

Here and there, in the most deeply placed cells, — those
which have wandered off a little from the main mass and
form the outposts of the aggregate, — there is a sli ght
indefiniteness of boundary owing to the peripheral portion of
the cell-body becoming disintegrated into granular matter.
But there is no formation of connective tissue ; the connective
tissue of the pharyngeal mass at this stage has therefore an
entirely different origin. This specimen agrees with the last
described in the nature of this connective tissue of the
deeper part of the mass, and of its nuclei; and also in the
absence or very slight and partial development of a capsule.

Summary of Appearances in Young Pheretimas. —
In successively younger specimens of Pheretima therefore:

(1) The cells are more and more confined to the superficial
portion of the pharyngeal mass. This is strongly suggestive
of a derivation from the peritoneum ; it is the opposite of
what, presumably, would happen if the cells were derived
from the pharyngeal epithelium.

(2) The disintegration and the transformation of the cells
into connective tissue is progressively less marked.

(3) The capsule is less differentiated ; the chromophil cells,
which in places even in the adult border the coelomic cavity

278

J. STEPHENSON.

without the intervention of a peritoneal layer, do so in the
younger stages almost over the whole surface of the mass.
In other words, the chromophil cells are not derived from a
previously differentiated flattened peritoneal layer ; the
chromophil cells, and the flattened peritoneal cells which
cover neighbouring structures, are equally specializations of
the lining cells of the coelomic cavity. The inference, drawn
from the appearances in the smaller masses of cells in
Helodrilus parvus, that the chromophil cells are derived
from the peritoneum, requires to be understood in the above
sense ; the often flattened cells of the peritoneal membrane,
which in the adult covers the greater portion of the mass,
are derived from the superficial cells of the chromophil
tissue, with which (cf. the description of P. ha way ana)
they may still be connected, rather than vice-versa.

Young Helodrilus Parvus. — Two small specimens, in
diameter ‘7 mm. in the anterior part of the body, were
examined; and several still smaller, *5 mm. in diameter;
even in some of these small specimens sexual organs, both testes
and ovaries, were beginning to form. Since the appearances
are merely, for the most part, confirmatory of what has gone
before, a short account will be sufficient.

The chromophil cells scarcely penetrate at all into the
muscular felt on the dorsum of the pharynx, and form only
the lobed masses round the muscular strands which emerge.
In the larger of these specimens these lobes extend back-
wards through segments Y and YI ; smaller patches of the
cells are present in VII, VIII, and IX on the walls of some
of the blood-vessels, on the septa, and in the angle between
the septum and the alimentary tube ; a few cells form a
flattish layer on the ventral vessel in segment X. In the
smaller specimens the lobes extend backwards, segmentally
arranged, as far as segment VIII ; they are as usual suspended
on muscular strands passing obliquely to the parietes, and are
also connected in a longitudinal series through the septa by
thick strands of connective tissue, which, piercing the septa
as cords, spread out somewhat in the lobed masses. The

PHARYNGEAL GLAND-CELLS OF EARTHWORMS. 279

connective tissue thus forms to a certain extent a central
axis for the whole, though it is not very distinct as such in
the middle of the lobes. While the appearances point, as
before, unmistakably to the derivation of this connective
tissue from the chromophil cells, that which sparsely penetrates
between the interlacing muscular fibres of the dorsum of the
pharynx has equally unmistakably another origin.

Nothing that can be called a capsule is visible ; the cells
form the surface of the mass. Here and there in the larger
specimens, in a prolonged search, are seen a few elongated,
or even flattened, nuclei on or near the surface; once a little
reddish (eosin) tinted material allowed a distinction to be
made between a superficial layer of tissue and the chromophil
cells beneath. But practically everywhere the surface of the
masses is the surface — it may be the irregular or disintegrating
surface — of the chromophil cells ; and where the interstices
between neighbouring cells come up to the surface they are
not bridged over.

These young specimens confirm in all respects what was
said previously regarding the relation of the smaller masses
to the septa and blood-vessels in this species. The cells
appear as developments of their peritoneal covering, the
place of which they take, and with which they are con-
tinuous.

The Cells in the Lumbricid Embryo.

An embryo Lumbricid, pretty certainly Helodrilus cali-
ginosus, about 2 mm. long, taken from the cocoon, yielded
interesting results. Younger embryos, of which several
were investigated, showed no trace of the chromophil cells.

The embryo was examined by transverse sections. Behind
the region of the as yet entirely separate and laterally
situated cerebral ganglia there is situated on each side, lateral
to the alimentary tube, a mass of cells which appear to be
dissolving into a reticular connective tissue, and amongst
which a few muscular fibres are becoming differentiated.

280

J. STEPHENSON.

This tissue is in two lateral masses, there being none covering
the dorsal vessel (here still double), which lies directly on the
gut. The tissue does not, as a whole, come in contact with
the inner surface of the parietes — i. e. it does not fill up all
available space between gut and body-wall, though connec-
tions with the body-wall exist in the form of strands of
reticular nucleated tissue. The masses I take to be the
dorsal mass of the oesophagus in an early stage.

At one place on the left side, at the periphery of this mass,
is an aggregate of a few cells which are distinguishable from
the rest (PI. 19, fig. 10). These cells, about a dozen in
number in the section which shows them best, and extending
only through a few sections, are mostly elongated in one
direction and 12-20 fx in greatest length. The nuclei are in
most of the cells somewhat obscured and difficult to see ;
they are spherical or ovoid, 3*5-4 y. in greatest measurement,
with a spherical homogeneous nucleolus of relatively con-
siderable size, surrounded, in the cases where it is best seen,
by a clear circular space ; nucleolus and clear space a, re
rather excentrieally situated. The peripheral chromatin is
distributed as distinct and fairly large granules. Some
nuclei have two nucleoli; in other cells two relatively small
nuclei are in close apposition ; but I could not discover any
mitotic figures (compare the various appearances of the nuclei
in PI. 19, fig. 10). The cytoplasm stains moderately deeply,
but not so deeply as the chromophil substance of the
adult cells; and not quite evenly, haviug a granular texture
which is closer and more homogeneous in some parts than
others.

These cells do not help to form the slightly pinkish (eosin
staining) reticulum into which the main portion of the dorso-
lateral pharyngeal masses seem to be dissolving. The cells
are in several cases connected together among themselves,
perhaps because nuclear division goes on in advance of division
of the cell -body (see the upper left-hand part of the figure).
No peritoneal membrane surrounds the mass ; while on the
body-wall the cells lining the ccelomic cavity are cubical with

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

281

spherical nuclei, or in places already flattened with elongated
nuclei.

The characters of the nucleus, and to some extent those
of the cytoplasm of these cells, resemble those of the
chromophil cells of the adult ; and it seems probable that we
have here the first appearance of the characteristic cells of
the pharyngeal mass. If so, they are evidently of mesoblastic
origin, and make their appearance at the periphery of the
pharyngeal mass.

Function of the Cells.

Though in the light of what has gone before we may
reject the usual supposition, that the cells pour a secretion
into the pharynx (or oesophagus, in the case of the smaller,
more posteriorly situated aggregates), it is not easj> to propose
another hypothesis to take its place.

That some of the chromophil cells on the dorsum of the
pharynx wander deeply into the pharyngeal mass in certain
species and there give rise to a fibrillar connective tissue,
seems plain. But this is obviously not the main function of
the cells ; nor does this change occur in the smaller, more
posterior aggregates.

That the main function of the cells is metabolic is, though
only a vague statement, perhaps as far as we are justified
in going. In this connection the following considerations
may be brought forward :

(a) Independently of the connective tissue change, the
cells are frequently, or usually in the adult, seen to have
indefinite outlines, and their margins appear to be dis-
integrating. This is visible even at the surface of the mass,
in the cells which border the coelomic cavity.

(b) The linear interspaces between the cells, always a
marked feature, evidently allow of the easy percolation of the
body fluids throughout the whole. Add to this the fact that
the peritoneal capsule is never complete, and often (and
especially in young specimens) largely absent, and we have the

282

J. STEPHENSON,

possibility, at least, of an extensive exchange between the
cells and the body-cavity fluid.

(c) The blood supply to the pharyngeal mass is extremely
rich ; this is a striking feature in the dissection of any earth-
worm in which the vessels of the anterior end of the body
happen to be engorged. Not only so, but in all the species of
Pheretima examined in the present paper, as well as in
certain others, there are present, within and immediately
behind the pharyngeal mass, large numbers of the structures
known as “ blood-glands These are spherical bodies with
an afferent and efferent vessel at opposite poles, containing
blood, but largely choked by a mass of blood -cells. IIow
widely these glands are distributed is not at present known ;
of the many score of species of Pheretima, for example,
by far the larger number have as yet only been examined
from a systematic point of view. Blood-glands have been
found in other genera of Megascolecidse also — in Acantho-
drilus (Beddard, 1), in Pontodrilus (first by Perrier, cf.
Eisen, 5), in Argilopliilns (=Plutellus, cf. Eisen, loc.
cit.) — as well as in Spar ganophil us among the Greosco-
lecidae (Eisen, 5) ; and they not improbably occur in other
genera also, where they will be revealed by a fuller examina-
tion than has yet been made. The situation of many of the
smaller aggregates of chromophil cells on the blood-vessels in
Helodrilus parvus may also be recalled in this connection.

(d) That active metabolism takes place in the pharyngeal
region is also indicated by the great development of the
nephridial tubules, in micronephridial genera, in some of the
most anterior segments. Here again we are dealing with a
character which is not of systematic importance, and which
has, therefore, seldom been recorded. Very noticeable
bunches of nephridial tubes opening to the exterior occur at
the sides of the pharynx in several species examined by
Miss Raff (7a). Bushy tufts, sometimes of relatively very
great size, and always in marked contrast to the minute
scattered tubules of more posterior segments, occur at the
sides of and immediately behind the pharyngeal mass in, for

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

283

example, Megascolides, Notoscolex, Megascolex,
Lampito, Pheretima, Ery thrseodrilus, Octochaetus,
Eutyphoeus, Eudi chogaster — to mention only genera in
which I have myself observed them. The nephridia of
meganephric forms are not enlarged in the pharyngeal and
immediately subsequent segments ; why no modification of
any kind occurs in them, when in other and sometimes closely
related genera a great multiplication and massing together of
the micronephridial tubules takes place in this region, I am
unable to say.

The chromophil cells do not stain with Lugol’s iodine
solution; glycogen seems therefore to be absent.

Summary.

(1) The “ pharyngeal gland-cells ” of earthworms are not
gland-cells in the usual sense, and do not communicate with
the pharynx ; the term “ chromophil cells ” is proposed for
them because of their intense coloration by haematoxylin
and similar stains. The so-called “ septal glands ” of earth-
worms are aggregations of similar cells at a more posterior
level.

(2) In the chromophil cells the deeply staining matter is
not equably distributed through the cell-body ; the peripheral
regions of the cells in general stain more lightly, and appear
to be disintegrating, or merge into an intercellular substance.

(3) While most of the cells form a more or less compact
aggregate on the surface of the pharyngeal mass, a number
penetrate inwards towards the pharyngeal epithelium, and
become progressively metamorphosed into fibrillar connective
tissue.

(4) A capsule of flattened cells covering the mass, though
present in part, is incomplete. The smaller masses of cells in
Helodrilus parvus are frequently continuous with the
peritoneal membrane, of which they appear as modifications.

(5) In Helodrilus parvus, and especially in all young
earthworms, the inwandering and the connective tissue change

284

J. STEPHENSON.

of the chromophil cells is less marked ; in very young specimens
neither has taken place. The capsule is also more and more
incomplete the younger the specimen.

(6) The cells are to be looked on as of peritoneal origin ;
that is to say, they are modifications of the original lining
cells of the coelomic cavity. Hence the absence of capsule
in the early stages; and hence the original limitation of the
cells to the superficial portion of the pharyngeal mass.

(7) The main function of the cells is probably metabolic ;
but it is at present impossible to particularise further.

References to Literature.

1. Beddard, F. E. — “ Contributions to tlie Anatomy of Earthworms,

with Descriptions of Some New Species,” ‘ Quart. Journ. Micr.
Sci.,’ (n.s.), vol. 30, 1890.

2. ‘ A Monograph of the Order Oligochaeta,’ Oxford, 1895.

3. Dobell, C. C. — “Cytological Studies on Three Species of Amoeba,”

‘Arch. Protistenkunde,’ vol. xxxiv, 1914.

4. Eisen, G. — “ Pacific Coast Oligochaeta,” ‘ Mem. Calif. Acad. Sci.,’

vol. ii, No. 4, 1895.

5. Ibid.. No. 5, 1896.

6. Hesse, R. — “ Zur vergleiclienden Anatomie der Oligochaeten,”

‘ Zeit. f. wiss. Zool.,’ vol. lviii, 1894.

7. Parker and Haswell. — ‘A Text-book of Zoology,’ London, 1910.

7a. Raff, Janet W. — “Contributions to our Knowledge of Australian
Earthworms. The Alimentary Canal ; Pt. 1,” ‘ Proc. Roy. Soc.
Viet.,’ vol. xxii (n.s.), pt. 2, 1910.

8. de Ribaucourt, E. — “ Etude sur l’anatomie comparee des Lom-

bricides,” ‘ Bull. Scient. France et Belg ,’ vol. xxxv, 1901.

9. Yejdovsky, F. — ‘ System und Morphologie der Oligochaeten,’ Prag.,

1884.

10. Yogt and Yung. — ‘ Traite d’anatomie comparee pratique,’ Paris,
1888.

PHARYNGEAL GLAND-CELLS OF EARTHWORMS.

285

EXPLANATION OF PLATE 19...

Illustrating Prof. J. Stephenson’s paper, “On the So-called
Pharyngeal Gland-cells of Earthworms.”

Fig. 1. — Pheretima lieterochseta ; dissection of anterior end.
c', Masses of chromopliil cells on pharynx; c2, upper, and c3, lower
cellular masses in segment V, behind pharynx ; d.v., dorsal vessel ;
m1, m2, muscular strands emerging from masses of chromopliil cells ;
n, masses of micronephridia ; 'pin., pharynx; 4/5, 5/6, the corresponding
septa, the latter turned back. X 8.

Fig. 2. — Chromopliil cells from the pharyngeal mass of Pheretima
posthuma. X 1250.

Fig. 3. — Individual chromophil cells from Pheretima hetero-
chseta. x 1650.

Fig. 4. — Portion of the surface of the pharyngeal mass in Phere-
tima lieterochseta, showing the general characters of the cells,
clefts between the cells, and, at this place, entire absence of capsule.
The surface of the mass is below in the figure. X 1000.

Fig. 5. — Chromophil cells at some depth in the pharyngeal mast
of P h e r e t i m a h a w a y a n a, undergoing transformation into connective
tissue, n, Nuclei whose stainable cytoplasm has undergone conversion,
and which are themselves becoming fainter ; m, mass of staining
material, apparently without nucleus. X ca. 1250.

Fig. 6. — Helodrilus caliginosus; dissection of anterior end
c'-c4, Lobular masses of chromophil cells on pharynx; m, muscular
strands emerging from the masses ; 5/6, 6/7, the corresponding septa
(the first few septa are absent or unrecognisable). X 6.

Fig. 7.— Helodrilus parvus, an approximately median longitudinal
section. c\ Lobular masses of chromophil cells in segment IV, on dorsum
of pharynx ; c2, the same in segment V ; c3, the same in segment VI ;
cv, a portion of the latter appearing ventrally ; c.g., cerebral ganglion ;
d, dorsal mass of the pharynx, consisting of connective tissue and
muscle strands; ces., oesophagus; pli. div., dorsal diverticulum of
pharynx; v.n.c., ventral nerve cord; 4/5, 5/6, 6/7, the corresponding-
septa. X 40.

Fig. 8. — Chromophil cells of Helodrilus parvus, x 1250.

Fig. 9. — Chromophil cells of non-sexual Pheretima at some depth
VOL. 62, PART 3. NEW SERIES. 21

28(3

J. STEPHENSON.

in the pharyngeal mass. The features are the large nucleus and the
relatively small amount of cytoplasm which is undergoing fibrillar
change. X 2000.

Fig. 10. — Certain cells at the periphery of the loose mass dorso-lateral
to the pharynx in a Lumbricid embryo, x 1650.

THE CHROMOSOME COMPLEX OE CULEX PIPIENS. 287

The Chromosome Complex of Culex pipiens.
Part II. — Fertilisation.

By

Monica Taylor, S.N.D., D.Sc.

With Plate 20 and 1 Text-figure.

* Contents.

page

Introduction . . . . . . 287

Material and Methods ..... 289

The Reproductive Organs .... 295

The Egg . . . . . -295

General . . . . • . 298

Summary ...... 299

Note . . . . . . 299

Introduction.

In the summary of a paper, entitled “ The Chromosome
Complex of Culex pipiens” (4), it was stated that:

(1) The somatic number of chromosomes is three, both in
the male and female.

(2) The number of chromosomes in the spermatogonia, as
well as in the primary and secondary spermatocytes and
spermatids, is three.

Two alternative suggestions as to the cause of this apparent
anomaly were offered :

(1) The non-participation of one of the gametic nuclei in
the formation of the “ zygote” in fc fertilisation.”

288

MONICA TAYLOR.

(2) The fusion of three pairs of homologous chromosomes
at some early stage in the life-history, this fusion remaining
permanent throughout later divisions. In the discussion
preceding the summary it was stated that the question could
not be settled until the fertilisation process had been
examined.

This work on the cytology of Culex, as stated in the intro-
duction of the above paper, had been undertaken because of
the importance of the conclusions given by Miss Stevens in
her paper, entitled “The Chromosomes in the Germ-cells of
Culex ” (3).

The second of the alternative suggestions offered above is
a modification to meet the particular needs of the case of
Culex pipiens of Miss Stevens’ statement that: “Para-
synapsis (parasyndesis) occurs in Culex in each cell
generation of the germ-cells, the homologous maternal and
paternal chromosomes being paired in telophase, and remaining
so until the metaphase of the next mitosis.”

Difficulties in obtaining the necessary material, and in tlio
technique connected with the food supply of the imagines,
delayed, for the time being, the examination of the fertilisa-
tion processes, and prevented the demonstration of the whole
history of oogenesis.

Dr. Woodcock’s paper “On ‘Crithidia’ fasciculata
in hibernating mosquitoes (Culex pipiens) and on the
question of the connection of this parasite with a Trypano-
some ” (5) has, however, incidentally filled up some of the
gaps left in the history of oogenesis, and, in this paper, the
technique of artificial rearing of Culex pipiens in all
stages of its life-history has been described.

I should like to take this opportunity of thanking
Dr. Woodcock not only for sending me a Reprint of this
paper, but also for giving me full details in writing of his
experiments, by which I have been enabled to repeat his work
with similar success, and also, as will be shown later, to con-
firm his statement that “Culex pipiens is essentially the*
British mosquito which likes Avian blood.”

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 289

In the ‘ Proceedings of the Royal Society/ 1915, a full de-
scription of the chromosome cycle of Coccidia and Gregarines
appeared (Dobell and Jameson) (1), a work which elucidates
a cytological condition of affairs superficially somewhat similar
to that which obtains in Culex pipiens. The authors of
that paper demonstrate the presence of the haploid number
of Chromosomes at every nuclear division in the life-history
of Aggregata and Diplocystis except in the zygote.

In f Chromosome Studies of Diptera 5 (2), July, 1914,
Charles W. Metz calls attention to the neglect of Diptera by
students of cytology — a neglect all the more pronounced when
contrasted with the great amount of energy expended upon
other insect-groups — notably Hemiptera, Orthoptera, Coleop-
tera. He offers, as a probable explanation of this neglect,
the unsuitability of Dipterous material for cytological study
and the great difficulties connected with such study. The
results embodied in Metz’s paper, as will be shown later, have
been helpful in interpreting the phenomena observed in
Culex and in showing that there is no essential difference
between it and other Diptera.

Dr. Woodcock discovered that, after the summer female of
Culex pipiens has fed once on the blood of a living bird,
the eggs attain their normal size and are ready for fertilisa-
tion. The raft is laid almost immediately after the second
feed — fertilisation taking place in the interval between the
two meals. This probably accounts for the fact that females
reared in captivity and fed on a fruit diet appear never to be
fertilised.

Material and Methods.

In the summer of 1915 cages similar to those described by
Dr. Woodcock were set up in the College Laboratory of Notre
Dame, Glasgow, young pigeons being employed as food. A
pigeon was also caged in the near neighbourhood of a
wooden tub stocked with larvse, and placed in a small garden
at Notre Dame. Control experiments, which will be described

290

MONICA TAYLOR.

later, were also started at Lady wood, Milngavie, the source
of the material for all these experiments being mainly a
rectangular iron trough near the farm-yard of Garscadden
Mains, Bearsden. Two rafts were found during the course
of the summer 1915 in the wooden tub, although all attempts
in previous years to induce the imagines to lay there, or to
lay in any of the aquaria on the premises, had failed. This
experiment would seem to show that wild birds are not so
easily bitten by the gnats as domestic birds, since many
sparrows, thrushes, and blackbirds visit the small garden in
which the tub is situated.

The season's experience of artificial rearing showed that
the number of egg-rafts produced under artificial conditions
was not likely to be sufficient for a thorough investigation of
the fertilisation processes. Moreover, unfertile egg-rafts
were frequently obtained by Dr. Woodcook from the arti-
ficially confined imagines, and I too obtained rafts which
produced no larvae; while, on the other hand, egg-rafts laid in
the open invariably produced larvae. For these reasons the
confinement of the imagines in netted chambers was aban-
doned, and the stocking of a pond at a convenient place in
the country was resolved upon.

In view of the above experiments, and of the fact that all
the sources of material already used had been situated in the
vicinity of farm-yards, it was decided to establish such a
pond at Lady wood, near a stock of poultry — these latter
birds being evidently easier of access than wild birds.
Earlier in the summer of 1915 several small troughs, 12 in.
in diameter, and about 6 in. in depth, had been stocked with
larvae to serve as control experiments to those being carried
on at Notre Dame. The results were disappointing. No
egg-rafts appeared on these small ponds (during the summer
of 1915). The number of female gnats seeking for hiber-
nating quarters in the autumn of that year around Ladywood
showed that the imagines, developed from the contents of
the small troughs mentioned above, must have found a pond
more suitable to the needs of their offspring than those pre-

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 291

pared for them in the garden of Ladywood. Mr. MacDougalTs
permission having been obtained, a hunt over his nursery
gardens revealed the secret of the non-appearance of rafts
on the Ladywood ponds. A large disused iron bath, elliptical
in form (36 in. X 26 in. X by 11£ in.), and containing stagnant
water, was swarming with larvae and pupae of every age
which were evidently supplying hibernating imagines. This
discovery was made too late in the year to be utilised for the
further production of rafts — but it showed that the situation
was a good one — the former experiments having possibly
failed because of the smallness of the troughs, and because
they were more or less concealed by the surrounding grass.

Early in 1916, thanks to the kindness of Mr. MacDougall,
four ponds, A, B, C, and D respectively, were prepared :
One (A), a large circular iron trough, diameter 30 in., greatest
depth 24 in. ; another (B), a wooden tub, 25 in. in diameter,
depth 16 in. ; the third (C) was the bath already selected by the
gnats in 1915 ; the fourth (D), a rectangular porcelain sink,
20 in. X 14 in. X 10 in.

The first two, A and B, were situated side by side, being
protected on the north by shrubs, and on the east by a wall.
The iron trough (A) was exposed to all the sunshine of
morning, afternoon, and evening; the wooden one (B), being
nearer the wall, was more shaded. Both, however, were
quite unhidden by vegetation. The tinned iron bath (C) was
surrounded on three sides by glass-houses, and exposed on
the southern side; the fourth (D) was placed in the uncut
grass of an open space.

On May 21st, 1916, fifteen egg-rafts were discovered in A
— there was thus no necessity to stock the ponds. No rafts
appeared on the water in the wooden pond until June 15th.
The preference shown by the gnats for the iron trough, and
for the tin bath, may be due to the higher temperature of
the water of these, or to the fact that they were in a more
exposed situation, and consequently more easily found. The
porcelain trough has never been popular, comparatively few
rafts being forthcoming.

O O

292

MONICA TAYLOR.

The rafts found in May and June were evidently those laid
by the hibernating imagines seen in the previous autumn, since,
after that date, eggs were not abundantly produced until the
middle of July, when a spell of exceptionally warm, moist,
weather conduced to an abnormally abundant supply. This
supply continued to be good until the middle of August,
when the spell of hot weather ceased. Odd rafts were
occasionally found until the end of August.

In one of his letters Dr. Woodcock expressed his opinion
that the rafts were deposited at about 5 o’clock in the
morning, and text-books also state that the early hours of
summer mornings are chosen by the gnats for the purpose
of egg-laying. On July 12th, 1915, an egg-raft, cream-white
in colour, was found at the Bearsden pond at 5 a.m. Portions
of this raft were fixed at intervals of a quarter of an hour,
and sections showed that it was quite young. After a care-
ful study of ponds A, B, C, and D it became clear that, at
Milngavie, most rafts are laid between the hours 9.30 p.m.
and 12 p.m., very few between 12 p.m. and 4 a.m., and some
between 4 a.m. and 6 a.m. As the season advances fewer are
laid in the morning hours. Damp heat conduces greatly to
the deposition of rafts, many more being forthcoming after a
day of calm, moist, close weather. Numerous imagines were
always hovering over the ponds in the evenings at a height of
about six feet. These disappeared during the day time.

No difficulty in rearing the larval and pupal stages of the
gnat occurred until the summer of 1915 — the critical period
in the life-history of the Culex being apparently the imago
period. Even in exceptionally favourable circumstances the
number of egg-rafts deposited at any one period bears a very
small proportion to the number of imagines that escape from
the pupal cases, and this has been very marked in a long
study of the naturally occurring ponds. However, in the
ummer of 1916 Daphnia were introduced into the gnat
cultures at Notre Dame, and since these, like the Culex larvas,
also flourish well on a diet of Chlamy dom on as, the tub
quickly became swarming with Daphnia, while the gnat

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 293'

larvge disappeared almost entirely. Perhaps this accounts
for the more frequent occurrence of Culex in the fairly
clean water of rain-barrels and drinking- troughs, in spite of
the fact that, as shown by long experience, the larvge grow
more quickly in a good culture of C hlamy domonas, and
that, on this account, one would expect the gnats to prefer a
more stagnant habitat.

The egg-rafts, for the cytological studies in question, after
being laid, which process takes about ten to fifteen minutes,
were isolated, along with sufficient water from the pond, in
Petri dishes, in which they underwent their subsequent
development. Thus it was possible to determine the age of
the eggs. Rafts just laid, and of ages ranging from half
hour after the deposition of the last egg to two and three-
quarter hours, were preserved. Imagines just before, during,
and after oviposition were also fixed. Many rafts of unknown
age were necessarily found during the season, their colour
indicating to a certain extent their age.

My best thanks are offered to Sr. Carmela, S.N.D., who,
during my absence in August, went on with the work of
tending the ponds and fixing the rafts at timed intervals.
She gives twenty minutes as the period required for the
laying of one raft.

Carnoy proved an excellent fixative for both rafts and
imagines. Bouin and Gilson-Petrunkewitsch were used as
controls. The rafts did not sink in the former fixative, hence
the results were not so good, but the Bouin material was
more easily sectioned. Portions of whole rafts were em-
bedded in paraffin, and sagittal sections of from 5/* -to 8//
made of the whole mass. Thus many eggs could be examined
at once. The funnel (see Text-fig. 1 , f) was removed before
•embedding.

Mr. P. Jamieson’s long experience in microtoming was
placed entirely at my disposal in the matter of sectioning* the
proverbially difficult dipterous egg, and I should like to express
my gratitude to him for this help, and also for actually
cutting many of the sections used in this investigation.

294

MONICA TAYLOR.

Text-fig. 1.

a

Sections through mature female gnat parallel to the sagittal
plane (b under higher magnification), cli. Chorion, e./. Young
egg follicle. /. Funnel, m. Micropyle. m. e. Mature egg.
o. t. Ovarian tube. v. m. Yitelline membrane.

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 295

The Keproductive Organs in the younger stages have
already been described. In the mature ovary (Text-fig. 1)
the egg is elongated, and is provided with a chorion, very
beautifully sculptured, and a second membrane — the vitelline
membrane — both of which are perforated before fertilisation
by the micropyle. Separating one egg from its neighbour of
the same size, or from a young egg follicle, is a chitinous
structure (see fig. 30, f Natural History of Aquatic Insects/
Miall.), like a funnel in shape, which remains attached to
the laid egg at its broad anterior end. This funnel is per-
forated above the micropyle, and apparently serves to guide
the spermatozoa to the micropyle. A somewhat similar
structure is shown in Sedgwick’s f Student’s Text-book of
Zoology/ vol. 3 (fig. 400), for the egg of Drosophila
cellaris. The future head end of the embryo lies in the
anterior position in the ovarian tube, as is usual in insects— the
hind end of the egg being the first to emerge from the imago.

Dr. Woodcock (5) expressed his opinion that an imago was
probably capable of depositing more than one raft in the
season. Sections of gnats, which were fixed immediately
after depositing their rafts, showed that the contents of the
spermathecae were by no means exhausted. Moreover, the
gonad showed some large eggs, as well as very many small
egg follicles, which evidence seems to support his opinion.
The gnats, flying away after laying eggs, were perfectly
vigorous, and showed no tendency to die.

The three spermathecae communicate with the hind end of
the common oviduct by three minute tubes, so that the
spermatozoa make their way into the funnel as the egg
passes out.

In view of the foregoing description and the fact that
eggs laid in captivity often produce no larvae, the first hypo-
thesis as to the origin of the haploid number of chromosomes
in the somatic tissues of Culex can be rejected, and this
is justified by actual observation of the two pronuclei in
the egg.

The Egg. — I have not been able to observe the formation

296

MONICA TAYLOR.

of any polar bodies. The egg nucleus lies in the middle of
the egg, and is a typical resting nucleus. It is surrounded
by the large yolk globules which constitute the greater bulk
of the egg. The cytoplasm, which is sparse, is densely
staining and provided with numerous deeply-stained particles,
as was the case in the tissues of the larva and pupa, and
there are large numbers of small bodies surrounding the
yolk globules which resemble chromatin in their staining
reactions. These chromatin-like bodies, “ yolk nuclei ”, seem
to be intimately concerned with the digestion of the globular
yolk-masses, and with their conversion into protoplasm, and
sometimes resemble very minute chromosomes (PI. 20, fig. 1).
When surrounded by the digestion products, which have
resulted from their activity, they do not stain so clearly, and
eventually they appear in the fully-formed protoplasm as the
particles already described. This quality of the protoplasm
greatly detracts from the beauty of the histology.

The sperm nucleus (male pro-nucleus) assumes the resting
condition very quickly. In one egg of a raft, fixed a few
minutes after deposition, the two pro-nuclei are already in
close contact. In one egg of a raft half an hour old, the
spermatozoan has reached the egg nucleus, but is still sperm-
like. In one-hour rafts there are several segmentation
nuclei. (It must be remembered that there is a difference in
age between the first laid egg of the raft and the last laid
egg, of from ten to twenty minutes, hence these times are only
approximate.)

The segmentation nuclei, which lie in little islands of
protoplasm (PI. 20, fig. 2) pass into a decided resting-stage
after dividing, and several blocks have to be sectioned to
make sure of obtaining mitotic nuclei.

The prophases (PI. 20, figs. 4-9) of the dividing segmenta-
tion nuclei of Culex show six chromosomes. In early
prophase a tendency to emerge in pairs from the resting
nucleus is evident (PI. 20, fig. 9). These six chromosomes
(PI. 20, fig. 10) arrange themselves on the equator of a
perfectly typical spindle, split longitudinally, and in anaphase

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 297

and telophase six thin chromosomes can be seen going to
each daughter nucleus (PL 20, figs. 11-13). In contrast to
former experience of larval, pupal, and imaginal material the
dividing nucleus is most frequently found in the metaphase
and anaphase stage in the segmenting egg of Culex.
What takes place in late telophase and in the passage of the
chromosomes into the resting nucleus recticulum is difficult
to follow.

There is thus no difficulty in demonstrating six chromosomes
in the segmenting nuclei of Culex pipiens — fertilisation
and the early stages of development are perfectly typical.
No reduction in the “ Zygote,” as found by Dobell and
Jameson in Coccidia and Gfregarines, takes place. The
second hypothesis set forth in the beginning, viz. “ the
fusion of three pairs of homologous chromosomes at some
early stage in the life-history, this fusion remaining perma-
nent throughout later divisions,” most probably explains the
case. Early prophases show decided tendency of the chromo-
somes to come out of the reticulum in pairs, full prophases
show six chromosomes, among which pairing can sometimes
be detected (PI. 20, figs. 4, 7, 8, 9). As has been shown by
Stevens and Metz a side-to-side pairing of homologous chromo-
somes is a characteristic of Dipterous cytology. Metz (2)
states that Miss Stevens records it in nine species of Muscidae,
and four species of Mosquitoes, and that he has verified it in
five of these species of Muscidae, and extended it to eight
others, in addition to species, of Drosophila. An increas-
ingly closer proximity of these homologous chromosomes one
to the other, would produce, eventually, an actual fusion of
the maternal and paternal constituents, with the result, in- the
case of Culex pipiens, that in full prophase three, and not-
six, would be the apparent chromosome number. The three
chromosomes that appear so persistently in the somatic tissue
of larvae, pupae, and imagines of Culex pipiens are, there
fore bivalent in character ; they are really three groups of
chromosomes, with two in each group. I have not been
able to trace out more fully the development of this tendency

298

MONICA TAYLOR.

of homologous chromosomes to fuse into an apparently
single chromosome. This would entail a study of rafts
ranging from a quarter hour to three and a half to four days
old. (The development of the larva in Scotland occupies
from three and a half to four days.) Moreover, the egg-
capsule becomes so hard and brittle as it changes from cream
white to dark brown, that a special technique would have to
be devised for sectioning these eggs without destroying the
nuclear detail. All that can be stated with certainty up to
the present is. that the homologous chromosomes have not
fused in segmenting nuclei — while this fused condition has
become a characteristic of Culex pipiens in the early larval
stage.

It would seem, then, that parasyndesis has reached its limit
in the somatic tissues of Culex pipiens, resulting in the
actual fusion of homologous chromosomes, and that extreme
parasyndesis is responsible for the apparent anomaly de-
scribed in the f Chromosomes Complex of Culex pipiens', I.

General.

There is general agreement that we are justified in
assuming (1) that hereditary qualities are represented by
material substance, and (2) that this substance is either
chromatin or is inextricably involved in chromatin. Granting
these two assumptions, we seem logically bound, by the
generally occurring accurate longitudinal splitting of the
chromosomes in mitosis, to admit that the patches of material
representing definite hereditary qualities are arranged in
linear fashion along the course of the chromosome or thread
of chromatin. But this involves necessarily a tendency of
the hereditary substance representing one particular quality,
or group of qualities, to segregate together. In other words,
there must be, in the case of hereditary substance, an
attraction of like for like. If this be so, there will be a
tendency for chromosomes composed of corresponding patches
of hereditary material arranged in like order, to come

THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 299

together side by side with homologous poles together, the
opposite of what happens in the case of magnetic attraction.

Such a hypothesis will account for the frequent occurrence
of parasyndesis, and for the further accentuation of this
into complete fusion as is supposed to take place in Culex
pipiens.

Summary.

(1) The egg-rafts of Culex pipiens are laid most
copiously between hours 10.30 p.m. and 12 p.m. They are
also laid between 4 a.m. and 6 a.m.

(2) Fertilisation in Culex pipiens is normal. Segmenta-
tion begins in less than an hour after the deposition of the
last egg.

(3) The chromosome number in the segmenting nuclei
is six.

(4) A tendency to parasyndesis is exhibited by the seg-
menting nuclei.

(5) Parasyndesis probably effects the condition of the
chromosomes in the nuclei of larva, pupa, and imago, i.e.
is responsible for the presence of the apparently “ haploid ”
character of the nuclei in the somatic cells.

Note. — After the completion of the foregoing paper,
Metz’s “ Chromosome Studies on the Diptera II.
The Paired Association of Chromosomes in the D p-
tera, and its Significance’, Mourn. Exper. Zool.’, xxi.,
came into my hands.

With regard to certain criticisms passed on my work,
I should like to state : —

(1) The fixatives employed Jay me were precisely those
employed by Metz (see p. 379, ‘Quart. Journ. Micros. Sci.’,
vol. lx).

(2) Although I have not specified this, all the precautions
recommended by Metz to secure good fixation and penetra-
tion were employed by me. I cannot, therefore, put my
results down to bad fixation.

300

MONICA TAYLOR.

(3) I have failed absolutely in my former work on Culex,
although I devoted much time and study to this point, to find
any figures resembling Metz’s figs. 166 and 167, or Stevens’s
figs. 8, 9, and 10. This failure may be attributed to differences
in the material studied. It could not be due to bad fixation.

(4) Setting aside the Diptera, my figs. 68 and 69 (4),
would be interpreted, by the vast majority of cytologists,
as a split spireme, as chromosomes precociously split for
metaphase.

(5) My conclusions in the present paper, made after a
study of fertilisation, do not differ in principle from those of
Miss Stevens. I state, for my variety of Culex pipiens,
that parasvndesis has resulted in actual fusion. Only the
assumption that some such fusion has taken place can account
for the striking recurrence of three chromosomes in the
prophase of somatic tissues in larval, pupal, and imaginal
material.

(6) In a preliminary investigation of Chironomus sp.,
an account of which I hope to publish later, I have found a
confirmation of the results obtained in Culex.

Literature List.

1. Dobell and Pringle Jameson (1915). — “ The Chromosome Cycle in

Coccidia and G-regarines,” ‘Proc. Roy. Soc.,’ vol. lxxxix.

2. Metz, C. W. (1914). — “ Chromosome Studies in the Diptera. I. A

preliminary survey of five different types of chromosome groups
in the genus Drosophila,” ‘ Journ. Exp. Zool.,’ xvii.

3. Stevens, N. M. (1910). — “The Chromosomes in the Germ Cells of

Culex,” ‘Journ. Exp. Zool.,’ viii.

4. Taylor, Monica (1914). — “The Chromosome Complex of Culex

pipiens,” ‘Quart. Journ. Micr. Sci.’ (n.s.), vol. 60.

5. Woodcock, H. M. (1914).— “ On ‘Crithidia’ fasciculata in

Hibernating Mosquitoes (Culex pipiens) and the Question of
the Relation of this Parasite with a Trypanosome,” ‘ Zool. Anz.J
Bd. xviii, 8.

THE CHROMOSOME COMPLEX OF CULEX P1PIENS. 301

EXPLANATION OF PLATE 20,

Illustrating Miss Monica Taylor's paper on “ The Chromosome
Complex of Culex pipiens. — II: Fertilisation.”

All figures were drawn with the Abbe camera to the given scales
(Leitz y1, oil imm., Zeiss compensating oculars).

Fig. 1. — Section through egg fixed Gil. Pet. Stained in thionin ;
differentiated to show “ yolk nuclei.” “ Yolk nuclei ” remain deeply
stained ; chromosomes lose stain when sections are left for some time
in absolute alcohol.

Fig. 2. — Section through egg of a raft laid at 4.15 a.m. ch. Chorion.
c. m. Closed micropyle /. Funnel. s. p. n. Segmentation resting
nucleus surrounded by mass of protoplasm, v. m. Vitelline membrane.

Fig. 3. — Resting nuclei ; daughter nuclei of fertilisation nucleus ;
“ yolk nuclei ” around yolk globules.

Fig. 4. — Prophase of segmentation nucleus ; egg one hour old.

Fig. 5. — Prophase.

Fig. 6. — Early prophase.

Fig. 7. — Prophase.

Fig. 8. — Prophase.

Fig. 9. — Early prophase.

Fig. 10. — Metaphase.

Figs. 11 and 12. — Anaphase.

Fig. 13. — Telophase.

YOL. 62, PART 3. — NEW SERIES.

22

VISCERAL ARCHES OE THE GNATHOSTOME FISHES. 303

The Homologies of the Muscles related to the
Visceral Arches of the Gnathostome Fishes.

By

Edward Phelps Allis, jr.,

Menton, France.

Witli Plates 21 and 22 and 1 Text-figure.

In 1874 Vetter published his well-known work on the
muscles related to the visceral arches of the Selachii, and,
clothed somewhat with Gegenbaur’s authority, it immediately
became the recognised standard of reference, and all later
work relating to the subject has apparently been greatly
influenced by it. Certain parts of Vetter’s descriptions have,
however, always been to me obscure, but I have attributed
it to my not being personally familiar with the anatomy of
the Selachii. Considering that this familiarity had been in
a measure acquired by my present work on the cranial
anatomy of Chlamydoselachus, I recently carefully re-read
Vetter’s descriptions, but I still found the particular parts
referred to neither precise nor clear. Tiesing’s (1895), Ruge’s
(1897), and Marion’s (1905) later works not helping to a
proper comprehension, I then had recourse to dissections of
such few specimens of the Selachii, other than Chlamydo-
selachus, as I had at my disposal. The result has been to
lead me to consider the particular parts referred to,, in the
several works above mentioned, incorrect, and it has also
unexpectedly led me to seriously question every one of the
several instances cited by Edgeworth (1911) in which one of
■these visceral-arch muscles of fishes is said to be innervated,

304

EDWARD PHELPS ALLIS.

in the adult, by the nerve of a segment of the body other
than that from which the muscle itself is developed.

The proof that Edgeworth offers that this change of
innervation has taken place in these muscles is : that certain
muscles that he finds in embryos are developed from certain
segments of the body ; that these muscles of embryos are
the homologues of certain muscles described by other authors
in the adults of the same fishes ; and that these latter muscles
of the adult are said to be innervated by nerves other than
those of the segments from which he (Edgeworth) finds the
muscles of embryos developed. If these several premises
were all correct, the important conclusion that Edgeworth
deduces from them would evidently also be, but it is equally
certain that there is the possibility of error in some one of
the premises. This seems not to have been given serious
consideration, and yet Edgeworth’s descriptions of t lie-
development of these muscles in the Selachii is markedly
different from Dohrn’s, to whose important work Edgeworth
makes no reference, and it is well known that the innervation
of these muscles in the adult has been frequently wrongly or
incompletely given. Furthermore, I now find that even the
descriptions of the muscles themselves in the adult Selachii
are, in certain respects, incorrect.

Certain anatomists hold that a muscle fibre is, from the-
earliest embryonic stages, connected with the central nervous
system by a protoplasmic strand, not yet demonstrable by
known microscopic methods, which represents a future fibio
of the motor nerve of the segment, or that a “ something ”
(Braus, 1905) else establishes that connection. Other anato-
mists claim that there is no such connection, and that the-
motor nerve grows outward, independently, from the central
nervous system, and in some unknown way finds its end organ.

According to the first of these two views a nerve should,
in normal conditions, innervate a muscle derived from the
myotome of its own segment and from that segment only.
According to the second view the nerve might, in slightly
changed, if not even in normal conditions, find its end organ

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 305

in a muscle derived from another segment ; and if this change
in innervation were then to be transmitted by inheritance, it
is claimed that it is fatal to the first-mentioned view (Johnston,
1906, p. 63). There would, however, seem to still be question
as to whether this inheritable mutation related to the directive
impulse assumed to reside in a nerve fibre, or to a proto-
plasmic strand, or a something else, that pre-existed and
determined the course of the nerve. The mutation might
evidently have related to the one or the other. Furthermore,
there is frequently question, in the cases cited of such a change
of innervation, as to whether the definitive innervation was not
in reality primary, being the only innervation that the parti-
cular fibres under consideration had ever had in ontogeny,
instead of being secondary in the sense of replacing an earlier
and normal innervation by the nerve of the segment to which
the muscle belongs.

My work, it may here be stated, [in no wa.y attempts to
solve this vexed and very complicated question. It does
however raise serious question as to several of the examples
that have heretofore been cited of the so-called secondary
innervation of a muscle, and it also quite unexpectedly adds
a series of instances in which there must be such an innerva-
tion if existing descriptions of the innervation are correct.

Before describing my own investigations, limited to a few
Selachii, it will be well to point out some of the inconsistencies
and contradictions in earlier descriptions of the development
and anatomy of the visceral-arch muscles in these fishes.

Branchial Arches.

Dohrn (1884, pp. 109-115) says that the myotome of each
of the branchial arches of selachians, meaning the Plagiostomi,
becomes flattened “ in the middle,” and is finally there
separated into two parts, and the descriptions and figures
both show that this flattening and subsequent separation
takes place antero-posteriorly along a dorso- ventral line
passing through the middle of the externo-internal depth of
the myotome. The myotome is thus here separated into

306

EDWARD PHELPS ALLIS.

deeper and superficial portions, which Dolirn calls respectively
the proximal and distal portions of the myotome, proximal
meaning nearer the pharyngeal cavity and distal farther from
that cavity. The separation of the myotome into these two
parts did not apparently extend, in the embryos examined by
Dohrn, the full dorso-ventral length of the myotome, for
Dohrn definitely says that the dorsal ends of the two portions
of the myotome remain attached by a thin intervening
portion. The complete separation said to be found in the
adult must accordingly take place in later embryonic, or
possibly in postembryonic stages.

The cartilaginous bar of the arch, when it first begins to
develop, is said by Dohrn to lie against the posterior surface
of the proximal portion of the myotome at about the middle
of its length, and the bar, as it develops, is more curved than
the myotome. The proximal portion of the myotome thus
stretches across the morphologically anterior but actually
lateral surface of the curved bar, projecting, in the middle
of its length, mesial to the bar, and there acquiring a position
internal instead of external to it. This middle section of the
proximal portion of the myotome is later cut out of the
myotome along the line where the myotome crosses the bar,
and from the portion so cut out the musculus adductor arcus
visceralis is said to be developed. The remainder of the
myotome is said to remain external to the branchial bar, and
from those parts of its proximal portion that lie dorsal and
ventral to the piece cut out to form the adductor, and hence
also dorsal and ventral to the curved branchial bar, the
musculi interarcualis and coracobrancliialis are said (loc.
cit., p. 115) to be respectively developed ; these two muscles
and the adductor thus being primarily continuous and forming
the entire length of the proximal portion of the myotome
(loc. cit., p. 117).

The dorsal and ventral ends of the myotome, including both
its proximal and its distal portions, are each said to bend
posteriorly across the dorsal or ventral edge, respectively, of
the next posterior gill-pouch (Kieinenspalte), and from that

VISCERAL AKCHES OF i HE GNATHOSTOME FISHES. 307

part of the distal portion of the myotome that lies between
these dorsal and ventral bends the musculus interbranch ial is
is said to be developed. The musculus interbranchialis, as
thus defined by Dohrn, is accordingdy that part of the distal
portion of the continuous myotome that lies in, and extends
the full length of, the related branchial diaphragm, that
diaphragm being limited dorsally and ventrally by the dorsal
and ventral edges of two adjoining gill-pouches. The inter-
branchialis of the adult Selachii, as defined by Vetter, lies
between the dorsal and ventral extrabranchials of the related
arch, and the distal ends of these extrabranchials lie, respec-
tively, ventral and dorsal to the dorsal and ventral edges of
the next posterior gill-pouch, as will be later fully described,
and as is imperfectly shown in Dohrn’s figures 1-4, PL 7.
The interbranchialis of embryos, as defined by Dohrn, is
accordingly not the same thing as the one defined by Vetter
in the adult. The difference is, in fact, morphologically
quite important, although it seems not to have been noticed
by Dohrn.

From the distal portion of that part of the myotome that
lies dorsal to the dorsal bend in the myotome, Dohrn says
(loc. cit. p. 113) that the musculus constrictor superficialis is
developed. What is developed from the distal portion of that
part of the myotome that lies ventral to the ventral bend is
not clearly stated. Dohrn says (loc. cit. p. 114) : “Wie an
der dorsalen Seite schlagt sich auch an der ventralen die
proximale Portion des Muskelschlauches um die Fortsetzu ng
des Knorpelbogens herum und bildet die tiefen Portionen des
Musculus constrictor superficialis (Vetter) ; diesen Namen
verdienen sie freilich nur cum grano salis, denn der Constrictor
superficialis sollte nur aus denjenigen Muskeln bestehen^
welche von den distalen Portionen der urspriinglichen
Muskelschlauche abstammen. In der That siud diese Muskeln
auch vorlianden, aber in der VettePschen Monographic falsch
gedeutet worden. Er beschreibt sie als einen Theil der M.
Coraoo-arcuales, unter dem Namen M. coraco-branchiales ; sie
haben aber urspriinglich nichts gemein mit M, coraco-

308

EDWARD PHELPS ALLIS.

hyoideus, setzen sich vielmehr nur an sie an, durch eine
Fascie von ihu getrennt. Der M. coraco-hyoideus ist ein
echter Korpermuskel, aus den Urwirbeln her stamm end, und
hat genetisch nichts mit der Visceralbogen-muskulatur zu
schaffen.”

The deeper portion of the constrictor superficial^ of Vetter's
descriptions, above referred to by Dohrn, is simply a bundle
of the proximal fibres of that muscle, and it is thus said by
Dohrn to be developed from the proximal portion of the
ventral end of the myotome of its arch. But Dohrn has
elsewhere definitely said, as just above stated, that this part
of the myotome of his embryos becomes the musculus coraco-
branchialis. These two muscles must then either have been
considered by Dohrn to be identical, or he overlooked the fact
that they were both said to be derived from the same part of
the myotome. The muscle developed from the remaining,
distal portion of the ventral end of the myotome is not
specifically named by Dohrn, and although he says that it
was wrongly called the coracobranchialis by Vetter, he never-
theless seems to refer to it in a later work (1885) by that
name, as will be later shown. How he came to the conclusion
that the coracobranchialis of Vetter’s descriptions of the adult
was developed from this part of the myotome, and that the
muscle was wrongly named by Vetter, is not apparent; but it
would seem as if it must have been because of Vetter’s figure
of Acanthias, which seems to show the ventral ends of the
constrictores superficiales wholly wanting excepting as they
are represented, in the first branchial arch, by a small bundle
of muscle fibres. Probably misled by this figure, and Vetter’s
descriptions of it, which give, as will be later shown, an
incorrect idea of the conditions, Dohrn seems to have con-
cluded that the ventral ends of the constrictores must also be
wanting in the adult Scyllium, and as he found, in his
embryos of that fish, a distal portion of the ventral end of the
myotome that had to be accounted for, he concluded that it
must be the coracobranchialis of Vetter’s descriptions of the
adult.

VISCERA!- ARCHES OF THE GNATHOSTOME FISHES. 309

But, whatever Dohrn may have considered to have been
developed from the distal portion of the ventral end of the
myotome of each branchial arch, it is certain that the
nmsculus interbranchialis was considered by him to be inter-
calated, in the embryos described by him, between dorsal and
ventral muscles, the dorsal one of which, alone, was the con-
strictor superficialis. This is, however, said by him (loc. cit.,
p. 144) to be a secondary condition, the constrictor superficialis
having certainly, in earlier phylogenetic stages, traversed the
related branchial diaphragm along with the interbranchialis ;
and it is here further said, in direct contradiction to the
statement made on p. 113 of his work and above referred to,
that the constrictor superficialis is in reality simply the distal
portion of the interbranchialis. How it was, or when, the
interbranchialis became separated from the portions of the
myotome dorsal and ventral to it, or that it ever became so
separated, is not said ; and as it is definitely said (loc. cit.,
p. 119) that none of the muscles of the arch are directly
inserted on any of the branchial rays, the extrabrancliials
expressly included, it is certain that these three parts of the
myotome were, in the embryos examined by Dohrn, simply
arbitrarily established regions of a single continuous muscle.

In the identification, in embryos, of the several muscles
above referred to, Dohrn makes frequent reference to Vetter’s
descriptions of the muscles in the adult Selacliii, the evident
inference being that, unless otherwise stated, the muscles
described by himself, in embryos, were to be considered to be
identified with the similarly named ones in Vetter’s descriptions
of the adult. To this Dohrn makes one exception, the coraco-
branchialis, which has been above referred to and explained.
No limitation or qualification of any kind is made by him in
his use of the term musculi interarcuales, and this term is
used, where it applies to the muscles of a single arch, both
in the plural (loc. cit., p. 113) and in the singular (loc. cit.,
p. 115). This muscle (or muscles) is said to extend from the
pliaryngobranchial of an arch to the corresponding element of
the next posterior arch, and also (“ resp.”) to the epibranchial

310

EDWARD PHELPS ALLIS.

of its own arch. Reference to Vetter’s figures then certainly
shows that Dohrn intended to include the musculus inter-
arcualis dorsalis I of Vetter’s descriptions in the muscles
termed interarcuales by himself, for that muscle is the only
one that extends from the pharyngobrancliial of one arch to
that of the next posterior arch. Fiirbringer (1897), however,
maintains that the interarcuales dorsales I were not intended
by Dohrn to be so included. Fiirbringer had previously
found (1895) that these musculi interarcuales dorsales I were
innervated by spinal or spino-occipital nerves, instead of, as
Vetter maintained, by branches of the related branch of the
nervus vagus, and he (Fiirbringer) proposed for them the
name musculi interbasales. In his later work Fiirbringer says
(1897, p. 406), in making reference to Dohrn’s work: aDer
Mm. interbasales thut er keine Erwahnung.” This is strictly
correct, but Dohrn also does not specifically mention either
the interarcuales II or III, and it is certain that if so careful
a worker as Dohrn had intended to exclude either of these
three muscles from the term interarcuales as employed by him
he would have definitely said so. This is all the more evident
from the fact that, without making reference to these musculi
interarcuales I, Dohrn himself says, in the work in question
(loc. cit., p. 117), that the musculus subspinalis of Vetter’s
descriptions, which is simply an anterior member of the
interarcuales dorsales 1 series (Allis, 1915), is probably derived
from trunk myotones.

Edgworth says (1911, p. 234) that, in 17 mm. embryos of
Scyllium, the lower end of each of the branchial myotonies
grows backward and becomes cut off from the remainder of
the myotome to form the coracobranchialis. The ventral end
of the remainder of the myotome is said to then grow ventrally,
external to the Anlagen of the several coracobranchiales, to
form the ventral end of the constrictor superficialis. It is
then said that : “ The upper ends of the myotonies, in embryos
between the lengths of 17 and 20 mm., increase in antero-
posterior extent, and, fusing together, extend backwards as
the trapezius to the shoulder-girdle. Below the Anlagen of

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 311

the trapezius each myotome forms a transversely broad plate
in the branchial septum. The part internal to the branchial
bar forms the adductor; the part external to the bar forms
dorsally the arcualis dorsalis, and below that the inter-
branchial, whilst the external edge forms the constrictor
superficialis.” The constrictor superficialis was accordingly
developed from that part only of the myotome that lay
primarily in the branchial diaphragm, and although a later,
ventral downgrowth of this muscle is said to take place, as
just above stated, no mention is made of any dorsal upgrowth
from that part of the myotome that lies ventral to the trapezius.
It is even definitely said (loc. cit., 251) that no levator
muscles are developed in the branchial arches of this fish.
Here the embryological conditions, thus described, do not
agree with the conditions found in a 42 cm. specimen of this
fish that I have examined, and which will be later fully
described, for in each branchial arch of this fish there is, as
in Heptanchus, a dorsal portion of the constrictor superficialis
which extends beyond the dorsal extrabranchial and overlaps
externally the musculus trapezius. In the hyal arch of
embryos of this fish Edgeworth himself describes this dorsal
prolongation of the constrictor, and he there not only calls it
the levator hyoidei, but says (loc. cit., p. 228) that it is
serially homologous with the levator muscles of the branchial
arches of the Teleostei. The levator hyoidei is a part of the
einuscl Csd2 of Tetter’s descriptions of the Selachii described
by him, and as this muscle, in Heptanchus, and in my
specimen of Scyllium, certainly has its serial homologues in
the dorsal ends of the constrictores of the branchial arches,
if the one is the homolpgue of the levators of the Teleostei
the others must evidently also be.

The term “ arcualis dorsalis” is said by Edgeworth (loc.
cit., p. 226) to be employed by him, as proposed by
Furbringer, to designate the iuterarcuales dorsalis II and III
of Tetter’s descriptions, one of which muscles is, however,
(Tetter, Furbringer) an interarcual and not an arcual muscle.
The interarcualis dorsalis I of Tetter’s description is called

312

EDWARD PHELPS ALLIS.

by Edgeworth, as by Fiirbringer, the interbasalis. This term
I shall also employ in the following descriptions and dis-
cussions, the other two muscles being called the arcualis and
the interarcualis. The term interbran chialis ” is said by
Edgeworth (loc. cit., p. 232) to be employed by him as
Vetter employed it, and it is said to lie wholly proximal to
the constrictor superficialis.

Dohrn does not specify to what particular fishes his
several observations apply, classing them all under the
general term “ selachians ” ; but the figures that accompany
his work are of Scyllium, Pristiurus, and Torpedo. Edgeworth
limits his observations on the Plagiostomi definitely to
Scyllium, but in his generalisations he considers them to
apply to all the Selachii, and apparently also to the Batoidei.
As both authors included Scyllium in their investigations it
is instructive to note certain marked differences in their
observations. According to Dohrn the dorsal end of each
branchial myotome gives origin both to a dorsal portion of
the constrictor superficialis and to the interarcuales dorsales
of the related arch. According to Edgeworth a dorsal
portion of the myotome of each arch is first cut off to form
the musculus trapezius, and it is from that portion of the
myotome that lies ventral to this dorsal portion that the
musculi constrictor superficialis and arcualis (interarcuales,
Dohrn) are developed. Dohrn accordingly entirely over-
looked the separation of the trapezius from the dorsal ends
of the several branchial myotonies, which would certainly be
a serious oversight on the part of so careful a worker.
According to Dohrn the interarcuales dorsales I are derived
from the branchial myotonies, while, according to Edgeworth,
they are of spinal origin. According to Dohrn the con-
strictor superficialis of each arch lies dorsal to the inter- •
bran chialis, or possibly both dorsal and ventral to that muscle,
and it is definitely said that it does not traverse the branchial
diaphragm. Accordiug to Edgeworth it is primarily limited to
the branchial diaphragm, there lying distal to the inter-
bran chialis ; it has no dorsal, levator prolongation and never

VISCERAL ARCHES OF THE GNATROSTOME FISHES. 313

acquires it, but it later acquires a ventral prolongation which
extends beyond the branchial diaphragm. According to
Dohrn the coracobranchialis is formed from the proximal
portion only of the ventral end of the myotome of each
arch, and the muscle so formed is not the coracobranchialis of
Vetter’s descriptions of the adults of other Selachii. Accord-
ing to Edgeworth the coracobranchialis is formed from the
entire ventral end of the myotome, and it is identical with
the muscle described by Vetter. Both authors maintain that
the coracobranchialis of Vetter’s descriptions is a muscle
of branchial, and not of spinal origin.

This comparison of these two embryological works, which
are the only ones I know of that pretend to give, in detail,
the development of these several 'muscles, thus certainly
shows that the published embryological investigations of
these muscles must be accepted with some reserve.

The descriptions of the adult may now be considered.

In the adult Heptanchus the constrictor superficialis of
each branchial arch is, as described by Vetter (1874),
practically a continuous muscle-sheet with a large angular
incisure in its proximal (actually anterior) edge. The dorsal
attachment, or origin, of the sheet is said to be partly in a
so-called dorsal superficial fascia, but mainly in thin tendinous
bands (Platten), which lie external to the musculus trapezium,
extend to the dorsal edge of that muscle, and represent the
greatly and progressively reduced posterior portion of the
superficial fascia above referred to (loc. cit., p. 431).
The ventral attachment, or insertion, of the sheet is mainly
in a mid-ventral fascia which lies external and ventral to the
ventral longitudinal or so-called hypobranchial spinal muscles.
The large angular incisure in the proximal edge of the sheet
is made by the articulating ends of the epibranchial and
ceratobranchial of the arch, and the ends of the muscle
fibres, on either side of this incisure, are inserted on tho.^e
two cartilages. The triangular piece so cut out of the con-
strictor forms the musculus adductor of the arch, but this

EDWAKD PHELPS ALLIS.

314

adductor muscle is much smaller than the incisure in the
edge of the constrictor.

The innermost (proximal) fibres of the dorsal portions of
the constrictores are said by Vetter to have their origins on
the inner ends of the “ ausseren Kiemenbogen,” that is, on
the dorsal extrabranchials. It is not said to which arch the
extrabranchial related to each muscle belongs, but the use
of the expression “ ausseren Kiemenbogen,” without qualifica-
tion, and the fact that there is no extrabranchial in the arch
posterior to the posterior constrictor (Fiirbringer, 1903, p. 432),
leads me to conclude that this origin of these fibres of each con-
strictor is on the dorsal extrabranchial of the arch to which
the constrictor belongs. In Heptanchus, the outer (distal)
halves of the dorsal extrabranchials are said, in a footnote
(loc. cit., p. 409), to be almost completely imbedded in the
fibres of the constrictores, and on a later page (loc. cit., p.
429) it is said that : “ Die sehr schwach ausgebildeten dorsalen
wie ventralen ausseren Kiemenbogen liegen, z. Th. in die
Muskeln selbst eingebettet, nahe deren obern und untern
Enden auf denselben.” This statement that the extrabranchials
lie “ on ” the constrictores is markedly indefinite, but as they
usually lie against the posterior surface of the constrictor of
the arch to which they themselves belong, and as Vetter says
that all of the branchial rays of this fish have that position,
one at first concludes that that must also be the position of
the extrabranchials. But in one of Vetter’s figures (loc.
cit., fig 1, PI. 14), six of these extrabranchials are shown
lying one on the anterior surface of each of the six con-
strictores of the branchial arches, and apparently slightly
imbedded in it.

This unusual position of these extrabranchials is one of
the points in Vetter’s description that I have never been able
to comprehend, and as I have two considerably dissected
heads of Heptanchus I have examined them with reference
to this. In each of these heads the outer (distal) end of the
dorsal extrabranchial of each branchial arch lies posterior
(internal) to the constrictor of the related arch, as it

VISCERAL ARCHKS OF THE GNATHOSTOME FISHES. 315

normally should. The outer ends of most of the branchial
rays of each arch, however, pierce the constrictor of their
arch, and from there onward lie against the anterior (external)
surface of that constrictor, imbedded in that surface and
covered by a thin sheet of connective tissue, which is strongly
attached to the anterior (external) surface of the muscle on
either side of the branchial ray. The muscle fibres pass
unbroken beneath (posterior to) this end of the branchial
ray, and none of them are inserted on it. There is no
slightest indication that the fibres have been cut in two,
and later grown together again. The conclusion is, there-
fore, inevitable that these branchial rays, in growing outward,
have pierced the constrictor, and so passed from its posterior
(internal) to its anterior (external) surface ; and it seems
probable that this is what happened with the extrabranchials
in the specimen of this fish examined by Vetter. It is,
however, singular that in my two specimens it should be
the branchial rays that so pierce the muscle, and not the
extrabranchials, while in Vetter’s specimen it was the extra-
branchials, and not the branchial rays. Vetter’s figure is,
in any event, misleading, if not actually incorrect, for no
part of the constrictor of any of the branchial arches is
shown lying anterior (external) to the related ex tr abranchial.

A deeper (proximal) bundle of the ventral portion of each
constrictor superficialis of Heptanchus is said by Vetter to
have its ventral attachment, called by Vetter its origin, on
a tendinous band related to the dorsal surface of the hypo-
branchial muscles. Running dorsally from there, this bundle
is said to either pass between two of the musculi coraco-
branchiales of his description, or to perforate one of those
muscles, and to be inserted on the ceratobranchial of its
arch. This bundle of fibres might accordingly be considered
to be a coracobranchialis, and Dohrn did actually so con-
sider it. No musculus interbran chialis is differentiated in
this fisli.

In Acanthias, as in Heptanchus, the proximal (anterior)
fibres of the constrictor superficialis of each branchial arch

316

EDWARD PHELPS ALLIS.

are said by Vetter to arise from the inner (proximal) end of
the dorsal extrabranchial, but it is here said that it is the
extrabranchial of the arch to which the constrictor belongs.
The next distal fibres are said to arise from a small and thin
tendon which perforates the musculus trapezius and the
dorsal trunk muscles to have its insertion on the vertebral
column, and the distal and larger part of the fibres to arise
from a narrow tendinous aponeurosis which extends dorso-
anteriorly from the top of the gill opening next posterior to the
muscle. Such a linear aponeurosis is found related to both the
dorsal and ventral ends of each of the first four gill openings,
and each pair of aponeuroses is said to unquestionably repre-
sent the lines where the dorsal and ventral portions of the
overlapping outer edge of a long and tall branchial dia-
phragm, such as is found in Heptanchus, has fused with the
anterior (external) surface of the next posterior branchial
diaphragm in order to form the small gill openings of
Acanthias. It is said that, as a natural consequence of this
method of formation of these aponeuroses, the extrabranchials
(ausseren Kiemenbogen) lie immediately beneath them, firmly
adherent to them. It is not definitely said to which arch the
extrabranchial related to a given aponeurosis belongs, but
it is said (loc. cit., p. 430) that each aponeurosis marks
the limit between the outer, visible, posterior portion of each
constrictor (Constr. super, s. str.) and the musculus inter-
brancliialis of the same arch, this latter muscle being covered
by the next anterior branchial diaphragm. It is accordingly
evident that Vetter considered the extrabranchial that under-
lies a given aponeurosis to belong to the posterior one of the
two branchial diaphragms that have fused to form the
aponeurosis.

Each of the linear aponeuroses of Acanthias is thus said
to be a persisting cicatrice formed along the line where two
adjoining branchial diaphragms have fused with each other,
and, that being the case, the cicatrice in each individual fish
must evidently have been formed during the life of that
particular fish, for that a so-formed cicatrice could have been

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 317

acquired, by inheritance, from an earlier form would probably
not have been maintained by Yetter. This cicatrice, important
as it is, does not involve the dermal tissues, nor are those
tissues even said to be adherent to it, as the extrabranchials
are said to be. This, in itself, is singular, as is also the fact
that whereas the distal fibres of each constrictor, in all the
Selachii, Acanthias included, always have, throughout their
entire course through the related branchial diaphragms, a
course parallel to the free edge of that diaphragm, they are
shown by Yetter running nearly at right angles into a line,
whether cicatrice or aponeurosis, that is said to represent a
part of the former free edge of that particular diaphragm.
Yetter has himself called attention to this, and has attempted
to explain it, but the explanation is not convincing. Further-
more, the conditions in specimens of Suyllium and Mustelus
that I have examined, and that will be later described, are so
decidedly opposed to this interpretation of the meaning ot"
the aponeuroses that I consider it wholly impossible that they
represent lines where adjoining branchial diaphragms have
fused with each other, and, in my opinion, the small external
gill openings of Acanthias are due simply to the retarded
development of the outer edge of a gill cleft as compared
with that of the inner edge of the same cleft.

From the several surfaces of origin above described by
Vetter, the fibres of each constrictor superficialis of Acanthias
run at first antero-ventrally, and the proximal (anterior) and
larger part of them are said to be inserted either entirely
(Yetter) on the next anterior aponeurosis, or partly also
(Marion, 1905) on the extrabranchial that underlies that
aponeurosis. This latter extrabranchial is, according to
Vetter’s descriptions (loc. c 1 1 . , p. 430), the one related to
the arch to which the muscle belongs. Hut there is then
confusion in the descriptions, for as Yetter has previously
said, as just above stated, that certain of the proximal fibres
of each constrictor have their origins on the extrabranchial
of the arch to which the muscle belongs, these fibres could
not have their insertions on that same extrabranchial. The
VOL. 62, PART 3. NEW SERIES. 23

318

EDWAllL) PHEU'S AELIS.

distal (posterior) fibres of the dorsal part of each constrictor,
misleadingly called by Vetter the “ untersten ” and by Marion
the “ ventral” ones, are said by both those authors to traverse
the branchial diaphragm and to be continuous with the cor-
responding fibres of the so-called ventral portion of the
muscle.

The distal and larger part of the fibres of the ventral por-
tion of the constrictor of each arch are said to have their
origins on the linear aponeurosis that extends ventro-anteriorly
from the ventral end of the next posterior gill opening. In
the first branchial arch, the remaining, proximal fibres of the
constrictor, here called by Vetter the “ untersten/’ and by
Marion the {! median ” fibres, are said to have their origins in
the mid-ventral line from the tendinous ventral surface of
the hypobrancliial muscles, and the corresponding fibres in the
second to the fourth arches to have their origins from a so-
called aponeurosis related to a fascia that lies dorsal to the
hypobranchial muscles and serves as surface of origin for
them. Running antero-dorsally, the proximal (anterior) and
larger portion of the fibres of each constrictor, including the
little proximal bundles above referred to, are all inserted on
the next anterior linear aponeurosis, while the remaining,
distal (posterior) fibres turn dorsally and are continuous with
the corresponding fibres of the dorsal portion of the muscle.
Excepting only the little bundle of proximal fibres in the
first branchial arch, the ventral ends of the constrictores
superfieiales of this fish thus only reach the dorsal or dorso-
lateral edge of the hypobranchial muscles, and comparison
with Heptanchus led Vetter to conclude that the ventral ends
of the constrictores of Acanthias had undergone marked
reduction. He calls especial attention to this, and says (loc.
cit., p. 441) that the disappearance of these ventral portions
of the constrictores of Acanthias is related to the great develop-
ment of the hypobranchial muscles, but he makes no mention
of what would seem to be a strictly similar disappearance of
the larger part of the corresponding dorsal fibres. This
assumed disappearance in Acanthias, and also in Scymnus, of

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 319

the ventral portion of each constrictor superficialis, is probably,
as already stated, what misled Dohrn in his interpretation of
the muscles in embryos of Scy Ilium.

Musculi interbranchiales, not found in Heptanchus, have
been differentiated in Acanthias. They are said to be found
in each of the first four branchial arches of the fish, but not
in the hyal arch. The muscle is said to form a thin muscle-
sheet which extends, in each arch, between the extrabranchials
and the inner cartilaginous bar of the arch, completely filling
the space between them. Marion says that “they are in no
sense superficial, nor circular in the same sense that the other
muscles are, and they lie in a different plane.” Huge (1897,
p. 219) says that they extend from the branchial bar of the
arch to the free edge of the related branchial diaphragm, and
form the middle part of a “ Muskel-Scheidewand,” thus lying
between dorsal and ventral portions of the muscles of the
arch and wholly separating them from each other. Ruge’s
conception of these muscles is thus totally different from
Vetter’s and Marion’s, but it agrees with DohnTs description
of the muscles in embryos. Vetter says that the interbran-
chialis of each arch lies close against the anterior (external)
surface of the branchial rays of the arch, and extends to the
outer ends of those rays, there passing insensibly, without
definite boundary, into that part of the constrictor superficialis
of the arch that traverses the branchial diaphragm. The
distal and larger part of the fibres of each interbranchialis
are said to arise, both dorsally and ventrally, in part from
the extrabranchial of the related arch and in part from the
linear aponeurosis that overlies that extrabranchial, while
the proximal (anterior) fibres arise, both dorsally and ventrally,
in part from a feeble ligament that extends from the extra-
branchial of the arch to the extrabranchial of the next anterior
arch, and in part from the latter extrabranchial.

In Mustelus, the proximal fibres of the constrictor super-
ficialis of each branchial arch are said by Tiesing (1895) to
arise from a dorsal fascia similar to that described by Vetter
in Heptanchus, while the distal (posterior) fibres arise, as in

320

EDWARD PHELPS ALLIS.

Acanthias, from a linear aponeurosis, called by Tiesing a
septum, said to be formed between it and the corresponding
muscle of the next posterior arch. Running antero-ventrally,
the proximal (anterior) fibres are said to be inserted on the
next anterior so-called septum, while the distal (posterior)
fibres traverse the branchial diaphragm and are continuous
with the fibres of the ventral constrictor superficialis. The
ventral constrictor superficialis of each arch is said to arise
from a median ventral superficial fascia, as in Heptanchus,
and, running antero-dorsally, to be continuous with the distal
fibres of the dorsal constrictor superficialis. No mention is
made of any fibres not continuous with those distal fibres of
the dorsal portion of the muscle, that large proximal portion
of the ventral constrictor that is found in the Selachii
described by Yetter thus not being accounted for in these
descriptions of Mustelus. It is said that no ventral septum
is found in this fish, Mustelus differing in this from Acanthias
and resembling Heptanchus. Ruge says that the fibres of
the ventral portions of the several constrictores all have a
parallel course, and, their edges being contiguous and inti-
mately bound to each other, form a single continuous muscle-
sheet.

The so-called septa of Mustelus are said by Tiesing (loc.
cit., p. 100) to be formed by the “ Yerwachsung derKiemen-
locher” between two adjoining arches, this agreeing with
Vetter’s conclusion. Ruge (1897, p. 225) also says that these
aponeuroses are found “an den Verwachsungsstellen tier
freien Rander der Kiemen-Scheidewande.” Tiesing says of
each septum that, “ nach innen und vorn zu befestigt es sieh
an dem betreffenden Kiemenbogen and schliesst den oberen
tiusseren Kiemenbogen ein.” But it is evidently impossible
that a septum, formed where the outer edge of a branchial
1 diaphragm has fused with the next posterior one, could be
attached to the inner branchial bar of either of those two
arches. Tiesing does not say to which arch the extrabran-
chiaT related to a particular septum belongs, but Ruge says
(loc. cit., p. 227) that the constrictor superficialis of the-

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 321

hyal arch of this fish is in part inserted on the extrabranchial
of the first branchial arch, which leads one to suppose that
that extrabranchial lies beneath the linear aponeurosis related
to the first gill opening.

