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QUARTERLY JOURNAL
OF
MICROSCOPICAL SCIENCE.
EDITED BY
pir RAY LANKESTER, K.C.B., M.A., D.Sc., LL.D., F.RB.S.,
NONOKARY FELLOW OF EXETER COLLKEGK AND HONOKAKY STUDENT OF CHRIST CHURCH, OXFORD;
MEMBER OF THK INSTITUTK OF FRANCK (ASSOCIE ETRANGER DE 1’ACADEMIE DES SCIENCES) 3
CORKKEKSPONDENT OF THE IMPERIAL ACADKMY OF SCIKNCKS OF 8ST. PETEKSBUKG, AND OF THR
ACADKMY OF SCIKNCKS OF PHILADELPHIA, AND OF THE KOYAL ACADEMY OF SCIENCES
OF TURIN FORKIGN MEMBER OF THK KOYAIL SOCIETY OF SCIENCES OF
GOTTINGEN, AND OF THER KOUYAL BOUEMIAN 8SO0CIKTY OF SCIENCES, AND
OF THK ACADEMY OF THE LINCEI OF KOMK, AND OF THK AMERICAN
ACADEMY OF AKTS AND SCIENCES OF BOSTON . ASSOCIATE OF THK
ROYAL ACADEMY OF BELGIUM. HONORAKY MEMBER OF THK
NEW YORK ACADEMY OF SCIKNCES, AND OF THK
CAMBKIDGE PHILOSOPILLCAL SOCIKRTY, AND OF
YUK ROYAL PHYSICAL SOCIETY OF EDIN-
BURGH, AND OF THE
BIOLOGICAL SOCIKTY OF PAKIS, AND OF THR CALIFOKNIA ACADEMY OF SCIKNCES OF SAN FRANCISCO, AND
OF THK KOYAL ZOOLOGICAL AND MALACOLOGICAL SOCIETY OF BELGIUM;
COKKESPONDING MEMBER OF TUK SENKENBERG ACADEMY OF FRANKFURT-A-Mj3
FORKKIGN ASSOCIATK OF TILK NATIONAL ACADEMY OF SCIENCES, U.S., AND MEMBER OF THK
‘ AMERICAN PHILOSOPHICAL SOCIRTY 5
HONOKARY FELLOW OF THE ROYAL SOCIETY OF EDINBURGH;
LATE PIKKCTOK OF THK NATUKAL HISTORY DEPARTMENTS OF THE BRITISH MUSEUM, LATE PRKSIDENT oF THE
BKITISH ASSOCIATION FOR THE ADVANCEMENT OF SCIENCR; LATK FULLRKIAN PRKOFRSSOR OF
PHYSIOLOGY IN THK ROYAL INSTITUTION OF GREAT BRITAIN,
LATK LINACKK PROFPKSSOR OF COMPARATIVE ANATOMY AND FELLOW OF MERTON COLLEGRK, OXFORDS
EMEKITUS PROFESSOR OF ZOOLOGY AND COMPARATIVE ANATOMY IN UNIVERSITY COLLEGEK, UNIVERSITY OF LONDON
WITH THE CO-OPERATION OF
SYDNEY J» HICKSON, McA. FR
BEYRR PROFKSSOK OF ZOOLOGY IN THK UNIVERSITY OF MANCHKRTKK,
E.. A. MINCHIN, M.A., F.RS.,
PROFESSOK OF PROTOZOOLOGY IN THE UNIVERSITY OF LONDON;
GILBERT C. BOURNE, M.A., D.Sc., F.R.S.,
LINACKEK PROPRKSSOK OF COMPAKATIVE ANATOMY, AND FKLLOW OF MERTON COLLEGE, OXFORD 5
D
J. GRAHAM KERR, M.A., F.R.S.,
KEGIUS PROFESSOR OF ZOOLOGY IN THK UNIVERSITY OF GLASGOW,
VOLUME 60.—New SeErRIEs.
| a
LONDON:
J. & A. CHURCHILL, 7, GREAT MARLBOROUGH S
1915.
‘REET.
CO NEE NTs:
CONTENTS OF No. 237, N.S., APRIL, 1914.
MEMOIRS:
On the Anatomy of Conus tulipa, Linn., and Conus textile,
Linn. By H. O. N. SHaw, B.Se., F.Z.S. (With Plates 1-6 and
12 Text-figures) 1
On the Relation between the smruchure and bie Denelontent of
the Centrifuged Egg of the Frog. By J. W. Jenxinson, M.A.,
oD
D.Se., University Lecturer in Embryology, Oxford ; Fellow of
Exeter College. (With Plates 7-12 and Text- Gears 1-18) GE
The Sporogony and Systematic Position of the Aggregatide. By
Hewen L. M. Prxetu-Goopricn, B.Sc., Beit Memorial Research
Fellow. (With Plate 13) . : : : 5 BY:
CONTENTS OF No. 238, N.S., JUNE, 1914.
MEMOIRS :
The Blastocyst and Placenta of the Beaver. By ArrHur WILLEy,
F.R.S., Strathcona Professor of Zoology in McGill University,
Montreal. (With Plates 14-21 and 6 Text-figures) . 5 ies
On Acrossota liposclera, a New Genus and Species of Alcyo-
narian with Simple Tentacles. By GiLtBerr C. Bourne, M.A.,
D.Sc., F.R.S., Linacre Professor of Comparative Anatomy and
Fellow of Merton College, Oxford. (With Plate 22) . . 261
The Proboscidian System in Nemertines. By Dr. Grrarpa
Wynuorr, Utrecht. (With 36 Text-figures) ; PAB?
CONTENTS OF No. 239, N.S., SEPTEMBER, 1914.
MEMOIRS :
Observations on the Gametogenesis of Grantia compressiz.
By Arruur Drnpy, D.Sc., F.R.S., Professor of Zoology in the
University of London (King’s College). (With Plates 23-26). 313
The Chromosome Complex of Culex pipiens. By Monica
Taytor, S.N.D., B.Sc. (With Plates 27 and 28 and 3 Text-
figures) ; : E ; > oat
iV CONTENTS.
PAGE
Studies on Avian Hemoprotozoa: No. III.—Observations on the
Development of Trypanosoma noctue (of the Little Owl)
in Culex pipiens; with Remarks on the Other Parasites
occurring. By H. M. Woopncock, D.Se.Lond., Assistant to the
University Professor of Protozoology. (With Plates 29-31 and
1 Text-figure) . ; : ; : ;
Studies in the Experimental Analysis of Sex. Part 11.—On
Stylops and Stylopisation. By Grorrrey Smiru, M.A., Fellow
of New College, Oxford, and A. H. Hamm, Assistant in the
Hope Department, Oxford. (With Plates 32-35) : . 435
399
CONTENTS OF No. 240, N.S., JANUARY, 1915.
MEMOIR:
The Rat-Trypanosome, Trypanosoma lewisi, in its Relation
to the Rat-Flea, Ceratophyllus fasciatus. By E, A.
Mrncuin and J. D. Tuomson. (With Plates 36-45 and 24 Text-
figures) s : : . 463
Tire, INDEX, AND CONTENTS.
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. ]
On the Anatomy of Conus tulipa, Linn., and
Conus textile, Linn.
By
H. oO. N. Shaw, B.Sc., F.Z.8.
With Plates 1 to 6, and 12 Text-figures.
Since 1895, few workers on the anatomy of mollusca have
devoted their attention to the genus Conus. Inthat year Dr.
Bergh (8) published an extensive memoir on a large number
of species in this genus, and his work may be considered as
the most complete, and embracing the greatest number of
species examined, though his description of each species was
not exhaustive. ‘T'roschel (20) devoted most of his attention
to the radulz of the different genera and species of which his
excellent work is composed, and although he gives a certain
number of figures with descriptions of various anatomical
points, these latter are for the most part of rather a crude and
diagrammatic kind.
While malacologists have done a certain amount towards
working out and elucidating the anatomy of various members
of this genus, the conchologists, as is generally the case, have
produced many excellent monographs, and such names as
Reeve, Sowerby, Tryon, Weinkauff and others will always be
remembered for the general excellence of their figures and
descriptions of the numerous species which are contained in
this genus. Various writers have essayed different forms of
classification, but for the most part on purely conchological
grounds, and when more is known about the inhabitants of
these shells, and their different points of resemblance to one
vot. 60, PART 1.—NEW SERIES. 1
2 H. 0. N. SHAW.
another, whether they vary as much or more than their shells,
and if the conchological grouping also holds good from an
anatomical point of view, we shall be on the high road to
founding a solid and logical form of classification.
The two species with which I shall deal in this paper are
Conus tulipa, Linn.,and Conus textile, Linn. The speci-
mens of both species were females. The Conidz belong to
the order Prosobranchiata of the class Gastropoda, are
dicecious, and according to the most widely accepted form of
classification, Conus tulipais included in that sub-genus, or,
as some would have it, section, of the genus Conus, called
Rollus by Montfort, while Conus textile forms the type
of the sub-genus or section Cylinder, of the same author.
Rollus was first described by Montfort in 1810 (15, p. 395),
with Conus geographusas the type, and the sub-genus was
characterised as follows: “ Shell light, sub-cylindrical, spire
short but pointed at the summit, whorls slightly coronated,
aperture effuse, emarginate in front, columella smooth, outer
lip with a wide but not deep notch at the suture.”
This group corresponds to Nubecula of Klein, 1753 ; but
owing to his being pre-Linnean and non-binomial, this designa-
tion cannot be accepted. Utriculus, of Schumacher, 1817,
and Tuliparia, Swainson, 1840, are synonymous.
Cylinder was described on p. 391 of the same work.
“Shell sub-conic, smooth, spire elevated, pointed, whorls
numerous, body whorl ventricose, notched at the suture,
aperture effuse at the fore part.”’
Textilia, Swainson, 1840,is a synonym.
One of the difficulties to be contended with in working on
the anatomy of tropical molluscs is the trouble in getting
sufficient material, and in a good state of preservation. The
two specimens here described are from the British Museum
(Natural History), and had been there in spirit fora good many
years, and I had started working on these before some recently
collected specimens came to hand.
My thanks are due to J. Hornell, Esq., Pearl and Chank
Fisheries, Tuticorin, for sending me specimens which I have
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 3
not yet worked out, also to E. A. Smith, Hsq., I.8.0., of the
Natural History Museum, for kindly allowing me to use some
of the Museum specimens. My especial thanks are due to
Prof. G. C. Bourne, Merton College, Oxford, for his kindly
help and advice on many points connected with this paper, for
which I am much indebted.
With the exception of Conus mediterraneus, which is
found all round the Mediterranean and west coast of Spain,
no other species inhabit European waters, though this large
genus is plentifully represented in the tropical seas, and round
the coasts of Australia, Japan and America. It generally
lives in fairly shallow water, and is found on reefs and in pools
under stones, corals, etc., and is supposed to be able to inflict
a poisonous bite. I have made inquires from those who have
collected and handled them alive. They tell me that they
have never had this experience.! The animal is extremely
timid ; on the slightest touch it withdraws itself into its shell,
and will remain in this retracted condition for a considerable
time.
The operculum is generally elongate or unguiform, and so
small that it is useless for closing the mouth of the shell when
the animal has withdrawn itself inside.
The shells are covered with yellowish periostracum, which
in some species is only a thin, smooth, transparent, but tough
coating. In others, as in C. tulipa, the periostracum is
exceedingly thick and of a dark-brown colour. It is rough,
furrowed longitudinally, and of a leather-like texture, and has
tufts or outgrowths disposed in even rows along its surface.
When dry, this thick periostracum becomes very brittle and
peels off the shell.
No doubt this shell covering, which, as I have said, in some
species is tufted and of a leathery formation, is both a
protection to the shell and also a form of protective colora-
tion for the animal. C.tulipa has one of the lightest and
' While this paper was in the press, a note appeared in ‘ The Nautilus,’
vol, xxvii, pt. 10, pp. 117-120, 1914, ‘‘ Poisoning by the Bite of Conus
geographus.”
4. H. O. N. SHAW.
thinnest of shells, and at the same time one of the thickest
and most tufted periostraca. As the animal grows, and
outer whorls are formed surrounding the inner ones, the walls
of the internal convolutions of the shell are so reduced in
thickness as to be hardly as thick as a sheet of paper, and
semi-transparent.
Description oF Conus TEXTILE.
Maximum length of shell 24} in., maximum breath 1¢ in.
Operculum long and narrow, and slightly reflected outwards
Trxt-Fic. 1.
inv:
Conus textile. The siphon, with one side reflected to show
ridges and furrows, and invagination (cnv.).
at its anterior end. The foot, which is large and muscular,
when withdrawn completely fills the opening of the shell.
Along the internal edge of the foot (next the columella) is a
row or ridge of large tubercles or nodules of a reddish-brown
colour. he opening of the branchial cavity extends along
the right-hand side of the animal, and the cavity is enclosed
on its dorsal surface by a thin covering of membrane which
along its outer or free edge is considerably thickened and
muscular for a space of about Jin. This covering is attached
at its posterior end to the body-wall, and has no attachment
forward till it reaches the siphon, round which itisfused. It
extends forward as a kind of flap beyond its point of attach-
ment round the siphon, and overhangs the latter about + in.
The siphon (Text-fig. 1)"is large, and has thick muscular
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 3)
walls which are folded over and form a funnel, open in front
and beneath. The funnel isin the shape of a triangle, with its
base above, and apex ventral, the apex being formed by the
two free edges of the siphon, which run parallel and slightly
to the right of the foot. The interior and dorsal surface of
the siphon has a number of deep grooves and ridges running
across and at right angles to its longitudinal axis, and also
extending down its sides. They start near the anterior and
free end, and continue about half its length backwards. At
its junction with the body, the ventral edge of the left-hand
fold of the siphon is sharply reflected upwards and at right
angles into the funnel, forming a V-shaped invagination or
elbow (wv.), which closes up more than half the passage, and
fits into a depression in the foot.
The eyes are situated on the tentacles, about the middle,
and on the outer side, this position being due to the tubercles
on which they were borne having fused with the tentacles.
The anterior end of the foot has a glandular groove running
at right angles and across it, with a reflex fold above the
groove. About a quarter of an inch behind this groove, and
on the ventral surface, and equi-distant from both sides of
the foot, is the orifice of the pedal gland, or pedal sinus.
When, as in PI. 1, fig. 1, the branchial cavity (br.c.) is opened
from above, with the siphon and buccal openings turned away
from the observer, by cutting through the mantle (ml.)
longitudinally and about three quarters of an inch to the left
of the branchial opening, the ctenidium (ct.) is seen starting
just behind the opening of the siphon (sz.) into the branchial
cavity, running backwards and across the latter in a curve to
its dorsal attachment to the mantle, with the “ fausse-
branchie” on the right, and inside the ctenidium. ‘The
“ fausse-branchie,” or osphradium, is trifid, the result of
specialisation from the more archaic form, where the osphra-
dium is merely a filiform epithelial ridge. This specialisation
is common to most of the Gastropod Toxoglossa.
Below the large branchial cavity, and separating it from
the body-cavity, is a thin membrane or covering, which
6 H. O. N. SHAW.
gradually becomes thicker on each side, where it is attached
to the body-wall. Immediately beneath this membrane, and
at the posterior end of the body-cavity, lies a large yellowish
mass, the poison gland (p.g.). Anterior to this latter are the
numerous coils of the duct (p.g.d.) leading from the gland
into the cesophagus (w.); and anterior again to these and
over-lapping them is the radula-sac (7.s.). The cesophagus
is continued past the radula-sac and so to the mouth (m. h.).
The duct of the poison-gland enters the cesophagus close
behind the opening of the radula-sac, and passes backwards
and to the right of the body-cavity, where it is twisted into a
large coil. After leaving this coil it runs forwards and
downwards parallel to the cesophagus, both it and the latter
organ being surrounded by the nerve collars. Having passed
backwards through the nerve collar, the duct is composed of
very numerous and tightly twisted coils and knots, which are
situated in front of and under the poison gland. The duct
then straightens, passes to the right across the body-cavity,
and enters the gland at its right-hand extremity. For about
half an inch before the opening into the gland the duct is
much constricted (PI. 1, fig. 2). The whole of the poison duct
is firmly bound together by connective tissue, which also sur-
rounds the nerve collars and nerves given off from it, and
binds all these organs tightly to the cesophagus. ‘The poison
gland (PI. 1, fig. 2, p. g.) isa long narrow mass, nearly circular
in section, pointed at each end, and slightly curved. It lies
directly across the body-cavity with its two curved ends
pointing downwards.
It is impossible to completely straighten out the numerous
kinks and twists of the poison duct (PI. 1, fig. 2) and so measure
its length accurately, 270 mm. being as near as possible correct.
The length of gland is 17 mm. and maximum width 5°5 mm.
The length of the duct is about five times the total length
of the animal, and the duct and gland together occupy the
greater part of the body-cavity.
The salivary gland (PI. 1, figs. 1, 2, s.g.) isa small yellowish,
rather oval-shaped body, situated to the left of the body-
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. a
cavity, and in front of the poison ducts. It is provided with a
pair of extremely fine thread-like ducts (s.d.), which open
into the right side of the gland, one above and the other
below. The lower one passes under the cesophagus, and the
other above, and both enter the base of the V of the radula-
TEXT-FIGS.
33. 4.
bo
The tops of teeth of Conus textile. bs. Barbs. 7.f. Row of
denticulations.
sac (7. s.), one above the other, as in the case of their gland-
openings. ‘These two ducts lie at right angles to the body axis.
The radula-sac is in the shape of a V; the right arm is
elongated and about twice the length of the left, and ends in
a cul-de-sac; the base is produced downwards and forms a
knob, the left arm being the opening into the cesophagus.
The long or right arm lies across the body-cavity and over
the cesophagus, and behind the nerve collars. It is joined on
the right by the left arm, which runs forward and downwards
8 H. O. N. SHAW.
where it joins the esophagus. The long arm is slightly
curved, and its length is 20 mm. The radula-sac is thick-
walled and muscular.
The radular teeth (Pl. 1, fig. 4) are long, thin-walled tubes
composed of chitin, and are very brittle, transparent, generally
Trext-FIGs. 5 and 6.
A view of the base of two teeth of Conus textile. b.a. Showing
basal attachments.
light yellow, but sometimes quite dark. When placed in
xylol to clear them for mounting purposes, they immediately
underwent various contortions, tying themselves in knots
and twisting into circles, etc. They are provided at their
anterior ends with a flat lance-like point (Text-figs. 2, 3, 4),
and on each side have a large and hooked barb (bs.), pointing
backwards. The teeth are slightly curved and the barbs are
placed one on each side of the curve, the one on the outside
of the curve being about equi-distant between the one on the
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 9
inside and the lance-point. The average total length is
8-10 mm., and the diameter at the base, above the attach-
ment,‘2mm. The attachment variesa good deal in shape, but
TEXT-FIG. 7.
Conus textile. The base of rostrum, proboscis, mouth and
esophagus. mh. Mouth, rm. rostrum, ps. proboscis (with
thick reflected lips), 0. .s. opening of radula-sac into esophagus,
e., 0.p.d. opening of poison duct (p.g.d.) into esophagus,
n.c. nerve collars (only one represented). The shortness of the
proboscis, and thick wall through which the radula-sac opens,
will be noticed. (Diagrammatic.)
is generally a swelling of the base (Text-figs. 5, 6, b.a.), and
thickened in parts. The points and barbs hardly vary at all.
The lance-points in the radulz I have seen, are not serrated
as shown by Bergh (8), tab. vi, figs. 143, 144,145. The
radular teeth are placed in two groups in the radula-sac with
their bases in the angle formed by the union of the two arms.
10 H. O. N.. SHAW.
They are so arranged that one group have their barbs in the
cul-de-sac of the long arm, while the points and barbs of the
other protrude beyond the radula-sac opening, and into the
cesophagus.
The teeth are fixed or anchored by means of an attachment
or ligament which is firmly connected to their bases, and also
to the wall of the radula-sac, but is of sufficient length to allow
each tooth to move backwards and forwards. The teeth are
placed in rows one behind the other for a short way up each
side of the radula-sac but are all of nearly equal length, and
are surrounded and connected with one another by a stout
layer of connective tissue.
The rostrum (Text-fig. 7, and Pl. 1, fig. 1, rm.) is non-
retractile, and forms what Gray (10) calls a veil ; it is thick-
walled, muscular, and longitudinally ridged. The open or
free end of the veil is provided with a single row of tentacles
(ts.), by means of which the animal can attach itself to its
prey. The posterior and internal wall of the veil is reflected
forward, and, with the mouth (mh.), forms a retractile
proboscis (Text-fig. 7, and Pl. 1, fig. 1, ps.). The edges of the
mouth or lips are thick, ridged and corrugated and reflected
internally (Text-figs. 7, 8). The retractile proboscis is in this
species short, and after passing through the posterior wall of
the veil, opens out into the cesophagus. The opening of the
mouth is in the shape of a funnel (‘T'ext-fig. 7), the outer edge
being formed by the thick lips, and as the cavity of the funnel
narrows down internally, so the walls get thicker.
The cesophagus commences behind the posterior wall of
the veil, where it immediately swells out, and as it increases in
size, the funnel opening inside becomes smaller and the walls
exceedingly thick and muscular, until at the base of the funnel
only a very small passage to the mouth is left. These thick
cesophageal walls end suddenly as though cut across at right
angles just in front of where the cesophagus is slightly con-
stricted owing to its being surrounded by the nerve-collars.
The walls now become quite thin, with the result that the
much constricted opening at the end of the funnel suddenly
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 11
opens out into a large cavity. The edge of this thick wall
forms a ring or lip round the opening which hangs down into
the cavity. The outside edge of the lip is reflected back-
wards, so forming a minute funnel. The radula-sac opens on
the right (0. 7. s.) in the thick-walled part, and the poison
duct (0. p. d.) opens immediately behind it on the same side
into the thin-walled cavity. Behind this last the cesophagus
TEXT-FIG. 8.
Conus textile. The proboscis and mouth showing thick
reflected lips. The rostrum has been removed. p.s. Pro-
boscis, mh. mouth, rv. s. radula-sac, p.g.d. poison gland duct,
e@. esophagus (see diagrammatic, fig. 7).
is shghtly constricted and the poison duct runs parallel and
close to it, and both are surrounded by the nerve collars
(n.c.). The cesophagus now expands into a large and thick-
walled dilatation which corresponds to the stomach; it is
nearly circular and passes backwards and dorsad of the liver
(Pl. 1, fig. 1, /. 1). Here it narrows down, descends over the
edge of the liver and turns with a sharp bend to the left, and so
to the anus, situated at the posterior and right end of the
recto-genital mass (7.g. m.). Where the stomach passes over
the liver there is a large depression on the dorsal surface of
the former, in which lies the left side of the poison gland.
12 H. O. N. SHAW.
The whole of the stomach, cesophagus and duct are pleated
or corrugated, with the plications running parallel to their
length, and the thick and deep ridges of the mouth are
simply a continuation of these ridges in the cesophagus
(Pls2, fig. 15).
HistToLoay.
On account of the age and state of its preservation this
specimen was not satisfactory for histological purposes. The
sections were very hard to cut on account of the preparation
being macerated, and were very difficult to stain. Various
stains were tried, and those which gave the best results were
Ehrlich’s hematoxylin and eosin, but even with this method
the nuclei were very badly defined, and in some cases were
unstainable.
Porson Guanp, oR GLANDE DE LerpBLEIn (PI. 2, fig. 9).
Down the centre of this gland runs a nearly circular canal
(cw.). The poison duct opens into this canal at the right-
hand extremity of the gland. The duct joins the gland in
front and at right angles, so that the opening into the canal
(Pl. 2, fig. 14, 0. p. d.) is in reality to the side of one of its
extremities and not at the end. A considerable portion of
the coils of duct lies under and so ventral to the gland.
The canal in the gland is lined internally with non-ciliated
epithelium (PI. 2, fig. 9, /.ep.}; this is surrounded by a thin ring
ot circular muscular-fibres (¢. m. s.), external to which is a
layer of longitudinal muscle-fibres and connective tissue
(J. m. s.). This is again surrounded by another layer of
circular muscular tissue (c.m.s.),of about a third the thickness
of the preceding one. The last or external layer (Ul. m. s.)
is about three times as thick as the other four layers taken
together, and is composed of longitudinal muscular fibres and
connective tissue. In the two layers of longitudinal muscles,
1. e. the external and the middle layers, the muscle-fibres,
though all running longitudinally and parallel to the length of
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 13
the gland, are disposed in bands of different depth and width
so that they converge and overlap one another (PI. 2, fig. 14).
These thick muscular layers forming the walls of the poison
gland serve to eject the secretion along the duct to its
opening in the cesophagus whenever its use is required.
Poison Duct (PI. 2, fig. 10).
The wall of this duct is built up of two layers: an external
layer (J. 1. m.) of longitudinal muscular fibres and connective
tissue, and an internal layer (l.c.m.) of connective tissue and
circular muscular fibres. The internal layer is about half the
thickness of the external one. The edges of these layers are
well defined and do not merge intooneanother. This internal
layer is again lined with a thick epithelium (ep. cs.), composed
of very elongated club-shaped cells having basal nuclei which
vary slightly in their position. The cells are filled with a
fine granular substance. Interspersed among the cells of the
epithelium are anumber of granular vesicles. The club cells,
which are extremely attenuated, are as much as ‘2 m. in length.
The average external diameter of the duct is “65 m. An
invagination of epithelium (nv. ca.) hangs down into the free
passage of the duct. ‘This passage or channel (ca.) along the
centre of the duct is of a very irregular shape, having deep
grooves or arms running into the epithelium.
The numerous coils of the poison duct are firmly bound
together by connective tissue.
SromacH anp (Hsopuacus (Pl. 2, fig. 15).
The stomach is nearly circular in section and the wall is of
considerable thickness. The exterior layer (J. 1. m.) is
composed of longitudinal intermingled with a few transverse
muscle-fibres and connective tissue. Beneath this layer and
running into it is a complex network of transverse and
circular muscular fibres (/.c.l.m.),intermingled with connective
tissue and a few longitudinal fibres. Between this last layer
and the thin coat of epithelium (J. ep.) lining the internal
14 H. O. N. SHAW.
surface of the stomach and cesophagus is a thick layer (J. 1. m)
of, for the most part, connective tissue and longitudinal muscle-
fibres. In this there are a certain number of circular and
transverse fibres disposed about the whole wall of the
cesophagus and stomach, forming a complex mass of muscular
fibres and connective tissue. These run to a certain extent
in layers, one outside the other, and in some places are more
distinct than in others, but have no definite margins, and
merge one into the other.
The epithelium is in a very macerated state, but is not
detached from the wall.
The projections or invaginations on the inner surface take
the form of rounded lobes or ridges and have no very deep
recesses between them.
The average external diameter of a transverse section is
2°75 mm., length4mm. Internal diameter of passage 1 mm.,
length 2°5 mm.
The sections were by far the worst of all, the stain hardly
taking, nuclei being invisible.
SALIVARY GLanp (PI. 2, figs. 16, 17).
This gland is a small yellowish-white looking body which
lies to the left of the cesophagus, and is connected with the
latter by two very fine and twisted ducts, which enter at the
base of the radula-sac. The maximum length of the gland is
approximately 4 mm.
The two ducts (s.d.) opening into it are lined with cubical
ciliated epithelium, and some little way after their entry into
the gland branch off into two smaller ducts. These ducts are
again split up into smaller ones, which in turn are divided up
into still smaller ducts, so that the whole of the gland has a
sponge-like appearance, and is composed of an extremely fine
network of glands and ducts. The two main ducts enter some
distance into the gland before receiving any branches.
In the ordinary way one would expect to find these minute
glands grouped together in little bunches which empty into
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 15
one duct, and on account of their resemblance to a bunch of
grapes are known as “acinous glands.”
In the case of Conus textile, however, this is not so; the
glands are unicellular, of various irregular shapes and sizes,
placed side by side, and each cell has a separate duct
(dt. op.), which empties into a larger one.
The cells are filled with a very fine granular substance
(g. c. c.), which is secreted by their lining, and discharged
through each individual duct (dt. op.) into larger ones, and so
eventually finds its way through one or other of the main
salivary ducts to the cesophagus.
This system of grouped unicellular glands is extremely rare,
the acinous arrangement being much more common. Owing
to the inferior state of the preservation of this material | have
been unable to work out this point as minutely as I could
wish.
Circutatory System or Conus Textite (PI. 5, fig. 23, and
Pl. 6, fig. 24).
Owing to my researches on the nervous system, and one
specimen only being available, I have been unable to do much
with regard to the blood circulation and supply. There is
one artery (g.), the only one I have been able to follow out,
which passes from the heart under the cesophagus in an
oblique manner to the right. The artery is of considerable
size, is oval in section, attached to the cesophagus by connec-
tive tissue, and passes forward with it through the nerve
collars. The artery now leaves the under-surface of the
cesophagus, passes to the right and across, anterior to the
left pedal ganglion, to divide slightly to the right of the latter
into three large branches, forming a cross, with its arms at
right angles. The left branch (z.) passes to the base of the
siphon, the central one (y.) plunges down to the anterior and
central portion of the foot, while that on the right (p. ft.)
proceeds to the extreme posterior and dorsal surface of the
foot, along the right side of the latter, and only a short
16 H. O. N. SHAW.
distance below the external surface. About one quarter of its
length from its posterior extremity a branch emerges from
this last artery and passes downwards into the foot. There
are no branches given off from the other two arteries, and in
all cases they end abruptly. Just before the main artery
passes through the nerve collar formed by the pleuro-pedal
connectives, a right-angled branch is given off which is
directed upwards. After a short space this branch is again
divided at right angles, the one (j.) passing backwards. This
latter runs parallel to the main artery (g.), and both are con-
nected to the esophagus, and, together with it, are surrounded
by the pleuro-subintestinal collar. The branch artery, having
passed backward for a short distance beyond this last nerve
collar, is sharply reflected to the left and then turns forward
again, forming aU, which in section is flat, and closely
attached to the cesophagus. From its base and extremity
three small branches pass over the surface of the cesophagus.
The artery (w.) forming the ri, it arm of the T, bifurcates,
and its two branches are attached to the radula-sae on its
ventral surface. Anterior to the nerve collars a large artery
(c. ft.) proceeds from the main artery at the point where it
leaves the cesophagus, passes to the right, across and dorsal
to the pedal ganglia, and plunges into the centre of the foot,
giving it the appearance of a pedal nerve. The arteries, as
I have already stated, are in section oval or flat externally
(Pl. 5, fig. 23), and are composed of an external sheath of
areolar and circular muscular tissue, called “tunica adven-
titia” (/.c.m.), with an internal lining (tunica media) (l.¢.1.m.)
of circular and longitudinal muscular tissue. This last layer
is internally coated with a thin lining of endothelium (end.)
Description or Conus Tura (Pl. 1, fig. 1).
Maximum length of shell 2 in., maximum breadth 1+ in.
Operculum oblong, small, and thinner than in C. textile.
The foot is larger and wider, and the row of tubercles are
not present ; body-walls thick and muscular.
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 17
The siphon is the same as in C. textile, but has not the
invagination or elbow, and is not so deeply ridged.
The rostrum is the same as in C. textile, having thick
muscular walls which are corrugated and ridged throughout
internally. There is also a single row of tentacles at the open
end of the rostrum. This latter is in the form of a funnel
with the constricted open end in front. The walls forming
the base of the funnel are curved inwards to the centre,
where they form a free and retractile proboscis ( ps.) which
is about two thirds the length of the rostrum, the mouth (mh.)
being situated in the centre of the free end. The rostrum,
which is annulate, has grooves and ridges of muscle running
circularly round it on the outside, while internally these are
lined with strong muscle bands attached to its anterior end
and to its base, by means of which it can be exserted or
contracted. The cesophagus (e@.) runs from the mouth back
towards the stomach in the centre of the ring of muscle
bands. The muscular bands; are bound firmly to the walls
and to the cesophagus by connective tissue.
The cesophagus, on its emergence from the posterior end of
the proboscis, bends at right-angles to the right side of the
body-cavity ; it is then sharply reflected on itself (PI. 1, fig. 3),
becomes slightly larger, and passes back to the centre of the
body-cavity, where it is constricted, and again takes a sharp
turn to the right and back to the left. It now rapidly
becomes larger, and the radula-sac opens on the right side,
and the poison duct on the same side and slightly behind.
Here it is again constricted, and, together with the poison
duct, is surrounded by the nerve collars. The cesophagus now
opens out into the stomach, which has the form of a flattened
tube running from the centre backwards to the left, lying
under the poison duct and gland on the floor of the body-cavity.
The canal is again constricted, and passes over the dorsal
surface of the liver (PI. 1, fig. 1, /. /.), where it expands, and
passes downwards and backwards over the back of the liver
to the anus. The liver, a large brownish mass, lies on the left
of the body and under the ctenidium (ct.). Where the stomach
vou. 60, part 1.—NEW SERIES. 2
18 H. O. N. SHAW.
passes over the liver, it has the same depression as C. textile
on its dorsal surface; in this depression lies the left end of the
poison gland (pg.).
The ctenidium is situated on the inside and dorsal surface
of the mantle (ml.) ; starting in front and on the left side, it
runs across the branchial cavity (br. c.) and then backwards,
forming a semi-circle, lying above the liver.
The osphradium, or “fausse branchie,” is, as in C. textile,
trifid, being parallel to and on the right of the ctenidium, and
runs backward about half its length.
The eyes are placed on tubercles which are fused with the
tentacles in the same way as in C. textile, one on each side
of the rostrum on its dorsal surface and on the external edges.
These tentacles are situated about =, in. behind the opening
of the rostrum.
The radula-sac (PI. 1, fig. 3, 7. s.) differs from that in C.
textile in the fact that the left-hand arm of the V, which
opens into the cesophagus (#.) through a very constricted
passage, is much shorter than in the latter species. Moreover,
the right arm is curved in two places in the shape of an S,
is shorter and thicker, and the base of the V, instead of being
expanded so as to form a kind of bulb, in C. tulipa is very
much larger and forms a sort of triangular hood (h.). The walls
of the radula-sac are of about the same thickness and texture
in both species, with this exception, that the hood-like process
in C. tulipa is much thinner than the rest of the sac. The
right-hand arm of the radula-sac lies above and across the
cesophagus.
Immediately behind and above the radula-sac, and running
across the body-cavity, are the coils of the poison duct
(p. g.d.). These coils are much shorter and less twisted than
in C. textile (Pl. 1, fig. 3). Behind them the poison gland
(p.g.) lies athwart the body. The poison gland is about twice
the diameter but shorter in length than that of C. textile.
The poison duct enters the right extremity of the gland at
right-angles in the same way in both species (Pl. 2, fio. 14).
In C. textile it is constricted for about half an inch before its
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 19
junction with the gland, while in C. tulipa it is only con-
stricted just at its entry (Pl. 1, fig. 3). The poison duct pos-
teriorly to its opening into the cesophagus passes backwards
through the nerve collars, and forms a large coil on the left of
the body-cavity ; it then passes across and in front of the gland
to the right side, where there is another coil. From thence it
passes under the gland and enters on the right. The length
of duct after unravelling the coils is about 115 mm., or 2,°,
times the total length of the animal. In C. textile the
length of duct is 270 mm., or considerably more than twice
as long. The length of the gland is 15°5 mm., diameter
8&3 mm., as compared to 17 mm. and 5°5 mm. in C. textile.
The salivary gland (Pl. 1, fig. 3, s. g.) lies to the left of the
cesophagus and opposite the radula-sac. Its two fine and
crenulated ducts (s.d.) pass in the same way, one above and
one below the cesophagus, and enter the hood of the radula-
sac one on each side, and above the nervecollars. The gland
itself is larger than in C. textile.
This gland, called by Bouvier (5) the “glande impaire,” is in
this species in such a macerated condition that I have been
unable to obtain any sections sufficiently good to warrant
description.
There is considerable difference between the cesophagus
and proboscis in C. textile and those in C. tulipa, as can be
seen from the descriptions and figures of each. In C. tulipa
the proboscis, which is conical and annulate, is two thirds the
length of the rostrum ; the cesophagus runs down its centre,
and the mouth is simply an opening at its anterior end. In
C. textile the proboscis is quite short (Text-figs. 7, 8), and
the mouth is provided with thick reflected muscular lips. The
cesophagus is here simply a canal surrounded by thick
muscular walls; while in C. tulipa it isa free duct surrounded
by muscle-bands, which in turn are enclosed by the wall of the
proboscis (PI. 1, fig. 1), the ducts and muscle-bands only being
held together and to the internal wall of the proboscis by con-
nective tissue. Again, the distance in C. textile between
the mouth and the opening of the radula-sac into the cesophagus
20 H. O. N. SHAW.
is short (Text-fig. 7), about a quarter of an inch, and the
passage straight, In C. tulipa (Text-fig. 9) it is about four
Trxt-Fia. 9.
Conus tulipa. The rostrum, proboscis, esophagus and radula-
sac. mh. Mouth, ts. tentacles, rm. rostrum, ps. proboscis, @. @so-
phagus, r.s. radula-sac, 0.7.s. opening of radula-sac, o. p. d.
opening of poison duct (p. g. d.), m. ¢. nerve collars, h. hood.
The difference will be seen between this species and C. textile,
in the length of proboscis and length of esophagus between
base of rostrum and opening of radula-sac into the csophagus,
which is thin-walled. (Diagrammatic).
times as long, orabout oneinch. The duct isalso thin walled,
and twice sharply reflected on itself. Lastly, the constricted
funnel opening with its very thick walls suddenly emerging
into a large cavity, is, im C. tulipa entirely absent. The
radula-sac (0.7.s.) and poison duct (0. p. d.) in the latter species
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 21
simply open into the straight-walled cesophagus, which is here
rather dilated.
Troschel (20, pl. vi, fig. 142) gives a drawing of the radula-
sac, thick and short proboscis, and part of the cesophagus of
TExt-FIG. 10.
ba.
The base of two teeth of Conus tulipa in natural position show-
ing (ats.) attachments to wall of radula-sac. 6. a. basal attach-
ments, 7. ¢. row of denticulations on side of teeth, ats. tooth
attachment to sac-wall.
C. textile, which resembles my dissection of this species,
though the cesophagus as shown by him is of greater size than
I found to be the case in my specimen of the same species.
Whereas in C. textile (Pl. 1, fig. 2) the esophagus and
stomach for the greater part of their length are large, thick-
walled and almost circular canals, being only constricted
A He O. N. SHAW.
where surrounded by the nerve collars, in C. tulipa (PL &
fig. 3) the walls are thin, the duct is three times constricted,
and never more than a third of the width of the former. In
©. tulipa the canal is flat instead of round, and barely one
tenth of the diameter of that of C. textile.
The radula-sac (r.s.) is, as in C. textile, thick-walled
and muscular, but the length of the right arm is only 11 mm. as
compared to 20 mm. in that species, and I have already
given the difference in the shapes of the two sacs.
The teeth are placed in two groups, with the barbs or free
ends of one group in the cul-de-sac of the right arm, those of
the other group protruding through the esophageal opening
asin C. textile. At the base of each tooth, and firmly con-
nected to it, is a roundish muscular attachment (Text-fig. 10)
fixing the tooth to the wall of the radula-sac. Each attach-
ment is generally about half the length of a tooth, and curves
forward from its base in such a way that the other end is
fixed to the sac wall on the side, and anterior to the base of the
tooth. Asin C. textile, the teeth are surrounded and con-
nected together by connective tissue, and are generally hollow,
and of a dark yellow colour. The anterior end of each tooth
(Text-figs. 11, 12) is provided with a lance point, while on each
edge behind this point is a hooked barb (bs.) pointing back-
wards, the barbs, as in C. textile, being so placed that the
distance between the lance point and the first barb is the same
as between the latter and the barb behind it on the other side.
The point and barbs, though resembling those in C. textile,
are, however, not nearly as large or stout, and the whole tooth
is much straighter. The base (b. a.) is generally slightly
swollen, and the chitinous walls are thicker than the rest of
the tooth.
The teeth seem very constant in size and shape. On one
side of each, and on the flat surface of the lance-point, and
therefore at right angles to each of the two barbs, is a row of
stout hooked denticulations (r.¢.) with their points curved
backwards and directed to the base of the tooth. These den-
ticulations commence anteriorly about midway between the
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 23
two large barbs, and extend backwards in a row for about
one third of the total length of the tooth. The denticulations
vary in number from 15 to 25, and also in size in nearly every
tooth, though 20 to 22 seems about the average number. In this
respect the teeth of C. tulipa differ from those in C. textile,
Trext-Fies. 1] and 12.
12
The tops of teeth of Conus tulipa. bs, Barbs, rt. row of
denticulations.
as the latter have not got this row of lateral denticulations.
The average length of the teeth is 4 mm., or less than half
the length of those in C. textile (8 to 10 mm.) ; while their
diameter at the base above the attachment is the same in both
species, viz. ‘2 mm.
HistToLoey.
The state of this material, though better than that of C.
textile, was not of the best, the specimen having been in
24. H. O. N. SHAW.
spirit for some considerable time. The same stains were
employed as in C. textile, but again the nuclei were only
partially defined.
Porson GLAND.
As I have already stated, this gland occupies the greater
part of the body-cavity and lies across it, with its right end
slightly in advance. The canal runs down the centre of the
gland, and the poison duct enters it from in front and at right
angles at its right extremity in the same way as in C. textile
(Pl. 2, fig. 14).
The canal (PI. 2, fig. 7) (c. a.) is oviform, and lined through-
out its interior with a thin layer of epithelium (J. ep.).
The wall of the gland is very thick and muscular (PI. 2, figs.
6, 7, 8). It is composed of a deep external layer of longitu-
dinal muscular fibres (J. m.s.), which run parallel with the
length of the gland and form a thick sheath. Beneath this
layer is a very much thinner layer (c. m.s.) (about one-six-
teenth the thickness of the former) of muscular fibres running
round the gland. A third layer (J. m.s.), in thickness about
one third of the outer sheath, has the same composition as the
outer sheath of longitudinal muscular fibres, and again runs
longitudinally. Between this layer and the lining epithelium
of the canal is a very narrow ring of muscle-fibres (c. m. s.)
which run circularly round the gland. When compared to
C.textile the poison gland is twice the diameter ; the external
sheath of longitudinal fibres is also twice as thick, the second
layer is the same thickness, the third layer slightly deeper.
The fourth or internal sheath of fibres running round the
gland is about half the thickness, the epithelium is the same ;
the canal is one quarter the diameter, and oval instead of
round. The muscle-fibres are disposed in the same irregular
layers (PI. 2, fig. 6) as in C. textile.
Poison Ducr (PI. 2, fig. 11).
The condition of these sections is not at all good, as the
lining epithelium is very much macerated. The outer wall is
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 25
formed of a sheath of muscle-fibres (J. /. m.) which run parallel
to the length of the duct; this is lined by a layer of fibres
(l. c. m.) which run round the duct and are half the thickness of
the external sheath. This last layer has an internal lining of
epithelium (ep.cs.), which, as in C. textile, is composed of
elongated club-shaped cells with basal nuclei. The cells,
which are not as long or as fine as in the last species, are also
filled with the same fine granular substance. The epithelium
varies in length in different parts of the duct. The external
surface of the poison duct in C. textile is circular, while in
C. tulipa it is of a more irregular shape.
The whole of the centre of the duct is occupied by an irre-
gular invagination of the epithelium (inv. ca.), which hangs
down into the duct, and is connected by a restricted attach-
ment to the lining epithelium. There is thus formed, with
the exception of the place of attachment, an irregular
circular canal (ca.) between the lining epithelium and the
invagination. Inthe right side of this latter is a deep groove
which runs in a curve upwards and then down to the centre.
The average external diameter of the duct is “65 mm., the
thickness of the wall ‘05 mm. and the epithelial lining ‘02 mm.
In U. textile there is only a slight invagination hanging
down into the centre of the duct, but owing to the epithelial
lining being much thicker in this species (‘2 mm.), the area of
the canal is about one sixth of that in C. tulipa in spite ot
the invagination in the latter.
SromacH anp CKsopuHacus (PI. 2, fig. 13).
The cesophagus and stomach are in section oval externally
with thin walls. heir internal surfaces are very deeply
crenulated and pleated, forming large folds and furrows.
This internal ridged surface is lined with columnar epithelium
(l. ep.) of considerable thickness, which, as can be seen in
the figure, has become detached from the muscle and con-
nective tissue of the wall that surrounds it, this effect being
due to its contraction in spirit, and where they are still
26 H. 0. N. SHAW.
attached, the epithelium is distorted owing to its greater con-
traction and tendency to pull away from the outer layer. As
far as I can ascertain from the sections (and, as I have
previously said, the material is not of the best) the epithelium
is non-ciliated, but appears to have the striated border which
is characteristic of columnar epithelial cells. Embedded in
this epithelial layer are a considerable number of what appear
to be sporozoon parasites (sp. pa.). These are large, generally
round or oval bodies, scattered all over the epithelium and
buried in it at different depths. Some are so large as to
extend through the whole depth of the layer, but never
beyond into the muscle or connective tissue. In EKhrlich’s
hematoxylin they stain a deep purple colour, and in Heiden-
hain’s hematoxylin a greenish-brown. It is impossible to say
much about them with any accuracy, as they are extremely
difficult if not impossible to define. From their appearance
and the fact that they are clearly foreign bodies introduced
into the epithelium, there is little doubt that they are parasites
such as occur not uncommonly in Molluscs. The external
layer of the cesophagus (J. c. m.) is composed of circular
muscle-fibres which run round and ensheath it, intermingled
with connective tissue and a certain number of longitudinal
muscle-fibres. Beneath this layer, but very much confused
and mixed up with it, is an imperfect sheath of longitudinal
muscular tissue. Between this last and the epithelial lining
of the oesophagus is a thick but irregular layer of longi-
tudinal granular muscular tissue (. 1. m.), intermixed with
transverse muscles and connective tissue which bind the
epithelium to the cesophageal wall. In some places the
epithelium has an internal lining of a granular appearance,
while in other places it is wanting. No doubt this is a
glandular secretion or mucus derived from the epithelial cells.
The average external length of a transverse section is
16 mm. with width -75 mm., the length of the cesophageal
canal being 1°15 mm. and width °15.
In C. textile the external length is 4 mm. and width 2°75
mm., length of canal 2°5 mm., width 1:0 mm. It will thus
CONUS 'ULIPA, LINN., AND CONUS TEXTILE, LINN. 27
be seen that all the measurements are far greater in C.
textile than in C. tulipa.
In C. textile the wall of the cesophagus and stomach are
thick, while in C. tulipa they are comparatively thin. In
the former the canal is nearly circular, while in the latter it
is oval.
In C. textile the lining epithelium is much thinner, and
the internal surface, instead of being deeply crenulated and
ridged, has large curved projections of the wall into the canal.
The eyes, as I have already mentioned, are situated on the
internal edge of the tentacles, at the extremity of a small
outgrowth produced by the fusion of the tentacle and tubercle.
In both species they are identical, so I shall only describe that
of C. tulipa, which is in the best state of preservation. The
eyes are enclosed by a thin transparent membrane or outer
corneal layer (Pl. 2, fig. 12, co. 0.), which on each side of the
eye is continuous with the coat of epithelium (ep.) covering
the tentacle. Beneath the outer membrane is an inner
corneal layer (co. 7.), which is considerably thinner in front
than at the back of the eye and composed of epithelium.
This inner layer forms a hollow vesicle or eyeball, with a
transparent cuticular lens (/e.) occupying its interior. ‘lhe
optic nerve (0.), which is of considerable thickness, does not
enter the retina, but expands out over the back of the eyeball
and chiefly on the right side. As I have said, the inner layer
of cornea is much thicker at the back of the eye, and is so
modified that retinal cells (ret.) are formed in the epithelium,
directed inwards to the hollow vesicle. These retinal cells
run across the back of the eye, and round each side for a
considerable distance, extending forward most on the right.
These cells, which are embedded in pigment, are longest at
the back of the eye, and gradually get smaller as they advance
forward on each side, till they disappear posterior to the junc-
tion of the inner and outer corneal layers. The eye, which is
highly developed as is the case in most molluscs, contains no
points of special interest. ‘he eye is easily discernible with
the naked eye, appearing as a black spot on each tentacle.
28 H. 0. N. SHAW.
Nervous System or Conus TUtipa.
I found that of the two specimens under discussion
C. tulipa offered the most advantageous dissection for the
nervous system, and I therefore described and illustrated this
species first. As a complete description of the same system in
C. textile would be for the most part simply a repetition of
the former, though both have been equally carefully dis-
sected, I shall content myself in C. textile with pointing
out the differences which exist between the two species.
Description oF THE NERVE CENTRES OF Conus ‘I'ULIPA
(Pio; tis, 21),
There are thirteen nerve centres or ganglia, a right and
left cerebral (C.), pleural (Pl.), buccal (B.), and pedal (P.,
P. 1), aright (V. 1), left (V. 3), and median (V. 2) visceral,
one sub-intestinal (S7.) and one supra-intestinal ganglion
(S.). The ganglia with their nerves and connectives are of
a whitish-yellow colour, and may be brought into prominence
by treatment with osmic acid. The right and left cerebral
ganglia (C.) are round in shape and fused together on their
adjacent sides, and each is posteriorly united to the left and
right pleural ganglia (PI.) respectively, the right pleural
ganglion being again attached on its posterior side to the
supra-intestinal ganglion (S.). All these ganglia are ex-
tremely hard to define by reason of their junctions being
indicated only by very slight constrictions and the whole
being bound to the cesophagus by connective tissue. These
ganglia lie on the cesophagus, where it is slightly constricted
behind the opening of the radula-sac and poison duct. This
position is seen in Text-fig. 9 (w. c.), but as this figure is purely
diagrammatic, only one collar in the form of a ring, and
without any ganglia, is shown. ‘To the right, and slightly
posterior, are situated the pedal ganglia (P., P. 1).
These ganglia lie acoss the right side of the body-cavity
under the radula-sac with their anterior extremities pointing
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 29
slightly forward. Both ganglia are closely connected to-
gether along their internal sides, being only distinguishable
at both extremities, and more so at their anterior ends. The
ganglia are slightly pyriform or attenuated anteriorly.
A cerebro-pedal connective (ce. pl.) passes from the left
side of the left cerebral ganglion under the cesophagus, and
enters the left pedal ganglia (P.) on its dorsal and posterior
surface. The left pleuro-pedal connective (pa. pl.) runs
under the cesophagus and parallel to and behind the cerebro-
pedal connective, and joins the left pedal ganglion behind
the latter. In like manner a right pleuro-pedal and cerebro-
pedal connective passes over the cesophagus, connecting the
right pedal ganglion (P. 1) to the right cerebral and pleural
ganglia. The cerebro-pedal connective is smaller than the
pleuro-pedal. Posterior to this last connective, and having
their origins one in the left and one in the right pleural
ganglia, are the pleuro-subintestinal connective (d.’) and the
zygoneurous conective (z.) respectively of the visceral com-
missure (d.). The pleuro-subintestinal connective (d.’) issues,
as I have stated, from the left pleural ganglion behind the
left pleuro-pedal connective, passes in an oblique direction
backwards and under the cesophagus, and enters the anterior
and left extremity of the subintestinal ganglion (Sv.). The
zy goneurous connective (z.) issues from the same place in the
right pleural ganglion, passes backwards and over the ceso-
phagus, and enters the right anterior extremity of the sub-
intestinal ganglion. This ganglion is pyriform, its attenuated
posterior end forming the origin of the right loop of the
visceral commissure (P]. 4, fig. 19). The left pleural ganglion
terminates posteriorly rather bluntly, being little attenuated,
while the right ganglion, lying between the right cerebral
and the supra-intestinal ganglia, is hardly distinguishable.
This last ganglion, which is pyriform, is continued back-
wards, forming the left loop of the visceral commissure. ‘This
latter (d.), after leaving the ganglion, passes over to the left
of the body-cavity, and through the wall where is situated
the left visceral ganglion (V.3). This ganglion ends abruptly
30 H. O. N. SHAW.
posteriorly, the commissure passing backwards till it reaches
the median visceral ganglion (V. 2), which is placed across
the body, anterior to the recto-genital mass, and is attenuated
at both ends, The visceral commissure leaves the right
extremity, passing forward and to the right, where it jos
the right visceral ganglion (V. 1). This latter is pyriform
anteriorly, while posteriorly it is produced where connected
to a large nerve (k.) and the visceral commissure, the latter,
after passing forward for some considerable distance, joining
the anterior of the right visceral ganglion, with the posterior
extremity of the subintestinal ganglion. The commissure 1S
shortest between the median and right visceral ganglia.
From the anterior edges of both cerebral ganglia (PI. 3,
fic. 18, and PI. 5, fig. 21) issue the cerebro-buccal connectives
(c. b.). Both pass round the cesophagus, one on each side,
and join the external edges of the right and left buccal ganglia
(B.) respectively. These ganglia are small, circular, flat bodies
lying under the cesophagus. They are united by two com-
missures, but are so close together that they appear almost to
touch. The anterior commissure is of extreme fineness, while
the posterior, which is of considerable stoutness, is the origin
of the large buccal nerve (s. 5) which leaves the commissure
close to the Jeft buccal ganglion and passes backwards.
The cesophagus and the poison gland duct (PI. 5, fig. 21) are
completely surrounded by four nerve collars, with ganglia at
each extremity of the collars, the most anterior being formed
by the two cerebral and two buccal ganglia with their
connectives. This collar is the smallest and lies close round
the cesophagus, to which it is attached by connective tissue.
Behind this comes the cerebro-pedal collar, and posterior again,
the pleuro-pedal, these last two being larger and not so closely
attached to the cesophagus, round which they lie obliquely,
owing to the pedal ganglia being slightly behind the cerebrals
and buccals on the right. The fourth and last nerve collar
is the pleuro-subintestinal, and owing to the latter ganglion
being situated still further backwards, this last collar is more
oblique with regard to the cesophagus than the second and
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 31
third, but is smaller in circumference. The left half of this
collar, which is the stoutest, is formed by the pleuro-sub-
intestinal connective of the visceral loop (d.’), while the right
half is the zygoneurous connective (z.).
The nerves issuing from the various ganglia, with the
description of their functions and the parts they innervate,
etc., will be dealt with under the various headings hereafter
mentioned.
TENTACULAR AND Optic Nerves (PI. 3, fig. 18).
The tentacular nerves (¢.), or, as they may be called,
cephalo-tentacular, are the largest nerves given off from the
cerebral ganglia (C.).. They issue from the dorsal and
anterior surface of the ganglia, that from the right supplying
the right half of the rostrum, while the left side is supplied
from the left ganglon. This left nerve, after leaving its
origin in the ganglion, passes to the right, and is then sharply
reflected back to the left, where, after proceeding a short way,
a stout nerve (¢. 1) is given off from its anterior side, which
passes over to the left and is ramified, splitting up into
numerous fine branches. Slghtly anterior to the base of this
nerve the main tentacular nerve is bifurcated, both nerves
passing underneath it, the left branch (¢. 3) being ramified in
the same way as the preceding one. The right branch (¢. 2)
is subdivided into three, of which the median is the optic
nerve (0.), while those on either side of it are divided up into
numerous fine nerves like the other tentacular nerves.
The optic nerve, which, as has been shown, is only a branch
of the tentacular nerve, is stouter than the branch on each side
of it. When it reaches the place in the rostrum immediately
below where the eye is situated, it turns at right angles
through the rostrum wall and so to the eye.
The right tentacular nerve on leaving its ganglion passes
to the right and slightly forward, where, after a short course,
it is bent sharply backwards and then forward again, forming
a kink. In the left nerve this kink is close to the left
82 H. O. N. SHAW.
ganglion, no doubt the duty of these two kinks being to
allow a slight contraction or extension of the thick muscular
walls of the rostrum. After its flexion or kink a fine nerve
is given off on the inside of the right tentacular nerve, which
supplies the rostrum base, and anterior to this latter the main
nerve is bifurcated, the left branch again dividing into two,
the left of these two (¢.1) being similarly split up. Both
these last give off fine and very numerous branches on their
course towards the tentacles, as is also the case with the night
branch (¢. 3) at the first bifurcation of the main nerve. The
right branch of the second division of the main nerve (f. 2),
after proceeding a short way, gives off a fine nerve, while the
main nerve of these two, the left, is the optic nerve (0.), which
proceeds to the eye in the same manner as the left optic nerve,
and has two or three small branches issuing from it.
In his description of Conus virgo, Bouvier (5) says that
the left optic nerve is given off from the tentacular nerve of
the same side sooner than is the case on the right side. In
my dissection of C. tulipa the reverse is the case, but, hike
Bouvier, I have been unable to ascertain whether the optic nerve
is simply a branch of the tentacular, or whether, though both
are in the same nerve-sheath, they really issue as two distinct
nerves from the ganglia. The latter seems improbable, as in
the case of the left optic nerve there are stout branches on
each side supplying the tentacles, the optic nerve issuing from
between them. All the tentacular nerves are extraordinarily
ramified, splitting up and giving off very numerous fine
branches. The ends of the nerves which supply the tentacles
themselves, before entering the latter, are generally divided
into at least three branches. Both the main tentacular nerves
proceed from the ganglia under the base of the proboscis.
A most remarkable fact in connection with the nerve system
of this species is the following: From the posterior surface of
the base of the kink in the right tentacular nerve a stout
branch (¢.p.) issues; it rans backwards, and is much crenu-
lated. This latter joins at right-angles a nerve (7.) running
across the floor of the body-cavity, thus forming an inverted
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 33
T. The right branch is ramified, and supplies the base of the
rostrum on the right, while the left branch proceeds to the
dorsal surface of the right pedal ganglion, thus forming
a connective between the tentacular nerve and the right pedal
ganglion. This nerve, as far as I have been able to ascertain,
has not been noticed before, and its presence is hard to
explain.
There is no nerve or connective of any sort given off from
the left tentacular nerve until the latter is bifurcated, as I
have shown, the right branch passing forward to the rostrum
and cephalic teguments.
Acoustic Nerves (PI. 5, fig. 21).
These nerves are extremely fine and very hard to follow,
on account of the great number of pedal nerves through which
they pass. The origin of the left nerve (ac. /.) is in the left
cerebral ganglion, immediately below the left cerebro-pedal
connective, and between the latter and the lett pleuro-pedal
connective. This nerve runs between these two connectives
to the left pedal ganglion (P.) along the anterior side of the
latter, passing between numerous pedal nerves, then slightly
backwards, and reaches the left otocyst (ot. /.) situated some
little distance to the right and slightly posterior to the pedal
ganglia. The right acoustic nerve (ac. 7.) issues from the
right cerebral ganglion at about the same place as does the
left, but descends to the right pleuro-pedal connective, and
runs along its anterior edge till both reach the right pedal
ganglion (P.1). ‘lhe acoustic nerve, after following the
posterior surface of the latter, passes along the pedal nerves
backwards and to the right, till it reaches the right otocyst
(ot. r.), which is situated in the muscles of the foot, and at a
considerable distance from the pedal ganglia and behind them.