The musculi interbranchiales of Mustelus are said by Tiesing
to be practically as described by Vetter in Acanthias.

These several descriptions of these visceral-arch muscles
thus, as already stated, do not give a clear and concise idea
of the conditions in these fishes, and I have accordingly, as
already stated, had dissections made of such specimens of the
Selachii as I have at my disposal, which specimens consist of
a single already partly dissected head of Triakis, a 42 cm.
specimen of Scyllium canicula, and a 43 cm. specimen of
Mustelus (species unknown) . The accompanying figures show
the muscles as found in the two last-named specimens. The
musculi constrictores superficiales, interbranchiales, and
coracobranchiales were alone particularly considered in the
dissections, but other muscles are also shown in the figures.
The constrictor superficialis of each arch will be considered
as a single continuous muscle, instead of as two separate
muscles, one dorsal and the other ventral. I retain the term
constrictor superficialis, but, as there is no constrictor pro-
fundus, it seems a needless distinction.

In my specimen of Scyllium (figs. 1-7), those portions of
the constrictores superficiales of the hyal and first four
branchial arches that lie dorsal to the gill openings appear
to form, in lateral view, a single continuous muscle-sheet.
Immediately dorsal to the gill openings the lines of separation
between adjoining constrictores are apparent, and, starting
from there, each constrictor can be easily lifted off the next
posterior one excepting at its dorso-posterior corner. At
that corner each constrictor is inserted on the external
surface of the next posterior one, but elsewhere its distal
edge simply overlaps and is closely applied to that muscle.
Ventral to the gill openings the constrictores are less closely
applied to each other, the lines of separation between

322

EDWARD PHELPS ALLIS.

adjoining muscles are there distinctly evident, and there is
no insertion, at any point, of one muscle on the next posterior
one. Both dorsal and ventral to the gill openings there are,
in each branchial arch, two or three long muscle strands
which start from the internal surface of each constrictor,
near the dorsal and ventral ends of the next posterior gill
opening, and running respectively dorsally and ventrally,
join the muscle strands of the next posterior contrictor.
They can, however, easily be lifted off that muscle, and
are accordingly not shown, as separate strands, in the
figures.

The fibres of the constrictores are everywhere grouped
into bundles, which in most places form lamellar bands which
occupy the entire thickness of the muscle. Where the muscles
are thin these flat bands become simple rounded strands, and
they can all be referred to, for convenience of description,
as strands of the muscles.

The dorsal edge of the hyal constrictor is nearly straight,
and reaches, or lies slightly ventral to, the latero-sensory
canal of the body. Its anterior half, approximately, lies
anterior to the musculus trapezius and external to the
anterior portion of the dorsal muscles of the trunk. Its
posterior half lies external to the musculus trapezius, and, at
its hind end, overlaps the constrictor of the first branchial
arch. The anterior portion of the muscle has its origin in
part on the tissues that surround the latero-sensory canal,
and in part, ventro-lateral to that canal, on a fascia that is
evidently the dorsal superficial fascia of Vetter^s descriptions
of Heptanchus, Acanthias, and Scymnus. The posterior
portion of the muscle has its origin mostly on the external
surfaces of the trapezius and the constrictor of the first
branchial arch, certain of the tendinous ends of the fibres
penetrating those muscles, but it has its origin also in part
on ventral prolongations of the tissues that surround the
latero-sensory canal of the body. The tissues that surround
the latter canal lie directly upon and are firmly attached to
the dorsal superficial fascia, that fascia lying directly upon

VISCERAL ARCHES OP THE GNATHOSTOME FISHES. 323

and being firmly attached to the external surface of the
dorsal trunk muscles, and as the line separating the dorsal
and ventral trunk muscles here lies considerably ventral to
the latero-sensory canal, and hence ventral to the dor>al
edge of the trapezius, the fascia lies internal to the latter
muscle as well as to the latero-sensory canal. Vetter says
that this fascia lies upon the external surface of the musculus
trapezius, and he so shows it in his figure of Heptanchus.
Tt lies internal to that muscle in my specimens of Scyllium.
There are however, in Scyllium, delicate tendinous lines
which lie on the external surface of the trapezius and extend
from the dorsal edge of the several constrictores to the tissues
that surround the latero-sensory canal, and they apparently
represent the tendinous bands (Platten) described and shown
by Vetter.

The dorsal edges of the branchial ■ constrictores are all
irregular, the most dorsal point of each constrictor lying
proximal (anterior) to the distal (posterior) edge of the
muscle. From this most dorsal point the dorsal edge of
each muscle descends anteriorly, crossing the external surface
of the trapezius, and, in the anterior arches, extending
ventro-anteriorly beyond the antero-ventral edge of that
muscle. Where they cross the trapezius the dorsal edges of
these constrictores are inserted on, or firmly attached to, that
muscle, certain of the tendinous ends of the muscles pene-
trating the trapezius. The distal fibres of each constrictor
are inserted, as already stated, on the external (anterior)
surface of the next posterior constrictor. The distal (posterior)
fibres of the fourth branchial constrictor cross the external
surface of the shoulder-girdle, and are inserted on the anterior
edge of that cartilage along with the fibres of the musculus
trapezius.

The dorsal portion of the apparently continuous muscle-
sheet that is exposed when the dermis is removed is thus not
at all a continuous sheet, and the dorsal edge of the sheet,
excepting in its hyal portion, is formed by the dorsal ends of
those muscle-strands, only, that lie in the distal portion of

EDWARD PHELPS ALLIS.

3?4

each constrictor. From this dorsal edge of the sheet delicate
tendinous lines cross the external surface of the trapezius
and extend to the dorsal edge of that .muscle, apparently
representing, as already stated, the tendinous hands there
described by Vetter in Heptanchus.

There are no linear aponeuroses, either dorsal or ventral,
related to the gill-openings, but the fibres of the proximal
(anterior) half of the hyal constrictor are interrupted, as in
many other Selachii, by an aponeurosis that lies approxi-
mately in the line of the middle line of the gill-openings.
This aponeurosis is attached anteriorly to the mandibular
cartilages, and covers the articulating ends of the hyal
cartilages.

The muscle strands in the proximal (anterior) portion of
the dorsal half of the hyal constrictor run antero-ventrally
and are inserted on the ventral half or two-thirds of the
hyomaudibula, the deeper fibres being shorter than the
superficial ones and having their origins on the dorso- lateral
edge of the chondrocranium. 'The proximal fibres of this
constrictor thus form a musculus levator hyomandibularis
with two heads of origin. The next distal (posterior) strands
of the constrictor are inserted in the aponeurosis, just above
described, that extends posteriorly from the articulating ends
of the mandibular cartilages. The distal (posterior) strands
traverse the branchial diaphragm of their arch and are
continuous from the dorsal to the ventral end of the muscle.
In the dorsal and middle parts of their lengths these distal
strands have a nearly dorso- ventral course. Ventrally they
spread posteriorly and extend nearly to the ventral end of
the shoulder-girdle, there lying external (ventral) to the
ventral end of the constrictor of the first branchial arch and
external also to the liypobranchial muscles. The strands,
excepting a few distal (posterior) ones, all reach the mid-
ventral line, and form, with the musculus intermandibularis,
a continuous superficial muscle-sheet extending to the sym-
physis of the mandibulse. In the posterior three-fifths of this
continuous sheet the strands of opposite sides are separated

VISCERAL ARCHES OE THE GNATHOSTOME FISHES. 825

by a median aponeurotic line. In the anterior two-fifths of
the sheet the strands of opposite sides are directly continuous
with each other.

A large bundle of the superficial fibres of the ventral
portion of the hyal constrictor, composed of a number of
muscle strands, separates from the deeper fibres and has its
origin on the articular end of the mandibula. Beneath this
bundle, and also for a certain distance anterior to it, the
deeper fibres of the constrictor have their origins on the
ceratohyal, and the so-formed musculus interhyoideus is
connected with the adductor mandibulseby a tendinous fascia
which passes internal to the large bundle of superficial fibres
and is apparently the homologue of the tendinous fascia
described by Vetter, in a similar but wholly superficial
position, in Acanthias. Internal to the dorsal edge of this
fascia the ligamentum mandibulo-hyoideum has its insertion
near the dorsal end of the ceratohyal. Anterior to the large
bundle of superficial fibres, the superficial fibres of the
continuous muscle-sheet all have their origins on the ventral
(morphologically posterior) edge of the mandibula, the line
of attachment of the muscle beginning immediately anterior
to the tendinous fascia just above described. The anterior
portion of this musculus intermandibularis is innervated by a
branch of the nervus mandibularis trigemini, and is hence of
mandibular origin. The posterior portion is innervated by
the nervus hyoideus facialis. The musculus interhyoideus
extends to the mid-ventral line and is wholly separate from
and independent of the superficial, intermandibularis layer of
the sheet. It extends anteriorly slightly beyond the anterior
end of the mid-ventral aponeurotic line that separates the
constrictor fibres of opposite sides from each other, and is
apparently not inserted in that aponeurosis.

The muscle strands of the constrictor of each of the
branchial arches all have an approximately dorso-ventral
course, and they lie, throughout much the larger part of
their length, in the related branchial diaphragm and upon
the anterior (external) surface of the next posterior gill-

326

EDWARD PHELPS ALLIS.

pouch, separated from it by the branchial and extrabran chial
rays of their arch. The fibres that form the distal edge of
the muscle, as they cross the dorsal and ventral edges of the
next posterior gill-pouch, are , strongly attached by connective
tissues to those edges, and ventral to the gill opening a
certain number of them unite to form a larger strand, which
then forms the distal edge of the muscle. By far the larger
part of the muscle strands of the dorsal half of the con-
trictor traverse the branchial septum, only a few of them,
one to three strands, being inserted on the epibranehial of
the related arch. In the ventral half of the muscle, on the
contrary, a considerable number of strands have their origins
from the related ceratobranchial, and the additional strands
having this origin apparently correspond to those fibres of
the dorsal half of the muscle that have been utilised to form
the musculi interarcuales dorsales II and III of Dohrn’s
descriptions, these muscles being represented in Scy Ilium by
a single muscle, the musculus arcualis dorsalis. A few strands
of the constrictor have their origins, dorsally, on the dorsal
extrabran chial of their arch, near the dorsal bend in the
extrabranchial, and a somewhat larger number of strands
are inserted, ventrally, on the ventral extrabranchial of the
arch, near its ventral bend. All of thejfibres of the muscle
that lie distal to those thus inserted on the extrabranchials
cross the external (anterior) surface of both the dorsal and
the ventral extrabranchials and, as shown in the figures,
are attached ventrally, by connective tissue, to the external
surface of the longitudinal hypobranchial muscles, none of
them reaching the mid-ventral line.

Proximal (anterior) to the fibres that have their origins on
the dorsal extrabranchial a few strands of the muscle in each
arch have their origins in loose connective tissues of the
region, and proximal to the fibres that are inserted on the
ventral extrabranchial quite a number of strands unite to
form a muscle bundle which, as just above stated, corresponds
to the musculus arcualis at the dorsal end of the arch. These
ventral strands have a different course and insertion in each

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 327

of the branchial arches. In the first branchial arch they
pass internal (dorsal) to the musculus coracohyoideus, between
it and the coracobranchialis I, and end, without reaching the
median line, attached to the muscles between which they lie.
They pass across the anterior edge of the ventral extra-
branchial of their arch at the point where that extrabranchial
bends posteriorly around the ventral edge of the next
posterior gill-pouch, and they are twisted upon themselves so
that their internal surface is presented externally. In the
second branchial arch these fibres form a flat band which
passes between the coracobranchiales I and II, and reaches
the median line. There it is inserted, with its fellow of the
opposite side, in a median aponeurosis which passes dorsally
between the coracobranchiales II of opposite sides and is
continuous with connective tissues that surround the truncus
arteriosus and the pericardial chamber. In the third branchial
arch the fibres separate into two bundles, one of which passes
between the coracobranchiales I and II, and the other between
the coracobranchiales II and III. The first bundle does not
reach the median line, but the second and larg-er one reaches
that line and is attached, with its fellow of the opposite side,
to connective tissues that are attached to the tissues surround-
ing the truncus arteriosus and pericardial chamber. Certain
of those fibres of this muscle that are inserted on the ventral
extrabranchial of their arch pass, with that extrabranchial,
between the coracobranchiales III and IY ; the ventral end
of this muscle thus being separated into three parts. In the
fourth arch the fibres here under consideration form a flat
band which passes dorsal to the coracobranchialis IY and is
inserted on the lateral wall of the pericardial chamber, these
fibres thus having the relation to the coracobranchiales that
the extrabranchial of the arch would have if it were present.
The proximal fibres of a constrictor thus tend to acquire a
position anterior to the coracobranchialis that is assigned, by
nomenclature, to its arch, the extrabranchial of the arch
lying posterior to that coracobranchialis. This tendency is
the more pronounced the more anterior the arch, and in the

328

EDWARD PHELPS ALLIS.

hyal arch the entire ventral end of the constrictor lies
anterior even to the coracornandibnlaris.

Most of the muscle strands of each constrictor lie every-
where anterior (external) to the extrabranchial and branchial
rays of their arch, but a few of them are, as above described,
inserted on the dorsal and ventral extrabranchials. An
angular piece has been cut out of the proximal edge of the
primitive constrictor by the articulating ends of the epi-
branchial and ceratobranchial of the arch, and the cut ends
of the muscle fibres are inserted on those cartilages ; the
angular piece so cut out forming the adductor of the arch, and
the dorsal portions of the cut fibres becoming the musculus
arcualis dorsalis.

The gill-pouches, with their enclosed branchial lamellae,
are thick, cushion-shaped structures. The external opening
of the pouch lies, in a state of repose, at the outer edge of
the posterior wall of the pouch, and it is smaller than the
interna], pharyngeal opening of the pouch. The dorsal and
ventral edges of the pouch are convex, the greatest height of
the pouch being in the line of the outer ends of the branchial
lamellae and not at the pharyngeal opening of the pouch.
The anterior wall of the pouch curves posteriorly over the
distal ends of the branchial lamellae, and the branchial rays,
lying against the anterior (external) surface of that wall, are
similarly curved at their outer (distal) ends. The constrictor
that lies anterior to a gill-pouch lies on the anterior surface
of these branchial rays, and, following the curve in the rays,
curves posteriorly over them, the distal portion of the muscle
lying in the plane of the external surface of the body, and
the proximal portion lying in a plane directed antero-mesially
at an angle of about 45° to that surface. The hyal con-
strictor is not so curved, there being no gill-pouch anterior
to it and the cartilaginous bar of the arch lying nearer the
external surface than do the bars of the branchial arches.

The posterior wall of each gill-pouch presses against the
anterior surface of the constrictor next posterior to it, and
forms a slight depression on that surface. The outer edge

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 829

of this depression corresponds to the outer edge of the pouch,
and not, as might have been expected, to the outer edges of
the branchial lamellae; the lamellae lying within the pouch
and not extending to its outer edges. The constrictor is
thinner in the depression than it is immediately beyond it.
This, and the insertion of certain of the fibres on the dorsal
and ventral extrabranchials of the arch, are the only indica-
tions of the differentiation of a musculus interbranchialis.

Dorsal extrabranchials are found in the hyal and first four
branchial arches, but ventral extrabranchials only in the first
three branchial arches. The dorsal extrabran chial of the
hyal arch is a small rod of cartilage lying near the outer,
distal ends of the branchial rays, and was not found by
either White (1896) or Fiirbringer (1903, p. 432) in the
specimens examined by them. The dorsal and ventral extra-
brachials of the branchial arches lie along the dorsal and
ventral edges, respectively, of the depression, just above
described, formed on the opposite side of the constrictor of
their arch by the pressure of the next anterior gill-pouch.
Each of the four dorsal extrabranchials, running dorso-
anteriorly from its distal end, reaches the curved dorsal edge
of the gill-pouch next posterior to it immediately proximal to the
highest point of that edge, and there turns sharply v en trail v
and but slightly anteriorly over that edge, and then expands
into a short spatula-shaped end (Fig. 7) which lies against
the posterior surface of the gill-pouch. The dorsal end of
each of these dorsal extrabranchials is thus crook-shaped,
the crook lying close to the dorsal edge of the constrictor of
the arch and embracing the dorsal edge of the next posterior
gill-pouch, that pouch there being firmly attached to the
extrabranchial by connective tissues. The dorsal half of
the flat spatula-shaped end of the first extrabranchial lies
against the external surface of the musculus trapezius, closely
attached to it by connective tissues, and its ventral half
against the external wall of the vena jugularis. The corre-
sponding ends of the other three dorsal extrabranchials lie
upon and are strongly attached to the musculus trapezius

330

EDWARD PHELPS ALLIS.

alone, the vena jugularis here lying along the antero-ventral
edge of the trapezius and partly internal to it.

Each ventral extrabranchial turns posteriorly, at its ventral
end, around the ventral edge of the next posterior gill-pouch
immediately proximal to the lowest point of that curved
edge., aud then spreads both anteriorly and posteriorly into
long pointed processes. The posterior process hooks around
the ventral edge of the next posterior gill-poucli and is
prolonged, along the posterior surface of that pouch, by a
line of fibrous tissue which lies in the line prolonged of the
shank of the next posterior extrabranchial, and is inserted
on that extrabranchial at the point where it bends pos-
teriorly around the ventral edge of the gill-pouch next
posterior to it. The anterior process of the extrabranchial
of the first arch runs antero-mesially between the coraco-
branchiales I and II, and nearly meets its fellow of the
opposite side in the median line, being separated from it
by the connective tissues that surround the truncus arte-
riosus. The anterior process of the extrabranchial of the
second arch is similarly related to the coracobranchiales II
and III and to its fellow of the opposite side, while the
anterior process of the extrabranchial of the third arch is
similarly related to the coracobranchiales III and IV, but is
separated from its fellow of the opposite side by a con-
siderable interval because of the intervening ventral edge of
the pericardial chamber. The antero-ventral ends of these
anterior processes of the extrabrancliialis approach somewhat
the inner branchial bars of their respective arches, especially
in the first arch, but they are not especially attached to
them by connective or ligamentous tissues. They do not
turn posteriorly toward the inner branchial bar of the next
posterior arch, and they are not connected with that bar by
special ligamentous or connective tissues.

The branchial constrictor that lies anterior to a given gill-
pouch, and the related branchial and extrabranchial rays,
are all quite strongly attached, by connective tissues, to the
anterior wall of that pouch, but the constrictor of the arch

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 331

is not so attached, in my specimens, to the posterior wall of
the next anterior pouch. The liyal constrictor and its related
branchial rays are but loosely attached to the anterior wall
of the first gill-pouch, this apparently being due to this
muscle being a thicker and stronger muscle than the branchial
ones.

On the external surface of each wall of each gill-pouch
there are tall and narrow U-shaped lines which mark the
lines of attachment of the branchial lamellae on the internal
surfaces of those walls. The loops on the posterior wall of
the pouch lie against the anterior surface of the constrictor
next posterior to the pouch, and, on that surface of that
constrictor, and extending from the outer (distal) ends of
the loops to the outer edge of the depression that lodges the
gill-pouch, there are, dorsal and ventral to the gill-openings,
several strands of a tissue that is largely fibrous, but that
shows, under the microscope, certain transverse striae. These
strands are radially disposed, as are the branchial lamellae;
they cross the fibres of the constrictor at right angles, and
they are closely attached to the anterior surface of that
muscle. Their position suggests both the supporting rods
of the branchial filaments in the Teleostomi and the radially
arranged muscles related to those rods (Allis, 1903), but it
seems improbable that they represent the beginnings of the
formation of either of them. On the posterior wall of each
gill-pouch, opposite the dorsal one of these two series of
fibro-muscular strands, there are one or two flat muscle
bauds which lie approximately parallel to the fibr.ous strands,
but closely attached to the wall of the pouch instead of !o
the anterior surface of the constrictor. Distally, these bands
pass over the outer edge of the pouch, close to the dorsal
end of its external opening, and, turning ventrally, join,
near its distal edge, the constrictor that lies along the
anterior wall of the pouch. Ventral to the gill-openings
similar strands are found, but they here lie upon the internal
(posterior) surface of the constrictor next anterior to the
puuch, close against the outer edge of the pouch, instead of

332

EDWARD PHELPS ALLIS.

on the posterior surface of that pouch (Fig. 3). The action
of these fibres, although feeble, must be to retract and
constrict the related gill-opening.

The dorsal and ventral edges of the gill-pouches are
wholly free, excepting where they are each attached, as
already described, to the dorsal extrabranchial of the next
anterior arch and to the internal surface of the constrictor
of that arch, and there are, as already stated, no linear
aponeuroses in any of the constrictores. It is accordingly
impossible that the dorsal edges of these pouches, in Scyllium,
have been formed by the partial fusion of the edges of a
taller gill-opening, as is maintained by Vetter, Tiesing, and
Ruge for the Selachii examined by them.

The coracomandibularis, coracohyoideus, and coraco-
branchialis I all have their origins on the musculus coraco-
arcualis communis, the other coracobranchiales having their
origins on the ventral end of the shoulder-girdle. The
coracomandibularis is inserted on the mandibula close to
the symphysis, the coracohyoideus on the anterior edge of the
ventral surface of the basihyal, the coracobranchialis I on the
dorsal surface of the postero-lateral process of the basihyal,
the coracobranchiales II-IV each on the hypobranchial of
the related arch, and the coracobranchialis V on the cerato-
brancliial of its arch. There are arcualis, but no interarcualis
muscles in this fish, each arcualis being a stout muscle,
evidently primarily continuous with the constrictor super-
ficialis of its arch, but lying ventral to the vena jugularis
instead of lateral to that vein. The primitive constrictor, in
growing dorsally, seems to have been split into two parts
when it encountered the vein, one passing ventral and the
other dorsal to it.

In the small specimen of Mustelus (PI. 22, figs. 8-10) the
constrictores superficiales of the hyal and branchial arches
together present, in lateral view, the appearance, ventral as
well as dorsal to the gill-openings, of a single continuous
muscle-sheet, the sheet being perforated by the first four gill-

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 333

openings and bounded posteriorly by the fifth opening. Both
dorsal and ventral to these gill-openings, the muscle strands
of the continuous sheet incline posteriorly at a marked angle
to the vertical line, this inclination being greater dorsal to
the openings than ventral to them, and greater for the
posterior strands of the sheet than for the anterior ones. A
narrow aponeurotic line extends dorso anteriorly from the
dorsal end of each of the four gill-openings that perforate
the sheet, but there are no corresponding ventral aponeuroses.
Most of the muscle strands on either side of the dorsal
aponeurotic lines are juxtaposed, and faint tendinous lines
cross the aponeuroses and connect them, this strongly sug-
gesting that the muscle strands were here primarily con-
tinuous, and that the aponeuroses are simply tendinous
formations, of secondary origin, that interrupt them. The
muscle-sheet, in situ, accordingly presents the appearance of
being formed by a series of contiguous constrictores, one
hyal and four branchial, fused by their adjoining edges both
dorsal and ventral to the gill-openings but separated from
each other as they pass between those openings. The sheet
is, however, not so formed, as will be immediately shown, but
this superficial appearance, as it applies to the ventral part
of the sheet, is doubtless what led Tiesing to say that, in this
fish, the entire ventral constrictor of each arch traverses the
related branchial diaphragm and is continuous with the dorsal
constrictor.

The anterior edge of the dorsal portion of the sheet is
considerably thickened, and this thickened portion is almost
completely differentiated, as in Scyllium, as a levator hyo-
mandibularis with two heads of origin, one arising along the
line of the latero-sensory canal of the body, continuous with
the fibres of the posterior portion of the sheet, and the other
from the dorso-lateral edge of the chondrocranium, as Tiesing
has already stated. Both portions are inserted, together, on
the ventral end of the hyomandibula.

If that part of the muscle-sheet that lies posterior to this
levator hyomandibularis be cut along its line of dorsal attach-

VOL. 63, PART 3. NEW SERI KS. 24

334

EDWARD PD ELDS ALLIS.

inent, or so-called origin, the entire sheet can be turned
downward a certain distance, disclosing portions of the dorsal
extrabranchials, the dorsal branchial rays of the hyal arch,
and the dorsal portions of the five branchial pouches. It is
then found that four thin sheets of muscle fibres still attach
the large muscle-sheet to underlying structures, and if these
thin sheets be cut the large muscle-sheet can be turned
downward to the middle line of the gill-openiugs, as shown
in PL 22, fig. 9. The entire distal portion of each doi>al
extrabranchial is then exposed, and it is seen that they each
lie against the anterior (external) surface of the gill-pouch
next posterior to the arch to which the extrabranchial
belongs, that this distal portion of each of the four branchial
dorsal extrabrauchials lies slightly posterior to and parallel
to the dorsal edge of the gill-poucli next anterior to its arch,
and that it extends ventro-posteriorly slightly beyond the
level of the dorsal edge of the external opening of the latter
pouch. The dorsal extrabranchial of the hyal arch is short,
and extends but a short distance along the anterior (external)
surface of the first gill-pouch.

The four linear aponeuroses of the large muscle-sheet are
now seen to extend entirely through the sheet and to lie one
external to each of the four branchial dorsal extrabranchials,
attached to them only by loose connective tissues, and com-
parison with Dohrn’s and Edgeworth’s descriptions of the
differentiation of the musculi adductores in embryos of these
fishes definitely shows what the aponeuroses are. According
to both those authors those primarily continuous muscle fibres
(strauds) of the constrictores that cross the inner branchial
bars of their respective arches acquire insertion on those bars
along the lines where they cross them, and, as a result of this,
the musculi adductores are cut out of the primarily long and
continuous fibres (strands) so concerned. But before that
insertion of these fibres (strands) was acquired it is evident
that the individual fibres concerned must first have become
tendinous along the lines where they were later to be cut in
two, this doubtless being due to the interruption of the

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 335

muscle substance of the fibres, because of pressure against
the branchial bars, and the consequent formation of a ten-
dinous membrane by the united sarcolemmae of the fibres so
interrupted. This same formation of a tendinous interval,
with later insertion, might evidently take place along the
lines where the muscle fibres of the primitive constrictores
crossed any other skeletal element, and in my specimen of
Mustelus it has quite certainly taken place where the fibres
of the continuous muscle-sheet crossed the dorsal extra-
branchials, the process, with these particular fibres, not being
carried to the point of section of the fibres with accompanying
insertion on the extrabranchials, while with other fibres this
section and insertion has taken place.

The distal end of each extrabranchial lies, as in Scyllium,
on the anterior (external) surface of the gill-pouch next
posterior to it, and when the dorsal edge of that gill-pouch
passes its highest point and turns antero-ventrally toward
the pharyngeal opening of the pouch, the extrabranchial also
curves antero-ventrally and lies along the edge of the pouch.
In each of the branchial ai’ches the proximal end of the extra-
branchial then there expands into a flat and thin plate which
projects ventrally along the posterior surface of the gill-
pouch, there lying either between the pouch and the vena
jugularis or between the pouch and the musculus trapezius, the
exact relation of each extrabranchial to the vena jugularis and
musculus trapezius not being traced. In the hyal arch there is
no plate-like expansion of the proximal end of the extrabran-
chial, this extrabriinchial being, as in Scyllium, simply a slender
rod of cartilage. In the second, third, and fourth branchial
arches each extrabranchial, at the point where it crosses the
dorsal edge of the related gill-pouch and curves antero-ven-
tfc-ally along that edge, is somewhat embedded in the musculus
trapezius, and it there gives insertion to what appear to be
superficial fibres of the trapezius, the number of these fibres
increasing progressively from the second to the fourth arches.
The fibres so inserted on each extrabranchial lie not only
parallel to the fibres of the musculus trapezius, but also in the

336

EDWARD PHELPS ALLIS.

lines prolonged of the fibres of the related portion of the
musculus interbranchialis of the arch to which the extra-
branchial belongs, the latter fibres being inserted on the
opposite, ventral, edge of the extrabranchial. In the fourth
branchial arch a considerable number of - the most proximal
fibres of the interbranchialis cross the anterior (external)
surface of the extrabranchial of their arch and join the fibres,
above described, that appear to form part of the musculus
trapezius, lying contiguous with them, along their anterior
edge, on the lateral surface of the trapezius. This definitely
shows that the fibres that are inserted on the dorsal edge of
the extrabranchial are not parts of the trapezius, as they
appear to be, but are the dorsal portions of certain muscle
strands of the constrictor that have been cut in two by
insertion on the extrabranchial, the part ventral to the extra-
branchial forming the musculus interbranchialis and the part
dorsal to the extrabranchial secondarily fusing with the
trapezius.

The musculus interbranchialis of each branchial arch has
its origin from that portion of the dorsal extrabranchial of
its arch that lies proximal to tbe point where it begins to
curve antero-ventrally along the dorsal edge of the next
posterior gill-pouch, and also from somewhat more than half
of that part of the same extrabranchial that lies postero-
ventral to the curve, and its dorsal edge is seen in fig. 9
lying between the extrabranchial and the dorsal edge of
the next anterior gill-pouch. The fibres distal to those
thus inserted on the extrabranchial of the arch form the
thin sheet of muscle fibres that had to be cut in order to
turn the large muscle-sheet of the constrictores superficiales
downward as shown in the figure. These distal fibres of
each interbranchialis, thus here apparently inserted on the
internal surface of the large muscle-sheet, must, in younger
specimens, have formed the interbranchial portion of long
and continuous muscle strands that passed over the anterior
(external) surface of the extrabranchial of their arch and
were continued, beyond that extrabranchial, to the dorsal

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 337

edge of the muscle. They formed the middle portion,
approximately, of each branchial constrictor, and as the
constrictores of this fish overlapped each other to a con-
siderable extent, they must have lain internal to certain
strands of the next anterior constrictor, and they and the
overlying fibres both became tendinous where they crossed
the extrabranchial. If, then, those portions of these deeper
fibres, or strands, that primarily lay dorsal to the extra-
branchial and the related secondarily formed aponeurosis
did not abort, they must still persist as a deeper component
of the large muscle-sheet.

Ventral to the gill-openings the conditions, aside from the
absence of linear aponeuroses, differ in minor details only
from those dorsal to the gill-openings. Here each musculus
interbranchialis, excepting only a few proximal fibres, is
inserted, its full length, on the ventral extrabranchial of
its arch, and in each of the branchial arches of my preserved
specimen there is a slight fold in the muscle just before it
reaches the extrabranchial ; the muscle passing, from above,
posterior (internal) to the extrabranchial, then turning
dorsally upon itself, and then again ventrally to its insertion
on the dorso-anterior edge of the extrabranchial (Fig. 10).
From the opposite, postero-ventral edge of the extrabranchial
a corresponding portion of the fibres of the constrictor of each
arch have their origins, and running ventro-posteriorly immedi-
ately join, on its internal surface, the continuous muscle-sheet
formed by the constrictores superficiales of the several arches.
These ventral fibres of the constrictor of each arch thus form
a component part of the large muscle-sheet, and they here lie
internal to certain fibres of the more anterior arches; but, after
crossing the next posterior extrabranchial, they lie between
those fibres and certain fibres of the next posterior arch,
internal to the ones and external to the others. Here there
can be no question as to the persistence of these ventral
portions of the fibres of each arch, for they are not here
interrupted by linear aponeuroses.

A few of the most proximal strands of the interbranchialis

338

EDWARD PHELPS ALLIS.

of each arch unite ventrallv to form a small bundle. This
bundle contracts to a small and pointed head, which, passing
anterior (external) to the ventral extrabranchial of its arch
and anterior to the musculus coracobranchialis of its arch,
between that muscle and the next anterior coracobranchialis,
is inserted on the dorsal surface of the hypobranchial muscles.
These bundles are always referred to, in all descriptions of
these muscles, as parts of the musculi interbranchiales, but it
is to be noted that they are in reality the ventral ends of
continuous dorso-ventral fibres of the constrictores, no inter-
branchiales having been cut out of these particular fibres by
insertions on the extrabrancliials. Convenience of descrip-
tion, however, requires that they be considered to form
parts of the interbranchiales. The more distal strands of
each musculus interbranchialis extend either from the dorsal
to the ventral extrabranchial of their arch, or from the
related dorsal linear aponeurosis to the ventral extrabranchial,
lying along the anterior (external) surface of the branchial
rays of the arch, between those rays and the posterior wall
of the next anterior gill-pouch. Certain of the branchial
rays, in certain of the arches, have cut through the muscle
in places, and there give insertion to the cut ends of the fibres.

Thecoracohyoideusand coracobranchialis I are both inserted
on the basihyal, the coracobranchiales IT, III, and IV each
mainly on the hypobranchial of the related arch, but also
partly, in arches II and III, on a small cartilage interpolated
between the hypobranchial and ceratobranchial, and corre-
sponding, in position, to the most dorsal one of the three
cartilages marked Hbr II in Furbringer’s figures of Torpedo
ocellata (1903, Fig. 21, PI. 17). In the fourth arch this
independent cartilage has either fused with the ventral end of
the ceratobranchial or has not separated from it, and the
coracobranchialis is accordingly there partly inserted on the
ceratobranchial. In the first branchial arch the cartilage is
found lying between the ventral end of the ceratobranchial of
that arch and the basihyal. The most anterior hypobranchial is
related to the second branchial arch, as shown in FurbringePs

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 339

several figures, and not to the first arch, as shown in Gegen-
baur’s (1872) figure of Galeus. The coracobranchiales I and II
arise from the dorsal surface of the hypobranchial muscles,
but the coracobranchiales III, IV, and V from the lateral
surface of the pericardial chamber.

An angular piece has been cut out of the proximal edge of
each constrictor, as in Scyllium, to form the musculus
adductor of the arch, and the cut ends of the fibres are
nserted on the related epibranchial and ceratobranchial.

In the large head of Triakis, the muscles were only super-
ficially examined. 'The constrictores superficiales here, as in
Mustelus, form a continuous muscle -sheet perforated by the
first four gill-openings, and there are dorsal aponeuroses,
but no ventral ones. The dorsal aponeuroses are not so
well developed as in my specimen of Mustelus, certain of
the superficial muscle strands of the constrictores crossing
the aponeuroses, and others there being simply pinched,
without being completely interrupted. The aponeuroses
here seem to have been in part formed by the invasion of
subdermal connective tissues, rather than by the interruption
of the muscle substance of the fibres. Where the fibres of
the hyal constrictor cross the extrabranchial of that arch
the fibres are also partially interrupted, and the beginning
of the formation of a linear aponeurosis is plainly evident ;
and here there is no invasion of connective tissues. That
part of each constrictor that lies dorsal to the dorsal extra-
branchial of its arch, excluding the first branchial arch,
joins, as in Mustelus, and even more markedly so, the mus-
culus trapezius.

Comparing the conditions in Scyllium, Mustelus, and
Triakis, as above described, with those described by Vetter
in Heptanchus, it is certain that the continuous muscle-
sheet formed by the constrictores superficiales in Mustelus
and Triakis has arisen by the fusion of the separate but
overlapping constrictores of Scyllium and Heptanchus. To

340

EDWARD PHELPS ALLIS.

what extent these overlapping and fused muscles have each
been preserved, or have aborted, is problematical; but it is
certain that the several sections of the continuous muscle-
sheet that are included between each two of the series of
dorsal aponeuroses each contains elements derived from at
least two adjoining arches. In my specimen of Mustelus
certain of the fibres of the constrictor of the hyal arch even
cross, in their dorso-posterior course, all of the dorsal extra-
branchials of the fish, including the somewhat rudimentary
extrabranch ial of the hyal arch. Those portions of the fibres
of the constrictores of Mustelus and Triakis that lie ventral
to the ventral extrabranchials are not interrupted by linear
aponeuroses, and there is hence no reason to suppose that
they there aborted, or even became tendinous. They must
simply have joined the overlying fibres of the continuous
muscle-sheet and persisted as part of it. There is, however,
no noticeable evidence of any thickening of the sheet at these
places. The muscle-sheet is, on the contrary, much thicker
in its anterior portion, where there is no overlapping of these
muscles, than in its posterior portion, where this overlapping
takes place.

This interpretation of the constrictores in these several
fishes, based wholly on anatomical investigation, finds un-
expected confirmation in Dohrn’s figures of sections of
embryos said to be of Scyllium canicula (1884, Figs. 1-4,
PI. 7). In those figures Dohrn shows the constrictores super-
ficiales overlapping each other to such an extent that three,
or even four, of them may be superimposed the one above
the others, and certain of them are even shown fused with
each other to form a single muscle-sheet. How sections of
a selachian, with the constrictor muscles arranged as described
by Dohrn, Vetter, and others, could be sectioned so as to
show these muscles in this relation to each other has heretofore
been to me incomprehensible, but sections of a fish in which
these muscles were as I have above described and interpreted
them in Mustelus could easily be sectioned to show them as
given in Dohrn’s figures. It is, however, to be particularly

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 341

noted that in my specimen of a small but adult Scyllium it
would be impossible to, cut a section that could show the
conditions given by Dohrn in embryos of that fish. The
constrictores superficiales of tliis fish are so nearly dorso-
ventral in position in their dorsal portions that no one of
them overlaps more than the next posterior muscle, the gill-
pouches elsewhere intervening and separating the muscles ;
and in their ventral portions the constrictores do not at any
point fuse with each other, as Dohrn definitely shows them
in his sections.

In the adult specimens of Acanthias and Scymnus that
were examined by Vetter and Marion, conditions strictly
similar to those above described in Mustelus and Triakis
must certainly have existed, but they were misinterpreted
by those authors. Minor differences, however, apparently
exist, as in Acanthias, where it would seem, from Vetter's
descriptions, as if the dorsal fibres of the constrictor of a
given arch, had largely aborted after they had crossed the
extrabranchial of the next posterior arch, had still more
largely aborted after crossing the extrabranchial next pos-
terior to that one, and had wholly aborted on reaching the
third posterior extrabranchial.

The constrictores superficiales of Mustelus, Triakis, Acan-
thias, and Scymnus, and of all other Selachii where the
conditions are similar, thus forming a continuous muscle-
sheet which is subdivided, by the transverse aponeuroses,
into what appear like several separate segments, the ques-
tion of the innervation of these several segments becomes
important. Vetter says that he could not satisfactorily
determine the innervation of these muscles in the branchial
arches either of Acanthias or Scymnus, but in the hyal arch
of those fishes he limits the distribution of the branches of
the nervus facialis strictly to the constrictor of that arch, as
that constrictor is defined by him. Tiesing says that, in
Mustelus, the branches of the nervi glossopharyngeus and
vagus are distributed only to those segments of the dorsal
portions of the constrictores of that fish that are included

EDWARD PHELPS ALLIS.

842

between the corresponding linear aponeuroses, while in the
ventral portions of the muscles, where there are no apo-
neuroses to interrupt the muscle fibres, those fibres are
innervated, their entire lengths, by the nerve of the related
arch. Ruge shows all those fibres of the hyal constrictor
that are not interrupted by the most anterior linear apo-
neurosis continuing onward to the dorsal edge of the con-
tinuous muscle-sheet, and his descriptions lead one to
conclude that these fibres are innervated, their full lengths,
by the nervus facialis, while the fibres interrupted by the
aponeurosis are only so innervated up to the aponeurosis.

If the innervations thus ascribed to these muscles by
Vetter, Tiesing, and Ruge are correct, and if my conclusions
regarding these muscles are also correct, the constrictores
superficiales of all fishes in which they are interrupted hy
linear aponeuroses thus unexpectedly present typical examples
of a muscle derived from one segment of the body being
innervated bv the nerve of another segment; and here, not only
would the innervation be definitely a secondary one, replacing
an earlier and normal innervation, but the change of inner-
vation would have taken place in definitely postembroyonic,
if not in practically adult, stages. Furthermore, the secon-
darily acquired innervation of different parts of the dorsal
strands, or fibres, of the constrictores of the hyal and first
branchial arches of Mnstelus would be by several different
nerves, while the ventral portions of those same strands, or
fibres, would be innervated by a single nerve. This certainly
seems improbable, if not impossible, and nntil Vetter’s,.
Tiesing’s, and Ruge’s statements have been properly con-
trolled, it seems proper to conclude that the innervations
given by them are incorrect, and that the muscle of each
arch retains its primitive and normal innervation. That
certain individual fibres of each constrictor are cut in two
where they are crossed by the linear aponeuroses is practically
certain, and in all such cases it is probable that that part
of each muscle fibre that thus lost its connection with its
motor nerve underwent paralysis and subsequent reduction.

VISCERAL ARCHES OF THE GNATHOSTOME FISBES. 343

or abortion ; this then in part accounting for the absence of
any undue or noticeable thickenings in the overlapping
portions of the muscles.

In the Batoidei, as in Acanthias and Scymnus, the con-
tinuous muscle-sheet, formed by the constrictores superficiales
is said to be separated by septa into separate muscle segments,
which are assigned one to each branchial arch and one to the
hyal arch ; but it is impossible to definitely determine, from
the descriptions, whether or not these so called septa of the
Batoidei are similar to the linear aponeuroses of the Selachii.
Dohrn’s figures (1884, PI. 7, figs. 5 and 6) of sections of
embryos of Torpedo would lead one to conclude that dorsal
to the gill-openings the conditions were as in Mnstelus, while
ventral to the opening the primitive constrictor of each arch
turned posteriorly and fused with the external surface of the
next posterior constrictor, not passing beyond the line of that
contact and fusion. The ventral septa, at least, of this fish
would then not be similar to those in the Selachii. There is,
however, certainly some error in the descriptions of these
fishes, for it is evident that the constrictor superficialis of a
given branchial arch could not lie anterior to the gill-opening
between that arch and the next anterior one, and yet that is
the position that these muscles have in both Tiesing’s (1895)
and Marion’s (1905) figures of these fishes.

The musculi trapezius, coracobranchiales, and adductores
arcuum branchialium may nowbe more particularly considered.

Of the trapezius Edgeworth says (1911, p. 257): “Levatores
arcuum branchialium are developed from the upper ends of
the branchial myotomes in Teleostomi, Ceratodus, and
Amphibia, but are not developed in Scyllium, Sauropsida,
rabbit aud pig. The method of development of the trapezius
— apparently a homologous muscle throughout the vertebrate
groups — is intimately related to these differences. It is
developed in Teleostomi and Amphibia from the fourth, in
Ceratodus from the fifth, levator, i. e. from the penultimate
or ultimate levator; whereas in Scyllium, Chrysemys, Gallus

344

EDWARD PHELPS ALLIS.

and rabbit, it is formed from the upper ends of the branchial
myotomes — five in Scyllium, four in Chrysemys, two in Gallus,
and three in the rabbit. In view of the facts that in Scyllium
the subspinalis and interbasales, developed from trunk-
myotomes, are attached to the pharyngobranchials, and that
the trapezius is innervated only by the Xlth — the most
posterior of the vagus roots — even though a constituent from
the glossopharyngeal (first branchial) segment takes part in
its formation, it is probable that the absence of levatores
and associated method of development of the trapezius in
Scyllium, Sauropsida, and rabbit are secondary phenomena,
and that the primitive condition is a series of levatores formed
from the uppermost portions of the branchial myotomes.”

The trapezius is thus said by Edgeworth to be a muscle that
is wholly of branchial origin but that varies greatly in the
branchial myotome or myotomes from which it is derived, and
in the adult Scyllium, at least, it is definitely stated that the
muscle is innervated by the eleventh nerve alone. It is also
elsewhere definitely stated (loc. cit.,p. 281) that muscles
derived from the trapezius are innervated in Lacerta by
spinal nerves alone, and in Gallus and the rabbit both by the
eleventh nerve and by spinal nerves ; and frequent references
to the trapezius being innervated by the eleventh nerve, or
by the eleventh spinal, leads one to conclude that Edgeworth
considered the muscle to be innervated by that nerve, or by
spinal nerves, in all vertebrates. The muscle is, accordingly,
one of those to which I made reference in the opening para-
graph of this paper as being said by Edgeworth to be inner-
vated by the nerve of a segment of the body other than that
from which the muscle is developed. The condition of this
muscle, as found in the adults of fishes, does not, however,
warrant this conclusion in so far as it applies to them.

In the adult Heptanchus certain fibres of the trapezius are
said by Vetter to be inserted on the branchial bar of the
most posterior, or seventh, branchial arch, the remaining
fibres of the muscle being inserted on the shoulder-girdle.
Between the seventh branchial bar and the shoulder-girdle

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 345

there is no gill-opening, and no musculus constrictor super-
ficialis is described in relation to this seventh arch.

In the adult Chlamydoselachus I find conditions strictly
similar to those in Heptanchus, excepting that in this fish
there are but six branchial bars and six gill-openings, the
sixth gill-opening lying anterior to the sixth branchial bar.
The distal, ventro-lateral end of the so-called epipharyngo-
branchial of the sixth arch lies close to the shoulder- girdle,
and that bundle of the trapezius that has its insertion on that
element of this arch extends nearly to its hind end. The
insertion of the trapezius on the shoulder- girdle begins
opposite the hind end of the sixth epipharyngobranchial,
and from there extends upward along the anterior edge of the
shoulder-girdle. The trapezius is overlapped externally by
the dorsal ends of all of the constrictores superficiales,
including the constrictor of the fifth branchial arch, and each
of the five branchial constrictores is similarly overlapped by
the next anterior constrictor, the muscles thus being, in this
respect, serially homologous.

In Acanthias and Scymnus the trapezius, as described by
Abetter, resembles that in Heptanchus and Chlamydoselachus
excepting in that its relations to the branchial bars are
modified by there being but five branchial arches in these
fishes, and in that the trapezius is here perforated by the
slender tendons that are said to alone represent the dorsal
portions of the four branchial constrictores superficiales. In
the adult Mustelus the trapezius is said by Tiesing to closely
resemble that in Acanthias, as described by Yetter, and
Tiesing makes no mention of any fibres of the constrictores
superficiales joining the trapezius, such as I find in my young-
specimen of this fish. In Chimasra (Yetter, 1878), there are
minor, and for my purpose, unimportant variations in the
muscle.

In Heptanchus and Chimsera, Aretter could not determine
the "innervation of the trapezius, but Fiirbringer (1897) has
since shown that in Heptanchus it is innervated by branches
of the nervus vagus. In Acanthias and Scymnus the muscle

346

EDWARD PdELPS ALLIS.

is said by Vetter to be innervated either by branches of the
nervus intestinalis vagi, or, possibly, by that nerve together
with delicate branches of other, more anterior portions of the
vagus. In Prion odo n glaucus, that anterior bundle of
the trapezius that has its insertion on the branchial bar of the
ultimate arch of the fish is said by Vetter (1878, p. 460) to
be innervated by a branch of the ri'ervus vagus quartus, the
remainder of the muscle being innervated by the nervus
intestinalis vagi. In Chlainydoselachus I find the muscle
innervated by a branch of the vagus that lies next posterior to
that branch of the nerve that is sent to the penultimate
branchial arch.

The anatomical evidence regarding this muscle in these
several fishes, taken by itself, would thus evidently lead to
the conclusion that the trapezius, in each of the fishes con-
sidered, is simply a differentiation of the constrictor super-
ficialis of the ultimate branchial arch of that particular fish —
the seventh in Heptanclius, the sixth in Chlainydoselachus,
and the fifth in Acanthias, Scynmus, and Mustelus — or,
possibly, of that arch and other more posterior arches if such
arches primarily existed and have successively disappeared
by reduction or transformation. This conclusion would then
differ from that arrived at by Vetter (1874, pp. 432-433)
only in that the trapezius is considered to be derived mostly,
or entirely, from the constrictor of the ultimate persisting
branchial arch instead of from the constrictor of a modified
and more posterior arch that is represented in the shoulder-
girdle. This derivation of the muscle would also explain, and
find confirmation in, DohriTs otherwise inexplicable failure to
find it developed from the dorsal ends of all of the branchial
myotonies, as Edgeworth maintains that it is developed; and
the partial fusion of the proximal fibres of the dorsal portions
of the constrictores superficiales of the second to the fourth
branchial arches with the trapezius, in my specimens of
Mustelus and Triakis, would probably explain how Edgeworth
came to consider the latter muscle to be developed from the
dorsal ends of all of the branchial myotonies. It is, however,

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 347

to be noted that this partial fusion of these fibres with the
trapezius is not found in my specimens of Scyllium, and it is
to embryos of this particular fish that Edgeworth’s descrip-
tions relate. This view of the development of the trapezius
from the constrictor of the ultimate branchial arch is also in
fullest accord with GreiTs very complete descriptions of its
development in Ceratodus.

In this latter fish, Ceratodus, a muscle called by Greil the
dorsoclavicularis is said by that author (1913, p. 1343) to
represent the phylogenetic beginning of a musculus tra-
pezius. This dorsoclavicularis is said by Greil (loc. cit.,p. 1139)
to be derived from a ventral process of the posterior half of
the second trunk myotome, which, forking over the fifth bran-
chial cleft, sends one process down anterior to that cleft and
the other posterior to it. The former process lies in the fourth
branchial arch, and from it is developed the axial mesoderm
of that arch. The posterior process is shown in GreiTs fig. 1,
plate 52, apparently lying posterior and partly internal to a
branchial pouch which is called, in the index lettering, the
u siebenten Schlundtasche,” that is, the sixth branchial pouch.
In figs. 3 and 4 of the same plate the process is shown lying
directly external to this sixth branchial pouch, close against
the posterior edge of the fifth branchial cleft. The sixth
branchial pouch never breaks through to the exterior.

The posterior fork of the ventral process of the second
trunk myotome of Ceratodus accordingly lies in the fifth
branchial arch alone, or both in that arch and the region
of a sixth branchial arch that never develops. The process
is said to separate into superficial and deeper portions. The
deeper portion grows inward, dorsal to the pericardium, and
forms a muscle which, although lying ventral to the pharyn-
geal cavity, is called the musculus dorsopharyngeus. This
large muscle is the exact serial homologue of a smaller
muscle, called the interbranchialis IV, which is developed
from the ventral end of the axial mesoderm of the fourth
branchial arch ; and both these muscles, lying dorsal to the
pericardial cavity and the tr uncus arteriosus, are serial

348

EDWARD PHELPS ALLIS.

homologues of the so-called musculi interbranchialis pos-
terior, interbranchialis anterior, and ceratohyoideus, which
are developed, respectively, from the ventral ends of the
axial mesoderm of the third, second, and first branchial
arches, but lie anterior to, and hence morphologically ventral
to, the truncus arteriosus.

The superficial portion of the posteiior fork of the ventral
process of the second trunk myotome grows ventrally
external and ventral to the pericardial cavity, and appa-
rently separates into three muscles, but there is some
confusion in the name given to them. One of them is
certainly the musculus clavicularis, or dorsoclavicularis,
which, as above stated, is said by Greil to represent the
phylogenetic beginning of a musculus trapezius (den ersten
phyletischen Anfang eines Trapezius holier stehendenFormen).
The second and third muscles are first called the dorso-
branchinlis and dorsohypobranchialis, but later the names
dorsobranchialis, dorsocleidobranchialis, coracocleidobran-
chialis, cleidobranchialis, and coracobranchialis are appa-
rently used either to designate those muscles themselves
or muscles derived from them.

The musculi dorsopharyngeus and dorsoclavicularis are
definitely said by Greil (loc. cit., p. 1249) to be innervated by
a branch of the nervus vagus given off close to the ramus
intestinalis vagi. The other muscles derived from the ventral
process of the second trunk myotome must then also, in
embryos, be innervated by the vagus, but I do not find
that this is so definitely stated. A muscle that Greil con-
sidered to be the homologue of the trapezius is, in any
event, said by him to be derived from the axial mesoderm
of the fifth (or fifth and sixth) branchial arch, and it is
innervated by a branch of the nervus vagus that has the
position, serially considered, of a nerve of that arch (or
arches) .

Froriep (Greil, 1907, Discussion) thinks that this origin
of the axial mesoderm of the fourth and fifth branchial
arches of Ceratodus from a trunk myotome needs confirma-

VISCERAL ARCHES OF THU GNATHOSTOME FISHES. 349

tion, and Edgeworth (loc.cit., p. 176) also questions it, but
as it is a question that relates primarily to the origin of the
mesoderm of these two arches, and involves that in the other
visceral arches also, the question of a secondary change in
the innervation of a muscle, as I am at present considering
it, is not involved.

Edgeworth (loc.cit., p. 243), in his own investigations,
finds the trapezius of Ceratodus “proliferated from the
outer side of the fifth levator ” arcus branchialis ; these two
muscles together thus strikingly recalling the trapezius of
the adult selachian. This origin of the muscle is thus in
accord with my contention that it is developed from the
primitive constrictor of the ultimate branchial arch of the
fish, and from that constrictor only, and that it accordingly
retains, in the adult, its normal and primitive innervation.

In the Tejeostomi the conditions are probably strictly
comparable to those in Ceratodus, but this cannot be
definitely established from the descriptions given. Edge-
worth says that the trapezius is developed, in all these
fishes, from the fourth levator arcus branchialis. In Amia
it is said (loc.cit., p. 239) to be represented by the fifth
external levator of my descriptions of that fish, a muscle that
I found innervated (Allis, 1897, p. 696) by a nerve that
arose either from the base of the post-trematic branch of the
third vagus nerve (nerve of the fourth branchial arch), or
from the main trunk of the vagus near the base of that vagus
nerve. In Acipenser the muscle is said by Edgeworth (loc.
c i t . , p. 236) to be found in 8^- mm, embryos* given off from the
fourth levator, but to be in process of disappearing in 11 mm.
embryos. In the adult it is said, on Vetter’s authority, to be
absent. The fifth levator of Vetter’s descriptions of this fish
is said to be developed from the fifth branchial myotome, and
although it persists in the adult, it is not considered by
Edgeworth to represent the trapezius, as it does in Amia.
This seems singular, for in Polyodon there is a well developed
trapezius (Danforth, 1913, p. 141), innervated by a branch
of the vagus, and but four levatores arcuum branchialium ;

VOL. 63, PART 3. — NEW SERIES. 25

350

EDWARD PHELPS ALLIS.

the trapezius thus apparently representing the fifth levator.
In Menidia, Herrick (1899, p. 117) found a trapezius inner-
vated by a branch of the vagus, and there are but four
levatores in that fish. In Scomber I found (Allis, 1903,
p. 207) five external levatores, the fifth one being inserted in
a membrane attached to the clavicle, and there is no trapezius
in this fish. In Trigla and Scorpaena I also found (Allis,
1909) five external levatores, and I now find that there is no
trapezius in either of these fishes. In Ameiurus there is a
trapezius innervated by a branch of the vagus (Herrick,
1901, p. 209), and the levatores of this fish are none of th m
inserted on the fifth branchial arch (McMurrich, 1884). In
Polypterus, Edgeworth (loc.cit., p. 241) finds a trapezius,
and there are but four levatores in this fish.

These several facts regarding these fishes, when compared
with Greil’s descriptions of Ceratodus, seem certainly to
warrant the conclusion that in the Teleostomi, as in Ceratodus,
the trapezius is developed from the fifth branchial myotome,
and that it always retains its normal and primitive innervation
by the nerve of that segment of the body.

The musculi coracobranchiales are said by Dohrn to be
developed, as already fully explained, from the deeper,
proximal fibres of the ventral portions of the constrictores
superficiales of the branchial arches and to be represented, in
the adult, by those fibres as described by Vetter. They are
accordingly said by Dohrn to be totally different muscle-s
from the coracobranchiales of Vetter’s descriptions of the
adult, which are said by Dohrn to be simply the distal fibres
of the ventral portions of the branchial constrictores super-
ficiales, misnamed coracobranchiales by Vetter. Edgeworth
says, as already explained, that the coracobranchiales are
developed from the entire ventral ends of the branchial
myotonies, and he considers the muscles so developed to be
identical with the coracobranchiales of Vetter’s descriptions
of the adult.

'Phe coracobranchialis of the first branchial arch of the

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 351

adult Heptanchus is said by Vetter to arise mainly from the
tendinous dorsal surface of the musculus coraeohyoideus ; the
coracobranchiales of the second to the fifth arches to arise
mainly from the dorsal surface of the musculus coracoarcualis
communis, but in part from a tendinous cord formed in the
mid-ventral line of a fascia that covers, ventrally, the peri-
cardial chamber ; the sixth coracobranchialis to arise in part
from the shoulder-girdle; and the seventh coracobranchialis
to arise entirely from the shoulder-girdle. Running forward,
these several muscles are all inserted mainly on the hypo-
branchial of the arch to which they are assigned, but certain
of the fibres of the muscle of the first arch are inserted on
the basihyal, and certain of the fibres of the muscles of the
second to the sixth arches, and all of the fibres of the muscle
to the seventh arch, on the ceratobranchial of the corre-
sponding arch. Although not so stated by Vetter, the
coracobranchiales must, because of these origins and inser-
tions, in a measure embrace the pericardial chamber, running
at first dorso-laterally and then dorso-mesially around it.
Deeper (proximal) and distal fibres of the ventral portions of
the const rictores snperficiales both coexist with the several
coracobranchiales.

In Acanthias and Scymnus the coracobranchiales, as
described by Vetter, seem to differ from those in Heptanchus
mainly in that they arise ventrally, in Acanthias, from the
outer edge of the fascia that covers the pericardial chamber
-and in Scymnus from the shoulder-girdle and a process of
the fifth ceratobranchial. Marion (1905) says that, in
Acanthias, the coracobranchialis is composed of five parts and
forms the lateral wall of the pericardial chamber. In both
-Scy Ilium and Mustelus, as I have already fully described,
musculi coracobranchiales coexist with both deeper (proximal)
-and distal fibres of the constrictores superficiales, and there
is every reason to believe that similar conditions are found in
both Acanthias and Scymnus.

In Chlamydoselachus I find the coracobranchiales of the
third to the sixth branchial arches all arising, as a single

352

EDWARD PHELPS ALLIS.

continuous muscle-sheet, from the lateral edges of a strong
median fascia which is attached posteriorly, On either side, to
the lateral edge of the ventral portion of the shoulder-girdle.
This fascia extends anteriorly, beyond the united ventral
ends of the shoulder-girdles, as a narrow median tendinous
band, and, lying close against the ventral surface of the peri-
cardial membrane, forms, with that membrane, the related
ventral portion of the wall of the pericardial chamber. The
fascia is apparently formed in large part by the tendons of
origin of the fibres of the muscle-sheet, and from there the
fibres run at first antero-dorso-laterally and then antero-
dorso-mesially, thus encircling and enclosing the pericardial
chamber and the truncus arteriosus. The musculi coracc-
branchiales of the third to the sixth branchial arches thus
have every appearance of having been developed in intimate
relations to the wall of the pericardial chamber, and of having
retained their relations to that wall. The first and second
coracobranchiales have become more or less independent of
the pericardial wall and its related fascia. In this fish, as in
Heptanclius, the deeper and distal portions of the ventral
ends of the constrictores superficiales both coexist with the
coracobranchiales.

The coracobranchiales of Chlamydoselachus are all inner-
vated, as they are said by Vetter to be in the fishes examined
by him, by a large nerve of spinal or spino-occipital origin,
the exact origin and composition of which I have not as yet
determined, this nerve also innervating the musculi coraco-
arcualis communis, coracohyoideus, and coracomandibularis.
This common innervation of these several muscles, their
practical continuity in the adult Heptanchus, and the fact
that, in Heptanchus, Chlamydoselachus, Scyllium, and
Mustelus, coracobranchiales, innervated by spinal or spino-
occipital nerves, coexist with both the deeper (proximal) and
the distal fibres of the ventral portions of the constrictores
superficiales, all of which latter muscles are innervated by
branchial nerves, give every reason to believe that both
Dohrn and Edgeworth have in some way misinterpreted the

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 353

muscles in embryos, and that the coracobranchiales of Vetter’s
descriptions are not each derived from the ventral end of the
corresponding branchial myotome, as they are said to be by
both those authors.

The coracobranchiales of these fishes must then either be
developed from trunk myotomes, as the eoracohyoideus and
coracomandibularis are said to be ; be developed, as in
Ceratodus (Greil), from the ventral end of the axial mesoderm
of the ultimate branchial arch, as will be later explained ; or,
possibly, be developed from some part of the coelomic wall.
Their innervation, as at present given, is decidedly against
the supposition that they are developed from the myotome of
the ultimate branchial arch, and in favour of their being-
developed from trunk myotonies, as the other hypobranchial
muscles are said to be. The conditions in Chlamydoselachus,
if this fish is as primitive a one as it is generally considered
to be, would favour their being developed from the coelomic
wall, and this derivation has been ascribed to them, in other
Selachii, by van Wijhe.

Van Wijhe says (1882 b, p. 16): “ Der Muse, coraco-

branchialis + coraco-mandibularis hat eine ganz andere
Entstehungsweise als der coraco-hyoideus. Er entwickelt
sich namlich aus der unpaaren vorderen Verlangerung des
Pericardiums, dessen Hohle, wie wir gesehen haben, im
Stadium J mit den Holden der Visceralbogen communicirt.
Nach dem Stadium K fangt diese vordere Verlangerung zu
obliteriren an ; die Zellen ihrer Wande werden Muskelfasern,
und im Anfang des 0 ist die ganze Hohle geschwunden ;
ihre muskelosen Wande sind zusammengekommen, undbilden
die Anlage des Muse, coraco-mandibularis -f coraco-bran-
chialis. In spaten Stadien ist derselbe immer leicht von dem
Muse, coraco-hyoideus zu unterscheiden. Die Nebenzweige,
welche ersterer zu den Visceralbogen abgeibt, sind aus den
Unterenden der Wande der Visceralbogenhohlen entstanden.”

The iC Nebenzweige ” above referred to by van Wijhe are
quite certainly simply the deeper, proximal fibres of the
ventral portions of the constrictores superficiales, which

854

EDWARD PHELPS ALLIS.

coexist, in Heptanchus, Scyllium, and Mustelus, with the
coracobranchiales and are inserted on them, but form no
part of them. The coracobranchiales would then not be
derived in any part from branchial myotonies, and their
primitive innervation would depend upon what nerve or
nerves innervated the parts of the pericardial wall from
which they were derived, and this might evidently be either
by branchial or postbranchial nerves. But if these muscles
and the coracomandibularis are both derived from cells of
similar origin, as van Wijhe states, and if the coraco-
mandibularis was primarily innervated by spinal or spino-
occipital nerves, the coracobranchiales must certainly also
have been so innervated, and even Edgeworth does not
question that the coracomandibularis was primarily as well
as actually innervated by those nerves. Edgeworth further-
more says (loc. cit., p. 178) that no muscles are directly formed
from the walls of the branchial portion of the cephalic
coelom, which, if correct, would indicate that the muscles
described by van Wijhe were developed in the postbranchial,
or spinal, region. The anatomical evidence is also all strongly
in favour of the similarity of origin of these muscles ascribed
to them by van Wijhe, and until the conflicting embryo-
logical evidence has been controlled it accordingly seems
proper to conclude that all the so-called hypobranchial
muscles are of similar origin, and that they were primarily,
as they are actually, innervated by spinal or spino-occipital
nerves.

In Chimaera coracobranchiales are said by Vetter (1878)
to be found, and to there closely resemble the muscles found
in the Selacliii. These muscles are thus found in all the
Elasmobranchii, and in all of these fishes they are said to be
innervated by spinal or spino-occipital nerves.

In the Teleostomi and Dipneusti, coracobranchiales have
been described as such in certain fishes, while, in other
fishes, muscles described under other names are said to be the
homologues of the coracobranchiales of the Elasmobranchii.

In Acipenser the coracobranchiales of the first three

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 355

branchial arches are said by Vetter (1878) to be repre-
sented by special tendons of the musculus coracoarcualis
anterior which are inserted, one each, on the hypobranchial
of the corresponding arch, the main tendon of the muscle
being inserted on the hypohyal. The coracobranchialis of
the fourth arch is said to be probably wanting. The coraco-
branchialis of the fifth arch is said to be represented by the
single tendon of the coracoarcualis posterior, which tendon is
inserted on a ligament which extends from the basibranchial
of the fourth arch to the ventral end of the rudimentary
branchial bar of the fifth arch. Furbringer (1897, p. 460)
found the tendon of the latter muscle separated into two
parts, one of which was inserted on the branchial bar of the
fourth arch and the other on that bar of the fifth arch.
Vetter says that all these muscles are innervated by spinal
nerves.

The coracobranchiales of the adult Acipenser are thus said
to be so completely fused with the coracoarcuales anterior and
posterior that they appear as simple tendons of those muscles,
and Edgeworth says (loc. cit., p. 235) that these tendons
are developed from downgrowths of the lower ends of the
first, second, third, and fifth branchial myotomes, while the
coracohyoideus, which is Vetter’s coracoarcualis anterior
together with the tendon inserted on the hypohyal, is said (loc .
cit., p. 268) to be of spinal origin. No downgrowth, giving
rise to a coracobranchialis, takes place in the fourth arch,
this arch thus forming, for some inexplicable reason, a marked
exception to the other arches.

This interpretation of these several muscles of Acipenser,
based by Vetter on anatomical and by Edgeworth on embryo-
logical investigations and considerations, may perhaps be the
correct one, but I strongly doubt it. Comparing the condi-
tions in this fish with those in Heptanchus, Scyllium, and,
Mustelus, as I have described them, it seems much more
probable that the so-called coracobranchiales of the first
three branchial arches of Acipenser are simply the homo-
logues of the proximal fibres of the ventral ends of the con-

356

KD WAR'D PHKLPS ALUS.

strictores superficiales of Heptanchus, Scyllium, and Mustelus,
and not the homologues of -the hypobranchial coracobran-
chiales of those fishes. The' musculus coracoarcualis posterior
of Acipenser, if its innervation by spinal nerves is correct,
might - be. the homologue of the coracobrancliiales of the
Selacliii, but this innervation needs confirmation.

In Ceratodus, coracobrancliiales are said by Edgeworth to
be developed from the ventral ends of the second, third, and
fifth branchial myotonies, but not from those ends of the first
and fourth myotonies. In later stages, still another coraco-
branchialis is said to be differentiated from the already
differentiated interarcualis ventralis of the first branchial
arch. Greil gives quite a different account of the origin of
these muscles. According to him (1913) there is but one
coracobranchialis on either side of the head of this fish, and
it is said, as already explained, to be developed from the
external one of two processes of the ventral end of the axial
mesoderm of the fifth branchial arch, that mesoderm being
derived from a ventral process of the second trunk myotome.
The muscle is said by Greil (loc. cit. , p. 1344) to grow forward
and separate into three or four heads which acquire insertions
on the ventral ends of the branchial bars. The ventral ends
of the axial mesoderms of the first to the fourth branchial
arches are said to develop, respectively, into the musculi
ceratohyoideus, interbianchialis anterior, interbranchialis
posterior, and interbranchialis IV, while from the deeper
one of the two processes from the ventral end of the axial
mesoderm of the fifth arch the dorsopharyngeus is said to be
developed ; all of these muscles being said to be serial homo-
logues one of the other and all wholly independent of the
coracobranchialis.

In Amia, Edgeworth says (loc. cit., p. 237) that only one
coracobranchialis is developed, the coracobranchialis V, and
this muscle, in 14 mm. embryos, is said to divide into the
pharyngoclaviculares externus and interims of the adult. In
the Teleostei the development of these muscles is not par-
ticularly described, but references made by Edgeworth to

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 357

those fishes make it certain that the conditions were there
considered by him to be similar to those in Amia. In Poly p-
terus senegalus the musculi pharyngoclaviculares are said
to be developed as in Amia, but from the ventral end of the
fourth instead of the fifth myotome.

Regarding the innervation of the coracobranchiales, Edge-
worth says (loc. cit., p. 253) : “ A. coraco-branchialis, or
pharyngoclavicularis externusandinternus, developed by back-
ward growth from the last branchial myotome, i. e. fourth in
Polypterus senegalus, fifth in Amia, Salmo, Menidia,
may either retain its original branchial innervation from the
tenth, e.g. Amia (Allis), Esox (Yetter), Menidia (Herrick),
Lepidosteus, Polypterus senegalus, or be innervated by
spino- occipital nerves, e.g. Amiurus (Wright), Salmo
(Harrison). When coraco-branchiales are developed from
all the branchial myotomes, they are innervated by the
spino-occipital nerves, e.g. Selachii (Yetter, Furbringer),
Acipenser (Yetter), Polypterus ? species (Furbringer), Cera-
todus (Furbringer) Y

Certain of the musculi coracobranchiales are thus, like the
trapezius, muscles said by Edgeworth to be innervated by the
nerve of a segment of the body other than that from which
the muscle is derived. The muscles said by him to be inner-
vated by the nervus vagus can be left out of account in this
respect, and the muscles in the Selachii, and the tendons that
are said to represent the muscles in Acipenser, have been
already considered. In Ceratodus, the dorsocleidobrancliialis
of GreiFs (1913) descriptions, from which the so-called coraco-
branchialis is said to be developed, is said by that author to
be innervated, in embryos, by the nervus vagus, and although
Furbringer (189?), who is quoted by Edgeworth, includes
this muscle of this fish in the hypocranial spinal muscles,
innervated by spino-occipital nerves, I cannot find that he
himself traced their innervation by branches of those nerves.
In Ameiurus, Herrick (1901, p. 209) says that the pharyngo-
claviculares are innervated by the vagus, and not by spinal
nerves as they were said to be by Wright, and I have con-

EDWARD PHELPS ALLIS.

858

trolled and confirmed this innervation by the vagus in
sections that I have of this fish. In sections of a 75 mm.
specimen of Polypterus senegalus I also find these
muscles innervated by a branch of the vagus and not by
spino-occipital nerves. In Polyodon, which is not cited by
Edgeworth, the pharyngoclaviculares are said by Danfortli
(1913) to be practically continuous, at their origin from the
shoulder-girdle, with the coracoarcualis,.and to be innervated,
as the latter muscle is, by spinal nerves ; and Danfortli adds :
“ I could trace no branches of the vagus into their upper
ends.” I however find, in a series of transverse sections of
a 141 mm. specimen of this fish, a branch of the vagus going
into the upper ends of these muscles and apparently inner-
vating them. The large spinal nerve that innervates the
hypobranchial muscles passes close to the ventral ends of the
pharyngoclaviculares, but no branch could be found entering
them. An artery that accompanies the large spinal nerve
leaves it and enters the pharyngoclaviculares.

The anatomical evidence regarding these muscles in the
Teleostomi and Dipneusti is thus, as in the case of the Elas-
mobranchii, against the view that they have undergone a
secondary change of innervation, but it is strongly in favour
of the view that there are, in these several fishes, two totally
different sets of muscles that have both been called coraco-
branchiales, one being of spinal or spino-occipital and the
other of branchial origin. The muscles of spinal or spino-
occipital origin are found in the Elasmobranchii, while those
of branchial origin are found in the Teleostomi and Dipneusti.
In the Teleostomi, with the possible exception of Polypterus
(Edgeworth), the muscles are derived from the ventral half of
the primitive constrictor superficialis of the ultimate branchial
arch, this portion of this constrictor thus being utilised, in
these fishes, for the secondary purpose of forming this muscle
just as the dorsal portion of this muscle has been utilised, in
the Elasmobranchii, for the secondary purpose of forming the
musculus trapezius. In the Elasmobranchii the muscles are
quite probably derived either from trunk myotonies or from the

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 359

walls of the coelomic cavity, and hence not from branchial
myotomes.

The development of these muscles in Ceratodus, as
described by Greil, may perhaps offer an explanation of this
difference of innervation and apparent derivation of these
muscles in these two large groups of fishes. The coraco-
branchiales of Ceratodus are said by that author to be
developed from a ventral process of the posterior half of the
second trunk myotome. From a similar ventral process of the
entire third trunk myotome a large muscle is said to be de-
veloped (1913, p. 1140) which acquires insertion on the hypo-
hyal and ceratohyal and hence is evidently a musculus
coracohyoideus, and from this muscle the coracomandibularis
is differentiated. Similar ventral processes of the fourth and
fifth trunk myotomes form the posterior portion of the
hypobranchial muscles, these portions evidently representing
the musculus coracoarcualis of the Selachii ; and these
muscles are all innervated by branches of a nerve formed by
the fusion of the nerves of the fourth and fifth trunk seg-
ments (myotomes). Of these nerves Greil says (loc. c i t .,
p. 1139) : “ Es besteht jedoch keine engere Zugeliorigkeit zn
den betreffenden Segmenten, jeder Nerv versorgt auch den
Myotomfortsatz des vorderen Segmentes, was sich schon
daraus ergiebt, dass der dritte Segmentalnerv in der Hegel
keinen ventralen Nerven an die hypobranchiale Musculatur
abgiebt.” Here it is said that the coracobranchiales and the
coracomandibularis + coracohyoideus are derived from ventral
processes of adjoining segments of the trunk which differ
only in that one of them becomes affiliated with the branchial
arches and acquires innervation by the vagus, while the other
retains its affiliation with the trunk myotomes and acquires
innervation by the nerve of the next posterior trunk seg-
ment. This change of innervation, based on embryological
evidence alone, I am always inclined to doubt, but it is to be
noted that if the process of the second trunk myotome had
retained its primitive relations to the other trunk myotomes,
instead of undergoing some sort of change because of its

360

EDWARD PHKLPS ALLIS.

affiliation with the branchial myotomes, the coracobranchiales
would have been innervated by a spino-occipital instead of by
a branchial nerve ; and this is possibly what has occurred in
the Selachii.

An adductor arcus branchialis is said by Vetter to be
found, in all the Selachii examined by him, in each of the
fully developed branchial arches, which would seem to
exclude the ultimate arch in each of these fishes, that arch
certainly not being fully developed. In Chimaera, Vetter
says that similar muscles are found in the first three
branchial arches, but that the corresponding muscles in the
fourth and fifth arches resemble the arcuales dorsales of the
Selachii rather than the adductores of those fishes. Tiesing
(1895) says that in Mustelus and the Batoidei there is an
adductor muscle in each arch, and I find an adductor in each
of the six arches of the one specimen of Chlamydoselachus
that I have examined for this purpose. Fiirbringer (1903,
p. 397) did not find an adductor in the first branchial arch of
his specimen of Chlamydoselachus. Vetter says that the
adductores in the Selachii, and also in Chimaera, are all
innervated by branches of the nervus vagus of the related
arch, but he does not give the course of those branches.
Tiesing gives the same innervation in the Selachii and
Batoidei examined by him, and he adds that the branch of the
vagus that innervates the muscle perforates, in each case,
the related epibranchial in order to reach the muscle. In
Chlamydoselachus 1 also find the nerve perforating the
related epibranchial, near its anterior edge. The muscle, in
all the Plagiostomi, and in the first three branchial arches of
Chimaera, arises from the internal surface of the epibranchial
of its arch and is inserted on the opposing, internal surface
of the ceratobranchial of the arch.

In the Teleostei, Vetter (1878) found no adductores arcuum
branchialium excepting in one large specimen of Esox, in
which specimen they are said to be represented by a few
scattered muscle fibres lying in connective tissue in the angle

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 361

between the epibranchial and ceratobranchial in each of the
first three branchial arches. These fibres being found only in
a particularly large specimen of this fish, does not favour the
view that they are persisting fibres of a muscle that is in
process of reduction ; for one would naturally expect to find
such a muscle relatively the more developed the younger the
fish. In Ameiurus, Wright (1885) did not find any of these
muscles, and they are said by Pollard (1892) not to be found
in Polypterus. In the Dipneusti, also, they are apparently
not found, for Fiirbringer (1904) makes no mention of them
in his descriptions of those fishes.

In Amia, I described (Allis, 1897) two adductores arcuum
branchialium, one related to the fourth aud the other to the
fifth branchial arch. The fourth adductor arises from the
internal surface of a posteriorly projecting process of the
fourth epibranchial, and is inserted on a similar process of
the fourth ceratobranchial, the muscle thus lying on the
posterior surface of the branchial bar. The fifth adductor
extends from the opposite side of the process of the fourth
ceratobranchial just above mentioned to the fifth cerato-
branchial, lies somewhat on the posterior surfaces of those
two cartilages, and is in part continuous, ventrally, with the
transversus ventralis posterior. The branch of the vagus
that innervates these muscles passes, in each case, over the
posterior edge of the related branchial bar.

In Polyodon, Danforth (1913) finds an adductor arcus
brauchialis in each of the first four branchial arches. Each
muscle arises from the flat posterior surface of the related
epibranchial, the surface of origin not approaching the margin
of the cartilage at any point, and the muscle is covered by a
tough aponeurotic sheet which binds it to the cartilage and
also serves as a secondary basis of origin. Running ventro-
laterally each muscle passes between the epibranchial and
ceratobranchial of its arch and is inserted on the anterior
surface of the latter cartilage. Branches of the nerrus vagus
of the related arch are sent to the muscle, passing, in each
case, over the posterior edge of the related epibranchial.

362

EDWAKD PHELPS ALLIS.

Fibres of the ramus post-trematicus internus of the next
posterior arch are said to also enter the muscle, but Danforth
could not determine whether they were motor or sensory nerves.

In Acipenser, Vetter (1878) found a small adductor in each
of the first three branchial arches.

Adductores arcuum branchialium are accordingly described
only in the Elasmobranchii and Ganoidei, and in these two
groups of fishes there is marked difference, not only in the
position of these muscles, but also in their manner of inner-
vation. The muscles in these two groups of fishes cannot,
then, be homologous if the innervation of muscles, and the
relations of nerves to skeletal structures, are as constant as I
consider them to be. That the nerves that innervate the
muscles in Amia and Polyodon have cut through the
related epibranchials, from their anterior to their pos-
terior surfaces, the perforation of the cartilage in the
Plagiostomi representing an intermediate stage in this
process, I, on principle, greatly doubt, and, furthermore,
DohriPs observations offer a different and more probable
explanation of the conditions in the latter fishes. According
to that author (1884, p. Ill), a concentration of mesoderm
cells takes place, at a certain stage in embryos of these
fishes, posterior to the proximal edge of the related myotome,
and soon afterwards a second concentration of similar cells
takes place anterior to the myotome. These two groups of
cells are said to represent the beginnings of the chondrifica-
tion of the branchial bar of the arch, but it is not said how or
when the two groups fuse. That part of the myotome of the
arch that lies between the two groups of cells is said to
later differentiate as the adductor of the arch, and it would
seem as if the nerve that innervates the muscle so differen-
tiated would of necessity lie between the two groups of ce Is,
and hence later perforate the branchial bar, and this seems
to find confirmation in conditions that I find in my 42 cm.
specimen of Scyllium. In this fish each musculus adductor lias
its insertion, at either end, in a pit in the related epibranchial
or ceratobranchial, and in each of the ceratobranchials this pit

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 303

m part perforates the cartilage, the muscle strands of the
adductor there being directly continuous with those of the
musculus interbran chialis of the arch. The adductor is thus
here not yet fully cut off from the primitive constrictor of its
arch, and if the epibranchial were similarly perforated in
younger stages it is certain that these particular strands
would be innervated by a nerve that traversed the perforation
of that cartilage.

If this be the explanation of the conditions in the Plagio-
stomi, it is quite certain that the conditions in the Ganoidei
were not derived from them. The conditions in these latter
fishes are associated with, and quite undoubtedly correlated
to, the presence of the straight form of branchial bar instead
of the sigma form, and to the absence of cartilaginous
branchial rays. Where these latter rays are found, the con-
strictor muscle of an arch could slip, or project, over the
anterior edge of the related branchial bar, as described by
Dohrn in plagiostoman embryos, and so give rise to an
adductor muscle, but it could not so slip over the posterior
edge of the bar. The ganoidean adductores could not,
accordingly, have been developed in a fish already possessed
of cartilaginous branchial rays, and it would even seem a< if
they could not have been developed in a fish already pos-
sessed of the cartilaginous or osseous rods that support the
branchial filaments in all the Teleostomi. These osseous rods
I have already described in Scomber (Allis, 1903), and I now
find similar supporting rods, of cartilage instead of bone, in
Amia, Polyodon, and Polypterus. In Amia they are not
evident until the fish is over 12 mm. in length. These rods
are found in two series, one along the anterior and the other
along the posterior edge of the branchial bar, and it would
seem as if a constrictor muscle, which must primarily have
occupied a position between their lines of attachment to the
branchial bar, could not, after their development, have
slipped over either edge of the bar. The Ganoidean adduc-
tores must accordingly have been differentiated before these
supporting rods were developed. In a specimen of Ceratodus

'364

EDWARD PHELPS ALLIS.

that has been long and not well preserved in alcohol, I do not
find any of these supporting rods, and descriptions of this fish
do not speak of them. There are nlso no adductores arcuum
branch ialium in this fish, but there are persisting remnants of
the constrictores superticiales, as already explained. In such
a fish as this, certain of the fibres of the constrictor of an arch
might slip over onto the posterior surface of its arch and so
give rise to the ganoidean adductor.

One other branchial muscle may here be mentioned, the
retractor arcuum branchialium, found in Amia, Lepidosteus,
and certain of the Teleostei, for this muscle is said bv Edsre-
worth to be developed from trunk myotonies and to later
acquire an innervation by a branch of the vagus. I have,
however, recently shown (Allis, 1915) that this muscle of the
Teleostei is quite certainly the homologue of a muscle, found
in Chlamydoselachus, which is simply a differentiation of the
* anterior end of the constrictor oesophagi. If I am right in
this conclusion, the innervation of the muscle of the Teleostei
is normal and primary, and not secondary.

From the embryological and anatomical facts above pre-
sented regarding the several muscles related to the branchial
arches, it seems quite certain that, in the gnathostome fishes,
the primitive condition of these muscles was, as Vetter long
ago concluded, a simple annular constrictor in each arch ; and,
to act as such a constrictor of the enclosed cavity, the muscle
must have been attached both dorsally and ventrally either to
some fixed structure or to its fellow of the opposite side. If
attached primarily, at either end, to the related branchial bar,
the muscle could not have acted as a constrictor.

The branchial bar, in this primitive condition, probably
lay directly internal to the constrictor muscle, DohriFs
assertion that it lies posterior to the proximal edge of the
myotome from which the muscle is developed probably
applying only to early stages in the Elasmobranchii. The
muscle and its relafed branchial bar probably lay primarily

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 365

in a plane perpendicular to the axis of the body, but this
plane later became inclined to that axis, the acute angle
lying posterior to the plane ; and still later it acquired, in the
Elasmobranchii, the well known sigma form. What impressed
this sigma form on these arches is not known, but it would
seem as if it must have been related to the relative lengths of
the pharyngeal cavity and the occipital portion of the chondro-
cranium. But, whatever the cause, this sigma form of arch
has definitely associated with it, in recent fishes, the presence
of cartilaginous branchial rays, of musculi constrictores super-
ficiales, and of musculi adductores arcuum branchialium of the
plagiostoman type; while associated with the other, or straight,
form of arch is the absence of the above cited features, the
presence of supporting rods in the branchial filaments and of
musculi levatores arcuum branchialium, and the occasional
presence of musculi adductores arcuum branchialium that are
innervated by nerves that pass over the posterior edge of the
branchial bar of the related arch.

In Ceratodus, it is probable (Allis, 1915) that there are
much reduced pharyngobranchials and that they are directed
postero-mesially, as they are in the Elasmobranchii, while the
hypobranchials are directed antero-mesially, as in the Teleos-
tomi ; and in this fish there are no adductores and apparently
no supporting rods to the branchial filaments, but there are
so-called musculi interbranchiales which are probably persist-
ing remnants of the plagiostoman constrictores superficiales.

This limitation of cartilaginous branchial rays or sup-
porting rods in the branchial filaments, together with certain
other associated and distinctive features, to the Elasmo-
branchii and Teleostomi respectively, and the probable
absence of both branchial rays and supporting rods in
Ceratodus, would seem to favour the view that the Teleostomi
were descended from a fish in which the cartilaginous
branchial rays had not yet been acquired. I have, however,
quite recently (Allis, 1915) concluded that the basal portions,
at least, of the cartilaginous extrabranchials are archaic struc-
tures, and that they are found, in modified form, either in the

VOL. 63, PART 3. — NEW SERIES. 26

366

EDWARD PHELPS ALLIS.

branchial arches, in the hyal and mandibular arches, or fused
with the neurocranium, not only in all living Teleostomi but
also in most, if not all, higher vertebrates. If this con-
clusion is correct, and if these extrabranchials are simply
modified branchial rays, as is generally accepted, then the
early ancestors of the Teleostomi must have possessed those
rays. But I have, since the publication of the paper above
referred to, found that Braus (1906) concludes, from con-
ditions found in embryos of Heptanchus, that the extra-
branchials belong to an independent category of skeletal
pieces. If this be so, it then seems probable that the early
ancestors of the Teleostomi possessed these particular carti-
lages, but not the ordinary branchial rays with which they
a, re usually associated.

The branchial muscles of the Selachii seem to be more primi-
tive than those of any other of the gnathostome fishes. When,
in the ancestor of the Selachii, the branchial arches acquired
positions oblique to the axis of the body, and later, or at the
same time, acquired the sigma form, the proximal edge of the
simple dorso-ventral Constrictor of each arch slipped, in the
middle of its length, over the anterior (actually lateral)
surface of the branchial bar of its arch, and the fibres of the
muscle, where they crossed the branchial bar, there first
became tendinous, by the interruption of their muscular
substance, and were then later cut through by acquiring'
insertion on the bar. A triangular piece was thus cut out of
the proximal edge of the muscle, and became the adductor
of the arch. The gill-pouch anterior to the arch, pressing
against the anterior surface of that part of the constrictor
that remained external to the branchial bar, first caused a
simple thinning of the muscle. The dorsal and ventral rays
of the branchial series were then modified, as extrabranchials,
in supporting relations to this thinned part of the muscle,
or these extrabranchials were otherwise and independently
developed for the same purpose, and at certain places in the
lines where the muscle passed over these cartilages, it again
became tendinous, or acquired insertion on the cartilages.

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 367

Two narrow and more or less extensive incisures were thus
made in the muscle, and that part of the muscle that lay
between these two incisures became the musculus inter-
branchialis. Where the muscle fibres were not thus cut
through, or did not become tendinous, the musculus inter-
branchialis was simply a thinner portion of the primitive and
continuous constrictor. That portion of the muscle that lay
distal to the extrabranchials remained intact, and formed the
continuous dorso-ventral fibres of the constrictor superficialis.
The dorsal and ventral ends of the constrictores had, in the
meantime, and in certain fishes, turned posteriorly, possibly
influenced by the sigma form of the branchial bars. The
arcual and interarcual muscles were then differentiated, and
this, together with the overlappings and fusions of the dorsal
and ventral portions of the constrictores superficiales with
each other and with the musculus trapezius, and the forma-
tion of tendinous aponeuroses where the muscle fibres
crossed the underlying extrabrancliials, produced the many
variations found in the adult. The constrictor of the ultimate
branchial arch was utilised to form the musculus trapezius.

In the Teleostomi, the straight form of arch was retained,
and correlated to this the constrictores superficiales did not
slip over the anterior edges of the related arches, but, in
•certain fishes, certain of them slipped over the posterior edge
of the related arch aud gave rise to the ganoidean adductors.
The constrictor of each branchial arch then apparently became
rudimentary in the middle of its length, doubtless because of
modifications in the branchial lamellae and the development
-of supporting branchial rods, but it was in part utilised to
form the delicate radial muscles related to the supporting
branchial rods. The dorsal and ventral portions of the primi-
tive constrictor then became the levatores, and the transversi
and obliqui dorsales and ventrales, and the ventral portion of
the constrictor of the ultimate arch became the coraco-
branchiales, or their homologues the pharyngoclaviculares.
The levator of the ultimate branchial arch became, in certain
•of these fishes, a musculus trapezius.

368

EDWARD PHELPS ALLIS.

Edgeworth comes to totally different conclusions regarding
the primitive condition and the later differentiations of these
muscles. He says (He. p. 259) : “The probable primitive
condition of each of the branchial myotomes was, from above
downwards, a levator, a marginalis, an interarcualis ventralis,
and (the lateral half of) a tranversus ventralis,” which would
seem to imply that there was, in this primitive condition as
conceived by him, no simple continuous constrictor extending
the full length of the arch. The interarcuales ventrales are
said by him to be muscles that extend between the ventral
ends of the branchial bars. The marginales are said (l.c_
p. 233) to be muscles found by Schultze in anuran larvae and
having their exact liomologues in what Edgeworth calls the
vertical muscles of Ceratodus. These vertical muscles of
Ceratodus are called by both Fiirbringer (1904) and G-reil
(1913) the interbranchiales, and they are said by Fiirbringer
to extend from the neurocranium to a process on the ventral
end of the ceratobranchial of the arch next anterior to the
one to which the interbranchialis belongs. It would,
accordingly seem as if these muscles must be remnants of
the primitive constrictor of the arch and not interbranchiales;.
and yet Edgeworth intercalates them, in each branchial arch
of the primitive vertebrate, between the musculi levator and'
interarcualis ventralis of that arch. Edgeworth (l.c. p. 178)
considers the branchial muscles in the Amphibia to repre-
sent the most primitive condition found in any vertebrate, and
he (l.c. p. 176) furthermore thinks it probable that there
were, in the primitive vertebrate, but two branchial arches, and
that where other arches are now found they have been,
subsequently added posterior to those two.

Hyal Arch.

Dohrn (1885) says that, in the hyal arch of selachians
(Plagiostomi), that proximal portion of the myotome (Muscu-
latur) out of which, in the branchial arches, the adductor is
developed is wanting, its formation having been wholly pre-

VISCERAL ARCHES OP THE GNATHOSTOME FISHES. 369

vented bv the one commissure formed, in this arch, in relation
to the efferent arteries. The musculi interarcuales are also
wanting in this arch, bnt it is said that in their place there is
a complicated system of ligaments. It is not said that these
ligaments are developed from any part of the myotome of the
arch, but this would seem to be implied, the ligaments then
representing the missing musculi interarcuales. In the
ventral part of the arch the muscles are said to be found,
undiminished in number, exactly as in the branchial arches.

The distal portion of the myotome (Musculatur) is said to
form the constrictor superficialis of the arch, which is richly
developed, especially in its ventral portion. Dorsally this
constrictor is said to turn posteriorly and fuse with the
corresponding portion of the muscle of the first branchial
arch. Ventrally, the distal portion of the constrictor is said
to fuse with a similarly named portion of a myotome (Muskel-
schlauches) which comes from the mandibular arch, tiie two
muscles, together, then running ventrally and fusing with
the fibres of the coracohyoideus and coracomandibularis
exactly as the “ other coracobranchiales ” do (in der Weise
der iirbigen M. coraco-branchiales). This expression evidently
affirms that the distal portions of the ventral ends of the
constrictores superficiales of the liyal and mandibular
arches represent the coracobranchiales of those arches, and
it would seem to imply that the coracobranchiales of the
branchial arches were derived from the corresponding
portions of the constrictores superficiales of their arches.
But as, as has already been fully explained, the coraco-
branchiales of DohriTs descriptions are said by him to be
developed from the proximal portions of the myotomes of
their respective arches, it must be that the coracobranchiales
here referred to are the muscles so named by Vetter, but
said by Dohrn to have been wrongly identified by him. What
becomes of the remaining, proximal fibres of the ventral
portion of the hyal myotome is not said, notwithstanding that
they have been said to exist exactly as in the branchial arches.
The descriptions are thus not clear, but it is important to note

370

EDWAKD PHELPS ALLIS.

that the ventral end of the constrictor of the hyal arch fuses
with a muscle developed from a corresponding portion of the
mandibular myotome.

Edgeworth says (1911, p. 206) that in 14 mm. embryos of
Scyllium, the ventral end of the hyal myotome becomes con*
tinuous with the lateral edge (morphologically the dorsal end)
of the future interhyoideus, this latter muscle being said to
be developed, as will be later explained, from the walls of the
ccelomic cavity and not from the hyal myotome. In 16 mm.
embryos the myotome is said to be partly continuous with the
interhyoideus and partly inserted on the lateral surface of the
hyal bar to form a levator hyomandibularis (levator hyoidei,
Edgeworth). In later stages the myotome becomes separated
from the interhyoideus, and the lateral edge (dorsal end) of
the latter muscle is inserted on the ceratohyal. A backward
extension of the myotome and the interhyoideus then takes
place, and a continuous dorso-ventral muscle-sheet is thus
formed, which lies posterior to the hyal bar, and is said to be
the muscle C3vd of Huge’s (1897) descriptions of the adult.
This dorso-ventral sheet is accordingly said to be formed, in
its dorsal portion, by fibres derived from the hyal myotome,
and in its ventral portion by fibres derived from the ccelomic
wall. The constrictor superficialis of the hyal arch is
accordingly not the strict serial homologue of the constric-
tores of the branchial arches, although this is not so stated
by Edgeworth. It is said that the primary form of the
interhyoideus, developed from the coelomic wall, would
appear to have been a transverse band connecting the two
hyal bars.

In the adult Selachii the muscles of this arch have been
described by Vetter, Tiesing, Ruge, and Marion, Ruge’s
descriptions being particularly complete. In this arch no
musculi interbranchialis, arcualis, or interarcualis are differen-
tiated ; but, according to Dolirn, the two latter muscles may
be represented by ligaments. A musculus interbasalis
(Interarcualis dorsalis I, Vetter) may be found extending
from this arch to the first branchial arch (Allis, 1915), but

VISCERAL ARCHES OF THE GNATHOSTOME FISH US. 371

this muscle is derived from trunk myotomes and not from
branchial ones.

In Heptanchus (Vetter), Hexanchus (Huge), and Chlamy-
doselachus the fibres of the constrictor superficial^ of the
hyal arch have a nearly dorso-ventral course, but in most
other Selachii that have been described the dorsal and ventral
ends of this constrictor are directed more or less posteriorly,
and it is probable that in all these latter fishes, as is certainly
the case in my specimen of Mustelus, the distal (posterior)
fibres cross, in their course, the extrabranchials of one or
more of the branchial arches. In certain of these fishes the
ventral fibres extend posteriorly nearly or quite to the ventral
end of the shoulder-girdle. The fibres of the muscle may
become tendinous where they cross the extrabranchials of
the branchial arches, particularly the dorsal extrabranchials.
They do not usually become tendinous where they cross the
extrabranchials of their own arch, nor are they inserted on
those extrabranchials, this doubtless being due to the absence
of an overlapping branchial diaphragm and gill-pouch, and
accounting for the absence of a musculus interbranchialis in
this arch. In the middle of the length of the constrictor,
opposite the hyomandibulo-ceratohyal articulation, the muscle
fibres are, probably in all Selachii, interrupted by a more or
less extensive aponeurosis. Dohrn describes this aponeurosis
even in embryos, but it is evident that the fibres must here
have been primarily continuous, and Ruge (1897, p. 224)
says that the conditions in the adult Hexanchus warrant this
conclusion.

In the proximal edge of the constrictor there is, as in the
branchial arches, a large angular incisure, and this incisure
is filled by the articulating ends of the epihyal and ceratohyal,
the cut ends of the fibres being inserted on those cartilages.
Comparison with the conditions in the branchial arches would
then seem to make it practically certain that a piece has here
been cut out of this hyal muscle, as it has been cut out of the
branchial muscles, and that the pieces so cut out of these
several muscles were all serial homologues. If this be so.

372

EDWARD PHELPS ALLIS.

some indication of the piece so cut out of the hyal muscle
should be found in some stage of development of these fishes.
According to Dohrn, it is not found in embryos, and he
further says that the conditions there are such as to preclude
the possibility of its development. There is, however, in the
adults of these fishes a large and important ligament, the
inferior postspiracular ligament, found in the hyal arch but
not in the branchial arches, and not accounted for in Dohrn’ s
descriptions of embryos.

In a recent work I suggested (Allis, 1915) that this inferior
postspiracular ligament of the Selachii was probably derived
from the musculus arcualis dorsalis of the hyal arch. I at
that time accepted the currently expressed opinion that an
adductor muscle was not differentiated in this arch, or that if
differentiated it had later completely aborted. My present
work leads me to doubt both these assumptions, and it now
seems to me much more probable that the ligament is derived
from the adductor of the arch than from the arcualis dorsalis.
The ligament is found in nearly all, if not in all, the Selachii,
and it is not found either in the Batoidei or the Teleostomi.
In the Teleostomi the plagiostoman adductores are not found
even in the branchial arches, as has been already fully
explained, this accounting for the absence of the ligament in
the hyal arch of these fishes; and the reason for its absence
in the Batoidei will be considered immediately below. In the
Selachii, the adductor, probably developed exactly as in the
branchial arches, ceased to be of functional value, doubtless
because of the intimate attachment of the cartilages of the
arch to those of the mandibular arch, and, travelling upward
along the epihyal until it reached and acquired insertion on
the chondrocraniuin, it became the inferior postspiracular
ligament. The relations of the ligament to the nerves,
arteries, and veins of the region, said by me to be in accord
with the derivation of the ligament from the arcualis dorsalis
of the arch, are equally in accord with its derivation from the
adductor of the arch, and, while the ligament might appa-
rently have been developed from either muscle, the derivation

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 373

from the adductor is much more in accord with the conditions
in the Batoidei.

In the Batoidei there is no inferior postspiracular ligament.
In these fishes the proximal portion of the myotome of the
hyal arch apparently passed over onto the anterior surface
of the cartilaginous bar of the arch exactly as in the branchial
arches, but, because of the marked change in the angle
between the epihyal and ceratohyal (see Parker, 1876, PI. 61,
fig. 4), and the separation of the epihyal from the pharyngo-
hyal, which latter element was utilised to form the hyo-
mandibula (Allis, 1915), the proximal portion of the myotome
here separated from the distal portion throughout its entire
length, and no small middle portion was cut out to form an
adductor. The proximal portion then acquired attachment
on the pharyngohyal (hyomandibula) ami gave rise to the
musculi levator and depressor hyomandibularis of Tiesing’s
(1895) descriptions, and possibly also to the depressor
mandibularis, which is said by Tiesing to be innervated by
the nervus facialis. The remaining, distal portion of the
myotome formed the constrictor superficialis. No adductor
muscle being differentiated in this arch in these fishes, an
inferior postspiracular ligament was naturally never de-
veloped.

Ruge says that, in the Selachii, the dorsal and ventral
portions of the hyal constrictor superficialis both Tend to
separate into superficial and deeper layers, the former
acquiring an insertion on the mandibular cartilages while the
latter retains its primary insertion on the hyal cartilages.
The insertion of certain of the fibres on the mandibular
cartilages he considers to be an ancient acquisition of these
fishes, and whenever it is wanting, in recent fishes, he con-
siders it to be due to retrogression. The nervus hyoideo-
mandibularis facialis is said to always lie external (anterior)
to that part of the hyal constrictor that is inserted on the
hyal cartilages, and to usually, but not always, lie internal
(posterior) to the fibres inserted on the mandibular cartilages.
In the region of the hyomandibulo-ceratohyal articulation,

374

EDWARD PHELPS ALLIS.

where the constrictor never separates into superficial and
deeper layers, the nerve apparently always lies on the external
(anterior) surface of the muscle.

In the several figures given by Ruge, the nervus facialis
is seen to lie internal to the dorsal portion of the hyal con-
strictor superficialis only in Heptanchus and possibly, in part,
in Spinax ; the nerve in the latter fish first lying on the
external surface of the muscle and then apparently piercing
it before it, the nerve, reaches the level of the hyomandibulo-
ceratohyal articulation. In all of the many excellent figures
of these fishes given by Luther (1909), the nerve lies internal
to this part of the constrictor only in Heptanchus, Hexanchus,
and Lamna. In the remnant of a head of Lamna that I have,
I find the anterior fibres of the proximal portion of this part
of the constrictor inserted on the palatoquadrate, but the
remaining proximal fibres inserted on the liyomandibula.
The nervus facialis lies internal to those fibres that are
inserted on the palatoquadrate, but, beyond those fibres, it
lies between the palatoquadrate and the hyomandibula, and
hence external to the fibres inserted on the latter cartilage.
In all the other Selachii figured by both Ruge and Luther,
the nervus facialis lies external to all the fibres of this portion
of the hyal constrictor.

In Heptanchus and Hexanchus the liyomandibula is
relatively slender and lies internal to the palatoquadrate
(Gfegenbaur, 1872). In Lamna I find the dorsal end of the hyo-
mandibula lying internal to the palatoquadrate. In all the
other Selachii figured by Ruge and Luther, the dorsal end of
the hyomandibula, so far as I can determine from existing de-
scriptions at my disposal, lies posterior to the palatoquadrate
and separated from it by a considerable interval, as shown in
Gregenbaur’s figures of Mustelus, Scymnus, Centrophorus,
and Heterodontus. This, then, probably gives an explanation
of the differing relations of the nervus facialis to the dorsal
portion of the hyal constrictor. Where the dorsal end of the
hyomandibula lies internal and close to the palatoquadrate,
the nervus facialis also lies internal and close to the

VISCERAL ARCHES OF THE G N AT H 0 s TO M E FISHES. 375

latter cartilage. The fibres of the hyal constrictor, all
primarily inserted on the hyomandibula, were then overlapped
externally by the palatoquadrate, and the dorso-posterior
edge of the latter cartilage lay posterior (distal) to the
nervns facialis. The superficial fibres of the hyal constrictor
then acquired insertion on the palatoquadrate along the line
where the dorso-posterior edge of that cartilage crossed them,
and so acquired a position external to the nervus facialis.
Other, deeper fibres of the muscle then followed and joined
the superficial ones. Where the dorsal end of the hyo-
mandibula lay at a considerable distance from the palato-
quadrate, the fibres of the constrictor simply pushed bodily
forward, carrying the nervus facialis with them, and so
retained their primitive position internal (posterior) to that
nerve.

In the Holostei and Teleostei the conditions are here quite
different from those in the Selachii. In the former fishes,
the epihyal does not acquire articulation with the neuro-
cranium; the posterior articular head of the hyomandibula
quite certainly being formed by the fusion of the supra-
pharyngobranchial of the arch, derived from the basal
portion of the extrabranchial of the arch, with the epihyal
(Allis, 1915). That part of the constrictor superficialis that
lay dorsal to the suprapharyngobranchial (extrabranchial)
must then have been cut off from the ventral portion of the
constrictor, and, lying between the suprapharyngobranchial
and the cranial wall, it became modified to form the inusculi
adductor hyomandibularis and levator and adductor operculi.
These three muscles of the Holostei and Teleostei are,
accordingly, together, the serial homologue of the levatores
arcuum branchalium in their own branchial arches, and
the homologue of the dorsal portion of the hyal constrictor
superficialis of the Selachii. The branch of the nervus
facialis that innervated these hyal muscles, lying primarily on
the anterior (external) surface of the constrictor of the arch,
would naturally have followed the muscles, and so come to
lie internal to the dorsal end of the hyomandibula. The

•376

EDWARD PHELPS ALLIS.

muscles would naturally retain tliei r primitive relations to
the vena jugularis, and when they acquired, by their dorsal
ends, insertion on the neurocranium, that insertion would be
dorsal to the vein; and such I find to be the relations of the
muscles to the vein in Amia, Lepidosteus, Polypterus,
Polyodon, and several Teleostei that I have examined for
this special purpose, with the single exception of Ameiurus.
In Ameiurus the vein passes over the posterior edge of the
adductor hyomandibularis, and then lies dorsal (external) to
that muscle, Ameiurus thus being exceptional in this as also
in several other cranial features (Allis, 1915, p. 566).

Vetter (1878, pp. 532-534), also, considered that these
muscles of the Teleostei were derived from what corresponds
to the dorsal portion of the constrictor superficialis of the
hyal arch of the Selachii, but he said it was difficult to con-
ceive the intermediate stages in such an extraordinary change
of position. The development of the hyomandibula in the
manner that I have suggested wholly removes this difficulty.
The levatores arcuum branchialium were considered by
Vetter to represent remnants of the musculi interbranchiales
of the Selachii, this conclusion being evidently based on the
assumption that the constrictores superficiales of the Selachii
had entirely disappeared in the Teleostei, as he had pre-
viously concluded that they had disappeared in Chimasra and
Acipenser. •

In an earlier work I said (Allis, 1897, p. 751) that : “ The
adductor hyomandibularis is probably developed from a
muscle comparable to one or more of the interarcual muscles
of the branchial arches of selachians, and is thus homodyn-
amous with the levators of the branchial arches of teleostomes,
and not with the adductor inandibulae. The adductor operculi
and levator operculi, at least the latter, are derived from the
interbrancbial muscles of their arch, and are thus homodyn-
amous with the levator arcus palatini, and not with the
levator muscles of the branchial arches. ” These conclusions
were based on my interpretation of Vetter’s descriptions of
the Selachii, and on the acceptance of his conclusion that the

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 377

constrictores superficiales had entirely disappeared in the
Teleostei, but as, as I have fully explained in preceding pa ges,
Vetter’s descriptions of these muscles are not wholly correct,,
my deductions from them were also not wholly correct. In a
recent work (Allis, 1915), still influenced by Vetter’s descrip-
tions, I suggested that the adductor hyomandibularis of the
Teleostei might be the homologue of the inferior postspiracular
ligament of the Selachii ; but as the adductor hyomandibularis
of the Teleostomi would then be the serial homologue of tlio
adductores arcuum branchialium of the Selachii, this cannot
be if my present conclusions are correct.

Edgeworth (l.c., p. 210) says that the retractor hyo-
mandibularis of Acipenser, and the adductor hyomandibularis
of Lepidosteus, Ami a, and Sal mo, are all derived from the
anterior portion of the constrictor superficialis of the liyal
arch of the Selachii, and that the musculus opercularis of
Acipenser and Lepidosteus, and the adductor and levator
operculi of Amia and Salmo, are derived from the posterior
portion of that constrictor of the Selachii ; which is in accord
with my present conclusions. The levatores arcuum branchi-
alium of the Teleostei are said by Edgeworth to be developed
from the upper ends of the branchial myotomes, which i&
evidently correct, but he then further says that, because of
this origin, these muscles of the 'Teleostei have no counter-
parts in the Selachii, unless it be in the musculus trapezius as
described by him, which I consider incorrect.

The proximal (anterior) fibres of the ventral portion of the
constrictor superficialis of the hyal arch must, primarily,,
have nil been inserted on the ceratohyal, and, in the Selachii,
they became connected with their fellows of the opposite
side by a median ventral aponeurosis, and so formed a musculus
interhyoideus which extended from one hyal arch to the
other across the ventral surface of the head. But a more or
less important portion of the fibres later here acquired, as in
the dorsal portion of the constrictor, a secondary insertion on
the mandibular cartilage of either side, and so became an

378

EDWARD PHELPS ALLIS.

intermandibularis. Whether or not this intermandibularis,
innervated by the nervns facialis, was overlapped externally
by an intermandibularis derived from the corresponding-
portion of the mandibular myotome, and innervated by the
nervus trigeminus, cannot be told from dissections of the
adult, but it is certain that, in the adults of living fishes, these
two muscles are indistinguishably continuous one with the
other. There is, accordingly, question as to where one
muscle ends and the other begins, and it is frequently asserted
that that part of the muscle that is of mandibular origin has
lost its primary innervation by the nervus trigeminus and
secondarily acquired innervation by the nervus facialis. It
is accordingly important to know the relations of the nervus
facialis to these muscles.

The ramus hyoideus facialis, as shown in nearly all of
Vetter’s (1874), Ruge’s (1897), and Luther’s (1909) figures of
the Selachii, leaves the external surface of the hyal con-
strictor to acquire a position between the musculi interhyoideus
and interinandibularis and does not reappear on the external
surface of the latter muscle. This is not, however, invariably
the case, for in one of Luther’s figures of Heptanchus (1. c.
p. 75) so-called motor branches of the^ nerve are shown
reappearing on the external surface of the intermandibularis
near its anterior end, and in the same author’s figures of
Chlamydoselachus, Heterodontus, Squalus, and Etmopterus,
small branches of the nerve are also shown reappearing on
the external surface of the muscle, but it is not said that they
are motor nerves, as in the case of Heptanchus. In Chlamy-
doselachus one of these small branches is shown re-entering
the muscle. Ruge found no branch of the nervus trigeminus
going to any part of the musculus intermandibularis in any
of the fishes examined by him. Luther, on the contrary,
found branches of that nerve going to, and apparently
innervating, the anterior part of the intermandibularis in all of
the Plagiostomi examined by him excepting only Chlamydo-
selachus and the Notidanidas. In his earlier work (1909)
he concluded, in accord with Fiirbringer (1903) and

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 879

Huge (1897), that when the intermandibularis is innervated
wholly by the nervus facialis, a muscle of facialis origin has
simply crowded out and replaced one of trigeminus origin,
but in a later work (1913, p. 46) he concluded that the
trigeminus muscle here persisted, but had secondarily
acquired innervation by the nervus facialis. Because of the
wide distribution of the innervation of certain fibres of the
intermandibularis by the nervus trigeminus, he considers
this to be an archaic feature in fishes.

In Chlainydoselachus, I find the musculi interliyoideus and
intermandibularis forming a single continuous muscle-sheet
which extends transversely from one side of the head to
the other, without the intervention of a median aponeurosis.
The posterior quarter, approximately, of this muscle-sheet
is inserted, on either side, on the corresponding ceratohyal,
while the anterior half is inserted wholly on the mandibula.
Between these two parts of the muscle-sheet, and lying im-
mediately anterior to a tendinous band which extends from
the musculus adductor mandibulm to the musculus inter-
hyoideus (see Luther, 1909, fig. 1), I find, in all my speci-
mens, the fibres of the remaining quarter of the sheet
separated, for a short distance along each lateral edge, into
deeper and superficial layers, the deeper (dorsal) fibres
being inserted on the ceratohyal and the superficial (ventral)
ones on the mandibula. The deeper layer lies external to
the proximal (anterior) portion of the ventral end of the
constrictor of the first branchial arch, but in large part
separated from it by the hyal branchial rays and the hyo-
branchial gill pouch. The constrictor of the first branchial
arch similarly overlaps and lies external to the constrictor of
the second branchial arch. This overlapping of these
muscles is well shown in Vetter’s figure of Heptanchus
(1874, PI. 15, fig. 7), where the proximal fibres of the con-
strictores superficiales of the first and second branchial
arches, are shown lying directly internal to the musculus
interhyoideus.

There are accordingly here, in Chlamydoselachus and

380

EDWARD PHELPS ALLIS.

Heptanohus, four muscle-sheets superimposed one above the
other, the two internal muscles being wholly independent
of each other and of the external ones, because of the inter-
vening branchial pouches, but the two external muscles being
fused to a greater or less extent with each other in the mid-
ventral line. It might then be assumed that these two
external muscles belonged the one to the hyal and the other
to the mandibular arch, and that they had, because of the
abortion of the intervening branchial cleft, partially fused
with each other, as the overlapping constrictores of the liyal
and branchial arches of Mustelus and certain other Selachii
have, and as has already been described. The conditions in
Chlamydoselachus and Heptanclius can, however, equally well
represent two different stages in the change of insertion of a
hyal muscle from the branchial bar (ceratohyal) of its own
arch to that (mandibula) of the mandibular arch, similar to
the change of insertion that takes place in the dorsal portion
of the muscle and has just been described. The first assump-
tion requires the further assumption that the overlapping
muscle of mandibular origin has wholly, or in large part,
lost its primary innervation by the nervus trigeminus and
secondarily acquired innervation by the nervus facialis ; and
Chlamydoselachus, generally considered to be the most primi-
tive of living Selachii, would present a more advanced stage,
not only in the fusion of these two muscles but also in the
secondary change of innervation, than any other selachian
that I know of. The second of the two assumptions entails
no secondary assumptions for its justification, excepting the
readily acceptable one that part of a hyal muscle has secon-
darily acquired insertion on a mandibular cartilage, and
Chlamydoselachus, if a primitive fish, would naturally show
an early stage in the process. The innervation of the muscles
in Chlamydoselachus favours the second assumption.

In this fish, Chlamydoselachus, in each of five specimens
that I have examined, the nervus hyoideus facialis at first
runs forward along the external surface of the posterior
portion of the interhyoideus, and there gives off two or more

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 381

branches. These branches also at first lie on the extern
surface of the posterior portion of the interhyoideus, and send
branches to that muscle and to the adjacent portions of the
continuous, dorso-ventral fibres of the constrictor super-
ficialis ; these branches anastomosing more or less with each
other. When the nerve and its branches reach the region
where the primarily single muscle- sheet separates, along its
lateral edges, into a superficial intermandibularis and a deeper
interhyoideus portion, they, in four of the five specimens
examined, all perforate the muscle-sheet, either at that line
of separation or immediately anterior to it, and acquire a
position between the two sheets. In this position the several
branches either remain independent or unite to form one or
two nerves, and, in one specimen which was examined simply
for the muscles and not the nerves, they are shown, in my
drawings, always lying between the two muscles, internal to
the one and external to the other. In the other three of these
four specimens, which were more carefully examined, the
nerve or nerves ran forward for a certain variable distance
between the two muscles, sending branches to them, and then
perforated the intermandibularis, this time from within out-
ward, and, reaching its external surface, there ran forward
nearly to its anterior end. At I his point, all the branches
again penetrated the muscle-sheet, which was here repre-
sented by the intermandibularis alone, and did not again
reappear on its external surface. In several instances the
mesial branches of the nerves of opposite sides fused in
the median line, anterior to the interhyoideus portion of the
muscle, to form a single median nerve which then entered the
musculus intermandibularis and was not farther traced.

On one side of the fifth one of the five specimens a large
branch of the nerve remained on the external surface of the
muscle-sheet, the main nerve perforating the sheet and
running forward in the manner above described. The large
branch crossed onto the external surface of the musculus
adductor mandibulae, sent a large branch to anastomose with
the nervus mandibularis trigemini, and then itself turned

VOL. 63, PART 3. NEW SERIES. 27

382

EDWARD PHELPS ALLIS.

antero-mesially to reach and penetrate the anterior portion of
the musculus intermandibularis. On the other side of the
head of this same specimen, what was apparently the corre-
sponding branch was given off while the main nerve was on
the internal surface of the intermandibularis, and, having
perforated that muscle from within outward, it joined and
anastomosed with the latero-sensory nerve that innervates
the sense organs of the hyomandibular line. No branch was
noticed later leaving the latero-sensory nerve to go to the
musculus intermandibularis, but as the dissection was made
without any thought of there being such a branch it is
probable that it existed but was overlooked. No branch of
the nervus trigeminus was found going to any part of the
muscle-sheet in any of my specimens. Luther (1909) shows
a branch of this latter nerve going to the anterior end of the
muscle, but he considered it to probably be a sensory and not
a motor nerve.

The nervus hyoideus facialis must primarily have lain, in
all the Selachii, along the anterior (external) surface of all
the muscles it innervates that nre derived from the myotome
of its own arch, that being the position in which all the
branchial nerves are found, and it does actually lie external
to the interhyoideus in all of the five specimens of Chlamydo-
selachus above described. It, however, lies, in four of those
five specimens, external to certain portions of the inter-
mandibularis, but internal to certain other portions. If this
intermandibularis muscle be developed from the myotome of
the hyal arch, this difference in the relations of the nerve to
the muscle can be naturally explained, as in the dorsal portion
of the constrictor of this arch, by the assumption that certain
of the fibres of the muscle, which were primarily inserted on
the ceratohyal, had secondarily acquired insertion on the
mandibula by passing external to the nerve as that nerve ran
forward near the ventral (morphologically posterior) edge of
the mandibula, while in other cases the muscle retained its
primitive position internal (posterior) to the nerve. This
explanation would, however, not apply if the intermandibu-

V ISCERAE ARCHES OF THE GNATHOSTOME FISHES. 383

laris were of mandibular origin, for the muscle would then
have lain primarily anterior and hence external to the nervus
facialis, and it is difficult to conceive how certain portions of
it, still retaining* their primary insertion on the mandibula,
could have shifted from this primarily anterior and external
relation to the nerve to a posterior and hence internal isola-
tion to it. And as the muscle in no way lies in the path of,
or interferes with the nervus facialis, it is difficult to conceive
a reason for the perforation of the muscle by the nerve.

The interhyoideus and intermandibularis muscles of
Chlamydoselachus could accordingly both be of facialis
origin, so far as the relations of nerve and muscle are con-
•cerned, but in all probability only that portion of the inter-
mandibularis that lies anterior to the point where the nervus
facialis definitely disappears from its external surface could
be of mandibular origin. And if this portion of the muscle
be of mandibular origin, as several authors have maintained,
I consider it certain that it is innervated by a branch of the
nervus mandibularis trigemini, and that that branch has
simply been missed in dissections, my own included. That it
is possible that this nerve has been so missed is shown by the
fact that in Heptanchus, where Furbringer and Luther both
found the intermandibularis innervated by the nervus facialis
alone, my assistant, Mr. John Henry, finds, on both sides of
the head of one of three specimens of this fish that were
•examined, a branch of the nervus mandibularis trigemini
going to the intermandibularis in a position strictly com-
parable to that shown by Luther in several of the Selachii
examined by him, while in the other two specimens it was not
found.

Edgeworth says that the musculi interhyoideus and inter-
mandibularis, apparently wherever found in the vertebrate
series, are not developed from the myotomes of their respec-
tive arches, but from related portions of the wall of the
coelomic cavity, and that they accordingly have no homologues
in the branchial arches. According to him (loc. cit., p. 178) ;
•“ The cephalic coelom disappears in the mandibular and hyoid

384

EDWARD PHELPS ALLIS.

segments early in development, and its walls develop into the
intermandibularis and interhyoideus, which are at first con-
tinuous with the mandibular and hyoid myotonies. The
lower ends of the branchial myotomes separate from the wall
of the branchial portion of the cephalic coelom, and they
develop into the branchial muscles. No muscles are directly
formed from the wall of the branchial portion of the cephalic
coelom, which subsequently retreats from the head.” This
strikingly recalls van Wijhe’s description of the development
of the coracobranchialis + coracomandibularis muscles in
these same fishes, but it seems certain that the observations
of these two authors do not relate to the same muscles. Of
the intermandibularis of Scyllium Edgeworth says (loc. cit.,
p. 180) : “ The intermandibularis (Cs2 of Vetter, C2mv of
Ruge) is formed from the ventral portion of the mandibular
cavity, which, as mentioned above, does not meet its fellow in
the mid-ventral line, but passes backwards ventro- median to
the ventral end of the hyoid cavity to open into the fore end
of the cephalic coelom.” Here, in Scyllium, the inter-
mandibularis is thus definitely said to arise from the
mandibular cavit}7 and hence not from a part of the cephalic
coelom, which would seem to be in direct contradiction to the
statement just previously made. Edgeworth then further
says: “It results from this that there is no developmental
stage in which the intermandibularis lies altogether in front
of the interhyoideus. It gradually extends backwards,
underlying the interhyoideus, so that in 23 mm. embryos its
hinder end lies posterior to the ventral end of the cerato-
hyal.”

Luther (1909, p. 97) has already made brief reference to-
this development of the intermandibularis and interhyoideus
from parts of the cephalic coelom, as set forth in an earlier
work of Edgeworth’s (1902) which I have not been able to-
consult, and he, Luther, expresses much doubt as to its being
correct, an opinion which I strongly share. My reasons for
considering it incorrect can be best explained by reference to
Scammon’s (1911) figures of the head somites in embryos of

VISCERAL ARCHES OF THE GNATHOSTOME FfSHES. 385

Squalus acanthi as. In fig. 20 of that work Scammon
gives a reconstruction of the head somites in a 9 mm. embryo
of Squalus, and I have reproduced it in the accompanying
Text-fig. 1. In this figure it is seen that while the coelomic
cavity might properly be considered to be prolonged into the
short united portion of the hyal and mandibular stalks, and
even beyond the hyal stalk for a short distance into the
ventral end of the mandibular stalk, it can no more be con-
sidered to be prolonged into the basal portion of the hyal stalk
than also into the basal portions of the stalks of the branchial
myotomes. The case is strictly similar to that of the truncus
arteriosus and the afferent arteries that arise from it. The
truncus arteriosus cannot be considered as in any way con-
tinued into any of these arteries excepting only into the
afferent mandibular artery. With regard to this latter artery
there is no line of demarcation to indicate where the truncus
arteriosus ends and the mandibular artery begins, and in my
discussion of these arteries in embryos (Allis, 1908) I assumed
that the basal portion of the afferent mandibular artery
represented an anterior prolongation of the truncus arteriosus.
The cavity designated as the hyal cephalic coelom in Edge-
worfhks Text-fig. 1, showing a transverse section of a 7 mm.
embryo of Scy Ilium, is then certainly a part of the hyal stalk,
and the fact that the interhyoideus muscle, developed from
this part of the stalk, is said to be at first continuous with the
hyal myotome would seem to be of greater significance than
the further fact, to which Edgeworth gives the greater signifi-
cance, that this part of the stalk does not separate from the
wall of the coelomic cavity before developing into muscle
fibres, as the stalks of the myotomes in the branchial region
are said to do. The so-called mandibular cephalic coelom
of this same Text-figure of Kdgewortlds might, however, be
considered to be a part of the cephalic coelom, for, as in the
case of the afferent mandibular artery, there is no line of
demarcation to indicate where the cephalic coelom ends and
the mandibular stalk begins. But if the musculus inter-
hyoideus is developed from the ventral portion of the hyal

386

EDWARD PHELPS ALLIS.

myotome and not from a part of the cephalic coelom, as above
explained, then the musculus intermandibularis must be
developed from a corresponding portion of the mandibular
myotome, for Edgeworth says (loc. cit., p. 226) that these two
muscles are serially homologous. This conclusion is inevitable
if the premises are correct, and the intermandibularis, although
lying actually, in sections, ventral to the interhyoideus, would
lie morphologically entirely in front of that muscle. This

Text-fig. 1.

explanation of Edgeworth's observations would also establish
that the intermandibularis and interhyoideus of his descrip-
tions could not be the coracobranchialis + coracomandibularis
of van Wijhe's (1882 b) earlier descriptions, notwithstanding
the marked similarity in the descriptions of their derivation.

Of fishes other than tlie Selachii Edge worth says (loc. cit.,
p. 209) that, in 8 mm. embryos of Acipenser, the hyal
muscles “ consist of a hyoid myotome, the anterior part of
which is inserted into the upper end of the hyoid bar, forming
a levator hyoidei, and the posterior part of which forms a
dorso-ventral sheet — homologous with C2vd of selachians —

VISCERAL ARCHES OF THE UNATHOSTOME FISHES 387

continuous with the posterior part of the interhyoideus,
whilst the anterior part of the interhyoideus is inserted
laterally into the hyoid bar.” And also (loc. cit., p. 210)
that : “ The fore part of the interhyoideus of Acipenser forms
the hyohyoideus inferior (Cs5 of Vetter), the hinder part,
i.e. the lower part of C2vd, forms a constrictor operculi
(Cs3 and Cs4 of Vetter). In Polypterus the condition is
similar. In Lepidosteus, Amia and Salmo, the fore part
forms the hyohyoideus inferior ; the hinder part becomes
attached laterally to the hyoid bar (only partially so in
Lepidosteus) and forms the hyohyoideus superior.”

In Amia, the superior or deeper, and the inferior or super-
ficial portions of the geniohyoideus of my descriptions of that
fish are respectively called by Edgeworth (loc. cit., p. 210)
the musculus hyomaxillaris and the musculus intermandibu-
laris posterior. The musculus hyomaxillaris, as above defined
by Edgeworth, is said by him to be differentiated from the
“ upper edge ” of the hyohyoideus inferior, but comparison
with the adult shows that this so-called upper edge of that
muscle must be the dorsal edge as seen in transverse sections
of embryos, and hence morphologically the anterior edge of
the muscle. In Lepidosteus and Acipenser these same fibres
of the hyohyoideus inferior are said to form a hyomaxillaris
ligament, and this ligament is said (loc. cit, p. 212) to be the
ligamentum mandibulo-hyoideum of van Wijhe’s (1882a)
descriptions of the adults of these fishes. But there is evi-
dently some error or oversight here, for a ligamentum
mandibulo-hyoideum is described by van Wijhe in Amia, as
well as in Lepidosteus and Acipenser, and hence coexists in
the former fish along with the musculus hyomaxillaris of
Edgeworth’s descriptions. A further difficulty is that the
musculus hyomaxillaris of Amia is said (loc. cit ., p.223) to be a
serial homologue of the interarcuales ventrales of the branchial
arches of that fish, notwithstanding that the former muscle
is said to be derived from the cephalic coelom, as already
explained, and the latter muscles to be developed from the
ventral ends of the branchial myotomes. Edgeworth calls

388

EDWARD PHELPS ALMS.

attention to this, and explains it by saying that the corre-
sponding muscle in Alytes, Rana, Pelobates, and Lepus is
formed from the ventral end of the hyal myotome, and
that this method of formation is probably the primitive one.

The intermandibularis of all teleostoman embryos is said
by Edgeworth (loc. cit. , p. 187) to form at first, with its fellow
of the opposite side, a transverse muscle attached laterally to
Meckel’s cartilage, and it is later said (loc. cit.,p. 202) that a
comparison of the various forms of the muscle shows that
this condition of a transverse sheet is the primitive one for
the muscle. It is, however, immediately afterwards said that
this condition of a transverse sheet persists (“ exists ”) only in
Salmo. Edgeworth further says (loc. cit., p. 280) : “ (3) The
intermandibularis anterior and posterior (the latter called
* inferior geniohyoid ’ by Allis) of Amia are innervated by both
the fifth and seventh (Allis). (4) The hyo-maxillaris of
Teleostomi, developed in the hyoid segment, is in some, e. g.
Menidia (Herrick), wholly innervated by the seventh ; whereas
in others, e.g. Esox (Vetter), Salmo, its hinder part is inner-
vated by the seventh and its fore part by the fifth ; and in
Amia (Allis) it is innervated by the fifth and seventh.”

These latter two statements would seem to imply that
certain of the individual fibres of the muscles referred to in
Amia were innervated at the same time by two different nerves,
and that they were in process of losing their normal innerva-
tion by the nerve of their segment of origin and secondarily
acquiring an innervation by a nerve of another segment. If
this be the meaning of the statements, the reference to Amia is
unfortunate, and is apparently based on the literal acceptance
of the heading of one of the sections of my work on that fish
without any consideration of the accompanying text. That
heading is (Allis, 1897, p. 559) : “ Muscles innervated by both
the Trigeminus and Facialis,” which, literally accepted, might
have the meaning that Edgeworth apparently gives to it. But
in the text (loc. cit., p. 613) it is carefully explained that the
muscles in question are innervated by branches of a nerve
formed by the anastomosis of trigeminus and facialis branches

VISCERAL ARCHES OE THE GNATHOSTOME FISHES. 389

which run directly into each other and so form a complete
circuit in which it is impossible to tell where the .one nerve
ends and the other begins. In the General Summary it is
further said (loc. cit., pp. 744-5) that the ramus maxillaris
inferior trigemini probably innervates the musculus interman-
dibularis and all, or a part, of the inferior division of the
geniohyoideus, and that the ramus hyoideus facialis probably
innervates the superior division of the geniohyoideus, and a
part, at least, of the inferior division of that muscle. The
innervation, in each case, is only given as probable, and
there is no slightest suggestion of any part of either of
the muscles being innervated, at the same time, by both the
nerves.

Mandibular Arch.

Dohrn says (1885, p. 13) that, in selachian embryos, a
muscle is developed from a myotome that comes from the
mandibular arch (eines Muskelschlauches welcher vom Kiefer-
bogen kommt), and that this muscle corresponds to the
ventral portion of the constrictor superficialis of the hyal
arch. There is, as already explained, some question as to
whether Dorhn considered a part of this muscle to be the
homologue of the coracobranchialis of the branchial arches,
but it is certain that the myotome, said to come from the
mandibular arch, must be the ventral end of the mandibular
myotome, and that the muscle said to be developed from it
must be that part of the musculus intermandibularis of the
adult that is primarily, if not actually, innervated by the
nervus trigeminus. Edgeworth (1911) says, as has already
been fully explained and discussed, that the myotome of the
maudibular arch only extends to the ventral edge of the
musculus adductor mandibulas, and that the musculus inter-
mandibularis is developed from the walls of the cephalic
coelom. There is thus here marked difference of opinion.

Accepting Dohrn* s observations as correct, and assuming
that there was primarily a premandibular arch separated
from the mandibular arch by a visceral cleft, there must

390

EDWARD PHELPS ALLIS.

have been primarily, in the mandibular arch as in the hyal
and branchial arches, a single continuous constrictor muscle
that had a dorso-ventral extent equal to that of the con-
strictor of the hyal arch. Branchial rays also probably
primarily existed in this arch as in the more posterior arches,
for remnants of them are said to be still found in certain
recent fishes. Branchial lamellee were then quite probably
also developed in this arch as in the more posterior ones, and
were probably found on the anterior as well as the posterior
surface of the arch.

The conditions in this mandibular arch would then have
been similar to those in the hyal and branchial arches, and,
such being the case, there seems no good reason why, when
the visceral arches all began to assume a position oblique to
the axis of the body, a small adductor muscle should not have
been cut out of the proximal edge of the constrictor of this
arch, as it is said to have been cut out of the constrictores of
the branchial arches, and as I assume that it was also cut out
of the constrictor of the hyal arch.

When the hyomandibular cleft later became reduced to the
small existing spiracular canal, the mandibular branchial
diaphragm, which must primarily have existed as in the hyal
and branchial arches, would necessarily have gradually
ceased to be formed excepting as it may still be represented
in some part of the anterior wall of the spiracular canal.
Because of this gradual reduction and final almost complete
disappearance of the branchial diaphragm of this arch, the
middle portion of the long constrictor superficialis of the
arch was necessarily forced over onto the anterior (lateral)
surface of the cartilaginous bar of the arch, and it carried
with it the nerve of the arch, and probably also the
anterior efferent artery of the arch (Allis, 1916), this nerve
and artery primarily lying' anterior to the constrictor muscle,
as they actually do in the branchial arches of living
Selachii. The afferent mandibular artery and the branchial
rays, both lying posterior to the constrictor muscle, were not
so carried forward, and retained their primitive positions on

VISCERAL ARCHES OF THE GNATHOSTOME FISHKS. 391

or near the morphologically external but actually posterior
edge of the cartilaginous bar of the arch. The posterior
efferent, artery, also, was not affected by this change in
position of the constrictor muscle, but it later underwent
reduction or abortion in its ventral portion, while dorsally it
persisted and retained its normal position posterior to the
spiracular cartilage, that cartilage representing either the
dorsal extrabranchial of the arch or one or more of the
branchial rays. That any of the branchial rays of this arch
could, in such a shifting of the constrictor, have acquired the
positions of the labial cartilages seems quite impossible.

The long musculus constrictor superficialis, having acquired
this position on the anterior (lateral) surface of the carti-
laginous bar of its arch, was later more or less completely
cut in two at the places where it crossed the palatoquadrate
and the mandibula. The portion so cut out of the middle of
the constrictor was then added to the small, pre-existing'
musculus adductor to form the large and powerful adductor
of the adults of living fishes, while the ventral portion formed
the intermandibularis and the dorsal portion the levator of
the arch. Certain of the fibres of the original constrictor
were, however, quite certainly not thus cut through at tlie
places where they crossed the palatoquadrate and mandibula,
for certain of them still extend, in the adults of living fishes,
the full length of the arch. This is markedly the case in
certain of the fibres of the musculus spiracularis of Astrape.
This muscle is said by Luther (1909, p. 14) to be developed
from the posterior fibres of the dorsal portion of the primitive
constrictor of the arch and to have a ventral prolongation
which lies along the internal surface of the mandibula
(kieferapparat) and extends as far as the symphysis of the
mandibulse, there uniting with its fellow of the opposite
side. This ventral prolongation is a feeble muscle, of no
apparent functional importance, and certainly cannot be a
secondary formation, as Luther considers it to be. It
must, on the contrary, represent a persisting remnant of a
distal part of the primitive constrictor which, when the

392

EDWARD PHELPS ALLIS.

branchial rays aborted, slipped onto the posterior, instead of
onto the anterior, surface of the cartilaginous bar of the arch,
and so, not crossing that bar, was not cut in two as the other
fibres of the constrictor were.

In certain other Batoidei, the musculus spiracularis is said
by Luther to have a less extensive ventral prolongation than
in Astrape, being said to extend either to the ventral end of
the hyomandibula, to the abdental edge of the mandibula, to
the ceratohyal, or to the dorsal fascia of the musculus coraco-
mandibularis. In Astrape and Torpedo, certain fibres of the
muscle are said to be inserted on, and others to arise from,
the spiracular cartilage, that cartilage thus lying between
dorsal and ventral portions of the muscle; and as I have
lately shown (Allis, 1915) that this cartilage of these fishes is
quite certainly the dorsal extrabranchial of the mandibular
arch, the muscle thus has the relations to this cartilage that
the branchial constrictores superficiales of certain Selachii
have to the dorsal extrabranchials of their respective
arches.

Other portions of the primitive constrictor apparently lost
only their ventral, intermandibularis, portion, retaining their
full lengths dorsal to that muscle. Such portions are
apparently found in the second and third divisions of the
levator maxillse superioris of my descriptions of Amia, and in
the levator labii superioris of certain of the Batoidei, all of
which muscles extend, with their tendinous ends, from the
neurocranium to the abdental edge of the mandibula. The
levator labii superioris of the Batoidei, called by Luther the
musculus prmorbitalis, is said by that author to usually extend
only to the angle of the gape of the mouth and to there be
inserted in the aponeurotic septum of the adductor mandi-
bulse, but it may have a ventral ligamentous prolongation, or
even a large muscle belly, which extends beyond the angle of
the gape and is inserted, with the mandibular portion of the
adductor, on the mandibula. In certain of the Batoidei it is
even said that the tendinous ventral end of the muscle is
practically continuous with the lateral edge, and hence

VISCERAL ARCHES OF THE GNATHOSTOME FISHES.

393

morphologically dorsal end, of the musculus intermandibu-
laris, the constrictor fibres in these fishes thus apparently
having retained their full primitive lengths.

Luther (1909, p. 49) considers the levator labii superioris
(praeorbitalis) to have been primarily simply a bundle of the
adductor mandibulae that had it£ origin at a high level on the
neurocranium, anterior to the eyeball. The more ventral
origin of this muscle, from the antorbital process, found in
Chlamy doselachus and certain other Plagiostomi, he considers
to be secondary and correlated either to an enlarged eyeball
or to a large gape of the mouth with the angle of the gape far
posterior, the muscle here secondarily becoming a “ Spreizer ”
of the articulating ends of the upper and lower jaws. The
eyeball is considered by him (loc. cit., p. 36) to have been
the chief one of these two causes of the splitting off of this
bundle from the remainder of the adductor mandibulae, and
if this be so, the eyeball thus being assumed to have lain in
the path of the muscle-fibres of the arch as they pushed dor-
sally to acquire insertion on the neurocranium, it would seem
as if this split in the muscle must have begun at the dorsal
end of the primitive constrictor and not at the dorsal end of
that middle portion of that muscle that is usually considered
to, alone, have given origin to the adductor mandibulae. The
dorsal portion of the praeorbitalis would then contain the
anterior fibres of the dorsal muscle, Csd2, of Vetter’s descrip-
tions, and where the praeorbitalis extends beyond the angle of
the ^ape the split that separates it from the adductor would
extend from the dorsal end of the constrictor as far at least
as the ventral end of the adductor. Such an extensive split
in this myotome can certainly not be explained simply by the
eyeball having caused the dorsal fibres of the constrictor to
diverge anteriorly and posteriorly in order to acquire a dorsal
attachment on the neurocranium, and if Luther is correct in
his conclusion that this muscle had primarily its origin at a
high level on the neurocranium, a much more rational expla-
nation would seem to be that this muscle belongs to a pre-
mandibular arch. The recorded innervation of the muscle.

394

ED W Alii) PHELPS ALLIS.

and the embryological evidence are, however, both against
this supposition.

Edgeworth (1911) derives both the levator labii superioris
of the Plagiostomi and the four divisions of the levator
maxillae superioris of my descriptions of Amia, directly from
the adductor mandibulae, and from that portion only of the
primitive mandibular constrictor. Luther also derives these
four muscles of Amia directly from the adductor mandibulae ;
and he proposes for the first and second divisions of the
muscle the name musculus adductor mandibulae parabasalis,
because of their partial origin from the lateral wing of the
parasphenoid (parabasalis, Gaupp), and for the third and
fourth divisions of the muscle the names musculus adductor
mandibulae praeorbitalis and musculus nasalis. Edgeworth
considers the muscles of Amia to all be upgrowths of the
internal and deeper portion, only, of the adductor of the
adult, and suggests that they be named in terms of that
internal adductor. The myotome of this arch is, according
to him, definitely and entirely cut into dorsal and ventral
portions where it crosses the dorsal edge of the palatoquadrate;
these two parts remaining always distinct and separate, while
the intermandibularis is, as already stated, developed wholly
from the walls of the cephalic coelom. Until the derivation
of these muscles is definitely known it would seem best not to
give them names based wholly or largely upon it.

There remains now only the aponeurotic septum of the
adductor mandibulae to be considered. Fiirbringer (1903,
p. 383), considered this aponeurosis to be of secondary origin
and of no great morphological significance, it being developed,
in fishes where the mouth opening had a pronounced posterior
extension, simply in order to give space for suitable develop-
ment of the belly of the adductor. Luther . (1909, p. 61)
thinks this an insufficient reason for the development of the
aponeurosis, and considers it to have been secondarily
developed, after the development of the levator labii superi-
ors (praeorbitalis), in order to furnish an attachment for that
muscle on the quadrato-mandibular joint, and so facilitate its

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 395

action as a protractor of the palatoquadrate and also as a
spreader (Spreizer) of the articular ends of the jaws. I
consider the aponeurosis to have been developed wholly
independently of either of these two functions. In my
opinion, a small adductor muscle had already been differenti-
ated in this arch before the remainder of the constrictor
began to slip over onto the anterior surface of the carti-
laginous bar of the arch. There was, at this period, quite
certainly not sufficient space between the relatively close
fitting integument of the arch and the cartilaginous bar to
permit this large and long constrictor muscle to immediately
assume the position that the adductor actually has in the
-adults of living fishes, and the small adductor already
occupied the angle between the two elements of the carti-
laginous bar. Certain of the fibres of the constrictor
accordingly quite certainly acquired attachment on the
internal surface of the dermis at the angle of the gape
These fibres would immediately act as an adductor when the
mouth was widely open, but when the mouth was closed, or
even nearly closed, they would act primarily as a protractor
anguli oris, and secondarily as an abductor of the arch ; for,
being attached to the dermis at the angle of the gape, and
the dermis being fixed, any contraction of the* muscle would
necessarily tend to open the mouth. This would evidently
be of advantage to the fish, for, in the early stages of the
development of the mouth, there was probably no other
abductor mechanism, the ventral longitudinal muscles not yet
having been developed. The fibres so inserted, increasing
in number and importance, would, as the adductor muscle
developed and a cheek was formed, pinch off the subdermal
tissues to which they were attached and an aponeurosis such
as is actual^ found in Chlamydoselachus, and will be fully
described in my later work, would almost inevitably arise.
If these fibres did not become attached to the dermis they
would, in certain cases, become tendinous as they passed
across the angle of the gape, as they do when passing
over the extrabranchials and the middle rays of the branchial

396

EDWARD PHELPS ALLIS.

series in the branchial arches of certain fishes (Vetter, 1874),
and the condition found in the adults of many fishes would
arise. If such fibres should then separate from the remaining
fibres of the adductor, a muse ulus praaorbitalis with dorsal
and ventral muscle bellies, such as is described by Luther in
certain of the Plagiostomi, would arise. And if the ventral,
mandibular portion of the muscle became wholly tendinous,
the condition found in the first and third divisions of the
levator maxillas superioris of my descriptions of Amia might
arise. The aponeurosis would naturally tend to be developed
only where the mouth had a marked posterior extension and
the opening of the gape was long. According to Luther
(loc. cit., p. 62) the aponeurosis and the muscle Addy of
Vetter’s descriptions vary inversely, a marked development of
the one being associated with a feeble development of the
other, and he attributes this to the fact that a strongly
developed Addy would act as a spreader of the jaws, and
the musculus prasorbitalis, being relieved of that func-
tion, there would be no call for an aponeurosis. As I
attribute the development of the aponeurosis to a totally
different cause it does not seem to me that this applies.

General Summary.

The primitive condition of the muscles related to the
visceral arches of the gnathostorne fishes was, as Vetter
long ago concluded, a simple constrictor muscle in each arch,
and associated with this muscle there was a branchial bar
which lay internal to the muscle.

From this simple primitive condition two distinctly different
lines of descent are indicated by later differentiations of the
muscles, and these differing differentiations are associated
with, and caused by, two distinctly different forms of branchial
bar in the branchial arches. One of these two lines of
descent is represented by the Teleostomi and the other by
the Plagiostomi, the Holocephali and Dipneusti apparently
occupying somewhat intermediate positions.

VISCKKAIi ARCHES OF THE GNATHOSTOM E FISHES. 397

In the Teleostomi, the four typical elements of each
branchial bar of recent fishes lie, approximately, in a single
plane, and this must have been their primitive relation to
each other. Primarily this plane must have been transverse
to the axis of the body, but it later became inclined to that
axis. Associated with this form of arch the branchial fila-
ments of the gill- bearing arches are supported by cartilaginous
or osseous rods. In the hyal arch there are, in addition,
osseous branchiostegal rays which lie anterior to the modified
constrictor of the arch.

In the Plagiostomi, the dorsal and ventral elements of
each branchial bar are directed postero-mesially at a marked
angle to the middle elements of the bar, these latter elements
lying, as in the Teleostomi, in a plane inclined to the axis of
the body. A sigma form of bar is thus produced, and asso-
ciated with it there are cartilaginous branchial rays in all the
gill-bearing arches. These cartilaginous rays all lie, primarily,
posterior to the constrictor muscle, of the related arch, bub
the muscle fibres may later become in part inserted on them.

In the Holocephali and Dipneusti, the dorsal elements of
the branchial bars are directed postero-mesially, as they are in
the Plagiostomi, while the ventral elements are directed
antero-mesially, as in the Teleostomi. In the Holocephali
there are, according to Vetter, cartilaginous rays both in the
hyal and the branchial arches, and the visceral muscles as
described by him seem plagiostoman in character. In the
Dipneusti there are cartilages in the hyal arch that are con-
sidered by Fiirbringer to be branchial rays, but there are
neither branchial- rays nor supporting rods to the branchial
filaments in the branchial arches ; and the branchial muscles
are teleostoman in character.

The constrictor muscle is found in a more primitive con-
dition in the Selachii than in any others of the gnathostome
fishes. Because of the sigma form of branchial bar in these
fishes, the proximal (anterior) edge of the constrictor of each
branchial arch has slipped forward over the anterior edge of
the middle, posteriorly-directed angle of the sigma, and
VOL. 63, PART 3. NEW SERIES. 28

EDWAKD PHELPS ALLIS.

backward over the posterior edges of the dorsal and ventral,
anteriorly-directed angles of the sigma ; and from the parts
of the constrictor that cross or span these three angles are
differentiated, respectively, the adductores arcuum branchi-
al ium, the arcuales and interarcuales dorsales, and the
coracobrancliiales of Dohrn's descriptions of embryos. These
latter muscles are simply the proximal (anterior) portions of
the ventral ends of the primitive constrictores of the branchial
arches, they are of branchial origin, are innervated by
branches of the nervus vagus of the arch to which they
belong, and they coexist, in the adult, with the coraco-
branchiales of Vetter's descriptions. The latter muscles are
said, by both Dohrn and Edgeworth, to be derived from the
ventral ends of the branchial myotonies, but their innerva-
tion, in the adult, by spinal or spino-occipital nerves, their
relations to the other hypobranchial muscles, and the marked
want of accord in the descriptions of their development, all
warrant the conclusion that they must be of spinal origin.

The distal (posterior) portion of the constrictor muscle of
each branchial arch of the Selachii, the so-called constrictor
superficial! s, lay primarily not only on the anterior surface of
the branchial rays of its arch, but also on that surface of the
extrabranchials of its arch ; ^and, in the adults of recent
fishes, its dorsal and ventral ends turn posteriorly, to a
greater or less extent, across the dorsal and ventral edges,
respectively, of the next posterior gill-pouch. When the
constrictor contracted, the muscle was accordingly stretched
across the extrabranchials of its arch, and certain of the
muscle fibres, in certain fishes, were there cut in two by
acquiring insertion on the extrabranchials. Other fibres
simply became tendinous where they passed over the extra-
branchials, and so there gave rise to more or less pronounced
linear aponeuroses, or so-called septa. That part of each
constrictor that lay between the dorsal and ventral extra-
branchials of its arch thus became more or less cut out of
the primarily continuous constrictor, and formed themuscuh s
interbranchialis. This muscle is never found definitely

VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 399

differentiated in the hyal arch, but indications of the begin-
nings of its differentiation may there be found.

In the hyal arch of the Selachii, an adductor muscle was
probably developed exactly as in the branchial arches, but it
was later transformed into the inferior postspiracular liga-
ment. Arcualis and interarcualis muscles are not found in
this arch of the adult, but Dolirn says that they are repre-
sented, in embryos, by ligaments, which he does not, however,
describe. The coracobranchialis of Dohrn’s descriptions of
the branchial arches is not differentiated in this arch. The
dorsal and ventral portions of the constrictor tend to separate
into deeper and superficial layers, as Huge has stated, the
deeper layer retaining its primitive insertion on the carti-
laginous bar of its arch, while the superficial layer acquires
a secondary insertion on the cartilaginous bar of the
mandibular arch.