ProgoscripEaN Nurves (PI. 4, fig. 19).
The nerves to the proboscis, or labio-proboscidean nerves,
are three in number on each side, and issue from the anterior
vo. 60, pART 1.—NEW SERIES. 3
34 H. O. N. SHAW.
edges of the cerebral ganglia. The nerves are stout, of a
white colour, not deeply imbedded in the muscular wall of the
proboscis, and run parallel to the cesophagus and at about
equal intervals round it, the central one (/. 1) being on the
ventral surface. The two central nerves (J. 1) are the longest,
and have the fewest nerves given off along their length, and
are the finest and most crenulated of the three sets. They
run to the anterior end of the proboscis, and are here broken
up into a number of fine nerves. ‘The second pair (/. 2) are
straighter, and the left member of the pair gives off a large
branch about one third of its length from the ganglion, while
in the right nerve the branch is half-way. The left nerve of
the pair (/. 3) is trifurcated, the right one bifurcated, all
branches being much ramified. In section these nerves are
nearly flat and much crenulated.
The walls of the rostrum and cephalic tegument are supplied
by three pairs of nerves (/. 4—l. 6) as well as by branches given
off from the tentacular nerves. As was the case with labio-
proboscidean nerves, these nerves to the rostrum are in three
pairs, and have their origin in the anterior part of the cerebral
ganglia (C.). The three pairs are nearly the same size, but
are very much finer and shorter than the three pairs of labio-
proboscideans. As was the case in Bouvier’s dissection of
C. virgo, so in this species, the first pair of nerves (1. 4) go
to the base of the rostrum wall, where they are ramified and
run forward, while the other two pairs on the right (J. 5, J. 6),
which are slightly finer, pass from the cerebral ganglion across
the cerebro-pedal connective, and so enter the muscular wall
of the rostrum, where they run anterior to the first pair (1.4).
Before they enter the muscular rostrum wall, and between here
and their ganglia, the nerves (J. 5, /.6) are very sinuous, as is
the case more or less with all the nerves to the rostrum and
also the labio-proboscideans. This is due to the fact that
nerves, being non-extensile or retractile, or nearly so, when
they enter a wall which may be exserted or contracted, as in
the case of the proboscis, and slightly so in the rostrum, are
supplied with enough slack nerve between the ganglia and
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 309
their entry to allow the wall to be fully extended without
pulling on the nerve. When fully contracted or abnormally
sO, as in spirit, the nerves have a very sinuous appearance,
Buccat Ganaiia AND THEIR Nerves (PI. 3, fig. 18).
The buccal ganglia (B.) are small, circular, flat bodies which
lie under the cesophagus, and their external edges are joined
by a stout connective to the anterio-dorsal surface of the
cerebral ganglia (C.). The two internal and adjacent edges
of the buccal ganglia are connected by two sub-cesopha-
geal commissures, but the ganglia are so close together that
they almost touch, the commissures being about a sixteenth
of an inch long. The anterior of the two is extremely fine,
while the posterior one is as stout as the cerebro-buccal
connectives (c. b.). These two ganglia, therefore, with their
connectives, form a complete nerve collar round the cesophagus,
the buccal ganglia being slightly posterior to the cerebrals.
The openings of the radula-sac and poison duct into the
cesophagus are anterior to the nerve collar.
For the sake of comparison I have used the same letters as
employed by Bouvier in his figures. From the posterior
surface of the right buccal ganglion a nerve (s. 1) is given off,
which innervates the anterior portion of the poison duct, till
a branch is given off to the latter from the main poison-gland
nerve. There is no corresponding nerve given off from the
left ganglion. The buccal proboscidean nerve (s. 2), which is
anteriorly ramified, supplies the wall of the cesophagus in the
proboscis, the wall of the proboscis being innervated from the
cerebral ganglia by the labio-proboscidean nerves. Here, in
like manner, I have been unable to find any such nerve issuing
from the left ganglion, nor was I able to find the nerve
mentioned by Bouvier as being given off by the side and pro-
ceeding to the cesophagus.
Two pairs of nerves (s. 3, s. 4), one pair issuing from each
ganglion, supply the wall of the radula-sac, those given off
from the right ganglion supplying the dorsal surface of the
36 H. O. N. SHAW.
sac, while the two from the left ganglion supply the ventral
surface. The main nerve (s.5) which innervates the poison
gland and duct, issues from the posterior commissure, and runs
backwards under the cesophagus and through the nerve
collars. This nerve, which is of considerable size, after pass-
ing backwards for some distance curves to the right, where a
branch is given off which supplies the poison duct and takes
the place of the first fine nerve (s.1). The main nerve now
doubles back to the left and then again to the right, three or
four slender nerves being thrown off to the coils of duct
among which the main nerve runs. This nerve, after passing
to the right, is trifurcated, the three branches being ramified
and supplying the walls of the poison gland, the branch
to the right entering the right extremity of the gland
by the duct opening. After the main poison gland nerve
has passed backwards from the buccal commissure, a fine
nerve (s.6) is given off on the left, which runs backwards
along the wall of the cesophagus and is much ramified. The
existence of this nerve was indicated by Bouvier (5) p. 341,
where he says: “ Dans un individu femelle, il me sembla qu’un
filet gréle se rendait de ce nerf a Vcesophage, en arriére des
colliers nerveux.” About this point he was not certain, and
mentions the difficulty experienced in removing the connective
tissue which completely surrounds and binds firmly together
the nerves, the cesophagus, and coils of poison duct; this I
also found most troublesome, but managed to free the nerve,
and ascertained that this nerve (s. 6) does run from the main
nerve to the cesophagus. As stated by Bouvier, the main
nerve (s.5), on account of both its position and origin, would
correspond to the cesophageal-aortic nerve in Buccinum, if a
branch was given off to the cesophagus, and this I have shown
to be the case.
Lert Pievrat GaneLtion and Nerves Issuine (Pl. 4, fig. 19).
This ganglion is attached to the posterior end of the left
cerebral ganglion, the end of the one and the beginning of
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 37
the other being only distinguished by a slight constriction
between them. The posterior extremity of this pleural gan-
glion (Pl.) is directed slightly to the left, while the ganglion
is very slightly pyriform and attenuated posteriorly. On the
left, and from the external and ventral surface, a stout con-
nective (d.’), the pleuro-subintestinal connective of the vis-
ceral commissure, issues, turns to the right, across the floor
of the body-cavity, runs slightly backwards and under the
cesophagus, and enters the ventral and anterior end of the
sub-intestinal ganglion (S7.).
Two columellar nerves (¢. 1, 7. 2) are given off from the
centre of the ventral surface of this pleural ganglion, while
none are present in the corresponding right ganglion. The
nerve on the right (7. 2) passes backwards and slightly to the
right, being much crenulated and unattached to the body-
cavity till it splits into four fine nerves, which diverge in the
columellar muscle. ‘The left of this pair (¢. 1) is twice the
size of the former, being sinuous or crenulated in the same
way, and runs directly backwards, being unattached to the
wall for some considerable distance. Shortly after its entry
in the wall it is bifurcated and descends almost vertically
into the base of the columellar muscle on the right, and
about level with the left visceral ganglion (V. 2). Both
nerves pass under the cesophagus, the left one running
parallel with it, and supply the anterior and left half of the
columellar muscle and base of the body-cavity. These two
nerves differ from those in Conus virgo, described by
Bouvier (5), in the following respects; they are shorter, supply
the columellar muscle anteriorly and to the left, instead of
posteriorly and on the right, do not pass through the nerve
collars or go anywhere near the right pleural ganglion or the
sub-intestinal, but are absolutely distinct and run directly
backwards from their origin.
Four nerves are given off from the postero-dorsal edge of
the left pleural ganglion, two being parietal, the other two
being the main and lesser pleuro-siphonal nerves.
The most anterior of these four (/. 1) is the lesser pleuro-
38 H. 0. N. SHAW.
siphonal nerve, the second (f.), the main pleuro-siphon, the
third and fourth (c. and c. 1) being parietal. All these four
nerves, after leaving their origins in the ganglion, pass directly
to the left, being suspended and quite unattached for some
considerable distance between the ganglion and their entries
into the base of the body-wall. The lesser pleuro-siphonal
nerve passes through the base of the rostrum and at once
runs downwards over the attachment of the base of the
siphon to the base of the rostrum. When it reaches the
siphon proper it gives off two or three small branches and
passes forward along the left siphonal wall. The main
siphonal nerve (f.), which is very much larger, runs parallel
but posterior to the former till it reaches the base of the
siphon, where it proceeds along the centre of the channel
and gives off fine nerves which run into the walls on
both sides. When one third of the length from the anterior
extremity of the siphon, the main nerve is trifurcated, each
branch ramifying in the sides and extremity. At its entry
into the base of the siphon, a stout anastomosis (a.) connects
it with the anterior branchial nerve (0.).
The parietal nerve (c.) enters the floor of the body-cavity
.on the left, just behind the main pleuro-siphonal nerve, giving
off fine branches to the floor and wall. The other nerve of
this pair (c. 1) issues from the ganglion behind the former,
and after a short distance passes forward under it, and also
beneath both the pleuro-siphonal nerves, entering the body-
wall anterior to the foregoing. Both parietal nerves are by
far the finest of the four nerves given off from this ganglion.
SUPRA-INTESTINAL GANGLION AND Nerves (P]. 4. fig. 19).
This ganglion (S.), which is closely connected to the
posterior extremity of the right pleural ganglion, serves as
origin for six nerves. There is hardly any constriction at
the junction of the two ganglia, which are, therefore, hard to
distinguish in a dissection. ‘I'he supra-intestinal ganglion is
pyriform in shape with its posterior extremity directed slightly
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 39
to the left. Bouvier (5) mentions only four nerves as coming
from this ganglion; of the two additional nerves that I have
recognised, one is only a parietal nerve and of little or no
importance ; the other will be mentioned in due course.
There are, then,as I have said, six nerves from this ganglion,
two being parietal, two branchial, one, the largest, the supra-
intestinal branch of the visceral commissure, and the last
supplying the inner edge of the branchial cavity. All these
six nerves lie above the two columellar nerves (7. 1, 7. 2).
All the nerves issue from the posterior extremity and
dorsal surface of the ganglion, the most anterior being the
main branchial nerve (b.), which is the stoutest of the six with
the exception of the supra-intestinal branch of the visceral
commissure.
The main branchial nerve (b.), after leaving the ganglion,
passes slightly backwards and to the left of the body-cavity
where it enters the wall running forward and parallel to the
pleuro-siphonal nerve (f.). In front of and above the anterior
end of the liver these two nerves come closer together, and,
as I have already stated, a stout anastomosis (a) connects
them before the branchial nerve reaches the mantle. The
branchial nerve is now reflected sharply to the left and passes
under the extreme anterior edge of the osphradium and
ctenidium, supplying fine nerves to each; the main nerve
ramifies in front and to the left of the ctenidium, and forms a
fine network, with numerous anastomoses, in the mantle.
Slightly to the left of where the branchial nerve les above
the left columellar nerve (7. 1), a stout branch (b. 1) is given
off which runs parallel to and almost touches the main nerve
for some considerable distance. After passing down through
the body-wall this branch lies across the anterior lobe of the
liver, whence it proceeds backwards and under the edge of
the osphradium, supplying this and the central portion of the
ctenidium.
The posterior branchial nerve (b. 2) is much finer than the
anterior, and crosses over to the mantle behind the branch of
the main branchial nerve which I have just described, being
40 H. O. N. SHAW.
practically parallel to it throughout its length. Having
passed under the posterior end of the osphradium and given
off two or three fine nerves, the main portion of the posterior
branchial nerve innervates the internal portion of the mantle
and the posterior parts of the ctenidium. According to
Bouvier the nerve with its fine branches and anastomoses
supplying the anterior edge of the mantle is given off after
the anastomosis (a.) between the anterior branchial nerve (6.)
and the main pleuro-siphonal nerve (/.), and is thus a product
of the pleuro-siphonal nerve. In my dissection this is not so,
for the branchial nerve bifurcates, the right branch, which is
the finer of the two, forms the anastomosis (a.), while the
left branch supplies the anterior portions of the mantle in
addition to the ctenidium and osphradium, so that this nerve,
which in both cases supplies the anterior mantle edge, is in my
dissection not pleuro-siphonal but branchial. The branchial
nerve, therefore, is of much greater length than that figured
by Bouvier.
Another curious point and one worthy of note is the
presence of the large branch (b. 1) issuing from the main
branchial nerve close to its origin. At first sight I was under
the impression that this was the posterior branchial nerve,
but closer inspection soon proved that this was not the case,
for it is undoubtedly simply a branch of the anterior nerve,
the posterior branchial nerve (b. 2) being quite distinct and
originating to the right of the former. The typical position
for the posterior banchial nerve would be slightly behind this
branch nerve, though not as far back as it is in this specimen.
There are, therefore, according to the parts they innervate,
three, and not two branchial nerves. The main and anterior
nerve is normal; its branch forms a median branchial nerve;
the posterior nerve proper is displaced backwards, and with
the aforesaid branch, innervates rather more than the area
covered by a normal posterior branchial nerve.
There is nothing of much interest about the two parietal
nerves (c.2,c,3). Both cross over to the left and supply the
body-wall. The anterior (c.2) leaves its ganglion between
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 41
the anterior and posterior branchial nerves, while the posterior
(c.3) has its origin between the visceral commissure (d.) and
the posterior branchial nerve. Soon after its entry in the
wall, this posterior parietal nerve expands out into a small,
ganglionic-looking mass, from which two fine nerves run
forward and three backwards into the body-wail.
The four parietal nerves, two from the left pleural ganglion
(c., c. 1) and two from the supra-intestinal ganglion (c. 2, ¢. 3)
innervate the body-wall and side, anterior to the left visceral
ganglion (V. 2).
The sixth nerve (c.4), which I have mentioned, leaves the
supra-intestinal ganglion on its right and postero-ventral
edge, runs backwards and to the left, and plunges into the
body-wall just in front of the left visceral ganglion, and after
running down through the wall, emerges above and passes
across the liver to the mantle base, where it ramifies and
supplies the mantle wall between the hinder portion of the
ctenidium and the base of the mantle. This nerve is not noted
by Bouvier in C. virgo, but in the specimen under discussion
it is as stout as the posterior branchial nerve, and from its
position is of some interest.
The remaining nerve, issuing from the supra-intestinal
ganglion, is the left or supra-intestinal branch of the visceral
commissure; this I shall discuss with the visceral ganglia.
Tue Viscerat Commissure anp Ganeuia (PI. 4, fig. 19).
There are three visceral ganglia. ‘The left one (V. 3) 1s
situated in the body-wall near the anterior portion of the liver
and above it on the right side. The right ganglion (V. T)as
found in the wall enclosing the posterior part of the body-
cavity and near its left extremity, while the median visceral
ganglion (V. 2) lies to the nght of the posterior part of the
liver, and in front of the recto-genital mass (r. g. ™.)
The left branch of the visceral commissure (d.) has its origin
in the posterior and dorsal surface of the supra-intestinal
ganglion, passes obliquely backwards and to the left, and so
42 H. O. N. SHAW.
into the body-wall, and enters the left visceral ganglion close
to the right edge of the liver. From the ventral surface of
this ganglion, two nerves issue, which might equally well be
called parietal or columellar nerves, since they supply the
walls and columellar muscle to the right and below their
ganglion and almost touch the left columellar nerve (t. 1)
which issues from the left pleural ganglion. The commissure,
after leaving the left ganglion, runs backwards and downwards
and slightly to the right, where it meets the median visceral
ganglion. Between these two last ganglia, and about one
third of the distance from the left visceral, a nerve (7.) issues
from the left side of the commissure, and runs through the
base of the mantle to the dorsal surface of the liver. This
hepatic nerve turns to the right and passes back through the
dorsal and right edge of the liver and emerges on the right
side, where it is trifurcated. The commissure itself does not
run out in a loop over the dorsal surface of the liver, but is
more normal, being situated in the thick tissue formed by the
junction of the mantle to the body-wall, and is thus on the
extreme right edge of theliver. The hepatic nerve (n.), which
I have just described, is peculiar both from its origin and
from the fact that it is of considerable size, and the only nerve
I have been able to trace which supplies the liver from the
commissure. Bouvier mentions the existence of a fine hepatic
nerve in C. virgo.
The median visceral ganglion is not so large as the left
visceral. Three nerves issue from it; that to the left (m.)
supplies the posterior lobe of the liver, while the central nerve
(m. 1), which is the largest of the three, is the visceral nerve,
innervating the heart as well as the genital organs and kidney.
The nerve on the right (m.2) is the genito-rectal nerve.
Between the median and right visceral ganglia, two parietal
nerves are given off to the body-wall from the commissure,
which latter, after leaving the median ganglion, passes forward
and to the right, and so to the right visceral ganglion. One
nerve (k.) arising from this last ganglion (V.1) is of con-
siderable size. As stated by Bouvier, this nerve is pleural,
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 43
and issues from the posterior and right side of the ganglion,
passes backwards and into the mantle, beneath the recto-
genital mass and up over the back without entering it, where
it again reaches the mantle, turns sharply to the right and is
ramified.
After leaving the anterior end of the right visceral ganglion,
the visceral commissure runs forward and to the right through
the body-wall till it reaches the posterior portion of the body-
cavity. Here it emerges from the body-wall under the
poison gland and duct, where it enters the hindmost part of
the sub-intestinal ganglion. I have been unable to find any
nerves issuing from the left visceral ganglion and passing to
the liver as indicated by Bouvier.
QuB-INTESTINAL GaneLion (PI. 4, fig. 19).
This ganglion gives off five nerves, four of which innervate
the right part of the body, while the fifth is the visceral
commissure. All these five nerves are unattached, and lie on
the floor of the body-cavity till they enter the posterior wall
of the latter.
The ganglion (Sz.) 1s pyriform and attenuated posteriorly
and slightly to the right, the visceral commissure (d.) entering
the posterior extremity. One nerve (¢.) issues from the left
and ventral surface of the ganglion, passes backwards and
slightly to the left, and enters the body-wall under the
visceral commissure, where, after its entry, it divides up into
numerous fine branches. The branches supply the body-wall
as well as the posterior muscles of the latter, which eventually
unite with the columellar muscle, so that this nerve, though
really parietal, is in part also a columellar nerve.
There are two true parietal nerves given off from the
ventral and right side of the sub-intestinal ganglion, both
being finer than the nerve just described, and lying to the
right of it. Both these nerves (e. 1, e.2) supply the hinder
portions of the body on the right, as also part of the right
side, but do not extend as far back as the parietal-columellar
4.4, H. O. N. SHAW.
nerve. The right of these two nerves (e. 1) issues from its
ganglion more anteriorly than does the left. Between these
two and starting more from the right side of the ganglion is
the largest of the four nerves, the right pleural nerve (e. 3).
This nerve passes backwards and slightly to the right, ranning
at no great depth through the body-wall till it reaches the
thick muscular ridge formed by the posterior edge of the
body-wall and bounding the anterior side of the anal channel.
On reaching this ridge, the nerve plunges straight down and
under the channel and to the right, where the nerve divides
into two, the left branch running back and ramifying in the
edge of the mantle to the right, while the right branch, curving
forward again, supplies the under-surface of the body by its
junction with the mantle. The zygoneurous connective
(z.) unites the right pleural ganglion (P/.) with the sub-
intestinal ganglion (Sz.). It is of considerable stoutness, and
issues from the ventral and right side of the former to pass
backwards and to the right, and is then bent abruptly
posteriorly and enters the sub-intestinal ganglion anteriorly
and on its dorsal and right side. This connective passes over
the cesophagus, while the pleuro-subintestinal connective (d.’)
of the visceral commissure is sub-cesophageal.
There are no nerves given off from the right pleural ganglion,
the right side of the body being innervated from the sub-
intestinal ganglion. Bouvier, in his figures and text, has
confused these two connectives, since he calls that between
the left pleural ganglion and the sub-intestinal ganglion the
zygoneurous connective, while he describes the one between
the latter and the right pleural ganglion as the connective
of the visceral commissure, whereas if is exactly the reverse.
Pepa Ganeuta (PI. 4, fig. 20, and Pl. 5, fig. 21).
These two ganglia are so closely connected together that
they look like one irregular ganglion. On closer inspection
it will be noticed that at both anterior and posterior extremities
there is a slight cleft or constriction between them, which is
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 45
most distinct at their anterior end. The ganglia lie under
the radula-sac on the floor of the body-cavity and are inclined
so that their anterior ends are slightly lower than the posterior.
The ganglia are so displaced that they lie across the body-
cavity instead of their longitudinal axes running parallel to
the foot, the anterior extremities being slightly in advance of
the posterior, the ganglia thus lying at a tangent to the
longitudinal body-axis and considerably to the right. From
this it will be noticed that owing to torsion, the symmetry
of the anterior part of the body has been entirely dis-
placed. Of this not only the pedal ganglia but also the
otocysts bear witness, for the latter are not really right and
left, but anterior and posterior, the right (ot. 7.) being
practically directly behind the left (ot. 1.), and both are
situated at the base of the foot and on the right side. Each
of the two pedal ganglia is connected with the cerebral and
pleural ganglia of its own side by the cerebro-pedal and pleuro-
pedal connectives. The cerebro-pedal connectives join the
pedal ganglia anteriorly to the pleuro-pedals, the latter
being the stoutest of the two pairs. These connectives with
their ganglia form two complete and wide nerve collars round
the cesophagus and lie obliquely round the latter, since the
pedal gangha are to the right, and shghtly posterior to the
cerebral and pleural ganglia.
The nerves given off from the pedal ganglia are extra-
ordinarily numerous, and for the most part of considerable
size. ‘hey issue from the sides and anterior extremities of
the ganglia. The posterior part of the foot is innervated from
the right ganglion, while the anterior half is supplied from
both right and left ganglia, the right ganglion sending nerves
to the anterior and right half, the left ganglion to the left
anterior portion.
There are many more nerves issuing from the right gan-
glion than from the left, the former giving off thirty, while
only eleven proceed from the left ganglion. As I have
already stated, most of these nerves are of considerable size,
some—8, 9, 14, 29—equal in stoutness the tentacular nerves ;
46 H. 0. N. SHAW.
thus the foot and right side of the body are very highly
innervated.
I do not propose to describe all these forty-one pedal nerves,
but they will be seen in Pl. 4, fig. 20, and the parts they
supply are described in the explanation of that figure.
The following nerves are worthy of a passing note. The
posterior portion of the foot is supplied from the right ganglion
by the nerves 29, 31 and 32, of which the first is the stoutest
while the latter pair run side by side for most of their length.
The right ganglion also innervates the central and ventral
parts of the foot, with the exception of the one bifurcated
nerve 23, which proceeds backwards from the left ganglion,
while the chief nerves from the right ganglion are 25, 27
and 28.
There are three main nerves to the anterior region of the
foot, of which 8 and 9 issue from the ventral and anterior
side of the left ganglion, and supply the left side, while 14
passes to the right side from the anterior edge of the right
pedal ganglion. An anastomosis exists between this nerve
and the smaller nerve 13.
One very sinuous nerve (1) after leaving the anterior and
ventral surface of the left ganglion, passes under the
cesophagus and cerebro-pedal and pleuro-pedal connectives,
directly over to the left side, and enters the floor of the body-
cavity immediately in front of the left columellar nerve (7. 1),
where it is trifurcated and innervates the floor under the left
pleural ganglion. A fine nerve, 41, issues from the ventral
surface of the right ganglion, and supplies the cavity floor
directly beneath the pedal ganglia.
From the foregoing account of the nervous system of this
species, several points of interest will be noticed, some new,
and others confirming the descriptions of previous writers on
this genus. Among the latter is evidence of the existence of
an oesophageal nerve given off from the poison gland nerve,
and indicated by Bouvier. Both my dissections have been of
females, as was Bouvier’s, and I have not yet been able to
dissect a male of either species owing to lack of material.
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 47
Perhaps the most interesting feature about the nervous
system of the above species is the connection between the
right tentacular nerve with the right pedal ganglion, by
means of a branch uniting the former to a nerve passing to
the body-wall from the right ganglion.
DIFFERENCES BETWEEN Nervous Systems oF CoNUs TEXTILE
AND CONUS TULIPA.
The nerve centres are situated in similar positions in both
species; in C. textile both the centres and their nerves are
of a reddish-yellow colour. The cerebral, pleural and supra-
intestinal ganglia are more closely connected, and even harder
to differentiate. The internal edges of both pleural ganglia
almost meet, and the constriction is hardly noticeable between
the cerebral ganglia, while the whole mass is covered by
a thick sheath of connective tissue. The supra-intestinal
ganglion is connected, as in C. tulipa, to the right pleural
ganglion, their junction being hardly determinable, while the
posterior extremity of the former is directed sharply to the
left, from whence the left-hand loop of the visceral commissure
issues. ‘This flexion of the ganglion is so abrupt as to bring
the posterior portion immediately behind the left pleural
ganglion, with the result that, at first sight, there appear to
be two supra-intestinal ganglia.
The visceral commissure and nerves issuing from the
supra-intestinal ganglion, owing to its peculiar shape, proceed
across the body-cavity to the left, and at right angles to its
axis, instead of obliquely backwards as in the former species.
The left pleural ganglion is more pyriform, but in other
respects is the same. The positions of the ganglia lying
around the cesophagus and also of the sub-intestinal ganglion
are similar, but the pedal ganglia lie further back than in
C. tulipa, and are not so attenuated anteriorly. These last
ganglia are attached with the cerebral and pleural ganglia
by the usual pairs of connectives, the anterior, the cerebro-
pedal, being very much finer than the pleuro-pedal con-
48 H. 0. N. SHAW.
nectives. Both run parallel and close alongside of each
other entering the sides of the pedal ganglia, and issuing
close together from the posterior and external edges of the
cerebral ganglia and the anterior edges of the pleural ganglia.
‘he pleuro-subintestinal and the zygoneurous connectives
are of about equal size. The right visceral ganglion, to which
the former proceeds, is not pyriform, but of an oblong shape,
and the connectives enter at the opposite extremities on the
anterior surface. The three visceral ganglia are situated in
about the same positions, but the commissure connecting
them is shorter. This latter has its origin in the posterior
and left side of the sub-intestinal ganglion behind the entry
of the pleuro-subintestinal connective.
The buccal ganglia are attached to the cerebral ganglia in
the same manner, but their connectives are almost twice as
long. The former ganglia are smaller, but globular in shape
instead of flat, while their commissures are much longer, the
anterior being almost as stout as the posterior. ‘I'he most
anterior nerve collar, the cerebro-buccal, is not closely
attached to the walls of the cesophagus, but is considerably
larger than the latter, beng very nearly as large as the
cerebro-pedal nerve collar. ‘his latter with the pleuro-pedal
are the same as in C. tulipa, with the exception that they
both lie more obliquely across the cesophagus. ‘he fourth
collar, connecting the pleural and the sub-intestinal ganglia,
like the second and third, is much more oblique.
The tentacular and optic nerves are the same, with the
exception that the former are not nearly as stout, and have
not the kink or elbow bend, the optic nerves issuing from
them as already described.
The connective between the right tentacular nerve and the
nerve issuing from the right pedal ganglion, and which, in
the former species, was so noticeable, is absent in C. textile,
there being no connection of any sort between the tentacular
nerve and the pedal ganglia.
The left or anterior otocyst is placed closer to the pedal
ganglia, while the right one is in about the same position.
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 49
The left acoustic nerve issues above the left cerebro-pedal
connective instead of below it and follows much the same
course, but runs along the floor of the body-cavity under the
anterior pedal nerves instead of through them. The right
acoustic nerve runs along the dorsal surface of the right
cerebro-pedal connective.
There is no difference of note in the proboscidean nerves.
I have already mentioned that the nerve collar round the
cesophagus formed by the two cerebral and two buccal ganglia
is of much greater diameter. Two nerves (s.3, s.4) issue
from the left of these latter ganglia and proceed to the
radula-sac in like manner, but are stouter, the fine nerve
(s. 2) running forward; the buccal-proboscidean nerve is
present, but only one nerve from the right ganglion to the
radula-sac, and the small nerve (s. 1) from the same ganglion
to the anterior part of the poison duct is also absent. The
main poison gland nerve (s. 5) is slightly modified, as shortly
after leaving the posterior commissure it is bifurcated, the
right branch innervating most of the poison duct. The left
branch, after proceeding backwards for some distance, is in
turn divided into two, these two innervating the poison gland
itself.
Lastly, the fine nerve (s. 6) from the main nerve (s. 5) to the
cesophagus, branches off from the former further back, and on
reaching the surface of the cesophagus sends one nerve forward
and one back over the surface of the latter. Though some-
what modified, the function of this nerve is the same, and its
existence is the chief point in question.
The two columellar nerves having their origin in the left
pleural ganglion are of equal size, and slightly stouter, and not
so sinuous. Both lie under the cesophagus, and innervate the
columellar muscle in much the same way. The four nerves
issuing from this ganglion—viz. the main and lesser pleuro-
siphonal and the two parietal nerves—differ but little, the
anastomosis between the anterior branchial nerve and the main
pleuro-siphonal being slightly longer.
I have already mentioned that the posterior portion of the
vot. 60, PART 1.—NEW SERIES. 4
50 H. O. N. SHAW.
supra-intestinal ganglion is reflected over to the left. There
are only three nerves besides the left loop of the visceral
commissure which proceed from this ganglion, namely, the
anterior and posterior branchial nerves and one parietal nerve.
With the exception that they pass straight across the body-
cavity to the left they are of no particular interest. The
branch given off from the main or anterior branchial nerve is
again present, but much smaller, with the result that the
anterior and posterior branchial nerves are closer together,
and, therefore, in more normal positions.
The sixth nerve (c.4) in C. tulipa is entirely absent in
this species.
The three visceral ganglia and their commissures require
no special comment, being much the same in both species, the
commissure being slightly shorter.
The difference in shape of the sub-intestinal ganglion has
already been noticed. There are only three nerves which
issue from the ganglion besides the right loop of the visceral
commissure. The nerve (e.) performing the functions of a
parietal and columellar nerve is absent.
The pedal ganglia are even more closely connected and
ensheathed by connective tissue. The nerves leave the right
ganglion, with one exception, from its anterior extremity only,
while they issue from the left side as well as from the anterior
of the left ganglion; tnus, more nerves are given off from the
left than from the right ganglion—the reverse of what occurs
in C. tulipa. In the latter there were 41 nerves altogether
from both ganglia; in this species there are 39—21 from
the left, 18 from the right ganglia, as compared to 30 and 11.
None of these 39 pedal nerves attain so great a size as the
chief ones in C. tulipa, and all enter the floor of the body-
cavity sooner. The right ganglion supplies the posterior
portion of the foot, while the central and anterior parts are
innervated from the left ganglion. It will thus be noticed
that the functions of the ganglia in the different species have
been reversed, since in C. textile it is the left one that is
the most important. The ganglia themselves do not lie at so
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 51
great a tangent to the longitudinal body-axis, but more at
right-angles to it.
There are no points of any great difference, or of special
interest, between the nervous systems of both species beyond
those already mentioned.
Crrcunatory Sysrem or Conus Toura (Pl. 5, fig. 22, and
Pie Gey fiers, 25):
As in C. textile, so in this species, I have only studied
the artery passing forward through the nerve collars. ‘This
artery (g.), after leaving the heart, proceeds forward and
obliquely to the right till it reaches the left branch of the
visceral commissure (d.) anterior to the left visceral ganglion.
For some considerable distance, both commissure and artery
are surrounded by one sheath of muscular and connective
tissue, the artery being on the right of the commissure.
Owing to the artery and commissure having been cut through,
where surrounded by this sheath, while removing the
cesophagus from the nerve collars, I at first mistook the artery
for a nerve given off from the commissure, and, indeed, had
dissected it out as such, being considerably perplexed by the
existence of such a nerve, till on having sections cut I realised
my mistake. It is owing to this error that I have been
enabled to work on the arterial system at all, for being then
engaged on the nervous system, had it from the beginning
clearly shown itself to be an artery, most probably it would
have been removed without much comment, and some
interesting facts remained unknown.
Having branched off from the visceral commissure, the
artery passes to the right almost at right angles to the longi-
tudinal body axis, and under the cesophagus. A_ little
distance after its separation from the commissure, a branch
artery (j.) is given off nearly at right angles and passes
upwards to form a loop which partly surrounds the cesophagus,
while the end of the loop is bifurcated, one branch running
up and the other down.
52 H. O. N. SHAW.
The main artery proceeds through the three nerve collars,
pleuro-subintestinal, pleuro-pedal, and cerebro-pedal, till it
arrives at the posterior cleft between the two pedal ganglia.
Here a stout but very short branch (op./.) opens into the
dorsal surface of the left pedal ganglion at its posterior edge.
‘The artery runs down the dorsal constriction between the
two ganglia and over the anterior edge. When about half
way along the depression between the ganglia, the artery 1s
slightly expanded, and two branches issue, that to the right
(op.r.) opening into the dorsal surface of the right pedal
ganglion, while the left branch (w.), which is much longer, 18
directed upwards and divides into two, each being again
bifurcated and spreading over the radula-sac.
The branches opening into each of the ganglia are of the
same size and length, but the left one enters the extreme
posterior edge of its ganglion, while the right one opens into
the centre of the dorsal surface of the right pedal ganglion.
After leaving the anterior edge of the ganglia, the main
artery curves to the left, becomes larger, and runs directly
forward, two arteries branching off from it on the right side,
then one on the left, and still more anterior to this latter, the
main artery is bifurcated.
The first branch (c. ft.) to be given off to the right, runs
backwards and through some of the pedal nerves, and enters
the centre of the foot, where it divides into two branches
supplying the central portions of the latter.
The second branch to the right (v. ft.) proceeds directly
downwards into the foot to its ventral surface, where it forms
a JT, one arm running forward and the other backward.
Slightly anterior to this artery, the main artery sends off the
only branch to the left (te.), which proceeds to the base of
the cephalic integuments. The left bifurcation of the main
artery (y. 1) runs forward through the dorsal portion of the
foot, while the right (y.), passing downwards into the foot,
attains the extreme right anterior basal surface of the latter.
In section, the main and branch arteries are for the most
part oval (Pl. 5, fig. 22), and are composed of two distinct
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 59
sheaths or layers, of which the inner (J, c. m.) consists of
circular muscular tissue lined with endothelium (end.), while
the outer sheath (I. m.s.), which is slightly thicker, is built
up of areolar and muscular tissue running longitudinally.
DIFFERENCES BETWEEN THE ARTERY IN Conus TEXTILE AND
Conus JTULIPA.
Although the position and functions of the artery in both
species are roughly the same, there are many points of great
difference between them.
In the first place, the artery in C. textile has no connec-
tion whatsoever with the visceral commissure, and lies entirely
under the esophagus. In C.tulipa, for some distance, the
artery and commissure are enclosed in the same sheath, and
the artery traverses the nerve collars in a direct line for the
pedal ganglia. In C. textile the main artery passes through
the nerve collars some distance to the left of these ganglia, and
a branch artery crosses the dorsal surface of the pedal gangha,
but has no connection with them. In C. tulipa this branch
is absent, but the main artery opens directly into the dorsal
surface of each ganglion, while from the same place as the
opening into the right ganglion a stout but short branch
proceeds to the radula-sac. |
The radula-sac of C. textile is supplied from a branch
which is given off from the main artery before the latter
passes through the pleuro-pedal nerve collar. This branch
is bifurcated, the left arm running backwards and through
the pleuro-subintestinal collar and» supplies the cesophagus,
while in C. tulipa the latter has a direct supply from the
main artery posterior to the last-named nerve collar. In this
latter species the main artery, after passing over the pedal
ganglia, becomes considerably larger, runs directly forward
and divides up into five branches, which supply the base of
the cephalic integuments, the anterior dorsal surface, the
anterior right and basal surface, the central ventral, and the
central portions of the foot respectively.
o4 H. O. N. SHAW.
In C. textile, anteriorly and to the right of the left pedal
ganglion the main artery forms a cross, the left arm running
to the base of the siphon, the central to the anterior and basal
surface of the foot, while the right arm, which is twice the
length of the other two, proceeds directly backwards to the
extreme posterior and dorsal surface of the foot. Lastly, in
section, the artery is of a different construction in each species.
In C. tulipa it is composed of two layers of tissue, of which
the outer, or tunica adventitia, which is slightly thicker than
the inner, is built up of areolar and longitudinal muscular
tissue. The inner layer, or tunica media, is composed of mus-
cular tissue running circularly round the arterial canal, and
having a smooth inner surface lined with endothelium, or
pavement epithelium. ‘he reverse in every point, except the
endothelial layer, is the case in C. textile. The outer layer,
which is half the thickness of the inner, is built up of areolar
and circular muscular tissue ; the inner layer is a confused
mass of circular and longitudinal tissue of the same sort ; while
the internal surface, which is lined with endothelium, is deeply
corrugated, aud a section looks more like a vein than an artery.
The total thickness of the arterial wall in this species is twice
that in C. tulipa.
Though superficially resembling one another, from the fore-
going comparison the many and great differences between
the arteries in the two species will be observed.
Poli (21), pl. xlv, fig. 18, and p. xxxix has illustrated and des-
cribed in Conus rusticus Linn. an artery which he calls
““Paorte abdominale,” which is similar to that in C. textile
in some respects. According to Poli’s figure, the artery runs
forward from the ventricle of the heart beside the left loop of
the visceral commissure, across the latter, and forward through
the nerve collars, where it is anteriorly split up into several
small branches.
In concluding this paper, it is hoped that the contents on
the anatomy of two species of the genus Conus may be of
use to workers on this group. Since I commenced my work
on this genus I have received from various sources a number
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 55
of different species, and I hope shortly to be able to start
work on them with a view to further determining the ana-
tomical variations between different species.
BIBLIOGRAPHY OF THE CHIEF Works CONSULTED.
1. Adams, H. and A.—‘ The Genera of Recent Mollusca,’ 1853-8,
London.
2. Amaudrut, A.—“ La partie antérieuse du tube digestif, et la torsion
chez les Mollusques gastéropodes,” ‘Ann. des Sci. Nat.,’ (8), vil,
1898, p. 1.
3. Bergh, R.—‘“ Beitriige zur Kenntniss der Coniden,” ‘ Nova Acta der
Ksl. Leop-Carol.,’ ‘ Deutschen Akademie der Naturforscher,’ Bd.
Ixy, Nr. 2, 1895.
4, Bernard, F.—* Recherches sur les organes palléaux des Gastéropodes
prosobranches,” ‘ Ann. des Sci. Nat.,’ (7) ix, 1890, p. 88.
5. Bouvier, E. L.—< Systeme nerveux, Morphologie générale et classifica-
tion des Gastéropodes prosobranches,”’ ‘ Ann. des Sci. Nat.,’ (7)
ili, 1887.
' 6. Cuvier, G.— Régne Animal. Mollusques,’ by G. P. Deshayes, Paris,
1836-49.
7. Eydoux, F., et Souleyet, L.—* Voyage autour du Monde sur la
corvette la Bonite,”’ ‘ Zoologie,’ vol. ii, pp. 632-3, pl. 45, Paris,
1852.
8. Fischer, P.—‘ Manuel de Conchylologie,’ Paris, 1887.
9. de Freycinet, L.—‘* Voyage autour du Monde,” ‘ Zoologie par Quoy et
Gaimard,’ pp. 437-440, pl. 69, Paris, 1824.
10. Gray, J. E—* On the Division of Ctenobranchous Gasteropodous
Mollusca into larger Groups and Families,’ ‘Ann. and Mag. Nat.
Hist.,’ 2nd series, vol. xi, No. lxii, paper xiii, 1853.
11. “On the Head of the Genus Conus,” ‘Ann. and Mag.,’ 2nd
series, vol. xii, No. lxix, paper xix, 1853.
12. ‘Guide to the Systematic Distribution of Mollusca in the
British Museum,’ Pt. I, 1857, London.
18. Jhering, H. von.—‘ Vergleichende Anatomie des Nervensystems
und Phylogenie der Mollusken,’ Leipzig, 1877.
14, “ Beitriige zur Kenntniss des Nervensystems der Amphi-
neuren und Arthocochliden,” ‘ Morph. Jahrbuch,’ iii, 1877.
15. “ Gibt es Orthoneuren ?” ‘ Zeit. f. wiss. Zool.,’ xlv, p. 499,
1887.
56 H. O. N. SHAW.
16. Johnston, G.—‘ Introduction to Conchology,’ 1850, London.
17. Lacaze-Duthiers.—* Otocystes ou capsules auditives des Mollusques
(Gastéropodes),” ‘Arch. de Zool. expér.,’ (1), i, 1872.
18. de Montfort, D—‘ Conchyliologie systématique et classification
méthodique des Coquilles,’ vol. ii, 1810, Paris.
19. Pelseneer, P.—‘* The Mollusca,” in a Treatise on Zoology, edited by
E. Ray Lankester, Part v, London, 1906.
20. Perrier, R.—* Recherches sur l’anatomie et l’histologie du rein des
Gastéropodes prosobranches,” ‘ Ann. des Sci. Nat.,’ (7) viii, p. 61.
21. Poli. J. X., et Delle Chiaje, S.— Testacea utriusque Sicilie,
eorumque historia et anatome,’ iii, Parme, 1826.
22. Quoy et Gaimard.—* Voyage de l’Astrolabe,” ‘ Zoologie,’ iui, Paris,
1834.
23. Simroth, H.— Gastropoda prosobranchia,’ ‘ Bronn’s Tierreichs.
Mollusea,’ 1896-1907.
24. Troschel, F. H.—‘ Das Gebiss der Schnecken,’ vol. ii, Berlin, 1866-
1893.
25. Woodward, S. P.— A Manual of the Mollusca,’ London, 1858, 1869,
1875.
EXPLANATION OF PLATES 1-6,
Illustrating Mr. H. O. N. Shaw’s paper ‘On the Anatomy of
Conus tulipa, Linn., and Conus textile, Linn.”
LETTERING IN ALL THE FIGURES EXCEPT NUMBERS IN Fie. 20.
a. Anastomosis between pleuro-siphonal and anterior branchial nerves.
ac. 1. Left acoustic nerve. ac.7. Right acoustic nerve. ats. Tooth
- attachments to sac wall. B. Buccal ganglia. 6. Anterior branchial
nerve. 6.1. Branch off anterior branchial nerve. b. 2. Posterior
branchial nerve. b. a. Basal attachment of radule. 6. c. Body-cavity.
br. c. Branchial cavity. bs. Barbs of teeth. C. cerebral ganglia. c.,
c.1, c. 2, c. 3. Parietal nerves to left of body. c. 4. Main and posterior
parietal nerve. ca. Canal. c.b. Cerebro-buccal connectives. ce. pl.
Cerebro-pedal connectives. c. ft. Artery to centre of foot. c¢.m.s,
Circular muscular sheath. co.z. Inner corneal layer. co. 0. Outer
corneal layer. ct. Ctenidium. d. Visceral commissure. d.' Pleuro-
sub-intestinal connective. dt. op. Duct opening. dts. Ducts from
unicellular glands. e., e. 1. Parietal nerves to right of body. e.2
« te
Columellar nerve. e. 3. Right pleural nerve. end. Endothelium. ep.
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 57
Epithelium of tentacles. ep. cs. Epithelial cells. f. Main pleuro-siphonal
nerve. jf.1. Lesser pleuro-siphonal nerve. ft. Foot. g. Artery from
heart through nerve collars. g.c¢.¢. Granular contents of glands. h.
Hood-like process of radula-sac. 7.1. Left columellar nerve. 7.2. Right
columellar nerve. inv. Invagination into siphon. div. ca. Invagination
into canal. y. Artery to the esophagus. k. Right pleural nerve. J/.1,
1.2, 1.3, 1.4, 1.5, 1.6. Proboscidean nerves. J.c.l.m. Layer of inter-
mingled circular and longitudinal muscle. J/.c.m. Layer of circular
muscle. Je. Lens. J. ep. Lining epithelium. J/./. Left lobe of liver.
L.d.m. Layer of longitudinal muscle. J/. m. s. Longitudinal muscular
sheath. m. Hepatic nerve. m.1. Visceral nerve. m. 2. Recto-genital
nerve. mh. Mouth. ml. Mantle. «. Hepatic nerve from visceral com-
missure. n.c. Nervecollar. nw. Nuclei. o. Opticnerve. @. Gsophagus.
o. p. d. Opening of poison duct. op. l. Artery opening into left pedal
ganglion. op.r. Artery opening into right pedal ganglion. 0.7. s.
Opening of radula-sac. of.1. Left otocyst. otf, r. Right otocyst. P.
Left pedal ganglion. P.1. Right pedal ganglion. pa. pl. Pleuro-pedal
connectives. p. ft. Artery to posterior portion of foot. p. g. Poison
gland. p.g.d. Poison-gland duct. Pl. Pleural ganglia. ps. Proboscis.
r. Nerve from right pedal ganglion joining tentaculo-pedal connective.
vet. Retina. +.g.m. Recto-genital mass. 7m. Rostrum. r.s. Radula-
sac. 7+.t. Row of denticulations. §. Supra-intestinal ganglia. s. 1.
Nerve to anterior portion of poison duct. s. 2. Buccal proboscidean
nerve. s.3,s.4. Nerves to radula-sac. s.5. Main nerve to poison gland
and duct. s.6. Fine nerve to esophagus. s.d. Salivary duct. s.g.
Salivary gland. 8.7. Sub-intestinal ganglion. sz. Siphon. — sp. pa,
Sporozoon parasites. st. Stomach. ¢. Main tentacular nerves. f. 1,
t.2, t.3. Branches of main tentacular nerves. te. Artery to cephalic
integuments. tent. mus. Muscle of tentacle. ¢. p. Tentaculo-pedal
connective. ts. Tentacles. w.g. Unicellular salivary glands. JV. 1.
Right visceral ganglion. V.2. Median visceral ganglion. V. 3. Left
visceral ganglion. v. ft. Artery to ventral surface of foot. w. Artery
to radula-sac. #. Artery to base of siphon. y. Artery to anterior base
of foot. y. 1. Artery to anterior dorsal surface of foot. z. Zygoneur
between right pleural ganglion and sub-intestinal ganglion.
PLATH 1
Fig. 1—Conus tulipa. A general view of the anatomy. The
mantle (ml.) has been cut open, and reflected to the left showing the
ctenidium (ct.) lying ina curve along its dorsal surface. The ospradium,
which is situated on the inside of the ctenidium, is hidden by the base
of the rostrum. This latter (rm.) has also been opened along its dorsal
58 H. O. N. SHAW.
surface, showing its thick muscular walls and tentacles (ts.) and the
proboscis (ps.) situated at its base. The dorsal wall of the body-cavity
has been partly removed, and the large poison gland (p. g.) will be seen
to occupy all the posterior portion. The poison gland duct (p. g. d.) is
coiled across and anterior to the gland, while the radula-sac (r. s.) is
anterior and partly dorsal to the poison duct. An incision has been
made in the base of the proboscis to show the cesophagus (@.) passing
up the centre to the mouth (mh.). (As the various organs are situated
in the same positions in both Conus tulipa and Conus textile, I
have used this figure to illustrate the description of the latter species, as
well as the one it depicts.)
Fig. 2.—Conus textile. A view of the poison gland (p.g.) and
duct (p. g.d.), esophagus (@.), salivary gland (s. g.), and radula-sac (7. s.)
removed and spread out to show the coils and length of poison duct,
stomach (st.), salivary ducts (s. d.)
Fig. 3—Conus tulipa. A view of the poison gland (p.g.) and
duct (p. g.d.), esophagus (@.), salivary gland (s. g.), and radula-sac (r. s.)
removed and spread out as in fig. 2, to show the difference in size of
poison gland, constricted opening of duct, which latter is shorter and
less coiled than in C. textile. Hood-like process (h.) of radula-sac,
not present in C. textile, stomach (s¢t.), salivary ducts (s. d.).
Fig. 4.—A tooth of Conus textile. Barbs (bs.), basal attach-
ment (0. a.).
Fig. 5.—A tooth of Conus tulipa. Barbs (b.s.), basal attachment
(b.a.), row of denticulations (r. t.).
PLATE 2.
Fig. 6.—Conus tulipa. A longitudinal section through poison
gland, showing layers of muscle-fibres, highly magnified.
Fig. 7.—A transverse section of same.
Fig. 8.—A transverse section of same, highly magnified.
Fig. 9.—Conus textile. A transverse section of poison gland, cu.
canal, c.m.s. circular muscular sheath, J. ep. lining epithelium of canal,
l. m.s. longitudinal muscular sheath.
Fig. 10—Conus textile. A transverse section of poison duct,
showing long club-shaped cells, and irregular surface of canal.
Fig. 11—Conus tulipa. A transverse section of poison duct showing
invagination hanging down into canal, c.a. canal, ep. cs. epithelial cells,
inv. ca. invagination into canal, lJ. c.m. layer of circular muscle-fibres,
1. l. m. layer of longitudinal muscle-fibres.
CONUS TULIPA, LINN., AND CONUS TEXTILE, LINN. 99
Fig. 12.—Conus tulipa. A transverse section through the eye, co. 7.
Inner corneal layer, co. 0. outer corneal layer, ep. epithelium of tentacle,
le. lens, 0. optic nerve, vet. retina, tent. mus. muscle of tentacle.
Fig. 13—Conus tulipa. A transverse section through the
cesophagus, showing sporozoon parasites in the lining epithelium, which
has shrunk away from the muscle wall. ca. Canal, l. c. m. layer of circular
muscle, /. ep. lining epithelium, J. 1. m. layer of longitudinal muscle-fibres,
sp. pa. sporozoon parasites.
Fig. 14.—Conus tulipa. A transverse section through the end of
poison gland, showing disposition of layers of muscle-fibres and opening
of poison duct (0. p. d.).
Fig. 15.—Conus textile. A transverse section through cesophagus,
thick muscular walls, with a layer of longitudinal and circular muscle-
fibres (l. c. 1. m.) intermixed in the centre.
Fig. 16.—Conus textile. A transverse section through salivary
gland showing entry of one of the two main ducts. dts. Ducts from
unicellular glands, s. d. salivary duct, w.g. unicellular glands.
Fig. 17.—The above highly magnified, showing a group of five
unicellular glands with nuclei and granular contents. g.c. ¢. Granular
contents of cells, dt. op. duct opening, nw. nuclei.
PLATE 3.
Fig. 18—Conustulipa. Thenervoussystem. Buccal ganglia
(B.) and nerves issuing therefrom, with the tentacular and optic nerves
given off from the cerebral ganglia (C.). The two pleural and the supra-
intestinal ganglia are not shown.
PLATE 4.
Fig. 19—Conus tulipa. Thenervous system.—The cerebral
ganglia (C.), pleural ganglia (Pl.), the sub (Si.) and supra-intestinal
ganglia (S.), with the nerves given off from each, also the visceral
commissure (d.), and the three visceral ganglia (V.1, V.2, V.3,). The
pedal ganglia, cerebro-pedal, and pleuro-pedal connectives are not
represented.
Fig. 20.—Conus tulipa. The nervous system. The pedal
ganglia P. and P. 1 with the nerves issuing, and the cerebro-pedal (ce. pl.)
and pleuro-pedal (pa. pl.) connectives. Parts of the foot and body to
which the nerves (1-41) proceed. 1 to anterior floor of body-cavity in
front of the columellar nerves (7. 1, 7. 2), 2 to centre of floor of body-
cavity, nerve much crenulated, 3 to left and anterior portion of foot, 4
and 5 to floor of body-cavity, 6 and 7 to left anterior side of foot, 8and 9
60 H. O. N. SHAW.
to left anterior extremity of foot (main nerves), 10 to central anterior
extremity of foot, 11, 12 and 13 to right anterior extremity of foot, 14
main nerve to right anterior extremity of foot, 15 to anterior and right
side of foot, 16 to right side of foot, 17 to extreme light and anterior
part of body-wall, 18 to anterior and right side of foot, 19 to extreme
right and anterior part of body-wall, 20 to right anterior side of foot, 21
and 22 to centre and right part of foot, 23 to ventral and central surface
of foot, 24 to right side of body-wall, 25 and 26 to centre and lower
surface of foot (25 main nerve), 27 and 28 to centre of foot, 29 main nerve
to extreme posterior end and base of foot, 30 to centre of foot, 31 under
operculum, 32 through right corner of body-wall, and to extreme end
and base of foot, 33 to right posterior corner of body-wall, 34 to floor
and body-wall, 35 to base of posterior wall of body-cavity, 36 to left
central side of foot, 37 to posterior wall of body-cavity, 38 to left and
central side of foot, 39 to base of posterior body-cavity wall, 40 and 41
to floor of body-cavity, under cerebral ganglia.
PLATE 5.
Fig. 21.—Conustulipa. Thenervoussystem. Nerve centres.
The ganglia with their connectives and nerves. Only nine nerves are
shown issuing from the pedal ganglia (P.and P.1). The buccal ganglia
(B.) with the cerebro-buccal connectives (c. b.), which lie parallel to the
cerebro-pedal connectives (ce. pl.), have in this figure, for the sake of
clearness, been brought forward. The left and right otocysts (of. L.,
ot. 7.) with their nerves (ae, l., ac. 7.) can be clearly seen, as also the
nerve (r.) issuing from the right pedal ganglion (P.1), and united by
means of the tentaculo-pedal connective (¢. p.) to the right tentacular
nerve (t.).
Fig. 22.—Conus tulipa. A transverse section of the artery to the
pedal ganglia.
Fig. 23.—C onus textile. A transverse section of the artery which
passes through the nerve collars.
Pia 6,
Fig. 24.—Conus textile. A general view of the nerve centres
(semi-diagrammatic), showing the position and branches of the artery
proceeding from the heart through the nerve collars to the foot, ete.
Fig. 25—Conus tulipa. A general view of the nerve centres (semi-
diagrammatic), showing the position and branches of the artery which
proceeds from the heart through the nerve collars to the pedal ganglia
and foot, ete.
CENTRIFUGED “EGG OF THE FROG.
+
On the Relation between the Structure and the
Development of the Centrifuged Egg of the
Frog.
By
J. W. Jenkinson, M.A., D.Se.,
University Lecturer in Embryology, Oxford; Fellow of
Exeter College.
With Plates 7—12 and Text-figs. 1—18.
ConTENTS.
I. INTRODUCTORY...-~
Il, THe EXPERIMENTS.
A. On the CentriPiged Egg
(1) Method’.
(2) Details of the Experiments
(3) The Effect of a Centrifugal Force on—
(a) The Egg Structure
_ (b) Segmentation .
— (e) Development
B. On € ntrifuged Egg-Pulp. The Guomcey of he
Constituents of the Cytoplasm ;
c. Conclusions to be Drawn from the eee Experi-
ments
III. On THE RELATION BETWEEN THE Gennes tana Sheree
OF THE EGG AND THE DEVELOPMENT OF THE EMBRYO IN
GENERAL .
I. INTRODUCTORY.
108
1167
117
130
140
144
Iv is to the structure and constitution of the germ-plasm, of
the cytoplasm as well as of the nucleus of the germ-cell, that
the path of inquiry into the internal causes of the development
|
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J
{
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62 J. W. JENKINSON.
of the organism, the causes which determine in each generation
the reproduction of the specific form, inevitably leads back.
It is not alittle curious that a method of experimental research
which has already given something and promises perhaps to
contribute still more to the analysis of the factors resident in
the egg-cytoplasm should owe its existence to those classical
experiments of the physiologist Emil Pfliiger, which led their
author to formulate the doctrine of the “isotropy ” of the
ovum, and to deny to the egg-cytoplasm any structure, or at
least any significant structure at all.
Normally, the eggs of the frog and toad—Pfliiger used the
ova of Bombinator—are not free to rotate inside their
tightly adherent jelly-membranes when first they are laid.
But shortly after and as a consequence of insemination there is
developed between the egg and the innermost layer of the jelly
a perivitelline space, inside which the egg then rotates until
its axis becomes vertical with the heavier yolk-pole below. It
will be recalled that Pfliiger succeeding in inhibiting the
formation of this space, though not the subsequent segmenta-
tion and development, by giving each eg’; only a minimal drop
of sperm-containing water. Such eggs ould be kept forcibly
inverted in any position in which the ex:perimenter chose to
place them, with the original axis making any angle with the
vertical, and, except when the inversion was absolutely complete,
were capable of normal segmentation and development. In
cleavage it was found by Pfliiger that the firs two divisions
were vertical and the third horizontal, just as in the undis-
turbed egg the first two (meridional) and third (latitudinal)
are also respectively vertical and horizontal, while later on the
dorsal lip of the blastopore appeared just below the actual
equator, and travelled downto the actual lower pole in the plane
including the actual (vertical) and the original (inclined) egg-
axes. The conclusion drawn from these facts—that the embryo
may be developed from any part of the egg, that the egg is
consequently isotropic, and only undergoes a specific develop-
ment because it is always brought under the same external
conditions—was not allowed to remain unchallenged for long.
CENTRIFUGED EGG OF THE FROG, 63
First Roux showed that the constant directive influence of
gravity might be easily eliminated by keeping the eggs in a
perpetual state of slov rotation, under which circumstances
their segmentation and development bear the same relation to
the original axis and polar structure as is ordinarily the case.
And secondly, Born’s examination of sections showed at once
that in these forcibly inverted eggs there takes place a re-
distribution of material, the hghter liquid plasma ascending,
the heavier yolk-particles descending until there is conferred
upon the ovum a new structure with a new axis, which is of
course vertical, and has unlike animal or protoplasmic and
vegetative or yolk poles. The pigment, however, is not wholly
shifted, though some is carried up into the lighter plasma.
The upturned white pole remains white, or at most becomes
greyish. To this new structure—which may have any relation,
make any angle with the original structure—the cleavage and
development of the egg has the same relation as normally.
With regard to the new axis the first and second furrows
are meridional, the third latitudinal, while the head of the
embryo appears near the new animal pole. The median plane
of the embryo is that plane in which both the original and
the new axes lie, for the streaming up and down of the plasma
and yolk take place symmetrically about that plane, and the
bilaterality so conferred upon the egg-contents persists as
that of the embryo.
The very experiments, therefore, which were vainly imagined
to prove the isotropy of the cytoplasm have only succeeded in
emphasising the significance of the structure of the ovum for
the development of the embryo.
This experiment can be performed in what is, I think, a
more convenient manner by substituting for gravity a cen-
trifugal force, greater than gravity, but not too large. As
soon as the eggs have been inseminated they are placed with
sufficient water to allow the jelly to swell and the perivitelline
fluid to be exuded, in the tube of the centrifuge, and centri-
fuged at once for a short time. The eggs, of course, lie hap-
hazard in the tube, with their axes making any angle with the
64, J. W. \JENKINSON.
direction of the force, and sihce they cannot move until the
perivitelline space has been de veloped their contents are re-dis-
tributed as in Pfliiger’s experimewt. Taey are then removed
from the machine. It wiil be found that they do not turn
over, that the first and second cleavages are parallel, the
third at right angles to the direction of the force, and that the
dorsal lip of the blastopore appears just on the centrifugal
side of the (new) equator of the egg.