In the Batoidei, an adductor muscle was not differentiated
in the hyal arch, and there is accordingly, in these fishes, no
inferior postspiracular ligament. The proximal (anterior)
portion of the primitive constrictor of this arch is differenti-
ated into the levator and depressor hyomandibularis, and
probably also the depressor mandibulae, these muscles
replacing the adductor, arcualis, interarcualis, and Dohrn’s
coracobranchialis of the Selachii. The so-called septa in
these fishes are probably similar to those in the Selachii,
but this cannot be definitely determined from existing
descriptions.

In the mandibular arch of the Selachii, a small adductor
muscle was probably developed exactly as in the more
posterior arches. Later, because of the suppression of the
branchial diaphragm related to this arch, excepting as it may
be represented in the anterior wall of the spiracular canal,
the entire constrictor muscle was forced, in its middle portion,
-onto the anterior surface of the cartilaginous bar of its
arch, and, acquiring insertion on the palatoquadrate and
mandibula, where it crossed their lateral edges, Avas added to
the small, pre-existing adductor, and so gave rise to the

400

EDWARD PHELPS ALLIS.

large adductor mandibulae actually found in the adult.
From those portions of the primarily continuous constrictor
that lay dorsal and ventral, respectively, to the palatoquadrate
and mandibula, the musculi levator maxillae superioris and
intermandibularis were developed. The musculus levator
anguli oris was probably derived from the anterior edge of
the united levator and adductor muscles before they became
separated from each other. The musculus intermandibularis
underwent relative reduction, and, in the adults of recent
fishes, is largely crowded out and replaced by those super-
ficial fibres of the hyal constrictor that have secondarily
acquired insertion on the mandibula. The relations of the
nervus hyoideus facialis to the muscle fibres thus inserted on
the mandibula is against the view that those fibres that are of
mandibular origin have lost their primitive innervation by
the nervus trigeminus and secondarily acquired innervation
by the nervus facialis.

The posteriorly directed dorsal and ventral ends of the
hyal and branchial constrictores of the Selachii always over-
lap, to a greater or less extent, the next posterior constrictor.
Where the ends of the constrictores are strongly inclined
posteriorly, they may overlap two or more posterior con-
strictores, the fibres of the muscles then, crossing the
extrabranchials of those arches and there tending to become
tendinous exactly as they do where they cross the extra-
branchials of their own arches; a series of tendinous
aponeuroses thus being formed in each constrictor. The
overlapping muscles then fuse more or less completely with
each other, and, as the linear aponeuroses related to each
extrabranchial are superimposed and transverse to the muscle
fibres, the continuous muscle-sheet formed by the fusion of the
several constrictores is cut up into what have heretofore
been considered to be separate segments, one related to
each branchial arch and developed entirely from the myotome
of that arch. These segments are, however, each formed by
muscle fibres derived from two or more consecutive constric-
tors, and hence from a similar number of consecutive myotonies-

VISCERAL ARCHKS OF THE GNATHOSTOME FISHES. 401

The dorsal portiou of the constrictor of the ultimate
branchial arch undergoes excessive development and becomes
the musculus trapezius.

In the Teleostomi, each branchial bar, although inclined to
the axis of the body as in the Selachii, continues to lie,
approximately, in a single plane, and the dorsal and ventral
ends of the constrictores do not turn posteriorly as in the
Selachii. The pull of the constrictor, when contracting, did
not, accordingly, tend to make the muscle slip, in the middle
of its length, over the anterior edge of the branchial bar of
its arch, but in certain of the branchial arches of the G-anoidei
the proximal edge of the muscle slipped over the posterior
edge of the branchial bar, and there gave rise to an adductor
that is the functional equivalent but not the homologue of
the adductor of the Selachii. The remaining fibres of the
middle portion of the constrictor either later aborted or,
possibly, became modified to form the radially arranged
muscles related to the supporting rods of the branchial
filaments. The dorsal and ventral ends of the constrictores
became the levatores and the transversi and obliqui dorsales
and ventrales. The levator of the ultimate arch is a slender
muscle, and may secondarily acquire insertion on the shoulder-
girdle. It is the homologue of the large musculus trapezius
of the Selachii.

In the hyal arch of the Teleostomi, the constrictor persists
to a greater extent than in the branchial arches. Its dorsal
portion becomes the adductor hyomandibularis and the
adductor and levator operculi, these muscles, together, being
the equivalent of the levatores of the branchial arches of the
Teleostomi and of the dorsal ends of the constrictores of the
Selachii.

The ventral portion of the constrictor of the ultimate, or
fifth, branchial arch of the Teleostomi is modified to form
the musculi coracobranchiales or pharyngoclaviculares, these
muscles of these fishes thus being branchial muscles, and
hence probably not the homologues of the coracobranchiales
of the Selachii. They always retain, in all fishes that I have

402

EDWARD PHELPS ALLIS.

been able to examine, their primitive innervation by branches
of the nervus vagus. The dorsal portion of the constrictor
of this arch forms, as already stated, the fifth levator muscle,
which is the homologue of the musculus trapezius of the
Selachii.

Literature.

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Spinal Nerves in Amia calva,” ‘ Journ. Morphol.,’ vol. xii.

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VISCERAL ARCHES OF THE GNATHOSTOME FISHES. 403

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nebst Bemerkungen iiber Pleuracanfhiden, Holocephalen und
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Luther, A. (1909).—“ Beitrage zur Kenntnis von Muskulatur und Skelett
des Kopfes des Haies Stegostoma tigrinum Gm. und der Holo-
cephalen mit einem Anhang iiber die Nasenrinne,” * Acta Soc.
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— (1913). — “ Uber die vom N. Trigeminus versorgte Muskulatur

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McMurricli, J. P. (1884).- — “The Myology of Amiurus catus,”
‘ Proc. Canadian Institute,’ vol. ii, fasc. iii.

(1885). — “Cranial Muscles of Amia calva, with a Considera-
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404

EDWARD PHELPS ALLLS.

Marion, G. E. (1905). — “ Mandibular and Pharyngeal Muscles of
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Parker, W. K. (1876). — 1,4 On the Structure and Development of the
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Pollard, H. B. (1892). — “ On the Anatomy and Phylogenetic Position
of Polypterus,” 4 Zool. Jahrb. Abtheil. f. Anat. u. Ontog ,’ Bd. v.
Jena.

Ruge, G. (1897). — “ fiber das peripherische Gebeit des Nervus facialis
bei Wirbelthieren,” ‘ Festschrift Gegenhaur,' Bd. iii, Leipzig.

Scammon, R. E. (1911). — “ Normal Plates of the Development of
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Tiesing, B. (1895). — “ Ein Beitrage zur Kenntnis der Augen-, Kiefer-
und Kiemen-muskulatur der Haie und Rochen,” 4 Jenaisclie
Zeitschr. f. Naturwiss.,’ vol. xxx.

Yetter, B. (1874). — 44 Untersucliungen zur vergleichenden Anatomie der
Kiemen- und Kiefer-musculatur der Fische,” 4 Jenaische Zeitschr.
f. Naturwiss.,’ Bd. viii.

(1878). — 44 Untersuchungen zur vergleichenden Anatomie der

Kiemen- und Kiefer-musculatur der Fische,” 4 Jenaische Zeitschr.
f. Naturwiss.’ Bd. xii.

White, P. J. (1896). — 44 Note on the Extra-branchial Cartilages o*
Scyllium canicula,” 4 Anat. Anz.,’ Bd. xii.

Wijhe, J. W. van (1882a). — -“ fiber das Visceral skelet und die Nerven
des Kopfes der Ganoiden und von Ceratodus,” 4 Niederl. Archiv.
fiir Zoologie,’ Bd. v, Hft. 3, Leiden.

(1882b). “ Ueber die Mesodermsegmente und die Entwickelung

der Nerven des Selachierkopfes,” Amsterdam.

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Amiurus,” 4 Proc. Canadian Institute,’ vol. ii, fasc. 3, Toronto.

VISCERAL ARCHES OE THE GNATi IOSTOME FISHES.

405

EXPLANATION OF PLATES 21 and 22.

Illustrating Mr. Edward Phelps Allis's paper on “The
Homologies of the Muscles related to the Visceral
Arches of the Gnathostome Fishes."

Reference Letters.

Add. Musculus adductor mandibulse. Add. br. I-IV. Musculi ad-
ductores arc. branch, of I-IV branchial arches, ahy. Afferent artery
of hyal arch. ap. I-IV. Linear aponeuroses related to the first to
fourth gill clefts. Arc. I-IV. Musculi arcuales of I-IV branchial
arches. bcl. I-V. First to fifth branchial clefts. BH. Basihyal.
bp. 1 V. First to fifth branchial pouches. BR. hy. Branchial rays of
hyal arch. Care. Musculus coracoarcualis communis. CB. I II.
Ceratobranchials of first two branchial arches. Cbr. I-V. Musculi
coracobranchiales of I-V branchial arches. CH. Ceratohyal. Chy.
Musculus coracohyoideus. Cmd. Musculus coracomandibularis.
Cs2 0. Musculi constrictores superficiales of second to sixth visceral
arches, ex. I-IV. Extra branchials of I-IV branchial arches, ex. h.
Extrabranchial of hyal arch. HMD. Hyomandibula. hmf. Nervus
hyoideo-mandibularis facialis. Ibr^-Q. Musculi interbranchiales of third
to sixth visceral arches. Ihy. Musculus interhyoideus. Imd. Musculus
intermandibularis. Ic. Lateral canal of body. Lhmd. Musculus levator
hyomandibularis. m. Dorsal muscles of trunk. MD. Mandibula.
mit. Nervus mandibularis trigemini, pc. Pericardial cavity. S.
Sho ildir-girdle. sp. Spiracle. Tr. Musculus trapezius. vj. Vena
jugularis. ,

PLATE 21.

Fig. 1. — Lateral view of the head of a 42-cm. Scyllium canicula, with
skin removed to show the branchial muscles, x 1L

Fig. 2. — The same. The constrictores superficiales cut along their
dorsal edges and turned forward and downward so as to expose the
underlying structures, x 12-

Fig. 3. — Ventral view of the same. The constrictor superficialis of
the hyal arch cut through in the mid- ventral line and turned forward
on the right-hand side of the figure. X \\.

Fig. 4. — The same ; a deeper dissection. On the left-hand side of
the figure the hyal constrictor has been cut through near its lateral
edge and turned forward. On the right-hand side it has been wholly
removed, and the ceratohyal turned slightly forward. On both sides

406

EDWARD PHELPS ALLIS.

of the figure the ventral portions of the constrictores of the first three
branohial arches have been cutaway up to the line of the extrabranch ial
of the arch, and on the right-hand side of the figure the ventro-posterior
portions of the gill pouches have been cut away so as to expose the
underlying constrictor of the next posterior arch. In the fourth
branchial arch a piece has been cut out of the constrictor of the arch
( Cs.6 ) so as to expose the fifth gill pouch. The musculi coracomandi-
bularis, coracohyoideus, coracobranchialis I, and coracoarcualis have
been cut through and removed. X 1§.

PLATE 22.

Fig. 5. — The same ; a still deeper dissection. The ceratohyal and the
extrabranchial of the first branchial arch both removed on the right-
hand side of the figure. X 1|

Fig. 6. — The same ; a still deeper dissection. The extrabranchial of
the second branchial arch also removed on the right-hand side of the
figure. X 1L

Fig. 7. — Lateral view of the branchial region of the same. The con-
strictores superficiales of the hyal and first three branchial arches, and
the four related gill-pouches removed, but the cut dorsal ends of the
extrabranchials of the first three branchial arches left in place. X 2.

Fig. 8. — Lateral view of the head of a 43-cm. Mustelus. The skin
removed so as to expose the constrictores superficiales of the hyal and
branchial arches. X 2.

Fig. 9. — The same. The continuous sheet formed by the constrictores
superficiales has been cut along its dorsal edge, and those fibres of
each musculus interbranchialis that are inserted in the related linear
aponeurosis ha*e also been cut close to their insertion on that aponeu-
rosis, and the entire muscle sheet, excepting the distal portion of the
constrictor of the fourth arch (Cs.6), then turned downward to the level
of the middle line of the gill openings. X 2.

Fig. 10.— Lateral view of the constrictor and interbranchialis muscles
of the third branchial arch of Mustelus. Dorsally, the continuous
muscle-sheet formed by the constrictores superficiales has been cut
along the line of the linear aponeurosis related to the arch, and also
slightly posterior to that aponeurosis. Yentrally, the muscle-sheet has
been cut immediately anterior to the line where the ventral portion of
the constrictor of the arch (Cs.5) joins it, and also slightly posterior to
that line. X 2§

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 407

The Cytoplasmic Inclusions of the Germ-Cells.

PART I. LEPIDOPTERA.

By

J. IS ro site Gtitenby, IS. A.,

Exhibitioner of Jesus College, Oxford.

With Plates 23, 24, and 25 and 5 Text-figures.

Table of Contents.

PAGE

1. Introduction • 408

2. Previous Work . . . 409

3. Technique and Material . 412

4. Nomenclature .... 414

5. Mitochondria (Male) . . .418

6. Comparison of Mitochondria of Several Species 421

7. Changes undergone by Mitochondria in Spermatocyte 423

8. Mitochondria in Spermatocyte Division . 424

9. Formation of Macromitosome (Nebenkern) . 425

10. Subsequent Fate of Macromitosome . . 429

11. The Micromitosome 430

12. Cell Processes of the Spermatocyte . 434

13. The Spindle Bridge ..... 435

14. The Probable Function of the Spindle Bridge . 436

15. The Precociously Formed Flagella . 436

16. Acroblasts and Acrosome .... 438

17. The Mitochondria of the Female . 442

18. Other Cytoplasmic Bodies .... 446

19. Abnormalities ..... 446

20. The Fixing Reactions of the Cell Inclusions . 449

21. The Probable Nature of the Micromitosome . . 450

22. Discussion ..... 450

23. Summary ..... 457

408

J. BRONTE GATENBY.

Introduction.

Since the numerous important papers on the spermato-
genesis of many animals have shown that the elaborate series
of events leading to the metamorphosis of the spermato-
gonium into the spermatozoon entail not only great changes
in the nucleus, but also the presence of a number of definite
eytoplasmic bodies whose movements and staining powers
are as regular as those of the nucleus and centrosomes them-
selves, it becomes increasingly evident that it is incorrect to
look upon the sperm as simply the vehicle for the chromatin
of the nucleus and for the centrosome. There are other bodies
whose behaviour merits the belief that they are concerned in
the transmission of some qualities to the egg, and therefore
to the offspring. The Lepidoptera have several such bodies
whose ultimate fate is obscure. Moreover, they are not
properly understood, and their relationship to chromatin, if
any, has not been determined. An examination of the ferti-
lisation of the Lepidopteran has not hitherto been carried out
with the intention of ascertaining what role these bodies play
in this important stage of the germ cell cycle, and such a
field would probably yield useful results. The spermatozoon
becomes in later stages of formation such a difficult object to
study that there is no doubt that the only manner in which
one could feel sure as to whether any of the several bodies
really reached the egg would be to examine fertilisation
stages fixed in suitable media, which should be absolutely
free from certain acids. This unfortunately introduces great
difficulties of technique, for the chorion of insect eggs is
extremely difficult of penetration by any fixatives except
those containing the ingredients, which experience has shown
are mostly hurtful to most cytoplasmic inclusions. Neverthe-
less, there are ways in which such difficulties can be over-
come, and subsequent research will be done on this work.

The subject of this research was suggested by Dr. E. S.
Goodrich, whom I have to thank not only for his suggestions*

CYTOPLASMIC INCLUSIONS OF THE GEKM-CELLS. 40$

but also for liis many kindnesses to me in the years during'
which, a student, I had the privilege of working under him.

This work was done in the Department of Physiology
during a part of the time 1 relieved Dr. Scott, and I owe
my warmest thanks 1 to Prof. Sherrington for the way in
which he facilitated my task and encouraged my work.

A good deal of my material was derived from Prof.
Poulton’s Department, and his assistants, Mr. Hamm and
Mr. Britten, aided me considerably in finding suitable species.

It had been intended in the first place to study the
chromosomes as well as the cytoplasmic bodies, but as the
work proceeded I found that the latter structures needed
examination more than the former, and it was not possible
to study both satisfactorily at the same time. This paper
therefore deals exclusively with plasmatic inclusions.

Previous Work.

It is not intended to give here an extended account of the
great body of work done on the spermatogenesis of the Lepi-
doptera. The oogenesis has hardly been treated at all by
those observers who have examined the spermatogenesis ;
the former provides problems somewhat more difficult to
follow out, and perhaps also less attractive, for in the early
stages it is difficult to find individual oogonia and oocytes
sufficiently clear for study, so crowded are they. In this
paper I have endeavoured, as far as I found possible, to find
homologous processes going on in the germ-cells of both sexes.
Of all the work done on the spermatogenesis of the Lepi-
doptera, that of Meves (1) stands out most prominently ; Meves
was not occupied so much with chromosomes as with the cell
inclusions and the metamorphosis of spermatid into sperma-
tozoon. In all his work on the spermatogenesis, Meves has
overlooked important facts concerning the cytoplasmic bodies,
some of which he has not found, and, moreover, his modified
Flemming iron heematoxylin technique is not calculated to

1 I have also to thank Prof. G. C. Bourne for some excellent
suggestions with regard to the text.

410

J. EiiOiNTE GATJBNBY.

give the best results if it is used exclusively. Examining
Meves’ beautiful plates one would be led to believe that the
subject of the spermatogenesis of Lepidoptera, in so far as it
touches upon the mitochondria and allied structures, has been
exhausted. This is not the case. Meves has described for a
number of species a remarkable dimorphism in the manner of
formation of the sperms. Apart from the ordinary method,
he describes how the chromosomes in the second maturation
division fail to come together to form the spermatid nucleus
but instead become at first vacuolated, and then finally recon-
stitute themselves each like a small spermatid nucleus. The
behaviour of the large mitochondrial body is fairly normal.
He calls the abnormal spermatozoa “ apyrene Spermien ” and
the normal “ eupyrene Spermien,” and shows that the sperm-
bundles of “ apyrenes ” are several times shorter than the
normal “ eupyrenes.”

He finds such dimorphism in Pygaera bucepliala,
Gastropacha rubi, Bombyx mori, and in Harpyia
vinula. According to Meves, who has some weighty con-
clusions to draw from the “ apyrene Spermien,” the latter are
able solely to cause segmentation of the egg, only providing
the centrosome, but not being able to carry paternal hereditary
factors to the egg s which they fertilise, since theyhave no nucleus.

Among others, the work of Dr. M. H. Cook may be men-
tioned. In this paper (2) the chromosomes of the Lepidoptera
have been examined successfully for the first time, and though
no serious attempt was made to follow out the cytoplasmic
inclusions, Dr. Cook’s work adds many facts of interest to
our knowledge of the spermatogenesis of the Lepidoptera.
I am unable to agree with some of this observer’s statements,
especially concerning the spermatogonium, but otherwise we
are generally in agreement. The remarkable bodies in Acro-
nycta, sp., which Cook describes from a single pupa, would
repay further observation ; I have difficulty in bringing one
large accessory body, described as “ chromatin granule,” into
line with any structures that I have been able to find in the
species that I have studied. (However, see p. 446.)

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 411

Henneguy, who, apart from his original work upon the
germ-cells of Lepidoptera (3), has given a good review of the
literature of the subject in that admirable text-book ‘ Les
Insectes,’ says, concerning the metamorphosis of spermatid
into spermatozoon, “ Le noyau de la spermatide subit, comme
les elements cytoplasmiques, pendant la formation du sperma-
tozoide, des modifications importantes qui n’ ont pas ete encore
suffisamment etudiees.” That this remark is correct is con-
firmed by the difficulty one has in reconciling the statements
of the various authors. The whole question of the correct
homology of the bodies present in the spermatogonium aud
metamorphosing spermatid is in a confused state. Meves
identifies in the spermatid, a mitochondrial mass, two centro-
somes, an idiosome, and a “ spindelrestkorper.”

In Text- tig. 1 are drawn Meves’ figures to illustrate his
view. I incline to the opinion that the archoplasmic idiosome
does not exist as such in the spermatogonium of moths, and
that the “ spindelrestkorper ” is of transitory nature. I also
consider that the “ spindelrestkorper ” is absent in the
spermatid about to metamorphose, and that there is no con-
nection between the bodies marked J. and S. in the sperma-
togonium and spermatid respectively. Moreover, Meves has
tailed to account for a characteristic body in the Lepidopterous
spermatid, and he has also overlooked the second centrosome
in all his diagrams of the spermatogenesis of the Lepidoptera.
My statements, be it noted, are derived from the Lepidoptera
alone, and I cannot reconcile Meves’ sketch in Text-figs. 1, II
with anything I have seen in my sections. This question is
dealt with more fully in the discussion.

For reasons which will be clear later on, Meves’ figures
and description of the behaviour of the mitochondria during
the later stages of spermatogenesis are not altogether correct.
He leaves a great gap in the description of the behaviour
of the centrosomes and quite overlooks the micromitosome.
(He, however, figures it in one place, but does not mention
it in the text.) Some of Platner’s (4) figures, executed
nearly thirty years ago, give a remarkably true picture of

412

J. BRONTE GATENBY.

these cytoplasmic bodies, but this cytologist unfortunately
gave a confused account of idiosome, small mitosome, and
centrosome. Nevertheless the figs. 8 and 10 are very
good, and I have adopted some of Platner;s nomenclature
(Text-fig. 1, V). Quite recently Doncaster has studied the
germ-cells of Abraxas and Pieris (5). With regard to
Meves’ two types, Doncaster mentions that Prof. E. B.
Wilson suggests that the “apyrene” type may be abnormal.
Doncaster says quite rightly, “ The suggestion of Meves
that “apyrene” spermatozoa are capable of fertilising an
egg, but not of transmitting the paternal hereditary
characters is not borne out by breeding experiments, nor
do these confirm the suggestion that the two types of
spermatozoa determine different sexes in the fertilised egg '*
(vol. i, p. 183).

Doncaster in this paper also figures abnormal mitosis of
the “ apyrene " spermatocyte divisions. On the whole
Doncaster is more concerned with the chromosomes and sex
than with cytoplasmic bodies.

Technique and Material.

Almost every modern observer of the germ -cells of
Lepidoptera has used the strong Flemming-iron haematoxylin
method. Some of my material was so treated, but I soon found
this method gave only a caricature of the cells. Many acids,
and especially acetic acid, either altogether destroy, or at
least distort most plasma structures ; though acetic acid
helps to give a clearly differentiated preparation, it should
be avoided altogether, as Champy has already pointed out.
Most of my material was fixed either in strong Flemming
without acetic acid, or in Champy's fluid. Flemming, with
reduced acetic acid, according to Meves, was also used. All
the other better known fixatives were tried, but were mostly
found useless for my purpose. Sections were stained on the
slide with either iron haematoxylin, Ehrlich's haematoxylin
and Orange Gr, methyl blue eosin. Mayer's acid haemalum.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 413

pyronin and methyl green, BreinTs process or the carmine
stains. Alizarin and crystal violet, and iron hasmatoxylin
were used especially for mitochondria.

Among fixatives I also used Regaud’s formol bichromate,
but with little success in the bulk. With smears I found
that this fixative gave an extremely useful result. Testes
were smeared on a slide, fixed first in osmic vapour and
then soaked for a short time in Regaucl. Afterwards they
were transferred to 90 per cent, alcohol where they remained
several hours. They were then stained in iron hgematoxylin
by the long method. As a rule in such preparations the
mitochondria alone were stained, astral rays of the spindle
and chromosomes remaining colourless, but sometimes the
nucleus in the growth stage took up the stain. As will be
seen later this useful reaction helped me to clear up some
doubtful points. Instead of further fixation in Regaud,
smears were often transferred from the osmic vapour bath
to water and alcohol and then stained. These also gave
useful results. Bismark brown smears were not of much
help. Under separate headings I have mentioned a few
other special methods used by me.

Of the species of Lepidoptera used, Smerinthus
populi, Pier is brassicae, and Orgyia antiqua were
most thoroughly studied in the order named. I carefully
examined many preparations of Porthesia similis, Pieris
rapae, Pygaera bucephala, Spilosoma lubricipeda
(Arctia), Euchelia jacobseae, Cossus ligniperda,
Bombyx 1 a nest r is and rubi, and Abraxas grossu-
lariata. In Euchelia, Cossus, and Bombyx no maturation
divisions were found, because my material was not sufficiently
advanced in development. In Spilosoma only the degenerate
u apyrene sperms ” were to be found because I took my
larvas too late. Smears of Vanessa, Orgyia, and Porthesia
were also examined.

In later stages of this work Bensley’s permanganate of
potash, acid fuschin stain was used, but no one stain was found
to approach iron haematoxylin for certainty and usefulness.

VOL. 62, PART 3. NEW SERIES. 29

414

J. bront£ gatenby.

Nomenclature.

In the spermatogonium of the secondary group four sets
of bodies can be found in the cytoplasm : —

(1) A centrosome.

(2) A spindle body (“ spindelrestkorper,” “reste fusorial.”)

(3) A body somewhat larger than the centrosome and

staining darkly (micromitosome).

(4) A cloud of granules (mitochondria).

The first needs little notice, but I should mention that I
have been unable to find an archoplasmic region surrounding
the centrosome. Dr. Cook mentions an archoplasmic region
after a spermatogonial division before the cells have com-
pletely constricted, but figures nothing resembling Meves’
spermatogonial idiosome, nor can I find any similar body.
The spermatogonial idiosome may be present in Paludina,
but I feel sure that the centrosome in the “resting”
spermatogonium is not imbedded in any such structure as
Meves shows in his schematic plan. Even in the case of
Paludina I do not think that Meves is justified in assuming
that the acrosome body in the spermatid is identical with
the idiosome body of the spermatogonium. His seemingly
careful figures of this spermatogenesis provide not a tittle
of evidence for this view, and I am at a loss to understand
on what grounds he comes to his conclusions.

The second body mentioned is that left by the spindle
when the two cells are constricting. This body is certainly
present in both secondary oogonia and spermatogonia, but
later becomes absorbed, at least in the case of the male
germ-cell. Its probable use and significance will be discussed
in a later stage of this work.

The third body is one quite overlooked by all previous
observers. It later forms what Platner calls the small
mitosome of the spermatid; Hemmgy calls it “la petite
mitosome.” In this paper it will -be called the micromito-
some.

The fourth number refers to a cloud of granules which

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 415

are the mitochondria. They need no further mention at
this juncture.

It will now be clear that my account of the bodies in the
cytoplasm of the spermatogonium differs from that of Meves
in my denial of the presence and significance of an idiosome
(archoplasmic zone) and in my account of the micromitosoine
overlooked by the German cytologist. I would like to make
it quite clear that if any small granules do possibly appear
around the centrosome I am convinced that they have no
connection with any other body in the spermatid, and that
in this case Meves and I would differ in the significance
we attach to such a zona. No other bodies can be seen with
certainty in the cytoplasm of the spermatogonium.

Almost all the work on the cytoplasmic bodies of the
germ-cells of insects consists in the description of these
bodies from the spermatid onwards, and it is the identification
and interpretation of these bodies in the spermatid which
have led to a great confusion. Meves has, as already stated,
overlooked some cytoplasmic bodies, both Munson (6) and
Platner are confused in their treatment of these structures,
and some other authors also seem to have failed to distinguish
centrosome from acrosome. The correct usage of the term
“ nebenkern ” is doubtful. According to Paulmier (12)
u nebenkern ” means a body formed from the spindle fibres
and yolk granules. In the text-book on ‘ Cytology 3 Wilson
(7) offers the following remarks : “ The foregoing account
shows that our positive knowledge of the formation of the
spermatozoon still rests on a somewhat slender basis. . . .
All agree, further, that the middle piece is of archoplasmic
origin, being derived, according to some authors, from a true
attraction sphere (or centrosome) ; according to others, from
a ‘ nebenkern 3 formed from the spindle fibres. The former
account of its origin is certainly true in some cases. The
latter cannot be accepted without reinvestigation, since it
stands in contradiction to what is known of the middle piece
in fertilisation, and is possibly due to a confusion between
attraction sphere and f nebenkern. ’ Similar doubts exist

416

J. BRONTE GATENBY.

in regard to the origin of the apex, which is variously
described as arising from the nuclear membrane, from the
general cytoplasm, from the ‘ nebenkern/ and from the
centrosome.” No author, I believe, has given the correct
version of what really happens in the formation of the
Lepidopterous spermatozoon, and the bodies confused
generally are micromitosome, acrosome, and centrosome.
In the glossary of his book Wilson gives the following
interesting definitions :

“ Mitosome (/li'itoq, a thread; crCofia, body), a body derived
from spindle fibres of the secondary spermatocytes, giving
rise, according to Platner, to the middle-piece and the tail-
envelope of the spermatozoon. Equivalent to the Nebenkern
of La Yalette St. George. (Platner, 1889.)

“ Nebenkern (Paranucleus), a name originally applied by
Biitschli (1871) to an extranuclear body in the spermatid ;
afterwards shown by La Yalette St. George and Platner to
arise from the spindle fibres of the secondary spermatocyte.
Since applied to many forms of cytoplasmic bodies (yolk-
nucleus, etc.) of the most diverse nature.”

I have been unable to find any body in the spermatid
formed from “ spindle fibres ” or “ yolk granules,” and I do
not intend to use the term “nebenkern,” which has been,,
and still is, used without discrimination for almost any
granule or body in a cell. For example, Hegner (8) lately
draws attention to the “ granules of Blochmann ” in the wasp
and two ants, which have also been called “ nebenkerne,”
quite regardless of whether or no they are of the same nature
as the original “ nebenkern ” of Biitschli.

In Text-fig. 1, I have drawn two figures (III and IY) to
illustrate the nomenclature used in this paper. The bodies in
the secondary spermatogonium have already been considered.
In the spermatid we have the following bodies :

(1) Two centrosomes without definite archoplasmic zone.

(2) The micromitosome (identical with that body in the
spermatogonium (M.) .

(3) The macromitosome or middle part, formed from tlie

Text-fig. 1.

I and II (after Meves). Shows Meves’ view of the relationship of the spermato-
gonial and spermatid bodies. C. Centrosome. I. Idiosome. MD. Mito-
chondrium. N. Nucleus. S. Spindle bridge (spindlerestkorper). Ill and
IY. Shows the view adopted in the present paper. According to this the
acrosome originates not from an archoplasmic zone, but from a definite number
of bodies probably present in the spermatogonium ( X ). These form the acro-
blasts (XX) in the spermatid. M'. The micromitosome. Y. The spermatid
according to Platner and Munson ; the acrosome has been mistaken for the
centrosome. The micromitosome has been figured. YI. The spermatid
according to Meves. The micromitosome has been overlooked, the second
centrosome not figured (though placed in Meves’ scheme in II), and the macro-
mitosome (M") has been distorted by the acetic acid fixation. Tim acrosome
is partially dissolved away (A.). YII. The spermatid according to the present
phper. The macromitosome is a spireme (M"). The acroblasts are figured as
well as tbe micromitosome and second centrosome. G. Excretory granules.

418 j. bront£ gatenby.

mitochondrial granules which have run together to form a
spireme ( M.D. ).

(4) Several spherical acroblasts,1 which soon unite to form
the acrosome (X.X.).

(5) A number of excretory granules (6r.).

The acroblasts can be found easily in late stages of
spermatocytes and in all probability are present in the sper-
matogonium. I therefore add them to my scheme of the
spermatogonium, only it should be understood that I could
not find them until the spermatocyte was fairly large. The
point which I particularly wish to emphasise is that the
acroblasts are separate bodies in the young spermatocyte,
and have nothing to do with the centrosome or archoplasm
(if demonstrable).

If the idiosome (arclioplasmic zone) of the spermatogonium
is identical with that (acrosome) of the spermatid, we could
only make certain of this in one way, that is, by following
this body right up through the growth period ; by explaining'
how the spermatogonia! centrosome needs' an arclioplasmic
zone and the spermatid centrosome not; by describing the
time and manner of separation of the centrosome from the
archoplasm (idiosome) ; and, finally, by showing why some
spermatids have several idiosome-like bodies instead of the
regulation one. This has not been done, and I venture to
say never will be.

The Mitochondria.

in the primary spermatogonium and oogonium the nucleus
is partially enveloped by a cloud-like body, which is formed
by a closely-massed collection of minute granules. There is
no doubt that at this stage these granules, which are the
mitochondria, bear some definite relation to the nucleus.
Almost always they lie in a crescentic cloud towards one side
of the nucleus. Whether at this period they bear a relation -

1 Acroblast : This useful term was suggested by Dr. H. D. King,
‘ Amer. Journ. Anat.,’ vii, 1907-8, and denotes a body which eventually
gives rise to the acrosome.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 419

ship to the centrosome it is impossible to say. It is certain
tli at either the mitochondrial granules are not of the same
size, or they have among them other larger granules of a
different nature ; but it is always possible to detect larger
masses here and there among the smaller granules. It is
these larger masses which make it so difficult to be certain
of the identity of any given body in a primary germ-cell of
either sex, and though the granule marked M in PI. 23, fig. 1,
is probably the micromitosome, it might quite possibly be of
another nature. When the primary germ-cell is dividing, the
mitochondria become disposed on one side of the spindle, as
in PI. 23, figs. 2 and 3, and in PI. 24, fig. 21. At this stage,
and for some time afterwards, especially in the female, the
mitochondria resemble in shape rods rather than spheres or
grains.

Viewed at the metaphase, the mitochondrial cloud is found
to form a halo surrounding from one-third to two-thirds of
the surface of the equator of the spindle ; it never, or very
rarely, forms a complete circle around the amphiaster, as the
mitochondria often do, in the spermatocyte divisions (PI. 23,
fig. 11). It is impossible to say with certainty whether the
amphiastral rays are concerned with the division of the mito-
chondria between the daughter cells, but I am inclined to
think that they are not. In fact, mitochondria always seem
to. clear a path for the astral rays instead of being directly
caught up in them, and my observations seem to favour the
view that mitochondria are partly distributed by cytoplasmic
currents.

PI. 23, fig. 3, shows a later stage in division; the distribu-
tion of the mitochondrial matter seems to have been equally
carried out, and I do not remember having seen any stage
of division in which one cell appeared to be receiving more
than its share. In all the spermatogonial divisions the cells
act in the same way, and after these mitoses are finished the
secondary spermatogonium about to become a spermatocyte
possesses a cloud of mitochondrial granules at present dis-
tributed towards one side of the cell. This side is generally

420

J. BRONTE GATENBY.

the one in which lies the spindle bridge (PL 23, fig. 4), or
“ reste fusorial,” which has been formed by a thickening
of the spindle fibres at the telephase of division (see pp. 435
and 436).

In the female the mitochondrial fibres always seem to lie
quite near, even enveloping the spindle bridge (PI. 24, figs.
25, 26, and 28), but in the male the mitochondria, even if at
first they always have this relation with the spindle bridge,
soon become more granular, and tend to spread around the
nucleus, as shown in PI. 23, fig. 5. At about this period
some changes come over the mitochondria ; heretofore they
resisted the action of acetic acid (PI. 23, fig. 3, is drawn from
a Flemming fixed cell), and in material fixed with acetic acid
preservatives these bodies are either hard to see or altogether
destroyed in later stages (compare figs. 8 and 18). It should
be understood that up to the beginning of the growth stage
of the spermatocyte and oocyte the mitochondria of both
sexes are apparently identical, but after the synizesis stage
and thence forwards the bodies in either sex behave quite
differently. The case of the male will be described first.
After synizesis the chromatin soon becomes arranged, as
shown in PI. 23, fig. 5, in the manner characteristic of the
entry upon the growth stage. The mitochondrial cloud,
which in some cases looks more fibrous than granular,
gradually thins out, and moves around the nucleus till it
eventually forms a complete outer sheath to the latter. In
PI. 23, fig. 6, it is in process of forming this sheath, and in
fig. 7 the layer is complete.

As this has been taking place the individual mitochondrial
granules have been changing. As far as one can ascertain
from the powers of the microscope at one’s disposal, the mito-
chondria in the stages drawn in PI. 23, figs. 1, 2, 3, and 4 are
solid, and from the point of view of staining, homogeneous,
but from stage PI. 23, fig. 5 and onwards in the male tlie
character of these grains is altered. Each individual mito-
chondrial granule has formed within it, or absorbs in some
way, a chromophobe substance. The mitochondrial matter

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 421

properly so called forms a cover for this central core, which
shows no affinity for the stains mentioned on p. 412— It will
be seen at a later stage that this colourless inner matter forms
the mechanism whereby the macro-mitosomal spireme is pro-
duced. In PL 23, fig. 7 the granules at M.D.T. have the
chromophobe core already clearly formed, while the grains at
M.D.X. still are in the midst of forming this core. By stage
PL 23, fig. 8, every mitochondrial granule consists of the
inner and outer parts. The inner part is consequently of new
origin, and has, I believe, no counterpart in the female. Up
to the end of the growth period the mitochondria increase in
size, but thenceforth I have not found that their size becomes
greater.

A Comparison of the Mitochondrial Bodies in the
Spermatocytes of Smerinthus populi, Pieris
brassicte, Spilosoma lubricipeda, Orgyia an-
ti qua, and Pygmra bucephala.

An examination of PL 24, figs. 19 (lubricipeda), 22
(brassicae), and 24 (antiqua)1 show that these granules
have a fairly characteristic shape in different families. Smer-
inthus populi, Pieris brassicae, and Spilosoma lubri-
cipeda have a mitochondrial body about the same size in
proportion to the cell contents, but both Smerinthus and
Pieris have a larger number than Spilosoma; the latter,
again, has a larger number than Orgyia antiqua. Pieris
brassicae has mitochondrial bodies similar to those of the
allied Pieris rapae, and as numerous. Cossus ligni-
perda and Pygaera bucephala both have mitochondria
very like those of S. populi. In some ways these structures
in Orgyia are remarkable. Reference to PI. 24, fig. 24 shows
that the mitochondria of this species are very large, perfectly
spherical, and of many sizes. In the material which I had

1 The Orgyia antiqua material was fixed in Meves’ fluid. From
preparations subsequently made with my own modification of Flemming,
1 believe that the mitochondria in my figs. 24 and 27 are abnormal-
This matter is now being examined.

422

J. BRONTE GATENBY.

preserved in Flemming with reduced acetic acid, and from
wliicli PL 24, figs. 24 and 27 were drawn, the inner substance
of the mitochondria appeared to stain a very faint greyish
tint after prolonged treatment in iron-alum haematoxylin, but
this is the only case I found in all my material. Judged from
seven species of Lepidoptera belonging to different families,
it seems that, generally speaking, the mitochondrial bodies of
the spermatocyte at the end of the growth stage are of the
same general shape (spherical to ovoid) throughout, but
differences exist more particularly not only in the propor-
tionate size, but also in the number of these cytoplasmic
structures in the cell. In not every species do the mito-
chondrial bodies resemble one another either in shape or
staining affinities. PL 24, fig. 22 depicts the spermatocyte of
Pieris brassicas. The mitochondria are relatively small
and spherical, being crowded towards one side of the cell.
Some of these are seen to be somewhat compressed and
irregular, and none is found in any. cell process. The darker
bodies, marked A.B., are the acroblasts, and they are more
conspicuous than the mitochondria.

Measurement showed that the size of the mitochondria of
the several species dealt with was as follows, the average
being taken in each case, for, as already pointed out, these
bodies vary in size :

Orgyia antiqua, diameter of average full-grown mito-
chondrial body, 2 microns (M eves’ fluid).

Sme rin thus populi, 1 micron (Champy).

Pygaera bucephala, 1*5 microns (Champy).

Pieris brassicae, 1 micron (Flemming without acetic).

Spilosoma lubricipeda, *5 micron (Flemming with-
out acetic).

Vanessa urticge, -5 micron (Regaud solution).

The above measurements give the average diameter, but it
should be remembered that one could easily find a mito-
chondrial body in Orgyia as small as the average of Spilo-
soma. Some forms show a great tendency to variation in-
size (Orgyia), whilst others are more uniform (Smerinthus),

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 423

but I do not believe mitochondrial bodies of the spermatocyte
will be found specifically different enough to distinguish
satisfactorily genera, families, or even, sometimes, orders of
Lepidoptera. Exceptions to this may be found ; for instance,
one could easily distinguish between Orgyia and Pieris, apart
from other cell peculiarities in the two examples, by means of
their mitochondria, but in all probability Orgyia antiqua
and Orgyia casnosa would be just as much alike as the two
Pierids mentioned in this paper. Then, too, it should be
noticed that the Sphingidae (represented by Smerinthus) have
the same type of mitochondrial body as the Pieridas — two
families in no way related. Before comparison can be carried
further, we must have at our disposal more work on the
subject, and I leave the matter at this point.

Changes undergone by Mitochondria in the Sper-
matocyte.

The mitochondrial bodies never remain unchanged. They
are able to move about in the cytoplasm, probably being
carried by cytoplasmic currents. But there are other facts to
be noticed. Near the end of the growth stage, and thence-
forth, one often finds that several mitochondria have coalesced
or run together and form a single, very large body as shown
in PI. 23, fig. 11, V.V., and PI. 25, figs. 32, 33, 34, and 43. In
such cases I believe the running together may be caused by
the close contact and subsequent fusion of the outer rim of
the body, and the final flowing together of the chromophobe
fluid core of adjacent mitochondria. It is often found that
the mitochondria, where they are densest, run together to
form cords or filaments, as shown in PL 25, fig. 31, and there
is some probability that these large mitochondria and fila-
ments are caused by the fixative. In my material of Vanessa
fixed in the bulk in Regaud solution the mitochondria are
bead-like, as is usually the case ; in smears fixed previously
in osmic vapour and then soaked in Regaud solution the
mitochondria are filamentous, or apparently a solid mass. It

424

J. BRONTE OATEN BY.

appears to me that such peculiarities are due to the effect of
the fixative on the matter around which the true mito-
chondrial substance is applied ; thus the filamentous condition,
of which PI. 25, fig. 31 is an example, is probably due to the
rupture of the outer layer by the “brutality ” of the fixative
and the consequent running together of the rims of the mito-
chondria. This last process is one which the mitochondrial
bodies always have a tendency to undergo. It may, then, be
stated that the running together, though a natural process in
later stages, is artificially hastened by the action of the
fixative. In my sections of Smerinthus populi, the cells
nearest to the periphery have a more vacuolated mitochon-
drial mass than the cells which are in the mid-region of the
gonad, and to which the fixing fluid (Champy) must have
taken longer to penetrate.

The Mitochondria in Division of Spermatocytes.

In PI. 23, figs. 9, 10, and 11, PI. 24, fig. 27, and in PI. 25,
figs. 32, 33, and 34 are drawn stages in division.

These stages seem to show that the ainphiastral rays may
be partly concerned in the division of the mitochondria
between the two daughter cells. These bodies generally
keep clear of and rarely become completely tangled in the
spindle fibres. The mitochondria near the poles of the
spindle in PL 23, fig. 9, are unusually near to the centro-
sotnes, but there is a clear space left around the latter. This
spermatocyte was drawn from material preserved in Champy,
and the astral rays stain very slightly. The mitochondria
generally become grouped to one side of the cell as in
PI. 23, fig. 9, but this is not the only state in which they are
found. Very often they form a complete circle, as shown in
an equatorial view in PI. 23, fig. 11, and PI. 25, fig. 33. It is
during division that it becomes very obvious that the indi-
vidual mitochondrial bodies are affected by what is taking-
place in the cell ; some mitochondrial bodies become elongated
in the longitudinal axis of the spindle, others run together

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 425

with their neighbours, and also become elongated in the
same direction. In PI. 23, fig. 10, this is clearly shown, and
in extreme cases the mitochondria form threads, as in PI. 25,
fig. 31, elongated in the same direction as the spindle ; this
takes place especially in the anaphase and telophase.

In PI. 25, figs. 32, 33, and 34, some stages are shown in
which the mitochondria are blacked in for clearness. PI. 25,
fig. 32, was drawn after focussing on the cell till only
the outer layer of mitochondrial bodies were in the field ;
by screwing the microscope tube further down one focussed
upon the spindle and chromosomes, as in PI. 23, fig. 10;
only in the latter the bodies are grouped to one side
of the daughter cells. PI. 25, fig. 33, is drawn from such
a section as that through X. — X. in PI. 25, fig. 32. The
clumped chromosomes are seen in the middle, and it will be
clear from these two drawings that the mitochondria often
form two funnel-like masses with their narrow ends applied
to each other, representing the region where the cells con-
strict. In PI. 25, fig. 32, the elongation of the mitochondrial
bodies is evident ; the figure suggests that such elongation
might be due to mere mechanical reasons, pressing of the
individual mitochondria one against the other.

In PI. 25, fig. 34, a second maturation division is shown, and
the tendency to a running together of the bodies is noticeable.

In both maturation divisions the behaviour of the mito-
chondria is similar, only in the second one the latter tend to
run together more. In PI. 25, fig. 27, I have drawn a first
maturation metaphase of Orgyia anti qua to show the hap-
hazard arrangement of the mitochondria and their inde-
pendence of the spindle.

The newly-formed spermatid is^a cell like that drawn in
PI. 23, figs. 13 and 14, the mitochondria* rarely surrounding
the nucleus, but being heaped to one side of the cell. In
neither of these cases had the mitochondria run together at
all. The mitochondrial bodies now proceed to form the macro-
mitosomal spireme as follows :

426

J. BRONTE GATENBY.

Formation of Macro mitosome (“ Nebenkern ” of
some authors).

Immediately after the second maturation division and just
as the nucleus becomes reorganised, the mitochondrial bodies
show a tendency to form a horn-shaped figure (PI. 25, fig. 35).

Text-fig. 2.

1. 2, 3. Supposed method in which spireme is formed from mito-
chondria. 1. Mitochondrial bodies agglomerated together, the
running together will begin at X. Y. 2. The mitochondria (X and
Y) and their neighbours have coalesced to form two loops confluent
on the other side of the membrane (31.), with other loops likewise
formed. 3. Spireme nearly formed, the bodies A and B in 2 have
run together. 0. Outer layer (mitochondrial matter). I. Inner
vesicle of chromophobe substance represented by cross hatching.

4. Spermatocyte nest cut across the middle, showing the manner in
which the flagellate organs (in black) project into the lumen \L.).
W. Wall of the follicle cells of the cyst. M.D. Mitochondria.

5. Much enlarged view of an acroblast in longitudinal and in trans-
verse section. C.P.Z. Chromophile zone. C.H.Z. Chromophobe
zone.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 427

This process sometimes goes no further than in PI. 23, fig. 13.
When this preliminary formation of a horn-like figure has
taken place, one end, almost always the broader, undergoes a
change. The mitochondrial bodies at this region flow
together, as shown in PI. 25, fig. 35, forming at first elon-
gated structures, then loops, and finally filaments, the latter
joining up gradually to form a tangled anastomosing figure
(PI. 25, figs .35, 36, 37, 38; and PI. 23, figs. 14, 15, and 16).
At X.X. in PI. 23, figs. 14 and 15, the mitochondria are still
beadlike, just as in PI. 25, fig. 36 X. In Text-fig. 2 I have
drawn a schematic plan of the manner in which I believe
these occurrences take place. The sum total of a number of
involved changes is the formation of a perfectly coiled
spireme (PI. 23, fig. 17 ; PL 24, fig. 23; and PI. 25, figs. 39,
40, 41, and 42). In Text-fig. 2 this process is seen to fall
under three stages: the first is the preparatory clumping
together of the mitochondrial bodies ; the second, the
initiation of a flowing together to form loops ; and the third,
the joining up of these loops to form a spireme. The most
important point to observe is that the spireme is formed
from the chromophile rim (outer layer) of the mitochondrial
body, while the substance, in which the spireme lies, is the
coalesced inner substance (chromophobe part) of the mito-
chondrial layer. In Text-fig. 2, 1-3 this is shown taking
place. The macromitosome so formed is drawn in PI. 23,
fig. 17 ; PI. 24, fig. 23 ; and PI. 25, figs. 39, 40, 41, and 42 ; it
consists of two parts, the inner chromophobe cores now com-
pletely run together, and the contained spireme, or true
mitochoudrial matter, derived from the outer chromophile
part of the body.

The coil so formed is of a slightly different appearance and
size in different moths. If the diameter of the spermatid
nucleus in a number of species be taken as 1, the ratios of
the nucleus to the macromitosome in these species is as
follows :

428

J. BRONTE G ATEN BY.

Spermatid nucleus

Macromitosome,

taken as 1.

greatest length.

Vanessa urticae

1

1*5

Smerinthus populi

1

1-8

Pieris brassicm

1

2-3

Orgyia antiqua

1

1*7

A comparison of PL 24, fig. 22, and PL 24, fig. 24, of
Pieris and Orgyia shows why the former should have a larger
macromitosome in proportion to its nucleus than Orgyia •
this is the difference in number and collective bulk of the
mitochondrial bodies. Up to a point this ratio comparison
holds good, and shows that the more numerous the mito-
chondrial bodies, the larger will be the macromitosome, but
it should be remembered that there are also variations in the
comparative sizes of the nuclei of the spermatids of the
several species, a fact that must be taken into account.
There is little doubt that the reason why the Pieris ratio is
so high is partly due to the smallness of the nucleus (vide
PL 2$, fig. 22, and compare with Pl. 2$, fig. 24, etc.). .On the
average, the nucleus is half the diameter of the macromitosome
(taken in its longest measurement, for it is at this stage ovoid,
PL 23, fig. 16). Moreover, the figures in Pl. 23, fig. 17, and
PL 24, fig. 23, show that the ratio of the mitochondrial
matter (chromophile) may differ from that of the inner
substance (chromophobe), for the spireme in PL 24, fig. 23, is
very loosely coiled.

There is a very important point which should be noted at
this stage, and which is responsible for the mistaken idea
(Platner) that the macromitosome (nebenkern) is derived
from the spindle fibres of the spermatocyte division. I
pointed out, when describing the mitochondria, that as soon as
the individual mitochondrial grains began to absorb and form
within them the inner chromophobe substance, their power of
resisting acetic acid fixatives become diminished. Near the
end of growth period, when the mitochondrial mass is large,
the acetic fixed cell looks like PL 24, fig. 18, the renin ins of
the mitochondria being still evident (M.L.). But it is when

CYTOPLASMIC INCLUSIONS OF THE GEItM -CELLS. 429

t^e . mitochondrial matter lias separated fro,m the chromo-
phobe substance (PL 23, fig. 16, etc.), that the former t again
becomes distinctly, visible to the eye, and I think that this
renewed power of resisting acetic acid is caused by some
definite but unknown change, similar to that already described
just at the beginning of the growth period. The acetic acid
acts so violently upon the mitochondrial matter that it pro-
duces figures like those in Text-fig. l,v after Platner and
Meves. Here, then, one realises more fully the faulty
technique introduced into such research by dependence on
acetic acid, however much reduced, and comparison of the
figures drawn in this paper from fixatives free from acetic
acid with those of other observers who have used acetic acid,
will show that the latters’ drawings are really caricatures
produced by distortion, and this remark can be readily con-
firmed by examining material in the fresh and with intra-
vitam stains.

There are two periods when the mitochondria of moths
resist acids : (1) the pre-growth period of the spermatogonium,
and (2) the period after the formation of the spireme; by
resist ” I mean to signify the power to resist becoming stained
and visible, not the power to resist distortion, for Meves’ figures,
-66, 67, 68, and 69 of Taf. XXV J I (in 1b), show resistance to
destruction but not to distortion. Acetic acid fixatives seem
to penetrate so violently that they cause the spireme to
collapse into a shapeless mass within the chromophobe
substance.

Subsequent Fate of the Macro mitosome.

The spermatozoon was traced up to the stage drawn in
PI. 23, fig. 17, or PL 24, fig. 23. At this stage the macromi-
tosome is a distinct spireme, though I do not feel able to say
whether the coil has free ends or whether it is not so pro-
vided.1 However, as spermatozoon formation goes on, the
macromitosomal spireme appears to become gradually divided
into two by the impressing of the axial filament upon the
1 See, however, my paper on the “ Apyrene ” spermatozoa, p. 470.
VOL. 62, TART 3. NEW SERIES. 30

430

J. BRONT& GATENBY.

envelope of this body. As this goes on the chromophobe
substance dwindles, and the spireme appears to break up
partially. In PI. 24, fig. 20, is a fairly late stage. The mito-
chondrial matter is filamentous, but one wonld hesitate to say
whether the filaments were intercontinuous. In later stages
of spermatozoon formation the middle region becomes so
attenuated that it is quite impossible to say whether or not
the macromitosome is absorbed or sloughed off. I think that
the macromitosome does probably persist, but its final fate
is difficult to ascertain, and I do not regard late spermato-
genesis stages as suitable material for finally settling this
point. The obvious course is to examine fertilisation, and to
follow out the sperm after entry into the egg. Concerning
these points I shall have a few remarks to offer in the
discussion (p. 450).

What I have said under this heading naturally applies ta
the micromitosome and second eentrosome as well, for all
attempts at finding either of these structures in the ripe
sperm have failed.

It may be that the macromitosome and micromitosome
resemble the nucleus and acrosome in that they may become
so condensed as to be easily overlooked, but may, neverthe-
less, be present, and be carried into the eggs.

The Micromitosoina of Sinerinthus populi followed
out in Material fixed in Cliampy.

In the primary spermatogonium the nucleus always is
wrapped around, especially on one side, by a half-moon of
granular substance (PI. 23, fig.^. This semi-lunar shaped
zone is made up of minute granules quite darkly staining,,
and as far as one can make out not of the same size ; but by
choosing favourable examples it is sometimes possible to find
two other bodies in the cytoplasm, one often quite near the
nucleus (but almost as often isolated towards one end of the
cell), which is the eentrosome ; the other distinctly larger and
staining very sharply, which is probably the inicromitosoma.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 431

The centrosome is never, as far as I have ascertained, sur-
rounded by a definite zone (archoplasm), and the structures
mentioned by Dr. Cook are almost certainly the mitochon-
drial granules.

Though it is a matter of great difficulty, and I confess of
some doubt, to detect these bodies (mitosome and centrosome)
in the cytoplasm of the resting primary spermatogonium,
their presence is indubitably confirmed when one examines
spermatogonia in process of division. In PI. 23, fig. 2, is
drawn a inetaphase from the side. On the left are the mito-
chondrial granules (MD), while on the mid-right of the cell
are seen two bodies marked M which are the micromitoso-
mata. This figure, and the drawings in PI. 23, figs. 3, 9, and
10 ; in PI. 24, fig. 18 ; in PI. 25, figs. 34 and 49, seem to show
that this body does divide, and apparently in the early
prophases of the cell division, but I found it very difficult
to make sure. I never was quite satisfied that it really did
divide, and the only confirmatory evidence is that found in
my figures, above mentioned. Bearing in mind that it is very
rarely that one finds more than one micromitosome in the
spermatocyte, and that almost without exception every sper-
matid has such a body, one is justified in concluding that
this body really does divide. Apparently the micromitosome
divides autonomously. Confirmation of this view is derived
from the behaviour of the micromitosome in the spermatocyte
divisions, mention of which will be made later.

Reference may be made to PI. 23, fig. 3; PI. 24, fig. 21,
which show these bodies in material fixed in strong Flemming
in which the glacial acetic acid has been reduced. In this
case the body does not stain so heavily ; spermatogonial
mitoses show that the position occupied by the micromitosome
is not definite. The most that can be said is that this body
more often than not lies between the chromosomes and the
equator of the spindle, rarely being found further towards
the centrosome.

During the growth period of the secondary spermatogonium
and spermatocyte the micromitosome, which apparently has

.432 J. BttONTlL GATENBY.

taken part in every cell division, lies in no definite position;
it grows a good deal, but its staining reactions do not appear
to alter at all. I mention this especially because there was
always the possibility that it might have become less or more
densely staining in sympathy with the chromatin of the
nucleus. In PL 23, figs. 4-8, and PI. 24, fig. 19, the body is
shown in the various positions in which one may find it. In
PI. 23, fig. 9, a metaphase of the first spermatocyte division
is ehown, the micromitosomata being far apart (31.). In
fig. 18 of PL 24 a Flemming-fixed spermatocyte in the early
prophases is shown, in order to illustrate the micromitosome
(M.) apparently in division; this body is constricting, and
before the asters have moved one on each side of the nucleus
it will have divided. In fig. 18 the micromitosome is swollen
by the acetic acid of the strong Flemming solution

This cell is particularly interesting, for it appears to prove
that the micromitosome does divide without the intervention
of the amphiastral rays. In the case of the spermatogonial
division it is fairly certain that the micromitosomata are not
directly affected by the astral rays, and the same may apply
in the case of the spermatocyte division.

One can find numerous cases where the micromitosomata
have reached the opposite ends of the cell before the two
daughter cells have begun to constrict. This seems to point to
the fact that these curious bodies are separated one from the
other by cytoplasmic currents. In PL 23, fig. 9, one focussed
down upon the micromitosomata before one came to the chromo-
somes. In the spermatocyte anaphase the micromitosome
generally appears to keep well within the zone of influence
of the centrosome, as PL 23, fig. 10, shows, and I do not
remember having seen these bodies very far removed from
the aster at this period. In the second spermatocyte division
(PL 25, fig. 34) the micromitosome behaves as in the first
maturation division, and in the newly-formed spermatid
always lies near the nucleus (PL 23, figs. 12 and 13).

When the spermatid begins to metamorphose the micro-
mitosome may be orientated in any direction ; e. g. in Pl. 23,

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 433

fig. 13 ifc is below the nucleus, in PL 23, fig. 16 it is above,
and in Pl. 23, fig. 14 it is at the side, assuming that the thick
line represents the head end of a sperm (see Pl. 23, fig. 17).
The mitochondria have by now run together to form the large
mitosome (or “ nebenkern”) of Platner, or the macromitosome
under my nomenclature, and the nucleus moves up near the
wall of the sperm cyst wall (PL 23, figs. 14 and 16). The
micromitosome leaves whatever position it hitherto occupied,
and becomes placed between the nucleus and the macromi-
tosomal spireme, as shown in Pl. 23, fig. 17, and in the figures
in PL 25.

Its further history is not at all easy to follow out, for the
sperm begins to lengthen and thin out, and the frequent
presence of several acrosome bodies contributes to the
confusion.

Though I was unable to make any smears of testes of
Smerinthus, examination of such preparations from other
species leads me to doubt whether the micromitosome always
divides. I have examined a very large number of spermatids,
and a few of them do not appear to possess this body ; more-
over, it seems that the size of the micromitosome is seldom
very regular. In some cases, especially in Vanessa, this
becomes very apparent on examining the spermatids smeared
from a single nest of cells. One counts four or five spermatids
with micromitosomata, and then often finds two or three quite
near in which no such body can be found. All of such
cases are not due to the micromitosome being hidden by the
macromitosome or by the nucleus. I would not feel justified
in stating that the micromitosome of one spermatid whose
immediate neighbour had no such body was twice as big
as the micromitosomes from a group of cells all of which
possessed the body. In Smerinthus I can say that on the whole
the micromitosomes are remarkably uniform in size and in
the constancy of their presence in the developing spermato-
zoon. In such a form as Euchelia I could not find a micro-
mitosome in any spermatogonial divisions or rest stages, and
it was not until the growth-period had begun that this body

434

J. bront£ gatenby.

became quite clear (PI. 25, fig. 45). Smerinthus is a very
favourable example for the study of micromitosome, both
because the individual cells are large and the cytoplasm is
not crowded with coarse granules, as is especially the case
with Orgyia and some others.

The Cell Processes of the Spermatocyte.

Not long after the beginning of the growth period, and at
a stage which varies a good deal in different species, the
spermatocyte shows a tendency to put out cell processes.
This tendency seems correlated with the advent of the centro-
some near the surface of the cell (PL 23, fig. 6) ; in PI. 23,
fig. 7, the centrosome has divided and the cell processes are
beginning to appear. In Text-fig. 2 I have drawn a group
of spermatocytes to show the relationship of the processes to
the lumen of the cell-nest. The latter forms a sphere whose
centre is hollow (L.) and into which the cell processes stretch.
The length and character of these processes differ widely ; in
Pierids they are extraordinarily large and clavate, in some
cases at least one half of the spermatocyte is taken up in the
formation of these very large finger-like projections. In
PI. 24, fig. 22, the Pierid spermatocyte is drawn and it shows
how extensive these processes are. In some cases it appears
as if the processes sprung from a centre which is formed by
the two centrosomes; in such examples the cell projections
are arranged fan-wise, the centrosomes forming the bottom of
the handle of the expanded fan. In most other species it is
difficult to imagine any relationship between processes and
centrosomes, as in Smerinthus populi. In Euchelia, especi-
ally in early stages, the spermatocyte projects into the lumen
in the characteristic manner (not well shown in PI. 25, fig. 45),
the cell often being extraordinarily attenuated. In these cases
the centrosome (at present undivided) may or may not lie in the
single large cell process. Acetic fixatives are rather unfavour-
able, for they tend to break up these delicate organs, and
I am inclined to believe that such fixatives cause the cell

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 435

processes to disappear partly or wholly, just as these solutions
sometimes affect the pseudopodia of protozoa. Freshly teased
preparations of testes failed to show any movement of either
flagella or cell process, even though a warm stage was used.

The Spindle Bridge (Le reste fusorial).

The secondary spermatogonia and young spermatocytes and
the secondary oogonia and young oocytes are connected one
with another by spindle bridges. Such structures are well
known and need not detain us longer than necessary to draw
attention to some peculiar facts worthy of notice. In PI. 25,
fig. 46, I have depicted a nest of secondary spermatogonia of
Euchelia jacobaese, showing the spindle bridges (S.B.),
the mitochondria (M.D.), and curious interconnecting bridges
of mitochondria joining the main mass of granules ( M.D .)
with the spindle remains. These peculiar interconnecting
structures ( I.B.M.D .) are always present and remarkably clear
in Flemming fixing material. At this stage the spindle bridges
stain very darkly with iron hasmatoxylin, showing that they
are very dense. When the spermatogonia enter^ the growth
stage as young spermatocytes the protoplasmic bridges are
not at first broken, but all the cells have common spindle
bridges. As the lumen appears in the middle of the nest of
cells the bridges become attenuated, and a central darker
part in the cell is connected to the central darker part of its
fellow’s spindle bridge by a paler structure. Soon the cells
become as large as shown in PI. 25, fig. 45, and towards the
end of the prophases the cells are interconnected by a very
small, rapidly dwindling bridge ; in PI. 25, fig. 45, the bridges
are at a stage when interconnecting parts between cells (I.P.)
are very distinct, and these parts will probably soon break
asunder. The cell is left with the central dark part ( C.D.P .)
which in the male soon disintegrates. Iu the female moth
the fate of the spindle bridge is different. I have not
described the case of the female because this has been done
by Miss Pauline H. Dederer in the f Journal of Morphology/

436

J. BRONTE GATENBY.

vol. xxvi. This observer sums up the situation in the case of
the female as follows : “A transfer of material takes place
from nurse-cells to the egg through connecting tubes derived
from the spindle remains of the final oogonial divisions.” In
this paper, of which Philosamia is the type, Miss Dederer
shows that the spindle remains in this species persist for a
long time. In some cases this happens in the male also, and in
all probability what Platner draws in his PI. XIII, fig. 4, in his
paper (4) is not the mieromitosome but the remains of the long
persistent spindle fibres. I have found similar occurrences in
several species in spermatocytes about to undergo the matura-
tion divisions. In Taf. XXVII, fig. 66 of Meves’ paper there
are figured two spermatids side by side, apparently joined by
a bridge of staining matter. Meves says that the spermatid
spindle bridge sooner or later disappears. I have been un-
successful in my search for such spindle bridges in the
spermatids of any of the species I have examined; there is
often a well-marked equatorial granule (or granules).

The Probable Use of the Spindle Bridge.

In the crowded spermatogonial nests some cells are liable
to get more nourishment than their fellows and would con-
sequently grow faster than would be desirable. Where two
nests touch the cells would be less well nourished than those
cells bordering ou the part of the nest more exposed to the
nutritive fiuid of the gonad. To facilitate intercommunica-
tion between cells and complete uniformity of growth the
spindle bridge is retained. In the case of the female, Miss
Dederer shows that the spindle bridge is also concerned in
passing on nutriment. The function of the bridges is some-
what different in each sex.

The Precociously Formed Flagella.

Meves long ago described these structures, and very little
remains for mention here, but I am able to add several facts

CYTOPLASMIC INCLUSIONS OF THE OERM-CELLS. 437

to the original description. As has been said, the advent of
the centrosome, or centrosomes (if the division has taken
place), at the edge of the cell (PI. 23, figs. 5 and 6) heralds
the appearance of4 the first cell processes. If the centrosome
has not already divided it now does so, and from these two
bodies there begins , to grow out flagella, two from each. In
Pieris, Smerinthus, arid Spilosoma at least, the outgrowing
flagella bear at their tips a remarkably large clavate structure
of a definite size. In PI. 25, fig. 44, these organs are seen
just as they begin to protrude from the surface of the sperma-
tocyte. The club-like end is, as far as I am able to ascertain,
not one of the cell processes carried out in a simple manner
by the growing flagellum, but really a definite organ formed
on and around the end of the filament. The very tip of the
outgrowing flagellum is occupied by a dark spot, at this stage
quite as darkly staining as the centrosome itself, and the area
of the filament between the clavate end and the centrosome
stains fairly densely. The filament can be seen passing
through the substance of the clubbed end, as shown in PI. 25,
fig. 50, which is drawn at a later stage when the club-shaped
organ is at its largest. The terminal body ( T.B. ), the coarse,
curiously vacuolated protoplasm of the clavate end (P.R.),
and the central vacuole often present ( V.P.R .) are shown in
this figure ; the filament ( F .) is seen to pass through the
clavate organ and to end in the terminal body ( T.B. ).
Reference may be made to PI. 24, figs. 19, 22, and 24, for
this clavate flagellum. In PL 24, fig. 24 (Orgyia), the terminal
club-like organ is small. In Spilosoma it is relatively huge.

By the time the second maturation division has occurred the
clavate end of the flagellum is smaller, and rapidly dwindles
during spermatozoon formation, When the spermatozoon is
at the stage of PI. 24, fig. 20, the clavate end is difficult to
find. I believe that the terminal organ is really a store of
nutritive matter, the terminal bodj a centre of metabolism
for this matter; therefore the dwindling of the clavate end,
and the later disappearance of the terminal body may be
correlated with the growth of the flagellum. The clavate

438

J. BRONlA GATENBY.

organ, therefore, according to this suggestion, is an
arrangement for enabling the filament to grow at both ends,
the centrosome end undoubtedly assisting also ; it is possible
that the terminal body ( T.B .) is in reality a part of the centro-
some, detached and carried out on the tip of the filament in
order to assist growth at the opposite end. The dark terminal
dot is the growing tip, the clavate organ the storehouse of
food matter.

The Acroblasts and Acrosome ( Smerinthus) . *

After the individual mitochondrial bodies have become
clearly visible, one notices other more darkly staining, slightly
curved, sickle-shaped bodies, much less numerous and more
definitely located. These structures are much denser than
the mitochondria, as is shown by their staining reactions with
iron hsematoxylin. At the end of the growth period these
darker bodies appear to have some relationship with the
nucleus ; the mitochondrial bodies may be scattered irregu-
larly, but the darker bodies, which, I believe, form the
acrosome of the sperm, are placed generally with their concave
surface towards the nucleus. Moreover, they nearly always
occupy the spaces clear of mitochondrial bodies (see PL 23,
fig. 8, A. B., 9, 10, 12, and 13).

When the maturation divisions take place, the acroblasts,
as they may be called, are even more definite in their
behaviour. They are almost invariably placed in a semi-
circular figure near the aster, as is shown in PL 23, figs. 9 and
10, PL 25, figs. 32, 33, 43, and 48.

In some cases the acroblast, which looks solid for most of
the growth period, is seen to be vesicular, only one side of the
vesicle is always more solid than the other. This is shown in
Text-fig. 2, 5.

The acroblasts are never quite equal in size, but the largest
ones are seemingly formed by the running together end to
end of two acroblasts. The latter, when not vesiculiform, are
more darkly staining than when they are hemispherical. In

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 439

the newly formed spermatid these bodies occupy the position
shown in Pl. 23, figs. 12 and 13. At the time the mito-
chondrial bodies begin to run together, the acroblasts, which
are about three or four in number, gradually become vesi-
cular. This process is quite easily followed out, and can
often be seen occurring in the several acroblasts in one
spermatid (see PI. 23, figs. 14 and 15, and the figures on
PI. 25). In fact this process rarely seems to happen quite
synchronously in one cell. When formed the acroblasts are
quite spherical, and their wall is of equal thickness, not more
bulging or thicker on one side than the other. Sometimes
very small acroblasts are found (PI. 25, fig. 39).

After the various other cell elements are arranged in final
order, as shown in PI. 23, fig. 17, the acroblasts approach, and
adhere to the nucleus very often at first as shown in PI. 23,
fig. 17. In PI. 25, figs. 39, 40, 41, and 42, the formation of
the acrosome is shown. PI. 25, fig. 41, is typical ; it shows two
large acroblasts adherent to the nucleus ; where they touch the
nucleus is a small, darkly-staining body, shown also in PI. 25,
figs. 38, 39, 40, and 42. In PI. 25, tig. 42, the dark body is large,
and the acrosome has been formed and is in its usual position.
The dark dot is always touching the nucleus, and probably
formed under its influence. One can sometimes find three or
four acroblasts all adherent to the nucleus, each containing
the dark granule. The question arises as to what occurs to
the several acroblasts in such cases. I think the acrosome is
finally formed by the running together of several acroblasts.
This explains how such a large cap as that shown in PI. 24,
fig. 20,- arises. At this stage it almost surrounds the nucleus.
In PI. 25, figs. 47 and 51, other stages are shown; the four
acroblasts are together forming one large acrosomic body, and
in PI. 25, fig. 47, the running together of these structures has
taken place.

It will be seen from this account that the acrosome is
formed from acroblasts, which can be found in the growth
stage of the spermatocyte, and which act definitely, especially
in division of the cells. In Text-fig. 3 this process is shown

Text-fig. 3.

CQ •

Diagrammatic scheme of the history of the acroblasts and centro-
somes in spermatocyte, permatid, and spermatozoon. For
explanation see p. 463.

CYTOPLASMIC INCLUSIONS IN THE GERM-CELLS. 44<1

in progress, and in Text-fig. ,4 there is drawn a. semi-diagram-
matic scheme to illustrate the various stages in the formation
of acrosome and the rotation of the nucleus.

It is a very difficult matter to make certain in sections as
to whether the acroblasts are equally divided among the
daughter-cells during the maturation divisions. They are
approximately divided, it is true, for the size of the acrosomes
in any given cyst is the sanje. In Smerinthus there are from
three to five acroblasts in the spermatid, and one can count
about sixteen to twenty in the spermatocyte; in Pierids the
number of acroblasts seems to be greater, but, except* for the
fact that they are slightly more vesiculiform, the two insects
have pretty similar acroblastic bodies. In Text-fig. 2, at 5 I
have drawn schematically what I believe to be the longitudinal
and transverse section of the acroblast at the end of growth
of the spermatocyte. In Text-fig. 5 the remarkable behaviour
of the acroblasts of Pieris is shown during metapliase of the
second maturation division. The two elements, mitochondria
and acroblasts, act quite differently. The former are passive,
and evidently not so easily managed in cell-division as are the
latter, which collect near the aster. In PI. 25, fig. 48, the
prophase of the first maturation division is shown. This cell
not only shows that the mitochondria are visibly affected by
the spindle-fibres in division, for those near the fibres have
run together (X.X.), but also establishes, as do most of my
other figures, that the acroblasts have some definite relation-
ship with the nucleus and aster. The orientation of the
acroblast with the concave surface towards the nucleus is
shown as well in PI. 24, fig. 22, PI. 25, figs. 30, 31, 32, 34, 43,
and 48, and as a rule is rarely departed from. The reason for
this curious relationship with the nucleus is unknown to me,
but it is worthy of notice that this orientation is clearly
marked from the time one can distinguish the acroblasts in
the cell, and finally culminates in the attachment of these
bodies upon the spermatid nucleus. The whole series of
events seems to show that the acroblast is, especially in celb
division, a more tractable cell element than the mitochondrial

442

J. BRONTti GATENBY.

body, and the latter never bears the same relationship in later
stages to the nucleus as the acroblastic body. In Orgyia
antiqua the acroblasts seem to be vesiculiform, but as my
material is fixed in Meves* fluid I cannot be quite sure. In
PI. 24, figs. 24 and 27, these bodies are shown, but the trace
of acetic acid used seems to have abnormally affected them.
They are less visible than after Champy or Flemming without
acetic fixation. In Spilosoma the cell was too small for me to
make sure of the presence of the acroblasts in the growth
period, though the mitochondria could be seen.

*

The Mitochondria and Other Bodies in the
Female.

In every way the cytoplasmic bodies in the oogonia and
younger oocytes resemble the same structures in the male;
this is a point which has hitherto escaped notice in the Lepi-
doptera, and it is one of significance. It is not till the growth
period has begun that the male cell becomes different from
the female. In the former I have found micromitosome,
spindle bridge, and mitochondrial cloud, and these bodies are
all present in the female, though they may be slightly
different in some ways. In the oogonium the mitochondria
are grouped just as in the spermatogonium, and the same
applies to the behaviour of these bodies during the oogonial
divisions. After divisions have ended, the prophases of tho
heterotypic division begin, and the cell looks like that drawn
in PI. 24, figs. 26 and 28. The spindle bridge at this period
may be relatively enormous, as in the latter figure, and it is
often surrounded by, or closely related to, the mitochondria
in some way or other (PI. 24, fig. 26) ; these cases vary greatly.
In PI. 24, figs. 25, 26, and 28, there is a body (M.) which i&
almost certainly the micromitosome ; in both Smerinthus and
Pygaera I am quite certain of its presence. If ovaries and
testes are preserved and stained at the same time these bodies
can readily be compared (see PI. 23, fig. 4, and PI. 24, fig. 25-
both oocyte and spermatocyte being at contraction figure),
The micromitosome of the female seems to stain less heavily

Text-fig.

CYTOPLASMIC INCLUSIONS OF THE GERM-CULLS. 443

444 j. bkonte , gatenby.

than that of the male, though this may be accounted for
when one remembers " that the fixing fluid has different
amounts of tissue to penetrate in the testis than in the ovary,
and length of fixation and accessibility to fixation will cause
great differences in staining.

I liave been unable through Jack of suitable material to
follow the micromitosome through oogonial divisions, but it
is quite possible that the nurse-cells differ from the oocytes
in that the latter may possess a micromitosome, the former
not. The search I was able to make showed that this view

may be true, but good enough mitoses were not available

to follow this body. It is very plain in Pygsera

bucephala, and this moth will make a good species for
study, because its cells are so large. In later oocytes
(PI. 24, fig. 29) one often finds a large vacuolated body
marked X. which I believe is the micromitosome; it seems
to break up about this period ; but my material is not
extensive enough to allow me to make a definite state-

ment.

In PI. 24, figs. 25, 26, and 28, the centrosome and
mitochondria are shown. The latter are somewhat different
from those of the male ; this difference may be due to the
accessibility to the fixative in the female, for in later stages I
know that the more peripheral spermatocytes and spermatids
are differently fixed from the medullary ones. In the male
the mitochondria are granular, in the female fibrous, but
exactly this difference in later stages can be demonstrated
in the male. (Compare PI. 25, figs. 30 and 31.) The
mitochondria of the female never become far removed from
the nucleus, are always minute, never have formed within
them the cliromopliobe medulla, and in late stages form a
cap on one side of the nucleus (PI. 24, fig. 29). Their
subsequent fate in the maturation divisions and in fertilisa-
tion is unknown.

It is noticeable that the semilunar cloud of granules near
the oocyte nucleus is not destroyed by acetic acid fixatives
-at this period, and in this way resembles the mitochondrial

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 445

Text-fig. 5.

MD

Outline drawing by camera lucida to show varying behaviour in mitosis of
mitochondria ( M.D .) and acroblasts ( A.B .) in the first spermatocyte
division of Pier is brassicse. The mitochondria are passive, while the
acroblasts are caught up in the asters, and are orientated with their concave
surface towards the chromosomes. X 4250.

VOL. 62, PART 3. NEW SERIES.

31

446

J. BRONTE GATENBY.

bodies in the spermatogonium and young spermatocyte
(see PL 25, fig. 45). It is only after the formation of
the chromophobe medulla that the acid completely destroys
the male mitochondria.

Other Cytoplasmic Bodies.

In Orgyia antiqua there are numerous large granules
which are drawn in PI. 24, fig. 24, X.G., and X.M.; these
are rarely of a size, bnt almost always there is a very large
granule ( X.G .) which goes to one or the other daughter- cell
in divisions. This large body always has a clear centre and
is a constant factor ; in the spermatid it becomes carried out.
to the end of the cell and finally sloughs off (PI. 24, fig. 23).
There are other bodies of smaller size (X.M.) which are probably
of the same nature as the last mentioned. Besides these
one finds much smaller granules (G), which are very evident
especially in division. For this reason Orgyia is unsuitable
material for following out any cytoplasmic bodies except
mitochondria, for there are so many granules that confusion
arises. Even in the sperm atogonial divisions large granules
are present. One concludes that these granules are excretory
in nature, and are therefore of little importance from the
point of view of this paper. Were further material at
my disposal it might be possible by suitable methods to
detect differences in these granules, but this has not been
possible with Flemming with reduced acetic acid and iron
hmmatoxylin. Probably Miss Cook's “chromatin granule"
in Acronycta sp. is an excretory granule, or at least a body
of the same nature as one of those mentioned above in
Orgyia. In nearly all spermatids in Pieris, Smerinthus
and others there is a mass of excreted matter sloughed off
(see PI. 23, figs. 15 and 17; PL 24, figs. 23, etc.) during
spermatozoon formation .

Abnormalities.

Though a very large number of gonads of y several species
were examined nothing abnormal in relation to the sex was

/

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 447

found. All larvae and pupae examined by me were either
males or females. With regard to cytological abnormalities,
one found nothing very remarkable. By means of smears1
fixed in osmic or in formalin bichromate or in both, it
is possible to find some curious facts in connection with
mitoses and mitchondria. With the exception of Munson,
who figures several abnormalities, observers have overlooked
these exceptional stages; the reason for this is that such
abnormalities can only be detected successfully in smeared
preparations, which most other observers of Lepidoptera
have not used. However, PI. 25, figs. 30 and 43, were
drawn from sections of Smerinthus. The former shows a
binucleate cell, with three sets of centrosomes, and two
micromitosomata ; the mitochondria and acroblasts are
normal; such cells seem to arise by the cell-plate not
forming during a spermatogonial mitosis. The latter figure
(PI. 25, fig. 43) shows a triaster mitosis.the twenty-eight chromo-
somes being distributed as indicated ; eight to one aster,
four to another, and sixteen to the other; each aster has its
acroblasts and the biggest aster has the most mitochondrial
matter near it. This was a probably first maturation division
and was found in a nest of second maturation divisions ; it
is larger than the usual spermatocyte at the end of growth
period and this accounts for the delay in - divisions ; I take
it to be a first maturation division by the fact that two of
its centrosomes each had two flagella. In PI. 25, figs. 52
and 53 are shown drawings from smears of Vanessa testis;
the first figure shows an accessory macromitosome ( M.D 4),
•evidently formed by some mitochondria being isolated during
spireme formation ; the second figure shows a double cell
in which one macromitosome has more matter than! its
share.

1 Too much reliance cannot be placed on abnormalities found in
smears, for during the process of spreading the gonad on the slide the
fresh cells often either burst or run together.

Nucleus and . '

centrosomes. Mitochondria. Micromitosome. Acrosome. Cytoplasm.

448

J. BRONTE G ATEN BY

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CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 449

The Fixing and Staining Reactions oe the Cell Elements
of Spermatocyte and Spermatid.

In table form I have set out on p. 448 the reactions of
the nucleus, centrosomes, mitochondria (macromitosome),
micromitosome, and acrosome to the various fixatives. Iron
haematoxylin and some other stains were used to judge
staining power, any staining method serving to standardise
a number of fixing methods.1

These results are mainly derived from Pieris, Smerinthus,
Porthesia, and Vanessa. A compromise between Fixatives 1
and 2 can be got by using reduced acetic acid, but as
Champy has insisted the use of acetic is not really indicated,
and constantly leads to faulty results. The micromitosome
in some forms seems to be more affected than in others,
and I am inclined to believe that this body is somewhat
different in different species. In Porthesia I believe the
micromitosome is constantly present, but as my material
was all fixed in Flemming, the body was very vague and
lightly staining. The Regaud reaction is a very useful
one, for from it we know that the acroblasts are in some
way different from the mitochondria. This applies also to
the centrosome ; the whole table of reactions shows that
the acetic acid favours nucleus, centrosome, and aster. The
mitochondria are different in this way, and again the
acroblasts are more delicate than the mitochondria. I
believe that acetic acid should never be used unless for
very special purposes. A great deal of the work at present
done on chromosomes is carried out with acetic fixatives;
these are unnecessary and probably destroy many features
of interest in the cytoplasm. Of course, in the cases where
chromosomes are very small acetic acid is an advantage for
it gives a sharp stain. A curious effect of Champy is
sometimes to cause the nucleolus to stain very black and

1 The time given to washing out of the material after removal from
the fixative is an important factor in staining.

450

J. BRONTE GATENBY.

the chromosomes hardly at all ; this might be a useful
reaction in some work (see PI. 25, fig. 48).

The Probable Nature of the Micromitosome.

Some observers have referred to this body as a “chromatin
granule.” If one stains in pyronin and methyl green the
micromitosome is red, and the chromatin green.1 Iron
Haematoxylin is not a specific stain for chromatin, and the
fact that it stains the micromitosome darkly, merely means
that the latter is of a dense nature. If the micromitosome
really divides autonomously it resembles the centrosome
somewhat in this way, but the Regaud test shows that the
centrosome will not stain where the micromitosome will.
According to the Regaud reaction the latter resembles
mitochondria more than chromatin, but the value of the
test is doubtful. The important fact to be noticed is that
very probably the micromitosome is altogether of a different
nature from chromatin, and should not be loosely called a
“chromatin granule.” Montgomery (9) calls certain bodies
he finds “ chromatoid granules,” which is non-committal.

Discussion.

The few facts brought together in this paper show that
Meves’s schematic comparison of the bodies in the sperma-
togonium and the spermatid probably does not hold good for
the Lepidoptera. In particular I have the greatest suspicion
of the view at present current, that the acrosome is derived
from the arclioplasmic idiosome detached from the centro-
somes. I believe that at present such a view cannot be
entertained for a moment in Smerinthus or any other Lepi-
dopteran upon which I have worked, and when the sperma-
togenesis of the mammal has been more carefully studied, I
think that the acrosome will be found in the growth stage, as
a body independent of the centrosome, at every stage of its
existence.2

1 This, however, does not show that the micromitosome is not
chromatin ; for in later stages (spermiogenesis) the nucleus also
becomes red for a time.

2 In Helix aspersa the acrosome has no relation to the archoplasm.

CYTOPLASM tC INCLUSIONS OF THE GERM-CELLS. 451

The behaviour of the mitochondrial bodies, their chromo-
phobe and chroinopliile zones, and the eventual formation of
the spireme, which is described here in Lepidoptera for the
first time, have not hitherto been properly examined.
Munson (6) seems to have been the latest observer to attack
the problem, but none of the above structures have been
correctly described. The formation of the acroblastic body
at the head of the sperm, from the several acroblasts, the
behaviour of these bodies in division of the cells, are all facts
which have either been altogether overlooked or incorrectly
described. The micromitosome has not been traced back to
the secondary spermatogonium, and the division of the centro-
some has been overlooked.

The fate of the middle piece (macromitosome) cannot be
successfully followed out in later stages in spermatozoon
formation, and until fertilisation stages have been examined
after fixation in suitable media, it will be impossible to tell
whether it even enters the egg. In the same way the behaviour
of the mitochondria in the egg at this stage will provide
interesting material for research. It can fairly be stated here
that it is most unlikely that such a body as the macromitosome,
bearing in mind its origin and remarkable formation, does not
enter the egg, for it would then seem that all the complicated
evolutions of the mitochondria were for nought. Theoretically
one might suppose that three bodies at least met and fused in
fertilisation — the pronuclei, the mitochondria, and the micro-
mitosomata. We know already that the nuclei do fuse, but
the case of the last two is less certain. In addition, of course,
one would find observers who would wish to suggest that the
two cytoplasmas (if the male does introduce cytoplasm) met
and fused ; but the sperm of the moth is too small for one to
judge, whether or no, cytoplasm is present in any appreciable
quantity.

The acroblasts and mitochondria are bodies of great
interest; in the newly-formed spermatid it has been shown
that each half-moon shaped acroblast becomes spherical.
This evidently takes place by the absorption of a chromo-

452

j. bront£ gatenby.

phobe fluid similar to the same substance inside the mito-
chondrial body; the ultimate use of this region in the
acroblast is also similar to that of the mitochondrial chromo-
phobe zone. It enables the acroblasts to fuse readily, and is
very probably in some way connected with the formation of
the acrosomic granule. The latter seems to be the essential
organ in the formation of the sperm head, and the acroblasts
are the vehicles whereby it is secreted. From the manner in
which the granule appears it is extremely likely that the
nucleus is the prime factor in the secretion of this body. The
manner of origin of the acroblasts is unknown to me ; they
can only be found with certainty at the time the spermatocyte
is fairly well advanced in growth period, but one is justified
in assuming that the matter or bodies from which the acro-
blasts originate is represented in the spermatogonium. It may
be possible, with improved technique, definitely to identify
acroblastic material in the latter cell ; exactly what becomes
of the acrosome in fertilisation is not properly understood, for
the current idea that it degenerates may not be true. Instead
one might be justified in assuming that the acid and alcoholic
fixatives destroy it, and cause it to be invisible just as the
same media do in growth stages of the male germ-cell. Until
this matter has been completely examined with suitable
technique we are not justified in paying no attention to the
acrosomic body after entry of the sperm. It may be true that
the acrosome persists in some way or other, but the point I
wish to emphasise is that we cannot tell whether it does or not
until we use proper methods.1 Carnoy, Petrunkewitsch and
such fixatives are not suitable for this work.

It has occurred to me that the acroblasts may be mito-
chondrial bodies of a modified nature, but I have little
evidence at present either way. The number of the acro-
blasts seems to vary somewhat, and they may well be mito-
chondria which have not absorbed the chromophobe zone.

1 For similar view see F. Payne, ‘ Journ. Morph.,’ vol. 28, No. 1,
p. 311.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 453

In later stages, after the assumption of the vesicular shape,
the acroblasts are readily mistaken for mitochondria.

Thos. H. Montgomery, jun. (9), in his paper on the “ Sperma-
togenesis of Euschistus,” figures in PI. I, fig. 1, a sperma-
togonium with “mitosome” and “ idiosome.” According to
my interpretation his “ mitosome ” is a spindle bridge, and
his “ idiosome ” a cloud of minute mitochondrial granules.
His mitochondria in late stages I believe to be run together
into threads by the acid fixatives he has used. His mito-
chondria possesses no chromophobe zone, they are all chro-
mophile, and his figures of maturation divisions give the
impression that the mitochondrium is being divided auto-
nomously. If one examines Henneguy’s figures of Pyrrho-
co'ris apterus, stained in violet dahlia intra vitam Les
Insectes/ p. 661), it will be seen that the mitochondria form
long fibres, but that each rod consists of a number of mito-
chondrial grains containing the chromophobe zone. I there-
fore believe that the mitochondria of Euchistus are really like
those of Pyrrhocoris, only Montgomery has used acetic acid
and produced threads.

We now come to the question as to whether the filament or
the granule is the true form of the mitochondrial body. My
study of the Lepidoptera causes me to believe that with
proper fixation the mitochondria will be found to be in the
form of grains, but that the grains often tend to lie in chains,
as Henneguy shows (f Les Insectes/ p. 661), and that brutal
fixation produced by acetic acid, or by other unsuitable
media causes the whole chain to coalesce. Even Champy and
Flemming free from acetic often cause distortion, if not
properly diluted to suit the material used.

I have at present very little evidence to offer concerning
the theories of division of mitochondria, and as to whether
.they are the " chromosomes of the cytoplasm,” as Meves
would say, but I feel sure that no division of the mitochondrial
body in the maturation divisions can be demonstrated with
any degree of certainty. The view I take is that the mito-
chondrial granules are not divided individually into parts, but

454

J. BRONTE GATENBY.

are distributed whole to one or the other daughter cell. I
have already shown that abnormalities may occur, and that
one cell may get more than its share of the mitochondrial
mass, but for Meves's conclusion that the mitochondria carry
the hereditary characters of the cytoplasm we need evidence
and not supposition. The view is interesting and worthy of
the most careful attention.

Meves (10) claims in Ascaris that the mitochondria of the
female fuse with those of the male, a view previously urged
by Zoja (11), but, as far as I know up to the present time,
nothing similar has been described elsewhere. With regard
to the question of the origin of the mitochondria in the young
germ-cells, I think that in moths they are already present
from the segregation of these cells, being passed on perma-
nently from parent to offspring, but in other forms there is
still the question as to the origin of the mitochondrial
granules from the nucleus.

We now come to a review of Meves’s “apyrene” and
“ eupyrene ” spermatozoa. Meves fully believed that the
“apyrene” sperms were functional, though he only had his*
theory to go upon. Doncaster (5) is sceptical of the truth of
Meves;s conclusion that the spermatozoa of the two kinds may
affect sex. The “ apyrene spermien ” I believe to be degene-
rate, and of no importance in fertilisation ; those who have
studied germ-cells even cursorily have noted that degeneration
takes place not at one stage but at every step. Meves’s
“ apyrene ” sperms, I believe, are examples of flagging
energy in cells, and are due to some cause unknown to us.
I have taken testes full of “ apyrene spermien ” and stained
fresh in osmic acid or scarlet red, and have found the walls
of the testis to be full of fat cells and globules. The
degeneration observed cannot be due either to a want of
nourishment or a diversion of such nourishment to other
sources. Whole nests of spermatogonia and oogonia in many
animals are to be found in process of degeneration. In some
way or other the “apyrene” chromosomes lack the energy,,
whatever that may be, to coalesce to form the nucleus.

CYTOPLASMIC INCLUSIONS OF THU GEltM-CELLS. 455

Meves does not describe the acroblasts, for he has not found
them, but it would be of great interest to know what happens
to these structures.1 We know that the macromitosome is
apparently normal in the “ apyrefpe ” sperm. This shows
that the nucleus is not concerned in the formation of this
cell organ ; probably the macromitosome, if I may use the
expression, is “ looked after ” by the centrosome or centro-
somes. The acroblasts, by their behaviour in growth and
division, seem to rely on the nucleus ; we thus get the interest-
ing state of different cell elements being independent inter
se and governed by special parts of the cell. The centro-
some governs the macromitosome, the nucleus influences the
acroblasts, and the centrosome keeps close to the nucleus ;
and so the elements are orientated correctly in the sperm.
Exactly what part each plays it is quite impossible to state,
and the extent in which cytoplasmic movements affect the
question cannot be decided at present.

The moth or butterfly never lacks sufficient normal sperma-
tozoa, and it is not until a great number have been formed
that the “apyrene” spermatozoa become formed in any
number; but in many species in later stages the abnormal
spermatozoa are exclusively found in process of formation.

Von N. Divaz, in a late paper (14) on the “ Spermato-
genese von Naucoris cimicoides,” treats of spermato-
genesis from the spermatocyte at the end of growth stage
onwards. He identifies, in the cytoplasm of the first
spermatocyte, some three bodies, which he calls Archoplasmaj
and several chromophile bodies (Chromatophile Korperchen).
Since he has partially destroyed the mitochondria with acetic
fixation, his account of the formation of the macromitosome
is inadequate. In PL 1, fig. 1, he depicts, but does not
mention, the mitochondria, which are in a dissolved con-
dition. His archoplasmic corpuscles are probably acroblasts,
and he figures them with a crescentic edge turned towards
the nucleus, in the opposite way to that in which I found
them in Lepidoptera. In the spermatid he believes that the

1 See my paper on the “ Apyrene spermatozoa.”

456

J. BRONTE GA TENBY.

chromatophile corpuscles join the archoplasmic bodies, and
that these two categories of structures together form the
acrosome. I have not examined any material of this Hemip-
teron, but I rather doubt this description. He does not
figure the centrosomes at any stage of sperm formation till
the tail is beginning to grow, and according to his figures the
centrosome does not divide.

Following von Divaz there are formed in the cytoplasm two
zones, peripheral and central, the latter forming the macro-
mitosome (nebenkern). I am unable to compare this view
with what I find in Lepidoptera. Divaz figures a macro-
mi tosome, but gives the name “ mitochondrial body ” to
another structure which is not of the same nature or staining
affinity as what is generally called “ mitochondrial; ” The
formation of the macromitosome and the appearance of what
von Divaz calls the mitochondrial body is peculiar, and should
be properly examined to ascertain its real nature, and its
origin. Dr. Goldschmidt (16) lately discusses the “apyrene”
spermatozoa from the fertilisation point of view, and, in
agreement with the view already stated above, declares that —
uWe think it not unsafe to conclude from these facts that
apyrene spermatozoa can not induce development, even if
they enter the eggs, which, however, also seems improbable.”

In the spermatogonium and early spermatocytes of moths
the mitochondria appear to be dividing autonomously as they
lie in the cytoplasm, but it would be impossible to come to a
decision from such evidence, for the appearance of being in
apparent division might be caused by the temporary fusion or
apposition end-to-end of two separate mitochondrial bodies.
Nevertheless I am inclined to believe that the mitochondria
divide at this period by simple constriction, and some recent
work which I have carried out on these bodies in the frog and
the snail leads me to think such a view worthy of considera-
tion. Future work on some form whose mitochondria1 are

1 In a forthcoming paper by me, two kinds of mitochondria are
described in Helix, as well as some other constantly occurring bodies
of doubtful nature.

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 457

large and not too numerous may finally settle this important
question. It might be assumed that the spermatocyte mito-
chondrium divided twice, so that each sperm finally possessed
a part of the original mitochondrium. From the method of
distribution of these cell inclusions between the daughter-cells
I believe that no such mathematically correct division takes
place in any mitosis, and the slightly varying size of the
macromitosoine does not give support to this view.

Summary.

(1) In Smerinthus populi, Pieris brassicae, and a
number of other species of moths and butterflies the cyto-
plasmic bodies have been followed out.

(2) The micromitosome has been followed from the sper-
matocyte back into the secondary spermatogonium. It is
very probably present in the primordial germ-cell.

(3) The micromitosome has been definitely found in the
female.

(4) The micromitosome seems to divide in all divisions, and
I consider that it is a constant factor in the spermatids of
Smerinthus.

(5) The probable nature and function of the micromitosome
is discussed.

(6) The mitochondria have been carefully examined in the
male and female germ-cell in all stages except in the matura-
tion division of the female and in fertilisation.

(7) It has been shown that in early stages the cytoplasmic
bodies of the female resemble those of the male.

(8) There is a definite period, judged to be about the
beginning of growth stage, when the subsequent fate of the
mitochondria in the male becomes different from that of
the female.

(9) The remarkable formation of chromophobe and chromo-
phile zones in the male mitochondrial body and the use of
these zones are described.

(10) The formation of the macromitosome from the mito-
chondria is described.

458

J. BRONTE GATENBY.

(11) The changes undergone by the macroniitosome in
sperm formation are followed out.

(12) The presence of the acroblasts in the fairly early
growth period of the spermatocyte is described.

(13) The complicated evolutions of these bodies in division
of the cells, their subsequent fate and their probable nature
are discussed.

(14) The staining and fixing reactions of the cytoplasmic
bodies are fully described.

(15) A number of abnormalities have been described.

(16) The centrosome has been shown to divide in the young
spermatid, and one centrosome is probably lost, but definite
evidence is not forthcoming.

Bibliography.

1. Meves, F. — “Ueber Centralkorper in den mannlichen Geschlechts-

zellen von Schmetterlingen,” ‘ Anat. Anz.,' Bd. xiv.

la. “Ueber den von v. La Valette St. George entdeckten

Nebenkern (Mitochondrienkorper) der Samenzellen,” ‘Arch. f.
mikr. Anat.,’ Bd. lvi.

lb. “ Ueber oligopyrene und apyrene Spermien un uber ihre Entste-

hung nacli Beobachtungen am Paludina un Pygsera,” ibid.,
Bd. lxi.

2. Cook, M. H. — “Spermatogenesis in Lepidoptera,” ‘ Proc. Acad.

Nat. Sci. Philadelphia,’ Bd. lxii.

3. Henneguy. — (See Bibliography in ‘ Les Insectes.’)

4. Platner, G. — “ Samenbildung und Zelltheilung im Hoden der

Schmetterlinge,” ‘ Arch. f. mikr. Anat., xxxiii.

5. Doncaster. — ‘ Journal of Genetics, vols. 1, 3, and 4.

<3. Munson. — “ Spermatogenesis of the Butterfly, Papilio rutulus,”
‘ Boston Soc. Nat. Hist.,’ vol. xxxiii.

7. Wilson. — ‘ The Cell.’

8. Hegner, R. W. — “ Studies on Germ-Cells,"’ ‘ Journ. Morph.,' xxvi,

1915.

9. Montgomery, T. — Ibid., vol. xxii.

10. Meves. — “ Uber die Beteilung der Plastochondrien au der Befruch-

tung des Eies von Ascaris,” ‘Arch. mik. Anat.,’ Bd. 76.

11. Zoja,. — “Uber die fuchsinopliilen Plastidulen,” ‘Arch. Anat. u.

Entwick.,’ 1891.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 459

12. Paulmier. — ‘ Journ. Morph..’ xv.

13. Dederer. — I bid., xxvi.

14. Yon Divaz. — ‘ Zool. Anzeig..’ 1914-1915.

15. Pantel et Sinety. — ‘ La Cellule,’ t. 23, 1906.

16. Goldschmidt. — “ The Function of the Apyrene Spermatozoa,”

‘Science,’ Friday, October 13th, 1916.

EXPLANATION OF PLATES 23 ^nd 24,

Illustrating Mr. J. Bronte Gatenby’s paper on “The Cyto-
plasmic Inclusions of the Germ-Cells.”

Explanation of Lettering.

(For x see text.)

A.B. Acroblast. A.S. Acrosome. A.F. Axial filament. C1. Proximal
centrosome. C2. Distal centrosome. C.O. Chromophobe zone of macro-
mitosome. C.P. Cell process. C.D.P. Central darker part of spindle
bridge. E.P. Equatorial plate granules. F. Filament. G. Excretory
granule. I.B.M.D. Interconnecting bridge of mitochondrial grains.
I.P. Intercellular part of spindle bridge. L.M.D. Mitochondrium
elongated by pressure. M. Micromitosome. M.D. Mitochondria.
M.D.X. Mitochondria without well-marked chromophobe medulla.
M.D.Y. Mitochondria with chromophobe medulla well marked.
P.B. Protoplasmic part of terminal body of flagellum. S.B. Spindle
bridge. S.P. Macromitosomal spireme. T.B. Terminal body of axial
filament. V. Chromophobe part of large mitochondrium formed by
coalescence of several individual bodies. X.G. and X.M. Excretory
grains (?).

PLATE 23 (Smerinthus populi).

With the exception of fig. 3, all figures drawn from material pre-
served in Champy and stained in iron hsematoxylin. Drawn with camera
lucida, at table level, with T^th oil immersion lens by Koristka and
12 compensating eyepiece. Magnification now 2250.

Fig. 1. — Primary spermatogonium showing mitochondria (M.D.)
applied to nuclear membrane. The centrosome, and what is probably
the micromitosome (M.), lie near.

460

J. BRONTE GATENBY.

Fig. 2. — Spermatogonial metaphase from side. On the left are th&
mitochondria and on the right are two equal-sized bodies which are
the micromitosomata. The side of the cell drawn in more thickly is
the morula- wall.

Fig. 3. — Spermatogonial anaphase from Flemming with reduced
acetic acid fixation. On right is in the lower cell a micromitosome
and on left are mitochondria, and in the upper cell the other micro-
mitosome. Between the cell are three small thickenings forming an
equatorial plate.

Fig. 4. — Contraction figure stage showing micromitosome (M.), centro-
some ( C .), mitochondria (M.D.), and the “ reste fusorial,” or spindle
bridge ( S.B ).

Fig. 5. — Young spermatocyte in growth stage showing mitochon-
drial cloud, micromitosome, and centrosome which has come to surface
of the cell.

Fig. 6. — Later stage, showing mitochondria, micromitosome, and
centrosome which has divided and from which four flagella begin to
appear. Cell processes (C.P.) appear.

Fig. 7. — Spermatocyte with mitochondria better defined, centrosomes
separated, and filaments longer. In some parts (M.D.Y.) the mito-
chondria are larger than in others ( M.D.X. ).

Fig. 8. — Almost at end of growth-period, showing well-defined mito-
chondria forming a crust around the nucleus. Micromitosome near
top of figure. Acroblasts (A.B.) with concave surfaces to nucleus.

Fig. 9. — First maturation division showing arrangement of mito-
chondria. Micromitosomata near centrosomes. Acroblasts near poles
of spindle.

Fig. 10. — First maturation anaphase, showing micromitosomata (M.),
equatorial plate thickenings {E.P.), and mitochondria. Right centro-
some dividing. Acroblasts orientated in the characteristic manner.

Fig. 11. — Equatorial view of first maturation division spindle show-
ing precocious running together of mitochondria (F.) and the twenty-
eight chromosomes.

Fig. 12. — Spermatid just after second maturation division, showing
mitochondria, micromitosome ( M .) and centrosome, which is just
dividing. One centrosome keeps its connection with the axial filament.
At A.B. are several acroblasts.

Fig. 13. — Spermatid at a little later stage ; only one centrosome and
five acroblasts could be found.

Fig. 14. — Spermatid showing the incipient formation of the macro-
mitosome (nebenkern); on the right a continuous chord is already

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 461

formed, while on the left the mitochondria are still separate. At A.B.
are two acroblasts.

Fig. 15. — Spermatid, with centrosomes, micromitosome, macromito-
some, acroblasts, and nucleus. At XI. the mitochondria have not yet
completely run together, and there is no true spireme in the macromi-
tosome. The nucleus lies towards the lumen of the spermatid group,
and will have to travel around as it is doing in the next figure.

Fig. 16. — Same stage as fig. 15, only the nucleus is nearer its goal.
The acroblasts are nearly spherical.

Fig. 17. — Nucleus in its definitive position. Centrosomes marked
Cl and C~, the latter now having passed towards the tail, not having a
definite connection with the axial filament. The acroblasts are pressed,
in part, between macromitosome and nucleus. Micromitosome at M.
The outline of the neighbouring spermatozoa is drawn at X.Y. One
acroblast has formed the acrosomic granule.

PLATE 24.

Figs. 18, 20, 21, 25, 26, and 28 drawn at the same magnification as in
PI. 1. Fig. 29 is drawn to the same scale, but has been reduced by one
half. All these figures are of S. populi. Figs. 19, 22, 23, 24, and 27,
drawn at table level with camera, lucida and with a y^th Koristka semi-
apocliromatic oil immersion and compensating eyepiece 18. Magnifica-
tion, 4250.

Fig. 18. — Spermatocyte of Smerinthus populi preserved over-
night in strong Flemming, to show effect of the acetic acid. Nuclear
matter has become resolved into chromosomes. The micromitosome
(M.) is much swollen and is apparently about to divide ; the amphi-
aster in is process of formation. (Compare with fig. 8, PI. 1.)

Fig. 19. — Spilosoma lubricipeda preserved in strong Flemming
without acetic acid. Spermatocyte near end of growth-period, from an
“ apyrene ” group. The filaments terminate in the large clavate organ

(C.O.).

Fig. 20. — Spermatozoon of S. populi well advanced in meta-
morphosis. Shows acrosome and granule (A.G.).

Fig. 21. — Metaphase of spermatogonial division in S. populi, show-
ing mitochondria ( M.D .) a micromitosome (M.), and the persistent
nuclear membrane.

Fig. 22. — Spermatocyte of Pier is brassicse at end of growth-
period, showing various cell details.

Fig. 23. — Young spermatozoon of Orgyia antiqua (Flemming
with reduced acetic acid).

VOL. 62, PART 3. NEW SERIES.

32

462

J. BRONTE GATENBY.

Fig. 24. — Spermatocyte of Orgyia at end of growth-period to show
mitochondria and other cell inclusions (Flemming with reduced
acetic acid).

Fig. 25. — Three oocytes of S. populi in contraction figure stage,
showing micromitosome (M.), spindle bridge ( S.B. ), centrosome ( C .),
and mitochondria ( M.D. ). Drawn to same scale as the spermatocyte
in PI. 23, fig. 4 (Champy).

Fig. 26. — Oogonium before contraction stage showing spindle bridge
in transverse section, surrounded by mitochondrial cloud. Micromi-
tosome (M.) near the upper end of the cell (Champy).

Fig. 27. — First spermatocyte division of Orgyia antiqua showing
spindle granules ( S.G .) and other cell structures (Flemming with
reduced acetic acid).

Fig. 28. — Oogonium of S. populi with spindle bridge in longitudi-
nal section. Shows also, micromitosome (M.), mitochondria ( M.D. ),
and two centrosomes (Champy).

Fig. 29. — Oocyte of S. populi showing appearance of cell inclusions
at later stage.

PLATE 25.

[All figures on left side of Plate 25 are drawn at table level with
camera lucida, with a TVtli Koritska semi-apochromatic oil immersion
lens and compensating eye-piece 18, and in reproduction reduced by
one half. In all these figures the mitochondria have been blackened in
order to show them more clearly. On the right side of the Plate,
figs. 43, 45, 46, 48, 49, 52, 53, 54, and 55, are at same magnification.
Figs. 44, 47, 50, and 51 are twice the magnification.]

Fig. 30. — Binucleate spermatocyte near end of growth stage. It
contains two micromitosomata, and has an accessory centrosome near
the top of the plate. The acroblasts are also shown.

Fig. 31. — Spermatocyte to show appearance of mitochondria caused
probably by fixative. The central bodies have run together to form
rods.

Fig. 32. — Spermatocyte in first maturation division showing mito-
chondria and acroblasts.

Fig. 33. — Section through X. — X. in previous figure.

Fig. 34. — Second maturation division showing mitochondria and
acroblasts.

Fig. 35. — Young spermatid, showing incipient formation of macro-
mitosome. Acroblasts becoming spherical.

Figs. 36, 37, 38, and 39, later stages in formation of macromitosome,
and acroblasts are shown.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 463

• Figs. 40, 41, and 42, show macromitosomatic spireme, and acrosome
)<rmation.

Fig. 43. — Abnormal triaster mitosis showing disposition of mito-
chondria and acroblasts.

Fig. 44. — Outgrowth of flagellar organ in Pieris brassicse. At X.
is a body whose significance has not been worked out. It may be
formed by the conglomerated acroblasts, or the remains of the spindle
bridge.

Fig. 45. — Three young oocytes of Euchelia showing the cytoplasmic
bodies.

Fig 46. — Spermatogonial nests of Euchelia showing spindle bridge
and mitochondria

Fig. 47. — Head of sperm just after acrosome is formed. A.G. Acro-
somic granule.

Fig. 48. — Prophase of first maturation division in S. populi showing
undoubted effect of spindle fibres on the mitochondria and acroblasts.

Fig. 49. — From a Regaud smear showing the staining effect with iron
hsematoxylin in the first maturation division (compare with fig. 32).

Fig. 50. — Detailed drawing of clavate flagellar organ of Pieris.

Fig. 51. — An earlier stage than fig. 47 to show several acroblasts
secreting the granule. In fig. 47 these have fused.

Figs. 52 and 53. — Two abnormal spermatid stages, showing peculi-
arities in the micromitosome.

Figs. 54 and 55. — Regaud smear preparations of the metamorphosing
spermatid. Nucleus dotted in, though it did not stain.

EXPLANATION OF TEXT-FIGURE 3.

(See p. 440.)

Figs. 1 to 9 illustrate (jiagrammaticaUy the behaviour of all the
plasmatic elements found in Lepidopterous spermatogenesis. The
acroblasts (AB) can be followed through cell division to the spermatid
(Fig. 3) ; later they become vesicular (Figs. 4 and 5), and finally fuse to
form the bladder-like body which forms the acrosomic granule. The
mitochondria in Figs. 3 and 4 fuse to form the macromitosomal spireme
( MD ). The division of the centrosome (Fig. 4) and the partial revolu-
tion of the sperm head are also illustrated. The apparent fragmentation
of the mitochondrial spireme in Figs. 8 and 9 may be an artefact.

■

DEGENERATE (APYRENE) SPERM- FORMATION OF MOTHS. 465

The Degenerate (Apyrene) Sperm-formation of
Moths as an Index to the Inter-relation-
ship of the Various Bodies of the
Spermatozoon.

By

.1. Bronto Gatenby, B.A.,

Exhibitioner of Jesus College, Oxford.

With Plate 26.

Introductory.

In late years much attention has been given to certain
atypical spermatozoa in some insects and molluscs. On the
latter especially a good many papers have appeared, and
there has been much conjecture as to the function (if any)
of these curious bodies.

In some molluscs (Strombus, Reinke (1)) the atypical sperm
is a most remarkable object, and many times larger than the
typical sperm.

The atypical spermatozoa have been called t( apyrene ” and
“ oligopyrene ” according as to whether they were thought
to contain no, or little, nuclear matter.

That in insects, and this applies to the Lepidoptera, an
atypic sperm so remarkably different from the typic as is the
case in many molluscs, is ever found is extremely doubtful.
But it is noteworthy that in such widely different forms
as Lepidoptera and Prosobranch Mollusca, an analogous
dimorphism should occur. We are tempted to inquire what
in common have the conditions of these forms, and why, if
these apyrene spermatozoa serve a special purpose, should
nearly identical conditions arise under such widely different

466

J. BRONTE GATENBY.

environments. In the Holometabola, we get remarkably
varying stages of life history, for which assuredly- we would
look in vain in Murex or Tritonium, and, while admitting
that in many Prosobranchs the case is so remarkable as to
permit speculation and theory, I feel that in the Lepidoptera
we cannot attach any importance to the atypical spermatozoa
other than that of a degeneration product.

The special purpose of this paper is an inquiry into these
degeneration products in moths and butterflies, but the apyrene
sperm of the mollusc has formed the subject of a note.

I have to thank Dr. Goodrich for his kind interest and
criticism. This work was carried out in the Department of
Physiology, and in this connection my best thanks are due to
Prof. Sherrington.

Previous Work.

Meves (2) alone has given a good account of these atypic
spermatozoa of moths. But the atypic sperms described by
him (Pygsera) are not the only kind of apyrene formation of
moths, and it has been the purpose of this paper to point out
that it would be a mistake to think that apyrenes arose exclu-
sively from the time of formation of karyomerites as seems to
occur in some molluscs (1).

Quite recently H. Federley 1 (3) has reported upon the
spermatogenesis of Pygaera anaclioreta, P. civitula,
and P. pigra, and has been able to describe as much as his
Carnoy technique would allow. Needless to say this fixative
has spoilt all cytoplasmic details except the amphiaster, and
his results from the point of view of this present paper have
been less successful than those of Meves. Federley has
given a completely satisfactory account of the apyrene
maturation divisions in so far as the behaviour of the chromo-
somes is concerned. This observer also gives an account
of the chromosomes of the hybrids of the above species of
Pygaera, which he has worked out well.

1 For the loan of this paper I am indebted to Dr. J. W. Heslop
Har rison.

DEGENE KATE (APYRENE) SPERM-FORMATION OE MOTHS. 467

Meves worked upon Pygaera bucephala, and has
given a correct account of every stage, except that he has
failed to note the behaviour of mitochondria in spireme
formation, of the acrosome and of the centrosome. These
important stages are considered in the present paper, and the
material used has proved more favourable than Pygaera.

In the Metazoa most of the latest work has shown that the
spermatid about to become a spermatozoon contains the
following bodies : Nucleus, centrosome (or centrosomes),

mitochondria, acroblast, and one or two basophil or siderophil
bodies besides. The latter, which might have some special
significance, are not dealt with here.

The special purpose of this paper is to attempt to analyse
the inter-relationship of the first four bodies in the spermatid.
We know that in spermiogenesis, and by this term I mean
the stages from spermatid onwards, the nucleus, centrosome,
acroblast, and mitochondria undergo a number of definite
movements and changes which finally culminate in the pro-
duction of that remarkable cell, the spermatozoon. In
moths, and may be in many other forms, degenerate stages
of spermatogenesis occur, and it has been by observing these
stages in the former that I have attempted to come to
some conclusion concerning the various functions and the
various influences which together go to produce the sperm.
If, in some cases, my conclusions be not clear, it is because I
haye found great difficulty in identifying the special cell
element which might be a centre of influence for another
cell body, but it will be seen that the evidence provided
permits one to make tentative statements.

The technique employed was similar to that used in my
previous paper (4). Smerinthus populi and Pieris
brassicae were most satisfactory forms upon which to work;
in addition Pygaera bucephala and Portliesia similis.
were examined. The character of the apyrene or degenerate
sperm-formation was . found to be somewhat different in
different species. Thus in my Pierid material almost, if not
invariably, degeneration, or the occurrences leading to the

468

J. BRONTE GATENBY.

formation of an apyrene sperm, began after tbe establish-
ment of the spermatid nucleus. In Pygaera the second
maturation chromosomes failed to fuse at all in most cases,
while, on the other hand, a transition between Pygsera and
Pieris was provided by Smerinthus where several nuclei
became formed in the spermatid from a number of success-
fully fused chromosomes.

The Nucleus in the Degenerate Stages.

In PI. 26, fig. 4, is drawn a fairly typical second maturation
division of the degenerate type which will lead on to the
abnormal sperm. The two cells are almost constricted from
each other, but the chromosomes ( C.H .) are straggling, as if
the centrosome and astral rays lacked the energy to finish
anaphase and telophase. The mitochondria are normally
constricting*. The acroblasts are not quite normal, but are in
the usual position in which they are found. The micro-
mitosomata are normal. Now this figure is typical of many
maturation divisions; it shows what most other degenerate
stages do — that the disintegratory process seems traceable at
first to the chromatin. It is almost, if not quite invariably
the nucleus which, in the abnormal growing spermatocyte,
shows signs of degeneration, and it is the nucleus in other
stages (PI. 26, figs. 6, 8, 10, etc.) which is the first cell
element to fail. Inspection of PI. 26, fig. 4, might lead one to
believe that the centrosome is at fault, but this seems nega-
tived when one remembers that in later stages the centrosome
still has the energy to keep its position at the head of the
cell, to undergo some normal changes, to form the axial
filament as usual, and finally to enter into its normal relations
with the mitochondrial spireme. This, at least, points to the
fact that one must be careful in imputing this abnormal telo-
phase to the amphiastral rays. In the telophase of normal
mitosis the chromosomes fuse, and afterwards a new nucleus
is formed. In these abnormal stages this process rarely takes
place in the usual manner. The chromosomes either do not

DEGENERATE (aPYRENE) SPERM-FORMATION OF MOTHS. 469

fuse at all, or only a limited number fuse. These latter, as
well as the isolated single chromosomes, endeavour to form
new nuclei, and one gets the sort of cell drawn in PL 26, figs.
1, 2, and 6. PI. 26, fig. 2, is quite typical. There were in the
field seven abnormal nuclei, none of which had succeeded in
forming a proper reticulum. In PI. 26, fig. 6, there were some
five nuclei, the two bottom ones of which were normal in
appearance. In PI. 26, fig. 1, the truth of these remarks is
also illustrated ; some nuclei were apparently normal, though
smaller; others were abnormal. Degeneration of the nucleus
takes place at every stage of spermiogenesis. Even when the
nucleus of the spermatid is apparently normally formed, it
may in some other way show that it is not properly func-
tional. PI. 26, figs. 3, 7, 8, 10, and 11, show degenerate
stages in which the nucleus, though seemingly normal, fails
to become attached to the centrosome, and drifts down the
growing sperm. The centrosome keeps its position. In
PI. 26, fig. 13, the several nuclei formed from the number of
chromosomes which failed to join up all together have under-
gone the usual staining change which takes place at this
period, but only one nucleus has managed to keep its position
at the head of the cell. In PI. 26, fig. 12, the very darkly
staining nucleus has drifted down the sperm, and has
attached to it a small acrosomic granule (6r.), which does not
seem to be quite normal. In PI. 26, fig. 9, the nuclei are
normally reconstituted, but, judged from the progress of the
acrosome, are late in reaching this stage. All the nuclei in
this nest were unable to keep their positions at the head of
the cells. The section is somewhat oblique.

With regard to the formation of the nuclei from single or
fused chromosomes, every conceivable stage can be found,
especially in Smerinthus and Pygaera, where failure to form a
proper spermatid nucleus is common ; this also applies to
Spilosoma, but in Pier is brassicae failure generally comes
at the stage when the spermatid nucleus should adhere to the
centrosome (PI. 26, figs. 10 and 11, etc.). PL 26, fig. 13,
allows what often occurs in Smerinthus and Pygaera.

470

J. BRONTfc G-ATENBY.

A very important point to notice is that degeneration of the
nucleus may occur at any stage, but in some species it tends
to .take place at certain special stages. It should be men-
tioned that in different bundles the individual spermatids all
tend to reach the same stage of development, degeneration
appearing at one and the same time throughout. Some
spermatozoa get further than others in development, and
apparent arrival at the last step of sperm-formation does not
necessarily mean that the canker of degeneration may not
appear later when the sperm has entered the egg. Thus
development might be initiated by such spermatozoa without
the male nucleus being able to fuse. But I do not believe
that the so-called “apyrene” sperm could enter the egg,
though the suggested effect of an a apyrene ” sperm might
be got by degeneration overtaking a normally constituted
spermatozoon at some stage in fertilisation. If a spermato-
zoon entered an egg, if its nucleus began to lag, as it often
does in spermiogenesis, and if the centrosome acted normally,
one might possibly get development initiated, and there is no
doubt that every sperm from the same bundle would act
likewise. There is nothing illogical in the thought that if
degeneration can overtake the sperm at any stage of sper-
matogenesis, it could also overtake it at any stage in fer-
tilisation.

In PL 26, fig. 14, I have drawn a degeneration stage in
PygBBra bucephala. The nuclei have shrunken into
darkly staining lumps, as is usually the case when any
nucleus degenerates completely. Even after this stage a
partial elongation of the cell may take place.

The Mitochondria.

In my previous paper I stated that it was impossible to
make out the relationship of the macromitosomal spireme
(nebenkern) and the centrosome. By means of degenerate
stages such as those drawn in PI. 26, figs. 3 and 11, it is clear
that the macromitosome consists of a spireme, the front free
end of which is attached to the centrosome. The abnormal

DEGENERATE (aPYRKNE) SPERM-FORMATION OF MOTHS. 471

sagging of the macromitosome has caused the method of
fixation to the centrosorne to be revealed, though how the
other end of the spireme is attached I do not know. In the
normal spermatid the stage drawn in PL 26, fig. 6, gradually
passes to the stage of a tangled spireme (PI. 26, fig. 2, etc.).
Now when the spermatid begins to elongate, the attached
front end of the spireme remains in its position, and the rest
of the mitochondrial matter becomes “let out ” or “ played
out” as the elongation takes place. This process has taken
place normally from C. to Y. in PI. 26, fig. 12, but somehow
or other the spireme subsequently became apparently refrac-
tory (M.), and was left in this position, while the lower part
(Z.) made an endeavour to form normally as the elongation
went on. In PI. 26, fig. 5,. another sort of abnormal formation
is shown : here the macromitosome, instead of pulling out in
a smooth, even manner, came out in lumps (ili.1— Iff.6) . In
PI. 26, fig. 3, the spireme is loosing its position en masse.
It seems that the retention of the spireme in its place, or at
least its completely normal elongation, rarely takes place
after the failure of the nucleus to act properly. This may be
due to the general abnormal condition in the cell.

The Aero blast and Acrosome.

This body can almost invariably be found in the early
spermatid, and in those cases where its presence cannot be
ascertained until sperm-formation is well advanced, special
refinements of technique may succeed in demonstrating it
early. In the case of the snail I have experienced unusual
difficulty in studying this body. I have found that bichro-
mate of potash is extremely favourable for fixing the acro-
some, and osmic-bicl) rornate fixatives are indicated in this
section of spermatogenesis studies. For instance, by smearing
the ovotestis of Helix on a slide, momentarily fixing in osmic
acid fumes, and then, after allowing the film to dry partially,
dipping into a 2 to 5 per cent, bichromate of potash solution,
an extremely intense stain of the acrosome is got if the slide
is so;iked long enough in the bichromate solution. My work

J. BttoNTti GATENBY.

472

has not yet been carrier! out far enough to allow me to attack
with any complete surety the view that the acrosome is derived
either partly or wholly from the sphere or archoplasm, but I
confess that 1 consider this suggestion improbable; not only
because I cannot allow that the observers who espouse this
view have shown clearly that such is the case, but also because
the fixatives most favourable, or at least favourable enough to
demonstrate archoplasm, at the same time uniformly unfavour-
able to acroblastic material. One result of this study of the
atypical spermatozoon has been the confirmation of a sugges-
tion advanced by me in a previous paper (4), that the acroblasts
in later stages are influenced by the nuclear matter alone.

Figures have already been given to show that in the
maturation divisions the acroblasts of Lepidoptera are
orientated in a remarkable and special manner in relation
to the chromatin, and it has been shown that this relation-
ship is rarely departed from ; a review of some of the best
work on the plasmatic structures in other orders leads me to
conclude that, just as in moths and butterflies, the acrosomic
material is definitely and undoubtedly subservient to the
nucleus at almost all stages. The acroblast (or acroblasts) is
almost always found to lie in the cytoplasm in the neighbour-
hood of the nucleus, and sooner or later takes up its position
upon the surface of the latter. Now, a very important ques-
tion to solve was whether this special orientation of the acro-
blast upon the surface of the nucleus could be brought about
by the instrumentality of any body other than the latter.

Meves, in his work on the “ apyrene ” spermatozoa of moths,
completely overlooked the acroblasts, and my own work on
the same species as he used leads me to believe that Pygasra
bucephala1 is not good material for this study. Pierids and
Smerinthids are very favourable. It is known that in the so-
called “ apyrene” or atypic spermatozoon the nuclear matter
fails to act normally and gradually becomes carried down

1 I have since ascertained that in P. bucephala the acroblasts are
difficult to discover because they so closely resemble mitochondria in
size and shape.

DEGEN EKATE (aPYRENE) SPERM-FORM ATtON OF MOTHS. 473

the body of the lengthening sperm. The chromatin is thus
removed from what is the head end of the spermatozoon ;
now, in the normal sperm the head end is formed by the
aerosome. In the atvpic sperm the centrosome forms the
head end, and a very remarkable fact is that the acroblasts in
the Lepidopterous apyrene sperm follow the chromatin down
in its path along the lengthening spermatozoon. In PI. 26,
figs. 8, 5, 7, 8, 11, 12, and 13, this is shown. If the centro-
some needs to be fairly near the body it influences, we would
be justified in assuming that the central corpuscle does not
influence the acroblasts, for they lie a considerable distance-
from this body. Reference to my previous paper will show
that the acroblasts of moths keep close to the spermatid
nucleus, and after approaching one side of the latter, fuse.
The fused or partially united acroblasts secrete a granule
which lengthens to form the aerosome. These events happen
synchronously with the special changes in the nucleus which
leads to the formation of the elongated, apparently solid,,
sperm nucleus; thus by the time the nucleus is elongated,
the aerosome is also elongated and pointed. It has already
been shown that the chromosomes in failing normally to come
together to form the spermatid nucleus, often tend to form
several nuclei of small and varying size. The nuclei fail to-
retain their position at the sperm head and drift downwards.
The acroblasts almost invariably follow at the same time, and
also tend to apply themselves upon rhe chromatic matter as
they do in normal spermatogenesis. We then come to realise
that though the chromosomes lack that power which normally
allows them to reunite to form the spermatid nucleus, never-
theless they still are able to influence the acroblasts. More-
over, one often discovers a spermatid in which one small
nucleus, probably formed from one chromosome, can be seen
shepherding several acroblasts which are stuck on its surface.
In these cases I do not think that a normal aerosome is found
in later stages. That this attendance of the acroblasts upon
the partially regenerated nuclei is not accidental, and caused
by the fact that any bodies tend to become cast down the-

474

J. BRONTE GAT ICN BY.

length of the sperm together, is shown by the fact that one
cannot find many straggling acroblasts (see PI. 26, fig. 10),
and that the latter are able to form an acrosome upon the
surface of the larger nuclei in later stages of degeneration.
Even in normal stages the steps which lead to the formation
of the acrosome from the acroblasts are never exactly syn-
chronous in a sperm nest, but they rarely vary beyond certain
limits. In the spermatocyte and spermatid the acroblasts are
semilunar in shape in normal stages, and this shape gives
way to a vesicular one. Now the acroblasts in abnormal
spermatids quite often show a tendency to shrink and become
darkly staining at a stage when in normal cells the acrosomic
granule is being secreted, and since these darker acroblasts
are often found removed from the nuclei, it might be possible
to assume that the changes leading to the formation of the
acrosome are not wholly dependent upon the nucleus. This
seems to be supported by the fact that the acroblasts, when
not in the immediate neighbourhood of the nucleus, may be
found even in normal stages of different degrees of meta-
morphosis towards acrosome. Another significant fact is
supplied by PI. 26, fig. 9, which shows a part of an abnormal
spermatozoon-nest in which the nuclei, though normally recon-
stituted after the second maturation division, have failed to
keep their position in the cell (see also PI. 26, figs. 3, 5, 7, 8,
10, 11, and 12), but on drifting down have been followed by
the acroblasts, which have formed complete acrosomes. The
latter (A.) are normal in every degree, but the nucleus has
failed to develop synchronously, and the curious condition is got
of one element racing another in metamorphosis. The fact to
be noted at present is that the acrosome can be formed without
the synchronous steps in the nucleus. The sharp acrosome in
PI. 26, fig. 9, belongs to a stage when the nucleus is also elongated
and when it has regained its affinity for nuclear stains. If the
metamorphosis of the acroblasts depended absolutely upon the
nucleus one would get these bodies still vesicular and just about
to apply themselves to the nucleus, but instead they are now
fully formed. I would interpret PI. 26, fig. 9, as follows :

DEGEN Eli AT K (APYRENE) SPERM -FORMAT I ON OF MOTHS. 475

The nucleus lacked the requisite force to place itself at the
head of the sperm, but retained its relationship to the acro-
blasts, which fused, secreted the acrosome, and gave rise to a
normal structure. But that missing power, whatever it may
be, still was wanting in the nucleus, and evinced itself by
causing a lagging in the proper formation of the elongate
sperm nucleus. This lagging, be it marked, did not affect
the acrosome, which became normally formed, and so the
condition in PI. 26, fig. 9, was reached. In PL 26, fig. 13,
each of the four nuclei is attended by a small round granule
( (jr . ) , which is probably an acrosome, but in the cell normal
acroblasts (A.) were still present. In PI. 26, fig. 12, the
nucleus had a granule applied upon its surface ( G .). It is
probable that if this body arises from an acroblast it is
abnormal, for I have not found like stages in normal nests ;
in normal stages such a granule would be surrounded by semi-
lunar acroblastic ring (see figures in previous paper). The
question of the attachment of rhe acrosome upon the nucleus
may now be dealt with ; in the snail I find that the acrosome
is embedded in the nucleus in a wedge-like manner. In some
other forms, as, for instance, in the rat (after Duesberg (5)),
the acrosome is plastered upon one side of the nucleus. In
some other Mammalia the acrosome seems to be fixed straight
across the square head of the nucleus. In fact, almost every
conceivable manner of joining between these two elements
can be found.

My study of Lepidopterous spermatozoa, leads me to
believe that the acrosome is sensitive to some influence
which orientates it. PI. 26, fig. 9, shows that each
acrosome, though it has no normal nucleus to fix upon, is
sensibly orientated towards the sertoli-cell or upper end of
the sperm-nest. It is quite probable that the front end
of this acrosome in Pi. 26, fig. 9, represents what would be
the front end of the acrosome in the normal sperm. Now
the acrosomes in Pi. 26, fig. 9, are not all resting upon the
nucleus, and the same applies to PI. 26, fig. 15, in which
the arrow denotes the head or front end of the nest. In

476

J. JBKONTE GATENBY.

PI. 26, fig. 13, the granule (6r.) it really an acrosoine
is not orientated towards the front of the nest, but
in the greater number of cases in all abnormal stages
the acrosome keeps towards the front end of the cell.
What cell element causes this sensible orientation ? Is it
centrosome or nucleus ? Tlmt it is not the former might
be concluded from the history of the normal sperm where
the centrosome is at the back of the nucleus, and in a
position which one might be justified in presuming, unable
to affect the acrosome. The facts of the influence of the
acroblasts upon the nucleus, and of their very apparent
inter-relationship leads me to believe that the nucleus might
have something to do with this orientation of the acrosome
in the direction of the head end, though more probably the
whole question should be considered in relation with the
growth of all the elongating spermatozoa in one direction,
which is a fact which has already attracted attention. In
the worm (Lumbricus) the spermatozoa grow outwards in
a beautifully regular manner to form a ball, and here there
is no sperm nest-wall. In several abnormal bundles of moth
spermatozoa I have found that this growing outwards in
a common direction by all spermatozoa is not completely
perfect. In the testis the bundles of normal spermatozoa are
cut across in all directions showing that this growing out
of all tails in one way is a matter concerning individual
bundles. I have not examined the part played by the sertoli
or nurse-cells with sufficient completeness to give even a
tentative statement, but I believe that this cell may be
intimately concerned in the orientation of sperm and sperm
elements.

The partial isolation of the acrosome from the nucleus in
abnormal stages shows that the element which causes the
adherence of one body upon the other is here lacking or
inefficient. Just as the nucleus is often unable to adhere
to the head centrosome, or vice versa, so the acrosome
sometimes may lie quite near, quite completely formed, but
lacking the power to become adherent. The manner by

DEGENERATE (APYRENE) SPERM-FORMATION OF MOTHS. 477

which the fusion of acrosome and nucleus is brought about
is difficult to understand. Whether it is caused by some
intermediate body, or by mechanical means can only be
ascertained with a very small degree of probability, but my
work on the snail and other forms leads me to believe that
even if adherence is brought about by some intermediate
substance, the efficiency of this latter substance is improved
by either the fixation of the acrosome upon the nucleus,
like the ferrule of a walking-stick, or by the wedge-like
embedding of the acrosome into a cavity in front of the
nucleus (Helix). One is led to conclude that in the moth
it is only after or when the nucleus of the sperm has become
elongated that complete fusion with the acrosome becomes
established. PI. 26, fig. 9 seems to show that this fusion
depends on the nucleus and not on the acrosome.

The Centrosome.

In the normal stages of spermatogenesis, the centrosome
from which the axial filament grows, becomes applied to
one side of the nucleus and in most forms becomes modified
in some special manner. It may become flattened upon the
nucleus, or squared off as in some mammals. In some cases
it may not appear to have actual physical continuity with
the nucleus. In Lepidoptera the centrosome becomes
cushion-shaped upon the nucleus and as the latter elongates,
the central corpuscle also becomes drawn out slightly. In
those cases where the nucleus fails to become fixed upon
the centrosome, the latter eventually forms the head of the
sperm (PI. 26, figs. 5 and 12.) As far as I can ascertain from
my sections the centrosome loses its spherical shape (see PI. 26,
fig. 2) and becomes elongate as in PI. 26, figs. 7 and 8. Meves>
figures of abnormal stages support this view. One concludes
from this that the centrosome per se, goes on with its
slight changes even though the nucleus has drifted aside.
This is in accordance with what happens in other cell
elements, which appear to go on developing with at least
partial independence. It is well to notice that after the

VOL. 62, PART 3. NEW SERIES 33

478

J. BRONTE GATENBY.

failure of the nucleus in the metamorphosis of the cell
elements of the spermatid the centrosome invariably retains
all the energy necessary to undergo its normal changes.
One is forced to believe that the central corpuscle of all the
plasmatic elements is the most resistant to those influences
which cause abnormality, and this should be borne in mind
in the discussions relative to any possible part played by
<c apyrene ” spermatozoa.

Discussion.

The main fact which we must keep before us in a review of
the evidence provided by these abnormal stages is that it
would be unwise to conclude, because one body is somewhat
far removed from another, that mutual influence is so pre-
cluded. Because the centrosome lies at the head of the
atypical sperm, and the abnormal nuclei and their acroblasts
far down, we are not justified in at once dismissing any
possibility of the one affecting the other. Nevertheless I
feel that we can conclude a good deal from such evidence as
we have at hand, though some suggestions must only be very
tentative. I have already shown that though the nuclear
matter is abnormally affected, still the acrosome may be
formed, and in the same way a normal macromitosome may
be developed from the granules. The tail of the sperm may
be normally formed by a thinning out and elongating of the
macromitosome, and otherwise, excepting the chromatin, the
cell elements may be quite normal. I believe that this shows
that we are concerned with two special occurrences in sper-
matogenesis : Firstly, the formation of the acrosome near the
nucleus, and the evident inter-relationship of these bodies ;
and secondly, the growth of the tail filament, and the special
grouping of, or formation of, an envelope from the mito-
chondrial granules. These two separate occurrences appear
to be able to take place with independence of each other, and
I think the two apparent free forces in the spermatid are
respectively lodged in the nucleus and in the centrosome

DEGENERATE (APYRENE) SPERM-FORMATION OF MOTHS. 479

The final linking up of all the occurrences in the sperm-cell
and their proper consummation depends on the correct
attraction between centrosome and nucleus at the time when
the various cell elements become formed up in line pre-
paratory to the elongation of the spermatid.

The growth of the sperm tail in length depends, I believe,
absolutely on the head centrosome. Now the centrosome also
is intimately connected with the macromitosome (nebenkern
of some authors), at least in the later stages, when the elon-
gation is taking place, and in the moths the centrosome is
certainly connected with one end of the mitochondrial
spireme. Yet we know quite well that in cellular division
the astral rays are certainly not connected with either single
mitochondria or groups of mitochondria. It therefore seems
that a new relationship betwixt centrosome and mitochondria
is brought about in later stages of sperm-formation. The
curious and definite groupings of mitochondria around the
filament in such a case as Enteroxenos might possibly be
otherwise accounted for, but I believe the centrosome to be
mainly responsible. The acroblast also offers curiously con-
tradictory evidence; it undoubtedly (in moths, at least)
becomes definitely oriented towards the nucleus in several
stages of spermatogenesis, and yet in mitosis it keeps appa-
rently completely within the zone of the astral rays, near the
centrosomes. From the maturation divisions and subsequent
spermiogenesis I conclude that the following inter-relation-
ships could be demonstrated between —

(1) Nucleus and acroblasts.

(2) Acroblasts and centrosomes (in cell division alone).

(3) Mitochondria and centrosome only in later stages (after
spireme formation).

(4) Centrosome and nucleus (not chromosomes) only in
later stages, when both bodies become adherent one to the
other. (I do not here refer to division of the cell.)

The question of the relationship of mitochondria to other
cell elements in cell division I believe to be settled by some
stages, especially in the prophases, where in moths the seed-

VOL. 62, PART 3. NEW SERIES. 33 §

480

J. BRONTE GATENBY.

like mitochondria tend to become fused fan-wise from the
zone of influence of the centrosome. It is nevertheless quite
true that the mitochondria, though often so affected, almost
invariably in later stages keepwell outside the amphiaster.
This seems to show, as well as much other such evidence does,
that the mitochondrial granules are not absolutely equally,
but only approximately equally divided. It is quite certain
that the mitochondria are never divided in such a correct
manner as the chromosomes.1 Some stages given by me
elsewhere show that this approximately even division of the
mitochondria may be departed from, resulting in a very
perceptibly uneven distribution. Even if the suggestion of
two special capital centres of force in the spermatid be not
completely substantiated, it is quite certain that spermio
genesis is the sum result of the separate workings of a
number of forces, and these latter, when not working in
unison, produce abnormal spermatozoa. The curious sper-
matogenesis of some hybrids might be traced to a want of
unison in the arrangement of the elements provided by both
sides. It has been suggested that the growth of tlie sperm-
tail depends upon the head centrosome. Whether this applies
to the alteration of shape in the nucleus and acrosome is,
indeed, difficult to say. The nucleus, after losing its place
at the sperm-head, is still able to become elongate, and the
same remark applies to the acrosome.

The main conclusion is that even though certain cell
elements in spermatogenesis become degenerate, others may
be able to go further on with apparent semi-independence,
and may even become normally formed.

The Degenerate Spermatozoon as a Probable
Special Sex Determinant.

Hertwig (6) suggested that the “ apyrene ” spermatozoon
might by fertilising an egg produce offspring of a different
sex from an egg fertilised by a “ eupyrene ” sperm. Other

1 See, however, E. B. Wilson on Centrums, ‘Nat. Acad. Sciences,’
yol. 2, June, 1916.

DEGENERATE (APYKENE) SPERM-FORMATION OF MOTHS. 481

authors have already pointed out that it has not satisfactorily
been shown that the “ apyrene ” is able to enter the egg.
The view taken in this paper is that the atypic sperm, in
moths at least, has no special significance beyond the fact
that it originates from a state of degeneration. I have shown
in the figures in this article that almost every conceivable
stage in degeneration from spermatocyte- onwards can be
found in lepidopterous spermatogenesis, and even if there
appears to be a special, true “ apyrene ” sperm, which I
doubt, this is because it is at one special stage that degenera-
tion is most prone to appear. I have already pointed out (4)
that degeneration cannot be due to a starved condition of the
larval or pupal moth or butterfly, but exactly why degenera-
tion should appear at all I cannot say. Nevertheless I
consider it certain that the appearance of “ apyrene and
oligopyrene ” spermatozoa is directly traceable to the same
forces which cause a whole nest of primary or secondary
spermatogonia to undergo degeneration. Even if subsequent
research should show that fertilisation by an atypic sperm is
possible, this cannot show that the latter has a special sig-
nificance from the sex point of view, though it would intro-
duce an element of very strong probability. I have already
remarked, in the section dealing with the nucleus, that a
sperm might be overtaken by degeneration after it had
entered the egg. This matter will easily be settled by a
cytological examination of enough fertilisation stages, together
with a comparison between the expected and the true sex
ratios in breeding experiments.

The “Apyrene” Spermatozoon of the Moths and
of the Molluscs.

Among later workers on the atypical spermatogenesis of
Mollusca there is a clear consensus of opinion that the
“apyrene” sperm could not fertilise the egg, and this seems
completely substantiated by Reinke's (1) inability to find any-
thing but eupyrenes in the receptaculum seminis of Strombus.

Goldschmidt (7) advances the view that the “ apyrene ”

482

J. BRONTE GATENBY.

spermatozoa of moths are reaction products, probably caused
by the changes in the chemical properties of the haemolymph
during metamorphosis. He says : “ In the case of Sarnia it
is easy to observe, without going into chemical details, that
the blood in old pupa), which produce the atypical sperma-
tozoa, has very different properties from those in the young.”
It must be admitted that there may probably be some truth
in this plausible explanation ; but, while believing that
“ apyrene ” spermatozoa may be caused by some subtle
alteration in the haemolymph, it is well at present to accept
Goldschmidt’s explanation with caution, and for the following
reasons :

(1) In different larvae and pupae of moths and butterflies,
though individuals may be sub-equal in size, the develop-
ment of the spermatozoa may have reached much later
stages in one than in another example. No rigid synchronism
here holds good.

(2) In some forms like Spilosoma, all sperm-formation is
finished by the time the larva is full grown. In other forms
which appear at the same time and have the same sort of life
cycle, sperm-formation takes place mainly in pupa).

(3) Apyrene spermatozoa may be the first kind to reach
development in the testes. Eupyrenes may only appear
much later.

(4) Apyrenes and eupyrenes may be found developing
side by side at what appears to be the same stage.

(5) Apyrenes appear in Molluscs where the suggestion of
altered conditions of a blood fluid would not seem to apply,
and where no metamorphoses takes place.

With regard to the statement in paragraph three, the follow-
ing is my experiment : A number of Pygaera bucephala
larvae pupated in September. The testes then contained
only spermatogonia and spermatocytes not quite grown to
their full size. The pupae were kept in the warm laboratory,
and near the end of October it was found that the testes con-
tained several bundles of spermatozoa and spermatids, all of
which were abnormal or apyrene. About Christmas the

DEGENERATE (APYRENE) SPERM-EORMATION OE MOTHS. 483

testes were found full of bundles of botli sorts, apyrene
and the normal or eupyrene metamorphosing side by side.
According to Goldschmidt, I suppose the explanation of this
would be that the haemolympli in early stages favoured the
formation of apyrenes, but that later in histolysis the fluid of
the body became adjusted suitably for eupyrenes. Whether
this explanation can be held good might be proven directly
by injecting some chemical substances into the pupa, and by
altering the haemolymph provide an easy basis for com-
parison with what occurs in the control pupae.

In molluscs it seems that in such a case as Paludiria, atypic
spermatozoa arise from ordinary spermatids, but in Strombus
Reinke traces the apyrenes from special cells not to be
identified as normal spermatogonia. How could Goldschmidt’s
explanation apply to either cass in molluscs ? I believe the
example of the moth testis, where both sorts of sperm ato,-
genesis goes on side by side is distinctly comparable to that
of the molluscs. Why does not the chemical unsuitability of
the hamiolymph apply alike to all spermatid bundles ?

In connection with these cases of remarkable dimorphism in
spermatozoa, due to degeneration, the noteworthy case of
Nerilla should be mentioned (Goodrich (8)). In this archi-
annelid the male has three genital segments : the first of
these, the seventh, alone produces normal spermatozoa, but the
eighth and ninth give rise to curious cells with granules;
the exact category under which these cells should be
placed, whether spermatogonia, spermatocytes, etc., and the
manner in which the granules appear have not yet been
elucidated, but there can be little doubt that the conditions
leading to abortive spermatozoa in segments eight and nine
are to be identified with the same forces which cause the
apyrene sperm of moths to appear.

Reinke (1) has some interesting suggestions to make
regarding the apyrene spermatozoa of Strombus. He says :
“ (a) They may serve as nurse-cells to the eupyrene spermatozoa
after copulation and before the latter reach the seminal
receptacle ; ( b ) they may, by liberation of some substance,

484

J. BRONTE GATENBY.

stimulate the eupyrene spermatozoa or the eggs or both during
fertilisation ; or by the liberation of some substance to which
the eupyrene spermatozoa are negatively chemotactic, they
may act as an aid in the final disposition of the latter.”

One remark naturally occurs on reading these suggestions
It is that the vast majority of the Metazoa have no such
noteworthy dimorphism in their spermatozoa as these Proso-
branchs, and they overcome any possible difficulty in the
nourishment, stimulation, and final disposition of their sperms
without recourse to any atypic formations. Then why should
certain molluscs depart from the usual methods in so far as
to need a special new sort of sperm ? Why should some
other molluscs not have such special sorts of sperms ? Experi-
ments have shown that the apyrene spermatozoa degenerate
sooner or later after undergoing katabolic changes in tlie
albuminous bodies, that they are not so greatly stimulated
by decaying tissue as are the eupyrene, and that the eupyrene
spermatozoa live longer than the apyrene in sea-water (1).
This all shows that the apyrene sperm lacks the energy of
the eupyrene, and that it is in this way degenerate. For
myself until further evidence of a more definite nature is
brought forward I prefer to believe that these atypic sperma-
tozoa are degeneration products produced by some altered
condition in certain cells of the testis.

Whether these abnormal conditions are due to some altera-
tion in the fluid nourishing and bathing the sperm-cells, and
why these conditions should apply to some cells and not to
others, are problems which in the case of the moths at least
might be solved by further experiment, in the line already
indicated. Finally, it should be remarked that Goldschmidt* s
hypothesis of altered conditions is to be regarded as more
likely than any sex theory, for I feel convinced that in moths
these spermatozoa arise out of a state of degeneration, and
the varying conditions which are undoubtedly to be found
during the metamorphosis of insects might possibly supply
the disintegratory stimulus. It has already been pointed out
where Goldschmidt’s hypothesis seems to me to be weak.

DEGENERATE (APYRENE) SPERM-FORMATION OF MOTHS. 485
Addendum.

With regard to the matter of degeneration in the testes
of moths, a very remarkable case has been brought to my
notice by my friend, Dr. H. Eltringham, of New College,
Oxford. A number of pupae of the Emperor moth (Satur-
nia carpini) were purchased May, 1916, and none emerged
that year. In 1917 about six moths emerged, paired, and
laid fertile eggs. The rest of the pupae failed to emerge.
This June a number of the male gonads of these pupae were
sectioned, and it was fonnd that they contained nothing
except undeveloped spermatogonia and quite degenerate
spermatocytes. There seems to be some correlation between
the state of development of the gonads and time of emer-
gence. The remaining pupae do not appear to be going to
emerge this year (July 29th, 1917).

Summary.

(1) In Lepidoptera degeneration of cell elements take
place at all stages of spermatogenesis.

(2) Degeneration of the chromosomes just after the second
maturation division leads to what has been called “ apyrene ”
spermatozoa.

(3) “Apyrene,” “ oligopyrene,” and “ eupyrene ” sperma-
tozoa are not separate kinds in Lepidoptera, but all inter-
mediate stages are to be found.

(4) It is suggested that in Lepidoptera these terms lack
the significance which has been attached to them.

(5) Degeneration may set in just when the cell elements
are about to be properly orientated before spermiogenesis,
and in such cases the nucleus and head centrosome fail to
join. The former sinks down the lengthening sperm.

(6) The acroblasts almost always accompany the nucleus,
and form a normal acrosome.

(7) In degenerate spermatids where the chromsomes fail
to join up normally, the macromitosome (nebenkern) may
be normally formed.

486

J. BRONTE GATENBY.

(8) The macromitosome may become normally elongated
in sperms in which the nuclei are degenerate.

(9) It is suggested that the abnormal sperms are unable to
bring about fertilisation.

(10) Individual nuclei can be reconstituted from separate
chromosomes.