Wetzel and Hertwig have recently employed this method in
the study of the particular case of complete inversion, which
does not apparently prevent the formation of the embryo, as
stated by Pfliiger.
The credit of subjecting the eggs of the frog to a still
higher centrifugal force belongs to O. Hertwig. He found
that the sezmentation of such eggs was meroblastic. A cap
of small cells or blastoderm was formed resting upon an
undivided though nucleated yolk, and these yolk-nuclei were
large and irregular, resembling the giant nuclei found in the
large-yolked eggs of fishes and other forms. If removed
from the centrifuge in time these eggs developed, though
monstrosities (spina bifida) were frequent.
More recently Morgan, Konopacka and McClendon have
all made contributions to the problem of the relation between
this disturbance of the egg-structure aud the development of
the embryo.
Morgan has shown that with a still higher speed (1600 revo-
lutions per minute for seven minutes, R = 5 in., f. = 370 g.)
the egg of the American species Rana sylvatica develops
a grey patch round the animal pole owing to the heavy
pigment being driven centrifugally into the interior of the
egg like a plate. In these eggs the first and second furrows
are approximately meridional, but those of the third phase
are abnormal in being again meridional. The dorsal lip of
the blastopore is in the normal position, but the yolk plug
may be pigmented. The tadpole is antero-ventrally unpig-
mented, but this defect may be made good in later stages.
Morgan refers to but does not follow up nor adequately describe
CENTRIFUGED EGG OF THE FROG. 65
an interesting abnormality which he says is not uncommon.
In this the front end of the nervous system is malformed, the
neural folds each terminating in a knobbed extremity. With
still longer exposures the yolk fails to segment, but ‘‘ the more
fundamental questions relating to the distribution of the
materials of the egg, and the interpretation as to whether
these visible substances are organ-forming or organ-deter-
mining have not been discussed. It is evident that the black
pigment has no such function, but further experiments will be
necessary in order to determine what value the other sub-
stances in the egg may have ””—a conclusion with which we
may cordially agree.
The principal object of the work of Konopacka (on Rana
fusca) is to discover whether there is any alteration in the
sensitiveness of the egg to this disturbing agent during the
early stages of fertilisation and segmentation, and it does
indeed appear that the number of abnormal embryos is greater
when the eggs are centrifuged during the early cleavages than
when the operation is performed upon unfertilised intra-uterine
eggs, or on eggs in process of fertilisation. Apart from this,
however, the paper contains a description and figure, the
first published, of the alteration in the structure of the egg-
cytoplasm, as well as an account of some of the monstrosities
produced. With short exposures to a fairly high acceleration
(f = 228 g.) the grey area of Morgan appears, and is seen in
sections as a layer of yellow vacuolated hyaloplasm sur-
mounting the inwardly driven pigment and the yolk. With
longer exposures a white hyaloplasmatic layer is interpolated
between the vacuolated and pigmented layers, while another
vacuolated layer appears between the white layer of hyalo-
plasm and the yolk while the first vacuolated layer becomes
folded. No attempt is made, however, to investigate the
gradual genesis of these layers, nor to ascertain the chemical
nature of the substances composing them. Amongst the
abnormalities described are numerous half-embryos, a portion
of the egg having remained unsegmented, embryos with
persistent blastopores, and tailed but headless monsters.
~
voL. 60, part 1.—NEW SERIES. 9)
66 J. W. JENKINSON.
The last, as I hope to show, are of particular. interest, but the
author figures only the external appearance of one, whose
development has been very much arrested, and gives no
detailed description of the anatomy of either this or other
stages of the malformation.
The most recent work of all, that of McClendon, marks a
very great advance, for here we have for the first time a
chemical analysis of the various layers into which the egg
cytoplasm is separated by the centrifuge. By the use of a
considerable force for a short time (f= 2771 g., for five
minutes) the eggs of Rana pipiens and Acris gryllus
become divided into zones. In the first there are three
zones, A, a yellow centripetal cap (corresponding to the grey
area of Morgan and the vacuolated hyaloplasm of Konopacka)
which consists of globules—soluble in oils and ether—which
are mostly fat but partly lecithin, inasmuch as there is some
phosphorus in the alcohol extract of this layer. The water
content of this layer is 50 per cent. The second layer, B, is
grey, of translucent protoplasm, 82 per cent. of which is water ;
there is also some fatand lecithin. The third layer, ¢, is black,
it consists of the yolk and pigment, contains 42 per cent. of
water, some fat, a good deal of lecithin, and a large quantity of
protein which is rich in phosphorus and therefore supposed to
be a nucleo-protein. In Acris the first layer consists of a
yellow cap and a white ring, while the pigmented portion of
the third is divided into three rings.
It goes without saying that the chemical composition
of the several layers could not have been ascertained from
any investigation of individual eggs. For this purpose a
whole mass of eggs was centrifuged, the layers separated
and analysed. The method was to take, not, of course, the
laid egg with its coating of jelly, but ripe ovarian eggs. In
actual practice the whole ovary was removed, including not
only the fully grown ova, but all the young ones as well,
washed and squeezed through bolting cloth to get rid of the
stroma. The pulp so obtained was then centrifuged. This
appears to me to introduce a certain error. I must also point
CENTRIFUGED EGG OF THE FROG. 67
out that McClendon has determined only the phosphorus
content of the ethereal and alcoholic extracts and of the
solid protein-containing residues, and has given no further
proof of the presence of lecithin and nucleo-protein. ‘These
points, therefore, and of course many others remain for investi-
gation, but McClendon’s work is certainly a beginning, and
the priority belongs to him.
I myself had frequently had occasion to examine, somewhat
cursorily, the development of the centrifuged eggs of the
frog, and it had occurred to me quite independently, as indeed
it would naturally occur to anyone with such eggs under his
eyes, and before I became acquainted with McClendon’s papers,
that the layers might be obtained in sufficient quantity for
chemical investigation if a mass of egg-pulp were centrifuged.
Moreover, if the abnormal development of the embryo really is
a consequence of the derangement of the materials of the
cytoplasm, it ought to be possible to relate a certain degree
of malformation with a certain degree of derangement by com-
paring on the one hand the composition of the several layers
in masses of egg-pulp centrifuged at different speeds, and on
the other the development of embryos from eggs centrifuged
at corresponding speeds. Such an investigation, though it
would not certainly lead to great results, yet seemed well
worth undertaking. But before that could even be possible
there was a preliminary question to answer. As a result of
similar experiments on the ova of various Invertebrates it has
been seriously suggested that the polarity of the egg, to
which the structure of the embryo bears such a very detinite
relation, is-not determined by the disposition of the various
visible and separable constituents of the cytoplasm, which,
indeed, so it is maintained, may be driven by the centrifuge
to any region of the egg, leaving the original polarity intact,
and without prejudice to the normality of development.
It became necessary, therefore, first of all to inquire into the
structure of the embryos produced from such ova, and the
relation of that to the derangement of the egg materials.
For only if it should turn out that the polar structure of the egg
68 J. W. JENKINSON.
to which the normal development of the embryo is related is
determined, in part at least, by a certain arrangement of the
visible materials which can be actually separated by the
centrifuge, would any attempt to ascertain their chemical
nature be of the slightest value, however successful. It was
with these objects in view that the experiments which are now
to be described were undertaken in March and April last.
I shall deal first with the structure and development of the
centrifuged eggs (which in any case have not yet been com-
pletely elucidated), and afterwards give such observations as
I have made on the chemical composition of the various con-
stituents of the cytoplasm.
A general discussion of these results and of their bearing
on the large problem of the nature and origin of the polarity
of the ovum and its relation to the development of the
embryo will be found in a final chapter.
T will only add here that I am under very great obligations
to my friend Dr. Ramsden, not only for permitting me to
work in the Laboratory of Physiological Chemistry, but also
for giving me the most valuable advice and much personal
assistance.
I am also very much indebted to Professor Dreyer for
allowing me the use of the centrifuge in his laboratory, and
to Dr. Scott for the loan of a freezing microtome.
Il. THE EXPERIMENTS.
(A) THe Structure anD DEVELOPMENT OF THE CENTRIFUGED
Kea or THE F Rog.
(1) Methods.—I have used the eggs of the common
English frog. The eggs were taken from the uterus, in-
seminated, and allowed to remain in water for about an hour
until the jelly had swollen and the perivitelline fluid been
exuded. The eggs turned into the normal position with the
axis vertical and the white pole below. They were then
CENTRIFUGED EGG OF ‘THE FROG. 69
placed on the centrifuge, which was at first set in gentle
motion to turn the axes of the eggs into the direction of the
force, and then more rapidly to bring about the desired effect.
The eggs were thus centrifuged in the direction of their axes
with the animal pole centripetal.
Various speeds were used, but I am unable to state in any
case the precise number of revolutions per minute.
In series G the electrically driven machine was used, and at
a speed of about 1500 revolutions. In the others a water-
driven machine was employed, at speeds varying from about
1100 to 3000 revolutions a minute. These speeds, which [
eall I, II, III and IV, I being the lowest, were determined by
the angle through which the tap of the apparatus was turned,
Owing, however, to the inconstancy of the water-pressure
they probably varied in the course of the experiments.
The radius in both machines was about three inches.
Different exposures were used, from five to thirty minutes.
After the treatment the ova were removed to vessels of
clean water.
(2) Details of the Experiments.
G.
Centrifuged 27 : iii: 13 on the electrically driven machine at bottom
speed.
1. One hour after insemination, centrifuged for 5 minutes.
A grey patch appears round the animal pole.
The first two segmentation furrows are normal.
28 : iii: °13.—The eggs have segmented normally.
The grey patch is no longer visible.
8:iv:°13.—The tadpoles have hatched out, but some are abnormal,
the yolk-sac being swollen, and are inert.
All preserved in picric acid.
Of these tadpoles 41 are apparently normal, i.e. like the controls |
12 abnormal. Of the latter 10 are of type (a), 2 of type (pb).
G.1. 8:iv:’13 (a) (Text-fig. 1, Pl. 11, fig. 19)—The operculum is
growing back over the external gills, the tail and fin well-developed.
The head is somewhat warty and wrinkled.
Sections show that the anterior head ectoderm is vacuolated, as are
also the olfactory pits, the brain, the Gasserian ganglia, the suckers
70 J. W. JENKINSON.
and the wandering mesoderm cells. The anterior end of the brain is
single.
The optic cup is some distance from the ectoderm ; its cavity is small
and encloses a blood-vessel (vitreous body).
There is no lens. The corneal mesoderm is present, but the con-
junctiva is not cleared. The infundibulum is present and the pituitary
body. ‘The auditory vesicles are well formed.
The notochord begins behind the infundibulum: its structure isnormal.
TExt-FIG. 1.
G.1. 8:iv:’13 (a). The lens-less eye. There is a blood-vessel in
the small cavity of the optic cup. Corneal mesoderm and con-
junctival ectoderm, choroid and sclerotic mesoderm are shown.
Differentiation of the skull and visceral arches has begun, the
labial cartilages, Meckel’s cartilage, the trabecule with the anterior
trabecular plate and cornua, the quadrate, the parachordals, the
hyoid and branchial arches being all present.
The thyroid is already separated, the trachea and lungs have been
formed. There are external gills, and the internal gills are beginning
to appear.
The heart is normal. Aortz and cardinal veins present.
The pronephros has the usual three funnels on each side and a
glomus; the ducts open to the cloaca.
CENTRIFUGED EGG OF THE FROG. yA
The gut is normal.
There is a blastema for the pelvis and hind limbs.
Germ-cells are present at the root of the mesentery.
The tadpole is clearly abnormal only in the vacuolation of the anterior
ectoderm and its derivatives and in the absence of the lens.
G.1. 8:iv:°13 (b) (Text-fig. 2, ad, Text-fig. 4, Pl. 11, figs. 20, 44a,
45, 47).—The tail isas well-developed as in the last, but the anterior end
TExT-FIG 2a.
Section of the series through the tadpole, G. 1, 8: iv: °13 (b).
Through the degenerate brain represented by a mass of pigment-
cells (br.). The rudimentary cranium (er.), the hyoid (hy.) and
parts of the branchial arches are alsoshown. Between the parts
of the skeleton are bundles of myoblasts (my.). The section
also shows gill-cleft, external gills, blood-vessels, a large lym-
phatie ventrally, and dorsally thickened and pitted ectoderm.
is seriously affected. The head appears to be abruptly truncated, the
gills far forwards. The body is swollen. There are no suckers.
Sections show the ectoderm of the head to be folded and much
vacuolated.
Olfactory pits, fore-brain, eyes, mid-brain, and part of the hind-
brain are all absent, or represented only by masses of degenerating cells.
72 J. W. JENKINSON.
In the front part of the head is found an aggregation of pigmented and
vacuolated cells, some still containing yolk-granules. These cells are
the disintegrated residue of the anterior part of the nervous system,
for some have the large pale nuclei characteristic of neuroblasts, others
the smaller, darker nuclei of spongioblasts. Groups of cells with nerve-
fibres proceeding from them are the ganglia of the fifth and seventh
TExtT-FIG. 2 b.
Section of the series through the tadpole, G. 1, 8: iv : 13 (b).
Hind-brain, with folded roof, auditory vesicles and ganglia,
cesophagus, trachea, heart, blood-vessels (aorte and cardinals).
No notochord.
nerves. One or two small vesicles of doubtful significance are also
found. In addition to the intact cells are cell-fragments, and chromatic
spherules, the remains of nuclear degeneration.
In and around this accumulation are mesoderm cells, some vacuo-
lated, others deeply pigmented (chromatophores).
The auditory vesicles are large and apparently normal; at their level
begins that part of the central nervous system which has escaped des-
CENTRIFUGED EGG OF THE FROG. 73
truction, namely the medulla, continued behind into the spinal cord.
Even this, however, is not normal. In outline it is circular, its lumen
crescentic. The thin roof is excessively folded, while in the thick floor
there is a continuous mass of white matter across the middle line below ;
next to this comes a layer of neuroblasts and next the lumen a layer of
TExtT-FIG. 2c.
Section of the series through the tadpole, G. 1, 8: iv: °15 (b).
Pronephros (one funnel on right), stomach, liver, intestine,
aorte, cardinals. Fusion of myotomes below medulla. No
notochord.
spongioblasts. The spinal cord presents the same histological char-
acters, the lateral tracts of white matter having been apparently forced
down into a median ventral position.
The ganglia of the eighth and ninth and tenth nerves are present.
The spinal ganglia are abnormal in position, being united ventrally
below the cord, an effect, presumably, of the same cause to which the
peculiar structure of the cord is due.
74 J. W.: JENKINSON.
The skull has also suffered, being represented only by a small bilateral
plate ventral to the degenerate brain, possibly the anterior trabecular
plate. Of the visceral skeleton there is a wedge-shaped piece in front
of the gut, which may be interpreted as first branchial arch, with
Text-FIG. 2 d.
Section of the series through the tadpole, G. 1,8: iv : 13 (D).
Pronephric ducts, primordial germ-cells, intestine. Spinal cord
abnormal, with white matter in a continuous ventral band,
and ganglia fused below. Myotomes fused. No notochord.
possibly mandibular and hyoid elements included in it. and behind this,
underneath the throat, a basi-branchial hearing two pairs of arches;
the relation of these to the gill-slits proves them to be the second and
third of the series. Attached to these skeletal elements are masses of
myoblasts.
CENTRIFUGED EGG OF THE FROG. 75
There is no stomodeum, and the fore-gut is in communication with
the exterior only by the gill-slits, of which the usual four are present,
the last three being open. There isarudimentary sixth cleft (pharyngeal
outgrowth). External gills are borne on the first three branchial
arches. j
The thyroid, still connected with the throat, lies in a depression of the
basi-branchial. The trachea and lungs have been formed. The heart
is normal, aorta and cardinal veins present. In front of the pericardium
TEXT-FIG. 3a.
i
i
ease
Vy
th
‘
iB
Wi
<=
tele)
90
~
(eXe)
OO %
Ks
(ola
ie
O50
2)
SE Res
7 Ah “ eS
se)
CESS RHA
OX
0
(e)
)
fe)
oO
BS
BS
=
%
fe)
Ly,
oe.
oS
pegeton Si
ce G, ereere 9
sremaasceseuans ceo
Optic vesicles and fore-brain. The pharynx below. i Dieisaviecks: (e):
Normal.
is a large sinus, lymphatic, partially divided by a septum. It causes
the body-wall to protrude. The gut is normal.
The pronephros is well-formed, but on one side there are only two
nephrostomes. The ducts open to the cloaca. There is a glomus.
The notochord is very imperfectly developed. The myotomes bend
round and fuse in a median mass of cells (myoblasts) below the spinal
cord; in this mass notochordal tissue may be here and there distin-
guished, but it is feebly differentiated. Even when present it is not
immediately below the spinal cord, but separated from the latter by
myoblasts. Germ-cells are found at the root of the mesentery.
2. One hour after insemination, centrifuged for 10 minutes.
The grey patch round the animal pole is more conspicuous.
76 J. W. JENKINSON.
Segmentation, the first two furrows, may be normal, but is sometimes
abnormal.
28 : iii: °15.—Segmentation has been normally completed. The grey
patch is still visible.
8:iv:'13.—Many of the tadpoles are retarded in development and
have failed to hatch. Those which have hatched are inert.
All were preserved in picric acid.
Of these tadpoles 28 are normal in appearance, 15 abnormal.
TEXT-FIG. 3 D.
O
= > Na
5}
2 0
2%
x Seeeraberc cis
I. 1,3:iv:13 (a). Optic vesicles and fore-brain. Lumen reduced.
The pharynx below.
Of the latter 6 of type (a) resemble externally G.1. 8: iv: 713 (a), 4 of
type (b) resemble G.1. 8:iv:°13 (b), while types (c), (d), (e), (f) and (g)
are represented by one each. Suckers and mouth are absent.
G.2. 8:iv:’13 (a) (Pl. 12, fig. 46)—The head ectoderm is con-
siderably vacuolated, and so are the mesodermal cells.
There are no olfactory pits, but two masses of vacuolated cells are
found, with nerve-fibres differentiated, each hollow, and connected with
the ectoderm by cell-strands.
The fore- and mid-brains and eyes are represented by paired and
CENTRIFUGED EGG OF THE FROG. (Wh
median masses of pigmented vacuolated cells. Some of the nuclei have
the characters of those of neuroblasts, and nerve-fibres can be seen.
The remains of the fifth and seventh ganglia and nerves are also distin-
guishable.
The brain begins as a solid mass with longitudinal and transverse
fibres at this level, and immediately behind, opposite the auditory
vesicles, a lumen appears. The hind brain and spinal cord have the
structure seen already in G.1. 8:iv:713(b). The spinal ganglia are
TEXT-FIG. 3c.
A
TEER
L. 1, 6:iv:13 (a). Optic vesicles and fore-brain. Very much
reduced. The pharynx below. The ectoderm (epidermis) is
thickened and pitted.
pressed down and may meet below. The ganglia of viii and ix and x
are present. The auditory vesicles are large, the cells vacuolated.
There is no trace of the trabeculz, but the visceral skeleton is repre-
sented by a median plate below the throat bearing three pairs of
branchial arches—better developed on one side than on the other—and
produced in front into a ring-shaped cartilage which seems to represent
the quadrate. To this skeleton are attached bundles of myoblasts,
especially anteriorly. Of gill-clefts, the hyo-mandibular and first two
branchials (the second open) are present on one side, the first three
branchials on the other (the second and third open). There are external
gills.
The trachea and lungs have been formed, the heart is small but
78 J. W. JENKINSON.
twisted. Aortze and cardinal veins are present. There are three pro-
nephric funnels on one side, but only two on the other. The ducts open
into the cloaca. The glomus is absent.
The gut is normal, but the intestine is not yet coiled. There are
germ-cells at the root of the mesentery. The myotomes bend down and
meet below the spinal cord. In this mass is the notochord, imperfectly
differentiated in front, more posteriorly vacuolated. More posteriorly
still the notochord is free and the myotomes separate.
The whole body is very edematous; the connective tissue and the
TEXT-FIG. 4.
Reconstruction of the cranium (er.) and visceral skeleton (hy. br. 1,
br. 2, br. 3) of G. 1, 8 : iv: 18 (b).
posterior cardinal vein round the pronephros suffer especially from this
accumulation of fluid.
G.2. 8:iv:’138 (b) (Text-figs. 8, 11)—Anteriorly the ectoderm is
highly vacuolated; so also are the mesodermal cells.
Ventrally the ectoderm is also folded for some little way back.
There are no suckers and no mouth. The gills are far forwards.
Olfactory pits, eyes, fore- and mid-brains represented only by a heap of
pigmented vacuolated cells, with traces of fibres.
The auditory vesicles with ductus endolymphatici. At this level the
brain begins. The hind-brain and the spinal cord have the same
characters as in (a), but the posterior part of the spinal cord is normal.
The auditory, vagus, and spinal ganglia are present, the spinal ganglia
being united ventrally.
CENTRIFUGED EGG OF THE FROG. 79
The visceral skeleton—the only skeleton developed—consists of a
median plate bearing three arches, the first, second and third, and an
anterior prolongation, situated in the front wall of the pharynx, which
represents hyoid and perhaps quadrate elements.
The hyomandibular cleft is absent. The first, second, third, and
fourth branchial clefts are present on both sides, the fourth being just
open on one side, while on the other the second and fourth are open.
TEXT-FIG. 5.
SA Ke
Mm ee
Hes. : 2:iv: 13 (b). Not very abnormal front end (section
oblique). Brain, neural crest, pharynx, one sucker cut.
There are external gills.
The trachea and lungs are formed, but the trachea is solid.
The heart is also solid, small, and hardly twisted. Aorte and cardinal
veins are formed. The pronephros has three funnels on each side, and
a glomus. The ducts open to the cloaca.
The intestine is not differentiated into regions, and in the yolk-cells
is a central mass where there are no cell-divisions and the granules are
fused into a coagulable liquid.
80 J. W. JENKINSON.
Germ-cells are found at the root of the mesentery. The myotomes
are fused below the spinal cord. The notochord is only distinguishable
behind the pronephros. Here it lies immediately ventral to the spinal
cord.
TEXT-FIG. 6.
G.2. 8:iv:'13(g). Degenerate brain (pigmented cells). The
auditory vesicles united (au.); below them the auditory and
vagus ganglia (vii, w) also united. (Esophagus, lungs, liver,
pronephros (one funnel cut on right). idematous connective
tissue.
In the ceelom and in the posterior cardinal vein round the pronephros
is an accumulation of fluid.
G2. 8:iv:’18(c) (Text-fig. 10, Pl. 11, fig. 21)—Body much
CENTRIFUGED EGG OF THE FROG. 8]
swollen; tail well developed. Neither mouth nor suckers. The head
ectoderm is vacuolated and the mesoderm. The mesoderm s edematous.
Olfactory sacs, eyes and front part of brain represented by an aggrega-
tion of pigmented and vacuolated cells, with traces of the ganglia of
v and vii, but no nerve-fibres. Hind brain, spinal cord and spinal
ganglia as in (a); the spinal cord abnormal to the end.
The auditory vesicles and the auditory and vagus ganglia are
present.
A small nodule of perichondrium in the front wall of the pharynx is
TEXxtT-FIG. 7.
Il. 4:iv:13(d). Auditory invaginations. The solid wedge
of cells in the middle of the larger endoderm cells is the de-
generating fore- and mid-brains. Ectoderm thick and pitted
(cut somewhat tangentially on the ventral side).
the only representative of the visceral skeleton, There is no trace of
the cranium.
The pharynx has an irregular cavity, but there are no gill-clefts.
The trachea and lungs are present, but the latter are short.
There is no heart.
The pronephros has only two funnels on each side, and there is no
glomus. The ducts are open to the cloaca.
The stomach and duodenum are very small.
Germ-cells are present in the mesentery.
von. 60, part 1.—NEW SERIES. 6
82 J. W. JENKINSON.
The myotomes, which are fused below the spinal cord, are poorly
developed in front.
There is no notochord except in the tail.
There is great edema of the connective-tissue spaces, of the posterior
cardinal veins round the pronephros, of the celom, and of spaces
(blood-vessels or lymphatics) around the stomach and duodenum.
The ectoderm is distended and flattened.
TExtT-FIG. 8.
G.2. 8 iv:13(b). Hind-end, normal, except for central mass
of undivided yolk in intestine and absence of lumen.
1G.2. 8:iv:’13 (d) (Text-fig. 14 a, b, Pl. 11, fig. 22).—The body
is very stunted; there is a short tail. The blastopore is widely open,
and the yolk-plug protrudes.
At the front end the ectoderm is folded, pitted and highly vacuolated.
Internally the front end is occupied by a large cavity with a lining
of flattened mesoderm cells. The cavity is probably persistent blasto-
cel. The hinder wall of this space is occupied by a mass of undiffe-
rentiated mesodermal and yolk-cells. Further back are traces of celom
and blood-vessels.
There is no sign of nervous system, notochord, or myotomes.
CENTRIFUGED EGG OF THE FROG. 83
G.2. 8: iv: 713 (e) (Text-figs. 12, 13, Pl. 11, fig. 23, Pl. 12, fig. 44 b).
—Body short; short tail; persistent large yolk-plug. No mouth; no
suckers.
Ectoderm of head folded and wrinkled ; highly vacuolated.
No olfactory pits nor eyes; no fore- nor mid-brain. These structures
are represented by a mass of pigmented cells.
Ganglia of v and vii not to be found. The auditory and vagus
ganglion pairs each united across the middle line.
TEXT-FIG. 9,
eo
ane
T
= PSU
() X a by,
ues
pO Sine
ai
secu:
I.1. 4:iv:’13(d). Thick and pitted ectoderm. Spinal cord
solid. Notochord not properly differentiated. Pronephric
ridge.
Auditory vesicles well formed, each constricted into two cavities.
The brain begins at the level of the auditory vesicles; it is solid, with
fibres ventrally.
The spinal cord has similar characters, but a lumen appears in it
here and there. The spinal cord does not reach the hind end of the
body. The spinal ganglia are united below.
Fore-gut, but no gill-slits. No lungs.
84 J. W. JENKINSON.
Gut not much differentiated. In the yolk-cells a central mass without
cell-boundaries ; here the yolk-granules are fused.
Blood-corpuscles are being formed from the yolk-cells ventro-laterally.
A pericardium is present, but the heart is represented only by a solid
cell-mass projecting into this cavity from above. Vitelline veins and
cardinal veins are present.
The pronephros has three funnels on each side, and a small glomus.
The ducts end blindly.
The pronephric tubules are enormously swollen.
TrExt-FIG. 10.
G.2. 8:iv:’13(c). Pronephros, one funnel cut on right.
Abnormal medulla. Fusion of myotomes; no notochord. Cso-
phagus. Lungs. Liver. Enlargement of posterior cardinal
vein and of lymphatics round liver. Cidematous connective
tissue.
The myotomes are united across the middle line by a mass of fusiform
myoblasts, some of which are vacuolated. No notochord, except for an
occasional vacuolation.
The peritoneal cavity is well developed.
G.2. 8:iv:’13(f)—Body very short and undifferentiated. At
one end, presumably anterior, the ectoderm is highly vacuolated and
wrinkled. At this end is what appears to be a yolk-plug, but is in
reality a yolk-burst.
CENTRIFUGED EGG OF THE FROG. 85
There is a small archenteron posteriorly opening by the blastopore,
a rudimentary pericardium, and the mesoderm is to some extent diffe-
rentiated—as myoblasts, connective tissue and blood-corpuscles.
No trace of nervous system, nor of sense-organs, nor of notochord,
nor of pronephros.
G.2. 8:iv:°13 (g) (Text-fig. 6)—Body short, neither mouth nor
Text-FIeG. 11.
cr
ae ‘| ar SS
L SerAatg
7
G.2. 8:iv:’13(b). Pronephros, one funnel cut on right,
glomus. Cclom much enlarged. Myotomes fused below
medulla. Ventral ectoderm folded.
suckers. Tail with dorsal and ventral fins. There is a side branch to
the tail but this does not receive any spinal cord.
The anterior ectoderm is much vacuolated. Neither olfactory pits
nor eyes. Fore- and mid-brains represented by a mass of vacuolated
pigmented cells. Ganglia of v and vii present, with the nerves
developing.
Auditory and vagus ganglion pairs each united below the brain.
The hind-brain, with fibres and neuroblasts stretching across the
86 J. W. JENKINSON.
thick floor, begins behind the auditory vesicle; the thin roof is much
folded. The spinal cord, which has similar characters and a small
lumen, is continued into the tail. The ganglia are united below the
cord.
The auditory vesicles are large and united, with their cavities also in
communication, above the hind-brain.
TrExt-Fic. 12.
G.2. 8:iv:°13 (e). Spinal cord nearly solid. Pronephric
tubules much swollen, especially on left.
There are external gills, and gill-clefts, two on one side, probably the
first and second branchial, the second being open, and three on the other
sides, probably the first, second and third branchial, the last two being
open.
The visceral skeleton is represented by two pieces: one, anterior to
the pharynx and even dorsal to it, probably represents the hyoid and
first branchial arches. Muscles (myoblasts) are attached to it. The
other is below the pharynx and bears a pair of arches, probably the
second branchial.
CENTRIFUGED EGG OF THE FROG. 87
Trachea and lungs.
Heart and pericardium, the heart straight.
Pronephros with three funnels on each side, the ducts open. The
glomus much enlarged.
Stomach and intestine differentiated, liver present, proctodeum
open. A central mass of fused granules in the yolk of the intestine.
The myotomes are united below the medullary tube by elongated
myoblasts, but there is no trace of a notochord.
Germ-cells at the root of the mesentery.
The connective tissue is edematous and the posterior cardinal veins
much distended.
3 One and a half hours after insemination, centrifuged for 174
minutes.
A whitish-yellow ring appears round the grey patch.
Segmentation in early stages is normal, except for the inequality of
the first or second division, or both.
98 :iii:°13.—A grey and yellow patch is still visible. The animal
hemisphere is segmented in some, but not in all. The vegetative
hemisphere is segmented in none.
8: iv:°13—All the eggs are dead, undeveloped.
4. One anda half hours after insemination, centrifuged for 28 minutes.
As the last; folds appear in the grey patch.
Segmentation is more abnormal than in the last.
28: iii :’13.—Like the last.
8: iv :’13.—All the ova dead, undeveloped.
H,
Centrifuged, 28 : iii : 13, on the water-driven machine, about one hour
after insemination.
1. For 10 minutes at speed III.
A grey and yellow patch, with folds, appears round the animal pole.
29 : iii :°13.—The yellowish patch is still present. The animal hemi-
sphere is segmented, but the vegetative is not; it is blotched with
pigment.
30 : iii :’13.—No blastopore has appeared.
2. For 10 minutes at speed II.
There is a grey patch surrounded by a yellowish ring.
29 : iii: °13—The animal hemisphere, which still shows the grey patch.
is segmented, but the vegetative is not; it is blotched with pigment.
30: iii: °13.—There is no blastopore.
3. For 10 minutes at speed I.
A grey patch appears round the animal pole, but there is no yellowish
margin.
29 :iii:’13,—The grey patch is still present.
88 J. W. JENKINSON.
The animal hemisphere is well segmented. The vegetative hemisphere
is also certainly segmented in some of the ova. It is blotchy.
30 : iii: °138.—A semicircular blastopore is present.
2: iv : 13.—Four of the embryos are dead in an early stage. Of the
others, some are normal, some abnormal, and of two types, (a) and (b). All
these embryos were preserved in picric acid.
H. 3. 2:iv:’18.—The normal embryos.
Trxt-Fie. 13.
G.2. 8:iv:18(e). Heart small, fore-gut solid. Auditory
vesicles well-formed. Ectoderm very thick and pitted.
There is a short tail stump. Stomodzeumand proctodeum. Suckers.
Sections show olfactory pits, optic vesicles, lens thickenings, auditory
vesicles in an early stage of invagination, endothelial cells of heart,
paired pericardium, pronephric ridge, sclerotome, notochord and sub-
notochordal rod. The only abnormality is the mass of fused granules
in the centre of the yolk-cells of the gut.
H.3. 2:iv:’18 (a) (Pl. 11, fig. 24)—The front end is normal,
with olfactory pits, optic vesicle, lens thickening, auditory vesicle,
stomodeum, pituitary body, suckers, paired pericardium, endocardial
CENTRIFUGED EGG OF THE FROG. 89
cells, pronephric ridge, sclerotome, notochord and subnotochordal rod.
In the centre of the yolk is a mass of fused granules. Posteriorly there
is an exposed yolk-plug bounded above by the tail—containing spinal
cord, notochord and mesoderm—and in front and on the left by a small
knob which may be a second tail, inasmuch as it contains two rounded
cell masses and some mesoderm.
H.3. 2:iv:’13 (b) (Text-fig. 5, Pl. 11, fig. 25)—The anterior end is
abnormal. The ectoderm here is highly vacuolated, the olfactory pits
TextT-Fic. 14a.
G. 2. 8:iv’13(d). Section of front end through the en-
larged head-vesicle lined by a thin layer of mesoderm and con-
taining some scattered mesoderm cells. This is the persistent
segmentation cavity.
very shallow, the fore- and mid-brain represented by a solid mass of
vacuolated cells, and there is no sign of the infundibulum nor of the optic
vesicles, The neural crest goes forwards into this region. The lumen of
the medullary tube appears first in the hind-brain just in front of the
auditory vesicles, and is continued into the spinal cord, which is normal.
The auditory vesicles are just invaginated, ductus endolymphatici
being present.
There is no stomodzeum ; the suckers are very far forwards.
90 J. W. JENKINSON.
Fore-gut with incipient gill outgrowths, but no clefts.
Heart-tube between the two sides of the paired pericardium. Slight
peritoneal cavity, pronephric ridge, sclerotome, notochord and subnoto-
chordal rod.
What appears to be an enlarged liver outgrowth extends forwards
below the heart.
A mass of fused yolk-granules in gut. Hind-end normal with tail gut
and neurenteric passage (a streak of pigmented cells).
TExt-FIc. 14D.
Posterior end of the same as Text-fig. 14a, through the yolk-
plug, with deep blastoporic involution. Some myoblasts and
blood-vessels have been differentiated.
ib
Centrifuged 31 : iii : 13 on the water-driven machine at speed IV.
1. Fifty minutes after insemination, centrifuged for 10 minutes.
A grey patch appears round the animal pole; it is surrounded by a
yellowish-white ring, and in its centre is a yellowish spot. Segmentation
is normal.
2:iv:13.—The grey patch is still visible. There is a crescentic
blastopore.
CENTRIFUGED EGG OF THE FROG. 9]
3:iv: 13.—The grey patch is still present.
Medullary folds formed, blastopore widely open and yolk-plug pro-
truding. Three embryos preserved, (a), (b) and (c).
I.1. 3:iv:’13 (a) (Text-fig. 36, Pl. 11, fig. 26).—The anterior
ectoderm thickened and vacuolated. The brain-cells are also vacuo-
lated. The optic vesicles are abnormally thick-walled, and have an
abnormally narrow lumen.
Nerve crest, sense-plate and gill-plate present. The notochord is
distinct.
In the yolk isa central mass of fused granules. The yolk-plug is
lateral (the blastopore has closed on the right, and the medullary tube
has grown back).
I.1. 8:iv:’18 (b) (Pl. 11, fig. 27)—Anterior ectoderm thick-
ened and vacuolated. The medullary groove shallow, deeper in front,
but the optic vesicles have not yet been evaginated. There is a neural
crest. The notochord is hardly distinct from the mesoderm.
There is a central mass of fused yolk-granules in the gut.
I.1. 3:iv:718 (c) (Text-fig. 3 a).—The anterior ectoderm and brain
vacuolated as in (a). The optic vesicles have a lumen of nearly normal
size. There is a neural crest, and the notochord is beginning to be
vacuolated. The vertebral plate is beginning to be separated from the
lateral plate.
There is a central mass of fused yolk-granules in the gut.
4: iv :°13.—Some are normal or nearly so, but many have large,
persistent yolk-plugs.
All these embryos were preserved.
Three are apparently normal, (a), ten abnormal. Of the latter there
are seven of type (b), and one each of types (c), (d) and (e).
I.1. 4:iv:’18 (a)—Normal except for the vacuolation of the
head ectoderm and the fusion of yolk-granules in the centre of the yolk-
cell mass.
Olfactory pits, optic vesicles, slight lens thickening, auditory invagina-
tions, stomodeum, pituitary body, notochord, pharynx, gill-outgrowths,
pericardium and heart-tube, suckers, liver, pronephric ridge, slight
peritoneal cavity, subnotochordal rod, proctodeum and neurenteric
streak of pigmented cells.
I.1. 4:iv:’18 (b) (Pl. U1, fig. 28)—Like (a), except for the
yolk-plug.
I.1. 4:iv:’18 (ce) (Pl. 11, fig. 29)—Head ectoderm vacuolated.
The front part of the brain degenerate, but the optic vesicles can be
distinguished as small, almost solid outgrowths.
Shallow olfactory pit, auditory invaginations. Suckers absent.
There are indications of a pericardium, but there is no heart.
The notochord is distinct. There is a pronephric ridge. The hind-
92 J. W. JENKINSON.
end is fairly normal except for the large yolk-plug. The spinal cord
ends in the middle line, it does not pass into the caudal swellings.
There is fusion of yolk-granules in the centre of the yolk-cells.
I.1. 4:iv:’18 (d) (Text-figs. 7, 9).—The head ectoderm is highly
vacuolated, thickened and pitted.
The fore- and mid-brains are represented only by a mass of pigmented
cells which are mingled with an aggregation of yolk-containing cells,
the front wall of the fore-gut.
In this region are found the two auditory ingrowths.
The medullary tube begins behind the latter asa solid cord, but
further back a lumen appears, nearer the dorsal than the ventral side.
TExt-FIG. 15.
L.1. 6:iv:713(c). Spinal cord very small. Notochord not
differentiated from the mesoderm. The mesoderm of the
vertebral plates united across the middle line by cells which
are beginning to elongate.
The spinal cord is similar, but anteriorly the lumen is only present here
and there; at the hind end it is better developed.
Suckers, heart, and pericardium are absent.
The neural crest is lateral, not ventral.
The pronephric ridge is well developed. There is no notochord; the
myotomes are united across the middle line by horizontally elongated
myoblasts. ‘This median cell-mass is, like the myotomes, segmented.
The gut hasa lumen. There is a mass of fused yolk-granules. The
yolk-plug protrudes.
Ti. 4:iv-*13\(e) (Text-fig. 16, Pl. 11, fig. 30).—The antenor
ectoderm is very thick, vacuolated and pitted.
CENTRIFUGED EGG OF THE FROG. 93
No fore-brain nor mid-brain, nor even auditory vesicles. There are
no suckers. The medullary tube is narrow, almost solid, and passes
into one lip of the widely open blastopore, round one side and there
ends. There is no notochord. Dorsal and ventral mesoderm are
present, but the former is not differentiated into vertebral and lateral
plates.
Neither heart nor pericardium are found, nor a pronephric ridge.
At the ventral lip of the blastopore is a short ectodermal involution,
presumably the proctodeum.
The gut has a narrow crescentic lumen.
2. Seventy-five minutes after insemination, centrifuged for 30 minutes.
There appears a yellow-grey patch round the animal pole; it is much
folded. Round it is a double whitish ring, and round this again a grey
ring.
Segmentation is abnormal. The first and the second divisions are not
meridional, and the grey patch may be cut off as a separate “ cell.”
Some fail to segment.
2: iv: °13.—Only one or two have segmented and developed further ;
the blastopore, if present, is a wide semicircle.
3: iv: ’13.—No further development. Cellular disintegration setting
in.
The zones are still distinguishable.
I. Controls, 2 : iv : °13.—Semicircular blastopore.
J.
Centrifuged, 1: iv :°13, on the water-driven machine, at speed I,
50 minutes after insemination.
1. For 10 minutes (PI. 7, fig. 7.)
2. For 20 minutes.
The results are similar in the two cases. A faint grey patch appears
round the animal pole with a margin of a rather lighter colour.
Segmentation is perfectly normal.
8:iv:°13.—The tadpoles are ready to hatch, and as normal as the
controls.
K.
Centrifuged 2:iv:’13 on the water-driven machine at speed II, 70
minutes after insemination.
1. For 10 minutes (P1. 9, fig. 13).
A grey patch appears round the animal pole with central, or sometimes
excentric, yellowish-white spots, and surrounded by a lighter marginal
ring.
Segmentation is normal.
94. J. W. JENKINSON.
2: iv: °13.—The grey patch is still visible. The ova appear to be as
well segmented as the controls.
3: iv :°13.—The grey patch is faintly visible. The blastopore is three
quarters of a circle.
4: iv :°13.—The medullary folds are closing. Fairly normal.
6 : iv : °13,—Fairly normal.
Text-Fic. 16.
‘
Haas
(/) TA
&
\
I
I.1. 4:iv 718 (e). Spinal cord reduced to a minimum. Gut
lumen very narrow. Dorsal and ventral mesoderm differen-
tiated, but no notochord. Ectoderm thickened and pitted.
Central undivided yolk-mass.
8: iv: °138.—Fairly normal, with stomodeum, suckers, nostril, gills,
tail and fin.
These tadpoles were not preserved.
2. For 20 minutes.
The grey patch becomes folded. The marginal ring is more marked,
and may be confluent with the spots inside the grey patch.
Segmentation is not normal, the first and second furrows not being
meridional, and the rate of division is retarded.
CENTRIFUGED EGG OF THE FROG. 95
2 : iv : 13.—The animal hemisphere alone is segmented. It is streaked
with white or grey. The vegetative hemisphere is blotched with
pigment.
3:iv:’13.—The blastopore has not yet appeared.
4:iv:°13.—The embryos resemble those of I. 1. 3:iv:°13. The
medullary folds are developed, the yolk-plug is exposed. The grey
patch is still to be seen at the anterior end.
6 : iv : °13.—Dead in an early stage.
These embryos were not preserved.
L.
Centrifuged 1 : iv : 13 on the water-driven machine at speed III.
1. Forty minutes after insemination, centrifuged for 5 minutes.
The grey patch round the animal pole has a whitish spot in the
centre and is surrounded by a faint marginal ring.
Segmentation is normal in form but slightly retarded on the controls.
2:iv:°13—The grey patch and whitish spot are still visible.
Segmentation has proceeded not quite as far as in the controls.
3: iv: 13.—The grey patch is still present. In some eggs the dorsal
lip of the blastopore has appeared.
4: iv : °13.—The blastopore is circular, the yolk-plug large and pro-
truding.
6: iv: °13.—There is a tail stump. The blastopore is open in many.
Twelve of these embryos were preserved.
Of these, 2 are normal, with nostrils, suckers, stomodzeum and in-
cipient gill-clefts, 9 are monstrous, and 1 undeveloped. Of the 9
monstrous embryos, | is of type (a), 1 of type (b), 1 of type (c), 2 of
type (d), 1 of type (e), 1 of type (f), and 2 of type (g).
L.1. 6:iv:’13 (a) (Pl. 11, fig. 31, Text-fig. 3c)—Anteriorly the
ectoderm is very thick and vacuolated. A solid in-growth of this
represents the fore-brain, produced into minute optic vesicles.
There are traces of olfactory pits.
The in-growth of ectoderm becomes grooved behind this point, and
the groove deepens: this is the mid-brain. Then the groove closes, in
the region of the hind-brain.
The auditory vesicles are in an early stage of invagination.
There are no suckers.
The heart and pericardium are not formed yet.
The notochord is normal, slightly vacuolated.
The mesoderm is also normal, with somites, lateral plate and pro-
nephric ridge.
There is a small lumen to the gut, open at the blastopore. There is
no proctodeum.
96 J. W. JENKINSON.
The medullary tube and notochord pass to one side of the protruding
yolk-plug. Neural crests small. In the yolk-cells there is a central
mass of fused granules.
L.1. 6:iv: 13 (b)—The vacuolated anterior ctoderm is very
thick and crinkled.
Neither nostrils nor suckers are present.
There is hardly any medullary tube in front of the auditory invagina-
tions, and that is solid.
The cells of the hind-brain are vacuolated, the spinal cord is normal.
The neural crests are small.
There are no gill-clefts.
There is a pericardium but no heart.
TExT-FIG. 17.
L.1. 6:iv:715(g) (1). No nervous system. No differentia-
tion of the notochord from the dorsal mesoderm.
Somites, lateral plate and pronephric ridge are present.
The notochord goes as far forwards as the auditory vesicle.
The proctodzum is open.
There is a mass of fused yolk-granules in the yolk-cells; the large
yolk-plug is one-sided.
L.1. 6:iv:’13 (ce) (Pl. 11, fig. 32, Text-fig. 15)—Anterior ecto-
derm thick and vacuolated.
No olfactory pits, no auditory vesicles, no suckers.
The medullary tube very asymmetrical in front; this is presumably
the hind-brain, but in the absence of the auditory vesicles it is im-
possible to say with certainty.
The lumen of the medullary tube is very small and absent in places.
Behind is an open medullary groove.
The notochord is not distinguishable in front, barely so behind.
There is a pericardium, but no heart.
CENTRIFUGED EGG OF THE FROG. 97
The pronephric ridge is indicated.
The fore-gut is much folded and crumpled.
The gut lumen disappears behind.
There is the usual mass of fused granules in the middle of the yolk-
cells, and a large yolk-plug.
L. 1. 6:iv:’13(d) (1) (Pl. 11, fig. 33)—Medullary tube and
notochord both absent. On one side of the large yolk-plug is a slight
protrusion: this is probably dorsal and represents the tail. The meso-
derm in this tail is continued forwards into the body; ventral meso-
derm is being differentiated.
There is no celom.
There are no suckers.
The anterior ectoderm is thickened, pitted and vacuolated, and there
is a central fusion of yolk-granules. The gut is limited to the small
archenteric cavity round the yolk-plug.
L.1. 6:iv:’13(d) (2).—This is similar to the last. In the dorsal
mesoderm there are indications of the differentiation of a median tract,
the notochord.
L.1. 6:iv:’13 (e) (Pl. 11, fig. 34)—Like (d), except that the
blastopore is closed, and the tail protuberance bilobed.
Of the two pits seen at the base of this tail one is quite superficial, a
mere fold of ectoderm, while the other is a deep involution which
passes in some way to end blindly. It is the proctodeum.
The vacuolated ectoderm is at the other (anterior) end.
L.1. 6:iv:’13 (f) (Pl. 11, fig. 35)—This is similar to (e) except
that the rudiment of the tail is not bilobed. Of the two depressions at
its base, one is a mere superficial folding, the other a deep passage
leading into a cavity lying behind the yolk-mass, a rudimentary archen-
teron.
L.1. 6:iv:’18 (g) (1) Text-fig. 17)—This resembles (d) (2).
There is an indication of the differentiation of the notochord—as a
median band of smaller cells—from the dorsal mesoderm. There is a
rudimentary archenteron opening under the dorsal lip of the blasto-
pore.
L.1. 6:iv:’13 (g) (2).—Similar to the last and to (d) (1). There
is no trace of a separation of the notochord.
8: iv: 13.—The remainder were preserved.
One is undeveloped, 2 are normal in form with the tail longer than
before, and 9 are monstrous. Among these tadpoles types (a), (b) and
(e), (f) and (g) are represented each by one, types (c), (d), each by two
specimens.
L.1. 8:iv:’13 (a) (PI. 11, fig. 36)—The anterior ectoderm is very
thick and pitted, and highly vacuolated.
There is no nervous system nor sense-organs. From the dorsal lip of
vot. 60, parr 1.—NEW SERIES. |
98 J. W. JENKINSON.
the blastopore a thick band of mesoderm stretches forwards ; in front
this becomes thinner. There is no trace of a notochord in it.
There is ventral mesoderm and the beginning of a pericardium.
TExT-FIG. 18;
Sez “i
Wy]
Wh
ae,
Oo
rr Al
sae
L.1. 8:iv:°13(g). Both tails are cut. Signs of segmentation
of the mesoderm in them. In the body mesoderm has been
differentiated from the yolk-cells, in which is a central undi-
vided mass of fused yolk-granules.
There is no sucker.
The blastopore is widely open; on one side is a deep involution, but
except for this no gut. The yolk-plug is large. In the centre of the
yolk there is a mass of fused granules.
L.1. 8:iv:’18(b) (Pl. 11, fig. 37).—The head ectoderm is
CENTRIFUGED EGG OF THE FROG. 99
vacuolated and thick. The fore-brain, with the optic vesicles and
infundibulum, is present. The mid-brain is also present, and the hind-
brain and spinal cord. All parts of the brain show cell-vacuolation.
Auditory vesicles, and ganglia, vagus ganglia, and ganglia of v and
vii all formed, and neural crest. The suckers are present.
The notochord, normally vacuolated, extends to in front of the auditory
vesicles. Both it and the spinal cord pass into the tail.
There is no stomodeum. The gill-slit outgrowths are solid.
The pericardium is large, but the heart rudiment is solid. The peri-
toneal cavity is still small. The pronephric tubules are developing, but
are still without lumina and funnels. There is a liver diverticulum, but
the lumen of the gut is obliterated.
There is fusion of yolk-granules in the yolk-mass, and a large yolk-
plug.
L1. 8:iv:’13(c) (1).—This resembles (a), but there is a deep
involution underneath each lateral blastoporic lip. Otherwise there is
no gut.
There are signs of the differentiation of a notochord,
There is no pericardium in the ventral mesoderm. The nervous system
is absent, the longitudinal pleats looking like medullary folds being
merely ectodermal ridges.
L.1. 8:iv:’13 (ce) (2) (Pl. 11, fig. 38)—Like the last, but a
rudimentary nervous system is present. A solid median mass of ecto-
dermal cells with paired appendages represents the brain and ganglia.
Imbedded in this are two small auditory vesicles. Behind this point a
solid spinal cord runs back and enters one of the caudal swellings.
The dorsal mesoderm is a thick plate ; it shows no signs of a notochord.
The ventral mesoderm is present, but neither pericardium nor heart.
The pronephric ridge is indicated on one side. The gut has no lumen.
In the yolk-cells a central fusion of yolk-granules.
L.1. 8:iv:’18 (d) (1).—The anterior end can only be determined
by the vacuolation of the ectoderm.
The segmentation cavity persists in front and dorsally. Below it is
the mass of yolk-cells; its roof isformed of a band of mesoderm cells
which passes backinto the dorsal lip. This band stops before the front end
is reached so that there the segmentation cavity is roofed only by
ectoderm.
Ventral mesoderm is being differentiated. There is an open blasto-
pore and persistent yolk-plug; the archenteric cavity is very small.
There is neither notochord nor nervous system.
L.1. 8:iv:’13(d) (2).—The segmentation cavity is obliterated
and there is an archenteron, narrow, but longer than inthe last. Other-
wise the same.
L.1. 8:iv:’13(e) (Pl. 11, fig. 39).—Body rounded, bearing a
100 J. W. JENKINSON.
short tail. No nervous system nor sense-organs. Dorsal mesoderm,
but no notochord. Dorso-laterally the mesoderm is thickened and these
two thickenings pass back into the tail. In each is a cavity (ccelom).
The ventral mesoderm is developed, but slight. There is an archen-
teron on the dorsal side with a postero-ventral extension. The latter is
in communication with the exterior by an ectodermal involution (proc-
todzeum).
The anterior ectoderm is vacuolated.
L.1. 8:iv:’13(f) (Pl. 11, fig. 40)—The body consists of a solid
mass of yolk-cells (in the centre of which is a region in which the
granules are fused together), enclosed by a vacuolated ectoderm. On one
side of the large yolk-plug is a tail. In this tail are paired segmented
somites, with indications of myocel. There is, however, no notochord.
On the opposite side of the blastopore is a much shorter tail-like structure.
The nervous system is completely absent. Dorsally and ventrally there
are thin layers of mesoderm.
L.1. 8:iv:’18(g) (Text-fig. 18, Pl. 11, fig. 41)—Body stumpy,
with two tail-like structures, one on each side of the large blastopore.
Each of these tails contains mesoderm, which shows signs of being
divided into pairs of somites. Otherwise this embryo resembles the
last.
The pit at the anterior end is a mere infolding of the ectoderm.
2. Forty minutes after insemination, centrifuged for 10 minutes.
The central yellowish spot is surrounded by folds of the grey patch,
the marginal white ring broader.
The first two furrows are normal in form, but retarded, not only on
the controls, but slightly on L. 1.
2: iv: °13—The grey patch and yellowish spot are still visible. The
animal hemisphere is as well segmented as in L. 1; the vegetative
hemisphere is, however, divided into only a few large cells.
' 3:iv:’13—The grey patch is still present. The dorsal lip of the
blastopore has not yet appeared.
4: iv : °13.—There are indications of a blastoporic groove at the edge
of the pigmented area.
6: iv: °13.—The yolk is still exposed.
3. Seventy-five minutes after insemination, centrifuged for 16
minutes.
The folding of the grey patch round the yellowish spot is more
marked ; the spot appears to sink in. The marginal ring is more
conspicuous and flecked with dark spots.
The first and second segmentation furrows may be normal in form,
but there are many irregularities; the furrows of the third phase are
also irregular, for instance, parallel to the first. These meridional
furrows fail to reach the vegetative pole.
CENTRIFUGED EGG OF THE FROG. 101
2: iv : °13.—The grey patch and yellowish-white ring are still visible,
but the foldings seem to have been smoothed out.
The animal hemisphere imperfectly segmented, the vegetative not at
all.
3: iv: ’13—No change.
4: iv : °13.—There is still no sign of a blastopore.
4. Seventy-five minutes after insemination, centrifuged for 25
minutes.
The marginal zone becomes broader still.
Segmentation as in 3, or even more irregular. The yellowish spot
may become cut off as a separate “cell,” the folds being incorporated
in the divisions or else entirely smoothed out.
2: iv : ’13.—The white ring is still visible.
Segmented in the animal hemisphere.
3:iv:’13.—As 3.
4: iv : °13.—There is no sign of a blastopore.
6:iv:°13.—The white ring is now very faint. No other change
(Pl. 9, fig. 14).
M.
Centrifuged 2 : iv : 13 one hour after insemination at speed II on the
water-driven machine.
1. For 10 minutes.
A circular grey patch appears round the animal pole, containing
sometimes a yellowish spot; it is surrounded by a paler border (PI. 7,
fig. 8a).
The first and second furrows are meridional as normally, but some-
times either of these divisions is unequal. They appear later than in
the controls.
The furrows of the third phase are abnormal in being meridional.
All these furrows reach the vegetative pole.
3: iv: °13.—Segmentation is completed ; the cells of the vegetative
hemisphere are rather large.
The grey patch has disappeared.
4:iv:°13.—There is a blastopore, three quarters of a circle or
circular ; it is rather large.
6:iv:°13.—The medullary folds are closing (when they have been
formed). The blastopore is still open in some cases.
Six embryos were preserved at this stage, one each of types (a) and
(b), two each of types (c) and (d).
M.1. 6: iv: ’13(a).—Rounded body, yolk-plug still exposed. A
slight back-growth of the body on the dorsal side of the blastopore is
the beginning of a tail. The archenteron is short and does not extend
in front of the middle of the body.
102 J. W. JENKINSON.
Fusion of yolk-granules occurs.
There is no medullary plate. The dorsal mesoderm is hardly differ-
entiated from the roof of the archenteron. There is no distinct
notochord ; ventral mesoderm is present.
Anteriorly the ectoderm is thickened but not so remarkably as in
later stages. It is slightly pitted.
M.1. 6:iv:’13 (b).—The anterior ectoderm is vacuolated. There
are no suckers.
Neither olfactory pits nor eyes are present. The auditory vesicles
are curved plates, slightly detached from the ectoderm.
A mass of pigmented cells in front probably represents the fore- and
mid-brains. Similar lateral masses would be the ganglia of this region,
possibly also the optic vesicles. The median mass is continued into a
fold, and this passes behind into a solid cord which presently enlarges.
The lumen of the hind-brain appears in this. The lumen is dorsally
situated. The hind-brain is continued in turn into the spinal cord, in
which the lumen is also excentrically dorsal ; posteriorly, however, itis
in the normal position.
The neural crests come fairly far down but do not meet below the
cord.
The vertebral plates are united across the middle line by horizontally
elongated cells. There is no notochord.
A pronephric ridge is present and there are indications of a
splanchnoceel in the lateral plate.
Ventral mesoderm is present but there is as yet no pericardium.
The enteron is narrow but extends far forwards.
There is a central fusion of yolk-granules.
There is a proctodeum, and a streak of pigment-cells indicates the
neurenteric passage.
The yolk-plug has been withdrawn.
M.1. 6:iv:’13 (c).—As (a), but there is no back-growth above the
yolk-plug, and the archenteron is shorter.
M.1. 6:iv:’13 (d).—As (b), but the medullary tube is smaller and
the lumen minute.
There is a yolk-plug.
8:iv:’13.—A tail is developed in most. The head is deficient.
Seven embryos were preserved, two each of types (a) and (c) and one
each of types (b), (d) and (e).
M.1. 8:iv:’13 (a).—Anteriorly the ectoderm is vacuolated. Here
there is a large cavity bounded by a layer of flattened cells. The cavity
is the persistent segmentation cavity, the lining cells mesodermal
elements which have been differentiated from the yolk-cells and passed
into the cavity, while the yolk-cells have remained in their original
position.
CENTRIFUGED EGG OF THE FROG. 103
Posteriorly the mesoderm cells pass into the main mass of yolk-
cells in which is the enteron.
There is a central mass of fused yolk-granules.
Dorsal mesoderm is present and differentiated into vertebral and
lateral plates. A median notochordal tract is distinguishable from the
vertebral plate in places.
Pronephric tubules are in course of formation.
Ventral mesoderm not well developed.
There is no nervous system. What appears from the outside to be
such is a median fold of ectoderm enclosing some mesodermal elements.
Proctodeum large but not open into enteron.
M.1. 8:iv:’13 (b) (Pl. U1, fig. 42)—Anterior ectoderm vacuo-
lated, enclosing a persistent segmentation cavity. In front this is
bounded by the ectoderm alone, but further back a thin layer of cells
appears lying inside the ectoderm. This layer is continued into the
mass of yolk-cells behind. In the latter a central mass of fused
granules.
The dorsal mesoderm passes back into the tail, and is there segmented.
There is no notochord. Ventrally the mesoderm is poorly developed.
The cavity of the enteron is small and short. The proctodzeum opens
into it.
There is no nervous system.
M.1. 8:iv:’13 (c) (Pl. 11, fig. 43)—As in (a) and (c) there is
anteriorly a persistent segmentation cavity. It is not, however, lined in
whole orin part by a sheet of mesoderm, but merely includes a certain
number of mesodermal cells.
The yolk plug is exposed, and there is a short archenteron opening by
the blastopore.
The tail springs from a thick mass of mesoderm lying beneath the
lip of the blastopore on one side; there is a similar mass of mesoderm
on the opposite side, and the two become confluent in front.