(11) At least partial inter-dependence of some cell ele-
ments is indicated by degenerate stages.

(12) Two centres of force, lodged respectively in the
nucleus and in the centrosome, seem to be present.

Department of Physiology, Oxford ;

March 19th, 1917.

Bibliography.

1. Reinke, E. — “The Apyrene Spermatozoa of S trombus bitu-

berculatus, Tortugas Laboratory, vol. vi.

2. Meves. — “ Ueber oligopyrene und apyrene Spermien und ueber

ihre Entstelmng nacli Beobaclitungen an Paludina und Pygsera,”
• Arch. Mikr. Anat.,' lxi, 1903.

3. Federly, H. — “ Das Verlialten der Chromosomen bei der Sperma-

togenese der Schmellerlenge,” etc., ‘ Zeit. f. unduktive Abstain.
Yererb.,’ Bd. 9, 1913.

4. Gatenby, J. Bronte. — “ The Cytoplasmic Inclusions of the Germ-

Cells.” Part I, Lepidoptera, ‘ Quart. Journ. Micr. Sci.,’ vol. 62,
1917.

5. Duesberg, J. — “ La Spermeogenese chez le nat,” ‘ Arch, Zellforsch.,’

1909.

6. Hertwig. — “ Ueber Correlation von Zellund Kerngroesse,” ‘ Biol.

Centrbl,’ xxiii, 1903.

7. Goldschmidt. — ‘ Science,’ vol. xliv, No. 1137.

8. Goodrich, E. G., ‘ Quart. Journ. Micr. Sci.,’ vol. lvii.

DEGENERATE (APYRENE) SPERM-FORMATION OF MOTHS. 487

EXPLANATION OF PLATE 26,

Illustrating Mr. J. Bronte Gatenby^s paper, “ The Degenerate
(Apyrene) Sperm-formation of Moths as an Index to
the Inter-relationship of the Various Bodies of the
Spermatozoon.”

Explanation of Lettering.

A. Acroblast or acrosome. C. Centrosome C.O. Chromophobe
vacuole of mitochondria. C.H. Chromosomes. F. Axial filaments.
F. IP. Follicle wall. G. Granule. M. Mitochondria (macromitosome).
M.I. Micromitosome. N. Nucleus. W.X. Nucleus being formed from
single chromosomes. X.Y.Z. Different regions in the macromitosome.

[All figures drawn from an 18-compensating eyepiece and a Koritska
Agth semi-apochromatic oil immersion, with a camera lucida, at table
level. In reproduction figures reduced by one half. Now x circa
2000 diameters.]

PLATE 26.

Fig. 1. — Four spermatids of Smerinthus populi showing ab-
normal multinucleate condition resulting from a failure of the chromo-
somes to fuse up normally after second maturation division. The
mitochondria in the top left-hand cell are less abnormal than those of
the other cells.

Fig. 2. — Spermatid of S. populi showing all cell elements normal
except the nucleus, which has not been reconstructed from the chromo-
somes. The latter, singly or in small numbers, are forming a group
of separate nuclei.

Fig. 3. — Young spermatozoon of Pieris, quite normal except that the
nucleus is falling away. At X. is the front end of the macromitosomal
spireme (January).

Fig. 4. — Second maturation division of S. populi, showing a lagging
in the chromosomes. The mitochondria are normal.

Fig. 5. — Advanced spermatozoon of Pieris brassicse, showing
abnormal elongation of macromitosome.

Fig. 6. — Spermatid of Smerinthus populi with five nuclei; other-
wise quite normal.

Figs. 7, 8, 10, and 11 show in P. brassicse the falling away of the
nucleus in the elongating spermatids. Latter otherwise normal
(January).

488

J. BRONTE GATENBY.

Fig. 9. — Oblique section of a sperm bundle of S. populi, showing
normal acrosome formed near lagging nuclei, all of which have fallen
away. Macromitosome normal.

Fig. 12. — Spermatozoon of S. populi, showing abnormal formation
of macromitosome. From C. to Y. is normal. The part M. contains
the main part of the mitochondrial matter, while at Z. there has been
an attempt at elongation.

Fig. 13. — Spermatid with four nuclei, all of which stain as they
do in normal stages. Cell otherwise normal, but each nucleus has a
granule, which may or may not be derived from acrosomic matter.
S. populi.

Fig. 14. — ’Spermatid of Pygaera bucephala, showing fairly normal
macromitosome, but quite degenerate nucleus (October).

Fig. 15. — Mid part of bundle of metamorphosing spermatozoa of
Pieris, showing abnormal acrosome not properly fused to the nucleus.

With Ten Plates, Royal 4 to, 5s. net.

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CONTENTS OF No. 248. -New Series.

MEMOIRS :

PAGE

Notes on the Morphology of Bathynella and some Allied Crustacea.

By W. T. Calman, D.Sc. (Published by permission of the Trustees
of the British Museum). (With 14 Text-figures) . . 489

On Oxnerella maritima, nov. gen., nov. spec., a New Heliozoon,
and Its Method of Division ; with Some Remarks on the Centro-
plast of the Heliozoa. By Clifford Dobell, Imperial College of
Science, London, S.W. (With Plate 27) . . 515

“Proboscis pores” in Craniate Vertebrates, a Suggestion concerning
the Premandibular Somites and Hypophysis. By Edwin S.
Goodrich, E.R.S., Fellow of Merton College, Oxford. (With
Plate 28 and 3 Text-figures) ..... 539

The Cytoplasmic Inclusions of the Germ-Cells. Part II.— Helix
aspjersa. By J. Bronte Gatenby, B.A., Exhibitioner of Jesus
College, Oxford (Dept, of Physiology). (With Plates 29-34 and 5
Text-figures.) ...... 555

Note on the Development of Trichogramma evanescens. By
J. Bronte Gatenby, B.A., Exhibitioner of Jesus College, Oxford . 613

Title, Index, and Contents

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 489

Notes on the Morphology of Bathynella and
some Allied Crustacea.

By

W. T. Caiman, D.Sc.

(Published by permission of the Trustees of the British Museum.)

With 14 Text-figures.

Contents.

page

I. Introductory ..... 489

II. External Characters of Bathynella . . 490

III. Internal Anatomy of Bathynella . . 500

IY. Development of Bathynella . . . 502

Y. The First Thoracic Somite in the Syncarida and

other Malacostraca .... 502

YI. The Mandibular Groove in the Syncarida . 506

YII. The Degeneracy of Bathynella . . . 508

YIII. The Classification of the Syncarida . . 509

IX. The Affinities of the Syncarida . . 512

X. List of Papers referred to . . 513

I. Introductory.

The recent re-discovery of Bathynella natans by P. A.
Chappuis (1914a, 1915)1 throws welcome light on one of the
most remarkable of living Crustacea. In the thirty years
that had elapsed since its first description by Vejdovsky
(1882), from two specimens found in a well in Prague, this

1 The numbers in brackets after names of authors refer to the list
of papers on p. 513.

VOL. 62, PART 4. NEW SERIES.

34

490

W. T. OALMAN.

minute form liad not been obtained by any other naturalist.
Some eighteen years ago a re-examination of the solitary
remaining specimen enabled me, in spite of its poor state of
preservation, to add some details to Vejdovsky’s account, and
led me to the conclusion that it was allied to Anaspides
(Caiman, 1899). The scantiness of information regarding it,
however, has caused most writers who have had occasion to
refer to Bathynella in text-books or elsewhere to suspend
judgment as to its affinities, and the late Geoffrey Smith
(1909) omitted it altogether from his revision of the Anaspi-
dacea. The detailed account of its structure now provided
by Chappuis from the specimens found in Switzerland entirely
confirms my earlier conclusions as to its systematic affinities,
and enables us to say that Bathynella is undoubtedly a
degenerate member of the Syncarida, a group of Crustacea
which has persisted from Carboniferous times, and of which
the only other living representatives are found in Australia
and Tasmania.

Certain features in the morphology of Bathynella seem
to me, however, to deserve somewhat more detailed con-
sideration than they have yet received, and on this account I
was particularly glad to have an opportunity of studying
three specimens that M. Chappuis kindly presented to the
British Museum (Natural History). From the small size of
the animal and the unusual delicacy of its cuticular covering
its investigation presents considerable difficulty, but I have
been able in great part to confirm and in some details to
amplify Chappuis’ account of its external structure. Since
his memoir, published in a German periodical, is likely for
the present to be difficult of access for many zoologists, it
seemed desirable to make the following account somewhat
fuller than might otherwise have been necessary.

II. External Characters of Bathynella.

Size. — The specimens examined by me measure almost
exnctly 1 mm. in length of body, and this is also the size

MORPHOLOGY OP BATHYNELLA ANL ALLIED CRUSTACEA. 491

given by Vejdovsky for the type-specimens. Chappuis states
that some of his specimens reached a length of 2 mm., but it
is not quite clear that this measurement excludes the an-
tennules. In any case Bathynella is one of the smallest
among the Malacostraca ; only some Asellota and Cumacea
are no larger, and a few Tanaidge are perhaps even a little
smaller.

Body. — The body (Text-fig. 1) is subcylindrical and ‘fully
segmented, and the general aspect of the animal approaches
that of the more vermiform of the Harpacticoid Copepoda.
The abdomen appears to be a little more bulky than the

Text-fig. 1.

thorax, and, according to Chappuis, it is slightly compressed
from side to side. The eight thoracic and six abdominal
somites are separated by well-marked grooves and appear
to be freely movable, but the cuticle is almost uniformly
thin, and there is difficulty in seeing the boundaries between
the tergal sclerites and the articular membranes connect-
ing them. The tergites of the posterior thoracic and the
abdominal somites overlap from before backwards, but in
the anterior three or four thoracic somites there is no over-
lapping.

Head. — The head (Text-figs. 2 and 3) is longer than wide.
It is truncated in front, with no trace of a rostral projection,
and behjnd it is sharply defined from the first thoracic somite
by an articulation exactly like those that separate the thoracic
somites from one another. There is no trace of eyes or of

492

W. T. CALMAN.

eye-stalks. Seen from the side, the lower margin is concave
in its anterior two-thirds, and in this part it overhangs the
bases of the mandibles and maxillulae in a very slight pleural
fold. On the side of the head a shallow groove, most clearly
seen in a specimen treated with caustic potash, runs obliquely
upwards and backwards, becoming almost imperceptible
where it joins with its fellow across the dorsal surface. At its
lower end, where it lies immediately above the basal articu-
lation of the mandible, the integument along the floor of the

Bathynella natans, $ . Head and anterior thoracic somites,
lateral view. a'. Base of antennule. a". Base of antenna.
md. Mandible, m. gr. Mandibular groove, mt. Metastoma or
lower lip. wx'. Maxillula. mx" . maxilla. thp\ Appendage
of first thoracic somite. I, II. Tergites of first and second
thoracic somites.

groove is slightly thickened and stiffened, and this thickening
spreads a little way in front and behind along the margin of
the pleural fold. The significance of this “ mandibular
groove ” is discussed below.

Telson. — The sixth abdominal somite is deeply incised
behind in the middle line where the anus opens, and on either
side of the incision, towards the dorsal side, a short appen-
dage is articulated, subcylindrical . or slightly flattened, and

Text-fig. 2.

TTi.gr.

II.

I .

MORPHOLOGY OP BATHYNELLA AND ALLIED CRUSTACEA. 493

bearing a group of spines and long setae (Text-figs. 4 and 5).
These appendages are not divided into two segments as
Vejdovsky described them, and one of the grounds for my
suggestion that they might represent the caudal furca is
therefore removed. 1 am now inclined to agree with

Text-fig. 3.

Bathynella natans, $ . Head and anterior thoracic somites,
dorsal view. a'. Base of antennule. m. gr. Mandibular
groove. I, II. First and second thoracic somites.

Chappuis that they represent the two halves of a deeply
divided telson, although I do not understand his argument
when he says — “ Da aber das Analsegment hier schon die
Uropoden tragt, so ist die Deutung der 2 Scliwanzplatten als
Furcalglieder ausgeschlossen.” In no other Syncarida does
the telson show even a tendency to division, and although in

494

W. T. CALMAN.

many Gammaridea the telson is split almost or quite to the
base, the two parts are never set wide apart as they are in
Bathynella.

Antennule. — The earlier accounts described the an-

Text-fig. 4.

Bathynella natans, $. Last somite, telson, and uropod,
lateral view, en., ex. Endopodite and exopodite of uropod. t.
One of the telson-plates.

Text-fig. 5.

Bathynella natans, $. Telson-plate of right side, dorsal view.

tennule as uniramous, but Chappuis has discovered that a
minute vestige of the inner ramus persists. According to his
interpretation this vestige is attached to the distal end of the
fifth segment. If this be so, it constitutes a very remarkable
exception to the rule that the peduncle of the antennule in
the Eumalacostraca consists of three segments. This rule is
only infringed, as far as I know, in certain species of

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 495

Apseude s, where, according to Claus, the rami are coalesced
at the base so as to form an apparent fourth segment of the
peduncle. In the Phyllocarida the peduncle consists of four
segments.

At first sight, the antennule of Bathynella (Text-fig. 6)
seems to bear out Chappuis* description. On the proximal
side of the vestigial inner ramus it presents three large
segments following upon two extremely short basal segments,
the latter together representing the first segment as figured
by Vejdovsky. A careful examination of these short seg-

Text-fig. 6.

Bathynella natans, $. Antennule of left side, dorsal view.
Drawn from a cleared specimen in which some of the finer
setae are missing, i. r. Vestige of inner ramus.

ments, however, leaves some doubt as to their being real
segments of the peduncle (Text-figs. 2, 3, and 6). The
proximal of the two forms a ring of chitin, not defined from
the exoskeleton of the head by any articulation or line of
suture, although it is overlapped above by a slight fold
forming the frontal margin of the cephalic tergite. It may
well be that this apparent proximal segment is simply the
everted margin of the socket with which the antennule
articulates. The second segment has a less firm outline than
the first and the succeeding segments; its surface (in the
single specimen in which I examined it closely) was irregu-
larly folded and wrinkled, and it may be nothing more than
the articular membrane of a joint that has more than the
usual range of motion. At all events there appears to be no

496

W. T. CALMAN.

reason for attributing any profound morphological signifi-
cance to these supernumerary segments of the peduncle.

Antenna. — Chappuis points out that the peduncle consists
of three segments, a short basal segment preceding the two
figured by Vejdovsky. In this case there can be no question
that Chappuis^ observation is correct, the additional segment
being well defined both proximally and distally (Text- fig. 7).
The character is possibly of importance, since in the other
living Syncarida, as in most Malacostraca, only two segments
are present. In this respect Bathynella agrees with the
Mysidacea and many other Peracarida. The third segment

Text-fig. 7.

Bathynella natans, $. Antenna of left side, dorsal view.

bears a small unsegmented exopodite tipped with two setae,
and is followed by a flagellum of five elongated segments.

Mouth -parts. — The mandible (Text-fig. 8, md.) has a
palp of three segments, of which the second is much the
longest. The oral edge is irregularly toothed, and its
proximal part, which would correspond to the molar process,
is thin and sharp-edged. The lower lip (Text-fig. 2, mt.) is
large, as in other Syncarida, and its lobes appear to terminate
each in a minute inturned point.

The maxillula (Text-fig. 8, mx/) is incorrectly figured by
Chappuis. It has two endites, the proximal small and
bearing two apical setas, the distal armed with a group of
spines. A rounded distal eminence on the outer side, bearing
three setas, no doubt represents a vestigial palp. The whole
appendage bears an unmistakable resemblance to that of
Koonunga as figured by Sayce (1908, PI. I, fig. 12). No

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 497

trace can be detected of the exite which is present in
Anaspides.

The maxilla (Text-fig. 8, mx.") has three endifces and a
short unsegmented palp. Here also a certain resemblance to
Koonunga may be traced in the fact that the endites
are directed inwards, and not crowded together as they are
in Anaspides.

Thoracic Appendages (Text-figs. 9 and 10). — With
exception of the last pair all the thoracic limbs are similarly

Text-fig. 8.

constructed, only the inner ramus becoming a little longer
and more slender in passing backwards along the series, and
the group of spines arming the terminal segment being
reduced to a single claw (with a minute seta at its base) in
the last three pairs. The coxopodite is short, and has two
vesicular epipodites on its outer surface. The basipodite is
long, and both exopodite and endopodite articulate with its
distal end. The exopodite shows an incomplete line of
articulation near the base and another beyond the middle of
its length, where there is a well-marked “shoulder” on each
side; it is not, however, distinctly divided into two segments
as described by Vejdovsky and myself. The endopodite
consists of four segments, the precise relation of which to the

498

W. T. CALMAN.

six segments present in Anaspides cannot be determined.
From tlie fact that the exopodite is attached to the distal end
of the second segment of the protopodite it may be inferred
that this segment represents the basipodite alone, and not
the coalesced basipodite and ischiopodite as it does in
Koonunga and in the posterior legs of Anaspides.

The appendages of the last thoracic somite are greatly

Text-fig. 10.

Text-fig. 9.

Fig. 9— Bathynella natans, ? . Thoracic appendage of third pair.

en. Endopodite. ep. Epipodites. ex. Exopodite.

Fig. 10. — Bathynella natans, £ . Thoracic appendage of sixth pair.
References as in Text-fig. 9.

reduced in size, and differ considerably in structure in the
two sexes. In the female the exopodite and endopodite are
short, unsegmented stumps, and a single epipodite is present.
In the male (Text-fig. 1) the exopodite is reduced to a papilla,
there is no epipodite, and the coxopodite is produced in-
ternally as a rounded prominence on which, or at any rate
very near it, is the opening of the vas deferens,

MORPHOLOGY OP BATHANELLA AND ALLIED CRUSTACEA. 499

While Vejdovsky and I had only described a single series
of thoracic epipodites, Chappuis has made the important
discovery that Bathynella, like all other living Syncarida,
possesses a double series of these appendages. The distal
epipodites are oval vesicles (not flattened, at least in pre-

Text-fig. 11.

Bathynella natans, $ . Uropod of right side, dorsal view.
en. Endopodite. ex. Exopodite.

served specimens) attached to the proximal segment of the
limb by a narrow base. Chappuis does not describe them as
showing any structural differentiation, but ih a cleared
specimen each is seen to be crossed by a fine suture-line
about the middle of its length. In the distal half of the
vesicle the cuticle is slightly thickened, forming a thimble-
shaped cap, while the proximal part has the cuticle thin and
flaccid. I formerly described the epipodite as “ borne on a

500

W. T. CALM AN.

short peduncle, from which it is separated by a transverse
articulation or suture.” It now appears probable that the
“ peduncle ” was formed by the collapse of the proximal part
of the epipodite in the shrivelled type-specimen. In reality
there is no peduncle, the epipodite springing directly from a
narrow base of attachment. The transverse suture suggests
comparison with the suture-line which I described near the
base of the epipodites of Anaspides, but in that case it is
the small basal portion which is more thickened, and the
distal part soft and membranous.

The epipodites of the proximal series are not constricted at
the base, but form merely lobular processes of the outer
surfaces of the coxopodites. They have a very delicate
cuticular covering and are not divided by suture.

Abdominal Appendages. — In the abdominal region the
only appendages present are those of the first and sixth
somites. The former (pleopods) are short, uniramous, and
consist of two segments ; they present no sexual differences
(Text-fig. 1). The appendages of the sixth somite (uropods)
(Text-figs. 4 and 11) are very stout, with the peduncle
laterally compressed, and armed with a row of spines on the
inner side ; the endopodite is sub cylindrical, and bears a
group of spines and long setae distally; the exopodite is
conical, much shorter than the endopodite, with two apical
setae.

III. Internal Anatomy op Bathynella.

As I have had no opportunity of studying the soft parts,
the following notes are based solely on the observations
recorded by Chappuis.

Alimentary System. — No masticatory stomach is des-
cribed, a smooth muscular oesophagus extending as far as the
sixth thoracic somite. This is followed by a widened portion
{“ Magen ”) reaching into the last thoracic somite, with
opaque glandular walls thrown into four longitudinal folds.
This may be supposed to represent at least a portion of the

MORPHOLOGY OF BATH YN EL LA AND ALLIED CRUSTACEA. 501

mid-gut, although Chappuis applies that name to the follow-
ing, still wider portion, which extends as far as the fourth
abdominal somite. In this region the dorsal wall of the gut
is thick and glandular, while the ventral wall is thin. The
short rectum is stated also to have a glandular structure.

The entire absence of hepatic or other diverticula of the
gut is a feature not paralleled in any other Malacostracan.

Circulatory System . — The short heart lies in the fourth
thoracic somite, and does not exceed in diameter the vessels
that come off from it in the middle line in front and behind.
No ostia have been seen. While the anterior vessel is no
doubt an aorta (arteria dorsalis, Chappuis), the posterior
vessel is described as a “ vena dorsalis ” collecting the blood
from the sixth abdominal somite and returning it to the heart.
Such an arrangement would be very unusual, if not unique,
among Crustacea, and perhaps the so-called dorsal vein
should be regarded rather as a backward extension of the
heart itself. In view, however, of the difficulties of investi-
gation to which Chappuis alludes, it seems possible that a
mistake has been made as to the direction of the blood-flow in
this region of the body.

Excretory System. — Chappuis describes in considerable
detail the remarkable structure of the maxillary gland. It
consists of an end-sac (coelomic sac), a looped canal extending
backwards into the fourth thorncic somite, and a terminal
vesicle with a slit-like opening ou the outer surface of the
maxilla. During life the terminal vesicle is thrown into
rapid pulsation, with opening and shutting cf its external
aperture, by a muscle attached to its wall. This pulsating
apparatus is compared by Chappuis with that found in the
maxillary gland of the remarkable Copepod Phyllogna-
thopus (Belisarius, Viguierella), where it was first
described by Maupas, and has recently been investigated by
Chappuis himself (1914b) in specimens found living in
company with Ba t h y nella . The similarity, however, appears
to be no more than superficial, either in structure or, probably,
in function, for while the pulsating vesicle of Ba thy nella is

502

W. T. OALMAN.

situated at the exit from the excretory duct, that of Phyl-
lognathopus is at its inner termination, and represents, in
all likelihood, a modification of the coelomic sac.

An excretory function is also ascribed to paired nephro-
cytes, or masses of them, in the head and body-somites, and
in the same connection there is described a pair of voluminous
glandular masses in the last somite, with ducts opening on the
uropods.

Nervous System. — The central nervous system is re-
markably bulky in comparison with the other organs. The
large brain shows no trace of optic lobes. The ventral nerve-
chain shows some degree of longitudinal concentration (not
very fully described), and the ganglia are indistinctly defined
from the connectives.

Reproductive System. — The reproductive system of
both sexes is simple. The gonads lie in the abdomen, and
their ducts run forwards to open to the exterior in the
positions characteristic of the Malacostraca, those of the
female on the sixth and those of the male on the eighth
thoracic somite.

IV. Development of Bathynella.

The only young stage observed by Chappuis (the size is
not stated) resembled the adult, except that the last four
pairs of thoracic limbs were rudimentary. The single pair of
pleopods and the uropods were fully developed. This is in
curious contrast to Koonunga, the only other Syncaridan of
whose development we know anything, where Sayce (1908,
p. 11) found a young specimen with all the thoracic appen-
dages fully developed while the pleopods were still un-
segmented buds.

Y. The First Thoracic Somite in the Syncarida and other
Malacostraca.

In discussing the structure of Bathynella in 1899 I
pointed out that it possessed eight free somites in the

MORPHOLOGY OF BATH YN ELLA. AND ALLIED CRUSTACEA. 503

thoracic region instead of seven as described by Vejdovsky.
This conclusion is fully confirmed by Chappuis and the
character is so unusual that it deserves further consideration.

I formerly stated (1899, p. 342) that “Nebalia and some
Stomatopods ” agreed with Bath ynella in having the first
thoracic somite free from the head. As regards the Stoma-
topoda, this statement was based on a remark of Claus,1 2 which
appears to be true only of the larvae. No adult Stomatopod
has the tergites of the first or second thoracic somites free
from the carapace, while those of the third and fourth are
only indistiuctly represented.3 * * *

InNebalia, the carapace envelops, but remains free from,
the thoracic tergites. The grooves separating these from one
another are distinct, but the anterior limit of the first tergite
coincides with the line along which the free carapace passes
into the dorsal integument of the head-region, and it is not
possible to say that the first tergite is defined from the head
in the same way as it is from the following tergite. Owing to
the small size of Nebaliait is difficult to obtain a clear view
of the parts in question, but it is comparatively easy to do so
in the case of the large Mysidacea of the genus Gnatho-
phausia, in which also the first thoracic somite has been
stated to be distinct from the head.8

If the free portion of the carapace be cut away on one side
of a specimen of Gnathophausia (Text-fig. 12) the tergites

1 “ Bei den Squilliden bleibt iibrigens die ganze Region der Kiefer-
fiisse vom Ruckenschilde getrennt, das Segment des ersten Kieferfusses
gelit hier nnterlialb der Schildplatte in die Innenlamelle fiber ” (Claus,
1876, p. 53).

2 Giesbrecht (1910, p. 9, pi. ii, fig. 9) states that the structures here
regarded as the tergites of the third and fourth somites represent
respectively the tergites of the first and second and of the third and
fourth somites fused together, but it is not clear on what evidence this
statement is based.

3 “In Sars’ figure of Gnathophausia longispina . . . the

first thoracic segment appears to be limited in front by a definite

groove, which would thus separate the cephalic and thoracic regions

(Lister, 1909, p. 436, footnote).

504

W. T. CALMAN.

of the thoracic somites are easily seen. The investing* cuticle,
however, is almost uniformly thin, and the tergites, which are
hardly to be described as sclerites, are defined from one
another only by superficial grooves. In the posterior part of
the thoracic region the tergites are regularly transverse, but
anteriorly the median portions are pushed backwards and
crowded, together owing to the backward extension of the
connection between the carapace and the body, which exten-
sion is visible externally on the dorsal surface of the carapace

Text-fig. 12.

Gnatliophausia zoea. Anterior region of body with free
portion of carapace cut away, from right side. c.f. Origin of
carapace-fold. ep'. Epipodite of first thoracic appendage
(maxilliped). ex2., ex*. Exopodites of second and eighth
thoracic appendages (that of the first is absent in this species).
gr. Groove marking boundary between maxillary and first
thoracic somites, uniting above with origin of carapace-fold,
ma:'. Palp of maxillula. mx" . Maxilla, pip'. Pleopod of first
pair. I, VIII. Tergites of first and eighth thoracic somites.

1*. Tergite of first abdominal somite.

as the “linguiform area” of Sars (1885, p. 22). This crowd-
ing makes it difficult to count the narrowed anterior tergites,
but by careful manipulation seven of them can be distinctly
seen to be continuous across the mid- dorsal line. The fore-
most of these, the second thoracic tergite, is defined in

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 505

front by a well-marked groove from the strip of cuticle which
is reflected to become the lining membrane of the carapace.
On each side this strip (Text-fig. 12, I) widens somewhat and
becomes what is clearly tlie lateral portion of the first thoracic
tergite. For the greater part of its length it is limited
anteriorly by the fold (Text-fig. 12, c.f .) which marks the
beginning of the free portion of the carapace. Towards its
lower end this fold turns forward to run horizontally where
the lateral, or pleural, margin of the carapace overhangs the
bases of the mouth-parts and antenna. No considerable part
of the lateral wall of the head is exposed between the origin
of this pleural fold and the attachment of the appendages
except in the case of the maxilla, where a short space inter-
venes. In front, this space is bounded by a cavity in which
lies the palp of the maxillula (Text-fig. 12-, mx) ; behind,
between the base of the maxilla and that of the first thoracic
appendage, a shallow and inconspicuous groove (Text-fig. 12,
gr.) can be traced running upwards for a little distance and
curving forwards to join the carapace-fold. It is this short
groove alone that can be definitely stated to mark the
boundary between maxillary and first thoracic somites, or, in
other words, between head and thorax.

With exception of the short groove just mentioned, which
I have not been able to observe in any other form, the con-
ditions found in Grnathophausia appear to be repeated in
all those cases where the eight thoracic tergites have been
stated to be free from the enveloping carapace (Nebalia,
larval Stomatopods, larval Decapods) ; that is to say, the
cephalothoracic tergal boundary is coincident with, and is
obscured by, the origin of the carapace-fold. Further, since
there is reason to believe that where the first thoracic somite
is coalesced with the head, as it is in Isopoda and Amphi-
poda, a vestigial carapace-fold is involved in the coalescence,
we arrive at the conclusion that Bathynella and the
Anostracous Branchiopoda are the only living Crustacea1 in

1 Possibly the Copepoda should be added, but tlie case is a little
obscure (cf . Caiman, 1909, pp. 6 and 73).

VOL. 62, PART 4. NEW SERIES.

35

506 .

W. T. CALMAN.

which the carapace is entirely absent. It is this that gives
special importance to the agreement between Batliynella
and some of the genera of fossil Syncarida (Uronectes,
Palasocaris) in which the first thoracic somite is similarly
free from the head.

YI. The Mandibular Groove in the Syncarida.

Anaspides was originally stated to have, as Batliynella
has, eight free thoracic somites, but I pointed out in 1896
that the supposed first thoracic somite was defined from the
head not by a movable articulation like those between the
following somites, but by a superficial groove in the integu-
ment, and that this groove, from its position immediately
behind the mandibles, probably did not mark the cephalo-
thoracic boundary.1 I later expressed some uncertainty as to
this interpretation, but it was strongly confirmed by the
discovery that in the fossil genus Palaeocaris (Text-
fig. 13, A) where eight free thoracic somites are clearly
defined, a short groove is present on the side of the head,
running upwards from the base of the mandible in exactly the
same position as the more strongly marked groove of Ana-
spides (Caiman, 1911, p. 489). To this groove, thus shown
to have nothing to do with the first thoracic somite, the name
of “ mandibular groove ” (Text-fig. 13, m. gr.) was given, and
it is of particular interest to find it present also in Bathy-
nella. In this genus the groove, although no more con-
spicuous than it is in Koonunga (Text-fig. 14) lias the same
relative position as, and is undoubtedly homologous with, that

1 The “ faint impressed line ” described (Caiman, 1896, p. 788) as
running behind the mandibular groove in Anaspides is very ill-
defined and inconstant, and no morphological significance can be
attributed to it. The “ horizontal groove ” is not so deep or so sharply
marked as the mandibular; it is still shallower in Paranaspides,
and in Koonunga I can only see a doubtful trace of it. It is asso-
ciated with a bulging of tfye side wall of the head in this region (shown,
for instance, in Sayce’s figures of Koonunga), which apparently marks
the position of the maxillary gland.

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA.

Text-fig. 13.

Head and anterior tlioracic somites, lateral view. A Palseo-
caris precursor (fossil, Coal-measures). B. Anaspides
tasm anise, a'. Antennule. a". Antenna, e Eye. h. gr.
Horizontal groove, md. Mandible, on. gr. Mandibular groove.
mt. Metastoma or lower lip. onx'. Maxillula. mx". Maxilla.
r. Rostrum. I, II. Tergites of* first and second thoracic
somites. (From ‘ Geol. Mag.,’ 1911, by kind permission of the
editor.)

Text-fig. 14.

II.

JKoonunga cursor. Head and anterior thoracic somites,
lateral view. a1. Base of antennule. a". Base of antenna.
md. Mandible, on. gr. Mandibular groove, mx'. Maxillula.
mx". Maxilla, thp1., thp 2. Appendages of first and second
thoracic somites. II. Tergite of second thoracic somite.

5-08

W. T. CALM AN.

of Anaspides; while, as in Palaeocaris, it co-exists with
a clear demarcation between the head and the first thoracic
somite. As was previously pointed out this mandibular
groove is, in all probability, to be identified with that named
by Sars “ cervical sulcus 33 in the Mysidacea, with the trans-
verse cephalic groove of the Anostraca and Conchostraca,.
and with the anterior transverse groove of the carapace in
Apus and other Notostraca. It is possible, but much less
certain, that it corresponds to a part at least of the
“ anterior cervical groove” of Decapoda. As regards its-
morphological significance, its position at the limit between
the naupliar and post-naupliar regions of the body suggests
that it may be of great phylogenetic antiquity and importance.
It is quite possible, however, that it may have rather a
mechanical and function^ meaning. In the Anostraca the
bottom of the groove is thickened to form a more or less
continuous chitinous bar connecting across the dorsal surface
of the head the points of articulation of the two mandibles.
The thickening is most marked, as it is in Bath yne 11a, at
the ends of the groove ; and there can be little doubt that,
whatever its origin, this groove hns, at least in these two-
cases, the function of giving the necessary support for the
articulation of the proximal condyle of the mandible.

VII. The Degeneracy of Bathynella.

The structure of Bathynella, as compared with that of
its immediate allies, is obviously, in many respects, simplified
or degenerate. Some of the evidences of degeneration are no-
doubt correlated with the habitat of the animal, particularly
the absence of eyes, which is almost universal in animals that
inhabit subterranean waters. It is quite likely, however,
that the simplification of structure is in great part a direct
consequence of unusually small size. Lankester (1880, p. 51)
long ago pointed out that “ the needs of a minute animal are
limited as compared with those of a large one,” and ho
enumerated as one of the causes of degeneration <c excessive

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 509

reduction of size.” Thus, we may suppose that the absence
of diverticula of the alimentary canal and the reduction of
the epipodial vesicles in Bathynella are due to the fact
that the necessary proportion of secretory, absorptive, and
respiratory surfaces can be attained without the need for out-
growths that are indispensable for more bulky organisms.
Apart from questions of adaptation, however, there are other
ways in which size greatly influences structure. As D’Arcy
Thompson (1917, p. 33) has recently reminded us, the physical
and mechanical conditions of growth may be profoundly
different in a small animal from what they are in a large one.
For example, we find in Bathyn ella and, I believe, in all
minute Crustacea, a certain clumsiness of modelling and a
tendency to rounded outlines in the smaller appendages such
as the mouth-parts which may be the result of the greatly
increased pressure due to surface tension on strongly curved
surfaces. With regard to some other characters we can only
dimly guess at the mechanical principles that may be involved.
It seems to be a general rule, to which Bathynella con-
forms, that in small Crustacea the setas on the limbs are
fewer in number and larger in relative size than in larger
species. It is rare, in very small Crustacea, to find any of
the appendages produced into long multiarticulate flagella.
Where such flagella are present, as in the antennas of the
males of some Cumacea hardly larger than Bathynella, the
segments of which they are composed are always much longer
than wide. So, in the antennules and antennae and in the
thoracic exopodites of Bathynella, the small number of
segments and their elongate form are very striking when
compared with the same appendages of Anaspides.

VIII. The Classification or the Syncarida.

If we compare the characters of the living genera of
Syncarida it is at once apparent that Anaspides and
Paranaspides are closely related, while Koonunga and
Bathynella differ widely from them and from one another.

510

W. T. CAL MAN.

There can, therefore, be no question as to the desirability of
recognising the three families Anaspididm, Koonungidae,
and Bathynellidae. When, however, we attempt to define
more closely the relationships between these families, and
especially when we try to frame a scheme of classification to
include the fossil genera as well, the matter becomes much
more complicated.

The attractive simplicity of a dichotomous classification is
always less easy to escape from and more likely to be mis-
leading when the forms to be classified are few and the'
characters available for their discrimination are scanty.
Such dichotomies have more than once proved a snare to the
taxonomist of Crustacea1 and they have already made their
appearance in the attempts to classify the Syncarida. Thus
Chappuis, in his preliminary paper on Bathynella (1914a,
p. 47) proposed to divide the members of the group into
Pleopodophora and Apleopodophora2 according as they possess
a full or a reduced series of abdominal appendages. In his
later paper (1915, p. 172) at my suggestion, he based his
division on the freedom or coalescence of the first thoracic
somite, naming the groups Bathynellacea and Anaspidacea.
Still more recently Vanlioffen (1916) 3 has separated the
genera that possess thoracic exopodites from the fossil genera
in which these appendages have not yet been discovered,
opposing the new name Duplicipoda to FritsclTs Simplicipoda.
It would be difficult, perhaps, to find a basis of classification
with less to commend it than that selected by Dr. Van-
lioffen.

1 We need only recall the false antitheses of Entomostraca and
Malacostraca, Phyllopoda and Cladocera, Edriophthalma and Podopli-
thalma, Macrura and Brachyura. In each of these cases one of the
paired groups has proved, on closer examination, to be a heterogeneous
assemblage.

2 Giving, by accident or by design, to the division that excludes
Bathynella the group-name originally devised by Vejdovsky (1899)
for Bathynella alone.

3 I am again indebted to M. Chappuis for the loan of Dr. Vanhoffen’s
pamphlet.

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 511

I am still of opinion, for the reasons given above, that the
freedom of the first thoracic somite is the most weighty mor-
phological distinction between Bathynella and the other
living Syncarida, and that it constitutes an important link
between that genus and the fossil Ur one ctes and Palaeo-
caris ; but so long as this evidence of affinity remains
unsupported by other characters I am doubtful as to the
desirability of establishing a new sub-order for these three
genera. It seems better to be content with a division of the
Syncarida (or Anaspidacea) directly into families, following
in this the example of Geoffrey Smith, although both the
definitions and the contents of the families recognised by him
require modification. Several of the fossil genera, such as
Nectotelson, Palasor chestia, and Gasocaris, are so
imperfectly known that it is impossible to be sure of their
place in any system. Pr aeanaspides, included by Geoffrey
Smith in the family Anaspididae, has proved (Caiman, 1911)
to be identical with Palaeocaris which he placed in the
Gampsonychidas, although evidently suspecting that the two
genera might be more closely related. With some changes
in nomenclature recently made by Cockerell, the classification
now stands as follows :

Division Syncarida, Packard, 1885.

Order Anaspidacea, Caiman, 1904.

Family Anaspididae (Anaspidae, Thomson, 1893).

Genera Anaspides, Thomson ; Paranaspides,,
G. Smith.

Family Koonungidae, Sayce, 1907.

Genus Koonunga, Sayce.

Family Acanthotelsonidae, Cockerell, 1916
(= Pleurocaridae, Chappuis, 1915).

Genera Acanthot el son , Meek and Worthen ;
Pleurocaris, Caiman.

Family Bathynellidae, Grobben, 1904.

Genus Bathynella, Vejdovsky.

Family Uronectidae, Cockerell, 1916 (= Gamps-
onychidas, Packard, 1885).

512

W. T. CALMAN.

Genera Uronectes, Broun ( = Gampsonyx,
Jordan nec Vigors = Gampsonychus, Bur-
meister) ; Palaeocaris, Meek and Worthen
(= Prseanaspides, H. Woodward).

IX. The Affinities of the Syncarida.

While the investigation of Bathynella throws little
further light on the systematic relations of the Syncarida as
a whole, a few comments may be made here on some opinions
recently expressed on the subject. Geoffrey Smith, accepting
the general scheme of classification adopted by me for the
Malacostraca, regarded the Syncarida as standing near the
direct line of descent of both Eucarida and Peracarida. He
assumed, however, that the carapace, possessed by the primi-
tive Eumalacostraca, was lost in the common ancestor of all
three groups, and redeveloped independently by the Mysi-
dacea on the one hand and by the Eucarida on the other.
This assumption is not only improbable, but unnecessary. In
the Carboniferous period there existed a considerable variety
of Malacostraca, regarding which we know little more than
that they possessed a carapace and the other characters of
the “ caridoid facies.” It is not at all unlikely that some of
these may have possessed all the characters that, in the
Syncarida, we regard as primitive, and that from them may
have been derived, by separate and diverging lines of descent,
the present-day Syncarida, Peracarida, Eucarida, and Hoplo-
carida.

Other authors who have discussed the systematic position
of Anaspides and its allies have been misled by the tradi-
tional classification of the Malacostraca into Podophthalma
and Edriophthalma (or Thoracostraca and Arthrostraca), and
have been unable to get rid of the idea that the group was in
some way related to the “ sessile-eyed ” Crustacea. Thus
Grobben (1904) places his group Anomostraca (a name that
has no sort of claim to supersede Packard's Syncarida)

MORPHOLOGY OF BATHYNELLA AND ALLIED CRUSTACEA. 513

between Thoracostraca and Arthrostraca,1 while Giesbrecht
(1913) even goes so far as to include it as one of the
divisions of the Arthrostraca. It may be worth while, there-
fore, to point out once again that the edriophthalmate orders
are unmistakably linked, through the Apseudidse and Cuma-
cea, with the lower Mysidacea (Lophogastridae), and that
in this series there is nowhere a place for the Syncarida.
This affiliation rests on the evidence, not of one, but of a
number of independent characters, which need not be re-
capitulated here, but which are in no way disposed of by
Giesbrecht’s bold assumption that the brood-pouch has been
independently developed in Mysidacea, Cumacea, and Arthro-
straca. As a matter of fact, although the Syncarida evidently
form by themselves a division of equal rank with the Eucarida
and Peracarida, the balance of characters inclines to ally
them rather more closely with the former than with the
latter.

X. List of Papers referred to.

Caiman, W. T. (1896). — “ On the Genus Anaspides and its Affinities
with certain Fossil Crustacea,” ‘ Trans. Roy. Soc., Edinburgh.'
xxxviii, pp. 787-802, 2 pis.

(1899). — “ On the Characters of the Crustacean Genus Bathy-

nella, Vejdovsky,” ‘Journ. Linn. Soc. Zool., xxvii, pp. 338-344,
pi. xx.

(1909). ‘ Crustacea.” In: ‘A Treatise on Zoology,’ edited by

Sir Ray Lankester, pt. vii, fasc. 3.

■ (1911). — “ On some Crustacea of the Division Syncarida from

the English Coal-measures,” ‘ Geol. Mag.’ (dec. v), viii, pp. 488-
495, 5 text-figs.

Chappuis, P. A. (1914 a). — “ Ueber die systematisclie Stellung von
Batliynella natans Vejd.,” ‘Zool. Anz., xliv, pp. 45-47, 1
text-fig.

1 Vanhoffen (1916) adopts Grobben's classification, but adds the
remarkable opinion “ dass die Ordnung [Anomostraca] keine natiir-
liche ist, dass wohl ahnliclie, aber nicht wirkliche nahe verwandte
Formen in ihr vereinigt wurden.”

514

W. T. CALM AN.

Chappuis, P. A. (1914 b). — “ Ueber das Excretionsorgan von Phyllo-
gnatliopus viguieri,” ibid., xliv, pp. 568-572, 4 text-figs.

(1915). — “Batliynella natans und ihre Stellung im System,"

‘ Zool. Jahrb., Abtli. Syst.,' xl, pp. 147-176, pi. iii, 17 text-figs.

Claus, C. (1876). — ‘ Untersucliungen zur Erforschung der genealogis-
chen Grundlage des Crustaceen-Systems.' 4to, Wien.

Cockerell, T. D. A. (1916). — “The Uropods of Acantliotelson
stimpsoni," ‘ Journ. Washington Acad. Sci.,' vi, pp. 234-236,
1 text-fig.

Giesbrecht, W. (1910). — “ Stomatopoden. Erster Thiel,” ‘Fauna u.
Flora d. Golfes v. Neapel,' Monogr. xxxiii.

(1913). — -“ Crustacea.” In : 4 Handbuch der Morphologie,

lierausg. v. Arnold Lang,’ iv, pp. 9-252, 356 text-figs.

Grobben, K. (1904). — ‘Lehrbuchd. Zoologie, begriindet von C. Claus,’
7 Aufl., lste Hiilfte, Marburg.

Lankester, E. Bay (1880). — ‘Degeneration: A Chapter in Darwinism,'
Nature Series, London.

Lister, J. J. (1909). — “Crustacea.” In: ‘A Student's Text-book of
Zoology,’ by Adam Sedgwick, vol. iii.

Sars, G. O. (1885). — “ Report on the Schizopoda,” ‘ Rep. Voy. Challenger,
Zool.,’ xiii, 228 pp., 3 8 pis.

Sayce, O. A. (1908). — “On Koonunga cursor, a Remarkable New
Type of Malacostracous Crustaceans,” ‘ Trans. Linn. Soc. (2)'
Zool.,’ xi, pp. 1-16, pis. i, ii.

Smith, Geoffrey (1909). — “ On the Anaspidacea, Living and Fossil,”
‘ Quart. Journ. Micr. Sci.,’ 53, pp. 489-578, pis. xi, xii, text-figs.

Thompson, D’Arcy W. (1917). — ‘On Growth and Form,’ Cambridge
Press.

Thomson, G. M. (1894). — “ On a Freshwater Scliizopod from Tas-
mania,” ‘ Trans. Linn. Soc. (2) Zool.,' vi, pp. 285-303, pis. xxiv-
xxvi.

Yanhoffen, E. (1916). — “ Die Anomostraken,” ‘ Sitz. Ber. Ges. natf. Fr.
Berlin,’ 1916, No. 3, pp. 137-152, 15 text-figs.

Vejdovsky, F. (1882). — ‘ Thierische Organismen der Brunnenwasser von
Prag,’ 4to, Prag.

(1899). — “Ueber die systematische Stellung des Brunnen-

krebses Bathynella natans” (Czech), ‘Sitz. Ber. k. bohm.
Ges. Wiss. Prag.,’ 1898, No. 14.

OXNERELLA MARITJMA.

515

On Oxnerella maritima, nov. gen., nov. spec.,
a New Heliozoon, and Its Method of Division;
with Some Remarks on the Centroplast of
the Heliozoa.

By

Clifford Dobell,

Imperial College of Science, London, S.W.

With Plate 27.

In 1913 I found a minute and pretty heliozoon in some
small tanks of sea-water in which I had been cultivating’
Foraminifera, Trichosphserium, and other marine Protozoa.
For some weeks the organisms multiplied rapidly and became
very abundant. I studied them alive as carefully as possible,
and made a number of fixed and stained preparations in
order to study their method of division in detail. I also
made a number of notes and drawings at the time, but was
too much occupied with other work to set them in order for
publication. As the organisms seem not to have been de-
scribed hitherto, and as their division presents certain
features of interest, I now take the opportunity of putting my
observations on record.

1. General Description of the Organisms.

Morphology. — The living organisms are typical sun-
animalcules of very small size. They are almost spherical,
with numerous filamentar pseudopodia radiating in all direc-
tions (see PI. 27, fig. 1). The pseudopodia are so extremely
fine that no internal structure can be made out in them, save

516

CLIFFORD DOBELL.

at their somewhat thicker proximal ends. Here it can be
seen that each is provided with a very slender axial rod or
fibre, which passes into the centre of the animal. Although
sometimes visible, with difficulty, in the living organism, the
pseudopodial axes are much more easily distinguishable in
fixed and stained1 specimens (see PI. 27, fig. 2), in which they
appear as almost immeasurably fine radiating lines. In short,
the pseudopodia appear to be, on a very small scale, axopodia
such as are characteristic of most Heliozoa.

The axes of the pseudopodia can be traced (PI. 27, figs. 1,
2) through a clear area in the centre of the animal to a
minute corpuscle — a so-called “ central granule ” — in which
they are rooted. This little body is not always easily seen in
living specimens, on account of its very small size, and on
account of the many food-bodies present in the surrounding
protoplasm. But in stained preparations it is always visible
(cf . PI. 27, fig. 2), and presents various appearances which
will be described below. The arrangement of the pseudo-
podia and “ central granule ” is similar to that already
described in Acanthocy stis, Wagnerella, and other
Heliozoa.

Many of the pseudopodia of a liviug specimen are ex-
tremely long, attaining a length equal to three or four times
the diameter of the animal's body (cf. PI. 27, fig. 1). In
fixed and stained specimens, however, they are usually much
contracted, and consequently appear shorter and fewer (PI. 27,
fig. 2). During life they are studded irregularly with nume-
rous minute granules (PI. 27, fig. 1), which constantly stream
up and down them. These streaming granules are already
well known in other Heliozoa.

With the exception of the clear area surrounding the

1 I fixed and stained the organisms in various ways. The best fixa-
tion was obtained with Bouin’s fluid and Schaudinn’s sublimate-alcohol ;
and by far the best of the stains which I tried was my alcoholic iron-
alum hsematein, which I have described elsewhere (Dobell, 1914). All
the figures here reproduced were stained by this method, which for
delicacy and detail can hardly be surpassed.

OXNERELLA M A ULTIMA.

517

central granule, all the protoplasm of the body is usually
closely packed with ingested food-bodies (PL 27, fig. 1). The
protoplasm itself is colourless. There is no obvious differen-
tiation of the cytoplasm into ectoplasm and endoplasm.

There is no contractile vacuole, no stalk, and no spicular
skeleton, either external or internal ; nor is a gelatinous
investment present.

There is a single relatively large nucleus (PI. 27, figs. 1, 2),
which is vesicular, with a large central karyosome. The
nucleus is excentrically placed, and usually ovoid, its more
pointed end being directed towards the centre of the organism
(PI. 27, figs. 1, 2). Those pseudopodial axes which lie in
contact with the nuclear membrane often stain more deeply
than the rest, and thus appear as dark lines traversing the
surface of the nucleus. (This can be seen in the nucleus
shown in PL 27, fig. 7.)

Most specimens are not perfectly spherical, and the in-
gested food-bodies often project above the surface of the
body, giving it an irregular contour. It is thus impossible to
measure the diameter of most individuals with great accuracy.
The diameter of fifty fixed and stained specimens (all nearly
spherical, and not dividing), measured as nearly as possible,
showed a range from about 10 fi to 22 /u , the mean being-
about 14 fi.

2. Systematic Position.

From the foregoing brief account of the structure of this
heliozoon, it will be clear that it belongs to the group of
naked forms (such as Actinosphaerium, etc.) which con-
stitute the order Aphrothoraca of R. Hertwig. Schaudinn
(1896) enumerates nine genera in this order, which all differ
in important particulars from the present form. Actino-
lophus, Zooteira, and Wagnerella (= Haeckelina)
are stalked; Actinosphaerium and Gymnosphaera aro
multinucleate, and possess sharply differentiated ectoplasm
and endoplasm ; Actinophrys is uninucleate, but possesses

518

CLIFFORD DOBELL.

no central granule; and the pseudopodia of Monobia,
Myxa strum, and Camptonema are so different in various
ways from those of the form under consideration that it is
impossible to place it in any of these genera. There is no
genus which combines the characters peculiar to my organ-
isms— namely, pseudopodia with axial fibres rooted in a
central granule ; a single nucleus ; no contractile vacuole ;
no distinct ectoplasm and endoplasm ; no stalk.

It therefore appears necessary to introduce new generic
and specific names for the present form, and I propose to call
it Oxnerella1 maritima, n. g., n. sp. It will be seen, I
think, that Oxnerella bears much the same relation to
Gy mnosp h sera that Actinophrys does to Actino-
sphserium; for Actinophrys is like a uninucleate Ac-
t inospliaeriu m just as Oxnerella is like a uninucleate
Gymnosphaera.

3. Habits, Habitat, Etc.

Of the habits of Oxnerella little need be said, for it does
not differ in any important ways from many familiar Heliozoa.
A few points are worth noting, however.

As the organisms occurred in cultures, I can only describe
their behaviour in these ; and it is possible that in nature
their habits are different. Nevertheless, they appeared so
healthy and normal that I do not suppose the differences can
be very great.

The animals often float freely in the water, but display a
special predilection for the surface film. Many of them were
always to be found also at the bottom of the culture -tanks
and on the sides, and in these situations they were generally
more or less firmly attached by their pseudopodia to the
substratum. When the cultures were well stocked, the
organisms could be “ fished ” with a pipette at all levels, and
were found attached to all the objects (algse, stones, etc.) in

1 In honour of my friend, Dr. Mieczjslav Oxner, of the Musee
Oceanographique, Monaco.

OXNERELLA MAR l TIMA.

519

the aquaria. Cover-glasses suspended at varying depths in
the water by means of cotton threads were generally found
to have many animals adherent to them after they had been
left for one or two days. As animals such as these are not
able to swim in any way, their distribution through the
motionless. water of a tank is probably brought about some-
what as follows : They lie first of all on the bottom — a
position which they always take up when removed from one
vessel to another. They then creep along the bottom, and up
the sides of the aquarium until they reach the surface film.
They crawl along this by means of their pseudopodia, and
then, becoming detached, they fall slowly towards the bottom
once more, alighting upon any foreign body which they may
encounter on the way. In nature, no doubt, they are much
more extensively distributed by the movements of the water.

The creeping movements of an Oxnerella may be easily
observed on a slide under the microscope. If it is not unduly
compressed by a cover-glass, it will begin to move about
slowly as soon as it has recovered from the shock of being
mounted in the preparation. It progresses by a gentle half-
gliding, half-rolling motion, dragging itself along by means
of its pseudopodia.

One of the features which first attract attention on closely
observing the living animal is the streaming of the minute
granules on the pseudopodia — of which mention has already
been made. Although this streaming is well known in other
Heliozoa, the mechanism by which it is brought about is still
unexplained, and the motions of the granules are really very
puzzling if observed for any length of time. The “granules”
are generally supposed to be little knobs or thickenings of
the protoplasm of the pseudopodium — an opinion shared by
Schaudinn (1896); but it is not impossible that they are
really adherent foreign particles borne along by mucus
currents. I have been unable to satisfy myself on this point,
either in the case of Oxnerella or of other forms displaying
the same phenomenon. I have observed that the “granules”
do not all stream in the same direction, even on the same

CLIFFORD DOBELL.

20

pseudopodium. They may at times be seen to meet one
another when coming from opposite directions, and, after
appearing to jostle one another for a moment, travel away in
opposite directions — sometimes returning along their former
paths, sometimes passing one another. In such circumstances
their movements appear curiously purposive, and they remind
me of the equally remarkable movements of the spindle-
shaped bodies (individual organisms ?) in the living network
of a Labyrinthula .

Oxnerella, like other Heliozoa, largely uses its pseudo-
podia for capturing its food. In the aquaria this consisted
almost entirely of the sw^arm-spores of green algae, which were
plentiful. The living animals were generally filled with the
bright green bodies of these (PL 27, fig. 1), in various stages
of digestion, and under a low magnification themselves
appeared to be green in consequence.

The water in which Oxnerella occurred was sent to
London from Plymouth. But it is quite likely that the
organisms are widely distributed and by no means uncommon
in the sea, and have hitherto escaped notice on account of
their minute size.

4. Division.

My chief reason for describing Oxnerella is to enable me
to record and figure in detail its method of multiplication b;y
division. I shall therefore enter into this matter now with
some particularity.

The two organs whose behaviour is of special interest in
division are the nucleus and the so-called “ central granule,”
to which the axial fibres of the pseudopodia are attached. I
shall therefore begin by describing these two structures in
the “ resting ” (i. e. not dividing) organism in greater detail.

The Nucleus. — On account of its somewhat large size
and its lack of colour, the nucleus of Oxnerella can usually
be made out quite clearly in the living organism. It lies
embedded among the bright green food-bodies present in the

OXNERELLA MARITIMA.

521

cytoplasm (see PL 27, fig. 1), and it is therefore sometimes
necessary to compress the creature slightly with the cover-
glass in order to see its nucleus distinctly. In the living
organism the nucleus appears as a relatively large vesicle
surrounded by a distinct nuclear membrane, and containing a
large central karyosome (PI. 27, fig. 1). Between the mem-
brane and the karyosome there is a clear zone, in which I
have not been able to make out any structure in the fresh
state (PI. 27, fig. 1). But in fixed and stained specimens
{PL 27, fig. 2) not only can all the structures just described
be seen, but in addition the clear zone appears filled with
minute chromatin granules supported on an indistinct achro-
matic network. These appearances are very constant after
fixation and staining in various ways, and I incline to the
view that the minute granules in the clear zone are actually
present — though invisible — in the living organism, and not
produced by fixation.

The karyosome appears to be almost homogeneous. It
stains uniformly with ordinary chromatin stains, but in iron-
haematein preparations it usually appears rather paler in the
centre than at the periphery (PL 27, fig. 2). It contains no
granules of any sort.

As already noted, the nucleus is usually oval in outline.
It may be noted farther that it appears to be rigidly fixed in
position. It is not possible to displace it by slight pressure;
and even if pressure so great be applied to an organism as to
burst it completely and set free most of its enclosed food-
bodies, the nucleus still remains behind in the debris of the
body. It is probable, I think, that the axial fibres of the
pseudopodia which cross the nuclear membrane, and stain
more deeply than the rest, are really attached to the nucleus
and serve to anchor it in position (see PL 27, fig. 7).

The nucleus in the resting state is about 4 /a-b /u in greatest
diameter, ranging from about 3*3 /x to 5*5 jx.

The “ Central Granule.” — As I shall often have to
refer to this important organ in the course of the ensuing
descriptions, it seems desirable to give it a name. The term

VOL. 02, PART 4. NEW SERIES. 36

522

CLIFFORD DOBELL.

“ central granule” is a periphrasis which does not
correctly describe its structure, nor indicate its most important
functions ; and for brevity, and also for other reasons which
will be clear later, I propose to call the organ in question a
centroplast.

In a living Oxnerella the centroplast is with some diffi-
culty visible as a minute spherical corpuscle lying at the
centre of the organism (PL 27, fig. 1). It is rather feebly
refringent, and is surrounded by a clear zone of protoplasm
quite free from all food-bodies or granules. The size of this
zone varies a good deal in different individuals. Radiating
from the centroplast and traversing the clear zone in all
directions can be seen the central ends of the axial fibres of
the pseudopodia.

In fixed and stained specimens all these structures can be
studied in greater detail. The centroplast itself (PI. 27,
figs. 2, 5) generally appears as a minute clear sphere with a
deeply-staining granule at its centre. The periphery of the
sphere stains deeply, as though it were clothed with a very
delicate membrane. The axial fibres, when traced through
the clear zone of protoplasm towards the centroplast, appear
to terminate in minute knobs or granules on its external
membrane, and cannot as a rule be traced beyond this to its
central granule (PI. 27, fig. 5).

The central granule of the centroplast stains deeply with
iron-liaematein, less deeply with carmine stains. In its
entirety the centroplast thus resembles a tiny nucleus with a
karyosome and nuclear membrane.

If one examines with care the centroplasts of a number of
different individuals, it can be seen that they do not all pre-
sent the appearances just described, though these are the
most usual. In certain individuals no differentiation into
central granule and surrounding membrane can be discovered.
The entire centroplast is a minute, deeply-staining, and
homogeneous dot (PI. 27, fig. 3), to which the pseudopodial
fibres appear to be attached directly. In other specimens
a very minute central granule appears to have become-

OXNERELLA M A K IT IMA.

523

differentiated in the centre of this minute darkly-staining
mass (PL 27, fig. 4) ; and still other specimens show gradual
transitions to the “ typical ” form of centroplast (PL 27,
fig. 5). Further, organisms can also be found in which the
whole centroplast seems to have greatly increased in size
(PL 27, fig. 6). The central granule is larger, and the clear
zone separating it from the membrane has also expanded.
In such specimens the axial fibres seem no longer to termi-
nate on the membrane, but to pass through it to the central
granule. I believe they are actually attached to this, but
from the very small size of all the structures it is hardly
possible to be absolutely certain of their relations. This last
condition (PL 27, fig. 6) affords an easy transition to that
first described (PL 27, fig. 3).

The appearances just described strongly suggest that
cyclical changes occur in the centroplast of the “ resting ”
animal. Similar appearances have already been described
and thus interpreted in Wagnerella (Ziilzer, 1909). Com-
parison, moreover, with the cyclical changes described in the
centrosome of the metazoan egg (Vejdovsky and Mrazek,
1903) at once suggests itself. Although both the interpreta-
tion and the comparison appear to me justifiable, I am not
completely convinced of their correctness ; and I am at a
loss to understand what function the “ cyclical changes ” in
Oxnerella can subserve. I thought at one time that they
might perhaps play some part preparatory to the division of
the organism — for the centroplast has, as will be seen laterT
an important function in this process. But since the structure
of the centroplast is “ typical” (PL 27, fig. 5) immediately
before division and immediately after, this seems to me im-
probable. There is no obvious reason why it should pass
through a cycle of changes — returning finally to its original
condition — during the interdivision period.

General Account of the Process of Division. —
Before describing in detail the successive phases of division
it will be convenient to give a brief general outline of the
process.

524

CLIFFORD DOBELL.

Division is, in all cases which I have observed, an equal
binary fission, resulting in the production of two small
organisms exactly like the parent organism in everything save
size. Although the organisms which are about to divide are
usually of large size, this is not always the case. I have seen
a number of quite small forms in various stages of the pro-
cess. Again, most dividing' organisms are filled with food-
bodies, but this is not always so. Illustrations of this will
be found in the figures.

During’ division the animal remains at rest, with all its
pseudopodia completely retracted. Not until the two
daughter-organisms are completely separated do the pseudo-
podia again make their appearance. I believe, moreover, that
the animal is always attached to some substratum during
division; that is to say, it does not divide while floating
freely in the water. I infer this from the fact that I never
obtained any division stages in organisms suspended in the
water ; whereas I obtained many in the organisms attached
to cover-glasses floating on the surface film or placed at
various levels in the aquaria. Most of the dividing organisms
were found attached to the surface film.

The nucleus divides by mitosis, the centroplast playing
the part of a centrosome. The whole process can really be
seen at a glance by inspecting the figures (PI. 27, figs. 8-22),
but the following brief account may be given in amplification.

Pro phases. — The centroplast is the first organ to divide.
Immediately before division it is in the “ typical” condition
(PI. 27, fig. 5), displaying a large central granule and a
distinct membrane. It then becomes elongated, and the
central granule divides into two (PI. 27, fig. 7). In the early
stages of division the central granule appears (see PI. 27,
fig. 7) as a minute cylindrical body with two deeply-stained
polar caps — presumably halves of the original granule. A
little later, however, the caps become minute spherical
granules, connected by a deeply-staining strand (PI. 27, fig. 8).
This appearance is exactly comparable with that of a dividing
.centrosome, at the stage when the daughter-centi osomes are

OXNERKLLA MAR IT LM A.

525

united by a centrodesmose. Up to this stage (PL 27, fig. 8)
the animal remains spherical, with a few pseudopodia still
visible. These are now completely retracted, and the animal
becomes drawn out into an elongate form. As it does so, the
daughter-centroplasts draw further apart, but still remain
connected by their “ centrodesmose,” which occupies the
centre of the long axis of the body (PI. 27, fig. 9). The
dividing centroplast at this stage appears, in consequence, as
a much attenuated dumb-bell. The centrodesmose is an
excessively fine but quite distinct line. Throughout the
division of the centroplast the central ends of the axial fibres
of the pseudopodia are visible, radiating through the cyto-
plasm exactly like the astral rRys of a centrosome (PI. 27,
figs. 8, 9).

The “ centrodesmose ” now vanishes, and the two daughter-
centroplasts are seen — each surrounded by a few short
“ astral rays ” — to occupy symmetrical positions at opposite
ends of the organism.

During the early stages of the division of the centroplast
the nucleus increases in size (PI. 27, fig. 8). As the animal
elongates, the nucleus gradually travels towards the middle
of the body, until it finally takes up a position midway
between the two daughter-centroplasts (PI. 27, figs. 8-11).
During this translocation the nucleus usually has an irregular,
misshapen appearance (PL 27, figs. 9, 10). Sometimes, also,
its karyosome becomes fragmented (PL 27, fig. 10). But as
soon as it reaches its station between the two centroplasts, it
recovers its spherical form and lies as a large and con-
spicuous vesicle at the very centre of the whole animal (Pl. 27,
fig. 11). The karyosome now begins to diminish in size
(PL 27, figs. 11, 12), and as it does so, the chromatin granules
in the rest of the nucleus increase in size and number. These
granules, therefore, are probably formed in part at the expense
of the karyosome.

During these nuclear stages, the centroplasts also undergo
changes. They become more or less elongated in a direction
transverse to the long axis of the dividing organism (PL 27,

526

CLIFFORD DOBELL.

figs. 11, 12). Their central granules thus appear as short
rods, sometimes slightly knobbed at the ends.1 The length
of these “rods” is different in different individuals (cf. for
instance, PI. 27, fig. 11, with PI. 27, fig. 12).

A definite spindle now begins to make its appearance
between the centroplasts. At first (PI. 27, fig. 12) a few
spindle-fibres only can be seen, stretching from the centro-
plasts to the nucleus. They gradually become more con-
spicuous and numerous, however, until a perfect spindle
figure is produced, with the centroplasts at its poles — exactly
like typical centrosomes (PI. 27, figs. 13, 14). Whilst the
spindle is forming, the nucleus undergoes further changes.
The karyosome disappears ; ‘the chromatin granules become
fainter, and chromosomes make their appearance arranged
transversely across the nucleus (PI. 27, figs. 12, 13). Mean-
while the nuclear membrane becomes less distinct, and the
nucleus becomes drawn out towards the poles of the spindle,
as though it were actively pulled by the spindle-fibres of the
centroplasts (PI. 27, figs. 12, 13). Finally, the nuclear mem-
brane vanishes, the chromosomes take up their position on
the equatorial plate, and a typical mitotic figure results
(PI. 27, fig. 14).

There are several details which I have not been able to
make out with certainty, chiefly on account of the very small
size of all the structures concerned. In the first place, the
mode of origin of the chromosomes in the nucleus is extremely
difficult to ascertain. The appearances are as I have just
described them, and the following seems the most plausible
interpretation. The chromosomes are formed chiefly (or
wholly) from the substance of the karyosome— as in certain
amoebae and other Protozoa (see, for example, Dobell (1914),
Jameson (1914), etc.). The “ chromatin ” granules surround-
ing the karyosome in the resting nucleus probably disinte-

1 It is probable, I think, that this appearance should be interpreted,
not as a rod with knobbed ends, but as an optical section of a disc with
a thickened rim, viewed edgewise. The structure is too small, however,
for me to speak with certainty.

OXNERELLA MARITIMA.

527

grate, and pass on to the spindle-fibres (PL 27, figs. 1 1-14),
which are very dark and granular between the “ asters” and
the equatorial plate (PI. 27, fig. 14). I have found no spireme
stage in the prophases, but am not prepared to deny its
occurrence.

I have not succeeded in counting the chromosomes on the
equatorial plate. They are extremely small and in the form
of short rods so closely packed together (PI. 27, fig. 14) that
their number can only be roughly guessed. As the equatorial
plate is probably in the form of a ring, and as rather more
than ten chromosomes can as a rule be counted across it when
presented edgewise, I estimate the chromosome number as
probably about twenty-four.

There are some peculiarities in the spindle which require
further notice. At all stages, after it is fully formed, it
shows a differentiation into two parts, which do not stain
alike. There is, first, an unstained or achromatic central
area immediately surrounding the equatorial plate (PI. 27,
fig. 14), so that the whole of the middle part of the spindle may
be compared with a tiny clear globe with the chromosome
ring forming its equator. Secondly, there is, on either side
of the clear central part, a more deeply-stained and granular
portion of the spindle which resembles a truncated cone with
its apex directed towards the centroplast (PI. 27, figs. 14, 15).
The cones become paler in the region of the centroplasts, and
the ends of the spindle-fibres appear to be rooted partly in the
central granules of the latter, and partly in their membranes
(PI. 27, fig. 14). There is no sharp demarcation between the
achromatic central part of the spindle and the stainable cone-
like ends, so that the two parts appear to be differentiations
of one and the same structure — the spindle — rather than
separate elements. They are, no doubt, respectively homolo-
gous with the so-called “ polar plates” and “ achromatic
cones” described in the mitotic figures of Actinosphaerium
(cf. Brauer (1894), R. Hertwig (1898) ).

Metaphase. — The chromosomes on the equatorial plate
now divide so that two daughter-plates are formed. It is not

528

CLIFFORD DOBELL.

possible to ascertain how the chromosomes divide, on account
of their extremely small size. But since the chromosomes on
the equatorial plate appear to be short rods (PL 27, fig. 14),.
and those at the metaphase smaller granules (PI. 27, fig. 15),
it seems probable that the cliremosomes themselves divide by
a transverse constriction into two.

Anaphases. — The daughter-groups of chromosomes now
move apart, gradually passing towards the poles of the spindle
(PI. 27, figs. 16, 17, 18). As they do so, they become still
more closely packed together, and individual chromosomes
can no longer be resolved. In the early anaphases (PI. 27,
fig. 16) distinct spindle-fibres can be seen between the
chromosome groups, but later the fibres become less distinct
and more irregular (PI. 27, figs. 17, 18). Simultaneously, the
differentiated structure of the spindle becomes less distinct,
and finally vanishes.

During the anaphases the centroplasts at the poles of the
spindle also undergo certain changes. Their central granules
are often much drawn out during the later stages, so that
they appear in optical section as fairly long rodlets (PI. 27,
figs. 17, 18). These then shorten, and thicken somewhat, and
they then become bent into the form of incomplete rings, open
on the side nearest the spindle (PI. 27, fig. 20). Finally, a
closed ring is formed, which becomes converted into a
minute, homogeneous, darkly- staining central granule in each
daughter-centroplast (PI. 27, fig. 19). All these changes
occur during the late anaphases and early telophases, and
they do not always synchronize with the nuclear changes.
For example, PI. 27, fig. 20, shows an earlier stage in the
reconstruction of rhe centroplast than PI. 27, fig. 19, though
the nuclear stage in PI. 27, fig. 19, is earlier than that in
PI. 27, fig. 20. Centroplast and nucleus are thus independent
of one another to some extent at this period. I have found a
good deal of variation in this respect in different individuals.
In at least one specimen I have seen the centroplasts com-
pletely reconstructed, and spindle-fibres no longer visible,
before the telophases had begun.

OXNEliELLA MAIUTIMA.

529

-Telophases. At the end of tlie anaphase stages each set
of chromosomes breaks up into a ball of deeply-staining
granules, with which probably a part of the substance of the
spindle becomes incorporated to form the daughter-nuclei
(PJ. 27, fig. 19). Between these two nuclei the spindle-fibres
rapidly disappear (PI. 27, figs. 19, 20). As reconstruction of
the nuclei proceeds, a part of the chromatin becomes aggre-
gated at the centre of each, forming the new karyosomes
(PI. 27, figs. 19, 20). The daughter-nuclei then soon assume
the structure characteristic of the ordinary resting nuclei.
Ihe stages in this process will be evident from the figures
without further description (PI. 27, figs. 20—22), since they do
not differ from the telophases of many other nuclear divisions

for instance, those in Amoeba gleba3, which I have else-
where described (Dobell, 1914).

Constriction of the protoplasmic body of the organism into
two begins during the anaphases (cf . PL 27, figs. 17, 18), and
is completed during the telophases (PI. 27, figs. 20-22). As
the two little daughter-individuals separate they gradually
form their pseudopodia, the axes of which can be seen to
spring from the new centroplasts (PI. 27, fig. 22). Each
animal very soon acquires the typical form of the adult, with
central centroplast, excentric nucleus in close relation to it,
and so on. Each daughter-organism also inherits ” about
an equal share of the food-masses of its parent.

It is perhaps worthy of note that the final constriction of
the body into two is preceded by a stage in which there is a
narrow strand of protoplasm connecting the two organisms.
(See PL 27, fig. 21. In later stages, before complete separa-
tion, the strand becomes much finer before it snaps.) This
detail in division distinguishes some of the free-living amoebae
— e.g. A. glebae and related forms — from others, in which
the constriction is effected more sharply, without the per-
sistence of a connecting strand (e.g. “A. limax” and
related forms).

I have not been able to watch the whole process of division,
from beginning to end, in the same living organism. I have

-530

CLIFFORD DOBELL.

found it impossible to study the dividing nucleus in unstained
specimens, on account, probably, of the small size of all the
structures concerned. Elongated organisms- — which are seen,
in stained specimens, to be at any stages of the prophases or
metaphase — complete their division in times varying from
about five minutes to a quarter of an hour. These are the
only living forms which I have been able to recognise as
forms about to divide; and they are actually, as will be
evident, already somewhat far advanced in the process. On
analogy with other organisms, and from the few observations
which I have been able to make, I should estimate the total
time taken for division at about twenty minutes to half an
hour.

5. Some Remarks on the Centroplast of the Heliozoa.

I propose to terminate this account of Oxnerella with a
brief consideration of certain problems presented by the
centroplast. Although this organ is probably peculiar to a
small section of the Heliozoa — for it is not known to be
present in any other organisms — a consideration of its
functions and homologies leads to some of the fundamental
problems in the morphology of the Protozoa.

The facts so far established concerning the centroplast may
first be enumerated before their interpretation is discussed.
They can, I think, be best reviewed in their historic order.

The centroplast appears to have been first seen in Acan-
thocystis by Grenacher (1869). He figured it, and described
it as “ a tiny pale corpuscle ” lying at the centre of the
organism. He believed, moreover, that the axial fibres of the
pseudopodia were rooted to it; but he was unable, as he says,
4t to prove a direct connexion.” The proof was, however,
soon furnished by F. E. Schulze (1874), R. Hertwig (1877),
and others. All later workers have confirmed their observa-
tions, and extended them to all the Heliozoa known to possess
■centroplasts.

The anatomical relations of the centroplast having been

OXNEEELLA MARLTIMA.

531

thus established, it became obvious that at least one function
of this organ is skeletal ; it serves as a central point of
attachment, or focus, for the axial fibres of the radiating
pseudopodia. But whether the centroplast possessed any
other function was not apparent from its morphological
relations, and had, therefore, still to be proved.