Correct orientation of this embryo is very difficult as there is neither
nervous system nor notochord, but assuming that the mesodermal
thickenings are lateral the tail is either right or left. The side on
which the two masses meet may then be dorsal.
M.1. 8: iv: ’13 (d).—The anterior vacuolated ectoderm encloses a
solid mass of yolk-cells, with peripheral mesoderm. The segmentation
cavity does not persist.
The mesoderm becomes concentrated posteriorly into three masses
which project wedge-like into the yolk-cells. At the hinder end these
three pass into the blastoporic lip, where they become continuous with
small cells on the surface of the yolk-plug. Enteron, nervous system
and notochord are all absent.
M.1. 8:iv:’13(e).—The anterior vacuolated ectoderm encloses a
104 J. W. JENKINSON.
solid mass of yolk-cells. Peripherally mesoderm is differentiated.
Posteriorly there is a short tail springing from what appears to be the
dorsal side, since the archenteron is on this and the proctodeum on
the opposite side.
The proctodeum ends blindly.
The tail contains mesoderm which springs from a dorsal median,
partly also from a lateral concentration of mesoderm.
Nervous system and notochord both absent.
2. For 20 minutes.
The grey patchislarger. A grey ring appears outside the white ring.
Segmentation as in 1 (PI. 7, fig. 8, b).
3: iv: 713.—The grey patch is still to be seen.
The vegetative hemisphere is imperfectly segmented.
4: iv :’13.—No blastopore has appeared.
3. For 30 minutes.
There is a yellowish central spot inside the grey patch.
Segmentation more irregular. The second furrow, for instance, may
be parallel to the first, and the divisions do not reach the vegetative pole.
3: iv : °13.—The grey patch is still visible. The vegetative hemisphere
is not segmented.
4: iv: °13.—No blastopore has yet been developed.
INE
Centrifuged 2:iv:°13 on the water-driven machine one and a half
hours after insemination at speed IV.
1. For 10 minutes.
The circular grey patch contains a yellowish spot surrounded by folds.
Round it is a yellowish-white margin.
By the time segmentation has begun the folds have disappeared and
the central spot is not very distinct.
The first furrow may be normal butis notalwaysso. The second also
may be meridional, but is not so always. Later furrows are irregular,
sometimes meridional, sometimes circular—that is, circumscribing a
small area in the animal hemisphere. The meridional furrows do not
reach the vegetative pole.
3: iv:713.—The animal hemisphere alone is segmented, and that
incompletely.
4: iv :’13.—No development has occurred.
2. For 20 minutes.
There is a grey ring outside the white margin.
The first two furrows may be meridional but are not necessarily so.
They do not reach the vegetative pole. Later furrows are very irregular.
3: iv: 13.—As 1.
CENTRIFUGED EGG. OF THE FROG. 105
4: iv : 13.—No development.
3. For 50 minutes.
The grey patch is surrounded by a groove. Outside this is first a
white, then a grey ring. Segmentation is very seldom regular, even in
the earliest stages. Divisions are often parallel to a meridian, and
circular furrows are common. The vegetative hemisphere is not seg-
mented.
ofiv: 13. j eo
ae ae
O.
Centrifuged 8 : iv : 13 on the water-driven machine at speed IV.
All these eggs were preserved in formol immediately or in early
cleavage stages.
1. One hour after insemination, centrifuged for 5 minutes (Pls. 7,
8, 10, figs. 1, 9, 16).
A circular grey patch appears round the animal pole; in it are radial
dark strive converging towards a central yellowish spot. The patch is
separated by a groove from the pigmented area.
The patch is sometimes grooved or folded. The first and second
furrows may be meridional, but are often parallel to a meridian. Later
furrows are very irregular. Some, but not all of the meridional furrows
reach the vegetative pole.
2. One hour after insemination, centrifuged for 10 minutes (PI. 7,
fig. 2).
The central spot is slightly depressed and surrounded by folds of the
grey patch. The patch is marked by radial striz, and surrounded by a
groove. It has a narrow, faint whitish border.
Segmentation as in 1.
3. One hour after insemination, centrifuged for 15 minutes (Pls. 7,
8, 10, figs. 3, 10, 17).
The central spot is sinking in and being covered over by the folds.
The radial striz are still present. The white borderis broader. Outside
the groove is a grey ring.
Segmentation is more irregular than in 2. The furrows hardly
_ reach the vegetative pole.
4. One and a quarter hours after insemination, centrifuged for
20 minutes (Pls. 7 and 8, figs. 4, 11).
The central spot is no longer visible, the folds having grown over it.
The grey patch is still radially striated. External to the marginal
groove is a second white ring, just above the grey ring and derived
from it.
The first furrow may reach the vegetative pole, the others do not.
Circular furrows are seen.
106 J. W. JENKINSON.
5. One and a quarter hours after insemination, centrifuged for
25 minutes.
The folds have nearly met and fused. Internal to the white border
of the grey patch is a yellowish ring.
The furrows, when meridional, do not pass beyond the pigmented
area (Pl. 7, fig. 5).
6. One and a half hours after insemination, centrifuged for 30
minutes (Pls. 7, 8, and 10, figs. 6, 12, 18).
The grey patch is now homogeneous, the radial strize having dis-
appeared. It is smaller, and immediately surrounded by a groove.
Outside this groove is a wide white ring which appears to be due to
the confluence of the two white rings of the earlier stage.
Then comes the grey ring subdivided into three zones, of which the
middle is darker.
Segmentation very irregular. The first furrow may begin at the side
instead of at the animal pole. Circular furrows are found.
(3) The Effect produced by the Centrifuge on the
Structure, Segmentation, and Development of
the Egg.
From the details of the experiments that have been given
in the preceding section it will be clear that the effect
produced by the centrifuge upon the structure of the ovum,
and upon its segmentation and development, varies with the
degree of acceleration and the length of exposure.
A strict comparison is, of course, only possible between
eggs of the same batch, centrifuged on the same machine, at
the same time, at different speeds and exposures. Obviously,
however, these conditions cannot always be observed, and
then, owing, as already explained, to diurnal, if not hourly
inconstancies in the water pressure, as well as to variations
in the eggs themselves, it may well happen that on some
occasion an exposure to a higher will not effect a greater
change, will effect, perhaps, even a less change than an’
exposure of the same length to a lower acceleration.
Fortunately, in my series of experiments, this has not
occurred, except, perhaps, once.
The accompanying table gives a résumé of the experi-
ments performed with the water-driven centrifuge :
CENTRIFUGED EGG OF THE FROG, 107
Exposure in minutes.
15 2¢
Speed. 5 10. 5. 20. 25. 30.
if Beef PEL ON a _— —
— . Jil — J.2 -- —
II — . He2 a a a a
= ed 0) re “2 — —
ee, Mid = Me 2 — M.3
BET Seen i de, — — — a
1 iva ie L. 3 L. 4 — —
Mesos ple bs —_—_ . — — i 2
— . N.1 Sp Nes 3 ae NGS
Ope. 10.2 On3si 22 OSAR En FOs oy ONG
On referring back to the preceding section it will be found
that in nearly all cases the same acceleration and exposure
produce the same, or nearly the same, effect upon the egg.
Thus in H. 3 and J. 1 there is a grey patch only, without a
markedly white border. In H. 2, K. 1 and M. 1 the patch is
surrounded by a lighter ring. In K. 2 and M. 2 the patch is
folded round a central lighter spot, but in M. 2 there is an
additional grey ring, which in K. 2 isabsent. In H. 1 and
L. 2 the patch is deeply infolded. In I. 1, N.1,and0O. 2 there
is a central yellowish spot in the grey patch, and the latter
has a lighter border. In N. 2 and O.4 the grey patch is
deeply infolded, the white border marked, while in I. 4, N. 3,
and O. 6 the grey patch is surrounded by a broad white
border, and this by a composite grey ring. When the same
speed is employed the effect is always increased by pro-
longing the exposure. Conversely, when the exposure is
constant the effect ought to vary with the acceleration, but
this has not proved to be the case invariably. ‘hus the
change in J. 2 is less than in K. 2, and less in K. 2 than in L. 4,
and similarly in the series J.1, K.1, L.2, and, again, less in
M. 3 than in O. 6, but H.1 and L. 2 are very like O. 4, L. 4 like
O. 6, so that here the case is realised of a lower acceleration
having caused a greater alteration than a higher in two
different lots of eggs, the exposure being the same.
The same discrepancy appears in the subsequent develop-
108 J. W. JENKINSON.
ment, the embryos of I. 1 being as well if not better developed
than those of L. 1 and M. 1 (which are muchalike). Develop-
ment only occurs in the series J.1 and J.2, H.3, I.1, K.1,
L. 1, M.1, and G.1 and G.2. The ova of the series H. 2 did
not develop though it would have been expected that they
should. O.1,N.1 and O. 2 would very possibly have developed
if they had been kept.
Of these J. 1 and J.2 develop best, then H.3, then G. 1,
then G. 2, then I. 1, L. 1 and M. 1. The position of the
embryos of K. 1 in the list is uncertain since they were
unfortunately not preserved. There is, however, sufficient
evidence that in the main the capacity to develop is progres-
sively decreased as the acceleration is raised and the exposure
prolonged. The experiments of the G series can only be
assigned a position amongst the others by the extent to which
the egg-structure and development are altered. In G. 1 and
2 there is a simple grey patch, and development is much
better than in any except J. 1 and J. 2. They probably lie
very near H.3. G.3 and G. 4 have the ring round the grey
patch and fail to develop.
We may now proceed to discuss in order the effects which
the operation produces upon the structure of the egg, its
segmentation and its development.
a. The Effect Produced upon the Egg-structure.
The Structure of the Normal Egg.—As is well
known, the spherical egg of the frog has a radially sym-
metrical structure about an axis, which axis has unlike poles.
The polarity is determined first by the distribution of the
superficial pigment, which only occupies about two thirds
of the egg-surface, and second, by the disposition of the plasma
(protoplasm) and yolk, the plasma being mainly, though not
exclusively, situated in the pigmented region, the yolk-granules
larger and more abundant in the unpigmented region, though
found, of course, in the plasmatic portion as well. The line
drawn through the centre (at the surface) of the pigmented
CENTRIFUGED EGG OF THE FROG. 109
and plasmatic region, the centre of the egg itself, and the centre
(at the surface) of the unpigmented yolky region is the axis,
and its unlike plasmatic and yolky poles respectively the animal
and vegetative. Internally there is also diffuse pigment,
aggregated a little more intensely in the axis and in the animal
hemisphere. The nucleus of the full-grown but immature
oocyte hes, of course, axially, but excentrically, in the animal
hemisphere, and it is in the same place that the pronuclei
meet in fertilisation. All this is, of course, a common place
of embryology, but the distribution of certain other substances
in the ovum has not, as far as I am aware, ever been described.
I allude to the fat (including lecithin, which, as I shall have
occasion to show later, is present) and the glycogen. The fat
appears to be uniformly distributed in the shape of small
globules, variable in size, lying in the plasma between the
yolk. These globules are easily demonstrated in sections of
eggs, preserved in formalin and frozen, by means of the fat stain
Sudan III. The yolk-granules remain uncoloured by the dye.
In respect of the glycogen the egg is, however, polarised,
for this substance, present in the form of small spherules, is
more abundant in the plasma of the animal than in that of the
vegetative region, the spherules being larger and more
numerous in the former, while in the latter they are excessively
minute and very scanty (Pl. 10, fig. 15).
The glycogen, therefore, like the plasma in which it is
embedded, gradually decreases in concentration from the
animal to the vegetative pole, while the concentration of the
yolk increases in the opposite direction!
This polar structure is seriously affected when the egg is
centrifuged, in my experiments initsaxis. Roughly speaking
what happens is that the lighter fat comes to the centripetal,
that is, the animal pole; the next lightest, the plasma with the
1 The distribution of glycogen in the course of development has not
yet been worked out. I have only observed that in tadpoles in which
the operculum is closing this substance is found only in the myotomes,
in the tubules and duct of the pronephros and in the roof of the
medulla.
110 J. W. JENKINSON.
glycogen, forms a layer next to the fat ; while the heavy yolk
and pigment are driven to the centrifugal, the vegetative pole.
Hence the various zones or strata into which these eggs
become divided. The somewhat complex details of the
changes are most readily made out in a series of eggs centri-
fuged at the same acceleration with successively longer
exposures, as, for example, the seriesO. The acceleration was
here considerable: the exposures were 5, 10, 15, 20, 25 and
30 minutes. The ova were preserved in formol; from this
material good sections are obtainable, either by the paraffin
method, or after freezing. The latter are necessary for the
study of the fat.
In O. 1 (Pl. 7, fig. 1, a, 6), there is developed round the
animal pole a circular grey patch radially striated with dark
lines converging towards a central yellowish spot. The patch
is separated by a groove from the surrounding pigment. The
radial striz are the lines along which the pigment is streaming
away from the centripetal pole, while the lighter fat and
plasma is moving in the opposite direction.
The yellowish spot may be excentric, and the grey patch
may be folded.
A meridional section shows, beginning at the animal pole
(Pls. 8 and 10, figs. 9, 16), (1) a superficial layer of rather
finely vacuolated plasma, staining violet with hematoxylin.
In it there is an occasional yolk-granule. Some of the
original pigment remains in this layer, being disposed in (a)
a denser sheet at the surface, (3) more diffusely below.
This layer is the grey patch seen from the surface.
(2) A more coarsely vacuolated layer of the same violet
staining plasmatic substance. In it is some pigment and a
few yolk-granules. At the level of this layer is one enormous
vacuole (v.) which presses out layer 1 on the centripetal side.
The floor of this vacuole is formed of the next layer. Both
the large vacuole and the smaller vacuoles of layers 1 and
2 contain fat, staining vividly with Sudan III, and it is the
large vacuole which is seen from the outside, shining through
the first layer, as the yellowish spot.
CENTRIFUGED EGG OF THE FROG. 1g
(3) A layer of a violet-staining plasmatic substance which
appears to have an alveolar structure (though this may be the
effect of one of the reagents used). his layer is composite,
there being denser sheets with a good deal of pigment running
through lighter patches which contain little pigment. Fat
vacuoles, singly and in groups, occur in this layer, and a good
deal of yolk remains in it.
Neither the second nor the third layer comes to the surface,
the first layer being here co-terminous with the fourth.
(4) The yolk and pigment.
Immediately below layer 3 is a sheet of pigment from
which dense streamers depend into the yolk below. The ori-
ginal sheet of superficial pigment remains at the surface.
(5) The unpigmented region round the vegetative pole
forms a fifth layer, but this, of course, was not produced by
the centrifuge.
A good deal of fat and plasma remain still in the yolk.
In O, 2 the central spot is slightly depressed, and a white
border is beginning to appear around the edge of the grey
patch (Pl. 7, fig. 2, a, b).
Sections show the following layers :
(1) With the same characters as in O. 1. Below it a large
fat-vacuole.
(2) With the same characters as O. 1, but with still less
pigment, hence the appearance of a white border round the
grey patch. This layer has probably been reinforced by
some plasma from the third layer seen in O. 1 from which the
yolk-granules have been drawn away.
(3) A broad band with numerous small yolk-granules—those
found in the third layer of O. 1—and some vacuolated plasma.
The latter has presumably just come up from the yolk below.
This layer lies immediately centrifugal to the layer of
pigment.
(4) The rest of the yolk with large granules. The super-
ficial pigment is unaltered. As layer—
(5) may be distinguished the original unpigmented area.
In O. 3 the central spot begins to sink in below the sur-
112 J. W. JENKINSON.
rounding folds of the grey patch, while the white border to
the latter is more distinct. Immediately outside this there is
in the pigmented area a grey ring (PI. 7, fig. 3, a).
The section shows the following layers (Pls. 8 and 10, figs.
VOGT):
(1) The coarsely vacuolated pigmented layer in which the
large vacuoles are embedded. This is the grey patch which
is folded. The folds appear to arise by the accumulation of
large globules or vacuoles of fat. As these are forced more and ©
more centripetally the folds which are caused by them pass
over the central depressed portion of the grey patch, which
itself appears yellow owing to the accumulation of fat there.
In some of the vacuoles of this layer there is a coagulated
fluid, which is stained pink with eosin. This appears to be a
protein distinct from the material of which the walls of the
vacuoles are composed. Whether the same vacuoles also
contain fat is not certain ; in that case this might be a protein
material accompanying the fat—the thin envelope, perhaps,
of the fatty globules. On the other hand, the eosinophilous
material may be normally associated with the other proteins of
the plasma, and only dissociated from it by a certain degree
of centrifuging. We shall see later that there is evidence for
the existence of more than one kind of protein in the plasma.
(2) A layer which appears homogeneous under a low,
finely alveolar under a high power. It stains violet with
hematoxylin. In it are a few fat-vacuoles, but it contains no
yolk-granules. Pigment is scattered through it, and the
remains of the superficial pigment is seen at its external
surface. It is the white border of the grey patch. This is
identical with layer 2 in O. 2, but is thicker, and has lost
nearly all its fat.
(3) Below this stretches a thin sheet of pigment, ana
immediately below this a broad vacuolated layer. The walls
of the vacuoles are formed of plasma—staining with hema-
toxylin—with yolk-granules embedded in it; the cavities
of the vacuoles are occupied by fat. Pigment is scattered
through this layer, but sparsely, and at the external surface the
CENTRIFUGED EGG OF THE FROG. 113
denseness of the original pigment sheet is much diminished.
Externally this appears as the grey ring.
This layer is evidently derived from layer 3 of O.2. What
has happened is that more fat-globules have been driven
centripetally and accumulated here in vacuoles underneath
the dense plasma of layer 2, through which at present they
are unable to pass.
In this layer are found local accumulations of deeply
staining (violet) plasma. The plasma is, of course, being
driven centripetally. There are also vacuoles containing
eosinophilous coagulum.
(4) The pigmented region of the yolk. The lower edge of
the pigment sheet is driven inwards.
(5) The pigment-free yolk.
O. 4. (Pl. 7, fig. 4, a, b). The folds of the grey patch
have nearly met. A second white ring, derived from the
grey ring, lies just external to the white ring of the grey
patch.
The section (Pl. 8, fig. 11) reveals the same layers as in O.
3. The third layer—of fatty vacuoles with yolk-granules inthe
walls—is emancipating itself from the sheet of pigment, and
sending streamers into the plasmatic layer (layer 2) above.
The yolk-granules are evidently adherent—for the moment—
to the fat-globules, and caught up in the centripetal move-
ment. This gives the white division of the grey ring. Layer
2 is now practically devoid of pigment. The other layers are
as in O. 3.
O. 5 (PI. 7, fig. 5, a) presents very little change beyond the
close approximation of the lips of the folds of the grey patch,
but in—
O. 6 great alterations have occurred.
The grey patch—no longer radially striated—is imme-
diately surrounded by a deep groove. Outside this lies a very
broad white ring, and then a grey ring, compounded of three
zones, the middle of which is darker than the others (Pl. 7,
fig. 6, a).
Sections (Pls. 8 and 10, figs. 12, 18) show that (1) the grey
vou. 60, part 1.—NEW SERIES. 8
114 J. W. JENKINSON.
patch is composed of the same coarsely vacuolated pigmented
material as before, and encloses a large fat-vacuole; (2) out-
side the groove which bounds this patch is another layer of
vacuolated material, but this includes but little pigment. The
vacuoles contain fat.
(3) There follows the finely alveolar plasmatic layer, almost
devoid of pigment, with a few yolk-granules, and here and
there a fat-vacuole. With the transitional condition O. 4 before
us it seems easy to understand what has happened. The fat
of layer 3, which was there beginning to penetrate the
plasmatic layer 2, has completely passed through the latter
and given rise to the present layer 2—a layer which, as we
should expect, contains but little pigment. In so doing it
has shaken off the yolk-granules, which have passed back in
the opposite direction, although a few remain entangled in
the third layer. The second and third layers then form
together the broad white wing. In this process the original
groove external to the white ring (layer 2 of O. 3 and O. 4)
disappears, while a fresh groove is formed round that portion
of the fatty layer in which a considerable quantity of pigment
is still entangled, namely, in the grey patch.
(4) Underneath the homogeneous plasma layer is a sheet
—the uppermost sheet of the yolk—from which a good deal
of the pigment has disappeared. ‘This is the grey ring.
In it a finely vacuolated layer can be distinguished above
from a layer which is not so vacuolated. The former is the
paler upper zone of the grey ring, and is due to a fresh
agglomeration of fat-globules in vacuoles below the plasma
layer. In other words, the process seen in O. 4 is about to
be repeated. It must be remembered that there is still a
good deal of fat left in the yolk. With Sudan III the yolk
stains a faint orange, and proper examination reveals the
fat-globules between the yolk-granules (PI. 10, fig. 18 ¢).
I have not succeeded in finding in the sections the lower
pale zone of the grey ring.
The other layers are as before.
We can now form some idea of the changes that take
CENTRIFUGED EGG OF THE FROG. 115
place when the egg is centrifuged at this high acceleration
for progressively longer periods.
First the pigment and yolk are driven centrifugally while
the fat and plasma are urged in the reverse direction.
The fat is, however, lighter than the plasma, so that the
former occupies the most centripetal position, At the
same time the globules become agglomerated into larger
masses, some of which are enormous; a certain amount of
pigment remains obstinately adherent to the fat. Hence the
grey patch and yellow spot. The plasma forms a layer next
to this, and gradually rids itself of fat and yolk, in opposite
directions. When it is free from pigment it appears as the
white border. At the same time the pigment is spread out
in a third layer, which sends streamers into the yolk below.
This fact, coupled with the frequent mottling of the original
unpigmented area, suggests that the pigment is perhaps
heavier than the yolk.
The supply of fat and plasma in the vegetative region is,
however, by no means yet exhausted, and a fresh accumulation
is soon spread out beneath the barrier, at present impenetrable,
of the plasma layer. But eventually this gives way, the fat
passes through, dragging at first some yolk-granules with it,
but these are quickly discarded and driven back. ‘The con-
dition seen in O. 6 is thus reached, though this is by no means
the final stage, since there are evident preparations for a
fresh conglomeration of fat. What the end would be is clear
enough. The fat—with adherent pigment and plasma—would
be centripetally disposed, the yolk and pigment centrifugally,
while the plasma, including, I may perhaps now say, the
glycogen, would lie between. A discussion of the chemistry
of the components of these layers must, however, be reserved
for a later chapter.
The same kind of effect is produced with a smaller accelera-
tion, but, as a rule, the white ring appears before the central
spot—as, for example, in series M (PI. 7, fig. 8). Probably
considerable force is required to make the fat-globules cohere
in one or more large masses.
116 J. W. JENKINSON.
A section of K. 1 (PI. 9, fig. 13) shows the grey patch as a
coarsely vacuolated layer, with pigment, the pale border as
a plasma layer, with some pigment, and below this a sheet of
pigment and the yolk.
When the acceleration is smaller still, as in series J (PI. 7,
fig. 7), the only alteration revealed by the sections is an
immigration of pigment round the animal pole. This causes
the faint grey patch seen in these eggs.
As a rule the various zones and rings become somewhat
confused, and the folds of the grey patch disappear after a
short interval; but a grey or blotched patch usually persists
for a considerable time, and may often be seen at the anterior
end of the embryo.
There is, in fact, a slight re-diffusion of the disarranged
materials through one another, but the normal arrangement
is never regained—not at least in eggs centrifuged so soon
before segmentation.
b. The Effect produced upon the Segmentation of
the Egg.
With the lower acceleration the segmentation of the egg is
normal except for a slight retardation, as for example in L. 1,
L. 2, M. 1 and M. 2 (PI. 7, fig. 8), but when greater force is
applied irregularities appear (Pl. 7, figs. 1 c, 2 c,d, 3b, 4c, d,
5 b,66,c). Even though the first two furrows are meridional,
those of the third phase may be meridional instead of latitu-
dinal (as in L. 4) or parallel to the first. The first and second
are often parallel toa meridian, and with greater accelerations
the normal sequence is almost entirely abandoned. The first
furrow may begin at the side instead of at the pole (O. 6), and
circular furrows, cutting off a small region of the animal hemi-
sphere completely or incompletely, are frequently observed
(O. 4, O. 6, L. 4). Many or all of the meridional furrows fail
to reach the vegetative pole, and as a result the segmentation
becomes more or less meroblastic ; in extreme cases, indeed,
‘
CENTRIFUGED EGG OF THE FROG. 1 Ly
it ends in the formation of a blastoderm resting upon an unseg-
mented yolk, as Oscar Hertwig pointed out; as mentioned
by the same author the nuclei in the yolk become enlarged,
irregular in shape and highly chromatic, so resembling the
yolk nuclei of Elasmobranchs and Teleostei, bodies which are
concerned in the elaboration of the yolk alone, and do not
play any part in the formation of the embryo.
A section (Pl. 9, fig. 14) through one of these eggs (L. 4,
6:iv:’13) shows that though five days have elapsed since
the operation no differentiation has occurred. The egg has
merely continued to segment slowly.
Some of the strata can still be recognised.
Round the animal pole is the grey patch, lightly pigmented
and vacuolated. This region alone is properly segmented, and
even here it is not possible to distinguish cell-boundaries
between the nuclei of the deeper layers. There are three or
four layers of nuclei in all. ‘
The grey patch passes into the remains of the plasmatic
layer, but this contains now masses of yolk-granules and a good
deal of pigment, both of which have evidently returned from
the inferior position to which they were driven. In this
layer are numerous large vacuoles, some clear (these evidently
contained fat), and some filled with a coagulated liquid which
stains with the plasma dye (picro-indigo-carmine). The fat-
globules are presumably due to the breaking up of the larger
vacuoles of the centrifuged egg—again a return of a substance
towards its original position. This layer contains nuclei,
many of which are homogeneous and highly chromatic, while
others are very large and irregularly amoeboid.
Below is the yolk, in the upper (centripetal) region of
which are a few nuclei, of a large but not excessive size.
Ge The Effect produced upon the Development of
the Embryo.
We have at our disposal embryos and larvee obtained from
the series J, H. 3, G. 1, G. 2, I. 1, L. 1 and M. 1.
118 J. W. JENKINSON.
In the series J the acceleration was small and the tadpoles
were normal.
In the series G. 1 a larger acceleration was (probably)
employed, and out of 53 tadpoles 12 were abnormal. ‘These
tadpoles were twelve days old when preserved. The H. 3 ova
were subjected to about the same force as those of G. 1, but
the embryos were killed at an earlier stage when five days
old. In G. 2 the same force was used as in G. 1 but the
exposure was longer: out of 43 larve (killed at 12 days) 15
were abnormal. The force employed and the effect produced
in the remaining series were greater, the degree of abnor-
mality being progressively larger in I. 1 (10 abnormal out of
13), L. 1 (17 abnormal out of 21), and M. 1 (all abnormal).
The numbers are, however, too small to be genuinely signifi-
cant, and all the embryos of these three series may be placed
in one class. Those of I. 1 were preserved on the third and
fourth days, those of L. 1 on the fifth and seventh days, and
those of M. 1 on the fourth and sixth days after the operation.
The available material should, therefore, provide an oppor-
tunity for the study of the genesis of these aberrations of
development.
The distortion of development produced by the centrifuge
is of a very striking kind, and is, moreover, one which cannot
be induced as far as Iam aware by any other method. It
consists essentially of, first, a disturbance at the anterior end,
which may manifest itself merely by a vacuolation of the
ectoderm and of the nervous system and other structures in
that region, but more usually takes the form of a total disin-
tegration of the front part of the head: the olfactory pits, the
fore-brain and eyes, and the mid-brain, the skull and the
mouth, all disappear as such, and the nervous system begins
in the region of the medulla. Secondly, the yolk is affected.
The only sign of any derangement may bea tract of undivided
yolk in which the granules have become fused into one mass,
but the closure of the blastopore is often prevented, or at
least delayed, and there is a more or less persistent yolk-plug.
When it is remembered that the yolk-plug is derived from
CENTRIFUGED EGG OF THE FROG. 119
material situated in the vegetative region of the egg while
the head end of the embryo is developed near the animal pole,
the significance of the relation between these malformations
and the structural derangement produced by the centrifuge
along the axis of the ovum will be sufficiently obvious. While
these abnormalities are proceeding in the head and round the
blastopore the middle region may be developing normally,
such structures as the auditory vesicles, medulla and spinal
cord, pharynx and gill-clefts, branchial skeleton (in part),
lungs, heart and blood-vessels, alimentary canal, pronephros,
germ-cells and tail may all be of ordinary appearance. (The
tail, I may remind the reader, though, of course, posterior, 1s
developed from the lateral lips of the blastopore—that is, from
material placed originally in the equatorial region of the egg.)
There is, however, one curious malformation in this region,
which may best be described as a fusion of paired structures
in the middle line. It is seen in the abnormal arrangement
of neuroblasts in the medulla and spinal cord, in the fusion
below the spinal cord of the paired spinal ganglia, sometimes
of the posterior cranial ganglia too, and in the fusion below
this, again, of the mesodermal somites, or rather of the myo-
tomes. The last leads to the obliteration of the notochord.
Exceptionally the auditory vesicles may unite above the
medulla. All these changes occur when the ova have been
subjected to a moderate degree of force (as in the G. series) :
when a greater force is employed, the derangement is more
serious; the whole of the nervous system and the organs of
the middle region of the body may disappear, leaving an
embryo with perhaps a very short archenteron and an un-
differentiated mesoderm. A longer or shorter tail may,
however, grow out ; sometimes it is double, and the mesoderm
contained in it may show traces of segmentation.
These monsters occur mainly in the L. 1 and M. 1 series;
that they are not merely retarded embryos of an early stage,
which might possibly have developed further, is indicated by
their age—four to seven days—and by the extreme irregu-
larity of their appearance.
120 J. W. JENKINSON.
The Vacuolation of the Anterior Ectoderm (Pl. 12,
fig. 44).
This alteration occurs at the front end, extending back
some little way on both dorsal and ventral sides, but never
reaches quite to the posterior end though the ectoderm is
here often folded. Where the centrifuging has been more
severe it is more extensive.
In slight cases the ectoderm remains two-layered and only
the epidermal layer is affected (asin G. 1,8: iv: 713 [a and
b]), but after a more violent operation the ectoderm becomes
thickened byan increase in the number of layers and at the same
same time folded and pittedand the cellsinall layersareaffevted.
At the same time the vacuoles are larger (as in G. 2, and
the embryosof the I.,L.and M.series). There canbe no doubt
that in the fresh condition these vacuoles were full of the fat
forced by the centrifuge to the animal pole. Other structures
at the anterior end may be similarly vacuolated ; the olfactory
pits (G,1, 8: iv 2713 [a]), the brain (G. 1, 8: iv -°loiee
J. 1,3: iv: 713 [a, band c]), the optic vesicles (I. 1,3: iv:
713 [a and c]), the auditory vesicles (G. 2, 8: iv: 713 [a]),
the ganglia of cranial nerves, and mesoderm cells (G. 1, 8:
iv: 18 [b], G. 2, 8: iv: 18 [a}).
The Degeneration of the Front Part of the Head.
InG. 1, 8:iv:713 (a) (PL. 11, fig. 19) the brain and skull are
normal and there isa mouth. The only abnormality, indeed,
to be noticed is the absence of a lens. The optic cup has a
narrow mouth and is some little distance from the ectoderm
(Text-fig. 1).
In such early stages as I. 1, 3 : iv : 713 (a, b, and oc),
H. 3, 2: iv: 718 (a) (PI. 11, figs. 24, 26, 27), both fore-brain
and mid-brain are present and apparently normal; while
normal olfactory pits are found in H. 3, 2: iv : 713 (a),
I. 1, 4: iv: 718 (a and b), and optic vesicles in H. 3, 2: iv:
715 (ay ki oe: LS (b):
CENTRIFUGED EGG OF THE FROG. 121
As a rule, however, these structures, together with the
front part of the hind-brain, suffer disintegration by the time
such stages asG. 1, 8: iv: 713 (b), G. 2, 8: iv: 718 (a, b, c, e,
and g) (Pl. 11, figs. 20, 21, 23) are reached. All that remains
of them is then a mass of pigmented vacuolated cells lying in
the front of the head (‘l'ext-fig. 2, a), some of which have the
large pale nuclei characteristic of neuroblasts (PI. 12, fig. 45),
and from some of which nerve-fibres proceed. Mingled with
the cells is a débris of cell and nuclear fragments (the
latter having undergone chromatolytic degeneration into
deeply staining spherules), yolk- and pigment-granules.
The ganglia of this region (v and vii) often preserve
their individuality more or less completely (G. 1, 8: iv :715
[b], G. 2, 8 : iv: 713 [a, c, g]); they appear as groups of
vacuolated pigmented neuroblasts, and the appropriate nerves
can often be traced (Pl. 12, fig. 46). In one case (G. 2, 8:
iv : 713 [a]) the olfactory sacs. remain as two vacuolated
fibrous masses, still retaining their connection with the ecto-
derm. No trace of the eyes is found in these embryos.
At an earlier stage the rudiments of these organs were laid
down; their degeneration seems to set in almost at once.
Mimsin H: 3, 2: iv: 713 (b), L. 1, 6:1v: 718 (a), the. front
part of the brain is a solid wedge of cells (the mid-brain
region in the last-mentioned is an open groove), in L. 1,6: iv :
°13 (b) the brain is very small, the olfactory pits are very
shallow in H. 3, 2: iv : 718 (b), I. 1, 4: iv: 713 (c), and the
optic vesicles thick-walled (I. 1, 3: iv : ’13 [a]) or much
reduced (L.1, 6: iv: 713 [a], I.1, 4: iv: 18 [c]) (‘Text-fig. 3).
Associated with débris of the nervous system and sense
organs are of course mesodermal elements, such as wandering
connective tissue cellsand chromatophores. Mesodermal cells
may suffer vacuolation (G. 1, 8: iv : 713 [b] (PI. 12, fig.
45 b,c).
The mouth is almost always absent. It is found in the
nearly normal G. 1,8: iv : 713 (a), and in early stages a
stomodeum is present (H. 3,2: iv: 713 [a] I. 1,4: iv: 713
{a and b]). The pharynx then communicates with the exterior
122 J. W. JENKINSON.
only by the gill-clefts, which are often well developed
(Text-fig. 2, a).
The head skeleton is also profoundly modified (except in
G.1, 8: iv: 713 [a]). Properly speaking, the cranium is
entirely absent, with the exception of a small plate of cartilage
situated below the débris of the brain, which appears to
represent some part of the cranial floor—perhaps the anterior
trabecular plate—in G. 1,8 :iv :’13(b). The branchial skeleton
is, however, better developed in a few cases. In G.1, 8: iv:
’13 (b) it consists of a wedge-shaped piece embedded in the
anterior wall of the pharynx, and a triangular plate placed
below the pharynx, with a median depression, in which the
thyroid is lodged, a backwardly directed apex, vertical lateral
edges, and two pairs of branchial arches. From their rela-
tion to the gills and gill-clefts these appear to be the second
and third branchial arches. ‘The anterior piece then represents
the first branchial and the hyoid, with, perhaps, an admixture
of mandibular (quadrate) elements. Bundles of myoblasts
connect these two pieces to one another and the rudiment
of the skull (Text-fig. 4).
In G. 2,8: iv:713 (a) there is a plate under the throat
bearing branchial arches, not symmetrically nor fully de-
veloped: on one side is a large third branchial and small
fourth, second and first branchials, and a hyoid, the last two
being united a little way from the median plate ; on the other
side are long second and third, short fourth and _ first
branchials, and a short hyoid. In front the plate is produced
into two curved pieces, which may be extensions of the hyoid.
Bundles of myoblasts pass from this anterior to the more
posterior parts of the apparatus.
In G. 2, 8: iv: 713 (b) there is a plate bearing three pairs
of short branchial arches, the first and second being united
on each side. In front, embedded in the anterior wall of
the pharynx, is a wedge-shaped piece, resembling that of G.1,
8:iv:713 (b), but here united to the main plate. It appears
to be hyoidean.
In G. 2, 8: iv: 713 (g) there is a similar anterior piece,
CENTRIFUGED EGG OF THE FROG. 123
with, however, irregular anterior prolongation. Behind, and
below the pharynx, is a plate with one pair of irregular
arches. lastly, in G. 2, 8: iv: 713 (c) the branchial skeleton
is reduced to a small nodule in the front wall of the pharynx.
Gill-slits are absent in this larva.
Suckers are almost invariably absent. They are found,
indeed, only in G. 1, 8: iv: 713 (a), L.1,8:iv: 718 (b) (PI. 11,
fig. 37), and in comparatively early stages, such as H. 3,
2:iv:713 (aand b), I. 1, 4: iv: 713 (a and b) (Text-fig. 5).
The Changes at the Yolk and Blastopore End.
SaoG- 2,8: iv:713 (b) (Text-fig. 8
which are as much or more malformed, a central region is
observable in the mass of yolk-cells in which cell divisions
are absent and the yolk-granules fused together. ‘This would
seem to be due to withdrawal of plasma. Another con-
sequence of the operation is the delay in the closure of the
blastopore, seen, for instance, in G. 2, 8 : iv: 713 (d, e), H. 3,
mee 13 (b), I. 1,.3:1v 2713 (a, b, c), 1. 1,4:1v2 718 (b,
c, d, e), and in most of the very severely affected embryos of
the Land M series (PI. 11, figs. 22, 23, 25, 26, 27, 28, 29, 30).
io, 1, 6: iv : 713 (e and f), in L. 1, 8: iv : 713 (e), m M. 1,
6: iv: 713 (b) and in M. 1,8: iv: 138 (b and e) the yolk-plug
is, however, withdrawn in spite of the very serious arrest of
development (PI. 11, figs. 34, 35, 39, 42).
), and in all embryos
/
Fusion of Paired Structures in the Middle Line;
the Hind-brain and Spinal Cord, the Myotomes
and Notochord.
In those embryos in which the front part of the nervous
system has disintegrated, the brain begins at the level of tlie
auditory vesicles or sometimes a little in front of or behind
this point (Text-fig. 2b). Inasmuch as the ganglia of v and
vii, or the remains of them, are found with the rest of the
débris in the head, we must suppose that part only of the
hind-brain has escaped destruction.
124 J. W. JENKINSON.
This part has the structure of the medulla with a thin roof
and a thick floor, but is still not quite normal since the floor
is excessively thick, often projecting into the lumen, while a
mass of white matter occupies the whole of its ventral side,
uninterrupted by any cells. The cells lie above this, the
spongioblasts next the lumen, the neuroblasts above the
fibres. The lumen is itself further dorsal than it should be.
The roof is excessively folded.
In this region are found the auditory and vagus ganglia;
they often approach one another in the middle line and may
meet (G. 2,8: iv: 713 [e and g]) (Text-fig. 6).
The auditory vesicles (Text-fig. 2) are well developed. In
G. 2,8: iv: 13 (e) each is constricted into two distinct parts,
and in G. 2,8: iv: 713 (g) the vesicles of opposite sides are
united in front of the hind-brain (Text-fig. 6). They are
found, of course, in earlier stages (H. 3, 2: iv: 713; 1.1,
4:iv:713 [d]) (Text-fig. 7). The spinal cord (PI. 12, fig. 47)
(Text-fig. 2d) has the same defects as the medulla—that is to
say, the lumen is driven dorsally by an excessive thickening
of the floor across which runs a continuous band of fibres;
next to this comes a layer of neuroblasts, and then the spongio-
blasts next the lumen (G. 1, 8: iv: 713 [b], G. 2,8: iv: 713
[a, b,c, e, g], M.1,6:iv:713 [b]). Posteriorly, however,
it may be normal (G. 2, 8: iv: 713 [b], M. 1, 6: iv: 713 [b])
(Text-fig. 8), and may be normal throughout (L. 1, 6: iv : 713
[a]; 8:iv:713[b]). The abnormality is interesting ; it is as
though the lateral tracts of fibres had come down, forced the
neuroblasts of the ventral cornua upwards towards the canal,
and then met in a continuous ventral band. In other words,
there has been a median fusion of paired structures, and the
same phenomenon is seen in the fusion below the cord of the
spinal gangha (G. 1, 8: iv : 713 [b], G. 2,8: iv +718 fa,
partial], [b, in front], [c, e, g]). The ganglia, however,
still retain their proper relations with the cord in that the
fibres of the dorsal root pass upwards from their cells to enter
the cord at the side.
Like the ganglia the myotomes unite below the cord
CENTRIFUGED EGG OF THE FROG. 125
(Text-figs. 2, c, d, 10, 11) in a mass of fusiform, horizontally
placed myoblasts (G. 1, 8: iv: 713 [b], G. 2, 8: iv: 713 [a,
not posteriorly], [b, c, e, g], I. 1, 4: iv: 713 [d],M.1,6:1v:
713 [b, d]). There is little doubt that these latter cells by
which the junction is effected are cells which should have, but
have not, given rise to the notochord ; for first the notochord
is absent in these cases or only represented by an occasional
vacuolation (as in G.1,8: iv: 713 [b], G. 2, 8: iv: 713 [eand
o]),except where some part of the original material has been
saved (as in G. 2, 8: iv : 7183 [b]), and a notochord is seen
lying above the median conjunctive mass, or where there has
been no fusion (as in the hind-end of G.2,8:iv; 713 [a]). In
the second place the beginning of the process is seen in
such early arrested stages as L. 1,6: iv :713 (c) (Text-fig. 15),
M. 1, 6: iv: 713 (b, d), where the vertebral plate mesoderm
of the two sides is continuous across the middle line in a
median mass of cells which are already beginning to elongate.
In early stages (of slightly centrifugalised eggs) there is a
normal notochord (H. 3, 2: iv:’13 [b]), and sometimes in
feel. 1, 62iv :713 [a,b]; 8:iv:713 [b] 5 1. 1,4:1v:713
fa. b; c]).
Lastly, in one case, already referred to, the two auditory
vesicles are united, while a fusion of the auditory and vagus
ganglia may also occur.
I am unable to suggest any explanation of this curious
change.
The Organs of the Middle Region of the Body.
With the exceptions already noted, the posterior part of the
nervous system, the auditory vesicles, and the muscles of the
back are well developed. The same may be generally stated
of the pharynx, lungs, alimentary canal, heart and blood-
vessels, pronephros, germ-cells and tail.
The pharynx is provided with gill-evaginations, some of
which may be open (Text-fig. 2,a). InG. 1, 8:iv:713 (b)
all five pairs are present, and the last three (second, third and
fourth branchials) are open. In G. 2. 8: iv: 713 (a) the first
126 J. W. JENKINSON.
three branchials are present on one side (the second and third
open) and on the other the second, third and fourth branchials
(the last two open). In G. 2, 8:iv:7’13 (b) the first, second,
third and fourth branchials are present on both sides (the
last two open on one side, the last hardly open on the other),
and in G.2, 8: iv :713 (g) the first, second and third branchials
are present on one side (the second and third open), while on
the other are the first and second branchials (the second open).
Thus the hyomandibular evagination is found only in the
first tadpole; in the remainder branchial clefts in numbers
which differ in individuals, and on the two sides of the same
individual.
The embryos of Series H and I are too young to show
gill-clefts. In L. 1, 8:iv:’18 (b) there are solid outgrowths,
but no perforations.
As already pointed out, there is a correlation between the
presence of clefts and the development of a branchial skeleton,
as would, of course, be expected.
External gills are found in G. 1, 8: iv: 713 (b), G. 2, 8: iv:
°13 (a, band g). They are placed very far forwards (PI. 11,
fie.20). In G.1, 8:iv:713 (b) there is a small opercular fold.
The trachea and lungs (Text-figs. 2b, 6, 10) are found
in the older embryos (G. 1, 8: iv: 713 [b]. G. 2, 8: iv:718 [a,
b, c, g, but not e]), whose development is not too much
arrested. The pericardium and heart (Text-figs. 2b, 13),
with the principal blood-vessels, aortee and cardinal and
vitelline veins, are found in the better developed embryos
and are usually well formed (G. 1, 8:iv:713 [b], G. 2, 8:iv:
13 °{a, b} 6, g|, lL. 1, 8:iv: 713 [b]), but in G. 2,8: 1y2 tae
the heart is small, in (g) not twisted, and in (e) and L. 1,8:
iv :713 (b) very small and solid. Aorte and cardinal veins
may be present when the heart is absent (G. 2, 8: iv: 713 [e]).
In G. 2, 8:iv:’13 (d and e) there are irregular blood-
vessels (l'ext-fig, 146) and a structure, which is possibly
the heart, lying on one side of the pericardial cavity. In the
younger but fairly normal embryos (H. 3, 2:iv:718 [a, b],
Tai 215 )a,eb)-c), lL. 1, 6:iv::713 [b, c]))-thegimee
CENTRIFUGED EGG OF THE FROG. 127
cardium is still small, and the heart in either a very early
stage or quite undeveloped.
The peritoneal cavity is frequently well formed (Text-figs.
feo. 8) (as 1n-G. 1,8: iv :7t3 [b], G. 2, dziv : 713
[a, b, c, e, g]), and in communication with the exterior by the
pronephros (Text-figs. 2c, 6,10, 11). The full number of
pronephric tubules and funnels is found in G. 2, 8:iv:715 (a,
b, e, g), but in G. 1, 8:iv:’18 (b) there are only two
funnels on one side, in G. 2,8: iv:’13 (c) two only on both
sides. The tubules are bathed, as usually, in the capillaries of
the posterior cardinal vein. The glomus is found (except in
G. 2, 8:iv:’13 [a andc]). The ducts open into the cloaca
(except in G. 2, 8: iv: 713 [e]).
In the younger embryos (H, I, L. 1, 6:iv:713 [a, b, ¢],
M. 1, 6:iv:’13 [b, d]), only the pronephric ridge is found
(Text-fic. 9). In L. 1, 8:iv:’13 (b) differentiation of the
tubules has not gone very far.
The gut is well differentiated (with stomach, liver and
intestine) (Text-fig. 2c); in G. 1,8:iv:'18 (b), G. 2,.8:1iv:
713 (c, g), less differentiated in G. 2, 8:iv:713 (a, b, e), L.
1,6:iv:’13 (c). Inthe H and I series little differentiation,
has occurred beyond the formation of the liver diverticulum,
and the embryos L. 1, 6: iv:713 (a), 8: iv: 718 (b), M. 1, 6:
iv:713 (b, d) are in the same early, probably arrested,
condition.
A proctodzum, not open to the gut in the early or arrested
stages, is found always except where the blastopore persists.
Primordial germ-cells are found at the root of the mesentery
Gl, S:iv:713(b), G. 2, 8:iv:713 (a, b, ¢, g) (Text-fig.
2d).
A tail is found, provided with a fin, in G. 1, 8:1iv:’13 (b),
G. 2, 8: iv : 7138 (a, b, c) (Pl. 11, figs. 20, 21).. In the young
embryos it is of course only a short stump.
(Edema.
Many of these tadpoles suffer from cedema or an accumu-
lation of fluid in the connective-tissue inter-cellular spaces,
128 J. W. JENKINSON.
or in cavities (Text-figs. 6, 10, 11, 12). Such an accumu-
lation is seen in the connective-tissue in G. 2, 8:iv:713 (a
and c), in the ccelom (G.2, 8: iv:’13 [b, c, g], in the pos-
terior cardinal vein round the pronephros (G. 2, 8:iv: 713
[a, g]),in the lymphatics or blood-vessels round the gut and
liver (G. 2, 8:iv: 713 [c]), and in the pronephric tubules
(G. 2, 8:iv:713 [e]). In G. 1, 8:iv:718 (b) there is a large
ventral cavity, in front of, but quite independent of, the
pericardium, partially divided by a median septum, which
is probably due to the same causes. In several cases where
the development has been more seriously interfered with, the
segmentation cavity persists. This, perhaps, belongs to the
same category. The persistent blastoccel may contain a
few scattered mesodermal cells, but otherwise retain its
original character, its front wall being formed of the small
ectodermal cells of the animal hemisphere, its hind wall of the
large yolk-cells (M. 1, 8:iv:°13 [c]), or the mesodermal cells
which have advanced into it may give it a lining of its own,
incomplete (M..1, 8: iv :713 [b], L. 1, 8:iv:713 fa ae
or complete (M. 1, 8:iv:’13 [a], G. 2, 8:iv:713 [d]) (Text-
fig. 14a). The cavity in question has no communication
with the alimentary canal, which is, indeed, in most of these
cases very small, or restricted to the blastoporic groove.
The Changes after Severer Treatment.
While it is thus possible, if the centrifugal force applied
be not too great, to obtain tadpoles which, though deformed
anteriorly and in the region of the blastopore, are yet
more or less normal in the middle portion of the body
and in the tail, in individuals which have suffered more
seriously there is witnessed a gradual loss of structure, until
eventually no more differentiation occurs than is involved
in the production of some dorsal and ventral mesoderm
and a slight blastoporic overgrowth.
The heart usually goes before the pericardium, or, to express
it in a better way, the latter may be developed while the former
CENTRIFUGED EGG OF THE FROG. 129
is not. Thus in L.1,8:iv:’13 (b) the heart is solid, the peri-
cardium large, and in I.1,4:iv:713 (c), L.1, 6:iv:713 (b,c),
the former is absent while the latter is present. In the more
seriously injured embryos neither is found, though blood-
vessels may be (G.2, 8:iv:’13 [d]). The pronephros holds
out perhaps a little longer, as it is formed not only in the em-
bryos just mentioned, but also in M.1,6:iv:713 (b,d), which
possess no pericardial cavity. The peritoneal cavity appears
to persist in these same embryos, but is not found in the more
degenerate individuals (except possibly in L. 1,8 :1v: “LS ifel}).
The absence of the heart and pericardium in L. 1, 6: iv :’13 (a),
while the nervous system and auditory vesicles are present, may
indicate that in general the former organs are affected before
the latter. The auditory vesicles are found almost as long as
the central nervous system persists. Indeed, in G. 2, 8:iv:
’13 (e) they are well developed, while the hind-brain and spinal
cord are reduced. On the other hand, in IJ.1,4:1iv:713 (e)
and in L. 1,6:iv: 713 (c) they have disappeared, while the
hind-brain and spinal cord have remained.
Thesuccessive steps in the degeneration of what is left of the
nervous system are easy to follow. The hind-brain has a very
small lumen in I. 1,4: iv : 718 (d), is nearly solid in I. 1,
4: iv :’13 (a, b, c, e), L.1,6:iv:713 (a), M. 1, 6:iv:713(b, d),
and quite solid in G. 2, 8: iv :’13 (e), L.1, 8 :iv:18 (e) (2). The
spinal cord is solid in the last-mentioned and nearly so in the
others; in L. 1, 6:iv: 713 (c) it is still open behind (Text-figs.
15, 16). In all other cases there is no sign of the nervous
system at all (Text-fig. 17). The gut may remain—even if
only as an archenteron—when the other organs have dis-
appeared. ‘Thus it is found asa narrow but fairly long cavity
in L. 1,6: iv :713 (c), M.1, 6:iv: 718 (b, d), which still possess
a nervous system (Text-fig. 16). In G. 2, 8: iv : 718 (f),
L. 1, 6:iv:718 (f) (Pl. 11, fig. 35), L. 1, 8: iv: 713 (d, e) (PL. 11,
fig. 39), M. 1, 6: iv:’13 (a,c), and M. 1,8: iv: 713 (a, b,c, d, e)
(Pl. 11, figs. 42, 43)—none of which have any nervous system—
it is a very short cavity, opening by the proctodeeum or by the
blastopore. In G.2,8:iv:713 (d) (Pl. 11, fig. 22), L. 1, 6: iv:
von. 60, PART 1.—NEW SERIES. 9
130 J. W. JENKINSON.
13 (d), 1, 8:iverls (a, b)~(Pl. 11, figs.'36, 37), (cytes
(Pl. 11, figs. 40, 41), itis reduced to the blastoporic involution
(Text-fig. 14), while in L. 1, 6: iv: 713 (e) it is altogether
absent, though a proctodzum is present.
he tail remains in many of these extremely stunted forms
as a longer or shorter stump, as in G, 2, 8: iv: 713 (d),
li. 1, 6: iv : 713,(f), lu. 1, 8: iv: 718 (a, b, e); M. Eibe ayaa
(a,b, d), M.1,8:iv: 713 (a, b,c, e), and it is sometimes double
(L. 1, 6: iv:’18[e], L.1,8:iv:’18 [f,g]). The double rudi-
ment of the tail is seen of course in those cases where it is
represented only by two caudal swellings (I. 1, 4: iv:718 [ce],
L. 1,6:iv: 713 [a], L. 1, 8: iv: 718 [e]) (figs. 2955 aah
These tails, though devoid of nervous system and notochord,
may yet display traces of a metameric segmentation of the
mesoderm (as in L. 1, 8: iv: ’13[f, g], M.1, 8: iv: 713 [b])
(Text-fig. 18).
In the rest there is not evena tail. Dorsal and ventral
mesoderm are, however, always developed. The dorsal meso-
derm may give indications of the differentiation of a median
notochordal tract (L. 1, 6: iv: 713 [g], 8: iv: 713 [e], [1]).
(ps) On tHE CHemicaL NatTuRE oF THE SUBSTANCES IN THE
Froac’s Eaa WHICH MAY BE SEPARATED FROM ONE ANOTHER
BY THE CENTRIFUGE.
The inquiry into the chemical nature of the substances
in the several layers which appear in the centrifuged egg can
only be prosecuted with any hope of success by centrifuging
a large quantity of egg-material in vitro. For this purpose
the eggs must be obtained free from their coating of mucin-
jelly, that is, before they have entered the oviduct.
Such eggs are not, strictly speaking, in quite the same
physiological condition as the fertilised eggs, imasmuch as
in them the maturation divisions have not yet occurred.
If, however, the eggs be taken after their release from the
ovary—while they are still in the peritoneal cavity—or
immediately before that release, it will be found that the
germinal vesicle has already broken down and dispersed its
CENTRIFUGED EGG OF THE FROG. 131
contents into the cytoplasm, while the first polar spindle has
come to the surface of the egg. In their cytoplasm such
eggs are probably not so very different from those that have
become completely mature.
My first idea was to employ only ccelomic ova, but I soon
found that I could not obtain anything like a sufficient
quantity of material in this way, since at any one moment
but few eggs are found in the body-cavity, some being still
retained in the ovary, while others are already in the oviduct.
I, therefore, adopted the plan of removing the ovaries, wash-
ing them well in Ringer’s solution to remove blood and
peritoneal fluid, and then leaving them in a quantity of the
same solution at a low temperature until the eggs dropped
out. I was able to get considerable numbers of eggs by this
method, which seems to me to be preferable to that employed
by McClendon; this, as already pointed out, involves the
inclusion of all the young ova with the old ones.
From the eggs so obtained the Ringer’s solution was poured
away as completely as possible. The ova were then ground
to a fine pulp in a mortar, and this egg-pulp was centrifuged.
In the first experiments a fairly high velocity was used (about
3200 revolutions a minute) for about twenty minutes; in later
experiments, to which I shall refer presently, different and
lower accelerations and exposures were employed and their
effects compared.
The centrifuged mass becomes separated into three distinct
layers :
1. A centripetal dark yellowish-grey layer.
2. A middle light grey or opalescent layer.
3. A very thick black centrifugal layer.
The first of these consists of fatty substances and some
protein, the second of proteins and glycogen, and the third
of pigment and yolk with an admixture of fatty substances.
Layer 1.
(i) This layer is slimy and viscid, hardened at its surface into a
thin crust.
(ii) Microscopical examination: Pigment-granules, refringent
12 J. W. JENKINSON.
globules entangled into angular masses in some other
material, and some fiuid.
The refringent globules are soluble in chloroform and
acetone, and blacken with osmic acid. They appear,
therefore, to consist of a fatty material. The material in
which the globules are entangled and by which they are
partly obscured is soluble in 1 per cent. NaOH. The
globules then become more evident.
Some of the globules are nearly or quite colourless, others
of a yellow colour. There are no yolk-granules in this
layer.
(iii) When this stuff is boiled for some time in alcoholic potash
a solution of soap is formed which may be salted out with
NaCl. By CaCl, the soap is precipitated, and on the
addition of acetic a layer of fatty acid rises to the surface.
(iv) A portion of the material was mixed with °75 per cent.
NaCl and filtered.
A. The filtrate—
(1) Filters quickly.
(2) Is opalescent.
(3) Gives Millon’s reaction slightly.
(4) Gives Heller’s reaction slightly.
(5) Gives the xanthoproteic reaction slightly.
(6) Gives a slight heat coagulum.
(7) Gives a slight glycogen reaction with iodine.
B. The residue—
(1) Gives Millon’s reaction.
(2) Gives the xanthoproteic reaction.
(3) Dried to a black colour and washed well withether. The
ether becomes yellow. The residue is now grey.
(a) This grey residue gives the xanthoproteic reaction.
(8) This residue was now washed with hot alcohol, dried
and incinerated. Theashes were dissolved in dilute
HNO.,. On the addition of ammonium molybdate
yellow crystals of ammonium phosphomolybdate
appear.
(v) Another portion of this material was placed in alcohol.
a. A flocculent coagulum appears at once. The coagulum gives
Millon’s reaction.
b. The material was then boiled repeatedly in alcohol. The
alcohol becomes yellow, and several large yellow globules
fall to the bottom without dissolving.
Filtered hot—
A. (i) The filtrate, which becomes cloudy on cooling, was now boiled
CENTRIFUGED EGG OF THE FROG. 133
for half an hour with BaOH, the baryta soap filtered off,
the barium removed by passing CO,, the BaCO, filtered off
and the filtrate evaporated to dryness.
A minute fragment of the evaporate placed on a slide
under a cover-glass in IKI showed clouds of black globules
and then rectangular black crystals. This is choline ennea-
iodide, and proves that part at least of the fatty material is
lecithin.
(ii) On evaporating a portion of the alcoholic filtrate a yellow-
brown residue is left. This residue is soluble in ether, but
not in acetone. A small piece placed in water slowly swells
and puts out finger-shaped processes.
When incinerated and dissolved in dilute HNO, it gives
crystals of ammonio-phosphomolybdate with ammonium-
molybdate and coffin-lid crystals of ammonio-magnesio-
phosphate with magnesia mixture.
B. The residue washed with ether. The ether becomes yellow.
(i) While acetone gives no precipitate with this yellow filtrate,
the choline reaction can nevertheless be obtained from the
dried residue.
(ii) The second residue, after washing with ether, was dried and
placed in 1 per cent. NaOH, in which it dissolves. The
solution—
(a) Gives the xanthoproteic reaction.
(3) Gives Millon’s reaction.
(y) Gives a pink biuret reaction.
(8) Does not give the iodine reaction for glycogen.
The centripetal layer therefore contains fatty substance,
protein and a little glycogen. Part of the fatty substance is
lecithin, which can be precipitated by acetone from alcoholic
but not from ethereal solution, which will give choline, and
from which phosphorus may be obtained. Part seems to be
fat, as some of the globules are soluble in acetone.
The proteins seem to include a globulin, but others are
possibly present ; for instance, the solid protein in which the
fat-globules are embedded, which may be the same as the
eosinophilous coagulum seen in the vacuoles of the egg-
cytoplasm. The phosphorus obtained from the protein-con-
taining residues may be due to inorganic phosphates or
perhaps to a phospho-protein ora nucleo-proteid. No purine
134 J. W. JENKINSON.
bases have, however, so far been satisfactorily demonstrated
in any constituent of this layer.
This layer obviously corresponds to the grey patch in the
highly centrifugalised eggs.
The admixture of a good deal of melanin pigment is an
additional difficulty in the investigation of the proteins of this
layer.
Layer 2.
A. (i) This layer is opalescent and liquid.
(ii) The greyish colour is not due to pigment, but to angular
masses enclosing refringent globules of fatty material.
These masses are not very numerous. They are soluble in
1 per cent. NaOH.
(iii) The liquid plasma is coagulated by alcohol, the coagulum
being finely granular.
(iv) Distilled water produces a finely granular precipitate, the
granules being arranged in a reticulum in which the angular
masses are emmeshed. The greater part of the plasma
remains liquid.
(v) The plasma may be coagulated by heat.
(vi) It may be precipitated by sublimate, and by 2 per cent.
acetic.
(vii) HNO, gives a white precipitate. This becomes yellow on
heating, and with NH, an intense yellow.
B. A portion of this layer is dissolved in distilled water and
filtered.
(i) The filtrate—
(a) Is perfectly clear under the microscope.
(b) Is slightly alkaline.
(c) Is heat coagulable.
(d) HNO, produces a slight opalescence.
(e) Gives the xanthoproteic reaction.
(f) Gives Millon’s reaction.
(g) Gives a purple biuret reaction.
(h) Gives a slight glyoxylic reaction (Adamkiewicz).
(¢) Boiled with 40 per cent. NaOH and treated with lead
acetate gives no sulphur reaction.
(ii) The residue, incinerated, gives the ammonium molybdate
phosphorus reaction.
c. A portion of this layer is dissolved in ‘75 per cent. NaCl and
filtered ; it filters slowly.
CENTRIFUGED EGG OF THE FROG. 135
(i) The filtrate—
(a) Is opalescent.
(b) Is alkaline.
(c) Gives a heat coagulum.
(d) Gives no precipitate with 2 per cent. acetic.
(e) Gives Heller’s HNO, reaction.
(f) Gives the xanthoproteic reaction.
(g) Gives Millon’s reaction.
(hk) When boiled with H,SO, the vapours (of furfural) turn
anilin acetate red.
(<) Acidified with acetic, iodine gives an abundant red colour,
which disappears on heating but reappears on cooling.
(j) This glycogen reaction is not given after the liquid has
been digested with saliva.
(k) After incineration gives the ammonio-phosphomolyb-
date and ammonio-magnesio-phosphate reactions.
(ii) The residue, dried and washed with ether—
(a) Gives the xanthoproteic and—
(b) Millon’s reactions.
(c) Washed again, in hot alcohol, the residue, incinerated
gives the two phosphorus reactions.
It appears, therefore, that the second layer contains pro-
teins, a good deal of glycogen, presumably in solution, and
a small quantity of fatty substance, of which a part may be
lecithin.
The evidence, so far as it goes, seems to point to the
existence of at least two heat coagulable proteins, of which
one is soluble in water, the other not. There is also the solid
protein, in which the fat-globules are entangled. The phos-
phorus may be due to inorganic phosphates, or to phospho-
proteius or to nucleo-proteins, but no satisfactory proof of the
existence of the last has been obtained.
This layer is represented in the eggs by the white circle
outside the grey patch.
Layer 3
A. (i) Is thick and pasty.
(ii) Microscopically examined, yolk-granules, pigment and fat-
globules are seen.
136 J. W. JENKINSON.
The yolk-granules are soluble in—
(a) NaCl 10 per cent., 5 per cent., 23 per cent., but in 14 per
cent. only swell a little and lose their refringency with-
out disappearing. In saturated solution the yolk-
granules swell and become less refringent, but remain
distinct.
(b) (NH,).SO, 2 saturated solution, but not in 3 saturated or
saturated solution.
(ec) NaOH 1 per cent.
(d) Na,CO, 1 per cent., 2 per cent.
B. A watery extract is made and filtered.
The filtrate—
(a) Is opalescent.
(b) Is alkaline.
(ce) Is coagulable by alcohol.
(2d) When acidified and boiled gives a heat coagulum.
(e) Gives Heller’s reaction.
(7) Gives the xanthoproteic reaction.
(g) Gives Millon’s reaction.
(hk) Gives a purple biuret reaction.
(it) Re-filtered, the filtrate still gives a heat coagulum, and
Heller’s, Millon’s, and the xanthoproteic reactions.
(k) Gives no glycogen reaction with iodine.
c. An extract is made in °75 per cent. NaCl and filtered.
The filtrate—
(a) Is opalescent.
(b) Is alkaline.
(c) Gives a heat coagulum when acidified and boiled.
(d) With HNO, gives a cloudy precipitate, which partially
clears on boiling and reappears on cooling.
(e) This turns yellow on addition of NH,.
(f) Gives Millon’s reaction.
(g) Gives a purple biuret reaction.
(h) Is precipitated by distilled water.
(z) Is precipitated by alcohol.
(j) Does not give the anilin acetate reaction.
p. An extract is made with 1 per cent. NaOH and filtered.
(i) The filtrate gives a precipitate with 2 per cent. acetic, com-
pletely when the liquid has been neutralised.
(ii) Re-filtered, the filtrate—
(a) Is coagulated by alcohol.
(b) Gives the xanthoproteic reaction.
(c) Gives Millon’s reaction.
(d) Gives a purple biuret reaction.
CENTRIFUGED EGG OF THE FROG. LS
gE. A quantity of the material is ground up in a mortar and washed
with ether. The ether becomes yellow.
It is then boiled in alcohol, which extracts still more fatty
substance.
The residue—
(a) Dissolved in 10 per cent. trichloracetic acid, and filtered.
The filtrate gives no phosphorus reaction.
(b) Incinerated and the ashes dissolved in dilute HNO, ;
(1) Ammonium-molybdate gives crystals of ammonio-
phosphomolyhdate.
(2) The crystals washed in water and dissolved in NH;.
Magnesia mixture gives crystals of triple phosphate.
(c) Boiled with H,SO;. The fumes do not give the anilin
acetate reaction.
F. A quantity of the stuff is treated with ether and hot alcohol
until the fat and lecithin has been extracted.
It is then subjected to the following treatment, borrowed with
slight modification from Fridericia, in order to see whether
purine bases are present :
[((1) Hydrolysed by boiling for fifteen hours with 1 per
cent. H,SO,.
(2) NH, added till alkaline.
(3) Boiled till no alkaline vapours are given off.
(4) Two per cent. acetic is added till the reaction is acid,
and the liquid is boiled.
(5) The coagulated proteins are filtered off.
(6) Equal parts of 40 per cent. sodium bisulphite and 10
per cent. copper sulphate are added. The whole is
boiled for three minutes.
(7) Filtered. The residue washed repeatedly with boiling
water until the water is no longer blue.
(8) Filter-paper and residue are now put back into the same
flask that was used for the copper precipitation, water
is added, the whole brought to the boiling-point, and
excess of sodium sulphide added.
(9) Acidified with acetic and the H,S boiled off; filtered.
The residue washed with boiling water which is added
to the filtrate.
In this filtrate are the purine bases if any. }
The copper precipitate obtained from the material of layer 5
was a bright Indian red colour. The final filtrate (9) gave the
following reactions :
(a2) NH,AgNO,added. A gelatinous precipitate came down
atonce. This is the silver compound of the purine base.
138 J. W. JENKINSON.
(b) The precipitate of (a@) was washed and dissolved in hot 33
per cent. HNO;. 2a
Diameter of placental groove in middle . O@s
a re » near omphaloid
end . ; : : 5 : oOo
Diameter of praanateid cavum. : ) ee
Thickness of placental mucosa. : . 4°00
- oe trophospongia . . | Bee
cs periplacental wall . : . 4°00
i obplacental wall. : 1:00
Both poles of the blastocyst are fixed to ae wall of the
omphaloid cavum. ‘Towards the posterior pole the adcarinal
BLASTOCYST AND PLACENTA OF THE BEAVER. 201
omphalopleure was greatly folded, while the folding of the
carinal trophoblast indicates the existence of a long keel like
that shown in PI. 15, fig. 17. As in the preceding substage,
we first meet the posterior U-shaped solid end of the meso-
blast, immediately in front of it the post-stylar coelom, and
then the massive exostylar tissue (Pl. 17, fig. 35).
This series is very useful for the migration of leucocytes
from the mucosa, through the uterine epithelium and across
the cavum uteri into the chromatophilous megalokaryocytes,
as well as for the ingestion of erythrocytes by the phagocytic
megalokaryocytes. The mechanism of ingestion of the leuco-
cytes seems to consist of an active immigration, the megalo-
karyocytes, so long as they retain a free, unattached outer
surface, remaining apparently passive. On the other hand
the phagocytic activity of the implanted megalokaryocytes
is often very obvious. Such cells, when engaged in the
ingestion of red blood-corpuscles, commonly possess several
large nuclei. The differentiation of the obplacental tropho-
blast into free leucocytophagous and attached erythrocyto-
phagous cells is a very singular and constant feature of the
beaver’s preplacental blastocyst.
In front of the region represented in Pl. 17, fig. 35, the
carinal epiblast is ruptured, and this has led, over a certain
extent of the series, to almost unaccountable confusion of the
layers. It can be made out that the mesoblast becomes
massed around the hypoblastic groove which dips into the
keel as usual and is lined by cubical epithelium. The
exoccelom becomes reduced to a linear cleft above the solid
exostylar mass, and its walls may be in contact so that no
Open ccelom appears in the section. ‘The carinal epiblast
becomes greatly flattened, and there is an abrupt transition
to the peripheral or adcarinal cubical epiblast (Pl. 17,
fig. 36).
The rupture of the carinal epiblast noted in preceding
sections culminates forwards in a notch at the apex of the
keel. If such a section is examined apart from the rest, the
bilobed apex of the keel might appear to have a special
202 * ARTHUR WILLEY.
bearing, whereas it is really an artefact. After a score of
sections from the last one figured, the primitive streak can
be recognised on one side of the keel not far from the free
distal edge. Its position is approximately in the centre of
the thickened formative epiblast which bends round the edge
of the keel (Pl. 17, fig. 37). On the left side of the figure
the formative epiblast is seen blending with the cubical
adcarinal trophoblast; on the right it passes abruptly into
what is left of the flattened epiblast of the keel.
The mesoblast continues to be massive on either side of
the primitive streak. At the proximal end of the keel on
the left side of the figure there is a small space limited
externally by cubical mesoderm, internally by columnar ©
mesoderm. I interpret this as part of the pericardial pri-
mordium, as will be expiained more clearly below. As we
pass forwards from this level the massive mesoblast gives way
to open ccelom. In the anterior region of the embryonic shield
the sections present the general appearance illustrated in Pl.
17, fig. 38. The keel is now embryonic; at its edges are
seen incipient amniotic folds. The hypoblastic groove is cut
tangentially near its anterior termination, and its carinal part
appears separated from the omphaloidean hypoblast. Another
part of the pericardial primordium is here seen in the meso-
blast distad of the hypoblastic groove.
In front of the embryonic shield the anterior terminal
portion of the exoccelom extends characteristically into the
proximal half of the keel, leaving the distal half free (Pl. 17,
fig. 39). Thus there is a very definite keel-formation near the
anterior pole of the blastocyst, well in front of the embryonic
shield.
The anterior and posterior trophoblastic portions of the
keel with their exoccelomic spaces correspond in position
with the ovate areas on the mature foetal sac which I described
(1912, p. 203) as fenestre pyriformes or umbilico-
placental areas. Between the pre-stylar and _post-stylar
keels occurs the exostyle ; between the anterior and posterior
pyritorm areas occurs the placenta.
BLASTOCYST AND PLACENTA OF THE BEAVER. 203
VIII. Susstrace E.
The series is cut transversely from before backwards, and
is numbered VIF in the Utrecht catalogue. There is noto-
chordal contact between hypoblast and formative epiblast in
front of the primitive streak.
Millimetres.
Height of gestation sac : : . about 10-00
Diameter a = : : : 727560
Height of cavum uteri. ' ; Sar tor 00)
Depth of placental groove . ; 2°79
Diameter “4 e : : 6: 50 to 0°80
Pe omphaloid cavum : : S) s;00
Thickness of placental mucosa. - about 3:00
s s trophospongia . > dred
53 periplacental wall . : ae)
sé obplacental wall : ' 1:00
The anterior pole of the blastocyst exhibits phagocytic
adhesion to the wall of the omphaloid cavum; the posterior
pole is free. Beginning from the front end, we find a long
keel composed of cubical epiblast with a hypoblastic groove
dipping into the proximal part of its cavity. Distally the
walls of the keel are agglutinated together so that the cavity
is occluded. After about twenty sections of the formation
just described, the mesoblast appears with a ccelomic cavity
embracing the sides and bottom of the hypoblastic groove,
with a Jong epiblastic keel beyond it. The hypoblastic
groove is lined by cubical epithelium with large round nuclei.
There is general phagocytic adhesion of the entire obplacental
hemisphere in the anterior region.
At length the anterior end of the folded embryonic shield
appears on the right side of the keel when the latter points
towards the observer under the microscope (Pl. 17, fig. 40).
Somewhat before this level is reached the coronal tropho-
blast begims to show patches of chromatophile cells alter-
nating with phagocytic megalokaryocytes. This leads on to
the condition of a continuous coronal chromatophilous calotte
204. ARTHUR WILLEY.
such as has been described in previous substages, the leuco-
cyte granules being extremely abundant. The mesoblast in
the region of the embryonic shield is nearly solid, with a
linear cleft representing the pericardial primordium,
Proceeding backwards with the sections, the solid meso-
blastic bands become separated at a certain spot, and the
embryonic hypoblast there comes into apposition with the
formative epiblast, its cells at the same time acquiring a
typical columnar form. It is the notochordal primordium
(Pl. 17, fig. 41). This figure shows the solid peripheral
swellings (sinus terminalis) of the mesoblastic bands
placed at slightly different levels and a little exoccelom
occurring between the swellings and the mass of the bands,
The notochordal primordium extends through about a dozen
sections of 10 u, and then it passes into the primitive streak
(Pl. 17, figs. 42 and 43). At this level of the blastocyst there
is a very extensive megalocytic attack in the coronal region,
flanked on either side by a pericoronal chromatophile band ;
beyond this there is a periomphaloid attack followed by peri-
carinal festoons — all in the typical manner previously
described (cf. Pl. 14, fig. 5).
At the posterior end of the primitive streak the massive
exostylar tissue commences and the mesoblastic bands enter
into close contiguity with the carinal epiblast over a wide
area (Pl. 17, fig. 44). The significance of the exostylar
portion of the keel with its massive mesoblast and superjacent
trophoblast (carinal epiblast) will be discussed below under
“Special Considerations.” In the mid-region of the exostyle
in this series the keel is damaged in nearly every section in
consequence of its having been torn from its viscid adhesion
to the uterine wall. The figures are further complicated by
the peculiar folding of the keel, which continues for some
distance behind the embryonic shield (Pl. 17, fig. 45).
Towards the posterior end of the exostyle the fold straightens
out and we have the appearance shown in PI. 17, fig. 46,
where the massive tissue is bordered by a simple keel with
juxtaposed walls. After this the post-stylar exoccelom opens
ig
BLASTOCYST AND PLACENTA OF THE BEAVER. 208
out in a typical manner (PI. 18, fig. 47). Finally, the two
sections of the primordial sinus terminalis meet together
(Pl. 18, fig. 48), and thereafter the mesoblast ceases.
Between the last two figures there are thirty-nine sections
of 10 wu.
Behind the posterior limit of the mesoblast we find for
some distance a didermic keel with trophoblastic extension
(Pl. 18, fig. 49). The balloon-shaped blastocyst is here free
from all adhesion to the uterine wall. Farther back, the
hypoblast withdraws from the keel (Pl. 18, fig. 50), and
eventually the section is exactly similar to that shown in
Pl. 15, fig. 17, which relates to the anterior pole of the
blastocyst in Substage B. The two poles of the blastocyst,
though differing in the extent of their adhesion to the uterine
wall, are naturally similar in other respects.
Apart from the broad difference between a free pole of the
blastocyst and a fixed pole, there is another more subtle
distinction which has been referred to incidentally and may
be exemplified once more in this series, namely, the difference
between a free and an implanted coronal disc. It may be
noted in passing that the term “ coronal” is not synonymous
with ‘ obplacental.’”” The whole of the wall of the omphaloid
cavum to which the omphaloid hemisphere of the blastocyst
may be attached is obplacental. At the level of Pl. 17,
fig. 44, the coronal region of the blastocyst in transverse
section exactly equals the diameter of the field of the micro-
scope when viewed under Zeiss oc. 4, obj. A; throughout
this extent the denuded mucosa is lined by a continuous
pseudo-epithelial sheet of megalokaryocytes closely attached
by their amoeboid peripheral ends to the decidual surface,
without any arcades. At one edge of this implanted area, a
gland opens into the pericoronal cavum at a true epithelial
surface ; at the other edge the pseudo-epithelium adjoins the
true cylindrical epithelium of the pericoronal cavum of that
side. ‘The implanted trophoblast does not sink into the
mucosa; in general the line of implantation is straight,
corresponding to the original basement membrane of the
206 ARTHUR WILLEY.
uterine epithelium. The subepithelial capillaries, at frequent
intervals, where they come into contact with the trophoblast,
actually open and discharge their red corpuscles directly
into individual megalokaryocytes. The obplacental glands
almost always open at an epithelial surface subtended by a
trophoblastic arcade, but in a few rare instances a gland is
seen to open directly upon the trophoblast, into which it
discharges a mass of intrusive chromatic granules. At the
level of fig. 47, instead of the implanted pseudo-epithehal
coronal trophoblast, we find the coronal region of the blasto-
cyst entirely free, composed of chromatophilous megalo-
karyocytes, as represented diagrammatically in PI. 15, fig. 24.
IX. Susstace F.
This excellent series shows very clearly the embryonic
shield with primordia of medullary groove, notochord, peri-
cardium and amniotic fold. It is cut from before backwards
and is numbered VI c in the Utrecht catalogue. Both poles
of the blastocyst are fixed to the wall of the omphaloid
cavum.
Millimetres.
Height of gestation sac : : : . Looe
Diameter of gestation sac. : , . 8:00
Height of cavum uteri . : : . ' oe
Depth of placental groove . ; : > Oe
Diameter of placental groove : . 0°75 to Ae
Gs omphaloid cavum. : . 9°00
Thickness of placental mucosa. : .- OG
= a trophospongia . - ee
of periplacental wall. 3°50 on one side,
4-00 on the other.
a obplacental wall =. . 1:00 to 1°85
These measurements, taken in conjunction with Pl. 18,
fig. 51, exhibit a placental groove which is considerably
wider and shallower than anything that has gone before.
This progressive expansion of the cavum uteri accords with
BLASTOCYST AND PLACENTA OF THE BEAVER. 207
the fact that the blastodise is farther advanced in develop-
ment, though the exostyle is still far from its destination on
the placental trophospongia. The estimated length of the
blastocyst is nearly 4 mm.
At the anterior pole the blastocyst is monodermic, and is
attached by the omphaloid hemisphere in broad arcades.
Then the hypoblast appears a short distance removed from
the pole; at first it stretches straight across the adcarinal
plane and a little further back dips down into the proximal
half of the keel, the distal half of which remains monodermic.
In previous substages it has been observed that in places,
the epithelium which lines the hypoblastic groove, i.e. the
carinal hypoblast, tends to break up into rounded cells that
he loosely in the cavity of the groove, but never escape into
the distal cavity of the keel. The extent and position of the
hypoblastic groove can always be made out, however dis-
connected its cells may be. This fact is due to the peculiar
behaviour of the basement membrane which exists between
the adcarinal epiblast and hypoblast.
At a certain point near the mouth of the keel the
adcarinal basement membrane can be seen to cross the
narrow interval that separates the epiblast from the hypo-
blast, and to attach itself to the latter, which it accompanies
into the keel. The trophoblastic cavity of the keel is thus
shut off from the omphalon by a delicate wrinkled membrane.
The keel-cavity contains a finely granular coagulum, which
differs slightly in its staining properties from the adjacent
coagulum in the adcarinal region, so that there is a sharp
contrast in the character of the coagulum on either side of
this structureless membrane (Text-fig. 2). The membrane is
inconspicuous and easily overlooked.
The anterior border of the mesoblast appears on one side
of the keel, viz. the embryonic side, as shown by subsequent
sections. In this region there is aremarkably deep epiblastic
keel extending far beyond hypoblast and mesoblast (PI. 18,
fig. 52). The mesoblast rapidly increases in volume as we
trace it backwards, and presently the anterior ccelom opens
VoL. 60, PART 2,—NEW SERIES. 15
208 ARTHUR WILLEY.
out with a deep extension into the keel. Produced beyond
this carinal ccelom there isa rather long and somewhat folded
epiblastic keel with its walls agglutinated so as to occlude
the potential trophoblastic cavity.
TEXT-FIG. 2.
Anterior region of keel, showing relation of basement membrane
to carinal hypoblast. 1. Interval between hypoblast and epi-
blast containing adecarinal coagulum. 2. Basement membrane
crossing the interval to join the carinal hypoblast. 3. Tropho-
blastic cavity of keel. 4. Hypoblastic groove.
The epiblastic keel soon begins to shorten, the carinal
coelom becomes reduced, and the cells of the hypoblastic
groove appear as a continuous epithelium on the embryonic
side. In the diagram (Text-fig. 3) I have reconstructed the
BLASTOCYST AND PLACENTA OF T'HE BEAVER. 209
hypoblast on the anti-embryonic side of the keel, where it is
evanescent in the actual section. Between the condition
itisp-crsinives 6}.
Appearance of the keel behind the anterior polar region. The
index numbers are placed on the embryonic side of the keel.
1. Exocelom. 2. Hypoblastic groove. 3. Shortened epiblastic
keel.
shown in Pl. 18, fig. 52, and that in Text-fig. 3 there are
thirty-nine sections of 10 . At the region of the blasto-
cyst which we have now reached the coronal disc is composed
210 ARTHUR WILLEY.
of large chromatophile cells freely subtending the coronal
cavum, which is filled with coagulum containing many
chromatic granules. Two or three sections after Text-fig. 3,
the front part of the embryonic shield begins to be cut
tangentially. Its abrupt appearance in section indicates a
much sharper definition than in previous substages.
Under the Zeiss microscope, when the keel appears to
point towards the observer, the embryonic shield is placed
on the right side of the keel. The plate-drawings were
reversed under the Edinger apparatus. For descriptive
purposes we may regard the edge of the keel as pointing
downwards towards the observer, although it really points
upwards towards the mesometrium. Above the embryonic
shield in the figure (Pl. 18, fig. 553) there is a somatopleuric
fold which looks like a lateral amniotic fold and is so inter-
preted. Below the shield in the figure there is the epiblastic
keel into the neck of which the exoccelom of that side is
produced, so that the material for the other amniotic fold is
in continuity with the keel. Underneath the thick formative
epiblast of the shield we find a thick-walled mesodermic
saccule, interpreted as the pericardial primordium of the
embryonic cceelom. The only ccelom at present existing in
the embryo is the pericardial ccelom. Against this the
embryonic hypoblast consists of a continuous cubical epithe-
lium, while on the opposite side the hypoblast is evanescent
and granular. Below the distal border of the hypoblast a
thin sheet of mesoblast intervenes between it and the epiblast
(Pl. 18, fig. 53).
Proceeding backwards, the pericardial saccule shortly
separates into two moieties. The portion which lies sub-
centrally under the thickened epiblast shows a character-
istic thicker inner wall of columnar cells and a thinner
outer wall of cubical cells. Between the two parts of the
‘pericardial ccelom the mesoblast thins out, and so the cubical
hypoblast comes nearer to the formative epiblast at this
point (PI. 18, fig.54). The next figure shows an accentuation
of the preceding features, and is introduced to exhibit the
BLASTOCYSI AND PLACENTA OF THE BEAVER. Past bl:
two diverging limbs of the pericardial ccelom which now
appear as antimeres (PI. 18, fig. 55).
As we approach the posterior quarter of the blastodisc, the
embryonic shield becomes larger, and a medullary groove
with subjacent notochordal plate appears in the position
shown in PI. i9, fig. 56. After another half-score sections
we have the structure represented in Pl. 19, fig. 57. The
medullary groove and the distal pericardial process have
ceased, and the lower part of the keel begins to be occupied
by massive mesoblast with a hypoblastic diverticulum extend-
ing into it. At the foot of the keel there is an open hollow
space containing coagulum ; it is possible that it is an incident
of growth rather than that it has any special significance, but
this may remain an open question. In this figure and the
preceding, the sinus terminalis is seen low down on the
anti-embryonic side of the keel; on the embryonic side it
occurs near the junction of the adcarinal and carinal regions.
This unequal behaviour of the parts of the sinus termi-
nalis has been observed in ali substages.
At the posterior end of the blastodisc, the primitive streak
in this substage is reduced both in extent and definition, and
it is evident that it would soon be given up. Its position and
the wings of massive mesoblast proceeding from it are shown
in Pl. 19, fig. 58. The hypoblastic groove lined by cubical
epithelium intruding into the distal mesoblast is conspicuous,
and the transition from cubical to flattened epithelium is
remarkably abrupt.
We are now rapidly nearing the region of the massive
exostyle which always follows behind the primitive streak.
In this substage the exostyle exhibits in section a broad distal
base with a small trophoblastic cavity and hypoblast pene-
trating into the massive mesoblast. ‘The epiblast on one side
of the keel is peculiarly folded. The distal lumen of the
hypoblastic groove is occluded, the occlusion taking place by
opposition of the walls accompanied by intense proliferation
of the massive mesoblast, whereby the axial hypoblast merges
imperceptibly with the surrounding mesoblast, without the
212 ARTHUR WILLEY.
intervention of a basement membrane (Pl. 19, fig. 59). The
large clear nuclei of the intrusive tongue of hypoblast contrast
with the more darkly stained mesoblastic nuclei in their
normal aspect, but they appear to blend with the latter at the
sides and apex. On the anti-embryonic side of the keel there
is unsplit mesoblast; on the embryonic side an exoccelom
whose epithelium is partly cubical. The anti-embryonic part
of the sinus terminalis lies low down, midway between
the foot of the keel and the position of the sinus on the
embryonic side. The massive tissue of the exostyle is con-
centrated within somewhat narrow bounds, covering about a
dozen sections of 10. This concentration and delimitation of
the exostyle are what must necessarily precede the initial
discoplacental adhesion.
A few sections behind PI. 19, fig. 59, the axial cavity re-
appears in the hypoblast and this marks the transition from
the exostyle to the post-stylar region. A deep and narrow
hypoblastic groove occupies the centre of the mesoblast ;
above the massive tissue the exoccelom opens out on both
sides to form a wide cavity. Below the mesoblast there is a
trophoblastic extension of the keel (Pl. 19, fig. 60).
At the region of the blastocyst corresponding approximately
with the course of the exostyle, the obplacental wall exhibits
the condition of a continuous megalocytic attack accompanied
by ingestion of red corpuscles in the coronal area, and a wide
pericoronal arcade subtending a pericoronal cavum on each
side. As already explained, the term arcade in this connec-
tion expresses typically a single bridge of chromatophilous
trophoblast with free outer surface washed by the uterine
fluid. The particular function of the obplacental arcades, in
addition to the ingestion of leucocytes by the chromatophile
cells, is to afford spaces for the openings of the persistent
obplacental glands.
Posteriorly the two halves of the exoccelom unite to form a
common cavity, the post-stylar coelom, which extends some way
into the distal cavity of the keel (Pl. 19, fig. 61). After this
the trophoblastic keel lengthens enormously (Pl. 19, fig. 62).
BLASTOCYST AND PLACENTA OF THE BEAVER. 213
About this region of the blastocyst the sections show demon-
stratively the ingestion of erythrocytes by the coronal megalo-
karyocytes and the ingestion of leucocytes with their lobed
chromatic nuclei by the pericoronal chromatocytes. In
addition to the coronal implantation and the pericoronal cava,
there is a typical periomphaloid adhesion and _ pericarinal
festoons.
The two sections of the sinus terminalis descend deeper
into the keel, and finally come near to each other at the bottom
of the carinal hypoblast as shown in PI. 19, fig. 63, which is
thirty-nine sections behind the preceding figure. At the same
time the post-stylar ccelom is reduced, and the trophoblastic
extension of the keel is devoid of a continuous axiallumen. In
Pl. 19, fig. 64, the two parts of the sinus nearly touch; and
fig. 65 shows the sigmoid confluence of the sinus, marking the
posterior limit of ccelom and mesoblast.
Next follows the posterior didermic region of the blasto-
eyst. The trophoblastic keel shortens, but the hypoblast
retains its carinal extension until near the posterior pole.
These features, as well as the relation of the keel to the
adcarinal omphalopleure, are portrayed in Pl. 20, fig. 66.
(Quite at the posterior pole we find in section a round didermic
blastocyst free on its mesometric side, adhering intermittently
elsewhere.
X. MATERNAL T'ROPHOSPONGIA.
Without undertaking a detailed discussion of the histology
of the mucosa, attention may be invited to certain interesting
changes which take place in the uterine mucosa of the
gestation sac, preliminary to placentation. ‘The perusal of
the foregoing pages will doubtless have left a vivid impres-
sion of the vital importance to the beaver’s blastocyst
of its obplacental implantation. One might have expected,
from the analogy of the rabbit, that this would involve
the degeneration of the obplacental uterine glands. On
the contrary, the obplacental glands retain their full func-
214 ARTHUR WILLEY.
tion throughout the period dealt with, and the trophoblast
gives free way to their crypt-like openings. It is only
in the walls of the mesometric or placental groove, which
have not yet entered into very close association with the
blastocyst, that the glands have already become atrophic.
The mucosa may be regarded as comprising two classes of
tissue: (1) Uterine epithelium and glands; (2) dermatic
connective tissue and capillaries. The fixation of the blasto-
cyst resolves itself into two periods: (1) Preplacental period ;
(2) euplacental period. The size of the gestation sac is
determined by two factors: (1) Dermatic proliferation; (2)
pressure of blastocyst.
In one of the uterine cornua belonging to a later phase of
gestation which will be described below, there was a slightly
swollen segment of the uterus, which looked as if it might
contain a very young blastocyst. After it had been sectioned,
it was found to be in a perfectly healthy condition, but non-
gravid. This series is numbered VII x in the Utrecht cata-
logue. The cavum uteri is a rather narrow lumen
bifurcated towards the mesometrium and lned by a high
columnar epithelium; opening into it on all sides there is a
profusion of glands (PI. 20, fig. 67). It contains a coagulum
with some dark-stained granules diffused throughout it. The
lumen is invested outside the epithelium by a narrow uni-
form zone of intense dermatic proliferation riddled with
capillaries; in this zone (not indicated in the figure) the
connective-tissue cells are more numerous and closer together
than in the deeper parts of the mucosa. On the mesometric
side the Jumen branches into two grooves, separated by a
ridge or dermatic cone covered by epithelium. Numerous
large vessels occur between the circular and longitudinal
muscles of the mesometric region; the circular muscle-ring
is broad and entire, except where penetrated by the vessels.
In contrast with the initial condition of uniform dermatic
proliferation and intact glands described above in a non-
gravid segment, we find in a gravid gestation sac belonging
to the preplacental period that all the mesometric and peri-
BLASTOCYST AND PLACENTA OF THE BEAVER. Palla,
placental glands are undergoing necrosis, being met and
vanquished by an epithelial proliferation operating centri-
fugally.- The dermatic proliferation acts centripetally. Mean-
while the dermatic proliferation in the future placental
region has outstripped the remainder, and now constitutes
what has been referred to as the placental trophospongia,
the term “‘trophospongia” having been introduced by
Hubrecht in 1889 to denote a vascular maternal proliferation.
In the beaver such a proliferation takes place all round the
cavum uteri, and is at first of uniform thickness.
The epithelial proliferation is a phenomenon of substitu-
tion accompanying the necrosis of the glands. It is paralleled
by a corresponding phenomenon described by Hubrecht
(1893) in the case of the shrew (Sorex), where the epithelial
proliferation leads to the formation of secondary crypts. In
the beaver’s gestation sac during the preplacental period,
the placental mucosa, towards the bottom of the placental
groove, exhibits, sometimes very clearly, radiating bands of
necrotic glands, with dark-stained shrunken nuclei (PI. 20,
fig. 68). These glands are to be replaced by an extensive
epithelial proliferation, which grows centrifugally into the
substance of the placental trophospongia in the form of two
lobulate wings, which correspond in their position with the
grooves on either side of the dermatic cone in the non-gravid
uterus (Pl. 20, fig. 67). Thus the wing-like epithelial pro-
liferations are preceded by necrotic belts. The cells multiply
by mitosis. In the niches between the lobes are found
subepithelial capillaries (Pl. 20, fig. 69).
In the obplacental region, where the subepithelial capil-
laries are also excessively abundant, the capillaries are con-
veyed by the centripetal dermatic proliferation. This happens
in the placental region as well, but in addition the centri-
fugal epithelial proliferation descends to meet the capillaries
and embrace them, thereby preparing a nidus for the true
placental implantation. At the beginning, the proliferating
cells retain their cell-boundaries (Pl. 20, fig. 70). Even-
tually the proliferation will become syncytial. At the level
216 ARTHUR WILLEY.
of the anterior part of the embryonic shield in substage F,
the capillaries which adjoin the inner borders of the wing-like
mesometric proliferations are greatly dilated, but there is no
trace of any endothelial proliferation (Pl. 20, fig. 71).
At the distal borders of the two principal mesometric pro-
liferations there are to be seen traces of the glands which
they have supplanted. There are no signs of glands between
the wings; the latter cause the more laterally placed meso-
metric glands to diverge widely, arching round the meso-
metric area, appearing healthy in their deep-lying portions,
but failing to reach the surface, their neck-portions having
been killed. Sometimes a transverse section of a glandular
tube is seen, the centripetal half of which is necrotic, the
distal half normal. In this way, centripetal glandular
degeneration and centrifugal epithelial proliferation take
place simultaneously. In the obplacental region the glands
persist and the uterine epithelium is largely replaced by
trophoblast ; in the placental region the glands degenerate
and are replaced by epithelial proliferations which are to
some extent moulded upon the pre-existing glands. At the
sides of the placental groove the necks of the glands some-
times widen out into large ampulliform dilatations with ill-
defined walls and wrinkled nuclei; such ampulle fail to open
into the cavum uteri. They may nearly open, but their
mouths are blocked, partly by their own degenerate cells and
partly by epithelial cells (Pl. 20, fig. 72).
There is not much to add to what has been said already
regarding the uterine epithelium. Where the trophoblast is
free and the epithelium consequently intact, the character of
the latter varies at different points without any regard to
symmetry. In one instance my notes record an epithelium so
flattened as to become a pavement epithelium, covering the
left side only of the omphaloid cavum. The cytoplasm of the
uterine epithelium stains dark yellow with orange G, and
thus offers a marked contrast to the adjoining trophoblast,
whose cytoplasm remains pale. J. W. Jenkinson (1902, p.
132) has noted the same distinction in the case of the mouse:
BLASTOCYST AND PLACENTA OF THE BEAVER. Pilea
“The cytoplasm [of the trophoblast cells] does not stain
intensely with acid stains, and may in this way be readily
distinguished from the cytoplasm of the maternal cells.”
Again, on p. 159, referring to epithelial cells which have
been ingested by the trophoblast, he says: “As for the cyto-
plasm, it stains brilliantly with plasma stains, and offers in
this respect a marked contrast to the cytoplasm of the
trophoblast by which it has been ingested.” This last is true
of the erythrocytes ingested by the megalokaryocytes in the
beaver.
There are signs that in the beaver the uterine epithelium
may not only disappear by direct substitution of the tropho-
blast in situ, but that, in a manner not unlike what P. Nolf
(1896) described in the bat, it may first be reduced to a
cubical and then to a pavement epithelium; or finally it may,
in places, be shredded off into the cavum uteri.
The migration of leucocytes from the mucosa across the
uterine epithelium into the omphaloid cavum and thence into
the chromatophilous megalokaryocytes, or, more briefly,
chromatocytes, is a phenomenon that requires further com-
ment. Granules staining darkly with hemalaun, occurring
commonly in triads and tetrads, are found deep down in the
stroma of the obplacental mucosa. From the deeper zone
they scatter through the intermediate stratum and are again
met with in great numbers in a subepithelial position, from
whence they pass into the epithelium. At first I supposed that
they might be derived from the nuclei of the decidual cells,
but was unable to find transitional forms. Not only do they
traverse the uterine epithelium, but they also pass in smailer
quantities into the lumina of the glands, and are then dis-
charged with the glandular secretion into the cavum uteri.
In much less numbers are they observed in the capillaries.
In places the obplacental mucosa showed an abundant
infiltration of undoubted leucocytes whose lobed nuclei were
these same chromatic granules of whose nature I had been
uncertain. When they reach the bases of the epithelial cells
and penetrate through the latter, their cytoplasm does not
218 ARTHUR WILLEY.
stain so well as it does in the middle of the mucosa, but
remains clear so that the granules often appear to be con-
tained in vacuoles. When they arrive in the uterine fluid,
which is represented in the sections by a coagulum staining
deeply with orange G, the cell-body is often invisible though
sometimes quite obvious. The marked tendency which they
exhibit to discharge themselves in volleys through the uterine
epithelium corresponds with their deep-seated gregarious
habit in the mucosa. ‘This infiltration of leucocytes is com-
parable with that observed by Nolf in the epithelioid tissue
and hypertrophied venous endothelium of the bat’s tropho-
spongia, although their fate is different.
XI. DiscopLaceNntaL ADHESION.
The stage of the incipient placentation is unfortunately
lacking in the material collected by me. In the keel of the
preplacental blastocyst four parts with independent destinies
may be distinguished: the anterior keel with anterior exo-
coelom, the embryonic keel, the exostyle, and the posterior
keel with post-stylar exoceelom. The first and last will be
employed in the formation of the umbilico-placental mem-
branes or fenestre pyriformes, consisting of diplotropho-
blast, i.e. trophoblast doubled by the somatic mesoblast of
the exocelom. The embryo will become folded off, and will
sink with its amnion into the exoceelom. ‘The exostyle will
furnish the material for the placental labyrinth.
But the chapter recording these events in their actuality
cannot yet be written. The groove at the foot of the keel in
Pl. 19, fig. 59, is ready to receive the dermatic cone of the
maternal trophospongia with its syncytial caps (cf. Pl. 20,
fios. 69-71), but we are not at present privileged to witness
the act.
XII. Tue EstaslisHep PLACENTA.
The material upon which the following description is
based consisted of six spherical gestation sacs about 25 mm.
BLASTOCYST AND PLACENTA OF THE BEAVER. 219
in diameter, obtained on two separate occasions—March 4th
and March 10th, 1913. It was on the latter date that I also
obtained the six youngest gestation sacs. Ina female beaver,
taken on March 4th, there were two spherical swellings, each
25 mm. in diameter, in the left uterus only, and two con-
tiguous corpora lutea in the left ovary. Above the upper
swelling there was a smaller one of 12°5 mm., upon which
ereat hopes were set; but it proved barren. The axillary
teats of the mother were distinct, but the post-axillary pair
was only found after the skin had been removed. The
trapper said it was a young female that would have been
three years old in the coming spring. In another female,
taken on March 10th, there was a single spherical sac in the
left uterus, and three in the right. From these data it may
be gathered that whilst the beaver produces a litter only once
a year in the spring, there isa considerable range of variation
with regard to the stages which may be observed upona given
date during the season of reproduction.
I opened one of these sacs in Prof. Hubrecht’s presence by
slicing away the antimesometric wall with a cartilage knife.
We were surprised to find that no liquid exuded when the
cavity of the omphalon was exposed; on the contrary it was
entirely filled by a translucent coagulum which cut like cheese
and showed no tendency to break away. One slice, about
2mm. thick, was cut through the middle of the gestation
sac; the coagulum, stretched from wall to wall like a sheet
of gelatine, retained its position exactly, and the cut surfaces
remained flat.
In the mesometric half the embryo could be seen through
the coagulum lying on one side, its left side being presented
towards the omphalon. The embryo lies in the exoccelom
between the placenta and the umbilical membrane. The
minimum thickness of the uterine wall exceeded 4 mm. ; the
maximum was nearly 6 mm.; the diameter of the cavum
uteri, and consequently of the omphalon that occupied it,
was about 12 mm. (PI. 20, figs. 73 and 74). The placenta,
measured in section, had a length of 4°25 mm., and a height
220 ARTHUR WILLEY.
of 225 mm. ‘The latter measurement signifies that the some-
what mushroom-shaped placenta projected freely to that
extent into the exocelom. After being reduced to sections
the body of the embryo was found to have shrunken rather
badly owing to the treatment it had received. When the
destination of such early stages is the microtome they should
not be cut open previously. Fortunately there was no lack
of good material.
In its shape and projection the placenta may be likened to
a cushion or a mushroom ora dome. At its edge it overlaps
the base of insertion, so that in sections passing tangentially
through the margin it appears detached from the uterine
wall. Such tangential sections are useful for determining
the essential structure of the placental labyrinth. There isa
straight trophoblastic base continuous with the rest of the
trophoblastic wall of the blastocyst, from which centripetal
trabecule ascend into the allantoic mesoblast. These tropho-
blastic trabecule are excavated by canals which carry
maternal blood, and are what M. Duval called sanguimaternal
lacunz in the ectoplacenta. The allantoic mesoblast conveys
foetal capillaries to the placental labyrinth. The allantoic
tissue appears in the form of centrifugal vill interlacing with
the centripetal trabecule, together establishing the placental
labyrinth (Text-fig. 4). Neither the trophoblast nor the
allantoic mesoblast remains passive in the growing placenta,
but they grow in opposite directions, the trophoblast centri-
petally, the allantoic mesoblast centrifugally.
After passing the point where the placenta is inserted into
and united with the uterine wall, the definite basal layer of
trophoblast with its sharp-demarcation disappears. Wherever
the trophoblast touches allantoic tissue it is composed of a
cellular layer corresponding to what E. van Beneden (1888)
called the cytoblast, subsequently lengthened into cyto-
trophoblast by J. H. Vernhout (1894). Towards maternal
blood and trophospongia the trophoblast presents a plasmodial
layer termed “ plasmodiblast” or ‘ plasmoditrophoblast.”
For the sanguimaternal lacune in the trophoblastic trabeculae
DID) |
BLASTOCYST AND PLACENTA OF THE BEAVER.
the plasmodiblast furnishes a pseudo-endothelium. The solid
buds produced at the growing edge of the placenta consist of
eytoblast which ascends into the allantoic tissue. The cyto-
blastic buds anastomose and so enclose allantoic islands. he
eytoblast is the formative layer of the trophoblast ; it may be
compared with the Malpighian layer of the skin, but instead
of giving rise to horny cells on its outer surface, it produces
plasmodiblast which is rooted in the vascular trophospongia.
The cytoblast itself is rooted in the vascular allantoic tissue ;
TEXT-FIG. 4.
Section through growing margin of placenta. Trophoblast
black, mesoblast dotted. The asterisks indicate the line where
the allantoic and somatic tissues meet; outside the asterisks
the double membrane is diplotrophoblast.
it does not give off any buds towards the maternal tissue.
The cytoblast is a growing tissue, the plasmodiblast is a
feeding organ, and the trophospongia is the nidus supplying
the nutriment.
The foregoing aphorisms are indited with special reference
to the conditions observed in the beaver, but they derive
support from the observations of others upon different
animals. In the rabbit, H. Schoenfeld (1903), confirming
Maximow, states that the plasmodiblast is supplied with
elements from the cytoblast, and adds that it has no growth
of its own; it moves about upon the surface of the cytoblast
222 ARTHUR WILLEY.
as described by A.Maximow (1900). Inthe squirrel (Sciurus),
F. Muller (1905, p. 560) says that the growth of the placenta
does not take place merely by the substitution of foetal for
maternal tissues, but by the progressive centripetal growth
of the ectoplacental mass, which thus surrounds the allantoic
villi more and more; nevertheless the greater part of the
uterine mucosa is supplanted by the placenta, because a
continual process of degeneration and resorption of maternal
tissue is taking place.
In the bat, P. Nolf (1896, p. 610) says that the increase in
thickness of the placenta is not the result of peripheral
growth at the expense of the maternal tissue. Almost all
of its secondary growth is centripetal ; the internal or foetal
face of the placenta grows towards and projects into the
blastodermic cavity. This is proved by the fact that the
vegetative epiblast throughout gestation is continued into
the placental cytoblast, not at the level of the internal sur-
face of the placenta, but at the level of its external surface.
This conclusion accords, he says, with those deduced by
Duval for Carnivora, by Hubrecht for the shrew, and by
Vernhout for the mole.
Tn all these instances the nutritive material for the centri-
petal growth of the ectoplacental trophoblast is furnished by
the maternal trophospongia, which forms a cushion upon
which the placenta rests. In the beaver this trophospongia
has a twofold origin in a vascular dermatic proliferation and
a lobular epithelial proliferation. The latter is now broken
up into polygonal blocks by the capillary network, producing
an areolated structure in section. Many of the epithelial
areole have degenerated into syncytia in which numerous
dark-stained nuclei are densely aggregated. Sometimes a
syncytial mass is extensive and then there appears a curved
band of nuclei in the midst of it. The epithelial areole, the
syncytia, and the aggregations of deeply staining nuclei are
very characteristic of the early trophospongia of the beaver’s
placenta. To these features should be added the presence at
certain points of brown granules chiefly surrounding large
BLASTOCYST AND PLACENTA OF THE BEAVER. 223
degenerating maternal nuclei. In the deeper zone of the
maternal trophospongia the cell-islands are not yet syncytial ;
some are entirely cellular, others are partly syncytial.
There are interesting analogies between events in the pre-
placental and euplacental periods, as under :
Preplacental period. Euplacental period.
1. Uterine glands partly normal, . Epithelial areole partly cellular,
partly necrotic. partly syncytial.
2. Centripetal dermatic prolifera- . Centrifugal allantoic prolifera-
tion conveying maternal capil- tion conveying fcetal capil-
laries. laries.
3. Centrifugal epithelial prolifera- . Centripetal ectoplacental pro-
tion into the dermatic tissue. liferation into the allantoic
tissue.
At the junction of trophoblast and trophospongia a
symplasma is formed. Schoenfeld (1902) discusses the
use of the terms syncytium and plasmodium which were
introduced by Prof. Haeckel; and symplasma suggested by
Graf Spee. A syncytium is immobile, a plasmodium is mobile.
In connection with the phenomena of placentation, Schoenfeld
applies syncytium to maternal formations, plasmodium to
foetal formations. Only when the fcetal plasmodium comes
into contact with the maternal syncytium does the latter
undergo degeneration and become converted into a symplasma
which is defined as a syncytium in retrogression. ‘The
symplasma is brought about by the incorporation of maternal
protoplasm and nuclei into the substance of the plasmodium.
Until the degeneration of the maternal elements is complete,
the peripheral zone of the plasmodium is a symplasma con-
taining active foetal nuclei and passive maternal nuclei. This
interpretation involves perhaps a slight extension of the
original meaning of the term symplasma. In the dog,
Schoenfeld described the fusion of decidual cells with the
plasmodium, thus converting the latter, for the time being,
into a mixed “ fceto-maternal plasmodium” ; but there can be
no harm in calling it a symplasma.! Good illustrations
of a typical symplasma in the sense here indicated were
' To avoid possible ambiguity perhaps the term “symplasmodium ”’
might he preferred.
VoL. 60, parT 2.—NEW SERIES. 16
224 ARTHUR WILLEY.
given by A. Maximow (1900, Taf. xxx, figs. 1 and 2) for
the rabbit.
At many points the junction of plasmodium and syncytium is
rendered conspicuous by the presence of the crowded syncytial
nuclei, which contrast in colour, size and shape with the
smaller, paler, oval, and more evenly distributed plasmodial
nuclei. The conjoint symplasma may be observed to surround
superficial capillaries of the trophospongia together with
adjoming decidual cells. At such places the plasmodial
nuclei may be seen intruding into the syncytium, and no
boundary can be drawn between the plasmodial protoplasm
and the syncytial protoplasm. The inclusion of maternal
capillaries seems to be effected by the tortuous growth of the
allantoic tissue which pushes the trophoblast before it into
the trophospongia, so that we find in the border zone between
trophospongia and placental labyrinth islands of allantoic
tissue surrounded by trophoblast, and outside that a mantle
of symplasma (PI. 20, fig. 75). In the diagrammatic figure
(Pl. 20, fig. 76), the allantoic villi, with their trophoblastic
investment, are seen to be tipped by syncytial groups of
nuclei, rendered in black. The characteristic trophospongial
islands or areole show, in the sections, various grades of
syncytiation at different levels, there being no regularity in
the distribution of the syncytia throughout the trophospongia,
except at the symplasma or zone of contact.
The trophospongial islands, whether syncytial or cellular, are
separated by capillaries with normal endothelium. At the
base of the trophospongial cushion, between it and the massive
dermatic proliferation, there is a basal sinus-like blood-space,
to which large capillary vessels pass vertically through the
dermatic tissue from the direction of the mesometrium. This
arrangement is indicated in PI. 20, fig. 76. Near each pole
of the placenta a large vessel is found receiving its affluents
from the sanguimaternal lacune in the swollen tips of the
trophoblastic trabecule ; from thence it descends into the
mucosa. The two polar vessels possess a proliferated endo-
thelium which they retain until they reach a point deep in the
BLASTOCYST AND PLACENTA OF THE BEAVER. 225
mucosa, where each of them is continued into a narrow vessel
with normal endothelium which passes directly and abruptly
into the proliferated walls (Pl. 20, fig. 77). There is an
equally abrupt transition from the proliferated endothelium
to the plasmodial pseudo-endothelium near the fcetal peri-
phery of the placenta. At one pole the vessel penetrates the
placenta near its right margin; at the other pole it enters
near the left margin.
The data at my disposal do not permit of a direct com-
parison of the placental circulation of the beaver with other
types which have been investigated. Such comparisons
should be made at equivalent stages. The circulation in the
early established placenta will necessarily differ in its details
from that in the mature finished placenta. As for the latter,
excellent figures have been given by Tafani of the injected
placente of various mammals, less successful perhaps as
regards the rabbit, but remarkably clear as regards rat,
guinea-pig, and bat. The placenta of the guinea-pig possesses
its own special features. That of the rat (Mus decumanus),
as represented in Tafani’s tav. v, fig. 2, 1s more like that of
the bat (Vespertilio murinus, tay. vi, fig. 2) than the
guinea-pig (Tav. v, fig. 1). In Mus and in Vespertilio
there is a central maternal artery penetrating through the
middle of the discoplacenta to its foetal aspect, where it
spreads out into the afferent sanguimaternal lacune.
To left and right of the placental insertion the areolated
trophospongia merges imperceptibly into a marginal zone of
hollow crypts, the walls of which are partly necrotic. In the
obplacental and periplacental regions of the gestation sac the
dermatic cells constitute an epithelioid tissue comparable to
Nolf’s epithelioid layer in the bat. In the mesometric region
the dermatic tissue, though proliferating, still retains a
primitive aspect, and does not present an epithelioid mosaic
pattern in section.
Median sections through the placenta show that the der-
matic tissue in which the vessels are lodged projects like a
226 ARTHUR WILLEY.
cone into the trophospongial cushion (PI. 21, fig. 78). After
leaving the basal trophospongial sinus, which now appears as
an arched line between the dermatic axis and the areolated
cortical substance, the vesse!s branch and enter into numerous
anastomoses with each other, forming a rete mirabile
within the dermatic cone continuous with the trophospongial
sinus, which is itself retiform. With a simple lens the
trophospongial crescent with its peripheral zone of syncytia,
its interstitial meshwork, and its basal sinus, can be seen to
perfection following the contour of the dermatic cone with its
rete mirabile. :
Another series, cut nearly longitudinally through the gesta-
tion sac, showed the placenta in a slightly more advanced
condition—4"50 mm. in length, 3°75 mm. high. In tangential
sections, i. e. such as do not pass through the central tropho-
spongial cone, the allantoic mesoblastic villi penetrate deeply
and tortuously into the embryotrophic cap of areolated
trophospongia, so that allantoic islands, surrounded by their
trophoblastic investment, appear in section amongst the areole.
Towards the centre of the placenta, the concentric strata of
which it is composed stand out very clearly. Beginning at
the foetal aspect, there is first a narrow zone of allantoic
mesoblast containing the superficial allantoic vessels; then
the swollen ends of the trabecule dilated with the sangui-
maternal lacune; thirdly, the labyrinth of anastomosing
trabeculze ; fourthly, the border zone of symplasma; fifthly,
the trophospongial areolz ; sixthly, the dermatic cone. Most
of these parts are to be found in figures already referred to,
and again in Pl. 21, fig. 79. Since the polar vessels enter
the placenta near its right and left margins, neither of them
is seen in a median section. Noteworthy is the abrupt
transition from the areolated trophospongia to the dermatic
tissue.
The position of the embryo in the blastocyst conforms to
the rule which applies to those Vertebrates in which there is
a great disparity between the animal and vegetative poles,
i.e. when an omphalon or a yolk-sac is present. It has been
BLASTOCYST AND PLACENTA OF THE BEAVER. Papa
mentioned above, and the figures show, that the umbilical
membrane or area vasculosa stretches across the foetal sac
midway between placenta and obplacenta. The body of the
embryo, surrounded by its amnion, lies sideways in the
exocceelom between placenta and umbilical membrane, with
its right side towards the placenta and its left side towards
the omphalon (Pl. 20, fig. 73, and Pl. 21, fig. 79). In the
Sauropsida, at the gill-slit or branchiotrematic stage, the
embryo also comes to he with its left side towards the yolk-
sac. In the rabbit, at the same stage, the forebody of the
embryo by reason of the cervical flexure projects into the
umbilical vesicle surrounded by the proamnion, or, more pre-
cisely, the proamniotic omphalopleure. In the beaver there
is no trace of a proamnion at this stage; and when it is
remembered that in the preplacental blastocyst the mesoblast
is continuous within the circuit of the sinus terminalis,
except at the notochordal contact, and that there exists
already an anterior as well as a posterior median extension of
the exoccelom, the formation of a proamnion at any stage
would seem to be excluded.
In Pl. 21, fig. 79, we see the amniotic membrane stretching
between the umbilical membrane and the placenta. In the
same figure the opening of the omphalomesenteric duct into
the omphalon and that of the allantoic canal into the flattened
allantois are indicated, though they do not occur actually on
the same section. The allantoic canal communicates with
the hypoblastic cloaca, into which the Wolffian ducts now
open, and forms the hypoblastic axis of a mesoblastic stalk,
accompanied by two arteries anda vein. At the distal end
of the stalk the canal] widens out as the allantoic sac which
is imbedded in the thickness of the mesoblast at the feetal
surface of the placenta, causing no protuberance. ‘This
flattened sac has a longitudinal extension of about 0°75 mm.
During part of its course from the cloaca to the sac, the
Jumen of the allantoic canal is occluded by cellular prolifera-
tion, so that it becomes solid; this condition has been
observed in two series.
228 ARTHUR WILLEY.
In one series (VIIa in the Utrecht catalogue) the omphalo-
mesenteric duct has a partially occluded lumen; but in
another series (V 8) the lumen is open, and contains maternal
blood-corpuscles, which it conveys from the omphalon to the
mid-gut. The presence of red blood-corpuscles in the mid-
gut could not easily be attributed to accident, and it appeared
at first a mystery how they came to be there. The clue to
the mystery was found in the behaviour of the obplacental
trophoblast, which at this stage consists of flattened megalo-
karyocytes closely applied to the uterine wall; here and
there they are greatly distended with erythrocytes, and there
is evidence of the transfusion of maternal corpuscles into the
omphalon. They pass into this cavity across the obplacental
trophoblast, and are in fact to be found scattered in the
midst of the coagulum.
I will conclude this chapter with some further details
concerning the embryo in order to define the stage of develop-
ment which it has now reached with greater precision. Its
actual age cannot be estimated, and can only be roughly
guessed at. If we assume the period of gestation to be
one hundred days, then these embryos will certainly fall
within the first twenty-five days, and probably within the
first fifteen days; the preplacental blastocysts are likely to
belong to the first ten days. The relative age is best
reckoned according to the size of the gestation sac.
The embryo is now in the gill-slit or branchiotrematic
stage; the mouth is open, but there is no anus. In section
the mouth-cavity, flattened dorso-ventrally, appears like a
pair of gill-slits, but this is entirely deceptive, since the
formation is that of the stomodeum with its pituitary
diverticulum meeting the blind end of the infundibulum.
The four pairs of true gill-pouches just fail to open to the
exterior, being closed over externally by a narrow cellular
bridge. The auditory sacs are closed; the optic stalk is
hollow, the optic cup and choroid fissure are formed, and
the lens invagination is still connected with the ectoderm.
Suitable sections show a length of the spinal cord flanked by
BLASTOCYST AND PLACENTA OF THE BEAVER. 229
somites resembling the corresponding figure of a 12 mm. pig
embryo in C. 8. Minot’s ‘ Laboratory Text-book of Embryology,’
p. 230, fig. 185. The buds of the fore-limbs are rather more
advanced than those of the hind-limbs, which appear as
broad crescentic thickenings.
XIII. InveRMepiate StaGces.
Two stages, intermediate between the establishment of the
growing placenta and the final period towards the term of
gestation when it ceases to grow, were obtained, but have not
yet been worked out in section. Their external characters
present two points of special interest concerning the early
relations of the chorion leve or diplotrophoblast of the
umbilico-placental areas, and the appearance of a spherical
allantoic vesicle. At the stage described in the preceding
chapter, the allantois occurred as a narrow tube communi-
eating proximally with the cloacal region and expanding
distally into a flattened sac immersed in the allantoic meso-
blast at the surface of the placental labyrinth, not causing
any additional protuberance into the exoccelom.
The next older stage is represented by three gestation sacs,
one in the right horn of the uterus, two in the left. These
were despatched to me in a tin containing 10 per cent.
formalin on March 23rd. The swellings were equal, some-
what shrunken, 60 mm. in long diameter. Upon cutting
through the antimesometric wall, 2°50 mm. thick, the
umbilical membrane (area vasculosa) was seen to be
expanded and in contact with the mucosa, though devoid of
attachment. Along the courses of the vessels low ridges are
developed, which press against the mucosa, so that the
impressions of the larger vessels are distinctly visible upon
the smooth inner surface. When first opened the mucosa
appeared dark reddish aud spongy, with white giant-cells
showing against the ground colour when viewed under a
simple lens.