About a quarter of a century ago, the centrosome, then
recently discovered by E. van Beneden, was occupying the
.attention of most cytologists. If an Acanthocy stis is
homologous Avith a metazoan cell — as it was generally sup-
posed to be — and its nucleus homologous with the cell’s
nucleus ; then if the centrosome is a permanent organ in the
cell — as was also then generally believed — there ought clearly
to be some corresponding organ in the Acanthocystis.
And seemingly there was : there was the centroplast, already
described and figured exactly like a centrosome. It was
therefore almost inevitable that someone should suggest
that centrosome and centroplast are homologous struc-
tures.

So far as I am aware, the first suggestion of this homology
to appear in print came from Biitschli (1892). It was,
however, nothing more than a suggestion based upon the
striking structural similarity of the two organs — a similarity
Avhich is, as I shall try to show, somewhat misleading.

At about this time a new fact was brought to light by
Sassaki. Whilst studying a new lieliozoon (G-y mnosphsera
albida), he disco\^ered that the division of the whole organ-
ism is preceded by a division of the centroplast — a fact Avhich
justified him, more than a mere structural resemblance could,
in homologizing this organ with the centrosome of a metazoan
cell. It should be noted that Sassaki’s observation had been
made and written down by him in 1891, 1 although it was not
published until 1894. To the Japanese zoologist, therefore,
belongs the credit, not merely of first suggesting that the
centroplast is the homologue of a centrosome, but of having

1 See footnote to Sassaki’s paper (p. 45) by R. Hertwig.

532

CLIFFORD DOBELL.

discovered an important fact upon which this homology could
be based.1

Now the heliozoon studied by Sassaki (Gfy mnosphsera) i&
multinucleate, though it possesses but a single centroplast ;
and he was unable to demonstrate that this organ plays the
part of a centrosome during the nuclear divisions. To this-
extent, therefore, the suggested homology of centroplast and
centrosome remained unsupported by facts.

This deficiency was soon made good. In a brief but
memorable paper, published in 1896, Schaudinn (1896a)
described the division of Acanthocy stis aculeata, a
heliozoon possessing a centroplast and a single nucleus.
“ From this brief account/’ he says, “it will, I think, be clear
that the nuclear division in the heliozoon investigated takes
place in essentially the same way as the typical mitosis of a
metazoan cell, and that the central granule [= centroplast]
corresponds to the centrosome of the metazoan cell.” Schau-
dinn published only six figures of division stages in Acan-
thocy stis, leaving many gaps which he promised to fill in his
full account, which was never published. He stated further
that he had observed similar phenomena in Acanthocy stis
turfacea, A. myriospina, Raphidiopliry s, Sphasras-
trum, and H e t e r o p h r y s . The division of all these forms is
still unknown — or, at least, undescribed ; and no full account of
the division of Acanthocy stis aculeata has ever appeared.

To my knowledge, only one partial confirmation of Schau-
dinn’s statements has hitherto been published. I refer to
Zuelzer’s description of Wagnerella (1909). Of several dif-
ferent methods of nuclear division described by the authoress
in this peculiar heliozoon, there is one in which the cen-
troplast appears to behave like a centrosome.2 But although

1 Another Japanese zoologist — Watase— put forward the same sug-
gestion in 1894. In the same year, also, Heider demonstrated the
centroplast of Raphidiophrys — previously described by F. E.
Schulze — and emphasized its resemblance to a centrosome. Both
these workers, however, knew beforehand of Sassahi’s results.

2 I refer to the multiple division of the “ head ” of the organism,
whereby a brood of young is formed. I do not consider the other

OXNERELLA MA ULTIMA.

533

the centroplast appears to divide before the nucleus, and to
take up the position of a centrosome during the division of
the latter, it is a curious fact — assuming the account to be
correct — that the nuclear division itself is apparently a kind
of amitosis. These observations do not furnish a very sub-
stantial confirmation, therefore, of Schaudinn’s statement that
the centroplast plays the part of a centrosome in the mitosis
of the heliozoan nucleus.

It has long seemed to me desirable that the behaviour of
the centroplast during the division of those Heliozoa possessing
this organ should be carefully studied and described. As
experience has shown, Schaudinn’s brief statements and
incomplete descriptions are not always to be accepted as
established facts ; for the magnitude of his mistakes is
beginning already to rival that of his successes.

It is for this reason that I have described the division of
Oxnerella in some detail. My own observations induce me
to believe that Scliaudimfis account of the division of Acan-
thocystis is almost certainly correct; and it therefore
appears to me justifiable to conclude that the centroplast
actually does behave during nuclear division, in those
Heliozoa which possess a single nucleus, precisely
like the centrosome in a typical mitosis in a metazoan cell.
We have still to learn, however, the part (if any) played by
the centroplast in the division of those Heliozoa which are
multinucleate.

Although I follow Schaudinn up to a certain point — as just
noted — I am unable to concur in his speculations. On the
evidence of the observations just considered, but more
especially from his observations on the origin of the centro-

methods of nuclear division in which a “ centriole ” is said to be
present, and later to become the centroplast of a new individual.
Although this is several times stated to occur, I can find no direct
evidence in support of the statement, which appears to be merely an
assumption based upon Schaudinn’s statements about Acantho-
cystis. Accordingly, I do not think Zuelzer’s assertions can be
regarded as confirmations of Schaudinn’s results.

534

CLIFFORD DOBELL.

plast in the young forms of Acanthocystis produced by
budding, he was led to advance certain views, now well
known, on the phylogeny of the centrosome. These views
depend, for their validity, upon the assumptions (1) that his
observations on the Heliozoa were correct (those relating to
the buds — the most important for his speculations — have
never been confirmed) ; (2) that the centroplast and the
centrosome are homologous organs ; (3) that the Protozoa,,
being “primitive animals, ” furnish us with data for deter-
mining the phylogeny of the Metazoa. As regards the first
assumption, I would merely note that the observations in
question have been confirmed in part only. As regards the
third, I will only say that I regard it, as I have elsewhere
pointed out (1911) as wholly unjustifiable. Concerning the
second assumption I shall here say a few words.

For reasons which I have given elsewhere (1911), I do not
regard any single protozoon as the homologue of a metazoan
cell. A single individual lieliozoon — e.g.an Oxnerella —
is, in my view, comparable with a single whole metazoon, and
not with one of the cells of which it is composed. An
Oxnerella is a whole non-cellular organism, whilst a meta-
zoon is a similar whole organism whose body is differentiated
into cells. The one displays a non-cellular, the other a
cellular, morphological composition. It follows, therefore,
that we have no reason to assume that those organs and
structures which are peculiar to the cell must have a morpho-
logical counterpart in the individual protozoon.

Now the centroplast of Oxnerella and other similar
Heliozoa is not an organ whose activities are limited to a
transient phase in the life of the organism — the momentary
process of division. It is, on the contrary, a complex struc-
ture, permanently present, which plays a skeletal part in the
organism as a whole, and in connexion with its organs of loco-
motion and prehension, and a totally different part as an
accessory to the process of division. It plays the part of a
centrosome, but it does much more. On the other hand, it
seems probable that the centroplast of multinucleate Heliozoa

OXNEKELLA MA1UTIMA.

535

(e.g. G-ymnosphaera) is only skeletal in function, and plays
no part in nuclear division. (This has still to be determined,
but it is not easy to conceive how a single centroplast could
act as centrosome to many different nuclei.) The centroplast
of the Heliozoa is thus closely comparable with the blepharo-
plast of the Mastigophora — an organ permanently subserving
a skeletal function to the organs of locomotion (the flagella),
and in some forms assuming the office of centrosome at
division, in others playing no part in this process (as in some
trichomonads and in Copromonas respectively, as I havo
shown in two earlier papers (1909 and 1908) ). To say that
either the centroplast or the blepharoplast is the homologne
of the metazoan centrosome, and to apply the same term to
all these structures, appears to me, therefore, inadvisable.
By giving so wide a connotation to the term “ centrosome ”
we shall effect an economy in words ; but we shall introduce
a confusion of thought by grouping together, as the same,
certain structures which are in many ways radically different.
In the language of the older morphology, I would say that
the centroplast and the centrosome may be analogous, but are
not homologous, organs.

For these reasons, in addition to those based on general
grounds, I consider that all phylogenetic speculations regard-
ing the centrosome, based on a comparison of the Heliozoa —
or any other Protozoa — with the cells of Metazoa, should bo
viewed with considerable scepticism. Opinions on such matters
differ, and always will differ; but for my own part, such
views as those of Schaudinn, and his many followers, on tho
“ phylogeny of the centrosome,” appear to be mere specula-
tions which are not only improperly called “ theories,” but
are even outside the limits of legitimate hypothesis.

6. Diagnosis of Oxnerella maritima.

In conclusion I will add a brief diagnosis of Oxnerellf»r
which will serve at the same time as a summary of the facts
recorded in previous pages.

Oxnerella maritima nov. gen., nov. spec. Aplirotho-

536

CLIFFORD DOBELL.

racan heliozoon of very small size (10 /u to 22 /u in diameter) ;
spherical, with numerous very fine radiate pseudopodia (axo-
podia) with streaming granules and with axes rooted in a
centrally placed centroplast ; no stalk, no contractile vacuole,
no gelatinous investment, no spicular skeleton, no sharply-
differentiated ectoplasm and endoplasm. Nucleus single,
large, excentric ; vesicular, with large central karyosome.
Reproduction by equal binary fission, in which nucleus divides
by mitosis, the centroplast playing the part of a centro-
sorne. Free-floating or creeping; solitary; marine. Food
chiefly vegetable matter (in cultures, swarm-spores of green
algae).

References.

Brauer, A. (1894).—' " Ueber die Encystirung von Actinosphserium
eichhorni Ehrbg.,” ‘ Zeitschr. wiss. Zool.,’ vol. lviii, p. 189.

Biitschli, O. (1892). — “Ueber die sogenannten Centralkorper der Zelle
und ihre Bedeutung,” ‘Yerhandl. naturhist.-med. Yer. Heidel-
berg,’ vol. iv (n.f.), p. 535.

Dobell, C. (1908). — “The Structure and Life-history of C opr onion as
subtilis, n. g., n. sp.,” ‘Quart. Journ. Micr. Sci.,’ vol. 52, p. 75.

(1909). — “Researches on the Intestinal Protozoa of Frogs and

Toads,” ibid., vol. 53, p. 201.

(1911). — “The Principles of Protistology,” ‘Arch. f. Protis-

tenk., vol. xxiii, p. 269.

(1914). — “ Cytological Studies on Three Species of Amoeba,”

ibid., vol. xxxiv, p. 139.

Grenadier, H. (1869). — “ Bemerkungen fiber Acanthocystis viridis,
Ehrbg. sp.,” ‘ Zeitschr. wiss. Zool./ vol. xix, p. 289.

Heider, K. (1894). — Demonstration of Sections of Raphidiophry s,
‘Yerhandl. deutsch. Zool. Ges.,’ vol. iv, p. 94.

Hertwig, R. (1877). — “ Studien fiber Rhizopoden,” ‘ Jena. Zeitschr.,
vol. xi, p. 324.

(1898). — “Ueber Kerntlieilung, Riclitungskorperbildung und

Befruclitung von Actinosphserium eichhorni,” ‘Abh. bayr.
Akad. Wiss.,’ vol. xix.

Jameson, A. P. (1914). — “A New Phytoflagellate (Parapolytoma
satura, n, g., n. sp.) and Its Method of Nuclear Division,”
‘ Arch. f. Protistenk.,’ vol. xxxiii, p. 21.

OXNERELLA MARITIMA.

537

Sassaki, C. (1894). — “ Untersuch ungen fiber Gymnosphaera albida,
eine neue marine Heliozoe,” ‘ Jena. Zeitschr., vol. xvviii, p. 45.

Schaudinn, F. (1896). — “ Heliozoa,” in ‘ Das Tierreich ’ (Berlin).

(1896a). — “Ueber das Zentralkorn der Heliozoen, ein Beitrag

zur Centrosomenfrage,” ‘Yerhandl. deutsch. zool. Ges.’ (Bonn),

p. 113.

Scliulze, F. E. (1874). — “ Rhizopodenstudien II,” ‘ Arch. mik. Anat.,’
vol. x, p. 377.

Yejdovsky, F., and Mrazek, A. (1903). — ■* Umbildung des Cytoplasma
wahrend der Befruchtung nnd Zelltheilung,’, ibid., vol. lxii,
p. 431.

Watase, S. (1894). — “Origin of the Centrosome,” ‘Biol. Lect., Wood’s
Holl.’

Zuelzer, M. (1909). — “ Bau und Entwicklung von Wagnerella
borealis, Mereschk.,’ ‘Arch. f. Protistenk.,’ vol, xvii, p. 135.

EXPLANATION OF PLATE 27,

Illustrating Mr. Clifford Dobell’s paper on “Oxnerella
maritima, nov. gen., nov. spec., a New Heliozoon, and
Its Method of Division ; with Some Remarks on the
Centroplast of the Heliozoa.”

[All figures depict Oxnerella maritima, n. g., n. sp., and are
drawn to the same magnification, 'which is approximately 1500
diameters. Except Fig. 1, all are drawn from whole specimens fixed
in Bouin’s fluid and stained with alcoholic iron-alum-haematein. They
were drawn under a Leitz 2 mm. apochromatic objective (N.A. = 1‘40),
with compensating oculars, using a Leitz achromatic condenser (N.A.
— 1’40) with critical illumination.]

PLATE 27.

Fig. 1. — Living organism, slightly compressed to show internal
structure. (The rounded masses in the cytoplasm in this and following
figures are ingested swarm-spores of green algae, in various stages of
digestion.)

Fig. 2. — Similar specimen, fixed and stained.

Figs. 3-6. — Central region in different individuals, showing various
appearances presented by the centroplast.

VOL. 62, PART 4. NEW SERIES.

37

538

CI-IFFORD DOEELL.

Fig. 7. — Central region and nucleus of an organism at a very early
stage of division of the centroplast. Note the attachment of the
nucleus to it by three of the axopodial rays.

Figs. 8-22. — Successive stages in division.

Figs. 8 and 9. — Division of centroplast.

Figs. 10-13. — Prophases.

Fig. 14. — Stage of equatorial plate.

Fig. 15. — Metaphase.

Figs. 16-18. — Anaphases.

Figs. 19-21. — Telophases.

Fig. 22. — Division complete, the two daughter-organisms completely
reconstructed.

(In Figs. 15, 16, and 19 the nucleus and surrounding protoplasm
only are shown.)

£; PROBOSCIS PORES ” IN CRANIATE VERTEBRATES. 539

“Proboscis pores” in Craniate Vertebrates, a
Suggestion concerning the Premandibular
Somites and Hypophysis.

By

Edwin S. Goodrich, F.R.S.,

Fellow of Merton College, Oxford.

With Plate 28, and 3 Text-fignres.

There is in Ampliioxus on the roof of the buccal cavity a
deep pit known as Hatschek's pit, from its discoverer. Its
blind, inner end extends upwards to the right of the noto-
chord, while the lining epithelium is continuous at the opening
of the pit with the areas of thickened ciliated epithelium
which spread over the roof and sides of the buccal cavity.
This ciliated organ is the wheel-organ of Johannes Muller,
and was shown by him to drive a current of water and food-
particles into the mouth. Van Wijhe (14) has since given a
detailed account of its structure. An unpaired dorsal region
extends backwards so as to surround the opening of the pit,
then divides into right and left tracts. The two branches run
towards the velum and then down the sides of the buccal
cavity, but do not meet ventrally. As shown in Text-fig. 1,
finger-shaped tracts extend forwards on the inner surface of
the oral hood. These structures become more complicated in
older specimens. The deeply staining epithelium of which
Muller's organ is composed is formed of very closely packed,
narrow columnar cells, whose nuclei form several layers, and
whose outer ends bear each one cilium. Good figures of
these cells have been given by Langerlians (8).

540

EDWIN S. GOODRICH.

Although the pit and its lining epithelium have been
described by several authors — Hatschek (6), Langerhans (8),
Willey (16), van Wijhe (14), Andrews (1) — the complexity of
its histological elements seems to have escaped the notice of
these observers, and its finer structure deserves further study.
In the adult the epithelium is composed of cells roughly
disposed in three layers (PL 28, figs. 12, 13, 14). The most
superficial cells are large, with a broad end reaching to the

Text-fig. 1.

Left side-view of the head of a young Amphioxus. The left
body-wall, oral hood, and wall of the pharynx have been
cut away, exposing the right half of the wheel-organ, w.o.
Hatschek’s pit, H.p., is seen by transparency, n.c. nerve
cord. n.p. Olfactory pit. n.t. Notochord, o.h. right oral
hood. pli . Pharynx, v. Yelum surrounding the true mouth.

free surface, and bearing a bunch of fine cilia generally
gathered together to form a flame-like tuft. Their nuclei are-
pale and rounded. The middle layer is composed of narrower
cells, with oval, deeply staining nuclei. Each of these cells is
prolonged to the surface and beyond it into a long, narrow,
stiff, rod-like extremity bearing a single stout cilium. The
third and simplest variety of cell forms the deepest layer next
to the basal membrane covering the organ. The peculiar
rod-bearing cells are arranged in four or five transverse rows-

“ PROBOSCIS PORES ” ]N CRANIATE VERTEBRATES. 541

alternating with the large ciliated cells, and also round the
opening of the pit. Here the rods become shorter and
shorter, until this type of cell passes into the more ordinary-
ciliated epithelium of the organ of Muller (PI. 28, fig. 12).

Hatschek, who noticed the rod-bearing cells, considered
the pit to be a sense-organ. But since no nerve can be
traced to it, this interpretation is probably incorrect. Andrews
states that in Asymmetron the pit secretes a mucous sub-
stance, which entangles food-particles and gets carried into
the mouth. He points out its relation to the blood- vascular
■“glomus,” and concludes that Hatschek’s pit is a slime-
secreting gland — a conclusion later supported by van Wijlie.

The story of the origin of Hatschek’s pit is one of the
strangest episodes in the strange history of the development
of Amphioxus. It is a mesoblastic structure formed from the
first mesoblastic somite of the left side. Hatschek (5) studied
the development of the anterior pair of pouches, and correctly-
described that on the right side as enlarging forwards and
downwards so as to give rise to the main head-cavity of the
larva. The left pouch he believed became constricted into
two portions. One, taking up a position on the right and
below the notochord, gave rise to Hatschek’s pit itself, while
the other opened to the exterior on the left side and gave
rise to the preoral pit of the larva (6). The larval preoral pit
subsequently becomes drawn into the buccal cavity at meta-
morphosis, and acquires its definitive position in the adult by
a process of shifting and overgrowth admirably depicted by
Willey (15). Hatschek's description of the early development
of the pit is by no means clear, and unfortunately is pub-
lished without figures (6). Some years later, Legros (9)
stated that the sac on the left side of the early embryo was
derived, not from a coelomic pouch, but from an invagination
of the ectoderm, and that from it were developed the pit, the
ciliated organ, and the anterior nephridium (Hatschek’s
nephridium).1 This interpretation of the origin of the sac

1 This nephridium is developed neither from a mesoblastic funnel,
as described by Hatschek (6), nor as an outgrowth from the second

542

EDWIN S. GOODRICH.

was disproved by MacBride (11), whose results were accepted
by vail Wijhe (14), and since Legros has recognised his
mistake the controversy may be dropped. As, however, it
seemed desirable to reinvestigate the whole question, an
account is here given of the development of the pit and
Muller’s ciliated organ from their first appearance to the
adult condition.

The first pair of coelomic sacs are given off as lateral'
pouches at the extreme anterior end of the archenteron, and
can be seen in embryos about twenty-four hours old still in
this condition (PI. 28, fig. 3). Later the pouches become
nipped off, and come to lie symmetrically on either side of the
notochord (PI. 28, fig. 2). Even in these very early stages
the left may have a rather thicker wall than the right sac
(PI. 28, fig. 1). At about the 30-ho.ur stage the right sac
begins to expand, it walls thin out, and later on it expands to
form the head-cavity of the larva. The left sac with its
thicker wall at first remains spherical, but later becomes
flattened, and takes up a position lying transversely between
the notochord above and a backward prolongation of the
right head-cavity below (PI. 28, fig. 5). Its outer end now
becomes applied to the ectoderm on the left side. Larvae
about fifty hours old show that an opening has been pierced
at this point of contact, placing the coelom in communication
with the exterior. At the same time the large cubical cells
lining the cavity acquire cilia (PI. 28, fig. 6). In larvae with
two gill-slits the left coelomic sac has enlarged, spreading
further towards the right side, while the ectoderm round the
opening has grown inwards, tending to form a depression

somite,, as described by Legros (10) ; nor, again, from the remains of
the communication of the coelomic pouch with the gut, as alleged by
MacBride (11), but from a little group of cells appearing quite early
just above the mouth. Several years ago I traced these cells to a stage
about thirty hours old, before the opening of the mouth ; but since I
was unable to find out their first origin, I refrained from publishing
my results. They are the cells figured recently by Smith and Newth
(PI. 18, fig. 4), who are indeed correct in their surmise that they may
represent the rudiment of the nephridium (13).

PROBOSCIS PORES ” IN CRANIATE VERTEBRATES. 548

(PL 28, fig. 7). Eventually this depression becomes largo
and deep, forming the preoral pit of the larva (PL 28, figs, 8,
9, 10). The lining epithelium becomes modified into tho
thick ciliated epithelium so conspicuous in later stages, and
in the adult wheel-organ developed from it. For it is this
preoral pit which is converted into the organ of Muller when
the buccal cavity is formed at metamorphosis. Whether the
thickened ciliated epithelium lining the preoral pit is actually
derived from the ectoderm and not from the HatschelPs pit it
is difficult to prove for certain, since the distinction between
the mesoblastic cells and the ectoblastic cells at the mouth of
the pit soon becomes indefinite. Moreover, there is an un-
fortunate slight gap in my series between the oldest stage
reared in the laboratory at Naples (51 hours) and the
youngest free-swimming larva with two gill-slits I was able
to obtain at Faro, and it is just at this stage that the
proliferation of cells at this point begins. Nevertheless, the
appearance in sections of these young larvae has convinced
me that the lining of the preoral pit is indeed of purely ecto-
dermal origin. How, then, can we account for the presence
of the rod-bearing cells in the lining of Hatscliek’s pit itself?
As mentioned above, they appear to be a specialised form of
the slender cells composing the epithelium of the preoral pit
(future wheel-organ). There can be hardly any doubt that
the rod-bearing cells invade Hntschek’s pit from the outside,
and are derived from the epithelium which grows in at the
open mouth of the sac. In young larvae they do not yet
occur among the lnrger mesoblastic cells; but in later stages
they can be seen in increasing numbers, first near the opening,
and then spreading over the inner surface of the sac.

To sum up concerning the history of the ciliated wheel-
organ of Muller and of HatschelFs pit in Amphioxus : The
first pair of coelomic sacs or somites develop as outgrowths*
which soon become nipped off from the anterior end of the
archenteron. They are at first symmetrical, but soon the
right enlarges to form the head-cavity, while the left, re-
maining comparatively small and thick-walled, acquires an

544

EDWIN S. GOODRICH.

opening to the exterior on the left side, in front of the mouth.
The ectoderm round the opening sinks in to form a deep
groove and depression — the preoral pit. The cells lining the
original left ccelomic sac, now known as Hatschek's pit, are
broad, with a rounded nucleus and a bunch of cilia. The

Text-fig. 2.

Reconstruction from transverse sections of a thick slice of the
head of an embryo of Torpedo, 10’5 mm. long. Anterior view.
f.b. Forebrain, h.b. Hindbrain, h.y. Hypophysis, n.t. Noto-
chord. o.l. Olfactory sac. o.p. Optic cup. p.c. Premandibular
somite, p.t. Premandibular tube, or canal opening into the
hypophysis.

cells lining the preoral pit are probably of entirely ectodermal
origin, and acquire a slender, elongated shape with an oval,
deeply staining nucleus and a single flagellum. In later
stages they appear to invade the pit of Hatschek, becoming
specialised into the rod-bearing cells. As the larval mouth

“ PROBOSCIS PORES ” IN CRANIATE VERTEBRATES. 545

becomes transformed into the adult mouth, and the lateral
flaps of the oral hood develop, the preoral pit is carried into
the buccal cavity, where it flattens out and spreads to form
the ciliated organ.

Now in Balanoglossus the first pair of ccelomic sacs arise in
essentially the same way, and acquire an opening to the
exterior known as the proboscis pore. As in Atnphioxus, so
in B. kowalevskii, the pore is formed only on the left side.
In B. kupfferi, however, both a right and a left pore are
present. More than thirty years ago Bateson, in his im-
portant papers on the development of Balanoglossus, com-
pared the opening of Hatschek’s pit in Amphioxus with the
proboscis pore (2), and further suggested that the proboscis
pore and gland of Balanoglossus correspond to the hypophysis
and pituitary gland of the Craniata. A discussion of the
latter interesting suggestion would require a detailed study of
the structure and development of these parts in Balanoglossus
— a subject into which we need not enter here ; but Bateson’s
oomparison of the pores seems to be strongly supported by
the facts mentioned above. The homology may, of course, be
•extended to the similar pores in Cephalodiscus, and to the
water pores of Echinoderms.1

Turning now to a comparison between Amphioxus and the
Craniate Vertebrates. That the hypophysis is an ancient
organ which must have been possessed by the ancestor of all
Craniates is shown by its constant presence and uniform
development. Invariably it arises as an ingrowth of ectoderm
just in front of the mouth and just anterior to the front end
of the archenteron.2 From the wall of the latter are here

1 A comparison with the Tunicata is much more difficult. We can
hardly avoid the conclusion that the subneural gland with its ciliated
duct and dorsal tubercle are homologous with the hypophysis ; but of
anterior ccelomic sacs and of proboscis pores in Ascidians we know
nothing as yet. On the other hand, it is possible that further research
may reveal traces of these organs in some of the less modified forms.

2 For an excellent account of the development of the hypophysis and
a review of the literature see the recent papers of E. A. Baumgartner
in the ‘ Journal of Morphology,’ vols. 26, 1915, and 28, 1916.

546

EDWIN S. GOODRICH.

produced the anterior extremity of the notochord, and the
lateral outgrowths which give rise to the first pair of somites
or anterior premandibular cavities of Balfour. In spite of the
doubts raised by Hatschek (6) and von Kupffer (7), it is now
generally admitted that the proboscis cavities of Balano-
glossus, the anterior sacs of Amphioxus, and the preman-
dibular cavities of Craniates are all homologous structures
•representing the first pair of coelomic somites (Willey (15),
MacBride (11)).

No satisfactory explanation of the origin of the hypophysis
has yet been arrived at. Beard, Dohrn, and others have
suggested that it represents a vestige of the original mouth,
a new mouth having been developed from gill-slits. But the
fundamental correspondence in the structure and relations of
the mouth and associated parts in the Ascidian, Amphioxus,
and the Ammocoete larva, and many other facts which need
not be mentioned here, render this view in the highest degree
improbable. Many authors have sought in Amphioxus for
the homologue of the hypophysis ; but, strangely enough,
most of them profess to find it in the neuropore or olfactory
pit of Koelliker. For this theory, suggested by Hatschek (6),
and strongly supported by Willey (15), there seems to be no
justification. Since both neuropore and hypophysis coexist
in the embryo Craniate, are situated widely apart, and are
related to quite different regions of the brain, it is difficult to
see how they could correspond to the olfactory pit. On the
other hand, the much more plausible comparison of the
hypophysis with the wheel-organ of Amphioxus has received
little attention. It is true that Legros at one time maintained
that HatschelCs nephridium, Hatschek^s pit, and the wheel-
organ correspond to the hypophysis and olfactory pit of
Craniates (9) ; but this view was based, as already men-
tioned above, on erroneous observations, and has since been
abandoned.

There is strong evidence to support the theory of the
homology of the hypophysis with the wheel-organ of Amphi-
oxus (the preoral pit of the larva). Were it not for tha

“ PROBOSCIS PORES ” IN CWANIATE VERTEBRATES. 547

excessive prolongation forwards of the notochord in Amplii-
oxus, they would both appear as ectodermal organs situated
below the brain and in front of the mouth. If we restored
the bilateral symmetry of the head in Ampliioxus, both the

Text-fig. 3.

Diagrams of transverse sections through the premandibular
region of the head of a. Amphioxus (restored to a bilaterally
symmetrical condition). b. Torpedo, and c. The Reptile
G-ongylus (from the figures of Salvi). H. Hypophysis. S1. Pre-
mandibular somite, or first anterior ccelomic sac.

right and the left anterior coelomic sacs would open into this
ciliated depression as shown in Text-fig. 3a; and there would
be two “ proboscis pores.” Now the suggestion I wish to

•548

EDWIN S. GOODRICH.

make in this paper is that there is direct evidence of the
existence of two such “proboscis pores” opening
into the hypophysis of the Craniate Vertebrates.
If the evidence be accepted it will, naturally, greatly
strengthen the theory that the hypophysis and the wheel-
organ are homologous structures.

Chiarugi, in 1898 (3), was, I believe, the first to mention a
•connection between the preman dibular somites and the hypo-
phj^sis in Torpedo. Since then Dohrn has carefully described
this connection in embryos of Torpedo ocellata and mar-
morata, and of Raja batis (4). It is a transient structure,
but when best developed consists of a tubular extension of
the premandibular somite passing downwards to the posterior
wall of the hypophysis, and placing the premandibular cavity
in communication with the lumen of the hypophysis (Text-fig.
3b). Just as in Balanoglossus, an Echinoderm, or Amphioxus,
the anterior coelomic sac grows towards and fuses with the
ectoderm to form the “proboscis” or “water” pore, so in the
Elasmobranch this tube grows out of the premandibular
somite and fuses with the hypophysial iugrowth. There may
be a right and a left tube, but — a significant fact — the left is
usually better developed and persists longer than the right.
In Text-fig. 2 a reconstruction is given from a series of
sections of Torpedo kindly lent to me by Prof. J. P. Hill, in
which Miss Fraser found the tube. In this case it appears to
be developed on the right side only. PL 28, fig. 15, shows,
on a larger scale, the opening into the cavity of the hypo-
physis.

Similar structures have been described in the Reptilia.
Already in 1888 Ostroumoff (12) mentioned a paired con-
nection between the premandibular somites and the hypo-
physis in Phrynocephalus, and the same structure has been
independently described in detail in the embryo of Gfon-
gylus ocellatus by Salvi (12a).

Want of material has prevented my confirming the obser-
vations of these authors, but, in the 3-day embryo of the
duck, I find the premandibular somites intimately connected

“ PROBOSCIS PORES” IN CRANIATE VERTEBRATES. 5491

with the hypophysis. Probably, if a careful search be made,
the premandibular tubes will be found to occur both in birds
and in mammals.

Finally, it may be urged that all these openings, water-
pores, proboscis pores, and premandibular pores are of the
nature of coelomostomes comparable to the excretory tubules in
the more posterior segments of the Craniates. It may also be
pointed out that the theory here advocated gives a clue to the
first origin and function of the hypophysis.1

Summary.

An account is given of the complex histological structure
of the epithelium lining Hatschek’s pit in Amphioxus, and of
the development of this pit and of the preoral pit from the
left anterior coelomic sac and an ectodermal ingrowth respec-
tively. The preoral pit becomes the wheel-organ of the adult.
The ciliated cells of Hatschek’s pit are of mesodermal origin,
but the rod-bearing cells appear to come from the ectoderm.
The evidence is strongly in favour of Bateson’s comparison of
the opening of Hatschek’s pit with the proboscis pore of
Balanoglossus and the water-pore of Ecliinoderms. All these
pores were originally paired. The anterior coelomic sacs of
Amphioxus are homologous with the premandibular somites
of Craniates. As shown by Ostroumoff, Dohrn, and Salvi,
these somites form tubular outgrowths opening into, or fusing
with, the hypophysis — a connection comparable with tlm
“ proboscis” pores of Enteropneusta, Cephalodiscus, and
Echinodermata. The premandibular, proboscis, and water-
pores are all of the nature of coelomostomes. It is concluded
that the hypophysis of the Craniata is represented in Amphi-
oxus by the wheel-organ situated in front of the true mouth,
and that its original function was probably to drive food into
the alimentary canal.

1 An abstract of this paper was read at the meeting of the Linnean
Society held on April 19th, 1917.

550

EDWIN IS. GOODRICH.

Postscript.

Since this paper was printed I have again come across
some interesting work which had unfortunately escaped my
memory, but to which attention must be drawn, as it has an
important bearing on the questions dealt with above. I refer
to the papers on “ Amia” by Phelps / Science/ vol. ix, 1899),
by Reigliard and Phelps Journ. of Morph./ vol. xix, 1908),
and by Eycleshymer and Wilson (‘ Biol. Bull./ vol. xiv, 1908),
and on “Polypterus” by Kerr /Budgett Mem./ 1907).
These authors trace the development of the adhesive or
•cement organ of the larva from paired diverticula of the
anterior end of the archenteron. Each diverticulum becomes
nipped off, and subsequently acquires an opening to the
■exterior. The adhesive organs of Lepidosteus and Acipenser
are probably of the same nature. Now, while Kerr is un-
willing to commit himself to any theory of the homology of
these organs, but nevertheless indicates “ the probability that
they correspond with the premandibular heud - cavities,”
Reighard and Phelps definitely compare the pouches which
-give rise to the adhesive organs to the so-called anterior
head-cavities found by Miss Platt in Acanthias, and supposed
to represent a pair of somites in front of the premandibular
somites of Balfour. Following Neal /Bull. Mus. Comp.
Zool./ vol. xxxi, 1898) they further homologise these anterior
head-cavities with the first pair of somites in Amphioxus (the
left one of which opens to the exterior), and suggest that they
and the adhesive organs may be homologous. This com-
parison, however, raises a serious difficulty. If the anterior
head-cavity really represents a separate segment, then the
segmental correspondence between the first pair of somites in
Amphioxus and the premandibular somites in higher verte-
brates would seem not to hold good. Since no somite has
been found in Petromyzon in front of the premandibular, we
may be forced to the conclusion that the whole cephalic
structure has been transposed one segment back in the
<Grnathostomata (see “ Segmentation and Homology,” this

“ PROBOSCIS PORES ” IN CRANIATE VERTEBRATES. 551

journal, vol. lix, 1913). Such a conclusion is by no means
inadmissible, but does not appear to be necessary. As a
matter of fact no definite trace of an anterior head-cavity has
been seen in any other group but the Selachii (adhesive
organs apart), and it is not constant even in them. On this
point the very careful work of Dohrn (24) seems to me
convincing. Many authors do not admit its homology with
a somite (v. Wijhe, Dohrn) ; rather would the walls of the
cavity seem to be derived from the premandibular segment,
and to represent merely a specialised region of its somite.

If, then, the anterior head-cavity really belongs to the
premandibular segment, and if it is represented in these
Teleostome larvae by the adhesive organ, the remarkable
conclusion is reached that not only in Cephalochordates,
Selachians, and Reptiles is there evidence that the first pair
of somites opened on the lower surface of the head (either on
or near the hypophysial depression), but that this is still the
case in some Teleostomes.

Literature.

1. Andrews, E. A, — “An nndescribed Acraniate, Asymmetron

lucayanum,'’ ‘Studies fr. Biol. Lab. John’s Hopkins Univ.,’
vol. V, 1893.

2. Bateson, W. — “Development of Balanoglossus kowalevskii,”

‘ Quart. Journ. Micro. Sci.,’ vol. 25, 1885.

3. Chiarugi, G. — “ Di un organo epithelioli . . . di Torpedo

ocellata,” ‘ Monitore Zool. Ital.,’ vol. xi, 1898.

4. Dohrn, A. — “ Studien z. Urgesch. d. Wirbeltiere,” ‘ Mitth. Zool.

St. Neapel,’ vol. xvii, 1904.

5. Hatschek, B. — “ Studien u. Entwickl. von Amphioxus,” ‘ Arb. Zool.

Inst. Wien,’ vol. iv, 1881.

6. “ Mitth. uber Amphioxus,” ‘ Zool. Anz.,’ vol. vii, 1884.

7. Kupffer, C. von. — “ Die Entwickl. d. Kopfes von Acipenser,”

‘Studien,’ Heftl, Leipzig, 1893.

8. Langerhaus, P. — “ Zur Anat. des Amphioxus,” ‘ Arch. f. Mikr.

Anat.,’ vol. xii, 1876.

552

EDWIN S. GOODRICH.

9. Legros, R. — “Cavite buccale de 1’ Amphioxus,” ‘Arch. d’Anat.
Micr.,’ vols. i and ii, 1897-8.

10. “ Sur quelqnes points de l’anat. et du devel. de l’Amphi-

oxns,” ‘ Anat. Anz.,’ vol. xxxv, 1910.

11. MacBride, E. W. — “ The Early Development of Amphioxus,”

‘ Quart. Journ. Micro. Sci.,’ vol. 40, 1898.

12. Ostroumoff, A. — “ Zur Entwickl. der Eidechsen,” ‘ Zool. Anz.,’

vol. xi, 1888 ; and ‘ Arb. d. naturf. Gesellsliaft zu Kasan,
vol. xix, 1888.

12a. Salvi, G. — “ Des cavites premandibulaires et des fassettes.
laterales de l’hypophyse cliez les Sauriens,” ‘Bibl. Anat./
vol. x, 1902.

13. Smith, K. M., and Newth, H. G. — “ Collar Cavities of the Larval

Amphioxus,” ‘ Quart. Journ. Micr. Sci.,’ vol. 62, 1917.

14. Wijhe, J. W. van. — “ Beitr. z. Anat. d. Kopfregion d. Amphioxus,”

‘ Petrus Camper,' vol. i, 1902.

15. Willey, A. — “Later Larval Development of Amphioxus,” ‘Quart.

Journ. Micro. Sci.,’ vol. 32, 1891.

16. “ Amphioxus and the Ancestry of the Vertebrates,”

‘ Columbia College Series, 1894.

EXPLANATION OF PLATE 28,

Illustrating Mr. Edwin S. Goodrich’s paper on “Proboscis
Pores in Craniate Vertebrates, a Suggestion concerning
the Premandibular Somites and Hypophysis.”

Reference Letters of Plate Figures.

a. Elongated rod-cell. ace. Anterior ciliated epithelium of wheel-
organ. acg. Anterior ciliated groove, b. Ciliated cell. blv. Blood-vessel.
ca. Premandibular tube or canal, cb. Basal canal. cHn. Coelom of second
somite, into which projects Hatschek’s nephridium. de. Ectoderm
dorsal to preoral pit. ebc. Epithelium of buccal cavity, end. Endostyle.
ent Enteron. Hn. Hatschek’s nephridium. Up. Hatschek’s pit. hyp.
Hypophysis, inf. Infundibulum, la. Left aorta. Ibf. Left buccal fold
or oral hood. lc.1 and lc. 2 First and second left coelomic cavities. Is.1
and Is.2 First and second coelomic sacs or somites, m.1 and m.2 First
and second myotomes. nc. Nerve cord. np. Neuropore. npl. Neural
plate, nt. Notochord, pee. Posterior ciliated epithelium of wheel-organ.
peg. Posterior ciliated groove, pm. Preoral muscle, pmd. Premandi-

PROBOSCIS PORES ” IN CRANIATE VERTEBRATES. 553

bular somite, pos. Preoral sense-organ, rbc. Roof of buccal cavity.
rbf. Riglitoral hood, rc.1 and rc.2 Right first and second ccelomic cavities.
rs.1 and rs.J Right first and second coelomic sacs or somites, ve. Ectoderm
ventral to preoral pit.

PLATE 28.

Figs. 1-14 are of Amphioxus.

Fig. 1. — Transverse section through anterior region and neuropore
of a 24 hours’ old embryo. Cam. W., 2 mm., oc. 3.

Fig. 2. — Similar section through a more advanced embryo, 24 hours
old. Cam. W., 2 mm., oc. 3.

Figs. 3 and 4. — Transverse sections through an embryo 24 hours old.
Fig. 3 shows the anterior somites, fig. 4 only the second pair of somites.
Cam. W., 2 mm., oc. 3.

Fig. 5. — Transverse section through an embryo 30 hours old. Cam.
W., 2 mm., oc. 3.

Fig. 6. — Transverse section through a larva 48 hours old, with
Hatschek’s pit opening to the exterior. Cam. W., 2 mm., oc. 3.

Fig. 7. — Portion of a transverse section of a larva with two open
gill-slits, showing Hatschek’s pit and the preoral sense-organ. Cam.
W., 2 mm., oc. 3.

Fig. 8. — Transverse section of an older larva. Cam. W., 2 mm., oc. 3.

Fig. 9. — Portion of a transverse section of an older larva, showing
the developing preoral pit and groove.

Fig. 10. — Section of the same larva farther forward. Cam. W.,
2 mm., oc. 3.

Fig. 11. — Portion of a transverse section of the buccal region of an
old larva in which the atrium has developed. Cam. W., 2 mm., oc. 2.

Fig. 12. — Longitudinal vertical section through Hatschek’s pit in an
adult. Cam. Z.D., oc. 2.

Fig. 13. — Transverse section through Hatschek’s pit in an adult.
Cam. Z.D., oc. 2.

Fig. 14. — Enlarged view of a portion of a section of the epithelium
lining Hatschek’s pit in the adult.

Fig. 15. — Portion of a transverse section of the head of an embryo
of Torpedo 10*5 mm. long. The entrance of the premandibular tube
or canal, ca, into the hypophysis is shown.

VOL. 62, PART 4. NEW SERIES.

38

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 555

The Cytoplasmic Inclusions of the Germ-Cells.

PART II. HELIX ASPERSA.

By

J. Bronte Gatenby, B.A.,

Exhibitioner of Jesus College, Oxford.

(Dept, of Physiology.)*

With Plates 29-34 and 5 Text-figures.

Table of Contents.

PAGE

1. Introductory ..... 556
•2. Previous Work ..... 556

3. Technique . . . . . . 562

4. The Germinal Epithelium .... 564

5. Cell Generations in the Snail . . 571

6. Manner of Metamorphosis of an Indifferent Germinal

Epithelial Cell into a Primary Spermatogonium . 578

7. Manner of Metamorphosis of an Indifferent Germinal

Epithelial Cell into an Oogonium . . . 587

8. The Fate of the Mitochondria and Nebenkern in the Egg 589

9. Manner of Metamorphosis of an Indifferent Germinal

Epithelial Cell into a Nurse or Yolk Cell . . 590

10. Description of the Scheme of the Ovotestis Lumen

drawn in PI. 29, fig. 1 . . . 591

11. Discussion under the heads of :

(a) Nebenkern ..... 594

(b) Mitochondria . . . .598

(c) The Determination of Oogonium Spermatogonium

or Nurse-Cell .... 599

(d) The Different Male Generations in the Ovotestis . 601

12. Summary .... 605

556

J. BRONTE GATENBY.

Introductory.

The first part of the present series of papers (1) dealt with
the cytoplasmic bodies in the gametogenesis of the Lepi-
doptera. In this paper I am publishing the results of a study
of the cytoplasm in the hermaphrodite. Helix aspersa.
Not only is the gametogenesis treated, but a careful study has-
been made of the problems surrounding the appearance of
eggs, spermatozoa, nurse-cells and follicle cells from the same
germinal epithelium. Special care has been taken in order to
get the very best preparations of all stages, and extensive
experiments were undertaken to improve the current tech-
nical fixing methods. This study proved more formidable
than at first seemed probable, and some of the minor problems
had to be imperfectly treated in order to keep the paper
within reasonable bounds. Where such minor problems
appear I have indicated in the text, and the snail still offers
many attractive lines of research on germ-cells. I have to
thank Dr. Goodrich for his kind and constant interest and
useful criticism. This work was done in the Department of
Physiology, and my warmest thanks are due to Prof. Sher-
rington .

Previous Work.

Apart from the ordinary study of the formation of the
gametes, the snail has provided the material for a host of
studies on hermaphroditism. The ovotestis, ever since its dis-
covery, has fascinated many observers, and the large number
of papers which have appeared on the hermaphrodite gland
of the Pulmonates is justified when one considers what a
remarkable phenomenon is the appearance of several distinct
categories of cells — sperms, eggs, and nurse-cells — from a
single indifferent pavement epithelium.

Those who would study the history of our knowledge of the
Helicid gametogenesis may consult Ancel’s excellent memoir,
wherein are mentioned the main contributions made by the
large number of observers on this subject. In this paper I

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 557

•only intend to review Ancel and the subsequent writers.
(Bolls Lee is mentioned in the text.)

Since I have not examined embryonic stages I give Ancel’ s
conclusions somewhat fully.

Ancel (2) describes the genital rudiment as appearing in
the embryo several days before hatching. At first the mass
of cells, quite solid, slowly elongates, and ultimately fuses
with the hermaphrodite canal. Soon afterwards a lumen
appears in the solid mass of cells, which now appear like an
irregular cubical epithelium lining an opening, and these cells
become covered on their outside by a layer of the mesoderm
cells in which they lie. At different points inside the lumen
the genital cells begin to divide mitotically, and in the region
of these buds secondary, tertiary, etc., culs-de-sac make
their appearance. In this way the hermaphrodite gland is
built up from a solid rudiment.

The cells which line this gland consist of a single layer.
Here and there some of the indifferent cells augment in
volume, the chromatin lumps of the nucleus fuse with one
another, and give rise to more or less round structures. Ancel
calls these cells “ cellules progerminatives indifferentes.”

Part of the chromatin of these cells loses its affinity for
nuclear stains, and the chromatin lumps become more
numerous and become rounded. They are now united two by
two by nuclein filaments.

All the chromatin of the indifferent progerminative cell
condenses into several round bodies. The whole cell grows
in size, the nucleus more than the cytoplasm. This cell is
called by Ancel the male progerminative cell. The latter
begins to divide indirectly, the chromosome number being
forty-eight. The products of these divisions drop into the
lumen of the ovotestis ; they are large and pedicellate (pedi-
cule), and have the nucleus in the larger part of the cell.
These primary spermatogonia go on dividing and give rise to
much smaller bodies — the secondary spermatogonia. The
products of this activity soon fill the lumen of the ovotestis,
and in the germinal epithelium changes take place. The

558

J. BRONTE GATE N BY.

cells become arranged in two layers, and the outer layer in
contact with the contents of the lumen becomes filled with
grains stained with osmic acid, and the nuclei of this outer
layer have their chromatin condensed into the form of
crowded blocks. These cells are the nurse-cells.

When the nurse-cells are formed certain elements in the
inner layer beneath the nurse-cells augment in size. They
undergo the progerminative indifferent stage, but then become
altered in a different manner. The chromatin lumps of the
indifferent cell break up and become oriented to the periphery.
The centre of the nucleus then looks clear, while all around
its periphery are collected little nuclein nucleoli (sic), united
one to another by chromatin filaments. In this cell it is the
cytoplasm which has grown most in size. Such is the history
of the oocyte. This element does not undergo division before
the maturation period. Ancel does not find in Helix the
stages of primordial ovum or oogonium described in other
oogeneses.

According- to Ancel the presence of nurse-cells is a sine
qua non for the formation of an oocyte, and is explanatory
of its development.

Concerning the growth period of the oocyte Ancel writes
in the following sense : The chromatin of the young oocyte
condenses into the form of little nucleoli, which become
disposed around the periphery of the nucleus. A network
becomes established at the expense of the nuclein nucleoli,
and a new network is formed afterwards from the chromatin.
In an involved statement Ancel describes the appearance of
parachromatic nucleoli, distinguished from the nuclein nucleoli
by double staining in haemalum and safranin.

In the oocyte at an early stage one can find filaments in
the cytoplasm. These filaments are originally dispersed
through the cytoplasm, but later condense towards a peri-
pheral zone, which diminishes more and more and disappears.

During this time the whole cytoplasrrf takes on an alveolar
appearance except at one place, where special bodies will
soon put in an appearance. Hsematoxylin or safranin prepa-

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 559

rations show that in this region one soon finds filaments of a
variable size. These rods or filaments are known as “ corps
intracytoplasmiques.” The latter seem to be either solid rods
or formed of moniliform structures placed in a series.

Ancel thinks that the young spermatocytes do not contain
a Nebenkern. Their cytoplasm is filled with short, colourable
filaments. The Nebenkern, which appears at the expense of
the cytoplasmic filaments, augments in size as these filaments
disappear. At the beginning of mitosis the Nebenkern frag-
ments and disappears. Ancel denies that the Nebenkern is
related either to the nucleus, the spindle, or the “ sphere
attractive,” and gives it as his opinion that this curious body
“ne represente, a notre avis qu'une phase de revolution des
formations iutracytoplasmiques.”

Elsewhere Ancel states : “ Le Nebenkern ne jouerait done
aucun role dans les cellules ; il ne serait que le produit de
transformation des filaments cytoplasmiques differences aux-
quels serait devolue une fonction speciale.”

R. Demoll (3) describes the fact that these are prophases
of the heterotypic division in the female, which was over-
looked by Ancel. He describes the Nebenkern somewhat
better than Ancel or previous authors and considers that this
body determines the sex of the differentiating cell.

Writing of the “ Bukettstadium in beiden Keimzellen,”
Demoll gives the following : “ Mit dem Ausstofen des Neben-
kernes wird sowohl die Wachstumsgeschwindigkeit als aucb
die Genese der chromatischen Substanz fur die beiden Arten
von Keimzellen eine spezifische. Oder : Der Nebenkern
bedingett erst die geschlecht liche Differenzierung der bis zum
Bukettstadium indifferenten Keimzellen.” In the discussion
it will be shown that for a number of good reasons DemolTs
sex determination hypothesis cannot be accepted (page 40).

Iw. Buresch (4) has also attacked the problem, but his
paper contains little of interest from the point of view of the
means whereby sex is determined. His work is not a great
advance on that of Ancel, written many years before, while
he has failed to study any cytoplasmic inclusions. Buresch

560

j. br*ont£ gatenby.

thinks the germinal epithelium is a syncytium, but as he
uses Zenker for fixing this is almost certainly due to the
brutality of fixation. Buresch gives a fairly good account of
the generations of egg-cells, nurse-cells, etc., and shows
how the presence of a large egg affects the surrounding cells.

Concerning forms other than Helix we have two late papers
by Terni and by Schitz. The latter (5) on Columbella finds
the Nebenkern rods in the spermatocyte at the beginning of
growth, and in later stages they form a sort of palisade
around the nrchoplasm (idiozome). In the prophases of the
maturation mitoses these rods disappear. The mitochondria,
before seed-like, become elongate as in the snail during
mitosis. The Nebenkern in spermatogenesis becomes passed
into the tail region where it “ ultimately degenerates/’ but
the author does not pay much attention to late stages.

Schitz somewhat obscurely describes the formation of an
acrosome from a “ grain siderophile,” derived from the idio-
zome (archoplasm), and to support his statement quotes
Champy’s demonstration of the fact that in Amphibia the acro-
some develops at the expense of one of the central corpuscles.
I find it difficult to accept Schitz’s claim, and his figures are
not conclusive enough. The mitochondria form a sort of
double sac-like structure, “ envelloppant le filament axile.”

Finally, Schitz speaks of the tail as “ constitute par le fila-
ment axile, entoure de sa gaine mitochondriale.” It will be
seen that, excepting the formation of the acrosome, the fate
of the Nebenkern and its behaviour throughout, according
to Schitz, corresponds to what happens in Helix.

Tullio Terni (6) in Geotriton carefully describes Nebenkern
and mitochondria. In divisions the Nebenkern fragments or
completely loses its original character. Terni having described
the fragmentation of the batonettes proceeds to discuss the
question as to the origin of the spindle. Nebenkern and
idiozome seem synonymous to him, for I find “II Neben-
kern dei Gasteropodi polmonati (Idiozoma).” He says :

“ Sostenendo l’origine endoidiozomica (Terni calls the
batonettes ‘formazioni periidiozomiche/ and elsewhere

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 561

‘ dittosomi ’) del fuso centrale, vengo ad ammettere che
1’idiozoma abbia un importante ufficio nella formazione del
fuso stesso : forse di significato assai piu profondo che non
quello di una semplice azione protettiva. Non ho perd dati
sufficients per definire la portata reale della partecipazione
materiale dell’idiozoma alia formazione del fuso centrale ;
credo tuttavia che la sostanza idiozomica non si esaurisca
tutta nella produzione del giovane fuso centrale.” In a foot-
note Terni informs the reader that in his work he could not
demonstrate the existence of “ due mezzi fusi periferici nella
profase dell prima divisione di maturazione.”

Terni definitely describes the formation of an acrosome
from a part of the archoplastn. In this he is in agreement
with what Schitz found in Columbella. As I have already
said I cannot find such a process in the snail, but I am quite
open to conviction that the idiozomatic body contains the
future perforatorium, though before coming to a conclusion I
should like to have further evidence either way. I am quite
certain that in the snail the archoplasm (and its Nebenkeru
rods) does not approach the head of the nucleus in the way
described by Schitz. In Term's case the nucleus seems to
revolve somewhat after the acroblast has become stuck on
one side. The acroblast is thus brought to the head end of
the cell. Term's case seems very clear.

Faure-Fremiet (7) in his comprehensive article, u Etude
sur les mitochondries des Protozoaires et des Cellules Sexu-
elles,'' gives figures and describes the mitochondria of Helix
pomatia and Arion rufus.

He fails to discover tbe micromitochondria, overlooks the
acrosome, and gives unproportionate drawings of Nebenkern
rods and chondriosomes. He definitely draws the batonettes
at the poles of the maturation spindle.

Though the minute cytology of the germ-cells of Helix lias
been unsuccessfully dealt with by Faure-Fremiet, the real
value of this observer’s work lies in his remarks on the
Nebenkern in fresh preparations. He says : “ II m’a semble
pourtant qu’ii (the Nebenkern) eta.it beaucoup plus facilement

562

J. BRONTE GATENBY.

altere que les mitochondries environnantes par les solvants
des graisses ; les mitochondries resistent parfaitement a ceux-
ci, et j’ai note qu’un spermatozoide d’Arion traite apres disso-
ciation par Talcool et le chloroforme et colore par la
mSthode de Mallory montre les deux cordons mitochondriaux
se colorant par la fuchsine acide avec energie.”

Technique.

All the earlier observers hardly without exception found
that Flemming’s strong formula gave the best results of any
fixatives at their disposal.

Murray (8) got good results with Perenyi. Flemming’s-
unmodified formula I consider out of date and worthless fora
study of the cytoplasm. Its fault lies in the presence of
acetic acid, a reagent which should never be used for work
on the plasma. As I explained in my last paper, a modifica-
tion, which I found best, was to cut out all the acetic acid.

Meves, in his great cytological work, used a Flemming of
this sort : 15 c.c. of chromic acid of 1 per cent., containing
NaCl of 1 per cent., and 3 to 4 c.c. of osmic of 2 per cent.>
with three or four drops of acetic acid. Benda used a
Flemming with only three to six drops of acetic acid to 15 c.c.
chromic and 4 c.c. osmic. I feel sure that it is dangerous to
try to temporise between two fixing solutions, as nuclear and
cytoplasmic, by simply cutting down the acetic. These
observers retain just enough acetic acid to distort the mito-
chondria, and the small improvement in the appearance of the
nuclei and in the penetrative power is quite outweighed by
this glaring fault. As I have mentioned before, the discussion
as to whether the mitochondria are rods or granules may be
largely a question of acetic acid and such-like injurious re-
agents.

Any fixative in general use will give a passable fixation of
the nucleus, but work on the cytoplasm is a very different
story. In the cytoplasm w.e have no bodies bounded by close
membranes, but structures which can be, and always are,.

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 563

blasted into fragments by many so-called “ admirable fixa-
tives.”

In this work on the snail I used the following fixatives :

(1) My modification of Flemming’s strong formula, in
whicn no acetic was used.

(2) The same fluid diluted one-sixth.

(3) Champy’s fluid.

(4) Flemming’s strong solution, in which the acetic acid
was re-placed by nitric acid.

All the better-known fixatives were also tried, such as
Flemming, Perenyi, corrosive sublimate, Carnoy, etc.

For staining I used the usual stains, such as Ehrlich’s
heematoxylin and eosin, Mayer’s acid heemalum, and iron
alum hsematoxylin with orange G., Bensely’s acid fuchsin-
methyl green, and alizarin-toluidin blue, etc.

Taking the reddish or orange alizarin or acid fuchsin as a
basis, the slides were often counterstained in toluidin blue,
crystal violet, methyl green, thionin, etc., and the same was
done taking iron heematoxylin as the first stain. Some slides
were mounted unstained in euparal for studying yolk.

Smears were tried, but failed to give very helpful results
except for studying later stages and ripe spermatozoa.

Of the large number of fixing and staining methods used, I
found that for the snail the following was the best : The

animals are anaesthetised in chloroform vapour, and as soon
as possible the last upper whorls of the shells are carefully
broken with strong forceps or by a blow with some hard
instrument. The shell is dipped in water, and the remaining*
pieces around the broken area are removed with forceps.
The ovotestis is cut out and laid on the table and superfluous
digestive gland is cut away, exposing the milk-coloured
ovotestis on every side. This piece is cut in half longi-
tudinally, and immediately thrown into a capsule of Flemming
without acetic acid, diluted one-sixth with distilled water.
Here the fragments are left overnight. They are washed for
at least two hours in running water next morning, and then
brought up from 50 per cent, alcohol and the other grades to

564

J. BRONTE OATENBY.

absolute and xylol. They are embedded as usual, cut into
6 fx sections, and stained in iron alum haematoxylin by the
long method.1 These sections are carefully differentiated, and
then tinged with orange G. or van Giesen. The mitochoudria
and Nebenkern are intense black and beautifully clear.

Neither Benda’s, Bensely’s, nor any of the other coloured
stains approach such preparations for definition, and for the
detail which is shown. As will be explained in another part
of this series, iron haematoxylin is not always indicated for
mitochondrial work, Bensely’s acid fuchsin having been found
better for some objects.

The Flemming solution, in which the acetic acid is sub-
stituted by nitric acid of 3 per cent., gives a very intense
stain when used on the mitochondria.

The Germinal Epithelium.

The germinal epithelium in its indifferent condition consists
of a row of flattened cells containing compressed nuclei. The
epithelium is not a syncytium, as has been stated by some
authors. In PI. 30, fig. 2, is drawn at a very high power
three cells of the epithelium in an indifferent state. The
nucleus is an oval, flattened structure, and as it is here cut
in its narrowest way it looks elongate. The chromatin is
arranged in a large number of irregularly triangular lumps,
which here and there are intercommunicating. The cyto-
plasm is not very large in volume, and consists of a wide
reticulum, which in some cases can be seen to be condensed
into a dark mass near one edge of the nucleus (see PL 30,
fig. 2, PI. 31, fig. 20, etc.).

The cells of the germinal epithelium rest upon a fibrous
layer, shown by Ancel to be of mesodermal origin (PI. 30,
figs. 2, 3, and 4, A.L.N., etc.). This layer contains nuclei,
which vary in size very greatly. In PI. 30, figs. 3 and 4, are
drawn quite typical examples of the germinal epithelium.
The germinal epithelial cells ( Gr.E .) are seen resting between

1 Ivon alum ten to twelve hours, hsematoxylin twelve to fourteen
hours.

GYTOPLASMIC INCLUSIONS OF THE GERM -CELLS. 565

the yolk cells (N.G.) and the layer of Ancel (A.L.N.).
According to the sort of sex cells in any given region the
germinal epithelium is characteristic. Where rapid pro-
liferation is taking place, where yolk cells are abundant, or
where a large oocyte is present, the epithelium lias a special
appearance. In PI. 30, figs. 3 and 7, and in PI. 31, figs. 9, 11,.
and 12, where an oocyte is in growth, the germinal epithelial
cells tend to either become atrophied or altogether pushed
aside. In Text-figs. 1, 2, 3, and 4 are drawn typical portions
of the ovotestis under different conditions. It will be seen
that the appearance of an alveolus of the ovotestis may vary
greatly. Not only does the wall differ at different stages, but
the contents of the lumen are rarely the same in appearance.
I believe that the varying different stages in the alveoli
can be classified for different seasons of the year, though
if the ovotestis is sectioned during hibernation it will
be found that all the various sorts of alveoli are present.1
Then one is led to inquire what causes individual alveoli of
the ovotestis to vary so remarkably in appearance as do those
drawn in Text-figs. 2 and 3. Ancel has given a description
of the metamorphosis of the alveolus in the young snail,
which, in view of his Flemming technique, is not very satis-
factory. Unfortunately his methods did not allow him to do
anything but describe the nuclei, but from his otherwise admir-
able description we know that the following events take place :

Firstly the male progerminative cells appear and drop into
the lumen. Then the germinal epithelium becomes arranged
in two layers, the inner of which remains indifferent, the
outer (next to the male cells) becoming converted into nurse-
cells. Thirdly, the inner layer of an indifferent nature
sporadically gives rise to oocytes. That this is reallv what
happens I have no doubt, and if one keeps this description
of the sequence of events in one’s memory, some difficult
problems with which one meets in studying the adult ovo-
testis become less hard to understand.

1 But the orders of cells differ in the seasons. This matter is dealt
with in a forthcoming paper.

566

J. BRONTfs GATENBY.

In the ovotestis of the hibernating snail where activity
is temporarily suppressed, just as in a summer example
where activity is very great, it is found that the diverticula
or alveoli in the hermaphrodite gland almost always are
better provided with yolk and younger cells at their upper
extremity than at tlie lower part of the finger-like alveolus
which joins the mouths of other diverticula. That is to say,
the higher ones penetrate into the diverticulum, the younger
and less differentiated are the elements. When one cuts a
transverse section across the upper part of an alveolus one
finds that the lumen is very small sind is choked either
with full yolk cells projecting from the walls, or closely
packed with spermatogonia and young spermatocytes. It is
rare to find an oocyte at these places, but it would be a
mistake to think that oocytes never occur in these regions.
In Text-fig. 1 is drawn the upper region of an alveolus.
Just as in the young snails described by Ancel, the first cell
elements to appear are almost invariably spermatogonia, but
I have found several instances where an oocyte had appeared
immediately after the yolk cells had been formed. In Text-
fig. 1, i, the germinal epithelium has already become
organised into two layers, an inner mass of yolk cells filling
the lumen and the lower indifferent germinal cells ( G.E. ).
At X. a germinal epithelial cell has grown in size, has lost its
flattened shape and is about to become a spermatogonium.
In Text-fig. 1, ii, this process has become more advanced and
the nurse-cells are becoming pressed apart at X. by a number

Text-fig. 1.

Fig. i. — Upper part of diverticulum of ovotestis showing yolk cells
(N.C.) and germinal epithelium (G.E.). At X. are enlarged nuclei,
which are in a progerminative stage. In the middle are some
spermatocytes. The vacuolised tissue outside consists of meso-
derm, which packs around the upper parts of the diverticula. X 800.
Fig. ii. — Another diverticulum cut near its upper end. At X. are
some primary male cells which as yet have no definite mitochondria
or Nebenkern. They will probably become spermatocytes directly.
At Y. is a pale cell beginning to enter a primary spermatogonial
stage. Figs, iii and iv are spermatids showing different Xebenkern
batonettes (N.K.). M1. — micromitochondria. M2. = macromito-

chondria. N.C. = yolk cell.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 567

Text-fig. 1.

Fig. ii.

568

J. BRONTE GATENBY.

of spermatogonia. This process takes place from different
parts of the alveolar wall and the lumen becomes quite choked
with cells. In Text-fig. 2 is drawn a lower part of the
alveolus showing that the proliferation of male cells from the
walls has stopped temporarily and that the yolk cells are
much reduced in size.

The consecutive stages from spermatogonium to spermato-
cyte are marked with figures. Quite often one finds oocytes
developing beneath the yolk cells. In such a case as the
group of spermatogonia marked i, in Text-fig. 2, many of the
cells may go on to the growth period and ultimately form
spermatozoa, but there is hardly any doubt that some remain
quiescent, or keep on dividing and give rise to the very small
spermatogonia with hardly any cytoplasm which are so
characteristic of the lower part of the ovotestis lumen. In
Text-fig. 3 another stage is reached. The yolk cells are-
either relatively few or small, or often not present at all, but
the lumen is generally strewn with a mass formed of the flot-
sam and jetsam of many generations of germ-cells of ripe
spermatozoa, of exhausted yolk cells or their nuclei, and of
many sizes of spermatogonia; also here and there around
the walls may be seen either an oocyte in process of growth,
often but not always accompanied by yolk cells, or a bunch of
spermatocytes, spermatids, or spermatozoa. If such a stage
as the contraction figure be taken, one is struck by the fact
that many sizes of cells are undergoing this nuclear change,,
often with a cytoplasm and nucleus varying remarkably in
size. In Text-fig. 2 there is no doubt that all the cells are of

Text-fig. 2.

Fig. i. — Slightly lower region than that drawn in Text-fig. 1, ii. All
cells belong to the same generation. Earliest stage at 1 has just
finished division. 2-7 are growth stages. 8 = spermatid. The
yolk cells ( N.C .) are beginning to become exhausted. The Neben-
kern rods of this generation were semi-lunar in shape. Fig. ii. —
Further down the diverticulum, where the yolk cells are more
exhausted or absent. The epithelium at the figures 1-5 is beginning
to produce another generation of male cells. 5 is about to enter
the prophases of mitosis. 7 = spermatocyte, 8 = spermatid. The
cell 2 is drawn in PI. 31, fig. 17.

570

J. BRONTE GATENBY.

the same generation. This is never the case further down
the lumen. We then come to one of the most remarkable
facts which I am able to point out in this paper. It is that
the spermatocytes, etc., in different parts of the lumen are of
different generations and derived in a varying manner, and
that they show this by their appearance, which is character-
istic in every case.

If PL 31, figs. 10, 11, 17, 19, 20, and PI. 32, fig. 24, be
inspected it will be seen that very remarkable differences
exist between the cells here drawn ; all these figures are to
the same scale and the cells were all found in the germinal
epithelium. The same remark about differences applies to
PI. 30, fig. 6, PI. 32. fig. 21, and PI. 33, fig. 30. These are in
a characteristic stage of the prophases of the heterotypic
division, and not only do figs. 6 and 21 differ markedly in
their nuclei, but the cytoplasmic inclusions are quite distinctly
unlike in each example.

In the same way, if the cell divisions drawn in PI. 32,
fig. 22, PL 32, fig. 28, and PL 33, figs. 34 and 39 be compared
it will be noticed that the cytoplasmic inclusions behave
differently and are different in size and shape. If the sper-
matocyte in PL 32, fig. 25, be compared with that drawn in
PL 33, fig. 32, and the spermatid in PL 32, fig. 24 a, with that
in PL 33, fig. 36, it will be seen that remarkable differences
exist. This brief recital of some of the curious facts which
are to be found in the ovotestis of Helix at once serve to
show that the problem of the derivation of the various sex
cells from the indifferent cell is a very difficult matter to
understand, and one which has been inadequately treated.

I should hasten to make it quite clear that this paper does
not by any means completely describe all the sorts of cells
found in the ovotestis. For myself, I consider that the many
months of close attention which I have devoted to this work
have only served to show me that a complete explanation of all
the various cell generations in the snail's gonad is not the
work of months, but of years. It will need the collection of
a complete series of sections of gonads for every month of

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 571

the year except the hibernatory ones.1 I have found that
properly to describe the remarkable facts concerning the
origin, function, and fate of the nurse or yolk cells alone would
need a separate paper, and should circumstances allow I hope
to apply myself to this task.2 Bolls Lee (9) was struck by the
number and varying sizes of the spermatogonia in the snail.
I can at present think of at least several sorts of spermato-
gonia ; by this I mean that it is quite possible to find a large
number of cells which are in the spermatogonial generation
of the male cells, and which differ markedly either in their
nucleus, their Nebenkern, or their mitochondria.

Cell Generations in the Snail.

The most important result of this study has been the
realisation that in observing the mixed mass of cells in the
lumen of the ovotestis, one deals not with one generation of
male cells derived in the same way, but with several genera-
tions whose origins are in certain ways considerably different.
It is not intended at this juncture to attempt any explanation
of this until the various cells have been described as well as
possible. From the excellent work of Bolls Lee (9) and
Ancel (2), not to mention some of the older writers, we are
fairly well acquainted with the appearance of the typical
lumen of the ovotestis. Inspection of the text-figures will serve
to show what these authors have failed to emphasise suffi-
ciently, viz. that the appearance of the various alveoli differs
greatly, not only individually, but just as importantly in the
contents of the alveoli which are different at different levels.

In Pl. 32, figs. 28 and 29, and PI. 33, figs. 30, 31, 32, 33,
34, 35, 36, and 37 I have drawn typical stages of the meta-
morphosis of a sperm from the loose cells lying in the open
lower region of the alveolus.

The spermatogonial division is drawn in PI. 32, fig. 28.
Typically one gets small mitochondria often so small as to

1 This has now been done.

2 This paper (Part iv) lias lately been finished.

572

J. bront£ gatenby.

be difficult to detect, and sometimes two other bodies of a
larger nature can be found. One, marked X.N.K., may be
the Nebenkern, but it is too small to be easily made out ; the
other is a large granule (S.G.), the history and fate of which
has been so ably described by Bolls Lee (9). Suffice to say,
this body is quite definitely seen in Flemming-fixed material
stained in iron alum haematoxylin, and persists for a long time
in the sperm cycle. The chromosomes in these divisions are
seed-like, slightly elongate, and much crowded. There are
considerably more than forty, and the correct number seems
to be forty- eight. As far as I can tell, the mitochondria in
this form of spermatogonial division do not alter in shape
during kinesis. In PI. 32, fig. 29, I have drawn a spermato-
gonium just after division has finished and when the nucleus
is properly reformed. At S.B. is a spindle bridge, at S.G-. a
siderophilous granule, towards one side of the nucleus the
small mitochondria are grouped into a conical heap, and
finally floating free in the cytoplasm is an apparently serrate
elongated body so plain and large as to be easily drawn in
with the camera lucida ( N.K. ). I feel quite sure that this is
the Nebenkern. When the spireme appears and the loops
become grouped to form the contraction figure the Nebenkern
takes up its position where the centrosome is known to be in
other cases. It should be stated that in the best preparations
I have, one is unable to see a centrosome at any stage until
near the maturation divisions. In PI. 33, fig. 30, the Neben-
kern (N.K.) appears to be broken into pieces but still has the
elongate rectangular shape. The mitochondria are now
becoming more loosely disposed, aud by the stage drawn in
PI. 33, fig. 31, are larger and dispersed throughout the cyto-
plasm. The Nebenkern is now quite distinctly formed of a
number of tiny intensely-staining rodlets.

By the end of the growth period the Nebenkern (PI. 33,
fig. 32) is seen to consist of elongate, slightly curved rodlets,
somewhat irregularly disposed, but often placed end to end,
as shown in the spermatid in PI. 33, fig. 35.

These rods are most easily described as banana-shaped.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 573

They lie in, or are grouped so as to enclose, an archoplasmic
region near the nucleus.

Despite Bolls Lee's assertion, I must confess that I am quite
unable to find a centrosome inside this archoplasm, but there is
little doubt that such a body may be embedded in this mass.1

When growth stage has finished the cell may be the size
drawn in PI. 33, fig. 33, at 4250 diameters. The first matu-
ration prophases are in progress. The chromosomes are
appearing, while the Nebenkern, as such, has disappeared.
In favourable examples it is found that small rodlets are still
visible here and there ( N.K . in PI. 33, fig. 33), and these are
almost certainly parts of the scattered Nebenkern. The
individual rods appear to break up into minor rodlets.

Bolls Lee (9) described in the maturation division a re-
markable centrosome structure. I found this quite easily in
some of my preparations, and these triradiate bodies are seen
at A.S. The striae figured by Lee in these bodies I did not
find so marked, but as our technique was different this is not
a matter of great importance.

The mitochondria are very dense and numerous. The
spindle now forms, with the disappearance of the astral body
(A.S.), and one gets a figure in which the mitochondria are
heaped around the spindle and elongated in shape as if
affected by some lines of force (see PI. 33, fig. 34).

Murray (8) figures the Nebenkern fragments as grouped
around the poles of the astral figure, but I am unable to come
to a definite opinion on this point. I am able to say that
occasionally one finds little rectangular bodies which might
be the Nebenkern, but unfortunately, as the mitochondria
also become elongate, it is difficult to come to a decision as to
the nature of these elements. Since mitochondrial stains also
tinge the Nebenkern, staining tests have so far failed. The
second maturation division closely resembles the first in so
far as the behaviour of the plasmatic bodies are concerned.
The spermatid then appears as drawn in PI. 33, fig. 35, when
it is just beginning to metamorphose. At what is later seen
1 Subsequent work shows that Lee is correct.

574

J. BRONTE OxATENBY.

to be the front end of the cell, the nucleus is found to be
covered at one side by a densely staining cap — the acroblast.
Despite especial work in this connection, I have been unable
to follow this body back into the spermatocyte. No staining
method of which I know will discriminate between acroblast
and mitochondrium, and it is obviously impossible to identify
this body until it has taken up its position next to the front
edge of the nuclear membrane. The spermatid,1 besides con-
taining the Nebenkern and the mitochondria, is seen to be
provided with a cloud of granules of a smaller size lying
behind the nucleus, near the locality from which the axial
filament presently begins to grow. The granules (AT.2) are
hardly demonstrable till the spermatid is in the stage drawn
in PL 33, fig. 35, but from thence onwards they are quite
easily found.