230 ARTHUR WILLEY.
When the feetal sac was cut into, the umbilical membrane
was found to be adherent to the amnion, from which it could
be peeled off. In the mesometric division of the fcetal
sac the amniotic membrane bends inwards to its insertion
upon the dome of the placenta, and here it becomes free from
the umbilical membrane, so that a considerable exoccelomic
cavity exists in this region. Thus the baggy amnion hangs
loosely in the mesometric exoccelom, but elsewhere adheres
by simple contact to the area vasculosa, obliterating the
exoccelomic cavity. A linea alba marks its insertion upon
the placenta.
By slicing away the wall of the gestation sac, which
becomes thicker (5 mm.) towards the mesometrium, and then
removing a portion of the vascular membrane, the fcetus,
38 mm. long, is exposed, loosely enveloped by the spacious
amnion. On the mesometric side, the area vasculosa ends
with a villous rim at the two connecting membranes which
surround the aditus of the interutricular segments. In this
region the mucosa presents a rugose or convoluted surface,
and the folds are continued as opaque whitish buttresses
from the mucosa to the connecting membranes, extending as
far as the edge of a narrow clear zone below the villous rim
of the umbilical membrane. At this stage, therefore, simple
dissection suffices to demonstrate the dual composition of the
definitive connecting membrane (PI. 21, fig. 80).
On laying open the whole cavity of the exoccelom the
polar areas of the foetal sac are exposed from the inside.
They cover over the aditus of the interutricular segments.
The dissection was made shortly after the preservation in
formol, and the polar areas (umbilico-placental areas) appeared
as clear, non-vascular membranes forming turgid protuber-
ances into the exocceelom near each end of the placenta, each
being surrounded by part of the sinus terminalis. The
pyriform caudad protuberance was 9 mm. high, 10 mm. long ;
the convex cephalad protuberance was about 5 mm. high
by 11 mm. long (Pl. 21, fig. 81). In another specimen
opened several months later the polar areas were flattened
BLASTOCYST AND PLACENTA OF THE BEAVER. Dene
out. In fig. 81 the caudad end of the fcetal sac is also the
caudad end of the gestation sac; the head of the embryo is
directed towards that segment of the uterus which leads to
the Fallopian tube. The cephalad protuberance lies over the
opening or aditus of that segment, while the caudad protu-
berance arches over the posterior opening. The placenta
measured 33 mm. in length, 21 mm. in width, and 21 mm. in
height. Besides its remarkable form and bold eminence, the
most noticeable feature was the absence of an allantoic
vesicle. The position of the allantoic sac at the placental
surface was indicated by a flat membrane without fluid,
situated beyond the centre of distribution of the vessels. In
a second specimen from the same tract there was a very small
incipient allantoic vesicle with a transparent wall. In both
of these instances the head of the foetus was directed towards
the oviduct.
The next intermediate stage is represented by four gestation
sacs, one right and three left, sent to me in 10 per cent.
formalin on April 6th, having an average length of 81 mm.
Soon after the arrival of the material I opened all the sacs
by cutting out an obplacental piece from each, and punctured
each vascular foetal sac, collecting the clear fluid that squirted
out. The amnion was again closely adherent to the umbilical
membrane, so that in puncturing the latter the amnion was
also pierced, and the clear fluid, which appeared faintly straw-
coloured en masse, was the liquor amnii. In the later
stages, when the foetus has nearly attained its limit of growth,
the amnion loses its adhesion to the umbilical membrane, so
that the exoccelom then becomes the main cavity of the feetal
sac. At the present stage the amniotic cavity is the main
cavity of the foetal sac, the exocceelom, even to the base of
the placenta, being temporarily obliterated.
The wall of the gestation sac has now become reduced to a
mean thickness of about 2 mm. _ In the first specimen
examined the head of the foetus was again directed towards
the Fallopian tube. The foetus as it lies in its amnion has a
Tae ARTHUR WILLEY.
length of 75 mm., measured from the crown of the head to
the base of the tail. The flattened tail is not bent under the
body, but simply curves round against the posterior end of
the foetal sac, extending a little beyond the hind feet; it
measured 15 mm. in length. The placenta now has a length
of 42 mm.
The most conspicuous difference between this and the
preceding stage is the presence of a spherical allantoic vesicle,
about 16 mm. in diameter, with thin pellucid wall, projecting
boldly into the amniotic cavity. It les just in front of the
centre of distribution of the allantoic vessels into the placental
labyrinth (PI. 21, fig. 82).
The three gestation sacs in the left uterus held their foetus
in the following positions: In the one nearest to the ovary
the head of the foetus is directed towards the cervix uteri;
in the middle sac the foetus lies in the same position, with
the head towards the cervix; in the last of the three, i.e.
the one nearest to the vagina, the head of the foetus is
directed towards the oviduct, the tail towards the cervix.
XIV. Tat Connecting MEMBRANE.
At the early placental stage represented in PI. 20, fig. 76
and fig. 79, the umbilical membrane is invaginated about
halfway into the omphalon and stretches like a diaphragm
across the blastocyst, separating the omphalon from the
exocceelom. The latter is limited towards the aditus of the
gestation sac by a sheet of somatopleure or diplotrophoblast,
which constitutes one of the umbilico-placental areas described
in my former paper (1912). At the poles of the feetal
sac, beyond the range of the placenta, this membrane
stretches free across the cavum uteri within the circuit
of the sinus terminalis (Text-fig. 5).
From the outer edge of the sinus terminalis two other
membranes arise—the umbilical membrane and the ompha-
lopleure. The short free zone of omphalopleure intervening
between the sinus and the uterine wall is the primordial
BLASTOCYST AND PLACENTA OF THE BEAVER. 233
connecting membrane. It is clearly derived from the
adcarinal membrane of the preplacental blastocyst; it is
what is left of this membrane after the peripheral encroach-
ment of the primordial area vasculosa has ceased. It
continues to grow in later stages, and, by accession of
material from the uterine mucosa, becomes converted into
the definitive umbilico-uterine connecting membrane. The
latter is therefore a composite membrane of dual origin,
TEXxT-FIG. 5.
Section through region of one of the aditus of the gestation
sac at the early placental stage. 1. Omphalon. 2. Umbilical
membrane. 3. Exocelom. 4. Sinus terminalis. 5. Con-
necting omphalopleure (representing adcarinal membrane). 6.
Diplotrophoblast. 7. Aditus.
as was indicated in the preceding chapter. This fact is
proved by its early history as well as by its histological
structure, into the details of which I will forbear to enter
at present, although illustrations have been prepared. The
manner of junction of splanchnopleure, omphalopleure and
somatopleure at the level of the sinus terminalis in the
early placental stage is shown in Text-fig. 6.
From the earliest to the latest days of placentation the
Sinus terminalis is situated at the junction of the three
membranes whose primary names have just been given. Their
234 ARTHUR WILLEY.
secondary designations are respectively—umbilical mem-
brane, umbilico-uterine membrane, and umbilico-placental
membrane.
The omphalopleure, as a whole, constitutes the inferior
(anti-mesometric) wall of the omphalon and of the entire
blastocyst. It is present in all its integrity during the early
placental period but disappears in later stages, with the
exception of the persistent connecting membrane. ‘The
origin of the latter is thus explained. It is a secondary
product of the primary obplacental implantation of the
TEXT-FIG. 6.
Part of periplacental wall of blastocyst, at the early placental
stage, to show the junction of membranes at the sinus
terminalis. 1. Cavity of omphalon. 2. Exocelom. 3.
Cavum uteri. 4. Mucosa. 5. Sinus terminalis. 6.
Splanchnopleure. 7. Omphalopleure. 8. Somatopleure.
blastocyst, and directly comparable with the adcarinal
omphalopleure of the preplacental blastocyst. The umbilical
membrane forms the major part of the outer wall of the mature
foetal sac. In my former paper on the beaver (1912, p. 207)
I called it the vascular chorion or endochorion of the rodent
blastocyst, not being at that time aware of the circumstance
that the more suitable name, umbilical membrane, had been
applied to it for the rabbit by E. van Beneden and C, Julin in
1884.
The umbilico-placental membrane is in its origin a non-
vascular somatopleure or diplotrophoblast, but in the mature
foetal sac there are intrusive capillaries proceeding into it
BLASTOCYS! AND PLACENTA OF THE BEAVER. 235
from the area vasculosa. They form anastomosing loops,
and bear a resemblance to the capillary loops in the chorion
leve of the bat’s blastocyst as figured by van Beneden and
Julin.
The reason for the existence of the connecting membrane
which supplements the placental implantation in the attach-
ment of the foetal sac to the gestation sac, may perhaps be
sought in the semi-aquatic habits of the beaver and in the
fact that the female retains her activity, leaving the lodge
and swimming under the ice to procure food from the sub-
merged stock of winter-provender, throughout the period of
gestation. Were it not for the additional support afforded by
the connecting membrane, the narrow deciduous root of the
massive placenta might easily be torn asunder. The con-
necting membrane must relieve the placenta of much of the
stress and strain to which it would otherwise be exposed.
The nearest approach to the condition of having a con-
necting membrane seems to be represented by a temporary
formation, which was described in the early gestation sac of
Sciurus by F. Muller in 1905. It appears that the peri-
placental mucosa forms a ring-shaped thickening to which
the thickened trophoblast adheres before the folding of the
amnion. In the following stage this ring-shaped zone otf
implantation, with the sinus terminalis close to it, con-
tinues to extend, leaving the placental part of the cavum
uteri still unoccupied, but with numerous crypts opening
into it.
In the young gestation sac of Sciurus, when the blasto-
cyst is attached to the obplacental wall, the cavum uteri,
as had been previously observed by Fleischmann, becomes
constricted by a circular periplacental thickening into a
smaller mesometric and a larger anti-mesometric portion.
The zone of adhesion or omphalo-placental ring (‘ ompha-
loiden placentatiering,”’ F. Muller, op. cit., p. 395) occurs at
the lip of the mesometric cavum. In this ring an epithelial
syncytium is formed, with which the trophoblast becomes
intimately united. At length the syncytium is destroyed and
236 ARTHUR WILLEY.
then the plasmodial union is exchanged for simple adhesion.
Later still even this is given up. This early periplacental
implantation occurs at the level of the sinus terminalis,
and is interpreted by Muller as a vestigial omphaloid placenta-
tion such as had not been described in rodents before. The
periplacental connection has nothing to do with the allantoic
placenta which forms subsequently. It extends below and
thus embraces the openings or aditus of the gestation sac.
Its limited centripetal growth and its transitory endurance
are its most remarkable features.
There can hardly be a doubt that the temporary omphalo-
placental adhesion of Sciurus is comparable with the per-
manent umbilico-uterine connection of Castor.
XV. SPECIAL CONSIDERATIONS.
The two leading characteristics of the preplacental blasto-
cyst of the beaver, the obplacental implantation with differen-
tiation of erythrocytophagous and leucocytophagous megalo-
karyocytes, and the placental keel, are of specific physiological
importance to the growing embryo, but they also have a
comparative value which can only be elucidated by a brief
discussion. In order that the reader may be orientated with
regard to the systematic position of the beaver, a few words
of introduction are desirable.
Although the beaver, in structure and habits, is unique
amongst the Rodentia, displaying in high degree the qualities
of intelligence and adaptability to local conditions, yet it would
be wrong to suppose that it is farthest removed from a primi-
tive organisation. On the contrary, in its monotrematous
cloaca and pentadactyle limbs, to mention these two external
features only, it retains the marks of a very ancient mam-
malian type. In my former paper (1912) it was suggested
that “the beaver occupies a position amongst Rodentia com-
parable with that attained by man amongst primates.” It is
well known that man has retained some primitive features in
limbs, teeth and digestive tract, as compared with many other
BLASTOCYST AND PLACENTA OF THE BEAVER. 237
mammals. Just as Hubrecht’s discovery of the blastocyst of
Marsius with its “ Haftstiel ” indicated an excessively remote
origin for the primate ontogeny, so the blastocyst of the
beaver with its exostyle may have an analogous bearing upon
rodent ontogeny.
It is generally agreed, under the support of such authori-
ties as Cope, Winge, and Tullberg, that the beaver family is
related to the squirrel family, both of these families being
associated under J. F. Brandt’s suborder, Sciuromorpha (1855).
Tullberg divides the Simplicidentata into two great tribes,
Hystricognathi and Sciurognathi. The latter comprises two
sub-tribes, Myomorphi and Sciuromorphi ; and the latter falls
into three sections, Sciuroidei, Castoroidel, and Geomyoidei.
Winge (quoted by Tullberg) held that rodents are to be derived
from primitive Mammalia resembling the least specialised
Insectivora, from which they diverged by cumulative merease
of the gnawing habit.
F. Muller (1905), adopting Haeckel’s (1895) generalisations
maintained that of all Rodents the Sciuromorpha have
diverged least from the ancestral type; and he added that the
genus Sciurus in particular occupies the most central position
and has preserved the most primitive form. On the other hand,
Max Weber (1892) regarded the scales on the tail of the beaver
as the remains of a primitive scaly covering of the body.
The preplacental blastocyst of Lepus and Sciurus is a
plano-convex blastocyst, that of Mus and Cavia is an
inverted blastocyst, that of Castor is an everted blastocyst.
The task before us is not to decide which of these three is the
most primitive type of blastocyst, but to consider which of
them offers the readiest comparison with the blastocyst of
Tarsius. It may be premised that in one respect the
euplacental blastocyst of the beaver is the most primitive
known amongst existing Rodents by reason of the persist-
ence of the umbilico-uterine connecting membrane which
is a consequence of the periplacental implantation of the
trophoblast.
238 ARTHUR WILLEY.
The keel extends from end to end of the elongated balloon-
shaped blastocyst, along its superior or mesometric side,
dipping into the deep placental groove. One of the funda-
mental relations of this remarkable formation is so obvious
that its importance in establishing the reality of the pheno-
menon might escape attention—namely, the coincidence in
the configuration of the wall of the blastocyst and that of the
cavum uteri or cavity of the gestation sac. The keel may
thus be accepted at once as a fact, and need not be regarded
with suspicion as an artefact.
There are four or five principal structures concerned in the
constitution of the keel: the epiblastic keel, the mesoblastic
keel, the hypoblastic keel and the embryomic keel, to which
may be added the exoccelomic keel. The interpretation and
comparison with other forms will hinge upon the massive
mesoblastic keel which follows behind the primitive streak.
It is convenient to anticipate conclusions to some extent by
assuming that the keel represents an ancient or primitive
mechanism, and that the exserted mesoblastic keel of the
beaver is comparable with the “ Haftstiel” of Tarsius,
monkeys and man.
History of the “ Haftstiel.”—So far as I have been
able to ascertain, the first use of the term “ Haftstiel” as
applied to the mammalian blastocyst occurs in Selenka’s
memoir on the opossum (1887, vide his text-fig. C, on p. 136
op. cit.). In this case the main cavity of the blastocyst is
the omphalon, into which the allantoic vesicle, surrounded by
a narrow exoccelom, hangs freely. In the early Primate
blastocyst the main cayity is the exoccelom, into which the
reduced omphalon hangs freely. These conflicting relations
depend upgn the varying degrees of development of the
exoccelom, allantois and omphalon respectively. In the
opossum the embryo with its allantoic vesicle and exoccelom
is suspended from the chorion or wall of the foetal sac into
the main cavity (omphalon) by a hollow exoccelomic stalk.
In the Primates the embryo with its umbilical vesicle is sus-
BLASTOCYST AND PLACENTA OF THE BEAVER. 239
pended from the chorion into the main cavity (exoccelom) by
a massive allantoic stalk. There is no massive allantoic
mesoderm in the opossum and no hollow allantoic vesicle in
ce P]
man. Just as the term “chorion” is often used to denote
the wall of the foetal sac, irrespective of the constitution of
its membranes, so the term “ Haftstiel” denotes the mecha-
nism by which the embryo is suspended within the cavity of
the foetal sac.
The real knowledge of the Primate “ Haftstiel”’ dates from
His’s third memoir on the ‘Anatomie Menschlicher Hmbryonen ’
(1885). I have not seen the original memoir, but the subject
is treated very fully by C. §. Minot in his contribution entitled
“Uterus and Embryo” (1889). His made what Minot
appraised as ‘‘the discovery of fundamental importance ”—
that in the early human blastocyst the allantoic sac appears
as a small endodermic tube lying in a posterior prolongation
of the body which His called the “ Bauchstiel,” and that at
this early stage the allanto-chorionic vessels already run to
and branch out upon the chorion. Thus “the allantois is,
from the first, continuous with the chorion” (Milnes Marshall,
1893).
F. Keibel has stated recently (1913) that the allantoic tube
in the human blastocyst is an entirely vestigial structure ;
he does not say explicitly of what it is a vestige, but the text
imphes clearly that he considers it to be a vestige of the free
allantoic vesicle of Sauropsida. The beaver may help us to
another explanation, namely, that it is a vestige of that
portion of the omphalon which descends into a keel-shaped
“Haftstiel”” or exostyle. The latter term can be used in
general as an equivalent rendering of ‘ Haftstiei,” its
etymology being analogous to that of exoccelom, both terms
referring to structures that lie outside the embryo.
Selenka’s memoir on the “ Affen Ostindiens” appeared in
1891. Inthis monograph it was shown that the characteristics
of the monkey’s blastocyst are: (i) The early separation of
the omphalon (‘ yolk-sac”’) from the chorion; it takes no part
in the nutrition of the embryo and must be regarded as a
voL. 60, parT 2.—NEW SERIES. Na
24.0 ARTHUR WILLEY.
vestigial structure ; it is scantily supplied with vessels and
floats as a small stalked vesicle in the exoccelom, until by the
expansion of the amnion it becomes pressed against the
chorion and finally succumbs to resorption.
(2) The spacious exoccelom acts as a reservoir of food-stuffs
until the allantoic vessels take over the function of fcetal
metabolism.
(3) After the formation of the amnion (the method of
which was not observed), the embryo retains its connection
with the placental chorion by a solid cord of mesoblast into
which a rudimentary allantoic tube penetrates. ‘lhis massive
cord with its vestigial endodermic cavity is the allantoic stalk
or “ Haftstiel,”’ the vehicle of the placental vessels.
(4) The main cavity of the early blastocyst is the exoccelom.
Subsequently the amniotic cavity dilates enormously and the
amnion finally fuses with the chorion, so that the cavity of
the foetal sac is then amniotic cavity.
From the. above résumé it is obvious that Selenka’s
‘‘ Haftstiel ” is identical with His’s ‘‘ Bauchstiel.” As an
English equivalent the expression ‘‘ body-stalk” has been
suggested by C. 8. Minot and adopted by J. W. Jenkinson ; but
if the comparison with the beaver is accepted, the need for a
more general term, e.g. exostyle, is indicated.
We must now refer briefly to Hubrecht’s classical paper in
Gegenbaur’s ‘ Festschrift’ (1896), entitled ‘ Die Keimblase von
Tarsius.” The chief characteristics of the Tarsius blasto-
cyst may be summarised :
(1) Rauber’s layer, or the trophoblast over the formative
epiblast, disappears in the very early stages, shortly after the
delamination of the hypoblast.
(2) The exoccelom has a remarkably precocious develop-
ment and the hypoblastic sac occupies only a small portion of
the spacious blastocyst.
(3) The blastodisce is at first excentric. The ectoplacental
proliferation of the trophoblast is situated some way behind
the blastodisc, not diametrically opposite to it.
(4) Between the ectoplacental proliferation and the posterior
BLASTOCYST AND PLACENTA OF THE BEAVER. 241
border of the blastodisc (embryonic shield), there extends a
solid mesoblastic tract which at the same time forms part of
the wall of the closed mesoblastic sac or exoccelom ; this solid
cord is the “‘ Haftstiel.’ At this stage there is no mesoderm
in the embryo and the region of the embryonic shield is still
didermic.
(5) The blastocyst is only attached to the uterine wall by
the ectoplacental disc; otherwise it lies free in the uterine
lumen.
(6) A complete and close-meshed area vasculosa
develops in the wall of the hypoblastic sac or umbilical
vesicle, and is filled with blood-corpuscles long before the
heart begins to beat; but vascular rudiments arise in the
“ Haftstiel ” before the appearance of the omphaloidean net-
work.
(7) The mesoblast of the “ Haftstiel ” is at first contiguous
with the adjacent trophoblast; but, pari passu with the
formation of the amnion, it becomes separated from the
trophoblast by insinuation of the exoccelom and so becomes
converted into the primordial umbilical cord.
(8) After the ‘“ Haftstiel”” has become vascularised and
enlarged, a tubular outgrowth from the umbilical vesicle
penetrates backwards into the connective tissue of the
“Haftstiel.” This is the allantoic diverticulum of the
umbilical vesicle.
From this brief tabulation of Hubrecht’s discoveries re-
garding the blastocyst of Tarsius, we gather that although
the allantoic tube is a secondary outgrowth, it belongs to the
omphalon. There is no question of its being a diverticulum
of the hind-gut at its first origin. Again, the “ Haftstiel”
is directly continuous with the primitive streak, and forms,
at the beginning, part of the wall of the blastocyst behind the
embryo. In 1889 Hubrecht, 4 propos of Hrinaceus, had
defined the “ Haftstiel” as a caudal mesoblastic cord which
grows backwards from the posterior end of the primitive
streak in order to promote the early vascularisation of the
chorion. To this definition Resink (1904) addedin italics: ‘‘ Der
242 ARTHUR WILLEY.
Haftstiel entsteht jedoch als die von Anfang an vorhandene
Verbindung des entypierten Keimfeldes mit dem Chorion.”
In the preplacental blastocyst of the beaver, the endo-
dermic allantois does not appear as an outgrowth, but is
actually a deep hypoblastic groove of the omphalon extending
into the keel. It is, therefore, from the first an integral part
of the omphalon. ‘lhe steps by which this hypoblastic
groove becomes narrowed down to the allantoic canal with
its distal flattened sac cannot be followed in the material at
my disposal. It must take place simultaneously with the
closure of the digestive tract of the embryo, which is
similarly associated with the narrowing down of the omphalo-
mesenteric duct.
With reference to the above quotation from Resink, a pupil
of Hubrecht, the important point in my estimation is his
insistence upon the primary character of the “ Haftstiel,”
which is not in its essence a secondary formation (cf. Milnes
Marshall, 1893). The flattened “ Haftstiel ” of Tarsius may
be directly compared with the keel-shaped exostyle of the
beaver. Both of these structures are organs of fixation, by
means of which the embryo attaches itself to the placental
complex. The ectoplacental disc is derived from the
proliferation of the exostylar trophoblast. The localisation
of the “ Haftstiel” in Tarsius and the exostyle in the
beaver affords strong confirmation of the view put forward
by Minot in 1889, that the discoidal placenta is probably a
primitive placental type. The chief characteristic of the
blastocyst of Castoris the possession of a massive keel-shaped
exostyle or “ Haftstiel” into which the endodermic allantois
extends as a primary groove of the omphalon; and the
exostyle itself is directly continuous with the proliferation
of the primitive streak. In his useful, though doubtless
ephemeral, speculations concerning the origin of the foetal
annexes of Mammalia, Resink admitted that he was unable
to explain the origin of the allantoic sac. Perhaps the
beaver may offer a new point of view from which this
problem may be envisaged.
BLASTOCYST AND PLACENTA OF THE BEAVER. 243
Within the limits of the order of the Rodentia, the con-
ditions preceding the ectoplacental implantation of the
beaver’s blastocyst are most readily compared with those
obtaining in squirrels. According to Fleischmann (1893), in
Spermophilus, where there is no preplacental keel, the
gestation sac is none the less subdivided into an omphaloid
cavum and a discoid placental cavum or “Scheibenhdhle.”
The young blastocyst is attached in the omphaloid cavum,
and is so orientated that the embryonic pole (blastodisc) lies
over the narrow passage (called ‘‘ Schlossspalte”’) connect-
ing the omphaloid cavum with the discoplacental cavum.
The latter is comparable with the deep mesometric groove in
the gestation sac of the beaver.
The isolated position and high standing of the beaver
amongst existing Rodents are prima facie evidence of an
exceedingly remote origin. Other facts of organisation and
paleontology are in harmony with such evidence. These
circumstances lend peculiar significance to the character ot
the preplacental blastocyst.
We may now give sume attention to the behaviour of the
inferior or obplacental hemisphere of the beaver’s blastocyst.
Without greatly enlarging the scope of the illustrations
accompanying this paper, it would be impossible to do
justice to the wonderful scenes of substitution of trophoblast
for uterine epithelium, which can be witnessed in almost
every section. In the preplacental blastocyst of the hedge-
hog, Hubrecht found a phagocytic trophoblast forming a
complete trophosphere round the blastocyst. The ecto-
placenta is a localised derivative of the trophoblast. Resink,
however, applied the term “ectoplacenta”’ to the entire tropho-
blast. On the present occasion I propose to confine my
remarks to Rodentia.
Obplacental ectodermal proliferations were described in
the blastocyst of the rabbit by A. von Kolliker in 1882.
According to Kolliker an event of fundamental importance
for mammalian embryology was Rauber’s discovery in 1875
24.4, ARTHUR WILLEY.
‘of the so-called ‘‘ Deckschicht’’ outside the formative epi-
blast of the rabbit’s blastocyst, showing that the traditional
view till then maintained by Coste, Hensen, and Kolliker, that
the wall of the monodermic blastocyst had the value of
embryonic ectoderm, was wrong. Kolliker called Rauber’s
“ Deckschicht ” the primitive ectoderm of the area embryo-
nalis. Of next importance was Lieberkuhn’s demonstration
in 1879 of the origin of the permanent or formative ectoderm
of the rabbit out of the inner cell-mass which lies against
the primitive ectoderm and splits into two layers—the
permanent ectoderm and endoderm. Kolliker’s primitive
ectoderm of the rabbit’s blastocyst thus prepared the way for
Hubrecht’s celebrated conception of the trophoblast of the
mammalian blastocyst. In the extra-embryonic primitive
ectoderm of the rabbit Kélliker discovered numerous ecto-
dermal proliferations in the form of elongate, villiform eleva-
tions due to local nuclear divisions.
These papille were next described by E. van Beneden and
C. Julin in 1884. On opening an eleven-day gestation sac of
the rabbit under picrosulphuric acid, by a crucial incision
through the antimesometric wall, a liquid escaped and a
coagulum was produced. ‘This was the fluid from the blasto-
dermic cavity, which becomes the umbilical or vitelline
vesicle or ‘‘ yolk-sac.” The cavity had been opened because
the obplacental wall of the blastocyst was intimately united
to the uterine mucosa. The union between the wall of the
umbilical vesicle and the mucosa is effected by means of the
epiblastic buds, which, as the authors stated, Kolliker first
noticed. These buds arise upon the whole surface of the
inferior hemisphere of the blastocyst, commencing about the
eighth day. The degeneration of the cells, whose prolifera-
tion engenders the buds, begins at the ninth day, and leads
to the rapid degeneration of the entire epiblastic membrane,
which, at the fourteenth to fifteenth day, detaches in shreds
from the mucosa.
M. Duval (1890, pl. i, fig. 28) described and figured the
bilaminar inferior hemisphere of the rabbit’s blastocyst at the
BLASTOCYST AND PLACENTA OF THE BEAVER. 245
tenth day, and observed the plasmodial swellings connected
by intervening thin tracts, but he let them degenerate without
having effected any useful purpose.
R. Assheton (1895) also mentioned and illustrated (‘ Quart.
Journ. Micr. Sci.,’ 37, Pl. 19, fig. 7) the obplacental papillee
of the rabbit. He says the first attachment of the blastocyst
takes place between the lower parts of the blastodermic
vesicle and the periplacental and obplacental folds of the
uterus. It is effected by means of epiblastic papillae about
the eighth day. Assheton suggested that the papillae became
wedged into the uterine epithelium by reason of hydrostatic
pressure from within the vesicle.
H. Schoenfeld (1902) made the first complete cytological
study of the festoon-like adhesions of the preplacental blasto-
cyst of the rabbit on the seventh and eighth days. At seven
days the zona pellucida becomes broken on the antimeso-
metric side of the blastocyst and multinucleated ectodermal
thickenings have appeared. At 7 days 4 hours the ecto-
dermal buds come into intimate adhesion with the uterine
syncytium and push into it with pseudopodium-like processes
so as to reach the maternal capillaries, thus fixing the blasto-
cyst to the mucosa in a manner comparable to the rooting of
a plant. From the seventh day of gestation until 8 days
4 hours, the blastocyst remains attached to the mucosa on the
obplacental side. At 8 days 6 hours numerous plurimitotic
figures occur in the epiblast of the obplacenta. At 8 days
22 hours Minot’s giant-cells appear in the obplacental mucosa.
They are of fcetal origin, resulting from retrogressive
changes of the obplacental multinucleated epiblastic buds,
now embedded in the mucosa. At their origin they are much
more voluminous than any elements belonging to the mucosa
or submucosa. According to Maximow, they reach their
maximum development, with a diameter of 100 yw, on the
sixteenth to twentieth days.
J. Rejsek (1904) describes the obplacental implantation of
the very young blastocyst of the Souslik (Spermophilus
citillus). It continues until the blastocyst acquires a
246 ARTHUR WILLEY.
diameter of 2 mm., when it is given up simultaneously with
the incipient placentation, which coincides in time with the
cervical flexure of theembryo. The obplacental implantation
is effected by means of a single multinucleate trophoblastic
plasmodial proliferation at the antimesometric pole, sending
processes into the mucosa. The number of nuclei increases
by amitosis from 22 in a blastocyst of 0°126 mm. to more
than 500. ‘This obplacental implantation of the souslik’s
blastocyst would seem to be a phenomenon sui generis, the
initial plasmodial thickening corresponding to a single
papilla of the rabbit’s blastocyst.
F. Muller (1905) also observed the antimesometric attach-
ment of the preplacental blastocyst of Sciurus (vide his
pl. vii, fig. 8).
For the characteristic features of the obplacental implanta-
tion of the preplacental blastocyst of the beaver, I must
refer the reader to the foregoing text. The obplacental
trophoblast is still connected with the uterine wall at the
early placental stage, so that the implantation endures for a
much longer period than in the rabbit. Eventually, when
the invagination of the umbilical membrane is completed and
the obplacental omphalopleure has vanished, giant-cells
remain in the mucosa, appearing as white specks peppered
over the internal surface of the gestation sac, scattered or in
groups, not evenly distributed.
In the preplacental stage the trophoblast, at the areas of
attachment, is not more than one layer in thickness and its
cells remain distinct, though they may have several nuclei.
It does not send processes to any depth into the mucosa, but
asa tule it has a flat insertion upon the decidual surface, the
maternal capillaries pressing towards the trophoblast rather
than otherwise. Wherever the uterine epithelium is displaced
by the obplacental trophoblast, it disappears without previously
forming a syncytium. It is surprising to find the trophoblast
planted like a pseudo-epithelium upon the decidual surface,
and, at its borders, normal uterine epithelium. This probably
means that the uterine epithelium becomes necrotic in
BLASTOCYST AND PLACENTA OF THE BEAVER. 247
advance of the trophoblastic attack and that the trophoblast
does not kill normal epithelium. When the mesometric
glands degenerate, they do not form syncytia in the beaver,
but the glandular epithelium becomes necrotic. The syncytia
in the trophospongia at the early placental stage arise froma
special proliferation of uterine epithelium, which takes place
by mitosis in the mesometric region at the preplacental stage,
before the ectoplacental proliferation of the trophoblast has
set in.
XVI. BrsLioGRAPHY.
[The subjoined references are supplementary to the list given by me
in 1912, and the items are not repeated here. Those marked with an
asterisk have not been seen. |
*1, Beneden, Edouard van.—* Sur les Placentas discoides,” ‘Comptes
rendus Soc. Biol.,’ November 9th, 1888.
and Charles Julin.—* Recherches sur la formation des
annexes fctales chez les Mammiféres (Lapin et Cheiropteéres).”
‘Archives de Biol.,’ v, 1884, pp. 369-434.
- Bischoff, Theodor von.—(1) ‘ Entwickelungsgeschichte des Kanin-
cheneies, Braunschweig, 1842. (2) ‘ Entwickelungsgeschichte
des Meerschweinchens,’ Giessen, 1850. (3) ‘Neue Beobach-
tungen zur Entwickelungsgeschichte des Meerschweinchens,’
Miinchen, 1866.
. Bonnet, Robert.— Lehrbuch der Entwickelungsgeschichte,’ 2° Aufi.,
Berlin (Paul Parey), 1912.
. Chipman, Walter.—‘ Observations on the Placenta of the Rabbit.
with Special Reference to the Presence of Glycogen, Fat and
Tron,” ‘Studies from the Royal Victoria Hospital, Montreal,
vol. i, No. 4, 1902, 261 pp., 8vo.
Fernandez, Miguel. Beitriige zur Embryologie der Giirteltiere.
I. Zur Keimblatterinversion und spezifischen Polyembryonie der
Mulita (Tatusia hybrida Desm.),” ‘Morph. Jahrb., xxxix,
1909, pp. 302-333.
7. Fleischmann, A.—‘ Embryologische Untersuchungen, III,” ‘ Die
Morphologie der Placenta bei Nagern und Raubtieren,’ Wies-
baden, 1893.
*8. His, W.—‘ Anatomie menschlicher Embryonen,’ i, 1
ili, 1885.
2.
*
ie)
nN
On
&
io 6)
CO
He
pas
—
19
19 6
tho
248
ARTHUR WILLEY.
9. Hubrecht, A. A. W.—(1) “The Placentation of Erinaceus euro-
pus, with Remarks on the Phylogeny of the Placenta,” ‘Quart.
Journ. Mier. Sci.,’ 30, December, 1889, pp. 283-404, London, 1890.
(2) “The Placentation of the Shrew (Sorex vulgaris L.),” op.
cit. 35, 1894, pp. 481-537. (3) “ Die Keimblase von Tarsius:
Kin Hilfsmittel zur schirferen Definition gewisser Saugetier-
ordnungen,’ ‘Gegenbaur’s Festschrift, pp. 148-178, Leipzig,
1896.
10. Jenkinson, J. W.—(1) “ Observations on the Histology and Physio-
logy of the Placenta of the Mouse,” ‘ Tijdschrift d. Ned. Dierk.
Vereen.” (2), vii, 1902, pp. 124-198. (2) ‘ Vertebrate Embryology,’
Oxford (Clarendon Press), 1913.
*l1. Jhering, H. von.—(1) “ Uber die Fortpflanzung der Giirteltiere,”
‘Sitzungsber. Kon. Preuss. Akad. Wiss.,’ Heft 47, 1885, p. 105.
(2) “Uber Generationswechsel bei Saugetieren,’ ‘Arch. f. Anat.
u. Physiol. Phys. Abth.,’ 1886, pp. 442-450.
12. Julin, Charles.—See Beneden, E. van, and C. Julin.
13. Keibel, Franz.—* Die Entwicklungsgeschichte der Wirbeltiere,”
pp. 333-398, in “ Zellen- und Gewebelehre, Morphologie und
Entwicklungsgeschichte, II,’ ‘Zoologischer Teil. Leipzig
(Teubner), 1913.
14, Kolliker, Albert von.—* Die Entwicklung der Keimblatter des
Kaninchens,” ‘ Wurzburg Festschrift,’ Bd. i, Leipzig, 1882.
15. Lieberkiihn, Nathanael. Ueber die Keimblitter der Siugethiere,”.
Marburg, 1879. [Abstract in ‘Zoologischer Jahresbericht’ for
1880, Leipzig, 1881}.
16. Maximow, Alexander.—* Die ersten Entwicklungstadien der Kanin-
chen Placenta,” ‘Arch. f. mikr. Anat.,’ lvi, 1900, pp. 699-740.
17. Minot, Charles Sedgwick—‘“ Uterus and Embryo: I, Rabbit.
IT, Man,” ‘ Journ. Morph.,’ ii, 1889, pp. 341-462.
i8. Muller, F.—‘‘ De Wederzijdsche Verhouding tusschen ei en uterus
bij de Knaagdieren meer in het bijzonder bij Sciurus vulgaris,”
‘Tijdschrift d. Ned. Dierk. Vereen.’ (2), ix, 1905, pp. 329-585.
19. Newman, H. H., and J. T. Patterson.—(1) “A Case of Normal
Identical Quadruplets in the Nine-banded Armadillo, and its
Bearing on the Problem of Identical Twins and of Sex
Determination,” ‘ Biol. Bull.,’ xvii, 1909, pp. 181-187. (2) “ The
Development of the Nine-banded Armadillo from the Primitive
Streak Stage to Birth; with Especial Reference to the Question
of Specific Polyembryony,” ‘ Journ. Morph.,’ xxi, 1910, pp. 359-
423.
BLASTOCYST AND PLACENTA OF THE BEAVER. 249
20. Nolf, Pierre.—* Etude des modifications de la muqueuse utérine
pendant la gestation chez le murin (Vespertilio murinus),”’
‘Archives de Biol.,’ xiv, 1896, pp. 561-693.
21. Patterson, J. T.—See Newman and Patterson.
*22. Rauber, August.—‘ Ueber die erste Entwicklung des Kaninchens,”’
‘Sitzungsber. Naturf. Gesell.,’ ii, pp. 103-109, Leipzig, 1875.
‘28. Rejsek, J.—‘Anheftung (Implantation) des Siaugetiereies an die
Uteruswand, insbesondere des Eies von Spermophilus
citillus,” ‘Archiv f. mikr. Anat.,’ lxiii, 1904, pp. 259-273.
24. Resink, A. J—‘‘ Die Stammentwickelung der embryonalen Organe,”
‘Tijdschrift d. Ned. Dierk. Vereen.’ (2), viii, pp. 159-201, Leyden,
1904.
25. Schoenfeld, H.—‘ Contribution 4 l'étude de la Fixation de uf
des Mammiféres dans la cavité utérine, et des premiers Stades de
la Placentation.” [Rabbit and Dog.] ‘Archives de Biol.,’ xix,
1903, pp. 701-830.
26. Selenka, Emil.—(1) ‘Studien iiber Entwickelungsgeschichte der
Thiere. Heft. 4, “Das Opossum (Didelphys virginiana),’
Wiesbaden, 1887. (2) Id., Heft 5, 1° Halfte, No. 4, “ Affen
Ostindiens,’ Wiesbaden, 1891.
27. Tafani, P. A—‘Sulle condizioni uteroplacentari della vita fetale,’
Firenze, 1886.
28. Tullberg, Tycho.—‘ Ueber das System der Nagethiere,’ Upsala, 1899.
29. Vernhout, J. H.—“ Uber die Placenta des Maulwurfs (Talpa
europea L.),’ ‘Anat. Hefte, V,’ Heft 1, 1894, pp.3-49.
30. Weber, Max.—* Beitrage zur Anatomie und Entwickelung des Genus
Manis,” ‘Zoologische Ergebnisse,’ Bd. ii, pp. 1-117, Leyden, 1892.
[See p. 15 and Taf. ii, fig. 17, for reference to beaver’s tail. |
31. Willey, A.—‘‘ Foetal Membranes of the American Beaver (Castor
canadensis),” ‘Zool. Jahrb., Suppl. XV, Bd. ii, pp. 191-218;
‘Spengel’s Festschrift, Jena, 1912. [With analysis of the
amniotic fluid by Professor R. F. Ruttan.]
EXPLANATION OF PLATES 14 ro 21,
Illustrating Mr. Arthur Willey’s paper on “ 'The Blastocyst
and Placenta of the Beaver.”
[Some of the drawings were reversed under the Edinger apparatus,
and others were not reversed. It has not been thought necessary to
250 ARTHUR WILLLEY.
mention this in each case. Figures executed by Mr. John Prijs are
accredited to him under the explanations. |
PLATE 14.
Fig. 1.—Section across middle of gestation sac with blastocyst in
sitt. The darker shading round the cavum uteri indicates maternal
proliferation. The upper circle surrounds the erypt-like mouth of a
gland near which there was a trophoblastic implantation. The lower
circle surrounds the keel which projects into the very deep placental
groove. (J. Prijs, del.)
Fig. 2.—Portion of pericarinal zone to show isolated intrusions of
trophoblast. 1. Hypoblast. 2. Trophoblast. 3. Interstitial substance
different from the coagulum within the omphalon. 4. Uterine epithe-
hum. 5. Subepithelial capillary vessel full of red blood-corpuscles.
The cytoplasm of the uterine epithelium stains darkly with orange G. ;
that of the trophoblast lightly.
Fig. 3.—Fragment showing phagocytic attack on the part of megalo-
karyocytes. 1. Uterine epithelium undergoing destruction. 2. Mucosa.
3. Capillary. 4. Megalokaryocytes. 5. Coagulum in cavum uteri.
6. Portion of gland. (J. Prijs, del.)
Fig. 4.—Portion of coronal region, showing complete substitution of
trophoblast for uterine epithelium on each side of the mouth of a gland
which retains its epithelium intact and contains groups of leucocytic
granules in its cavity. The abruptness of the cellular changes is not
exaggerated. Note the flat contact between trophoblast and denuded
surface of mucosa. 1. Obplacental gland. 2. Obplacental trophoblast.
3. Flattened trophoblast bridging mouth of gland. 4. Two trophoblast
cells charged with maternal erythrocytes.
Fig. 5.—Plan of regions of blastocyst and mucosa about the middle
of the gestation sac. The hypoblast is indicated by a broken line
(substage C). 1. Coronal region. 2. Pericoronal cavum. 3. Peri-
omphaloid zone. 4. Pericarinal festoons. 5. Adcarinal omphalopleure.
6. Keel. 7. Bottom of placental cavum at the mesometric side of
gestation sac. 8. Cavity of omphalon. 9. Peripheral limit of mesoblast
or sinus terminalis. 10. Obplacental glands. 11. Uterine epithe-
lium. 12. Megalokaryocytes (trophoblast).
Fig. 6 (substage A).—Keel in anterior region of blastocyst. 1. Peri-
pheral trophoblast. 2. Hypoblastic groove. 3. Mesoblast. 4. Formative
epiblast rounding edge of keel, showing scattered vesicular growing cells.
Fig. 7—Same. Near front end of primitive streak. Formative epi-
blast and mesoblast tinted. 1. Hypoblast doubled by unsplit mesoblast.
2. Primitive streak.
BLASTOCYST AND PLACENTA OF THE BEAVER. 745 |
Fig. 8—Same. Wedge-shaped proliferation of formative epiblast in
the primitive streak.
Fig. 9—Same. A cleft appears in the centre of the primitive streak.
- Fig. 10—Same. Embryonic area 50 » farther back. Cell outlines
are visible in the formative epiblast, but not rendered in the drawing.
Mitoses in the primitive streak. The asterisk (*) is placed at the limit
of the formative epiblast on that side of the keel; there is a vesicular
cell at that point.
Fig. 11—Same. The peripheral limit of the carinal half of the
formative epiblast is nearing the edge of the keel. An intermittent
groove is seen upon the surface of the primitive streak.
Fig. 12—Same. The peripheral limit of the carinal half of the
formative epiblast has reached the edge of the keel. The mesoblast
band of that side has shortened and become thicker. Another groove
is seen at the primitive streak.
Fig. 13.—Same. Plan of cavum uteri with contained blastocyst.
Drawn freehand as seen under microscope. The other sections of this
series were outlined under Edinger’s apparatus with slide reversed.
The blastocyst is only attached at the pericoronal angles. The hypo-
blast is indicated by a broken line. (1) Coronal uterine epithelium.
(2) Coronal cavum with coagulum. (3) Leucocyte in coronal cavum.
(4) Coronal chromatophile trophoblast. (5) Omphalon. (6) Embryonic
shield with primitive streak and mesoblast. (7) Placental groove.
Fig. 14.—Same. Massive tissue at posterior end of primitive streak.
This is the commencement of the “ haftstiel”’ or “ exostyle ” formation.
Fig. 15.—Same. The last of the mesoblast, a thin band on one side
of the hypoblastic groove, behind the primitive streak.
PLATE 15.
Fig. 16.—Sub-stage B. Anterior region of blastocyst, lying free.
(1) Omphalon. (2) Trophoblast. (3) Hypoblast.
Fig. 17—Same. Farther back in the anterior region, the blastocyst
still lying free. (1) Omphalon. (2) Peripheral trophoblast composed
of megalokaryocytes. They are more numerous than in the figure. (3)
Hypoblast passing over the cavity of the trophoblastic keel. (4) Keel
composed of cubical trophoblast, its walls agglutinated distally. (5)
Chromatophile megalokaryocytes. «. Peculiar plasmodial effect at the
pericarinal region, overlapping the cubical trophoblast. Semi-dia-
grammatic.
Fig. 18.—Posterior half of embryonic region showing deep groove
over the primitive streak. Formative epiblast tinted dark, mesoblast
952 ARTHUR WILLEY.
lighter. The half of the formative epiblast which surrounds the keel
is longer and thinner than the portion which lies above the groove in
the figure, i.e. it is more stretched out, the proportion being 550 pu to
350 nu. The epithelial change at (*) consists in this, that the nuclei of the
peripheral trophoblast lie at one level instead of at different levels, and
they are usually stained paler than those of the formative epiblast.
There is a potentiai linear celom indicated at the top of the massive
carinal mesoblast on the left of the figure.
Fig. 19. Same. Carinal region in the posterior part of the blasto-
cyst showing relations of trophoblast (black), mesoblast (pale), and
hypoblast.
Fig. 20.—Substage C. The didermic keel in the posterior region of
the blastocyst lying freely between the walls of the placental groove.
Subepithelial maternal capillaries indicated in black. (J. Prijs, del.)
Fig. 21—Same. The tridermic keel in the posterior region. (1)
Posterior end of mesoblast. (2) Loose hypoblast.
Fig. 22—Same. The keel with the posterior exocelom. The right
side of the figure is the embryonic side of the keel. The slide was
reversed under Edinger’s apparatus. (1) Solid proliferation marking
the peripheral limit or sinus terminalis of the mesoblast. (2)
Hypoblast (represented by broken line). (3) Posterior exoceelom.
Fig. 23.—Same. Portion of obplacental trophoblast in the coronal
region with adjoining part of the cavum uteri and uterine epi-
thelium. (1) Uterine epithelium (nuclei omitted). (2) Coagulum in
cavum uteri. (3) Trophoblast. (4) Leucocytes in mucosa crossing
uterine epithelium, lodged in uterine coagulum, and entering the
trophoblast.
Fig. 24—Same. Plan of cavum uteri and blastocyst at the level
of fig. 23. A band of slime extends from the edge of the keel nearly
to the bottom of the placental groove. (1) Coronal epithelium. (2)
Coronal cavum. (3) Coronal trophoblast. (4) Omphalon. (5) Keel.
(6) Slime.
Fig. 25——Same. Part of obplacental region showing flattened
megalokaryocytes with ingested leucocytic nuclei appearing as dark-
stained granules, sometimes surrounded by a vacuole. (1) Uterine
epithelium. (2) Coagulum in cavum uteri. (3) Chromatophile
trophoblast.
PLATE 16.
Fig. 26.—Same. Section through keel in region of massive exostyle.
The cubical epiblast changes its character abruptly, so that the exo-
style proper is not covered by a cubical epithelium. (1) Sinus
BLASTOCYST AND PLACENTA OF THE BEAVER. Zoo
terminalis on embryonic side. (2) Reduced exocelom. (3) Hypo-
blastic groove. (4) Exostyle. (5) Uterine epithelium.
Fig. 27.—Same. Section through exostyle in siti, showing detached
‘shreds. The piece of uterine wall to the right of the figure is drawn
nearer to the keel than it was in reality when seen under the
“Zeichenapparat.” The position of the nuclei opposite the lower shred
seems to indicate traction or stress. (1) Gland opening into placental
cavum. (2) Space at apex of keel. (3) Placental cavum. (4) Uterine
epithelium on wall of placental cavum. (5) Section of maternal capillary.
Fig. 28.—Same. Section through middle of exostyle, which is here
nearly solid throughout, with a linear interrupted cavity towards the
apex. (1) Exocelom. (2) Hypoblastic groove. (3) Cavity of exostyle
shut off at this level from (2). (4) Uterine epithelium. (5) Slime at
apex of exostyle.
Fig. 29.—Same. Anterior region of exostyle, showing central cavity
continuous with the omphalon. (1) Omphalon. (2) Sinus terminalis.
(8) Exoccelom on embryonic side of keel. (4) Cavity of exostyle. (5)
Uterine epithelium. (6) Loose hypoblast.
Fig. 30.—Same. The keel in the region of the primitive streak and
formative epiblast. The asterisks mark the limits of the formative
epiblast or embryonic shield. (1) Exoccelom. (2) Primitive streak.
(3) Hypoblastic cavity. (4) Sinus terminalis; unsplit mesoblast
extends a short way beyond it. (5) Anti-embryonie trophoblast. (6)
Anti-embryonic unsplit mesoblast. N. B.—On the embryonic side the
sinus terminalis lies at a higher level, out of the range of the
drawing.
Fig. 51—Same. The keel in front of the primitive streak. (1) Peri-
pheral trophoblast. (2) Parts of the celom. (3) Proximal groove on
embryonic face of keel. (4) Hypoblastic cavity. (5) Anti-embryonic
part of the marginal proliferation of mesoblast or sinus terminalis.
(6) Epiblastic cavity of keel.
Fig. 32.—Same. The keel at the level of the anterior limit of meso-
blast, in front of the celom. (1) Sinus terminalis. (2) Hypoblastic
cavity. (3) Carinal trophoblast.
Fig. 33.—Same. Proximal groove of the epiblast in front of the
mesoblast, showing proliferation with mitoses.
TEI ANIM DH Alz/e
Fig. 34.—Same. Keel in front of the mesoblast.
Fig. 35.—Substage D. Section through keel in the post-stylar region
cutting the exostyle tragentially. The ccelom appears in two discon-
tinuous cavities, owing to the intrusion of the massive mesoblast.
254 ARTHUR WILLEY.
(Freehand.) (1) Sinus terminalis. (2) Posterior exocelom. (3)
Exostylar mesoblast. (4) Unsplit mesoblast. (5) Hypoblastic groove.
Fig. 36.—Same. The keel in the region of the exostyle. Fifty-five
sections of 10, intervene between this section and that drawn in
fig. 35; in this interval the exostylar mesoblast has been increasing in
amount. Note the abrupt passage of cubical trophoblast into the
flattened epiblast of the exostyle. The exostyle does not exhibit such
a luxuriant development in this series as in others. (1) Sinus
terminalis. (2) Exocelom. (3) Exostyle. (4) Hypoblastic groove.
(Freehand.)
Fig. 37.—Same. Section through keel in region of primitive streak.
There are twenty-six sections of 10 between this and fig. 36. (Free-
hand.) (1) Exocelom. (2) Pericardial primordium bounded by cubical
epithelium outside, columnar on the inner side. (5) Regular hypoblast
co-extensive with the formative epiblast. (4) Primitive streak. (5)
Distal limit of formative epiblast, with abrupt epithelial transition.
Fig. 38—Same. Anterior region of embryonic shield. In the next
section the two sections of the ccelom fuse over the carinal hypoblast,
which then ends in a tangentially cut mass of cells. (Freehand.) (1)
Amniotic folds. (2) Carinal embryonic hypoblast. (3) Pericardial
primordium. (4) Omphaloidean hypoblast. (5) Exocelom. There are
thirty-nine sections of 10, between this and fig. 37. For position of
embryonic shield with reference to the keel-formation, compare Pl. 14,
figs. 6 and 7.
Fig. 39.—Same. Anterior end of pre-embryonic ceelom with epiblastic
keel-extension. Freehand. (1) Exocelom. (2) Broken line indicating
omphaloidean hypoblast.
Fig. 40.—Substage E. Section through the keel at the level of the
anterior region of the embryonic shield. (1) Sinus terminalis. (2)
Linear occluded exocelom. (3) Formative epiblast. (4) Pericardial
primordium. (5) Cubical embryonic hypoblast.
Fig. 41.—Same. Keel at level of notochordal primordium (1). The
hypoblastic epithelium changes its character on each side of the noto-
chordal contact zone. The asterisk denotes the proximal limit of
embryonic shield.
Fig. 42.—Same. Keel at the anterior part of the primitive streak (1).
(2) Epiblastic keel.
Fig. 43.—Same. Keel about middle of primitive streak.
Fig. 44.—Same. Post-embryonic keel at the level of the anterior end
of the exostyle. (1) Exostyle or “ Haftstiel” tissue. For the relations
of the surface membrane at such places of close union, compare PI. 16,
fig. 29.
BLASTOCYST AND PLACENTA OF THE BEAVER.
bo
Or
Or
Fig. 45.
Fig. 46—Same. Posterior end of exostyle. The whole extent of
mesoblastic bands is shown. (1) Sinus terminalis. (2) Exoccelom.
(3) Hypoblastic groove. (4) Massive mesoblast. (5) Exostyle. (6)
Trophoblastic keel.
Same. Mid-region of exostyle.
PLATE 18.
Fig. 47—Same. Region of post-stylar celom. (1) Post-stylar exo-
celom. (2) Hypoblastic groove.
Fig. 48.—Same. Posterior termination of mesoblast and exoccelom.
The hypoblast has been reconstructed in the drawing. (1) Sinus
terminalis. (2) Exocelom. (3) Uterine epithelium.
Fig. 49.—Same. Didermic keel behind the mesoblast. The flattened
epiblast cells graduate into flattened megalokaryocytes (not shown).
Fig. 50.—Same. Towards posterior pole of blastocyst. The hypo-
blast withdraws from the keel. (1) The last of the carinal hypoblast.
Fig. 51.—Substage F. Section through entire gestation sac with
blastocyst in sitt. (J. Prijs, del. (1) Omphalon. (2) Omphaloid
cavum; the keel is seen to the left of the index number. (3) Placental
groove; to be compared with Pl. 14, fig. 1. (4) Placental tropho-
spongia. (5) Periplacental wall; the fine tubes in the walls are partly
glands, partly capillaries. (6) Obplacental wall. (7) Mesometrium.
Fig. 52.—Same. Appearance of the keel in the anterior polar region
of the blastocyst. About 90 sections of 10 precede this in the series.
(1) Commencement of mesoblast on embryonic side. (2) Hypoblastic
groove. (3) Trophoblastic cavity. (4) Trophoblastic keel.
Fig. 53.—Same. Keel at level of anterior part of embryonic shield.
In an earlier section the pericardium communicated at its upper end
with the exocclom. (1) Sinus terminalis; the exocclom, real or
virtual, extends up to the sinus terminalis on each side, but the solid
mesoblast may go a short distance beyond it. (2) Amniotic fold. (3)
Embryonic hypoblast. (4) Formative epiblast. (5) Pericardial pri-
mordium. (6) Distal exoccelom.
Fig. 54.—Same. The pericardial primordium is separating into two
parts. (1) Proximal moiety of pericardium. (2) Distal moiety of same.
Fig. 55—Same. The pericardium consists of right (proximal) and
left (distal) halves. (1) Proximal half of pericardium. (2) Distal half
of same. (3) Sinus terminalis.
PLATE 19.
Fig. 56.—Same. Posterior quarter of embryonic shield. (1) The
two limbs of the pericardial primordium. (2) Notochordal plate. (3)
VoL. 60, PART 2,—NEW SERIES. 18
256 ARTHUR WILLEY.
Medullary groove. (4) Sinus terminalis. (5) Unsegmented em-
bryonic mesoblast.
Fig. 57—Same. Near posterior end of embryonic shield. (1) Noto-
chordal plate in front of the primitive streak. (2) Coagulum in hollow
at foot of keel.
Fig. 58.—Same. Through the primitive streak. (1) Primitive streak.
(2) Dense coagulum in hollow at foot of keel. (38) Hypoblastic groove.
(4) Exoccelom.
Fig. 59.—Same. The keel with the massive exostyle. (1) Exocelom
on one side only; the sinus terminalis of this side lies beyond the
range of the drawing. (2) Axial hypoblast. (3) Distal trophoblastic
cavity. (4) Epiblastic folds. (5) Sinus terminalis. (6) Proliferating
mesoblast of exostyle; this is typical ‘* Haftstiel” tissue.
Fig. 60.—Same. Transition from exostylar to post-stylar region. (1)
Exocelom. (2) Hypoblastic groove. (3) Massive mesoblast. (4)
Trophoblastic extension of keel.
Fig. 61—Same. Anterior part of post-stylar region. (1) Sinus
terminalis. (2) Hypoblastic groove with cubical epithelium. (3)
Post-stylar eelom. (4) Trophoblastic keel, somewhat folded.
Fig. 62.—Same. Middle of post-stylar region. (1) Post-stylar ccelom.
(2) Very long and straight trophoblastic keel. (3) Mucus at edge of keel.
Fig. 63.—Same. Near the posterior end of the post-stylar region.
(1) Sinus terminalis. (2) Post-stylar celom. (3) Trophoblastic
keel with agglutinated walls.
Fig. 64—Same. The two portions of the sinus terminalis nearly
meeting below the hypoblast. (1) Sinus terminalis. (2) Straggling
mesoblast cells.
Fig. 65.—Same. Posterior extremity of mesoblast; the sinus ter-
minalis confluent below the hypoblast.
PLATE 20.
Fig. 66—Same. Posterior didermic region of blastocyst. The
cubical carinal hypoblast passes gradually into the flattened adcarinal
hypoblast. (1) Adcarinal omphalopleure. (2) Carinal omphalopleure.
Fig. 67.—The lumen of a non-gravid segment of the uterus from a
cornu which contained young embryos with established placenta. The
pits are the openings of uterine glands. The dermatic cone on the
mesometric side is free from glands. (J. Prijs, del.) This is the stage
when the connective tissue is uniformly proliferated round the lumen,
but this zone containing numerous subepithelial capillaries is not ren-
dered in the drawing.
BLASTOCYST AND PLACENTA OF THE BEAVER. 257
Fig. 68.—Part of section through the posterior end ofja preplacental
gestation sac to show the general aspect of the mesometric glands
which have become atrophic. They reach the epithelial surface though
devoid of a lumen. (J. Prijs, del.)
Fig. 69.—Part of section through a preplacental gestation sac showing
epithelial proliferations at the sides and bottom of the placental groove.
Between the lobular growths capillaries are lodged. Capillaries are
also seen ascending towards the surface. (Freehand.)
Fig. 70.—Detail of epithelial proliferation at bottom of placental
groove. A few mitoses are seen.
Fig. 71.—Epithelial mesometric wings (tinted) with dilated capillaries
charged with red corpuscles. Substage F. (1) Bottom of placental
groove. (2) Dermatic cone. (3) Dilated capillary. (4) Epithelial
proliferation.
Fig. 72.—Ampulla of a periplacental gland failing to open into the
placental groove. From substage B.
Fig. 73.—Gestation sac opened from the antimesometric side showing
embryoin sittt. (J. Prijs, del.)
Fig. 74.—The same viewed from the side to show how the adjacent
segments of the uterus are inserted into the mesometric half of the
gestation sac at this stage. (J. Prijs, del.)