As the spermatid lengthens, the nucleus becomes shaped as
shown in PI. 34 , fig. 41, the acroblast lying as a thickened
area laterally. In PL 33, fig. 36, I have carefully drawn a
spermatid at this stage. The nucleus has become blackly
stained with iron hsematoxylin, while the small granules,
which will be’ called micromitochondria, are closely grouped
behind the nucleus. The other mitochondria, which, to dis-
tinguish them, will be called the macromitochondria, lie
further back. At the letter C .2 is a structure, constantly
present, formed of a large and a slightly smaller granule.
The larger granule is probably the second centrosome ; the
other might be a mitochondrial granule, but I cannot advance
any definite evidence as to its nature. A stage just before
this is drawn in PL 34, fig. 42, and the nucleus does not yet
stain entirely basophil. In PL 34, fig. 43, a still later stage
is drawn. The micromitochondria ( M .2) are now grouped
around the axial filament a good part of its way, and the
centrosomic structure is still visible.

In Text-fig. 4 iii a later stage is drawn ; in this (preparation
the two bead-like bodies in the tail of the sperm were par-
ticularly obvious.

1 See Addendum A.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 575

In Pl. 34, fig. 44, at a still later stage, the Nebenkern,
which hitherto kept its rectangular figure, has become col-
lapsed by the pressure of the narrowing space in which it lies,
and its individual parts are better revealed. In some cases,
at least, it seems that the Nebenkern elements at this stage
do really enclose an archoplasmic region, or at least a denser
region of the cytoplasm. In PI. 34, fig. 44, the mitochondrial
elements are becoming larger, and they now form a rough
coat to the axial filament. First are seen the micromito-
chondria, which now form an undoubted covering, while
behind is generally seen the Nebenkern. Behind this lie the
macromitochondria, which are less evenly disposed than the
micromitochondria. By PI. 34, fig. 45, the micromitochondria
have disappeared as such, but if the axial filament is carefully
examined, it will be seen that it lias increased both in thick-
ness or bore, and in its affinity for basic dyes. Followed
further down to the macromitochondrial region the filament
gradually resumes its original staining powers and propor-
tionate size. These areas of the filament are marked X ,
upper, Y, where the intermediate region lies, and Z, lower,
where there is a slight but hardly perceptible thickening.
In PI. 33, fig. 37, at a higher power is drawn the front region
of a metamorphosing sperm just before the stage described in
PI. 34, fig. 44. The disposition of the elements is quite
typical, while the Nebenkern is crumpled up and is seen to
consist of nine batonettes or rods, which together formed the
rectangular structure drawn in the previous figure (PI. 33,
fig. 36). In PI. 33, fig. 37, these rods seemed to be sur-
rounded by a clear zone.

As metamorphosis goes on the mixed-up macromitochondria
and batonettes become further and further removed from the
head of the sperm, while the former of the two become
somewhat larger and increasingly fewer in number. They
seem to be absorbed finally into the sheath of the tail, and if
anything is cast off it must be a very small portion indeed.1

1 A residuual bead is always cast off, and it always contains some
mitochondrial grains.

576

J. BRONTE GATENBY.

As the mitochondria slough down the sperm tail, the mito-
chondrial sheath becomes thicker and more darkly staining in
the region just cleared of the chondriosomes, the natural
inference being that the latter form the sheath. This seems
supported by such a stage as that drawn in PL 34, fig. 45, at
X, Y, and Z.

In the areas where the cells of the male generation are
densely packed one often finds variations in Nebenkern,
mitochondria, and nucleus. Without at present going into
the question of the cause of these variations, whether technical
or inherent in the snail, it is proposed to describe them. In
PI. 32, fig. 25, is drawn a spermatocyte near the end of
growth period. The mitochondria are seen to be small,
hollow spheres, scattered throughout the cytoplasm in much
the same way as in the spermatocyte in PI. 33, fig. 32k

The Nebenkern elements are much more numerous than in
the cases already described, where there are generally thirty
rodlets in the spermatocyte, and from six to twelve in the
spermatid. In the other spermatocytes, such as that in PL 32,
fig. 25, the rodlets are always almost completely circular, that
slight curve or banana shape being here much exaggerated.
The circular rodlets generally are placed as shown in PL 32,
fig. 25, but it will be noticed that they are almost invariably
placed so that their outer, thicker edge lies outermost, and
their centre is in contact with the archoplasm. This is very
well seen in PL 32, fig. 24 a. Re-examination of the Neben-
kern elements in PL 33, figs. 32, 35, and 36 shows that in this
variety of cell the elements are banana-shaped, and the
convex surface or back of the rodlet is turned inwards —
exactly the opposite of what is found in PL 32, figs. 25 or 24 a,
etc. Now when the spermatocyte breaks into the prophases,
the cell looks like PL 32, fig. 27. The chromosomes are
appearing ; the Nebenkern elements have become disposed
into two groups, one on each side of the nucleus, evidently
being influenced by the centrosome. Below the nucleus lie
several other rods, curved so as to form a circle, but with one
side especially thickened. It is quite characteristic of these

CYTOPLASMIC INCLUSIONS OF THE GKRM-CELLS. 577

stages that at this period one has difficulty in distinguishing
the mitochondria, which are sometimes quite big and ring-
shaped, from the Nebenkern elements.

As will be seen on inspection of PL 32, fig. 24 a, the mito-
chondria may be quite large and appear as hollow spheres.
When the cell division is in the metaphase the mitochondria
and Nebenkern elements become mixed, and I am unable to
say that the asters in any way affect the rodlets, as has been
claimed by Murray.1 In some cases one seems to be satisfied
that the spindle-fibres do exert some influence; in others the
opposite seems to be the case. In almost, if not all, meta-
phases I have examined, I feel with regard to this question
that the Scotch verdict, “ not proven,” is the safest view to
take. Should a specific stain either for mitochondria or for
the rodlets of the Nebenkern be found, it may be possible to
throw some clearer light upon this question.

In PL 32, fig. 22, a second maturation division is shown.
The mitochondria are hollow spheres, appearing as ringlets,
and here and there lie the elements of the Nebenkern, which
I can positively identify. The spermatid of this small-rodlet
generation almost always has a nucleus which contains a
greater number of karyosomes2 than the sort drawn in PL 33,
fig. 35. The Nebenkern elements in the example drawn in
PL 32, fig. 26, were very small, and at this stage evidently
just collecting after cell division. It will be noted in all these
stages except the one drawn in PL 32, fig. 27, that the cell
is angular in shape, from the abutting neighbouring cells
pressing on each side.

When the spermatid begins to metamorphose tke micro-
mitochondria appear, and the Nebenkern is formed as shown
in PL 32, fig. 24 a. Curiously enough, the sperm head in the
early stages of its transformation is generally irregular in

1 I have since found that in division the batonettes do lie in the zone
of the asters.

2 See PI. 34, fig. 48 a, b, c, where spermatid nuclei from different
regions are drawn to show variation in karyosomes. The bulk of the
karyosome matter does not vary much, only number.

578

J. BRONTE GATENBY.

shape — probably traceable to the cramped quarters in which
the cell is forced to metamorphose.

In Text-fig. 1, iii, iv, the two extremes in these various
kinds of cells are drawn. Fig. iii shows the typical Neben-
keru formed of many curved rods, while fig. iv shows the
typical rectangular Nebenkern formed of fewer elements.

The remarkable differences which are found in the later
spermatids are illustrated in PI. 33, figs. 37 and 40. These
are not quite at the same stage, the latter being slightly
younger. In this form the Nebenkern is very large and
peculiar, differing most markedly from that in PL 33, fig. 37.
This also applies to the mitochondria, which, while being
larger in PI. 33, fig. 40, and fewer. But, as will be noticed in
PI. 32, fig. 26, largeness of mitochondria and smallness of
Nebenkern batonettes do not necessarily go hand in hand.

The Manner of Metamorphosis of an Indifferent
Germinal Epithelial Cell into a Primary Sper-
matogonium .

Many of the older writers thought that the main factor of
change in the epithelial nucleus was the appearance, or at
least the exaggeration, of the chromatin lumps. PL 31,
figs. 11 and 19 show this. In one case we deal with a sperma-
togonium (the latter), and in the other with a oogonium. But
it should at once be pointed out that, from a careful study
of these stages, I have concluded that the amount of real

Fig. i. — Part of the lumen less well supplied with nutriment. The
epithelial cells have here and there grown into progerminative cells
(X), passed to the prophases of the heterotypic division ( C.F .) and
afterwards have begun to drop off (X.Y.). (Text-fig. 4.) At X. and
E.N.C. are yolk cells in stages of disintegration. At N.C.N. is a
bare yolk cell nucleus. At S.N. are many sperm atogonial nuclei
lying in a syncytium (R.), a quite characteristic occurrence in these
localities of a lumen. As at S. and in other cells there is a
distinct cloud near the nucleus. (See PL 31, figs. 19, 20 ; Pl. 32,
figs. 21 and 23.) Fig. ii. — Passage of a male progerminative cell
(primary spermatogonium) from its place on the germinal epithelium

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 579

Text-fig. 3.

(£.) into the lumen of the diverticulum. At S.P.S. are secondary
spermatogonia derived from a division of such a cell as S.P.P. The
arrow points to tlie inside of the lumen.

580

J. BBONTffc GATENBY.

variation is quite remarkable, and no two epithelial cells
metamorphose in quite the same way.

In PI. 30, fig. 2, the upper nucleus has to its right a small,
darker area in the cytoplasm, and the same applies to the
upper part of the middle nucleus in this figure. Not all cells
possess this cloud — or, more correctly, I should say that I
have been unable to find the cloud in every epithelial cell.
The cytoplasm of the germinal epithelial cell is of the open
variety, and is best seen in preparations stained in iron alum-
alizarin-toluidin blue, where it is a purplish-blue in shade.

In some cases, when the cell is about to abandon its in-
difference, the nucleus, at first a depressed oval, becomes
spherical or semi-spherical, as shown in PI. 31, figs. 17 and 19.
I believe that of the many variations which one finds, all fall
roughly under three heads : In the first, one has a cell in the
positions marked X. in Text-fig. 1, i, growing into fig. ii, X.,
in the same text-figure. Also in other cases one finds such an
example as that of Text-fig. 2 at 1 and 2. While, again, one
always finds cases such as that drawn in Text-fig. 3, i, X.Y.,
and Text-fig*. 4, i, and in PI. 29, fig. 1, at the roman figures (i,
ii, iii).

In the other cases one finds the progerminative cell as in
Text-fig. 3, ii, S.P.P. Also it seems that these arbitrary
classes are linked up by other classes, such as the cell 3 b in
PI. 29, fig. 1 (explained in “ Discussion ”).

In every case of an epithelial cell of the snail meta-
morphosing into a germ-cell of either sex one finds two
constant facts : one is the appearance of a fine cloud in the
cytoplasm, which follows after the other — an enlargement of
the nucleus. It is only by fine fixing reagents that this cloud
is shown, but it has never before been described simply
because the workers on the snail destroyed it with acetic acid
or alcohol.

I am unable to say why this cloud, which is shown in PL 31,
figs. 11 and 19 in a very early stage, should with absolute
constancy be found to one side of the nucleus. It may be
that it is on the side near which lies the centrosome if tliaf be

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS.

581

present, but I am unable to advance any other opinion
beyond this : I believe the cloud is formed by a growth and
enlargement of the zone already indicated as being present in
the cytoplasm of at least some germinal epithelial cells (PL 30,
fig. 2, Y. and X.), which is quite possibly a conglomeration of
some material around or in an attraction sphere. But so
difficult is it to study these early stages that I cannot advance
any evidence based on actual knowledge of this small aggre-
gation in the cytoplasm.

One significant fact I can, however, point out : it is that
the growth or the appearance of this faint cloud is subsequent
upon a change in the size and often of the staining power of
the nucleus. The latter moves first ; the cloud then appears.

In every case the germinal nucleus at first becomes round
or oval, and the chromatin lumps, before connected here and
there by bridges, become spherical and isolated. After this
the cloud in the cytoplasm becomes marked. From this
stage onwards there is a difference in the behaviour of the
kinds of male cells derived from such progerminative cells.
In the case of one generation of male cells, shown in Text-
fig. 3, the chromatin lumps, after some slight changes, break
into a spireme, and the prophases of the heterotypic division
are undergone while the cell still adheres to the germinal
epithelium ( C.F . in Text-fig. 3, i). The same sort of occur-
rence invariably happens in the oocyte, where a spireme
gradually appears and the prophases take place in situ. But
in the case of certain cells shown in Text-fig. 2, ii, and 3, ii,
the behaviour of the chromatin is different. It can at once
be explained that this different behaviour is due to the fact
that such cells are going to undergo mitosis. For this to
happen the chromatin must come into a resting stage for the
formation of a reticulum, which soon breaks up into chromo-
somes. (See Text-fig. 2, ii, at 5.)

In this last cell the chromosomes are beginning to appear.
It has been customary for many writers on this subject to
describe minutely changes in the nucleus which herald either
the formation of a spermatogonium or an oogonium, whichever

582

J. BRONTE GATENBY.

the case may be. I do not fail to recognise the splendid
labours of such observers when I state that I cannot
accept anything in their descriptions of such changes. There
may be very slight differences, but I have failed to find any
upon which one could reliably base a dogmatic statement. The
differences between the individual behaviour of the chromatin
in the nuclei of a number of progerminatives of the same
probable future sex, are so wide as to cover the statements
depending on size, staining power, and arrangement of chro-
matin lumps, upon which these descriptions are based.

To return to the cells which I have mentioned as about to
undergo mitosis, Text-fig. 3, ii, S.P.P., shows a section
through a region from which male cells were appearing when
the snail was killed. The cell S.P.P. is leaving its place in
the epithelium marked L and is pushing out. This cell has a
nucleus, oval in shape and containing its chromatin in faintly
staining lumps. There is a cloud in the cytoplasm containing
dark bodies (PL 34, fig. 49). Inspection of Text-fig. 2 at the
figure 2 shows another cell, but in a different locality, free

Text-fig. 4.

Fig. i. — Shows dropping off of the male cells, about to finish growth
stage. At Y. the cell is becoming detached, at X. it is already in
the lumen. The cell Z. may belong to this generation, or from the
generation to which the boquet stages ( BS ) are derived. There is
generally a mixture of various generations in this lower region of
the diverticulum. X 900. Fig. ii. — Bichromate smear of adult
sperm head. A. = acrosome, N. = nucleus, C. = centrosome,
M. = mitochondrial sheath. Iron hsematoxylin. x 2000. Fig. iii. —
Group of metamorphosing spermatids showing definite position of
Nebenkern ( N.K. ). Also the curious arrangement of two granules
(0.) where the hind centrosome lies. (Compare PI. 34, fig. 44.)
X 1000. Fig. iv. — A spermatogonial group with Nebenkerne
( N.K. ), spindle bridge ( S.B. ), mitochondria (M.), and a yolk cell
(. NC .). (Compare PI. 29, figs. 1, 2, and PL 32, fig. 29.) Fig. v. —
Several batonettes greatly enlarged showing difference in shape and
size, and in one the relations of the archoplasm ( A.R .) with the
inside of the batonette. (See Pl. 32, figs. 24 a and 25.) Fig. vi. —
The large variety of Nebenkern rod at same magnification. (See
PI. 33, fig. 36.) Fig. vii. — Three spermatogonia attached to a cell
with a germinal epithelial nucleus, but with the cell granules
grouped together at X. What these are, whether Nebenkern
batonettes or mitochondria, was not ascertained, x 2000,

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 583

Fig. i.

Text-fig. 4.

Fig. ii.

Fig. vi.

Fig. vii,

Fig. v.

584 J. BRONTE OATEN BY.

from much yolk. This cell is drawn in PL 31, fig. 17, at a very
high power.

The nucleus has the same general appearance as in Text-
fig. 3 at S.P.P., though in the latter the chromatin lumps
have not broken up so much as yet, as in PI. 31, fig. 17.
Both these cells will ultimately divide and give rise to some
secondary cells. The answer to the question why there
should be differences in these two cells, however small, is
that they originate from parts of the germinal epithelium
which are in a different nutritive condition. Cells like these
soon divide many times and give rise to bunches of spermato-
gonia such as those shown in PI. 29, fig. 1, at the outer 2, or
in Text-fig. 4 at iv. The connection between the yolk cell
and the male progerminative cell is sometimes severed, some-
times retained.

Now, if attention be centred on the cytoplasmic cloud in
these progerminative cells it will be noted that apart from the
apparent denseness of the cytoplasm which causes the cloudy
appearance one finds distinct granules. These are apparently
the first signs of the mitochondria. The size and number of
these granules are very variable. In PI. 31, fig. 17, they were
very large and distinct.

Towards the time when the lumps of chromatin in the
nucleus have fragmented to form a fine clear granular struc-
ture these cytoplasmic bodies become dispersed more and
more from the zone from which they originally appeared.

The chromosomes soon appear and a cell division takes
place, the cytoplasmic bodies being visible and scattered here
and there around the amphiaster. As far as I have been
able to ascertain, these mitochondria do not lose their rounded
shape during division. Such a series of divisions finally give
rise to cells such as those drawn in Text-fig. 3 at S.P.iS.

After a division is finished, and these secondary cells have
regained their resting nucleus an examination of the cyto-
plasm generally shows that two sets of bodies are present :
one, the mitochondria, are scattered indiscriminately, the
other is a dark structure lying close to the nuclear mem-

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 585

brane. In some generations of secondary spermatogonia this
latter structure is not demonstrable till later. This body is
the first visible sign of the Nebenkern of Pulmonates, and it
may possibly have been present in the stage drawn in PL 31,
fig. 17, but may not be plain enough to make its detection
possible.

In PI. 32, fig. 24, is drawn a spermatogonium of the genera-
tion shown in Text-fig*. 2, fig. ii, at 2. The Nebenkern ( N.K .) is
quite easily seen to consist of several straight rods. In PI. 32,
fig. 29, the Nebenkern of another generation of spermatogonia
is shown. The fate of the Nebenkern and mitochondria of
these generations has already been followed out.

Another sort of male generation of cells appears in the
following way : In Text-fig. 3, i, is drawn a lumen in which
nearly all the germinal epithelial cells have thrown off their
indifference and have become large peculiar cells. The
epithelium from which such cells arise consists of a single
layer of cells, yolk cells being either exhausted or completely
absent. This is shown in the plan on PI. 29, fig. 1, at the
Roman figures i-iv.

In PI. 31, fig. 19, the cell at ii in PI. 29, fig. 1, is drawn
at high magnification. It lies on the epithelium projecting*
into the lumen in a different way from PL 31, fig. 17.

The nucleus still has the chromatin lumps which stain some-
what lightly, and there are two nucleoli which are hollow
spheres, as far as one can ascertain. At any rate, their
centre does not stain so heavily as the periphery. The lumps
gradually become elongated and join up to form a wide reticu-
lum (PL 31, fig. 20). Now, the plasma in fig. 19 is seen
to contain to one side of the nucleus, the usual zone
which here and there has a granule. In the next stage the
granules have become quite plain and are seen to consist of
the ring-like mitochondria already described from the figures
in Pl. 31. In fig. 20 the zone of thickened cytoplasm has
spread right around to the opposite side of the nucleus but
as yet no distinct mitochondria have appeared on this side (Z.)

There is rarely any other structure to be made out in the
VuL. 62, PART 4. KEW SERIES. 40

586

J. BRONTE GATENBV.

cytoplasm, but in a few cases one finds what seems to be an
archoplasmic mass ( A.R .) upon which may be stuck from two-
to four Nebenkern rods. This is so seldom found at this
stage that I do not think it is the rule. The wide open
reticulum now breaks into a loose spireme and a contraction
figure is shown in PI. 32, fig. 21. 1 In this cell the mitochon-
dria were rather irregular in shape, here and there undoubtedly
ring-like, but they were all collected to one side of the
nucleus towards where the chromatin filaments converged.
It will be noticed that the nucleoplasm in this kind of male
cell generation is abnormally large for the amount of chro-
matin spireme. The diplotene stage and the rest of the pro-
phases soon take place and the growth period begins. The
Text-fig. 3, i, shows that at X. Y. and at other parts these cells
are losing their attachment to the walls. In Text-fig. 4, i,
the cell Y. is just falling into the lumen, while X. has already
arrived there. The contraction figures below belong to
another cell generation. In PI. 32, fig. 23, a typical cell just
after the beginning of the growth stage is shown. The
attachment to the germinal epithelium consists only of a
small area (Xj) which will soon part. The mitochondria are
dispersed in the cytoplasm, and outside the nuclear membrane
(at N.K.) is a cloud in which can be seen embedded a very
large number of small Nebenkern rods. These are slightly
curved. This is the usual way in which the Nebenkern
appears in this generation. PI. 32, fig. 25, is a later stage.
Subsequent stages are drawn in PL 32, figs. 22, 25, 26, and
27, and have been described.

It seems that in this generation the nucleus after the pro-
phases collapses somewhat in size, proportionate to the normal
amount of chromatin contained therein. It will be noted that
no spermatogonial divisions take place.

Pachynema.

CYTOPLASMIC JNCLUSIONS OF THE GEKM-CELLS. 587

The Manner of Metamorphosis of an Indifferent
Germinal Epithelial Cell into an Oogonium.

It has been agreed almost unanimously among workers on
the ovotestis that the presence of a group of yolk cells is the
determining factor in the appearance of the female cell. I
consider this explanation inadequate, for it can be shown that
spermatogonia appear in regions choked with yolk cells, and
oogonia may appear in regions where little or no yolk is present.
I feel that it would be quite a mistake to entertain the view
that a yolk cell exclusively determines the awakening of an
indifferent cell into the egg generation. But it might be
true to say that the presence of abundant yolk was the sine
qua non for the successful growth of an oocyte to maturity.
It would also be quite true to say that in the majority of cases
the transition from an indifferent cell to an egg took place
behind or between some yolk cells. Despite careful observa-
tion of a large number of cases I find it difficult to formulate
a statement of any differences between male and female
nuclei till after the growth stage begins. This is not the case
with the cytoplasm. The drawing in PI. 30, fig. 6, was
thought by me to be an oogonium in contraction stage, firstly
because it was so closely embedded behind yolk cells that its
exit into the lumen would have been difficult, and secondly
because the epithelium in this region was seen to be producing
many oocytes.

What I take to be the Nebenkern (N.K.) is very like that
in the male cell drawn at a little later stage in PL 33, fig. 31,
while the mitochondria are not very characteristic. The
nucleus is unlike the male generation nucleus drawn in PI. 32,
fig. 21, but almost identical with that drawn in PI. 33, fig. 30.
Apropos of the statement that the spermatogonia appear in
yolk regions compare Text-fig. 1, x, and especially Text-
fig. 3, ii, at S.P.P. On the other hand, in an epithelium like
that in Text-fig. 2, ii, a solitary oocyte may grow. This all
shows that the matter is somewhat more complicated than at
first supposed. Having shown that to the end of the pro-

588

J. BRONTE GATENBY.

phases the nucleus does not give us any very characteristic
evidence we will turn to the cytoplasmic bodies. In PL 31,
fig. 11, is drawn a cell thought to be an oogonium. I believe
it to be such for the same reasons which I brought . forward
for fig. 6 of PL 30. The cytoplasmic cloud does not differ
markedly from many another example known to be a male —
it contains the same lumps and is isolated to one side of the
nucleus in the same way. Pl. 30, fig. 6, is thought to be a
later stage. A Nebenkern has appeared. Fig. 10 of PL 31
is a still later stage — the nucleus is losing its chromatin loops,
while the mitochondrial mass looks less granular and more
flocculent. At one side is an undoubted Nebenkern, but of a
slightly different type, the rods being straighter. Fig. 10 of
PI. 31 really corresponds to the same stage in the male drawn
PL 33, fig. 31.

I have already mentioned what great variation was found
in the appearance of the male cells. This, I think, applies
even more strongly to the case of the female cells, but a
difference which seems to exist between all the female cells
and the male cell is that in the latter the mitochondria are
from the first to the last granular and comparatively large,
while in the former the mitochondria, even if at first
granular, rapidly become flocculent and lie like a cloud, as
shown in PL 31, figs. 10 and 12, and in later stages in PL 30,
figs. 3, 5, 7, Pl. 31, figs. 9, 13, 14. Later, the mitochondria of
the egg seem to become granular, spherical, and often some-
what larger than the male, but there is then no possibility
about making a mistake as to the sex at this period.

Pl. 31, figs. 12 and 9, show stages in the mitochondria.
In the former figure, which is drawn one-half the size of
fig. 10, the mitochondria are dispersing, not, however, from a
centre as they are in fig. 9. In this figure there is a
centre from which flocculent masses of mitochondria radiate.
It will now be clear that the most certainly diagnostic
evidence for difference between the oogonium and spermato-
gonium is to be found in the mitochondria, which early behave
differently in either sex.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 589

The Fate of the Mitochondria and Nebenkern in

the Egg.1

The flocculent mitochondria gradually become dispersed
somewhat unevenly throughout the cytoplasm as shown in
PI. 30, figs. 3 and 7, and in PI. 31, fig. 9. If these mito-
chondria be examined it will be found that they consist of
masses of exceedingly fine grains (PI. 31, fig. 13). In later
stages these grains become here and there mixed up with
la.rger granules as shown in PL 31, fig. 14. In a still later
stage no flocculent masses remain, there being now only large
mitochondria. The latter seem to be derived from the floccu-
lent masses as shown in PI. 31, fig.* 14, and in different eggs
are of different sizes as is the case with the mitochondria of
the male. Soon after this, vacuoles appear in the cytoplasm
and the mitochondria lie in the trabeculas between these
alveoli (PI. 31, fig. 16). Yolk disclets sooner orlater partially
fill these spaces (PI. 31, fig. 16, Y.), but are not to be con-
fused with the mitochondria at any stage. In later stages
the cytoplasm becomes divided into a cortical layer without
yolk vacuoles, and an inner layer like that drawn in PL 31,
fig. 16.

After the stage drawn in PI. 31, fig. 10, the Nebenkern
seems generally to become obliterated by a curtain of mito-
chondria, but as shown in Pl. 30, fig. 5, it may still be quite
plain. Its subsequent fate is difficult to follow, for it cannot
be found in every oocyte. In PL 31, fig. 22 (at X.N.K.) are
round bodies which are almost certainly separate parts of the
Nebenkern. I have found many oocytes showing these ring-
like structures.

In later stages there appears a clearer zone near one side
of the nucleus, and in this zone appear several blocks of
darkly-staining matter as shown in Pl. 31, fig. 18; the two
left-hand pieces are drawn at a high power in PL 30, fig. 8,
and consist of more or less solid matter, which generally con-
tains cavities. The nature of these structures and their con-

1 See Addendum B.

590 J. BRONTE OATEN BY.

nection, if any, with the mitochondria or Nebenkern, is
unknown to me.

The Differentiation of an Indifferent Germinal
Epithelial Cell into a Nurse or Yolk Cell.

It is quite usual to find small yolk granules in any indifferent
epithelial cell, and when the cell becomes either an oogonium
or a spermatogonium, these soon disappear at first (see PI. 30,
fig. 6). The growth of an indifferent cell into a yolk cell is
accompanied by the appearance of abundant yolk disclets, and
a change in the size and in the disposition of the chromatin of
the nucleus. In the latter the chromatin bridges between the
angular lumps if present (PL 30, fig. 2) become lost and the
chromatin becomes formed into little round structures. Syn-
chronously with the appearance of more and more yolk, the
nucleus grows larger and larger till it may be 35 jj. in length.

These changes are easily noticed and have already been
described well (2). A more difficult problem is what happens
to the plasmatic bodies of the modified cell. Does a Nebenkern
appear ? What happens to the mitochondria ? It is not at
all easy to find answers to these questions. In PI. 33, fig. 38,
is drawn a cell with a yolk cell nucleus gathered into
numerous chromatin lumps. In the cytoplasm there were no
yolk disclets, but the reticulum was of the very wide kind,
peculiar to yolk cells. Here and there were small masses
containing mitochondria (. M .), and to the left of the nucleus
was what I took to be an attraction sphere, inside which were
embedded a number of dark bodies, probably the Nebenkern.
In many ways this cell is intermediate between the yolk
cell and the indifferent epithelial cell. In the full yolk
cell it is probably not possible to discover attraction sphere,
Nebenkern, or mitochondria. But after many of the yolk
discs have been absorbed by neighbouring cells, it is generally
possible to discover bodies shaped like those drawn in the
oocyte in PI. 31, fig. 12, at X.N.K. What these are, subse-
quent work will be needed to show, but I liave done enough

CYTOPLASMIC INCLUSIONS OF THE GERM-CELL8. 591

to think that modified representatives of Nebenkern and mito-
chondria may be found in the cytoplasm of the yolk cell.1

After some time the yolk cells become exhausted completely,
and the wide reticulum breaks up and the nuclei float out into
the lumen of the ovotestis diverticulum as shown in Text-
fig. 3, i, N.C.N., E.N.C . These nuclei do not degenerate, as
might be supposed. They lie there in the midst of a mass of
live cells and degenerate yolk, and seem to undergo further
changes which need not detain us at present, but I should
say that it is my firm opinion that these nuclei regain a cyto-
plasm and become spermatocytes.

Description of the Scheme on PI. 29, fig. 1.

In this figure I have united my final conclusions concern-
ing the processes which go on in the various regions of diver-
ticulum of the ovotestis of Helix. All cells have been drawn
in to scale with a camera lucida, and in the majority of cases
are the same as those on the other plates drawn at a higher
power. Great care has been taken to show the germinal
epithelium in its true state; thus, for instance, the region of
the right lower edge marked by the Homan figures is the
same kind as that drawn in Text-fig. 3, i. The left lower
half appears also in Text-fig. 2, i, and so on.

In the following description, after mentioning each series
of cells, I will indicate where they are drawn at a higher
power in the other plates. If not the identical cell, I will
show this by adding the letter W. to the bracket.

The Genesis of the Egg.

A =■ Differentiating germinal cell embedded in yolk cells

(Pi. 31, fig. li-ir.).

B = Young oocyte (PI. 30, fig. 6- TP).

C — Older oocyte (PI. 30, fig. 3, 7, etc.- IF.).

D — Older oocyte (PI. 31, fig. 9-TF.).

1 More work on a dozen species of Pulmonates shows that the true
yolk cells do not contain mitochondria or Nebenkern.

592

J. BRONTE GATENBY.

The Genesis of the Sperm.

In the Roman figures to the right I have indicated the sort
of male cell generation in which no spermatogonia! divisions
take place. (See also Text-fig. 5.)

I. Earlier stage than drawn in any other figures.

II. Progerminative cell (PI. 31, fig. 19).

III. Mitochondria appearing in spermatogonium. Rare

kind of Nebenkern (PI. 31, fig. 20).

IV. Spermatocyte, with Nebenkern just appearing (PI. 32,

fig. 23). Usual Nebenkern.

V. End of growth stage. Cytoplasm differs greatly in
size in different examples (PI. 32, fig. 25- TV.).

VI. Maturation division. Spindle curiously orientated
with relation to cell, first maturation. (Second
maturation drawn in PI. 32. fig. 22- TV.).

VII. Spermatid (PI. 32, fig. 26).

VIII. Later spermatid (PI. 32, fig. 24 a- TV.).

It will be noted that on the left bottom region at the
Arabic numbers 1 and 2, there is another source of male cells.
The two sources of cells, marked in Arabic and in Roman
numerals respectively, are often mixed indiscriminately and
later stages are hard to distinguish. Thus the bouquet stages
marked 3 a are certainly derived from such a source as 1, 2,
on the left, because in the generation derived directly as
shown at the Roman numerals the contraction figure and
other stages up to the beginning of growth take place directly
on the wall (see PI. 32, fig. 21, which was stuck on the wall,
and Text-fig. 3, i).

In the case of the cells marked V 4 it was not possible to
tell whether they were derived as in the Roman numerals or
as in the Arabic. Thus the Roman numerals beyond VI are
probably of uncertain derivation. VI itself, on account of its
great size, is probably of the Roman numeral generation.
Almost invariably the two generations, derived respectively
from the bottom left corner at 1 and 2, and from the bottom
right at the Roman numerals have a Nebenkern formed of

CYTOPLASMIC INCLUSIONS OF THE GERM-0 EELS. 593

numerous curved elements as in PI. 32, fig. 25. Moreover,
the size of the entire spermatocyte, though not of its nucleus,,
varies greatly (VI, V, V-4). The spermatocytes are either
crowded as in V-4 to VIII or loose as at V. Crowded sper-
matocytes have walls like that in PI. 32, fig. 25, etc., loose
ones like that in PI. 32, fig. 27. Nebenkern in each may be
the same. The spermatid may be a large vacuolated cell,
though the sperm has the same end result.

At the Arabic numerals are drawn all the various genera-
tions which have male progerminatives which divide to give
rise to secondary spermatogonia and even tertiary generations
of spermatogonia.

At the mid left-hand side at 1 is a primary spermatogonium
dropping into the lumen. The male progerminative cell at II
on the right at the Roman numerals is derived from a different
kind of epithelium with a semi-empty lumen. At 2 (left-
side) this primary spermatogonium gives rise to many
secondary cells. Some, as at 2 a, go on dividing till they
become very small. Others form the bunch drawn at the
outer middle 2 (Text-fig. 4, iv) .

By 3 the prophases of the heterotypic division have begun.
One sometimes finds cells like that drawn at 3 6. Isolated
cells like this are found in connection with a nurse-cell and
may form a link between the generations at 1 in Arabic
numerals and between the other in Roman. These cells, such
as that at 3 6, seem to be primary spermatogonia which have
failed to divide but have entered the prophases.

At 4 are the spermatocytes ; in this generation they never
vary so greatly as in the generation derived from the epithe-
lium, as at (I, II, III) or as in the crowded cells. At 5 is a
first maturation division, which can be compared with the first
maturation at VI.1 The spindles are the same size.

The following indicates where these cells are drawn at a
higher power :

1 in PI. 34, fig. 49 (IF.).

2 in PL 32, fig. 29.

1 Such cells are not all so large as this example.

594

J. BRONTE GATENBY.

2 b in PI. 32, fig. 24 (IP).

2c in PI. 32, fig. 28 (IP.).

3 in PI. 33, fig. 31 (IP.).

4 in PL 33, fig. 32 (IP).

5 in PI. 33, fig. 34 (IP).

6 in PL 33, fig. 35 (IP).

7 in PL 33, fig. 36 (IP).

8 in PL 34, fig. 45 (IP).

9 in PL 34, fig. 46 (LP).

Discussion.

(a) Nebenkern. — Sex determiner, spindle former, de-
generation product, cliromidium, are only a few of tlie
different characters supposed to be fulfilled by this curious
aggregation of stick-like structures, known as the Neben-
kern.

In the first place we will examine the evidence that the
Nebenkern determines the sex of a differentiating cell.
Demoll, tracing the Nebenkern from about the time of the
“Bukett stadium ” in the prophases of the heterotype divisions,
thought that the subsequent development of this body was the
lever which turned the cell to oogonium. It is quite true
that it is at or immediately after the bouquet stage that the
first definite evidence for the female sex1 can be found, but
had Demoll succeeded in following out the mitochondria he
would have seen that the latter may show a differentiation
towards one sex before the Nebenkern becomes in any way
characteristic. DemolPs reservation that the “ sex chromo-
some 33 probably influences the Nebenkern to act in a way
productive of one sex or the other is interesting, but supported
by no evidence.

Briefly stated, I should think the following disposed of the
u Nebenkern and sex 33 hypothesis1 :

1 Sex, female or male, I have used somewhat loosely instead of
metamorphosis of indifferent cell into oogonium or spermatogonium ’>
respectively.

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 595

(1) The mitochondria in the egg are often diagnostic before
the Nebenkern rods alter.

(2) The Nebenkern varies more in individual spermatogonia
than between Demolhs spermatocyte and oocyte examples.

(3) The whole train of events leading to the appearance of
undoubted Nebenkern and of mitochondria is so liable to
variation that it would be impracticable to look to such struc-
tures as sex determiners.

(4) The Nebenkern may be late in appearance, even after
the cell is undoubtedly male.

(5) Bouquet stages are not rightly to be considered as
definite milestones, parallel in the sex development of either
sperm or egg cells, for the bouquet stage may appear in the
male at a time when it is known that the sex1 has been deter-
mined cell generations ago. I refer in this to the secondary
spermatogonia which go on dividing and which sporadically
enter growth stage.

(6) The amount of Nebenkern material in the bouquet
stages of the male alone varies from “ none demonstrable ”
to a large amount.

It will be seen, therefore, that lack of proper examination
and of sufficient knowledge of the Nebenkern has led Demoll
to suggest a theory unsupported by evidence of any descrip-
tion.

What would certainly be more logical would be to say that
the alteration in the Nebenkern was the result of and not the
reason for the appearance of a definite sex. As far as
diagnosis goes the mitochondria are better objects for building
up theories, but for many reasons the last paragraph about
the Nebenkern also applies to the mitochondria. As for the
suggestion that the sex-chromosome guides the Nebenkern in
its “choice of sex 33 I have no evidence either for or against.
Demoll's idea that up to the appearance of the Nebenkern
the cell may be considered indifferent is disproven by every
species of evidence.

The only other likely suggestion as to the function of the
Nebenkern is one which has been freely supported by certain

596

J. BRONTE G ATEN BY.

observers. It is that these rods are the condensed spindle,
and that their supposed disappearance before mitosis is due
to the fact that they have gone to form the astral figure.

As far as the snail is concerned 1 am somewhat doubtful as
to the validity of this interesting suggestion. Without
definitely condemning or upholding this view I think that
the following facts should be borne in mind. In the
Helicids :

(1) It is, if not absolutely unproven, at least extremely
doubtful whether the rods do disappear1 (PI. 32, figs. 23 and
27).

(2) The substance of the rods differs in bulk, as also do the
number of the rods. Variation in the size of the spindle is
almost negligible (Figs. 24 a and 36, Figs. 25 and 32, etc.).

(3) In the prophases the rods can undoubtedly be found
lying here and there in disorder, not as if they served a defi-
nite function in the formation of the amphiaster.

(4) In many spermatogonial late telophases the Nebenkern
appears in a position removed from the centrosome, archo-
plasm, or spindle bridge. Not in any case as if it appeared
to be reinstated by a condensation of any part of the amphi-
aster (PL 32, fig. 29).

(5) In numerous other animals the spindle appears and
disappears without a Nebenkern (batonettes).

(6) Bolls Lee and I describe a triradiate structure from
which the astral rays arise, and which is undoubtedly un-
related to the Nebenkern (PI. 33, fig. 33).

(7) Spermatogonial generations are to be found in which
the cells before entering mitosis had no definite Neben-
kern.

Before the spindle-forming function of the Nebenkern can
be proven all these objections must be met. I do not think
any of them can be explained satisfactorily from the point of
view of the observers who espouse the theory. Term's state-
ment already mentioned, that he did not believe that all the

1 In a forthcoming paper I have described how to fix and stain so as
to show these rods during metaphase.

CYTOPLASMIC INCLUSIONS OP THE GERM-CELLS. 597

i'Hozomatic material was taken up in the formation of the
spindle, I think rather vitiates the evidence for the view that
it is necessarily the Nebenkern part of the idiozome (archo-
plasm) which goes to form the middle spindle. Term's
material seemed to admit of clearer study of the Nebenkern
than mine, because in the snail the mitochondria do not
appear to become localised to one side of the cell as often
happens in Geotriton, according to Terni. This localisation
often clears the area around the Nebenkern, and assists
observation. (For further evidence against the “ ISTebenkern
spindle” view see Faure-Fremiet, page 580 and fig. 33).

To return to the Nebenkern of Helix aspersa, it may be
worth while to attempt to analyse some of the variations so
strikingly evident.

The Nebenkern rods differ in number without a doubt;
they likewise differ in size, and their shape is seldom the
same. Neither the number nor the great difference in size
can be the result of varying conditions of fixation. For the
shape I cannot speak with such confidence. It is quite
possible that some small variation either in the fixative or the
•condition of the cell might produce distortion of the rods.
To test this I examined a great deal of material by the intra-
vitam methods. Janus green of the strength of 1 in 30,000
and neutral red about the same strength were used. These
did not give very good results. After many experiments I
found that infra- vitam methods would not properly settle the
question, because the rods of the Nebenkern never seemed to
take the stain heavily enough.

I devised the following “ fresh method” : A small part of
the ovotestis was smeared on a slide, and a little 1 per cent,
permanganate of potash was added. A coverslip was then
placed over this, and the preparation, after about sixty
seconds, showed the cell inclusions a brown colour, and they
were very easily studied. The mitochondria were remarkably
•clear, and were of the spherical type, not stick or rod-like.
The Nebenkern was found to vary as in my drawings — rod-
shaped or curved. In this permanganate method the cells are

598

J. BRONTE GATENBY.

killed instantly. I found the oocytes stained just as shown in
my diagrams.

The Probable Function of the Nebenkern.

It will be seen that for some reasons I am at present unable
tc accept the view that the Nebenkern has anything to do
with the formation of the astral figure. Term's important
paper, I feel, reinforces me in this (see especially his figures
15 to 18).

I do not find myself in a position better than that of many
of my predecessors in-so-far as an analysis of the function of
the Nebenkern is concerned. But I feel sure that its real role
has not been pointed out. It seems evident that it is, in
molluscs, a piece of cell mechanism as definite as the mito-
chondria. I have shown that, like the mitochondria, it is
liable to variation in time of appearance, in size, and in its
general behaviour. It generally stains like the mitochondria,
it is destroyed by the fixatives which also destroy mito-
chondria, and it is seen best in preparations which show the
latter best. I therefore think that the Nebenkern rods in
Helix are to be classed with the two sorts of mitochondria
described by me as 'definite plasmatic elements whose exact
function is still unknown, but which have nothing to do with
the amphiaster. The aster and centrosomes can be followed
out best in material unfavourable for a study of the mito-
chondria or Nebenkern.

With regard to staining reactions, Faure-Fremiet says :
“ Si Foil isole des spermatocytes dans le serum au chlorure
de manganese additionne de violet dahlia ou de violet de
gentiane, les mitochondries se colorent assez rapidement en
lilias, tandis que le Nebenkern reste quelquefois plus long-
temps avant de se colorer."

b. Mitochondria.

It is not intended to discuss these bodies at length, but it
may be pointed out that in the snail one finds a remarkable

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 599

phenomenon. It is the presence of two definite kinds of
mitochondria, which apparently have a definite location in
every sperm tail. This is a fact of cardinal importance, and
seems to show that there is a division of labour or function;
between the mitochondria of the spermatid. The position of
the micromitochondria is more definite than that of any
plasmatic structure other than centrosomes or perforatorium.

Terni figures and describes his mitochondria throughout as
rods, remarkably equal in size and length. He has observed
fresh material, and therefore apparently this rod-like structure,
which can be artificially produced in Helix by bad fixation, is
the true state of affairs in Geotriton.1

c. The Determination of Oogonium, Spermato-
gonium, or Nurse-Cell.

In the following, the words “ determination of sex ” are
used for the above for convenience. I have been unable to
produce any definite evidence regarding the determination of
sex. This paper deals almost exclusively with the plasma,
but I have come to certain conclusions with regard to this
important matter. In short, I am convinced that since it is
the nucleus which is the first part of the indifferent cell to
begin differentiation, we are justified in believing that the
nucleus is the prime factor in the production of any one sex.
The nucleus enlarges, its chromatin undergoes changes, a
cloud appears at the edge of the nucleus, evidently under the
influence of the latter, and every step in the metamorphosis
of the cytoplasm is preceded by one in the nucleus. If we
saw the nucleus remaining as it was for some time, waiting
till the Nebenkern or mitochondria appeared, we would
naturally think that the latter structures, after appearing,
stimulated the nucleus. As it is the other way about, I feel
justified in entertaining the view that it is not the plasma,

1 Since this paper was written I have come to the conclusion that the
rod-like mitochondria (PI. 33, fig. 34) where they occur are artefracts
produced by bad fixation, but as I have not observed Triton I cannot
venture to question Term's results.

600

J. BRONTE GATENBY.

but the nucleus, which induces differentiation along a special
path. The natural outcome of this suggestion is the word
“ sex-chromosome/’ Curiously enough, after examining many
hundreds of sections I have never seen mitosis in the germinal
epithelium. When I consider that I only found very few
cases of divisions of the primary spermatogonia, I am re-
luctant to make a statement which might be injudicious, but
I cannot overlook the fact that there appears to be abundant
evidence that the germinal cells divide amitotically, and no
evidence for mitosis. It was my desire to examine the cyto-
plasm of a germinal epithelial cell in mitosis, if it could be
found, for at this stage many bodies become revealed.

Any evidence that one might have for believing that all
nuclei in the germinal epithelium of the ovotestis are endowed
with the same potentialities — that is, for maleness, femaleness,
or for becoming a nurse-cell — and that this power is directed
in any one of the three channels by purely external or
environmental conditions, is somewhat hypothetical. The
favourite view, that abundant yolk cells (nutriment) affects
the decision, seems negatived by the fact that male pro-
germinative cells can, and do, appear in areas choked with
yolk. A number of cells stuck upon the germinal epithelium
seemed too big to be male cells, and their cytoplasm recalled
that of the spermatocyte. I was much puzzled by these, and
it has occurred to me that they might be intermediates
between spermatocytes and oocytes. They contained distinct
Nebenkern batonettes of the semi-lunar type, and mito-
chondria of a fine nature, but not flocculent. The cells were
much larger than a full-grown spermatocyte, and were located
in a yolkless area of the epithelium. Could it be possible that
the indifferent cell is affected by stimuli sent forth by the
presence of yolk cells, by crowded spermatogonia, and by the
general condition of that area in which it lies ? It seems
certain that the matter is complicated, and cannot be reduced
to the bald statement that oocytes appear because of abundant
nutrition.

I think that there are a variety of conditions which act on

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 601

the cell ; the latter is, so to speak, hanging in the balance
when it begins to differentiate. If the stimulus is not definite
enough an intermediate might be formed, and later degene-
rate, as many cells do. For the moment I can think of
several possible sources of stimulation. Abundant and dif-
ferentiating male cells in the lumen might send the balance
towards femaleness. Absence of any cells in the lumen might
stimulate towards maleness. As a matter of fact, here often
enters a direct contradiction of the real facts. I have several
cases where a lumen, quite or almost empty, is producing either
male or female cells alone. It should be said, however, that
in the latter case the lumen wall still had some few yolk
cells.

Finally, I believe that the nucleus of the indifferent cell
may be stimulated by a variety of external agencies to tend
towards one sex, and that the nucleus is the cell organ
responsible for the differentiation of the plasmatic elements.
I conclude that the plasmatic elements do not influence the
nucleus in this matter.

d. The Different Sperm Generations in the
0 votestis.

The ovotestis lumen is continually giving rise to new sperm
cells. The conditions of nutriment alter so much that very
few of these generations arise under the same stimuli.

The number of times which the primary spermatogonium
divides, and the number of times the secondary cells continue
fission, depends on the nutrimental conditions of the lumen.
I think that the reasons for variation of the generations are
as follows :

(1) Spermatogonial divisions of variable number, leading to
a variation in Nebenkern and mitochondria. It is a fact that
such variation does arise through differing numbers of
divisions.

(2) The conditions of nutriment profoundly affect the
appearance of the plasmatic structures in spermatogonia or

VOL. 62, PART 4. NEW SERIES. 41

602

J. BRONTE GATENBY.

male progerminatives, and are probably instrumental in
checking or stimulating spermatogonial divisions.

(3) The nutrimental conditions of the wall producing a
male generation is hardly ever the same.

(4) Finally, it will be seen that the spermatogonium begin-
ning growth period may have had a very variable history.
This is demonstrated by the variation in not only its plasma,
but often in its nucleus.

It can safely be said that in hardly any other group are
the conditions under which the male cells arise so open to
variation.

Difference in the mitochondria of the various generations is
principally due to the fact that a much divided line of sper-
matogonia contains but few mitochondrial granules. When
growth begins, in every case it seems that the mitochondria
increase in number by dividing ; but there is some difficulty
in observing this, though one knows this process must happen
by the fact that spermatogonia have fewer mitochondria than
spermatocytes, and the question is not merely a matter of
increase of size of the granules in the growth period. Thus
the spermatogonium, which has, so to speak, originated at the
tail end of a large number of divisions, starts out on growth
with fewer mitochondria. These divide, but never give rise
to the same number . of granules as one finds in other “less
divided” generations; but paucity in numbers is almost
always balanced by increased size of individual granules
(compare PI. 33, figs. 37 and 40).

In many of the drawings in this paper mitochondria are
shown as hollow spheres, while in others they look quite solid
(compare PI. 32, figs. 25 and 26). The most natural explana-
tion of this is, that for some reason the stain is more easily
extracted out of some granules than out of others, and that
all the mitochondria are really hollow spheres, though over-
stains tinge the medullary zone. To test this I took a slide
with “ solid ” mitochondria and extracted more of the stain,
examining it at intervals. The half of the slide which was
ever-differentiated showed most of the mitochondria as hollow

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 603

spheres. But not everywhere. Those parts where the mito-
ohondria still seemed solid were generally seen to have their
•cell elements slightly distorted or run together. I conclude
that there is good evidence for believing that the mito-
chondria of Helix consist of an inner, somewhat chromophobe
substance, and a cortical stainable area.

In Text-fig. 5 is drawn a diagrammatic scheme of the
derivation of the various cell elements in the ovotestis from
the indifferent epithelium. At S.P. 1 is the generation in
which no spermatogonial divisions occur, the cell dropping
into the lumen just when the growth stage has been entered.
At S.P. 2 is a progerminative cell, which only divides once.
Its elements may go on to the growth stage, or may (asatX.)
divide again several times. At S.P. 3 the much-divided
generation of spermatogonia is shown. At Y. the spermato-
gonia are still dividing, and have become very small. Accord-
ing as to the stage when the spermatogonial cells enter the
growth period their cytoplasmic bodies have various more or
less evident differences. The connecting link between S.P. 1
and S.P. 3 is provided by such a cell as that in S.P. 2, whose
divisions maybe curtailed. The diagram is based on evidence
deduced from observation of the spermatogonia, but there is
no manner of finding out how many times such a spermato-
gonium as that at S.P. 3, Y, has divided.

Below S.P. 3 are drawn the derivations of egg, yolk cell,
and follicle cell from the epithelium. The latter is not
proportionate in size to any of the cell elements in the figure.

It has been shown that the number and size of the Neben-
kern batonettes vary greatly. In some cases, such as the
generation marked in PI. 29, fig. 1, by Roman numerals, the
large number is never reduced by spermatogonial divisions,
since these do not occur. The spermatocyte, therefore, has
the original large number which appeared in the progermi-
native cell. It seems certain that the individual batonettes
do not increase in number before a spermatogonial division,
so that after such a division their number is halved approxi-
mately.

604

J. BRONTE GATENBY.

Text-fig. 5.

Scheme showing generations of spermatogonia ( S.P . 1, 2, and 3). At
X. generation 2 and 3 are interconnecting, at Y. are the much
divided, small spermatogonia. The four small cells to the right of
the spermatogonial generations are spermatids. Below are the
derivations of egg, yolk cell, and follicle cell from the germinal
epithelium ( G.E. ).

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 605

In the spermatogonia derived after many divisions the
batonettes ultimately form the rectangular figure drawn on
PL 34. They are fewer in number, but, as often happens
with the mitochondria, generally bigger, though not in-
variably (compare PL 32, fig. 24 a, and Pl. 33, fig. 36).

Curiously enough, in different examples the arclioplasm
seems to be variably demonstrable, and these differences do
not depend altogether on the depth of penetration of fixative,
and the consequent possibility of different degrees of staining.

Murray (8) considers that the archoplasmic and batonette
material are interconnecting, the rod being, as it were, a
thickened edge of the idiozome. This is apparently true in
some cases (see Text-fig. 4, V.A.R.). In others the batonette
is separate. 1 do not think that this exceptional apparent
intercommunication between idiozome and Nebenkern rods is
evidence in favour of the view that the batonettes are a part
of the spindle apparatus.

Summary.

(1) The ovotestis of Helix aspersa is formed of finger-
like diverticula. The latter are hollow at their lower ends,
which connect to the hermaphrodite duct, while the upper
ends contain more yolk, and are filled completely with meta-
morphosing male cells.

(2) According to the manner of derivation — that is, the
nutrimental conditions of the locality from which new cells
arise, and the number (if any) of times which these new cells
divide — there are quite wide differences in the individual
generations derived from and under such varying conditions.

(3) These differences are found in nucleus, mitochondria,
Nebenkern, and general cell volume.

(4) The mitochondria vary in size and number, and such
variation seems to be caused by the varying number of
spermatogonial divisions in different regions of the ovotestis.

(5) In the early spermatid smaller mitochondria, the micro-
mitochondria appear in an unknown way near the region
from which the axial filament takes its origin from the centro-

606

J. BRONTE GATENBY.

some applied to the nucleus. These micromitochondria are-
about one-fourth the size of the other, or macromitochondria.
No perceptible variation in size of the micromitochondria of
various generations has been found.

(6) The micromitochondria form the front sheath of the
sperm ; the hind region of the micromitochondrial sheath
intercommunicates with the macromitochondrial sheath, which
follows behind.

(7) The Nebenkern does not apparently become absorbed
into the substance of the mitochondrial sheath. A sloughing
off appears to take place.

(8) The minute cytology of the derivation of the sperms,
eggs, and nurse-cells is described.

(9) The determination of the sex of the indifferent cell
seems to be brought about by a variety of causes. The
explanation of femaleness by presence of yolk cells is held to-
be inadequate, for male progerminative cells also appear in
regions choked with yolk.

(10) The probable function of the Nebenkern is discussed.

Addendum A.

With regard to the body in the spermatids (PI. 32, fig. 24 a,.
and PI. 33, fig. 35) marked P.N.A. it has lately been found
that this structure is derived from a number of grains, which
in the case of Arion I have traced back to the young sperma-
tocyte. In Helix aspersa these granules could not be found
in the spermatocyte. P.N.A. stands for post-nuclear apparatus,
from the position of this structure. The latter is fully con-
sidered in a forthcoming paper.

Addendum B.

With regard to the cytoplasmic bodies in the egg, some
late papers by Schaxel (f Zool. Jahrb./ Bd. xxxiv, etc.) are of
interest. Schaxel claims that the nucleus emits chromatin
nto the cytoplasm at a brief period after the prophases of
the heterotypic division. He gives several stages (primary
chromasie, chromasie post emission, etc.) during which the

CYTOPLASMIC INCLUSIONS OF THE GERM-CELLS. 607

egg is being formed. He finds no cytoplasmic bodies till after
the bouquet stage, an obvious error of observation, and an
error committed by Miss Beckwith (‘ Journ. Morph./ xxv) as
well. His figures of emission are (in Aricia) merely late
stages in formation of the mitochondria (compare PL 30,
fig. 3 of this paper and Schaxel's PI. 16, figs. 9, 10, etc., in
his paper ‘ Zool. Jahrb / Bd. xxxiv). He overlooks the early
stages of mitochondria formation, while his description of
the “ extra-nuclear chromatin” is apparently in some of his
papers merely a misinterpretation of later stages of the
formation of the mitochondria.

Miss Beckwith, in a paper quoted above, throws doubt on
the “ Chromatin-emission” of Schaxel. Miss Beckwith says :
“ There is no evidence of formed material passing through
the nuclear membrane into the cytoplasm either early
(Schaxel) or late (Smallwood) in the growth period.” It will
be seen that Schaxel's work needs confirmation. To me this
observer's papers appear to be written in a partisan manner,
simply to bolster up the “ chromidia hypothesis ” in its appli-
cation to the Metazoa. For an excellent review of this
matter see Dobell, f Quart. Journ. Micr. Sci./ vol. 53.

Bibliography.

1. Gatenby, J. Bronte. — “ The Cytoplasmic Inclusions of the Germ-

Cells,” Part I, Lepidoptera, ‘ Quart. Journ. Micro. Sci.,’ No.
247, 1917.

2. Ancel, P. — “ Histogenese et structure de la glande hermaphrodite

d’Helix pomatia,” ‘ Arch, de Biol.’ t. xix, 1902.

3. Demoll, B. — “ Uber Geschlechtsbestimmung im allgemeinen und

uber die Bestimmung der primaren Sexualcharaktere im beson-
deren,” ‘ Zool. Jahrb.,’ xxxiii, 1913.

4. Buresch, Iw. — “ Untersuchungen liber die Zwitterdru.se der Pulmo-

naten,” ‘ Arch. f. Zellforch.,’ Bd. vii, 1912.

5. Schitz, Victor. — “ Sur La spermatogenese chez Columbella rustica

L.,” ‘ Arch. Zool. Exper.,’ t. lvi, 1916.

6. Tullio, Temi. — “ Condriosomi, idiozomse formazioni periidiozomiche

nella spermatogenesi degli Anfibii,” ‘ Arch. f. Zellforch,’ Bd. xii,
1914.

608 J. BRONTE GATENBY.

7. Faure-Fremiet.— liltude sur les mitochondries des Protozoaires et

des cellules sexuelles,” ‘ Arch. d’Anat. micr.,’ t. xi.

8. Murray, J. A. — Contributions to a Knowledge of the Nebenkern

in the Spermatogenesis of Pulmonata,” ‘ Zool. Jahrb./ Bd. xi.

9. Lee, Bolls. — “ La Cellule,” ts. xi, xiii, xx, xxi, various papers on the

cytology of Helix.

EXPLANATION OF PLATES 29-84, 1

Illustrating Mr. Bronte Gatenby’s paper on “The Cytoplasmic
Inclusions of the Germ-Cells” : Part II, Helix aspersa.

Explanation of Lettering.

A. Acrosome (perforatorium). A.R. Archoplasm (idiozome). A.L.N.
Nucleus of Ancel’s layer (mesoderm). A. S. Astral sphere (from which
each aster arises). C. Centrosome. C.W. Cell wall. F.N. Follicle
nucleus. G.E. Germinal epithelium. M. Ordinary mitochondria
(macromitochondria). M 2. Smaller mitochondria (micromitochondria).
N.C. Nurse- (yolk) cells. N.K. Nebenkern (rods or batonettes). S.B.
Spindle bridge. S.G. Siderophilous granule. S.P.G. Spermatogonium.
V.A.C. Alveoli in substance of cytoplasm of egg. Later become filled
with yolk discs. X.N.K. Body thought to be Nebenkern rod. Y. Yolk
disclets. (Only in certain figures, for others see text.)

All figures drawn with camera lucida, paper at table level. Koritska
T’gth semi-apochromatic oil immersion and compensating eye-pieces were
almost invariably used. In most cases the figures are reduced. The
arrow points to the inside of the lumen.

F.W.A. Fixation in Flemming without acetic acid. C. Fixation in
Champy’s fluid. S. Bichromate osmic smear.

PLATE 29.

Fig. 1. — For an explanation see the text page 36. x 800.

1 In such a figure as that in PI. 32, fig. 24 a, the mitochondria seen at

a lower focal level have been drawn in palely, as is a usual cytological
convention. In PI. 33, fig. 33, the white line in each mitochondrial
grain represents the “ light.” Hollow mitochondria are as drawn in
PI. 33, fig. 40.

CYTOPLASMIC INCLUSION OF THE GERM-CELLS. 609

PLATE 30.

Fig. 2. — Germinal epithelium showing a cell of the mesoderm (Ancel’s
layer, A.L.N.) and three germinal nuclei. At X. and Y. are what are
probably attraction spheres. X 4250, F.W.A.

Fig. 3. — Oocyte lying in germinal epithelium, mitochondria beginning
to disperse (M.). X 2000, F.W.A.

Fig. 4. — Part of germinal epithelium showing yolk cells ( N.C. ), a
germinal nucleus ( G.E. ), and a spermatogonium (S.P.G.). X 2000,
F.W.A.

Fig. 5. — Upper part of an oocyte showing the Nebenkern ( N.K .) and
the dark mass of mitochondria just before dispersal in cytoplasm (M.).
x 2000, F.W.A.

Fig. 6. — Bouquet stage of a supposed oocyte. This cell was deeply
embedded in yolk like that in PL 29, fig. 1 a or b. x 4250, F.W.A.

Fig. 7. — Another oocyte showing mitochondria (M.) dispersing and
yolk being deposited (Y). x 2000, F.W.A.

Fig. 8. — Represents the two left-hand bodies marked X. in PI. 31,
fig. 18. x 4250, C.

PLATE 31.

Fig. 9. — Oocyte showing dispersal of mitochondria from a centre (c)
near the nucleus. X 650, C.

Fig. 10. — Oocyte just after the stage drawn in PI. 30, fig. 6. X 4250,
F.W.A.

Figt 11. — Supposed oogonium embedded in yolk cells like that in
PI. 29, fig. 1 a or b. x 4250, F.W.A.

Fig. *12. — Oocyte at time of dispersal of flocculent mitochondria,
showing several bodies ( X.N.K .) supposed to be Nebenkern elements
X 2000, F.W.A.

Fig. 13, 14, 15, and 16, several stages in the development of the
•cytoplasm of the egg. The upper white area in each figure represents
the nucleus. Fig. 13 was drawn from the same egg as that in fig. 9 ;
this was about 100 n in length. Egg in fig. 14 was 140 /x. Fig. 15 about
150 /*. Fig. 16 about 120 /x. X 2000, F.W.A. and Champy.

Fig. 17. — Male progerminative cell showing development of mito-
chondria. X 4250, C.

Fig. 18. — Oocyte near end of growth stage, to show the location of
the bodies drawn in PI. 30, fig. 8, at a higher power. X 510, C.

Fig. 19. — Male progerminative cell showing appearance of cloud and
granules, x 4250, C.

610

J. BRONTE GATENBY.

Fig. 20. — Later stage. Archoplasm ( A.B .) and Nebenkern rodlets of
fairly rare type. Usual sort is shown in PI. 32, fig. 23, and appears,
later. Z. Zone of cytoplasmic activity which has spread around the-
nucleus. x 4250, C.

PLATE 32.

Fig. 21. — Bouquet stage of generation drawn in the two preceding-
figures. M. Mitochondria. X 4250, C.

Fig. 22. — Second maturation division showing probable Nebenkern
rods ( X.N.K .) in cytoplasm mixed with mitochondria, x 4250, C.

Fig. 23. — Later stage, showing appearance of Nebenkern ( N.K .) and
dispersal of mitochondria ; at X. the cell is losing its place on the
germinal epithelium. X 4250, C. (Compare Text-fig. 4, i.)

Fig. 24. — Secondary spermatogonium showing Nebenkern (N.K.) and
mitochondria, x 4250, F.W.A.

Fig. 24 A. — Spermatid with ring-like Nebenkern (N.K.) and large mito-
chondria. x 4250, F.W.A.

Fig. 25. — Spermatocyte near end of growth showing Nebenkern with
many curved rods. X 4250, C.

Fig. 26. — Spermatid with largish mitochondria and small curved
batonettes in Nebenkern. Nucleus has large number of nucleolL
X 4250, C. (For P.N.A. see Addendum A.)

Fig. 27. — Spermatocyte in prophases. Ring-like Nebenkern rods
scattered somewhat haphazardly. At X., X., region of centrosomes.
X 4250, F.W.A.

Fig. 28. — Spermatogonial division with seed-like chromosomes^
X.N.K. Probable Nebenkern. x 4250, F.W.A.

Fig. 29. — Spermatogonium (secondary) with Nebenkern (N.K.).
x 4250, F.W.A.

PLATE 33.

(All F.W.A. x 4250.)

Fig. 30.— Bouquet stage, showing loops radiating towards Nebenkern.
Rather small example.

Fig. 31. — Growth stage of spermatocyte. Mitochondria dispersing.
Nebenkern at NK.

Fig. 32. — Near end of growth period. Nebenkern rods banana-
shaped. Between thirty and forty in number.

CYTOPLASMIC INCLUSIONS OP THU GKRM-CELLS. 611

Fig. 33. — Prophase showing astral body (.4.$.), which will give rise to
part of the spindle. Mitochondria at this stage often rod-like. Neben-
kern has lost its original disposition.

Fig. 34. — First maturation division metaphase, showing shape of
mitochondria and general difficulty of detecting Nebenkern rods.

Fig. 35. — Spermatid with small mitochondria and straight or slightly
curved Nebenkern rods. Nucleoli few in number. (For P.N.A. see
Addendum A.)

Fig. 36. — Spermatid of same generation.

Fig. 37. — Later spermatid showing collapse of Nebenken structure.

Fig. 38. — Probable nurse-cell showing nucleus and cytoplasmic bodies.
No yolk disclets remain (or have been formed). The exact history of this
cell is difficult to make out (see p. 591).

Fig. 39. — Spermatogonial division with few large mitochondria.
Compare PI. 32, fig. 28.

Fig. 40. — Spermatid with ring-like Nebenkern rods and large mito-
chondria.

PLATE 34.

Figs. 41, 42, 43, 44, 45, and 46. x 2000.

Figs. 40 to 45 stages in the formation of the sperm.

Fig. 46. — Sperm from smear drawn to scale of the foregoing figures

(S-).

Fig. 47. — Spermatid X 2000, showing manner in which axial filament
grows in cramped quarters. The micromitochondria keep their definite
position.

Fig. 48. — Spermatid nuclei X 2000, showing variations found in
chromatin nucleoli. These hold good without much variation for every
nucleus in the bunch of spermatids.

Fig. 49. — Male progerminative cell (primary spermatogonium) of the
generation drawn in Text-fig. 3, ii, S.P.P. X 4250.

NOTE ON DEVELOPMENT OF TRICHOGRAMMA EVANESCENS. 613

Note on the Development of Trichogramma
evanescens.

By

J. Bronte Cratenfoy,

Exhibitioner of Jesus College, Oxford.

The purpose of this short note is to correct some errors
which were overlooked in a recent paper by me. These
mistakes, which are in my text references to the figures of
the development of Trichogramma, are as follows, and occur
in my paper on “The Embryonic Development of Tricho-
gramma Evanescens, a Monembryonic Egg Parasite of
Donacia Simplex,” (‘ Quart. Journ. Micr. Sci./ vol. 62r
part 2, February, 1917).

Page 149, line 9, “ Compare p. 20 ” should be “com-
pare p. 168.”

Page 158, line 24, “fig. 20” to be “fig. 26.”

Page 161, line 21, “fig. 8” to be “fig. 14.”

Page 1 62, line 15, “ fig. 12 ” to be “ fig. 13.”

Page 164, line 1, “ varies little ” to be “ varies a little.”
Line 6, “ fig. 12 and fig. 13” to be “fig. 13 and
fig. 14.”

Page 165, line 5, “ N” to be “ N.N.”

Line 30, “fig. 9 ” to be “ fig. 15.”

Page 166, line 20, “fig. 18” to be “fig. 24.”

Page 170, line 5, “ p. 30” to be “p. 178.”

Line 17, “ fig. 18 ” to be “ fig. 24.”

Line 19, “ N-.G.N” to be “ N.C.”

Line 20, “fig. 21 ” to be “fig. 27.”

Line 22, “fig. 22” to be “fig. 28.”

<614

J. BRONTlS GATENBY.

Line 23, insert the figures “12” after the letters
“ Pl.”

Page 1 7 1 , line 12, “ figs. 6 a and 6 b” to be “figs. 5 a
and 5 b.”

Line 16, “ figs. 15 a and b ” to be “ 5 a and 5 b.”
Line 25, “ p. 26 ” to be “p. 175.”

Page 179, line 28, “ p. 11 ” to be “ p. 159.”

Page 184, line 27, “Extended mass of cytoplasm” to
be “ Extruded mass of cytoplasm.”

In the “Lettering” insert “ E.X.N. means extruded
chromatin nucleolus.”

In connection with SilvestrLs excellent work on Oophthora
the Rev. J. Waterston, B.D., very kindly informs me that
Oophthora is a synonym for Trichogramma, so that the para-
sitic forms examined by Silvestri and by myself are different
species of the same genus.

INDEX TO VOL. 02,

NEW SERIES.

Actinian larval parasitic in a Rhizo-
stome, by C. Badham, 221

ALcyonium digitatum, develop-
ment of, by Annie Matthews, 43

Allis, E. Phelps, junr., on the homo-
logies of the muscles related to the
visceral arches of Fishes, 303

Allis, junr., E. Phelps, on the labial
cartilages of Raia clavata, 95

Amphioxus, collar cavities of the
larval, by K. M. Smith and H. G.
Newth, 243

A pyrene sperm formation of Moths,
by Gatenby, 465

Badham, Charles, on an ichthyob-
dellid parasite of the Australian
Sand Whiting, 1

Badham, Charles, on a larval actinian
parasitic in a Rhizostome, 221

Bathynella, morphology of, by W. T.
Caiman, 489

Caiman on the morphology of Bathy-
nella and some allied Crustacea,
489

Cartilages, so-called labial, of Raia
clavata, by E. Phelps Allis, junr.,
95

Centroplast of Heliozoon, by Clifford
Dobell, 515

Cephalodiscus (of the Cape), de-
velopment of, by J. D. F. Gilchrist,
189

Chromosomes of Culex pipiens,by
Monica Taylor, 287
Crustacea allied to Bathynella, by
W. T. Caiman, 489
Culex pipiens, chromosome com-
plex of, by Monica Taylor, 287
Cytoplasmic inclusions of the germ-
cells. Part II. Helix aspersa,
by J. Bronte Gatenby, 555

Development of Alcyonium digi-
tatum, by Annie Matthews, 43
Development of the Cape Cephalo-
discus, by J. D. F. Gilchrist, 189
Dobell, Clifford, on a new Heliozoon,
515

Earth-worms, pharyngeal gland-cells
• of, by Stephenson, 253

Fishes, muscles of visceral arches, by
E. Phelps Allis, junr., 303

Gatenby, J. Bronte, the cytoplasmic
inclusions of the germ - cells.
Part II. Helix aspersa, 555
Gatenby, J. Bronte, on the cyto-
plasmic inclusions of the Germ-
cells. Part I, Lepidoptera, 407
Gatenby, J. Bronte, degenerate sperm
formation of Moths, 465
Gatenby, J. Bronte, note on the sex
of a tadpole raised by artificial
parthenogenesis, 213

616

INDEX.

Germ-cells, cytoplasmic inclusions
of. Part I. Lepidoptera, by J.
Bronte Gatenby, 407

Germ-cells of Helix aspersa, by
J. Bronte Gatenby, 555

Gilchrist, J. D. F., on the develop-
ment of the Cape Cephalo-
discus, 189

Goodrich, Edwin S., on proboscis
pores in craniate vertebrates, 539

Harmer, Sidney F., on Phoronis
ovalis, 115

Heliozoon, a new, by Clifford Dobell,
515

Helix aspersa, germ-cells of, by |
J. Bronte Gatenby, 555

Homologies of muscles of visceral
arches of Fishes, by E. Phelps
Allis, junr., 303

Hypophysis and premandibular som-
ites of Craniate vertebrates, by
E. S. Goodrich, 539

Ichthyobdellid parasite of the Aus-
tralian Sand Whiting, by Charles
Badham, 1

Index to the f Quarterly Journal of
Microscopical Science/ July, 1888,
to July, 1916, inclusive, separately
paged in Part 1 of present volume.

Lepidoptera, cytoplasmic inclusions
of germ-cells of, 407

Lepidosiren and Protopterus,
early development of the spleen in,
by G. L. Purser, 231

Matthews, Annie, on the develop-
ment of Alcyonium digitatum
43

Nerve fibres, nuclei of, by Henry E.
Reburn, 217

Newth, H. G., and K. M. Smith, on
the collar cavities of the larval
Amphioxus, 243

Nuclei of nerve fibres, by Henry E.
Reburn, 217

Oxnerella maritima, nov. gen.
et spec., by Clifford Dobell, 515

Parasite of the Sand Whiting (Sil-
lago), by Badham, 1
Parasite, larval actinian, in a Rhizo-
stome, by C. Badham, 221
Parthenogenesis, tadpole raised by
artificial, note on, by J. Bronte
Gatenby, 213

Pharyngeal gland-cells of Earth-
worms, Stephenson on, 253
Phoronis ovalis, by Sidney F.
Harmer, 115

Proboscis pores in Craniate verte-
brates, by E. S. Goodrich, 539
Purser, G. L., on the early develop-
ment of the spleen in Lepido-
siren and Protopterus, 231

Raia clavata, labial cartilages of,
by E. Phelps Allis, junr., 95
Reburn, Henry E., on an easy way of
demonstrating the nuclei of nerve
fibres, 217

Smith, K. M., see Newth.

Sperm formation of Moths, 465
Spleen, early development of, in
Lepidosiren and Protopterus,
by G. L. Purser, 231
Stephenson, J., on the so-called
pharyngeal gland-cells of Earth-
worms, 253

Tadpole raised by artificial partheno-
genesis, note on, by J. Bronte
Gatenby, 213

Taylor, Monica, on the chromosome
complex of Culex pipiens, 287

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