Fig. 75.—Detail from the border-zone between trophospongia and
placental labyrinth, showing chain of syncytial nuclei (black). (1)
Trophoblast. (2) Maternal capillaries containing red corpuscles. (3)
Fetal capillaries containing nucleated red corpuscles and surrounded
by allantoic tissue. To right of figure the latter appears as an island
surrounded by trophoblast. (4) Symplasma with many syncytial
nuclei and a few intrusive plasmodial nuclei (seen to right of figure).
(5) Trophospongia. One maternal capillary is included, the other is
being included within the trophoblastic labyrinth.
Fig. 76.—Semidiagrammatic section through blastocyst and placenta
between one end and the centre of the latter. The hypoblast, indicated
by broken line, becomes indistinct in the obplacental region. The
height of the blastocyst, measured from the fcetal surface of the placenta
to the obplacental wall, was 8mm. (1) Omphalon filled with dense
coagulum, 7 mm. wide. (2) Obplacental wall of blastocyst, applied
to obplacental wall of uterus. (3) Umbilical membrane (area vascu-
losa). (4) Sinus terminalis. (5) Umbilico-placental membrane
(diplotrophoblast). (6) Opening (aditus) of interutricular segment of
uterus into gestation sac. (7) Section of embryo in its amnion in
the posterior region. (8) Placenta showing diagrammatically radial
allantoic villi (tinted) alternating with radial trophoblastic trabecule
258 ARTHUR WILLEY.
excavated by sanguimaternal lacune. Height of placenta, 3°5 mm.
(9) Areolated trophospongia with capillary network and subjacent
capillary vessels in the dermatic tissue. (10) Primordial connecting
membrane.
Fig. 77.—Section through placenta near one of its poles to show the
polar vessel entering it excentrically. (J. Prijs, del.) (1) Polar vessel
with proliferated endothelium. (2) Diplotrophoblast. (8) Areolated
trophospongia. (4) Sinus terminalis. (5) Placental labyrinth. (6)
Allantoic mesoblast at foetal aspect of placenta.
PLATE 21.
Fig. 78.—Section through middle of placenta, showing the ecto-
placental labyrinth resting upon the areolated trophospongia, which is
supported by the dermatic cone containing branching and anastomosing
vessels. (J. Prijs, del.)
Fig. 79.—Diagram of sagittal-section through gestation sac. The
amniotic cavity extends between the umbilical membrane and the
placenta. (1) Opening of omphalomesenteric duct into the omphalon.
(2) Sinus terminalis; the index numbers are in the exocelom. (3)
Amnion. (4) Allantoic canal opening into a flattened allantoic sac.
(5) Umbilico-placental diplotrophoblast ; the index numbers are at the
openings of the gestation sac. (6) Mesometrium. (7) Omphalo-
mesenteric vein. (8) Omphalomesenteric artery. (9) Omphalopleuric
primordium of connecting membrane.
Fig. 80.—Fetus of 38 mm. lying in its baggy amnion, exposed by
cutting away the wall of the gestation sac and removing part of the
umbilical membrane. (1) Cut surface of wall of gestation sac, 5°25 mm.
thick behind. (2) Cephalad connecting membrane. (3) Rugose roof of
gestation sac. (4) Caudad connecting membrane. (5) Linea alba,
marking the insertion of the amniotic membrane upon the placenta.
(6) Cut edge of umbilical membrane. Note: At this stage the foetus
already possesses two pairs of mammary pits.
Fig. 81—Same. The fetus has been removed, and the amnion,
detached from its adhesion to the umbilical membrane, has been cut
short and drawn taut, the object being to exhibit the polar umbilico-
placental protuberances. (1) Cut edge of amnion. (2) Outer surface
of umbilical membrane; the inner surface is shaded. (3) Cut edge of
ditto. (4) Linea alba or insertion of amnion upon placenta. (5)
Cephalad protuberance. (6) Caudad protuberance.
Fig. 82.—Later stage; fetal length 73 mm. View of placenta and
allantoic vessels from the side after removal of the foetus. There are
three layers of vessels at the surface of the placenta; superficial allan-
BLASTOCYS! AND PLACENTA OF THE BEAVER. 259
toic veins (black) ; allantoic arteries (clear) in the middle level; maternal
vessels (tinted) at the lowest level. The main allantoic vascular trunks
are two arteries (clear) and one vein (black). The amnion, together with
the vascular area (umbilical membrane), has been cut open and reflected.
The vessels of the area show through the amnion, but this is only
indicated in the figure at the region of distribution of the omphalo-
mesenteric vessels. (1) Cut edge of amnion together with umbilical
membrane. (2) Inner surface of amnion. (3) Allantoic vesicle with
vein in the thin wall. (4) Surface of placenta projected into the
amnion. (5) Cut surface of umbilical cord. (6) Omphalomesenteric
vein. (7) Omphalomesenteric artery.
ACROSSOTA LIPOSCLERA. 261
On Acrossota liposclera, a New Genus and
Species of Alcyonarian with Simple Tentacles.
By
Gilbert C. Bourne, M.A., D.Sc., F.R.S.,
Linacre Professor of Comparative Anatomy and Fellow of Merton
College, Oxford.
With Plate 22.
Tue Alcyonarian Colony to be described in the follow-
ing pages was collected by Prof. A. Willey in the
d’Entrecasteaux group of islands, British New Guinea, and
sent to me, | am ashamed to say how many years ago, along
with a collection of Actiniz and Zoanthee. Partly because
of the difficulty and uncertainty of identifying spirit pre-
served actinians, partly because I had turned my attention
to another group of animals, I have neglected this collection
until, a short time ago, 1 began to prepare such specimens
as were suitable for cutting into sections with a view of
identifying and describing them. Among the Zoanthee I
found a colony of small translucent polyps apparently grow-
ing in a bunch attached to the fronds of a species of
Halimeda. Some of these polyps were partly expanded and
exhibited a few simple digitiform tentacles, and this
character, together with their general appearance, suggested
that they were non-incrusted Zoanthids. On closer examina-
tion I found that the polyps arose at intervals from a long,
sparsely branched adherent stolon, and that the apparently
single colony consisted of several such stolons so closely
intertwined that the polyps arising from them were closely
262 GILBERT C. BOURNE.
bunched together, giving the deceptive appearance of a
compact encrusting colony. And, as I shall describe in
detail, I found that the tentacles, though simple finger-
shaped structures, are always eight in number, and that the
structure of the polyps, in almost all other respects, presents
the well-known characteristic Aleyonarian features.
The entire absence of lateral pinnee on the tentacles of the
autozooids of an Alcyonarian has not, to my knowledge, been
previously recorded, and as my colony presents some other
interesting characters which differentiate it from any
described species, I must make a new genus for its reception.
It seems necessary also to make the genus the type of a new
family, for the possession of simple digitiform tentacles is a
unique feature among the Alcyonaria. But, as will appear
from what follows, the general characters of the colony and
polyps place the new family in the order Stolonifera, in
close juxtaposition to the Cornularide and Clavularide.
The specimen may be described as follows :
Order—Stotonirera, Hickson.
Family—Acrossotide, the tenacles simple, digitiform, with-
out lateral pinne.
Genus—Acrossota! n. gen.
Zooids borne at intervals on a simple sparingly branched
radiciform adherent stolon; no spicules or calcareous
skeleton of any kind; tentacles digitiform, without lateral
pinne.
Acrossota liposclera n. sp.
Stolon subcircular where free, flattened where adherent,
its cavity formed by a single solenium, but traversed by
_mesogloeal trabecule ; the stolon branched and the branches
1 d, without ; cpooowrdc, fringed.
ACROSSOTA LIPOSCLERA,. 263
intertwined but not forming a network by anastomosis.
Zooids subcylindrical, of various lengths, frequently giving
off stolonar outgrowths from their proximal moieties, the
distal moiety of each zooid invaginable within the proximal
moiety. Tentacles digitiform, completely invaginable.
Outer wall of stolon and zooids strengthened by a gelatinoid
supporting tissue formed by the ectoderm, and in addition a
thin parchment-like external cuticle, the latter forming the
chief supporting tissue in the basal parts of the zooids, the
gelatinoid tissue more largely developed in the subtentacular
region and in the stolon; the walls everywhere relatively
thin, and not differentiated to form calices into which the
zooids can be withdrawn. Stolon and zooids translucent and
colourless in spirit.
Locality, d’Entrecasteaux Group, British New Guinea.
The form and habit of the specimen collected by Prof.
Willey are so irregular that they are difficult to define. The
central part of the specimen consists of several relatively
large zooids, measuring 5 mm. in length and 1°75-2 mm. in
diameter and rather closely crowded together. From the
bases of these zooids several stolons are given off which creep
along the frond of a Halimeda and are closely attached to it
at intervals. Where attached the stolons are flattened and
tape-like, but in the greater part of their courses they are
round and simply twined round their support. The stolons
branch and the branches may subdivide several times in an
irregular manner, but the branches do not anastomose with
one another. The branches and their subdivisions are twined
round one another and tangled together, and bear other
zooids at irregular intervals. From place to place a branch
of the main stolon or a stolonar outgrowth of one of the
zooids projects for some distance from the support as a long,
free, thin-walled tube, near the end of which a zooid is
developed, and from this zooid other stolons are given off,
which may in turn bear zooids of various sizes. These free
tubular outgrowths are frequently constricted in places, and
it is probable that they are eventually separated off from the
264 GILBERT C. BOURNE.
parental colony at the points of constriction, and so give rise
to new colonies, for the specimen included several short
lengths of stolon bearing two, three, four or more zooids, and
quite independent of the maincolony. But these and the stolons
of the main colony were all tangled together and with the
Halimeda, and it required some care to disentangle them.
Acrossota, therefore, is remarkable, though not unique,
amoung Alcyonaria for producing independent colonies by a
form of gemmation. The stolons usually end in blunt, slightly
swollen extremities, but sometimes are fixed by one extremity,
which is then closely flattened against the surface of support
(fig. 1). Evidently each stolon is at first a simple tubular
outgrowth of the proximal part of the wall or of the base of
a zooid ; this outgrowth is lined by endoderm and forms what
I have called a “solenium.”! In Cornularia? the stolons
are simple solenia, but in Clavularia, Sarcodictyon and other
members of the Stolonifera the stolons are compound and
contain several solenia. In Acrossota the stolons are not as
simple as those of Cornularia, nor so complicated as those of
Clavularia. The simple cavity is largely occluded, especially
in the older parts of the stolons, by the formation of a spongy-
looking tissue within the originally simple cavity. This
tissue, as is shown in fig. 3, is formed by numerous fine
branching trabecule, which pass from wall to wall and
anastomose with one another. The trabecule are composed
of a core of mesogloea, covered by endoderm, and are more
or less flattened in section. Some of the larger trabecule
contain strings or islets of endoderm cells embedded in the
mesogloea, and the branching of the trabecule is apparently
brought about by the formation of cavities in these intrusive
endoderm cells, but there is no evidence of the formation of
definite solenial outgrowths of the endoderm penetrating into
the mesoglcea and forming compound stolons. The trabeculez
' G. C. Bourne, “ Anthozoa,” in Lankester’s ‘ Treatise of Zoology,’
Part II, 1900, p. 16.
? G. von Koch, “ Die Aleyonacea des Golfes von Neapel,” ‘ Mitth. a. d.
Zool. Stat. zu Neapel,’ ix, 1891, p. 645.
ACROSSOTA LIPOSCLERA. 265
become less numerous in the terminal and most recently
formed portions of the stolons, and are absent or very few in
number and imperfectly developed in the swollen extremities
(fig. 3). They are also scantily developed in the long free
outgrowths which give rise to separate colonies. On the
other hand, in the flattened adherent portions of the stolons,
the trabecule tend to fuse together to form lamine, dividing
the lumen of the stolon into several canals, running, on the
whole, parallel to the long axis of the stolon.
It is assumed rather than demonstrated that the compound
stolons of Clavulariide are formed by the union of the walls
of a close-meshed reticulum of simple solenia such as is found
in Cornularia. It is probable that many band-shaped or flat
encrusting stolons are formed in this manner, and all
encrusting stolons must be formed in part by the fusion of
the walls of radial outgrowths containing solenia from the
bases of the zooids, but it is equally probable that a large part
of the inosculating channels seen in sections of such stolons
have been formed as in Acrossota, by the ingrowth of
trabecule from the walls of a primitively simple solenium.
The general structure of the zooids, with the exception of
the tentacles, conforms to the Aleyonarian type.
The Actinopharynx (as van Beneden! has renamed the
** Stomodeum ” of the Anthozoa) is long, and its upper third
is lined by a ciliated epithelium, of which the character is
shown in fig. 5. The epithelial cells are elongated and
attenuated, their nuclei stain deeply and appear closely
crowded together in sections. The cilia take their origin
from minute deeply staining granules, which give the free
border of the epithelium a striated appearance. In this region
there isa distinct sulcus or siphonoglyphe in which the cilia
are specially long. But in the lower two thirds of the actino-
pharynx (fig. 6) the siphonoglyphe dies out; the epithelium
is composed of less elongated prismatic ciliated cells, the
nuclei are no longer crowded together and stand nearly on
1 E. van Beneden, “ Die Anthozoen d. Plankton Expedition,” ‘ Ergeb-
nisse d. Plankton Expedition des Humboldt-Stiftung,’ ii, 1897.
266 GILBERT C. BOURNE.
the same level; the cilia are short and of uniform length.
In contracted specimens this region of the actinopharynx is
thrown into longitudinal folds and its lumen diminished, but
the lumen is always considerably larger than in the upper
third. At the lower border of the actinopharynx the. pris-
matic ciliated epithelium passes rather abruptly into the
endoderm, but is clearly continued down the edges of the two
“ dorsal” or asulcar mesenteries to form the grooved filament,
which in section presents precisely the same characters as
those figured by Hickson! for Alcyonium. In the grooved
filaments the ciliated cells again change their character,
becoming small and narrow, with small deeply staining nuclei.
The remaining mesenteries have thickened edges covered by
endoderm cells, but no distinct filament. As is invariably the
case in Alcyonarian zooids, the two “ dorsal” asulcar mesen-
teries bearing the grooved filaments extend much further
down in the body of the zooid than the remaining six.
‘he section represented in fig. 4 shows that in other respects
the mesenteries present the usual Alcyonarian features. The
mesogloeal thickenings forming the muscle-banners are feebly
developed, and only distinguishable in the region of the
actinopharynx, but there they can be seen to be borne on the
“ventral” or sulear faces of all eight mesenteries.. The
peripheral parts of the mesenteries are very thin. Only one
of the Zooids that I used for cutting sections was sexually
mature. In it testes, in the form of small spherical follicles,
were borne on short pedicles near the free edges of all but
tle dorsal asulear mesenteries.
The tissues of the specimens I have examined were only
moderately well preserved, and the cell elements so minute
that I have not been able to work out histological details to
my complete satisfaction. The structure of the body-wall in
particular has proved very difficult to interpret, and the
structure of the various regions into which the body of the
zooid may be divided appears to differ in invaginated and
1 §. J. Hickson, “The Anatomy of Alecyonium digitatum,”
‘Quart. Journ. Mier. Sci.,’ 37, 1895, p. 343.
ACROSSOTA LIPOSCLERA. 267
extended specimens, from which fact I infer that the tissues
are very elastic and extensile, capable of being drawn out
into an exceedingly thin layer or contracted into thicker
sheets, according to the state of contraction of the animal.
From a study of longitudinal and transverse sections of
both extended and retracted specimens it appears that the
following regions may be recognised: (1) The tentacles and
the oral disc. (2) The portion of the body of the zooid
immediately below the tentacles. This portion is invaginated
in retracted specimens, but in both extended and retracted
examples the body-wall in this region is moderately thick,
and the tissues, both ectodermic and endodermic, are fairly
clearly differentiated. In extended specimens this region is
approximately of the same length as the actinopharynx, but
in retracted specimens the latter structure is pulled down into
the lower part of the ccelenteron and les below the tube
formed by the invaginated distal portion of the body-wall of
the Zooid. (3) The middle and basal region of the Zooid, in
which the body-wall is extremely thin. (4) The stolons, in
which the mesoglcea is thickened, the endoderm fairly con-
spicuous, but the ectoderm reduced or absorbed in the
formation of a gelatinoid supporting tissue.
The first two regions are clearly the seat of the chief phy-
siological activities. The cavities of the tentacles, the walls
of the actinopharynx and the central moieties of the mesen-
teries where attached to the actinopharynx are clothed with
a thick highly vacuolated endoderm containing a profusion
of zooxanthellee (fig. 4). The mesoglea is a thin layer exhibit-
ing a faint striation, but not including any cellular elements.
The ectoderm of the tentacles is formed by a layer of small
cells, cubical in the extended but more columnar in the
invaginated tentacles. The ectodermal muscular fibres are
but feebly developed in the tentacles, the circular layer of
endodermic muscular fibres being rather better represented,
but still feeble. Minute oval refracting bodies can be recog-
nised in the ectoderm of the tentacles, and I interpret them
as nematocysts, but I was unable to resolve their structure
268 GILBERT C. BOURNE.
with the highest powers of the microscope at my disposal.
The ectodermic lining of the actinopharynx has been described
already. The ectoderm of the body-wall below the tentacles
is illustrated in fig. 7, The continuous layer of cubical cells
of the tentacles and.oral disc here gives place to what I can
only describe as a reticulum of vacuolated protoplasm con-
taining nuclei, but in which the limits of the cell bodies are
with difficulty or not at all recognisable. ‘he body-wall is
raised into a number of thickened ridges, one of which is
shown in fig. 7. In this figure the mesoglcea is seen as a
thin but distinct structureless layer, produced on the inner
side into small processes to which the muscular fibres of the
vacuolated endodermic cells are attached. On the outer side
of the mesoglcea is an irregular aggregation of what may be
called ectoderm cells, though, as mentioned, the cell outlines
are not distinguishable. Some of these abut on the meso-
gloea, without, however, forming a continuous layer. Others,
more peripherally situated, form a discontinuous lining to the
thin darkly staining external cuticle. The intervening space
is filled up by a homogeneous more or less vacuolated sub-
stance which seems to be formed by the dissolution and
conversion into supporting tissue of the ectodermic cell-
fusion. The homogeneous supporting substance stains more
faintly than the mesogloea, but is probably of the same or
similar chemical composition, for in older and more differen-
tiated parts of the body-wall the ectodermic cell-fusion
becomes scantier, the homogeneous substance increases in
amount, and eventually fuses with the mesogloea, and can only
be distinguished from it by its staining less deeply. Usually,
however, tracts of ectoderm looking like cell ingrowths are
included in the homogeneous supporting tissue.
Fig. 8 represents a portion of the extremely thin body-
wall of the lower middle and basal part of the zooid. In com-
parison with fig. 7, it is seen that the endoderm is reduced
to a very thin non-vacuolated flattened epithelium. The
mesogloea is a distinct, very thin structureless layer. In one
part of the section it seems to consist of two layers, between
ACROSSOTA LIPOSCLERA. 269
which are a few flattened nuclei with remains of cytoplasm
surrounding them. ‘The outer layer, however, is nothing
more than the deeper part of the homogeneous supporting
tissue formed by the ectodermic cell-fusion, and the nuclei
between it and the mesogloea are ectodermic nuclei. Ex-
ternally may be seen a few nuclei of ectodermic cells which
have obviously been used up in the formation of local
thickenings of the supporting tissue, and externally is the
thin but tough deeply staining cuticle.
In the stolons the endoderm again becomes thicker and
vacuolated, but does not contain zooxanthellae. The external
cuticle remains, but the nuclei of the ectodermic cell-fusion
have almost entirely disappeared, and the ectoderm appears
to have been nearly wholly used up in the formation of
supporting tissue, which is now so closely applied to and
fused with the mesogloea as to be scarcely distinguishable
from the latter. One would say at first sight that the section
shows only endoderm, mesoglea and cuticle, the ectoderm
having disappeared altogether. In this region strings of
endoderm-cells make their way into the mass formed by the
fusion of mesoglcea and supporting tissue, and these in-
growths extend into the trabecule, which are themselves
formed as local out-growths of mesogloea covered by endo-
derm. ‘l'his ectodermic gelatinoid supporting tissue of
Acrossota is comparable in essential respects to the support-
ing tissue of Stereosoma celebense as described and
figured by Hickson.! But there is this difference: that in
Stereosoma there is a distinct external layer of cubical
ectoderm cells, but no cuticle, whereas in Acrossota the
definite external ectodermic epithelium is absent, but there
is a distinct and continuous cuticle.
There are no spicules in Acrossota. J have searched for
them in teased-up specimens, in sections, and in the residue
left after boiling in caustic potash. A felt-work of filamentous
algee enclosing numerous diatoms, sponge spicules, and here
' §. J. Hickson, “ A Revision of the Aleyonaria Stolonifera,” ‘ Trans.
Zool. Soe. Lond.,’ xiii, 1895.
270 GILBER1 C. BOURNE.
and there an alcyonarian spicule, covers the older parts of the
stolons and the basal moieties of the older zooids. But all
these spicules are adventitious and le external to, though
often closely adherent to, the cuticle.
It may be concluded, from the foregoing description, that
Acrossota is an Alcyonarian exhibiting primitive Cornularian
characters in the mode of growth and habit of the colony, in
the simplicity of its stolons (which, however, are not so simple
as those of Cornularia), and in the absence of calcareous
spicules, the last character being shared by such clearly
primitive forms as Cornularia and Protocaulon molle,
and also by Stereosoma celebense, Clavulariareptans,
Clavularia celebensis, and the variety of Clavularia
australiensis described as variety B by Hickson! The
absence of spicules may be regarded as evidence of primitive
organisation, but not certain evidence, for the spicules
present in variety A appear to have been lost in variety B
of Clavularia australiensis. The absence or disappear-
ance of spicules appears to be correlated in the Clavulariidz
with the formation of a supporting tissue derived from vacuo-
lated branched ectoderm cells, and Acrossota shares this
feature with Stereosoma and Clavularia australiensis,
var. B. The feature peculiar to Acrossota is the absence of
tentacular pinne, and it is a moot point whether this may
be regarded as a primitive character. Stereosoma has but
few pinne, and those spaced at considerable intervals along
the tentacles. Further reduction of such pinne might lead
to their ultimate disappearance, and the tentacles would then
be simple and digitiform. On the other hand, it may be
argued that simple tentacles must have preceded pinnate
tentacles in phylogeny, and we have in Acrossota an otherwise
primitive Alcyonarian with simple tentacles. The evidence
seems to point to this being a primitive character, but in the
absence of any criterion which shall enable us to distinguish
between what is primitive and what is secondarily acquired
by degeneration in such structures as these, one cannot be
Siprde
ACROSSOTA LIPOSCLERA. 271
dogmatic. ‘lhe one thing certain is that in the possession of
simple tentacles Acrossota stands as an exception to a rule
otherwise universal for Aleyonarians, and it was this that led
me to make as exhaustive a study as I could of its structure.
EXPLANATION OF PLATE 22,
Illustrating Mr. G. C. Bourne’s paper ‘‘On Acrossota
liposclera.”
[All the figures refer to Acrossota liposclera nov. gen. et sp.|
Fig. 1.—An independent stolon attached by one of its extremities to
a fragment of Halimeda. The stolon bears four zooids, three of which
are partially expanded and exhibit the simple tentacles.
Fig. 2—Two zooids from a specimen stained in hemalum and mounted
in oil of cloves. One of the zooids is expanded, and exhibits the eight
simple tentacles; the other is retracted, and shows the invaginated
tentacles. d.m. Dorsal or asulcar mesenteries.
Fig. 3.—A retracted zooid and part of a stolon from a specimen stained
in hemalum. The body-wall of the zooid and part of the wall of the
stolon nearest the observer have been cut away. d.m. Dorsal mesen-
teries. st. Stolon. ¢. The invaginated tentacles. tr. Trabecule in the
cavity of the stolon.
Fig. 4.—A transverse section passing through the upper part of the
actinopharynx of a retracted zooid, showing the actinopharynx with its
sulcus, the eight mesenteries with the muscle banners, the eight invagi-
nated tentacles and the thin external body-wall. The endoderm clothing
the actinopharynx, the central moieties of the mesenteries and the
tentacles is thick, and contains numerous zooxanthelle. aph. Actino-
pharynx. d.m. “ Dorsal” mesenteries. v.m. “ Ventral” mesenteries.
t.t. The invaginated tentacles.
Fig. 5.—A_ section through the upper third of the actinopharynx,
highly magnified, showing the elongated ciliated cells, the sulcus or
siphonoglyphe, and the vacuolated endoderm. z. Zooxanthelle. Other
letters as in fig. 4.
Fig. 6.—A section through the lower part of the actinopharynx,
showing the disappearance of the suleus and the changed character of
the ciliated epithelium. Lettering as in the preceding figures.
voL. 60, parT 2.—NEW SERIES. 19
pay Ps GILBERT C. BOURNE.
Fig. 7.—A longitudinal section through part of the subtentacular
region of the body-wall, magnified 750. cu. The external cuticle. ee.
Ectodermic cell-fusion with nuclei. en. Endoderm. h.s. The homo-
geneous supporting substance formed by the ectoderm. mg. Mesoglea.
Fig. 8.—Part of a longitudinal section through the body-wall of the
middle of the zooid, magnified 750. Lettering as in fig. 7.
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 273
The Proboscidian System in Nemertines.
By
Dr. Gerarda Wynhof,
Utrecht.
With 36 Text-figures.
Tae proboscis, together with its sheath and the rhyn-
chodeeum, are among the most characteristic features in the
anatomy of Nemertines, so characteristic even that the system
is not absent in any known species, neither of Anopla nor of
Enopla. And wherever it is found, the whole system shows
the same advanced development of construction; all three
organs are completely developed, the proboscis being an intro-
vertible tube, which is fastened to the body-wall at the
anterior end of the rhynchoceel, and is connected posteriorly
by a retractor muscle to the wall of the sheath. The rhyn-
chodzeum is a kind of atrium to the apparatus, through which
the proboscis is everted (l'ext-fig. 1). Both proboscis sheath
and proboscis itself possess a muscular wall, and the ]Jumen of
the sheath is lined by an endothelium. The cavity of both
rhynchodeum and proboscis is lined by continuous epithelium
that shows a differentiation of glandular elements in different
parts of the system. The proboscis, therefore, consists of
three layers—an epithelium, a muscular coat and an endo-
thelial layer ; the wall of its sheath of two—an endothelium
and a muscular coat. The whole system is imbedded in the
body-parenchyma. It has for a long time been assumed
that the proboscis had developed out of a retractible part
274. DR. GERARDA WYNHOFP.
cf the head independently of corresponding structures in
other invertebrates. Hubrecht (16) shared this opinion,
and tried to explain, by the histological facts shown by
the “Challenger” material, that the sheath had developed out
of muscular elements in loco, and was an independent
structure. However, Salensky (25) had published another
hypothesis, comparing the whole proboscidean system of
TEXxT-FIG. 1.
i
1 \\
1 is 8 prob.w.
i iH iE
At HE
i \ " rhynch.
ie ‘ia
I
le
al
Schema of the proboscidian system in Nemerteans after Salensky
(26). rhynch. Rhynchodeum or proboscis introvert cavity.
rhynch. c. Rhyncho-celomic cavity. prob. Epithelium of the
introverted proboscis. prob. w. Proboscis wall. rhynch. w.
Wall of the proboscis cavity (rhynchocel). retr. Retractor
muscle of the proboscis. Compare with Text-fig. 36.
Nemerteans with the proboscis of the Rhabdocclida
proboscida. He founded his theory on certain embryo-
logical facts denied by Hubrecht (15), who tried to find
support for his view in anatomical facts. Biirger (8) looked
for a homolegue of the whole system in the pharynx and
pharyngeal sac of the Polyclads, but neither Birger nor
Hubrecht could gain the agreement of Salensky, who published
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 275
two articles in 1909 (27) and 1912 (28) in which he maintains
his views of 1884 (25). As Salensky only gives embryological
facts, we shall have to look for further judgment to the
anatomical data. Wishing to draw my own conclusions as
to the value of Salensky’s theory, I have compared whatever
was known about rhynchodeum, proboscis and proboscis
sheath in Nemerteans.
TExtT-Fic. 2.
Schema of the proboscis of Paleonemerteans. p.e. Proboscis
epithelium. o.c.m. Outer circular musculature. i.1.m. Inner
longitudinal musculature.
The first fact that came to light was the great difference
in structure between the proboscis of armed and unarmed
Nemerteans. For the difference between the two does not
consist only in the presence or absence of the so-called stylet ;
if is shown also in the structure of the muscular coat, and in
the differentiation of the tube into different parts. It is even
possible in Hnopla that the armature has got lost, as seems
to be the case with the genus Planktonemertes (10a). Mala-
276 DR. GERARDA WYNHOFF.
cobdella also does not possess an armed proboscis. The
Anopla show a great variety of structure for such a plainly
built organ. The tube always consists of two parts, the
anterior part sometimes possessing a differentiated region
near the insertion to the body-wall. That a difference of
cpithelial glands exists between these parts is nearly certain ;
the exact nature of this difference, however, is not sufficiently
known. Very often the beginning of the hinder part is
TEXT-FIG. 3.
Section of the proboscis of Carinoma after Bergendal (6, text-
fig. 55). prob.n. Proboscidian nerve. p. end. Proboscidian
endothelium. o.¢.m. Outer circular musculature. 7.l.m. Inner
longitudinal musculature. m. cr. Muscle crosses.
marked by a constriction. The principal seat of the variety
of structure, however, is the muscular wall of the proboscis.
In all Paleeonemerteans the muscular sheath of the proboscis
consists of two layers, a circular and a longitudinal, of which
the circular layer is situated directly beneath the epithelium
(Text-fig. 2).
Exactly this type of proboscis is found in several species of
Tubulanus (9), in Procarinina atavia (5), in Carinina (9),
Hubrechtia (9) and Hubrechtella (3). he other genera
possess a more complicated proboscis, though, along the
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. DAY
greater part of each, the two muscular layers described above
are developed. Moreover, Bergendal described in Carinoma
(6) a proboscis in which the circular muscle-fibres show a
tendency to divert into the longitudinal layer and build mus-
cular crosses (Text-fig. 5). A circular layer outside the longi-
tudinal fibres is, however, not present. As the tendency to
form crosses is also known in the circular fibres of the body-
wall, their presence in the proboscis of Carinoma cannot alter
our opinion that only two muscular coats are present.
The genera Cephalothrix (83), Cephalotrichella (83) and
TEXT-FIG. 5.
TEXT-FIG. 4.
Text-FIG. 4.—Proboscidian section of Carinesta anglica,
Wynhoff. p.e. Proboscis epithelium. 7. 1. m., prob. n. and
p.end, as in Text-fig. 3.
TEXtT-FIG. 5—Proboscidian section of Carinesta orientalis,
Punnett. .e., prob.n., p.end. and 7.1. m. as before. par.
Parenchyma. constr., Constrictor muscle.
Procephalothrix (88), Callinera (2), Carinesta and Carino-
mella (12) differ more from the above-mentioned type. In all
of them the circular muscle-fibres are wanting in the portion
directly behind the insertion of the proboscis (Text-fig. 4). In
the family Cephalotrichidee, as well as in the three other genera,
this part is followed by a conspicuous thickening of the
circular layer, which acts as a constrictor (Text-fig. 5). At
different places in this neighbourhood, in Cephalotrichidee just
before (Text-fig. 6), in Callinera behind (Text-fig. 7), in Cari-
nomella (T'ext-fig. 8) before and behind the constrictor muscle,
the parenchyma is particularly well developed, causing a regres-
278 DR. GERARDA WYNHOFF.
sion of the muscular fibres. he longitudinal fibres, however,
are present over the whole length of the proboscis (‘Text-figs.
6 and 7), though often much reduced, as at the places men-
tioned above. In Carinomella (‘Text-fig. 8), however, the
whole circular musculature has degenerated in the anterior
part of the proboscis, and instead of four bundles of muscle-
TEXT-FIG. 6. TEXT-FIG. 7. TEXT-FIG. 8.
p-e--:
constr.-
par
TEXxT-FIG. 6.—Schema of the proboscis of Cephalothrix. pe.,
par., 0.¢c.m., i. 1.m., and constr. as in Text-figs. 3 and 5.
TExt-FIG. 7.—Schema of the proboscis of Callinera. Letters as
in Text-fig. 6.
Text-FIG. 8.—Schema of the proboscis of Carinomella. Letters
as in Text-fig. 6.
fibres, as in Cephalotrichide and Callineride (Carinesta [Text-
fig. 4] and Callinera), Coe (12) found only two longitudinal
muscles in the cavity of the sheath, connecting the wall of the
sheath with the anterior part of the proboscis (Text-fig. 9).
The other parts of the proboscis, however, consist, in all these
genera, of the same layers as in Tubulanus (Text-fig. 2); it
THE PROBOSCIDIAN SYSTEM 1N NEMERTINES, 279
seems, therefore, not too presumptuous to say that in all Paleeo-
nemertines the muscular coat of the proboscis is composed of
two layers, a circular and a longitudinal, the latter being
situated beneath the endothelial surface. he deviations from
this type, found in the families of Cephalotrichidie, Calli-
neridee, and the genus Carinomella, have merely been brought
about by the disappearance of the circular fibres in the region
directly behind the insertion, accompanied in Carinomella by
the egression of the longitudinal fibres.
Uop-arsinices, 8).
Section through Carinomella after Coe (12), fig. 54. 1. m. Longi-
tudinal muscle. rhynch.m. Rhynchodeal musculature. c¢./.m.,
Central long musculature. 7. ¢. m. Inner circular musculature.
bl. v. Blood-vessel. dig. Digestive tract. par. Parenchyma.
In Heteronemertea the proboscis shows a much greater
variety of structure. A certain number of species possess a
proboscis exactly like the Paleonemertea ; the outer layer of
the ejaculated duct consists of the glandular epithelium, next
to it is the circular muscle-layer, limited at the other side by
the longitudinal fibres; such are the proboscides in Micrella
rufa, Punnett (22), Oxypolia beaumontiana Punnett
(22), and in Huborlasia (9). The genera Lineus, Cerebratulus
and Micrura, which are so closely connected, that they cannot
even be distinguished from each other by anatomical features
280 DR. GERARDA WYNHOFF.
alone, show a marked resemblance in their proboscides too.
In all three genera species are known with two muscular
layers, others possessing a third muscular coat (Tex-figs. 10
and 11). Representative of the Paleeo-type are Cerebratulus
urticans (J. Mill.) (9), and C. greenlandicus Punnett
(23), Lineus scandinaviensis Punnett (24), and L.
Trext-Fic. 10.
|
Schema of the proboscis of Heteronemerteans (Cerebratulus
marginatus). o.l.m. Outer longitudinal musculature. p. e.
Proboscis epithelium. 0. ¢. m. Outer circular muscular layer.
i.l.m. Inner longitudinal muscular layer.
bilineatus McIntosh (9), Micrura varicolor Punnett
(24), M. bergenicola Punnett (24), and M. atra Punnett
(24). Variations on this scheme are found in Heteronemer-
teans as in Paleeonemertines. Miss Thompson (80) described
the interesting genus Zygeupolia. The circular muscle-layer
fails in the region behind the insertion, exactly as in Cephalo-
trichide and Callineride (Text-fig. 12). Other genera and
species possess three muscular layers in the proboscis; to this
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 281
group belong the Cerebratulus-, Lineus-, and Micrura-species
as Cerebratulus marginatus Ren. (9), and C. eisigi
Biirger (9), Micrura fasciolata Ehrenberg (9), and
Lineus versicolor Birger (9). Two of these three layers
consist of longitudinal fibres and they are separated by the
circular muscle-fibres (‘l'ext-fig. 10).
The presence of this third layer in the proboscis of Hetero-
TErxt-rie. 11.
Weare
°
eee
qe.
*
Cae.
Section of the proboscis of the introverted Cerebratulus mar-
ginatus after Birger (9, taf. 23, fig. 1). m.ecr. Muscle
crosses. 0. ¢.m. Outer circular muscular layer. 0.1. m. Outer
longitudinal muscular layer. 7.1. m. Inner ditto. n. 1. Nerve
layer. p.l. Epithelium (outer surface) of the proboscis.
nemerteans ts rather striking. The acquisition of a longitu-
dinal muscle-layer between the epidermis and the circular
muscle-layer is characteristic of the body-wall in this group
of Nemertines. The new layer in the proboscis wall, there-
fore, being also a longitudinal layer, can immediately be com-
pared with the outer longitudinal layer of the body-wall, and,
as its surroundings are exactly the same, it is laid down as
part of the outer longitudinal muscle-layer. The circular
coat of the proboscis in that case must be the outer circular
muscle-layer of the body-wall, and the original longitudinal
282 DR. GERARDA WYNHOFF.
layer is part of the inner longitudinal layer of the body-wall.
The Paleotype of proboscides consists of the epithelium, the
outer circular and inner longitudinal muscle-layers, that also
form part of the body-wall. The newly obtained layer of the
Heteronemerteans has not yet been acquired by all pro-
boscides ; those with the Paleotype have not got it at all, the
genus Parapolia Coe (11), is in the act of acquiring it (Text-fig.
13). In the hinder part this proboscis shows the Paleotype,
in the anterior part the Heterotype.
A pecuharity of the circular muscle-layer of Hetero-
nemertine-proboscides is shown in fig. 11. Some circular
Trext-Fic. 12.
Section of the proboscis of Zygeupolia after C. B. Thompson (80).
p. end. Proboscis endothelium. J. m. Longitudinal muscles.
p.e. Proboscis epithelium. probn. Proboscis nerve.
fibres are seen to divert from their original stratum and to
form crosses by entering the inner longitudinal coat, as I shall
henceforth call it, conformable to the corresponding layer of
the body-wall. These fibres of the muscular crosses continue
even outside the longitudinal layer, constituting something
like a very thin and very incomplete circular layer just
beneath the endothelium. As Zygeupolia has muscular crosses
in its proboscis, the thin circular coat beneath the endothelium,
described by Miss Thompson (80), might be comparable with
these circular fibres.
Still another type of proboscis is found in the genera Baseo-
discus (9) and Joubinia (9), at least in two of the three
species belonging to this genus. The muscular coat consists
again of two layers, one with longitudinal and the other with
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 283
circular fibres. Instead of the circular fibres lying beneath
the epithelium as in Paleonemertines, the longitudinal
muscles are found there (Text-fig. 14). The explanation would
have been difficult, for either the layers might have been the
inner longitudinal coat with a better-developed new circular
layer as described in Lineids and Zygeupolia, or the outer
TExt-FiGc. 13.
p-e. ----
o.l.m -
Schema of the proboscis of Parapolia. p.e. Proboscis epithelium
(outer surface). 0.1. m. Outer longitudinal muscular layer.
o.c. m. Outer circular ditto. 7. 1. m. Inner longitudinal ditto.
longitudinal and the outer circular muscle-layer, had not a
form hke Joubinia rubens Coe (11) or Oxypolella (3)
existed. Both species show a different arrangement of the
muscular fibres in the two parts of the proboscis (‘l'ext-fig. 15).
The second part possesses the three muscle-layers character-
istic of the body-wall of Heteronemertines. In the anterior
part, however, the inner longitudinal fibres are absent, the
wall showing the Baseodiscus type. ‘The process of disappear-
284 DR. GERARDA WYNHOFF.
ance of these longitudinal fibres, actually seen in Joubinia
rubens, culminates in their total absencein J. longirostris
and blanca and in Baseodiscus.! Soif the Paleotype consti-
tutes the first stage in its development, the three-layered
muscular coat of Cerebratulus marginatus gives the
second stage, Baseodiscus the third.
Putting all genera of Heteronemertines whose proboscis
structure is known together in one list, we may find the links
between the three above described stages.
Paleotype (Text-fig. 2): Micrella rufa Punnett, Oxy-
TrxtT-Fic. 14.
So
Fo.
ay
Section through the introverted proboscis of Baseodiscus after
Birger (9, taf. 23, fig. 2). p.e. Proboscis epithelium. 1. L.
Nervous layer. ¢.m. Circular musculature. l,m. Longitudinal
musculature.
polia beaumontiana Punnett, Huborlasia, Cerebratulus
urticans (J. Muiler) and other Cerebratulus species, Lineus
1 Bergendal (4) describeda Valencinia longirostris in which the
so-called circular layer shows an interlacing of longitudinal and cireular
fibres something like the proboscis sheath of Drepanophoride. The
principal difference between Bergendal and Birger (9), who gave this
peculiarity of the proboscis of Valencinia longirostris in his figure,
loc. cit., taf. 23, fig. 9, comes to this: that the circular fibres, according
to the first-named author, are arranged in two planes, neither coinciding
with the horizontal section of the organ, and that Birger had not
observed this fact. This, however, seems not to be of any importance
to our conclusions, since both writers agree as to the circular nature of
this musculature. The interlacing of fibres is, as has often been stated
THE PROBOSCIDIAN SYSTEM IN NEMERTINES., 285
bilineatus McIntosh, and other Lineus species, Micrura
atra Punnett and other species; variations on this type, Para-
lineus (29) and Zygeupolia (Text-fig. 12).
Transition to Heterotype: Parapolia (Text-fig. 13),
Heterotype (Text-fig. 16): Cerebratulus marginatus
Renier, and other species, Lineus versicolor Birger and
TExt-F1IG. 15. TExt-FieG. 16.
=|
pet
=
=
olm.=S
=
oc.m- =
=
= .
=|,
tI
=
I i
=i
2 El:
ulm i -
Text-Fic. 15.—Schema of the proboscis of Joubinia rubens.
Letters as in Text-fig. 13.
TExt-FIG. 16—Schema of the proboscis of Joubinia longi-
rostris and Basiodiscus. Letters as in Text-fig. 15.
related species, Micrura fasciolata Hhrenberg Langia;
variety Joubinia longirostris Berg.
by other investigators, the result of the dissolving of muscular crosses,
and seems therefore not to be of any consequence to our explanation.
More compromising is the fact that in Baseodiscus the nervous layer,
which I have not taken into consideration in this article, but which gives
only support to my views, does not lie in the expected place between
longitudinal and muscular fibres, but beneath the epithelium, as in
Paleonemertines. Is this a reminiscence of a more primitive state ?
286 DR. GERARDA WYNHOFF.
Transition to Baseodiscus type: Oxypolella and Jou-
binia rubens (Text-fig. 15).
Baseodiscus type (‘l'ext-figs. 14 and 16): Baseodiscus
species, Joubinia longirostris and blanca Birger.
Two genera of Heteronemerteans, Poliopsis and Valencinura
have not been mentioned yet. As regards Poliopsis this is
due to our ignorance about its proboscis; in all the specimens
which I know, it has been thrown off before capture. As
Trext-FIG. 17.
Section through the introverted proboscis of Valencinura
bahusiensis after Bergendal (4, taf. 1, fig. 15). p. end.
Proboscis endothelium. p.c. Proboscisepithelium. n.l. Nerve
layer. Other letters as in Text-fig. 13.
regards Valencinura its proboscis has been described accu-
rately by Bergendal (4). The anterior two parts of this organ
possess a muscular coat very much like the anterior part of
Parapolia’s proboscis. The third part, however, lacks all
circular musculature. From the description given by
Bergendal I cannot possibly make out whether the longitudinal
fibres of this last part are the continuation of the outer or of
the inner longitudinal layer. Two figures are given of the
region in which the circular musculature is getting thinner
before it disappears.
The order in which different coats are arranged is: Epi-
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 287
thelium, outer longitudinal layer, nervous layer, circular
muscles, inner longitudinal muscle layer and endothelium
with endothelial circular fibres (Text-fig. 17). The next section
that is figured, however, gives: Epithelium, circular fibres,
nervous layer, longitudinal muscle layer and endothelial circu-
lar fibres (Text-fig. 18). Probably, therefore, both outer cir-
cular and outer longitudinal muscle-fibres have disappeared,
or, as in Parapolia, the outer longitudinal muscle-fibres have
not yet been developed in the hinder part of the proboscis, and
the outer circular muscular layer has disappeared, as it does in
TExtT-FIG, 18,
Section through the proboscis of Valencinura bahusiensis
after Bergendal (4, taf. 1, fig. 14). e.m. Outer longitudinal
muscles. e. p. Proboscis epithelium. Other letters as in
Text-fig. 17.
the other Nemertines. The place of Valencinura in our list
should then be next to Parapolia; the nervous layer in
changing places with the circular muscle-fibres, however,
makes one cautious as to this supposition.
The facts taught by comparative anatomy, therefore, lead
us to the conclusion that the proboscis of the Anopla is a
structure of the body-wall in which both epithelium and
muscular coat have taken part. We shall now have to con-
sider whether all muscle-layers or only part of them helped to
form the proboscis.
The body-wall of Anopla is, as Miss Thompson (80) tried to
make certain, composed of four different muscle-layers. Of
these all Paleonemerteans, with the exception of Carinoma,
VOL. 60, part 2.—NEW SERIES. 20
288 DR. GERARDA WYNHOFF.
only possess three, namely, the outer circular, the inner longi-
tudinal and the inner circular muscle coat. In Heteronemer-
teans, as in Carinoma, an outer longitudinal layer of fibres
has been developed. In the proboscides of Palzeonemerteans
we have found traces of no more than two of the three layers
of the body-wall. Of the third or inner circular muscle-layer
I have not been able to detect any traces in the proboscis of
any Heteronemertean either, endothelial circular muscle-
fibres being of anentirely different nature. Here embryology
comes in to tell us where to look for the missing part.
TrExt-FiG. 20.
TExT-FIG. 19. cer Migs
eer
LM Sw
eb 10eceee-—--———
Pye Ly
NY
TExT-FIG. 19.—Longitudinal section through an embryo of
Prosorochmus after Salensky (28, taf. 3, fig. 27.4). p.mes.
Proboscidian mesoblast. ent. Entoblast. prob. e. Proboscidian
ectoblast. ep. Epiblast.
TEXxtT-FIG. 20.—Schema of an embryo of Lineus obscurus
after Hubrecht (15, fig. 101). rh.w. Rhynchocelomic wall.
mes. Mesoderm. cer. Cerebral ganglia. jp. e. Proboscis epi-
thelium. Other letters as in Text-fig. 19.
A description of the development of the proboscis and its
sheath is given by Salensky (25, 26, 27, 28), by Hubrecht (15),
Birger (7), Lebedinsky (18, 19, 20),and Arnold (1). Of these
authors, Hubrecht, Biirger and Arnold studied the embryology
of Lineus ruber, Salensky (26,28) Pilidium gyrans (pro-
bably the larva of Cerebratulus marginatus) and Pili-
dium pyramidale. The Hoplonomertea subject to these
investigations were Prosorochmus viviparus (Salensky,
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 289
25, 27), Prostoma vermiculum (Lebedinsky, 19, 20),
and Drephanophorus spectabilis (Lebedinsky, 18, 20).
All these investigations are essentially in agreement with each
other as regards our subject, with the exception of Hubrecht’s.
This author, like the others, described the origin of the pro-
boscis as an invagination of the ectoderm lying between, or,
according to other authors, being part of, the two ectoblastic
discs which give rise to the epidermis of the adult worm.
All authors agree also as to the fact that this ectoblastic
invagination, never becoming separated from the epiblast,
TEXT-FIG. 21.
Section through the proboscidian system of an embryo of Lineus
after Arnold (1, taf. i, fig. 18). retr. Retractor muscle. ect.
Eetoblast. ent. Entoblast. Other letters as in Text-fig. 20.
gets its own mesoblastic investment, which is ectodermic in its
origin (Text-fig. 19). At a later stage the ectodermic invagi-
nation of the proboscis has got two mesoblast layers separated
from one another by a space. Hubrecht (15) considered that
this second layer had grown out from the mesoblastic layer
of the body-wall, cutting off part of the body-cavity, or,
according to Hubrecht, part of the archoccel (T'ext-fig. 20).
Later investigations, however, have shown that Hubrecht made
amistake. Biirger (7) studied Hubrecht’s sections, and came
to conclusions in perfect accordance with those of Salensky
and all later investigators. The second mesoblastic layer takes
its origin from the first one by delamination. Lebedinsky has
described the presence of two “ urmesoblasts” at the hinder
290 DR. GERARDA WYNHOFF.
border of both layers. ‘Iwo mesoblastic sacs take in this way
the ectoblastic proboscis between them; when these sacs
reach each other behind the proboscis, the retractor muscle
is formed at the place where the walls fuse (Text-fig. 21). The
inner layer gives rise to the muscular layers of the proboscis-
wall; the outer to the wall of the sheath. Originally, how-
ever, these two structures belong together, their separation
being brought about by a splitting of the mesoblast.
TExT-FIG. 22.
Section through Carinina grata after Biirger (9, pl. xi, fig. 3).
rhynch. Rhynchocelomic cavity. rh.c.m. Rhynchoccelomic
circular musculature. xhynch. end. Endothelium of the rhyn-
chocel. dig. Epithelium of the digestive cavity. 7. ¢.m. Inner
circular muscle layer. nephr. Nephridium.
Here we have found the evidence we looked for on p. 4.
The proboscis has revealed itself as a part of the body-wall,
of which the innermost layer of the muscular coat, the mner
circular musculature, is absent. Embryology teaches that
the proboscis sheath belongs to the proboscis, as both take
their origin from the same tissue. We must, therefore, look
for the missing part in the rhynchoccelomic wall. If the
splitting took place between longitudinal and inner circular
muscle-layer, the sheath must consist of a circular layer alone ;
if the longitudinal layer was the seat of these changes,
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 291
the wall of the rhynchoccelom should have an inner longi-
tudinal and an outer circular muscular coat. The following
facts anatomy discloses:
Procarinina possesses a proboscis-sheath, consisting of
nothing but a very thin layer of circular fibres (5); in
Carinina nothing but circular fibres are found (Text-fig. 22),
Tubulanus linearis has a rhynchocelomie wall, as Pro-
carinina (9); in Tubulanus polymorphus inside this layer
Text-rie. 23.
‘a
‘2
y
34,
TEXT-FIG. 23.—Part of a section through Procephalothrix
linearis. rh.c.m. Rhynchocelomic circular musculature.
rh.l.m. Rhynchocelomie longitudinal musculature. — ¢.l. m.
Central long. musculature. 7.1. m. Inner, and ec. 1. m. central
longitudinal muscles. par. Parenchyma. ent. Entoblast.
Trext-FIG. 24.—Section through Carinesta anglica. rhynch.
caw. Rhyncocelie cavity. rh.c.m. Rhyncocelomic muscu-
lature. 7z.c.m. Inner circular muscles. prob. Proboscis.
bl. v. Blood-vessels. dig. tr. Digestive tract.
one row of minute longitudinal muscle-fibres can be found.
Cephalotrichide (Text-fig. 23) agree with Tubulanus poly-
morphus, and so does Hubrechtia; but the Callineridz (2)
do not possess any longitudinal muscle-fibres inside the
circular layer of the proboscis-sheath (Text-fig. 24).
The body parenchyma, in which the proboscidian system
is imbedded, develops, as a rule, longitudinal fibres in the
292 DR. GERARDA WYNHOFF.
Paleonemerteans. A localisation of these fibres is known to
develop in several cases as the longitudinal septum between
sheath and intestine, but also, as I have tried to demonstrate
(31), as a longitudinal muscle-layer around intestine and
proboscis-sheath, or as longitudinal. muscle-bundles around
the latter. In Procarinina these fibres have no connection at
all with the sheath; neither have they in Carinina, in which
a septum has developed. ‘hey seem to fail in Hubrechtia,
Trxt-rre. 25,
Section through Cephalotrichella after Birger (9, taf. 11, fig.
14). rh.c. Rhynchocelomic cavity. long.m. Outer rhyncho-
cclomic longitudinal musculature. J.n. Lateral nerves. es. n.
(Esophageal nerves. m. Mouth.
but are present in Carinina in the septum, as in some
Cephalotrichide. In Callinera and some Tubulanide longi-
tudinal fibres are present between the inner circular muscle
coat of the body-wall and the rhynchoccelom, without build-
ing a distinct layer, as is the case in Cephalotrichella
signata (Text-fig. 25), Carinomella (‘l'ext-fig. 9), and Cari-
nesta (Text-fig. 26).
In all these cases they are regarded as being a new
acquisition of the proboscis-sheath, developed out of the
central longitudinal musculature. A strong support to this
opinion is given in the behaviour of this musculature in the
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 293
family Cephalotrichidz (81). Even different species of one
genus as the two so nearly related species, Procephalo-
thrix filiformis and P. aliena, show the development of
a longitudinal musculature in the first and an ordinary
septum in the other (81, 33). The Tubulanide show the same
differences in one genus.
TEXT-FIG. 26.
Section through Carinesta anglica. rhyne. Rhynchodeum
rh. c.m. Circular muscles of same. 1. m, Longitudinal muscles
of same. dig. Digestive canal. nephrid. Nephridium. 0. ¢. m.
Outer circular muscular layer of body. 7. c. m. Inner ditto.
i. l.m. Inner longitudinal ditto. bl. v. Blood-vessel. 1. n.
Lateral nerve.
Therefore, if a longitudinal muscle-layer is developed out-
side the circular musculature of the proboscis-sheath, we
must regard it as a secondary one, that has not been present
ab initio, and not as an inherent part of this organ.
In Heteronemertini the two layers are noted. The circular
muscle-layer is always present, an inner longitudinal coat has
been recorded for Euborlasia, several Lineus and Cerebratulus
294. DR. GERARDA WYNHOFF.
species, Joubinia longirostris, Micrura and Langia,
Valencinura, Parapolia, Zygeupolia, ete.
A secondary longitudinal muscle-layer does not seem to be
frequent; as far as I know it is not present in any Hetero-
nemertean. Therefore we may conclude that as a rule the
proboscidian sheath of the Nemertea anopla consists of
two layers, an inner longitudinal layer, which may be absent
TEXxT-FieG. 27.
rad 7
rhb H 1
: 1 vlan
ch.c ty, -
oo
0 OR
oO
oo”
°
6
pee
L}
o
2 45 go8
Section through Emplectonema gracile after Birger (9, pl.
xv, fig. 27). rad. m. Dorso-ventral musculature. gl. Glands.
Other letters as in other figures.
(as in some Paleesonemerteans), and has sometimes the thick-
ness of one layer of fibres, and an onter circular muscle-coat.
If ever theory were in accordance with the facts it is m
this case. We were led to suppose that the proboscis was an
inverted part of the body-wall by anatomical facts. One
layer, however, failed, and embryology taught us that we
might find it in the proboscis sheath, the two structures
belonging together and being one in ontogeny. The missing
part is, as a matter of fact, found in the proboscis sheath,
and this consists of the two layers we knew a priori it must
consist of, if the supposition were right. We conclude,
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 295
therefore, that in Nemertea anopla the proboscis and its
sheath have phylogenetically the same origin, both being
part of an inverted portion of the body-wall; that the two
structures became separated by a rent in the muscular coat,
which took place in the inner longitudinal or between the two
inner muscle-layers. The probosvis has, at least, three layers,
TEXT-FIG. 28.
Longitudinal section through the proboscis of Prosorochmus
after Birger (9, pl. xxiii, fig. 14). st. Stylet. gl. Glands.
constr. Constrictor. c.m. Circular muscle layer. Other letters
as in previous figures. F
if reduction does not take place: the epithelium, the outer
circular and the inner longitudinal muscle-layer, an outer
longitudinal layer not necessarily developing, if it is present
in the body-wall. The proboscis sheath has two or one
muscular coat, an inner longitudinal layer, if present, part of
the inner layer of the body-wall, and an outer circular muscle
layer, the inner circular muscle-coat of the body-wall.
296 DR. GERARDA WYNHOFF,
I have purposely not confused these facts and conclusions
in Anopla with those in armed Nemerteans, for the develop-
ment of the armature in Hoplonemertea has so specialised
this organ, that we must expect great deviations from the
original conditions.
The genus Malacobdella also lives under such unnatural
circumstances for a Nemertine that ail kinds of anomalies
may be expected.
Of the three original muscle-layers of the body-wall, the
TEXT-FIG. 29.
Section through the proboscis of Amphiporus pulcher after
Birger (9, taf. 23, fig. 3). Letters as in other figures.
inner circular fibres, as such, are never present; we have to
look for them, as has been demonstrated by Miss Thompson (80),
in the dorso-ventral musculature of the body (Text-fig. 27).
The outer longitudinal muscle-layer of Heteronemertini has
nowhere developed, and we can recognise the other longi-
tudinal layer by the nervous system being imbedded in it.
Therefore we shall have to look for three muscular layers in
the proboscidian system: a circular and a longitudinal layer,
the latter possibly giving notice of its nature by the seat
of the nerves, both in the proboscis, and probably remnants
of the same longitudinal layer, and a circular layer in the
wall of the sheath.
Now let us examine the facts. Malacobdella has a proboscis
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 297
as described above, an epithelium with a circular muscle-
layer underneath, and the longitudinal layer divided into two
parts by a nervous layer. The endothelium, which lines both
proboscis and sheath, is in the latter structure followed by a
circular muscle-layer, as the presence of longitudinal muscle-
fibres between them seems to be doubtful (9). These facts
are in perfect accordance with those in Anopla.
In Hoplonemertea some diversity exists as to the structure
of the sheath. In all species, however, the wall of the pro-
boscis has developed in avery similar way. As a rule we can
TExtT-FIG. 30. Text-Fia. 31.
ulm.
_ eric
Text-Fic. 30.—Section through the hinder part of the proboscis
of Amphiporus marmoratus after Birger (9, pl. xxiii, fig.
18). p.n. Proboscis nerves. Other letters as in previous
figures.
Text-FIG. 31.—Section through the rhynchocelomie wall of Dre-
panophorus after Birger (9, taf. 23, fig. 37). Letters as before.
divide the proboscis into three parts (Text-fig. 28), the
middle part being the seat of the characteristic structures.
Of these parts the first region has a wall consisting of three
muscle-layers, two of which are circular coats separated by
the only longitudinal coat in which the nervous layer is present
(Text-fig. 29). The longitudinal coat, therefore, must be
regarded as the inner longitudinal layer of the body-wall, and
naturally one looks upon the circular layer between epithelium
and longitudinal fibres as the outer circular muscle-coat. The
other circular layer is present throughout the whole length
298 Dk. GERARDA WYNHOFF.
of the proboscis, as is the longitudinal layer. The outer cir-
cular muscle-layer, however, has absolutely disappeared in
the hind part of the proboscis (Text-fig. 30), and is found in
the middle part in the shape of two distinct sphincters (Text-
fig. 28). One might feel inclined to regard the second cir-
cular layer as the inuer circular coat of the body. ‘This,
however, is not in accordance with the embryological facts,
that taught us to regard the sheath as part of the body-
wall. Moreover, this sheath has a musculature exactly like
that of Anopla. In all Hoplonemertea two muscular layers are
present—an inner longitudinal layer often feebly developed,
and an outer circular layer (Text-fig. 29). There is no
reason not to regard them as identical with the same layers
in Anopla, and therefore the inner circular muscle-coat of
the body-wall is represented by this circular layer of the
sheath, the longitudinal fibres being part of the inner longi-
tudinal musculature. The family Drepanohporide, however,
has a differently constructed sheath, in which, as in the
hinder part of the same structure in Carinoma, longitudinal
and circular fibres have become interlaced (Text-fig. 31).
We have already discussed a similar process in connection
with the proboscis of Joubinia longirostris. As two
kinds of fibres are present, and in all related genera these
fibres are situated in the way stated above, we might regard
their arrangement in Drepanophorus as a complication of
that stage, which cannot disturb our view of the facts.
So homologues of all layers of the body-wall are found in
Hoplonemertea in exactly the same place as in Bdellonemertea
and in Anopla; the second circular layer of the proboscis,
however, cannot be homologised with any part of it, neither
do we know any such layer in Anopla. However, two cases
are known, in which a tendency seems to exist to form a new
circular layer underneath the endothelium; Bergendal (6)
described the presence of circular fibres at that position,
caused by the existence of faint muscle crosses (Text-fig. 3).
C. B. Thompson (80) showed the presence of a layer of
endothelial muscle-fibres. Though both are probably of a
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 299
totally different nature, we may look for an analogy between
the development of this second circular muscle-layer in the
proboscis of Hoplonemertea and the above described
structures. Moreover, the Hoplonemertea have such a highly
developed proboscis with a stylet, reserve stylets, the presence
of the whole middle part (Text-fig. 28) with its glands and
ejaculatory duct, etc., that the obtaining of a new muscle-
layer seems nothing compared with the development of the
TEXT-FIG. 32.
Soy, vhynch .cav,
oo 2
= 3 ? = ea a ea
7 2 oy BEE d.t.comny,.
2 : Pokaan ae ‘
S 5 : ee = S25 525000 S50Esemnoe
. = 7 va . 5 a E a
ae 2 . So Le Ge = =e 4 IT, rhynchy.
‘ pit Oy aia . . dy Be
x 2 « a= , : ‘ o : : 2, ras . ewe >
2S ROA Ug eee ee oe oe eas
a Nos ° - sos - - Seine
| TT Poo Wooo ae _ & — ee a
=a aa se ie
= 2S oe eo eS eee 2 SS ee we
Schema of a longitudinal section through Cerebratulus margi-
natus after Birger (9, pl. xxi, fig. 1). d.n. comm. Dorsal nerve
commissure. v.n.comm. Ventral nerve commissure. rhynch.
Rhynchodemu. dig. Digestive tract. prob. Proboscis. rhynch.
cav. Cavity of the rhynchoceel.
armature. And if we regard this layer as an acquisition of
the proboscis itself, the facts found in Hnopla are in perfect
accordance with those in Anopla.
After all, it seems to me that the structure of the proboscis
in all Nemerteans together with the structure of the rhyncho-
coelom can only lead to this conclusion, that they both took
their origin from the body-wall, a separation being brought
about in the musculature, which, as in embryology, caused the
development of a proboscis and of a sheath.
Proboscides have been described in many invertebrates ;
300 DR. GERARDA WYNHOFF.
they are known in Cestodes, in Turbellaria, in Hirudinea, in
Acanthocephala, in Sipunculids and Priapulids, in Echiurids
and in Kinorhyncha, and in Nemertines. There can be no
doubt that these proboscides are not homologous with one
another. In Kinorhyncha, Priapulids and Sipunculids the
so-called proboscis consists of the anterior part of the body,
bearing the mouth at its top. The body-cavity extends into
this part, and muscles extend from this region of the body-
wall to the wall of the trunk, causing the invagination of
the anterior part as a prolongation of the pharynx.
TErxtT-FIG. 33.
tf / d TE. comm.
fe = sro >
[rit sanee 2982 S0Re= === 2S SSae Bae - ood
Schema of a longitudinal section through Malacobdella grossa
after Birger (9, pl. xvill, fig. 2). atr. Atrium. rhynch.w.
Rhynchocelomic wall. d. n. comm. Dorsal nerve commissure.
v.n.comm. Ventral ditto. dig. Digestive tract.
In Hirudinez the so-called proboscis is quite another
structure. The wall of the pharynx of Rhynchobdellida
shows a protrusible part, which bears the opening of the
intestine at its extremity when erected, in the manner of the
pharynx of Turbellaria. Now, Birger (8) suggested that
the pharynx of Turbellaria is homologous with the pro-
boscidean system of Nemertea. Both structures, therefore,
must be considered more closely in this connection.
Biirger was brought to this conclusion by certain very
characteristic features in Enopla. For, though in all Anopla
the proboscis is extruded through a separate proboscis-pore
situated in front of the brain (J'ext-fig. 52), and the mouth is
THK PROBOSCIDIAN SYSTEM IN NEMERTINES. 301
always found behind the cephalic ganglia, digestive and
proboscidean systems having no connection whatever with
each other, they are closely connected in all Enopla (Text-
figs. 33 and 34), with the exception of Drepanophoride.
The genus Malacobdella, in many features so widely different
from all Hoplonomertea, shares this character, though the
way in which digestive tract and proboscidian system are con-
nected is quite exceptional in Malacobdella (Text-fig. 33). The
mouth is situated at the anterior extremity of the head, being
TExT-FIG. 34.
oes.
Schema of a longitudinal section through Nemertopsis
peronea after Birger (9, pl. xv, fig. 1). rhynch. Rhynchodeum.
rhynch. w. Rhynchoccelomic wall. prob. Proboscis muscles.
oes. cesophagus.
wide and slit-like as in many Heteronemerteans, and giving
entrance to the large cesophagus which lies underneath the
cerebral ganglia, and into which the intestine proper opens.
The rhynchodeum, which, in fact, is so small that as such
it does not seem to exist, opens into the dorsal wall of the
cesophagus, the anterior part of the latter being called atrium
because of this fact.
In Hoplonomertea exactly the reverse of this condition is
found (Text-fig. 34); here the proboscis-pore opens to the
exterior, giving entrance to a well-developed rhynchodeum,
in the hinder part of which the proboscis is inserted. Through
_Pproe. rhynsh,.w. rhynch.
ee
ays
Mi
S 5 - LT
He - >.
aA. - re F H
Eee ee =sssa0 ; ee TESS y
ee ate = : 7 &
SSeS —_— 2S ee J
oan ae Soe aa ii
; 2 cw
ars)
302 DR. GERARDA WYNHOFF.
an opening in the ventral wall of the rhynchodzeum the ceso-
phagus gets an open connection with the rhynchodzum.
Biirger took the rhynchodwum to be the homologue of the
atrium of Malacobdella, and declared the proboscidian system
to be a kind of appendix of the digestive organs. As both
cesophagus and rhynchodeeum originate by invagination of
the ectoblast, lined by the mesoblastic layer which gives rise
to the muscular coat of these organs, Biirger’s hypothesis
seems to be not impossible.
Biirger (8), who stands for the Turbellarian relationships of
Nemertea, compared the intestine of both classes, and found
a pharynx in the first and a proboscidian system in the other
class without any homologues. He then suggested that they
were themselves homologous, both being derived from the
epiblast and the mesoblast underneath. ‘The pharyngeal sac
of Turbellaria should be the homologue of the atrium s,
rhynchodeum in Nemertea. The great difference between
the two organs, however, consists in their different relation to
the cesophagus, which passes right through the pharynx, and
has no direct connection with the proboscis; for Birger
suggested, as we have done, that a splitting must have taken
place in the muscular coat of the pharynx, giving rise to a
rhynchoceelom. This seems altogether very plausible, but
how is the emancipation of the pharynx from the digestive
tract to be explained? Biirger says: ‘‘ Denken wir den
Pharynx nicht in den Vorderdarm gestiilpt, sondern tiber
oder vor der Anlage des Vorderdarms in das Parenchym
gvewachsen und dann einen Spalt im Mesoderm des so ver-
schobenen Pharynx entstanden, dieses in zwei Blatter teilend,
so bekommen wir den Pharynx in einer vor oder iiber dem
Vorderdarm befindlichen Héhle mit mesodermaler Wandung
zu liegen, welche dem Rhynchocélom homolog sein wiirde,
der Pharynx selber aber verhielte sich vollstandig wie der
Nemertinenriissel.” The way in which the cesophagus must
attain a direct communication with the pharyngeal sac is
rather absurd: ‘“ Es ergiebt sich ohne Weiteres, dass wir in
dem Pharynx ein Zuviel haben, denn nur seine Tasche, die,
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 303
nachdem wir den Pharynx exstirpiert haben, direct
mit dem Darm communiciert, entspricht dem Nemertinen
vorderdarm.” Even if Biirger did not suppose this mode of
arriving at the described situation to have really taken place,
as I am sure he never did, I cannot see in what other way
the pharynx could be emancipated from the digestive tract.
It is quite impossible to picture the way in which these
changes had to take place without any such lesion, and on
this ground alone the theory of Biirger seems to lose vitality.
But there is more. Birger gives three reasons to support his
views. ‘The first one we have already given above. The
second support is found in embryology: ‘‘ Der Nemertinen-
riissel entsteht stets aus einer Hctodermeinstiilpung, die
mit einem diese umgebenden Mesodermwulste verschmilzt.
Die Anlage des Riissels erfolgt bei den Metanemertinen am
selben Orte wie die des Vorderdarms und mit ihr gemein-
schaftlich.” ‘he similarity of histological structure of the
two parts is the third one. As to the latter, the histological
resemblance of pharynx and proboscis consists only in the
presence of an epithelium and the muscular layers of the
body-wall, and therefore the same statement as before, that
both are structures of the body-wall, must be repeated.
Real histological resemblance there cannot possibly be, as
the epithelium of the proboscis in Nemertines shows a great
variety of elements in different parts and different species.
That the muscular tissue of Nemertines and Turbellaria agree
in structure is too well known to have any proving impor-
tance in this case, for the proboscis musculature is built up
in exactly the same way as the muscles of the body-wall.
Biirger’s third reason, therefore, is not at all convincing.
The embryological facts, which follow in the second place,
have since been contradicted by Lebedinsky and Salensky.
In the first place, Biirger compares the invagination of the
proboscis-ectoderm with the extirpation of the pharynx-
ectoderm; then the pharyngeal sac is the primarily planned
organ, the pharynx developing afterwards at the bottom.
In Nemertines the proboscis is the organ that appears first,
VoL. 60, PART 2,—NEW SERIES, 21
304 DR. GERARDA WYNHOFP.
a later invagination of ectoderm giving rise to the rhyncho-
dzum, the homologue of the pharyngeal sac.
But even if one takes into consideration the plain embryo-
logical facts in Nemertines, it seems difficult to come to the
same conclusions as Biirger. For in what way do Nemerteans
get a mouth? In Anopla the blastopore sometimes, after
becoming closed, is transferred to the inside of the animal by
the invagination of the cesophagus, In Knopla the facts are
TEXT-FIG. 35.
Longitudinal section through Prosorochmus after Salensky (27,
fig. 8). @s.atr. Hsophageal atrium. rhynch. Rhynchodeum.
vh.c. Rhynchocelomic cavity. gl. Glands. dig. Digestive
canal. prob. Cavity of inverted proboscis. rhe. Rhyncocel.
less simple. Drepanophorus, the genus in which cesophagus
and rhynchodezum open separately, shows no connection at
all between the two systems, not even in embryology ; for
here the blastopore is closed, the narrow entodermic part
giving rise to the blind gut by being removed forward. The
primary ectodermic cesophagus invaginates near the probo-
scidian system, but perfectly separate. The place where
cesophagus and entoderm communicate in Drepanophorus
has, therefore, nothing to do with the same spot in Hetero-
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 305
uemerteans. In all other Hoplonemertea the primary ceso-
phagus originates in exactly the same way ; the mouth closes
afterwards, and the primary cesophagus gets a new opening
to the exterior through the rhynchodeum. lLebedinsky
described in Tetrastemma vermiculus how the rhyn-
chodzeal epithelium grows towards the cesophagus, a bifurca-
tion of the cavity being produced. Salensky (27), however,
is of opinion that in Prosorochmus the communication is
brought about by the cesophagus, which has produced through
histological differentiation an atrium of its own (Text-fig. 55).
The difference of opinion between these two authors does not
matter in the least for our decision, for it is evident that the
conditions in Drepanophorus are primitive, and that the facts
on which Birger based his theory are not primitive at all, as
the Drepanophorus stage is passed through in ontogeny.
This fact, the difference between the communication in
Malacobdella and Hoplonemertea, the impossibility of a
pharynx developing into a proboscis, the difference of origin
between the two organs, the differences in the connection
between pharyngeal sac and pharynx at one side, proboscis
and rhynchodeum at the other, the insufficiency of the grounds
on which Biirger bases his theory, make me very sceptical as
to its value. In fact, I cannot find any other point of resem-
blance between the two organs than that they are derivatives
of what the Germans call die Hautmuskelschlauch.
There are, however, other organs in Invertebrates, even in
Platyhelminthes, which have a similar origin, and we shall
have to look for comparison between them. For example, the
proboscis of Echiurids is a structure of the body-wall—at least
it consists of an epidermis and muscles only ; moreover it is
situated in front of the mouth, and is known to be very
elastic. As, however, this proboscis is supposed to be the
preoral lobe of the larva and cannot be retracted into the
body, it does not give any special indication of affinity to
the same structure in Nemerteans. In Acanthocephala (14) a
proboscis is found, which can only partly be retracted into
the body. The proximal part is surrounded by a sheath of
306 DR. GERARDA WYNHOFF.
muscles, the circular fibres of which act as protractors; the
retractor muscle, which retracts proboscis and sheath, the
two parts being continuous, is inserted at the hind end of the
sheath, and extends through the body-cavity to the body-wall.
Though some agreement in these structures in Hchinorhynchus
and Nemertea cannot be denied, I do not suppose there is
any relationship between them. The proboscidian system
of Acanthocephali is entodermic in origin and only secon-
TEXT-FIG. 36.
prob. atr.
Schema of the proboscis of Macrorhynchus croceus after
von Graff (18, pl. x, fig. 12). prob. atr. Proboscidian atrium.
prob. Cavity of the introverted proboscis. prob. w. Probos-
cidian wall. vad. m. Radial musculature. rhynch. w. Rhyn-
chocelomic wall (Muskelzapfen). Compare with Text-fig. 1.
darily gets its epidermis. This fact alone seems to exclude
all possibility of homology with the proboscis of Nemer-
teans.
Coming to the consideration of two other structures, both
in Platyhelminthes, the proboscides of Tetrarhynchus and
of Proboscida, the four proboscides of the characteristic
genera of Cestodes superficially show a great resemblance
to those of Nemerteans. The proboscis is an introvert,
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 307
being retracted by a muscle-bundle at the bottom of the
tube just as in Nemertines. A muscular sheath is present,
separated from the proboscis by a cavity filled with a liquid.
Histologically, however, a’ great difference seems to exist.
For instance, the proboscis itself has no muscles at all, if
I understand Pintner (21) right; the wall of the sheath is
differently constructed at its anterior and posterior part,
a muscular sheath only existing in the hinder part, the
anterior being a derivative of the ectoderm. Moreover the
proboscides of these Cestods are paired, those of Nemertines
being unpaired.
Much more striking is the likeness to the proboscis of Tur-
bellaria proboscida. Salensky (25 and 26) was the first
to compare them, and though he already proclaimed them
homologues in 1884, nobody seemed to agree with his opinion.
Hubrecht (16) raged against it, and afterwards Biirger (8)
gave a new theory. Salensky, however, was not convinced,
and tried to give fresh support to his hypothesis in his
articles of 1909 and 1912 (27, 28).
The proboscis, which is found in certain genera of Rhabdo-
ccelida, is localised at the anterior end of the head. Von
Graff (13) described the organ in this way: “Der Probisciden
riissel ist nichts weiter als eine bleibend gewordene Einstiil-
pung des Vorderendes, wie wir sie voriibergehend bei Meso-
stomum rostratum entstehen sehen.” It consists of two parts,
the proboscis and a kind of atrium, through which the pro-
boscis is everted (Text-fig. 36). The cavity of both is lined by
_ the somewhat changed epiderm. The wall of the atrium has,
like the body-wall, a muscular coat, which is the direct con-
tinuation of the muscular layers of the body-wall. At the base of
this sac or atrium the proboscis is inserted. Here the muscular
coat is broken up into two layers; the inner layer continues
along the epidermis, the other one forms the outer lining of
the proboscis. A thick layer of radially placed muscle-fibres
fills the space between the two parts of the muscular coat of the
body-wall. In Nemertines (Text-fig. 1) we find the same
arrangement: an atrium to the proboscis, being part of the
308 DR. GERARDA WYNHOFF.
body-wall, that has grown inward and is called rhynchodzum.
The continuation of the epithelium is found in the epithelium
of the proboscis. The muscular coat of the body-wall is split,
and has given rise to the muscular wall of the proboscis and of
the sheath. The cavity of the rhynchoccel is even traversed by
a band of radially placed muscle-fibres, the retractor muscle.
So Salensky (26) proposed the following homologies.
Rhabdocéles probosciféres. Némertiens.
‘1. Poche de la trompe. 1. Vestibule de la trompe.
2. Epithélium de latrompe. 2. Epithélium de la trompe.
3. Couche interne dela calotte 3. Couche musculaire de la
musculaire (Muskelzapfen trompe.
v. Graff).
4, Couche externe dela calotte 4. Parois de la gaine de la
musculaire. trompe.
5. Muscles radiaires de la 5. Bride musculaire.
calotte.
When von Graff (18) described the structure of the pro-
boscis of Macrorhynchus and other Rhabdoccelida the origin
of the proboscis and sheath in Nemertines was not known.
He could not find any homologue of the sheath in his genera,
aud on this ground denied any relationship between the
proboscides in these two classes of Platyhelminths. Another
ground for denying it was found in the division of the pro-
boscis of Nemertines into two parts, the posterior glandula
part not being present in Turbellaria proboscida. This
ground, however, seems not to be very important; the pro- —
boscis of Rhabdoccelida represents rather a simple stage of
organisation in comparison with the same structure in Nemer-
tines. It does not seem to be necessary to go into such details
of. structure, especially not since we know what a great
variety of epithelial structure is to be found in different
genera of Nemertea.
Salensky looked for support for this theory to embryology.
And certainly the fact that the muscular coats of rhyncho-
ccelomic and proboscidian walls take origin out of the same
THE PROBOSCIDIAN SYSTEM IN NEMERTINES. 309
layer of mesoblastic elements, a splitting giving rise to the
cavity of the sheath, is highly in favour of his views. It was
exactly on this point that Hubrecht did not agree with him ;
therefore Hubrecht, who, moreover, claimed the Annelida-
relations of Nemertea, could not possibly follow Salensky. As,
however, later investigations proved that Salensky was right,
this reason for disagreeing with his theory gave way. But
Hubrecht gave other reasons. In the ‘ “Challenger”’ Report’
he wrote, p. 104: ‘Salensky would probably not have made
his startling hypothesis above alluded to, based on ontogene-
tical observations of a scission in the proboscidian wall, by
which (1) a muscular proboscidian sheath surrounding the
proboscis becomes separated from, and independent of, the
musculature of the proboscis itself, and (2) an isolated ccelome
—the proboscidian cavity—is originated, if he had been as
well acquainted with the comparative anatomy of the animals
about which he writes as he is with certain details of their
ontogeny.” The anatomical facts, that tell against Salensky,
Hubrecht summarises in this way (loc. cit., p. 103): “There
can hardly be any doubt, when we take into consideration all
the morphological data at our disposal, that the muscles com-
posing the proboscidian sheath gradually took their origin by
the increase and modification of pre-existing muscular elements
which belonged to the body-wall and to the body-parenchyma
before the proboscis, modified from a tactile organ, as it
appears to have primitively been, had yet become evolved,
through the growth inwards of the anterior tip of the body,
into an aggressive weapon with stylet or nematocysts, etc.”
Since this was written many facts have been disclosed as to
the anatomy of the proboscidian system, and we have seen
that they lead us to the conclusion, that the sheath is
part of the body-wall, and has not originated in loco out
of muscular fibres in the parenchyma of the body. The
central parenchyma does not possess any circular muscle-
fibres. Wherever other than longitudinal fibres have been
found in it they have been demonstrated to be part of one
of the layers of the body-wall, as a rule the inner circular
310 DR. GERARDA WYNHOFF.,
muscle-layer. Longitudinal muscle-fibres may be present in
the central connective tissue, and we have already seen that
in some cases they are arranged around the proboscis sheath
so as to form a secondary longitudinal coat. This, however,
is not the rule, not even in Anopla, where a central longitu-
dinal musculature is developed in the majority of genera.
Our conclusion is exactly the reverse of the above statement
of Hubrecht; the facts described in the first part of this
article seem not to allow the denial of our conclusion. Both
on anatomical and embryological grounds we must declare the
muscular coats and walls of the proboscidian system of Nemer-
tines to belong together ab origine; the facts put forward by
Hubrecht against the theory of Salensky have been refuted by
later embryological investigations. I hope to have given a
new support to Salensky’s ingenious theory by this compara-
tive study of the anatomy of the proboscidian system. The
fact, stated by Hubrecht, that “we find the shorter pro-
boscides and the less significant proboscidian sheaths among
the more primitive genera of Nemertea,” is, though mentioned
by him as telling against the ‘'urbellarian relations, in perfect
accordance with, if not in favour of, Salensky’s theory.
LITERATURE.
Arnold (1898).—* Zur Entwicklungsgeschichte des Lineus gesse-
rensis O. F. Miller,’ ‘Trav. Soc. Imp. Nat. Pétersb.,’ vol.
xxviii, livr. 4.
P
2. Bergendal (1900)—‘Callinera biirgeri Bergendal, en rapre-
sentant for ett afvikande sligte bland Palaionemertinerna,”
‘Lunds Univ. Arsskr.,’ Bd. xxxvi, Afd. 2, No. 5.
3h (1902).—** Zur Kenntniss der nordischen Nemertinen ITI,”
‘Bergens Mus. Aarbog.,’ No. 4.
4. (1902).—** Valencinura bahusiensis Brgdl., ein Beitrag
zur Anatomie und Systematik der Heteronemertinen,” ‘ Lunds
Univ. Arsskr.,’ Bd. xxxviii, Afd. 2, No. 3.
5. (1902).—* Eine der construirten Urnemertine entsprechende
Paleonemertine aus dem Meere der schwedischen Westkiiste,”
‘Zool. Anz.,’ Bd, xxv.
THE PROBOSCIDIAN SYSTEM IN NEMERTINKES. 31]
6. Bergendal (1903).—‘* Beobachtungen iiber den Bau von Carinoma
Oudemans nebst Beitrage zur Systematik der Nemertinen,”
‘Lunds Univ. irsskr.,’ Bd. xxxix, Afd. 2.
7. Burger, O. (1894).—* Studien zu einer Revision der Entwicklungs-
geschichte der Nemertinen,” ‘ Ber. Naturf. Ges. Freiburg i B.,
1894.
8. (1895).—* Die Verwandtschaftsbeziehungen der Nemer-
tinen,’ ‘ Verh. Deutschen Zool. Ges. Strassburg.’
9. (1895).—** Die Nemertinen,” Fauna u. Flora Neapel., Mon.
22.
10. (1897-1907).—** Die Nemertinen,” ‘ Bronn Klass. u. Ordn.
Tierreich.’
10a (1900).—** Die Nemertinen,” ‘ Wiss. Erg. D. Tiefsee Ex-
pedit. Valdivia,’ Bd. xvi, Lief. 2.
1l. Coe, W. R. (1895).—* Descriptions of Three New Species of New
England Paleonemerteans,” ‘ Trans. Conn. Acad.,’ vol. ix.
(1905).—‘*‘ Nemerteans of the West and North-west Coasts
of America,” ‘ Bull. Mus. Harv. Coll.,’ vol. xlvii.
13. von Graff, L. (1882).—‘ Monographie der Turbellarien.’ I, ** Rhab-
docelida,” Leipzig.
14. Hamann, O. (1891).—‘* Monographie der Acanthocephalen,” ‘ Jen.
Zeitschr. Naturw.,’ Bd. xxv.
15. Hubrecht, A. A. W. (1885).—‘ Proeve eener ontwikkelingsgeschie-
denis van Lineus obscurus, Utrecht, Leeflang.
16. ——— (1887).—** Nemertea,” ‘ Rep. Sc. Res. Challenger Zool.,’ vol.
abe
17. Joubin, L. (1890).—‘* Recherches sur les Turbellariés des cétes de
France (Némertes),” ‘ Arch. Zool. expér. gén.’ (2), Bd. viii.
18. Lebedinsky (1896).—* Zur Entwicklungsgeschichte der Nemer-
tinen,” ‘ Biol. Centralbl.,’ Bd. xvi.
(1897).—Ibid., Bd. xvii.
(1897).— * Beobachtungen iiber die Entwicklungsgeschichte
der Nemertinen und Nachtrag.,” ‘Archiv Mikr. Anat. u. Entw.
gesch.,’ Bd. xlix.
20. —
21. Pintner (1895).—“ Versuch einer morphologischen Erklirung des
Tetrarhynchenriissels,” * Biol. Centralbl.,’ Bd. xvi.
22. Punnett, R. C. (1901).—*On Two New British Nemerteans,”
‘Quart. Journ. Mier. Sci.’ (N.s.), vol. 44.
(1901).—**On Some Arctic Nemerteans,” ‘ Proc. Zool. Soe.
Lond.,’ 1901, vol. ii, Part I.
VOL. 60, pAkT 2.—NEW SERIES, 22
23.
351
24
2 DR. GERARDA WYNHOFF.
. Punnett, R. C. (1903).—* On the Nemerteans of Norway,” ‘ Bergen
Mus. Aarbog.,’ 1903, No. 2.
25. Salensky, W. (1884).—‘* Recherches sur le développement du Mono-
pora vivipara,” ‘Arch. de Biologie,’ v.
26. (1886).—** Bau und Metamorphose des Pilidium,” ‘Z. W. Z.,
Bad. xliii.
2 i (1909).—** Ueber die embryonale Entwicklung des Pro-
sorochmus viviparus,” ‘ Bull. Acad. Imp. Sciences, Petersb.,
1909.
28. (1912).—** Ueber die Morphogenese der Nemertinen. I.
Entwicklungsgeschichte der Nemertine im Pilidium,” ‘Mém.
Acad. Imp. Sciences, Pétersb.,’ vol. xxx, No. 10.
29. Schutz, V. (1912)—‘*Paralineus elizabethe,” ‘Z. W. Z.,
30
31.
Bd. cio. t
. Thompson, C. B. (1901).—‘“* Zygeupolia litoralis, a New Hetero-
nemertean,” ‘Proc. Acad. Nat. Sc. Philad.,’ vol. liii, p. 2.
Wynhoff, G. (1910).—** Die Gattung Cephalothrix und ihre Bedeu-
tung fiir die Systematik der Nemertinen; I, Anatomischer Teil,”
‘Zool. Jahrb.,’ Bd. xxx, Abt. f. Anat.
32. (1912).—** Die Systematik der Nemertinen,” ‘Zool. Anz.,’
Bd. xl.
23h (1913)—** Die Gattung Cephalothrix, ete.; II, Systema-
tischer Teil,” * Zool. Jahrb., Bd. xxxiv, Abt. f. Syst.
GAMETOGENESIS OF GRANTIA COMPRESSA.
313
Observations on the Gametogenesis of Grantia
compressa.
By
Arthur Dendy, D.Sc., F.R.S.,
Professor of Zoology in the University of London (King’s College).
With Plates 23 to 26.
CoNtvTENTs.
) GENERAL INTRODUCTION
) MarerraLt AND METHODS OF INVESTIGATION
a THE BREEDING SEASON AND LIFE-CYCLE
(D) THE DISTRIBUTION OF THE GERM-CELLS IN THE See
(E) THE RELATIONS BETWEEN THE DIFFERENT TISSUE-
ELEMENTS
(F) aa
(a) Historical . :
(b) Origin and Growth of the ftine: Casati
(c) Multiplication of the Odgonia
(d) Growth and Feeding of the Young Onestes in the
Flagellate Chambers 2
(e) Growth and Feeding of the Oacee in the Mesc glea .
(f) Maturation of the Odcytes
(g) The Nurse-cells and their Origin ; Pliers eens
(h) Summary and General Remarks on Odgenesis .
(G) SPERMATOGENESIS
(a) Historical . :
(b) Origin and Growth of the Berrerey cnauaeane
(c) Formation of the Spermatocysts or Cover-cells
(d) Development of the Sperm-morule from the Primary
Spermatogonia : ;
(e) The Formation of Speniioraa Comparison with
Sycon, ete.
(H) FERTILISATION OF THE Ovum:
(k) List oF LITERATURE REFERRED TO
(L) EXPLANATION OF PLATES : : :
VOL. 60, PART 3.— NEW SERIES. 23
314 ARTHUR DENDY.
(A) GeneraL Intropuction.
Tue problem of the gametogenesis of Sponges is one which
has attracted a good deal of attention from time to time, but.
it can hardly be said that our knowledge of the subject is as
yet by any means in a satisfactory condition. As far back as
1851 Huxley described the occurrence of supposed sperma-
tozoa in Tethya, and in 1854 Carter did the same for
Spongilla, but Haeckel is probably right in supposing that.
both these authors were in reality describing flagellate collared
cells, a mistake which, owing to the difficulty of detecting the
collar in certain conditions, it’ is very easy to make.
Haeckel, indeed, claims for Lieberkiihn the original discovery
in 1856 of the sexual differentiation of sponges and of the
ova and spermatozoa (in Spongilla).
Haeckel points out the great difficulty in finding the
spermatozoa of sponges, a difficulty which has been, and
doubtless still is, experienced by many spongologists, and
which arises chiefly, no doubt, from their extremely minute
size, the fact that they occur scattered throughout the
sponge and not segregated in definite gonads, and their
lability to confusion with other tissue elements. The ova, on
the other hand, in their later stages of growth, are readily
recognisable by their large size, and, at certain periods, their
conspicuous vesicular nuclei, although these again are not
collected in definite ovaries, but occur scattered through the
sponge-tissues.
The spermatozoa of calcareous sponges were discovered by
Haeckel himself in 1871, and at about the same time by Eimer.
In his well-known monograph, “ Die Kalkschwimme ” (1872)
the former author gives an account of all that was previously
known of the germ-cells of sponges, and describes his own
observations and conclusions with regard to the Calcarea.
He finds reasons for deriving the spermatozoa from
collared cells of the so-called entoderm (gastral layer), and
gives several figures of sperm-morule lying amongst the
ordinary collared cells in the gastral layer of several species.
GAMETOGENESIS OF GRANTIA COMPRESSA. ey
With regard to the ova he remarks that the question of the
origin and of the original position of the egg-cells is the
most difficult and darkest part in the histology of calcareous
sponges, an opinion with which those who have studied the
question will hardly feel inclined to quarrel. Haeckel himself
at first regarded the ova as originating in the “ exoderm,”’
in which he, of course, includes everything but the “ ento-
derm,” but his more mature conclusion is that they really
originate in the “ entoderm.” He says (1872, p. 159): “ Hin-
zelne Geisselzellen des Entoderms vergréssern sich, ziehen
thren schwingenden Geisselfortsatz ein, umd entwickeln sich
direct durch Aufblaihung des Kernes und bedeutende Volums-
Zunahme des Protoplasma zu Hizellen.”
Since Haeckel wrote, opinion as to the origin of the germ-
cells in sponges has changed, and though very few writers
have recorded any detailed observations, it is generally held,
in accordance with the teachings of F. E. Schulze and others,
that both ova and spermatozoa arise from amceboid wandering
cells in the mesoglcea, which, of course, is part of Haeckel’s
‘*‘exoderm.” No one, since Haeckel’s time, appears to have
seen the sperm-morule lying amongst the collared cells in
the walls of the flagellate chambers, and no one has traced
the stages by which collared cells might be supposed to have
been converted into spermatogonia. Haeckel himself seems
to have been by no means satisfied that his own observations
on the subject were conclusive, and Poléjaeff, writing ten
years later (1882), evidently regarded them with grave
suspicion.
Nevertheless, I believe that Haeckel was essentially right
in maintaining that odgonia and spermatogonia both arise
from collared cells. It is, at any rate, quite certain that
bodies resembling spermatogonia are frequently to be found
in small morula-like clusters amongst the collared cells in the
walls of the flagellate chambers of Grantia compressa,
very much as figured by Haeckel for other Calcarea, except
that I have never observed the tails of the spermatozoa in
this situation, and, indeed, have only seen the sperm-morule
316 ARTHUR DENDY.
in properly prepared, stained sections. Between the original
collared cell and the mature germ-cell of either sex amceboid
stages intervene, which are frequently to be found in the
mesogloea, at any rate in the case of the odgonia. It is an
easy matter to trace the growth of the amceboid odgonia into
mature ova, but the question is—Whence come the earliest
amoeboid stages ?
The modern answer to this question is that they are
derived from primitive amceboid cells (“ archzocytes ”’), but
I cannot help suspecting that this answer is merely an echo
of Weismann’s well-known views as to the early segregation
of the germ-cells and the continuity of the germ-plasm. It
depends, so far as | am aware, not upon direct observation,
but upon a process of reasoning by exclusion. The germ-
cells are supposed to remain over after the somatic cells have
been subtracted from the sum total of cells derived from the
segmenting ovum.
I believe that this particular theory of the origin of the germ-
cells in sponges originated with Dr. Otto Maas (1894), who
sums up his conclusions as follows (p. 35) :
“© (1) Wir koénnen hier eine directe Abstammung der Keim-
zellen der einen Generation vom Ei nachweisen, indem durch
Subtraction aller somatischen specialisierten Hlemente schli-
esslich eine Anzahl indifferent gebliebener Elemente iibrig ist,
die Urgeschlechtszellen.
“ (2) Der Hauptunterschied zwischen den somatischen und
den Geschlechtszellen zeigt sich vom Anfang wie spater im
Kern, und zwar in der Quantitiét und Anordnung des Chro-
matins.”
Anyone who has studied the gametogenesis of sponges knows
how greatly the condition of the chromatin varies in different
stages, and I do not believe it is possible to indicate any nuclear
character by which primordial germ-cells can be definitely
distinguished from somatic cells, at any rate inthe present state
of our knowledge. The alleged distinction between the two
groups of cells is a purely theoretical one. Indeed, the latest
writer on the subject, Max Jérgensen (1910), has already come
to the conclusion that the distinction which Maas endeavours
GAMETOGENESIS OF GRANTIA COMPRESSA. Bays
to draw between germ-cells and somatic cells cannot be
maintained in sponges, and “ dass es sehr wohl zur Bildung von
Geschlechtszellen aus somatischen Zellen kommen kann, wie
man dies ja bei dem primitiven Charakter der Schwamme von
vornherein erwarten wiirde” (p. 170). This author derives the
odgonia of Sycon from ordinary ‘“‘mesoderm”’ cells rather than
from primitive undifferentiated amcebocytes (archeocytes),
though he thinks that the latter may also give rise to odgonia.
He does not deal with the spermatogenesis.
My own attention was first particularly directed towards
the problem of the origin of the germ-cells in calcareous
sponges under the following circumstances. Some years ago
Prof. Herdman invited me to write a memoir on the common
British species, Grantia compressa. In the course of
this work it became evident that although the ova of this
sponge were easily recognisable in the maternal tissues in
various stages of growth, and also embryos in varicus stages
of development, no one had ever been able to find the sperma-
tozoa, though they were searched for repeatedly by Mr. Carter
(1875, p. 25). For the sake of completeness it appeared
highly desirable that this gap in our knowledge should be
filled.
In April, 1912, accordingly, I paid a visit to the laboratory of
the Marine Biological Association at Plymouth, and although
I was unable to find spermatozoa in the living specimens, of
which I examined a large number, I preserved material which
on subsequent investigation furnished the clue to the solution
of the problem. I found that the sponge is hermaphrodite,
producing both male and female germ-cells simultaneously,
but that the spermatozoa are produced in comparatively small
numbers, the minute sperm-morule being scattered here and
there, enclosed in spermatocysts, between the collared cells of
the chamber walls, and also occurring free in the flagellate
chambers. The spermatogonia in these sperm-morule are
extremely minute, andI am unable to say anything with regard
to their mode of division. Apparently they are sometimes
transferred as sperm-morule to the inhalant canals of the
318 ARTHUR DENDY.
Same or of another individual, where they break up into
Spermatozoa, but it is probable that they may sometimes
break up into spermatozoa before leaving the parent sponge.
The evidence on these points is, however, curiously scanty,
and I conclude that the spermatozoa are rarely, if ever,
liberated in large numbers.
With regard to the process of odgenesis, on the other hand,
I found a number of very interesting stages, and with the
exception of the second maturation spindle (described by Max
Jorgensen in Sycon), am able to give a fairly complete
account. My observations agree in many respects with those
recorded by Jérgensen on the oogenesis of Sycon, but I am
able to add a good deal to his account, and in some respects
my interpretations are different. ‘Though I have repeatedly
observed mitotic figures of various types I have always found
the chromosomes very small and ill-defined, and I cannot
pretend to give such precise descriptions of the mitotic
phenomena as Jérgensen has done. It is very difficult to
understand his account of these phenomena, or to harmonise
it with currently accepted views, but I must leave to specialists
in cytology the detailed criticism of his work in this respect.
I may perhaps say, however, that many of his figures appear
to me to be very diagrammatic.
One of the most remarkable phenomena observed during
my investigations is the nutrition of the growing odcyte by
means of phagocytic nurse-cells. Very extensive phagocytosis
has also been observed in the case of certain large amcebo-
cytes, which seem to devour the young germ-cells in some
cases in a wholesale fashion.
Though well aware that I am not able to give by any
means a complete account of the history of the germ-cells in
Grantia compressa, I hope that the following pages may
not only help to fill a conspicuous gap in our knowledge of
this common British sponge, but also throw some new light
on the difficult problem of gametogenesis in sponges generally.
I must take this opportunity of thanking my friends at the
Plymouth Laboratory, especially Dr. Allen and Mr. Orton,
GAMETOGENESIS OF GRANTIA COMPRESSA. a19
for their hospitality and assistance during my visit. I am
also greatly indebted to the University of London for the use
of their table at the Laboratory.
(sp) MarertaL and Meruops or INVESTIGATION.
My observations at the Plymouth Laboratory, made from
April 10th to April 22nd, 1912, were mainly directed towards the
discovery of the spermatozoaof Grantia compressa. ‘The
sponge may be obtained in large numbers close to the Labo-
ratory, at low water, and I also received a number of fresh
specimens from Drake’s Island and Rum Bay. They varied
greatly in size, from quite small to as much as 80 mm. in
height by 18 mm. in breadth (a specimen from Rum Bay).
A large number were microscopically examined in the hving
condition, either by teasing or by means of hand sections, or
by pipetting out the contents of the central gastral cavity,
but my search for living spermatozoa was fruitless.
I preserved a considerable number of specimens, however,
for future examination, and the results recorded in the present
paper are based almost entirely upon the study of these by
means of paraffin sections.
The material that turned out satisfactorily was fixed either
in strong Flemming’s solution, or in a mixture of Fiemming,
formol and sea-water. In the former case it was graded up,
after washing, to 70 per cent. alcohol; in the latter it was
preserved in formol sea-water.
The sections were, for the most part, cut of a thickness
of 5y and stained on the slide. I found that iron-brazilin
gave excellent results, but iron-hematoxylin was also used.
For staining in bulk borax-carmine or paracarmine was
employed, the latter being sometimes followed on the slide
by picro-indigo-carmine, but without much effect.
I desire to express my thanks to my skilful laboratory
assistant, Mr. Charles Biddolph, for the care which he has
taken in preparing the sections.
Although many specimens were preserved, only five have
320 ARTHUR DENDY.
actually been used for the purposes of the present investiga-
tion, numbered in my notes 11, 21, 22, 25 and 24 respectively.
It will save repetition if I give particulars concerning
these specimens at once, and of the mode of treatment of the
material.
No. 11.—A very large specimen from Rum Bay, 80 mm.
high by 18 mm. in breadth; brought in about mid-day (April
15th) and examined the same afternoon (about 1 o’clock).
When examined a vigorous stream was coming out from the
main vent. A section of the living sponge from near the base
showed large ova, but no embryos were seen. A section near
the vent showed smaller germ-cells. The specimen was cut
in half lengthwise, and half fixed in strong Flemming’s solu-
tion (in the dark) for half an hour, then washed for an hour
or more in tap-water and graded through 30 per cent. and
50 per cent. to 70 per cent. alcohol.
No. 21.—A rather small specimen, about 14 mm. high,
collected about mid-day on April 18th, and examined the same
afternoon. There was an active current issuing from the
vent, bringing with it a quantity of fine yellowish-grey sedi-
ment, which collected at the bottom of the glass dish. In
hand section numerous large rounded cells were seen free in
and projecting into the flagellate chambers, apparently
actively moving, but probably only owing to the movements
of the flagella of the collared cells. Fixed in strong Flem-
ming’s solution and preserved in 70 per cent. alcohol.
No. 22.—A moderate-sized specimen, about 25 mm. high
by 18 mm. broad ; collected about mid-day on April 18th and
examined the same aftrnoon. Fixed entire in Flemming and
sea-water formol' for about a quarter of an hour, then washed
in formol and sea-water and preserved in same.
No. 23.—A moderate-sized specimen, collected on April
18th and examined and fixed on the 19th, having been kept in
the circulation of the aquarium overnight. Stream coming
from osculum when examined. Found no amecebocytes or
1 Take 10 c.c. commercial formaldehyde in 90 ¢c.c. sea-water and add
20 c.c. strong Flemming’s solution.
GAMETOGENESIS OF GRANTIA COMPRESSA. 321
other cells in water pipetted from gastral cavity. On exa-
mination of living section found very numerous rounded
amoebocytes with coarse granules, and a few with pseudo-
podia, in the chambers, some, if not most, attached to walls
by short peduncles. Half of specimen fixed in strong Ilem-
ming’s solution for about an hour, washed and graded up to
70 per cent. alcohol. Another part fixed in absolute alcohol.
No. 24.—A good-sized specimen from Rum Bay, examined
and preserved on April 22nd, after having been kept in the
aquarium circulation since April 15th. The collared cells were
found to be still active and did not show the characteristic
signs of suffocation. Bulk of specimen fixed in Flemming
and sea-water formol.
(c) THe Breeprna Season anvD Lire-Cycie.
There is strong, indeed, I think conclusive, reason for
believing that Grantia compressa is an annual sponge,
growing rapidly during the winter and spring and breaking
up and perishing in the autumn, after producing numerous
embryos. I have already given particulars as to the sizes of
some of the specimens met with at Plymouth in April, 1912,
and my colleague, Mr. R. W. H. Row, tells me that about the
middle of August, 1913, when he visited Plymouth, they
were already breaking up, and it was difficult to obtain a good
specimen of any considerable size—indeed, most of them had
apparently already disintegrated.
The breeding season at Plymouth would seem to begin in
the first half of April; germ-cells are then being produced in
enormous numbers, but comparatively few embryos are found.
At least that was my experience in 1912.
Previously, in 1911, I had observed mature embryos being
shot out of the osculum of a specimen which I examined in
the laboratory in the first week of June. I also find plenty of
advanced embryos, along with germ-cells in various stages of
growth, in a specimen collected for me by Mr. Row about the
middle of August, 1913. The germ-cells (ova), however, are
nothing like so abundant as in material taken in April.
322 ARTHUR DENDY.
It seems, therefore, that the breeding season lasts through-
out practically the whole of the spring and summer.
According to Mr. Orton, who kindly allows me to make use
of information about to be published in the ‘Journal of the
Marine Biological Association’ (1914), there are really two
breeding seasons at Plymouth for Grantia compressa. In
June embryos are discharged from large specimens (which sub-
sequently disintegrate). These embryos develop into indi-
viduals which, while still very small, produce numerous
embryos in October. Mr. Orton has also obtained data sup-
porting the view that the same specimen may breed twice
during its life-history—once in late autumn and again in the
following summer.
(D) THe DisrrrpuTion or THE GERM-CELLS IN THE SPONGE.
As a result of my observations I think I have been able
to establish the fact that Grantia compressa, unlike
certain non-calcareous sponges, such as Oscarella lobularis
(Schulze, 1877), is hermaphrodite, producing male and female
gametes simultaneously. In the case of Sycon raphanus,
Poléjaeff, as far back as 1882, came to the same conclusion,
but considered that that sponge afforded an example of in-
complete sexual separation, some individuals being predomi-
nantly male and others predominantly female. The former
were extraordinarily rare, but produced an immense quantity
of spermatozoa, as well as numerous ova. The latter pro-
duced very few, or even (apparently) no spermatazoa, but a
large number of eggs.
It is quite possible that the same relations may exist in
Grantia compressa, but if so I have never been fortunate
enough to find the predominantly male individuals. All
that I have examined appear to be predominantly female,
the sperm-morulz occurring only in comparatively small
numbers.
Poléjaeff derives both the sperm-morule and the ova (in
Sycon) from ordinary amceboid wandering cells, and figures
GAMETOGENESIS OF GRANTIA COMPRESSA. 323
them scattered in the mesogloea, apparently without any
special arrangement.
Goérich (1903) also accepts the usual views as to the origin
of both male and female germ-cells from amcebocytes, but he
states that in Sycon raphanus ova are produced in the
lower two thirds and sperm-cells in the upper third of the
sponge. He has not, however, followed the spermatogenesis
beyond its earliest stages. The only generalisation that |
can make about the distribution of the germ-cells in Grantia
compressa is that the younger parts of the growing sponge,
towards the osculum, only contain immature germ-cells, exactly
as might be expected.
It may be admitted that in Grantia compressa also the
germ-cells, both male and female, can be traced back to
amoeboid wandering cells, but, according to my own obser-
vations, these amcebocytes can, in their turn, be traced back
to collared cells of the gastral epithelium lining the flagellate
chambers. Presumably any collared cell may become directly
transformed into a primary odgonium or spermatogonium,
losing its collar and flagellum and becoming ameeboid. The
evidence for this statement will be presented in the next
section.
Having become ameeboid, the young germ-cells are free to
wander about. The primary odgonia first migrate into the
mesogloea from the gastral epithelium, and later on, when
fully grown, they migrate back into the chambers, where
they undergo repeated division and give rise to small odcytes.
The young oécytes remain, feeding and growing, for some
time in the chambers; then they migrate once more into the
mesogloea, where they undergo enormous growth, followed by
maturation and fertilisation.
The migrations of the spermatogonia appear to be of a less
extensive character. I have reason to believe that they may
migrate into the mesogloea and there become provided with
their cover-cells or spermatocysts, but they appear to spend
most of their existence in, or attached to, the walls of the
fla ellate chambers.
324. ARTHUR DENDY.
It is easy to observe, in hand-cut sections of living specimens
in the early part of the breeding season, that the flagellate
chambers contain large numbers of amcebocytes hanging, as
it were, from their walls. These are, for the most part,
germ-cells of both sexes in various stages of growth, although
other amcebocytes may also occur in the chambers.
There is thus no localisation of the germ-cells in Grantia
compressa, nothing that can be spoken of as gonads,
neither ovaries nor testes. Just before undergoing matura-
tion, however, the relatively enormous ova withdraw their
pseudopodia and round off, each one taking up a definite
position behind the gastral epithelium of an adjacent chamber,
and causing the layer of collared cells to bulge out into the
chamber. Here fertilisation and the earlier stages of de-
velopment take place, the embryo becoming surrounded by
an endothelial capsule, derived from the mesoglcea, during the
latter process. Finally the ciliated amphiblastula breaks
through the layer of collared cells into the chamber cavity,
and is discharged into the sea through the central gastral
cavity and vent.
The sperm-morule are also discharged into the flagellate
chambers, and doubtless find their way out through the vent.
I have found them, not only in the chambers, but also
adhering to the outer surface of the sponge and in the
inhalant canals, though, except in the chambers themselves,
only in very small numbers. I have also found some evidence
of their breaking up into spermatozoa in an inhalant canal.
There can be little doubt that fertilisation is effected by
spermatozoa which enter the sponge (perhaps as sperm-
morulz) through the dermal pores with the inflowing stream
of water, but whether these spermatozoa are derived from
the same sponge as the eggs which they fertilise, or from
another individual, would seem to be a matter of pure
chance.
In a paper on the ‘Anatomy of Grantia labyrinthica,
etc.,”’ published in 1891, I expressed the opinion that the ova
migrated through the walls of the inhalant canals and were
“<>
a
re
j
;
GAMETOGENESIS OF GRANTIA COMPRESSA. 325
fertilised while suspended in the inflowing stream of water,
subsequently migrating back to undergo their development
in the mesoglea. I certainly did observe amcebocytes sus-
pended in this position, but I now realise that they were far
too small to be mature ova, and must unreservedly withdraw
my interpretation of the observation. It is evident that
maturation and fertilisation (in Grantia compressa) both
take place after the ovum has taken up its definitive position
in the mesoglcea behind the gastral epithelium. Possibly the
spermatozoon has to penetrate a thin layer of dermal epithelium
and mesogloea in order to reach the ovum, or perhaps the
presence of the enormous ovum causes some rupture in the
wall of an adjacent inhalant canal. It is impossible to say
exactly what takes place.
One more point may be mentioned in this section, and that
is the tendency of particular stages of gametogenesis, or at
any rate of odgenesis, to occur in large numbers in certain
specimens, or in certain parts of the sponge, while more or
less completely absent from others. Instances of this pheno-
menon will be given in the followimg pages; it seems to
indicate that the odgonia are produced in successive crops
which go through their developmental stages synchronously.
(e) THe RELATIONS BETWEEN THE DirrerENT TissuE ELEMENTS.
There can be no doubt that the tissues of sponges are far
less definite and less permanent than those of typical Metazoa.
Without going back to the old view that the sponge is nothing
more than a colony of Protozoa, which seems to be negatived
by the degree of histological differentiation that they exhibit
and by the facts of sponge embryology, we may safely say
that the individual cells of which the sponge is composed
often exhibit a remarkable power of changing their relative
positions and also a high degree of polymorphism. Thus it
will be remembered that Minchin (1898) has shown that the
cells (scleroblasts) which secrete the triradiate spicule-systems
in calcareous sponges migrate into the mesoglwa from the
326 ARTHUR DENDY.
dermal epithelium (“ectoderm”), and that the porocytes in
Leucosolenia, when the sponge contracts, migrate through the
gastral epithelium and fill up the central cavity (Minchin,
1900). It is also well known that the epithelial cells of
the so-called ectoderm are highly contractile and capable of
great change of shape, and Maas has shown (1900) that in
the developing Sycon the epithelial cells lining the central
gastral cavity are derived by immigration from the dermal
epithelium on the outer surface of the sponge.
In the case of Grantia compressa it is hardly possible to
speak of permanent tissues at all. According to my observa-
tions any of the constituent cells cf the sponge may become
amceboid and wander off to some new situation. This may
very easily be demonstrated for the collared cells by examin-
ing teased preparations of the living sponge in sea-water,
when the collared cells can be seen putting out long, hyaline,
finger-shaped pseudopodia in an extremely characteristic
manner.! The fact that the collar may still be present along
with the pseudopodia, as shown in fig. 1, affords unmistakable
proof of the origin of these amceboid cells in teased prepara-
tions. In stained sections we sometimes see something of
the same kind, and fig. 2 represents a collared cell sinking
into the mesogloea from between its fellows of the gastral
epithelium. In this case pseudopodia and flagellum are seen
to be present simultaneously but the collar is not visible. In
fig. 3 an amcebocyte, probably derived from acollared cell, but
apparently without collar, flagellum and pseudopodia, is seen
lying in the mesoglcea behind the gastral epithelium.
Appearances such as are represented in figs. 4,6 and 7
also indicate very clearly that the cells of the so-called ecto-
derm are not only contractile, but may become converted
into amcebocytes and wander off into the mesogloea. In fig.
4 is shown one of the epithelial cells lining the central gastral
cavity in the contracted or “ flask-shaped ” condition (a), and
in the subjacent mesogloea a typical amcebocyte (b). Fig. 6
shows how such amcebocytes may be directly derived from
1 Compare Carter (1875, p. 22) for a similar observation.
GAMETOGENESIS OF GRANTIA COMPRESSA. o27
epithelial cells which have migrated inwards, the identity of
the two being clearly indicated by the presence of the
numerous darkly stained granules that characterised the
epithelial cells of the central gastral cavity, at any rate in
this specimen. Fig. 7 shows a similar relation between the
much less granular epithelial cells and amcebocytes around an
inhalant canal.
The mesoglcea of Grantia compressa contains, of course,
a large number of amcebocytes, and there is no need to
suppose that all of them are merely amceboid phases of either
collared or pavement epithelial cells (compare figs. 58-61).
Sometimes small amcebocytes may appear to form connective-
tissue networks of stellate cells, but I doubt very much if they
really do so, at any rate more than temporarily, and even in
this condition they bear such a close resemblance to the
epithelial cells lining the inhalant canals that I entirely fail
to see how they can be distinguished cytologically.
Under these circumstances it is, of course, quite impossible
to say what cells of the adult sponge are derived from each
of the cell-groups recognisable inthe larva. It also seems quite
inadequate to say that the germ-cells are derived from
wandering cells in the mesoglcea.
The one constant and characteristic feature about sponge
histology is, of course, the collared cell, and that is only
constant in the sense that its typical form is that which
possesses collar and flagellum. The sponge is, after all, not
very much more highly advanced in organisation than a
colony of choano-flagellate Protozoa. In such a colony we
should certainly, I think, expect the germ-cells to be derived
from collared cells, either directly or indirectly through an
amceboid phase, and in a later section I hope to be able to
show that this is how they actually originate in sponges.
There is one feature about the collared-cells which appears
to have attracted but little attention from sponge histologists
but which deserves notice in this connection. I refer to the
accumulation in them of what appear to be granules of reserve
food-material. I have observed these as a very constant
328 ARTHUR DENDY.
feature in sections stained in a variety of ways, as polygonal
bodies scattered more or less abundantly in the cytoplasm
(figs. 2, 3, 8, etc.). They vary much in size and in the
intensity with which they stain. In sections of material
(spec. 23) fixed in absolute alcohol and stained with borax-
carmine followed by picro-indigo carmine they are distinctly
recognisable, and stain a pale, greyish colour. In sections of
Flemming material without further staining they can easily
be detected, though only very lightly stained.
They are quite distinct in Flemming material stained with
iron-brazilin, but, I think, more so after counter-staining with
picro-indigo carmine, when they appear of a dark grey colour
(spec. 11). They are hardly affected by nuclear stains and
are evidently not chromidial in nature. The depth to which
they stain exhibits a curious variation in some cases, as will
be seen by reference to fig. 8, from a section of material (spec.
21) fixed in strong Flemming and stained with paracarmine and
picro-indigo carmine, where variation in this respect is visible
even in one and the same cell. They are usually most
abundant in the lower part of the cell (figs. 3, 8).
These observations on their staining reactions appear to
me to be quite in harmony with my view that the bodies in
question are ‘‘reserve granules.” The question next arises,
Are such granules characteristic of the collared cells, or do they
occur also in the other tissue-elements? We have already
had occasion to notice the presence of numerous granules
in the epithelial cells lining the central gastral cavity and
in the amcebocytes derived from these (fig. 6). ‘These granules,
it will be observed, are of a different character from those now
under consideration, being smaller and more highly refractive.
My observations lead me to believe, however, that granules
similar to those in the collared cells may also occur in the
epithelial cells both of the gastral surface and of the inhalant
canal system, though perhaps less abundantly. They
certainly occur in many of the amcebocytes, and I had at first
hoped that their presence might have served as a means of
distinguishing those amcebocytes that originate from collared
GAMBTOGENESIS OF GRANTIA COMPRESSA. 329
cells from those that do not. I fear, however, that this hope
must be largely abandoned, though the granules in question
may perhaps serve as supplementary evidence of origin in
certain cases.
Another point to be noticed in connection with the collared
cells concerns the condition of the nucleus. Usually in my
sections this appears to be darkly and almost uniformly
stained, but frequently it exhibits a distinctly reticulate
character, with small, scattered, chromatin granules at the
nodes of the reticulum, while intermediate conditions occur
between the two extremes. As all conditions may occur close
together in the same preparation it is difficult to account for
the differences, but such variations have to be borne in mind
in considering the origin of the germ-cells as indicated by
nuclear characters.
Miss Muriel Robertson and Prof. Minchin (1910) have
given an account of the division of the collared cells in
Cilathrina coriacea, and Miss Robertson (1911) has dealt
with the corresponding phenomena in Grantia compressa
and Sycon sp. Though figuring the collared cells in detail,
neither of these: authors refer to the “reserve granules,”
which I find to be such a constant feature in Grantia. This
is perhaps due in part to the fact that their preparations
were stained especially with a view to demonstrating the
phenomena of mitosis. They figure much the same variations
in the appearance of the nuclei of the collared cells as I have
seen, but I cannot agree with their views as to the general
occurrence of a single, relatively large karyosome (nucleolus).
I have myself only occasionally seen such a body in these
nuclei and do not attribute any special importance to it.
Alike in Clathrina, Sycon and Grantia the division of the
collared cells was found by Robertson and Minchin to take
place longitudinally and to be accompanied by a typical
mitosis. In Clathrina the number of chromosomes was found
to be “ about sixteen,” but in Grantia and Sycon the chromo-
somes are said to be “ not very distinct,” and the number is
not given. Miss Robertson’s figures, however, especially
voL. 60, PART 3.—NEW SERIES. 24
330 ARTHUR DENDY.
fig. 12 on Pl. 19, suggest eight or ten as the number rather
than sixteen. I have myself hardly ever observed mitosis in
the collared cells, but my fig. 9 represents a probable case,
and it will be seen that so far as my very limited observations
go they agree with those of Miss Robertson.
I do not, of course, profess to give a complete account of
the collared cells in this place; much more might be said
about them, but the only thing needful here is to emphasise
those points which have a direct bearing upon the problem of
the origin and maturation of the germ-cells.
(r) OOGENESIS.
(a) Historical.
By far the most complete account that has yet been given
of the odgenesis in any sponge is contained in Dr. Max
Jérgensen’s memoir (1910). As this account refers to a type
(Sycon) closely related to Grantia compressa, I propose
to give a brief review of Jérgensen’s results before passing on
to describe my own.
As already noticed, this author derives the primary o6gonia
from so-called mesoderm cells, either resting stellate con-
nective-tissue cells, or amcebocytes, between which he con-
siders that no essential difference exists. The mesoderm cells
multiply mitotically and become converted into odgonia of the
first order. These increase in size and presently wander into
the flagellate chambers, where they undergo mitosis and divide
into odgonia of the second order. The latter divide again
into oédcytes. The two o6dgonial divisions are said to be
“atypical,” but quite similar to one another. During the
mitosis eight chromosomes make their appearance “in Form
von 'l'etraden”’ ; these are believed to be formed by fusion of
several segments of a segmented spireme. Lach “ tetrad” is
figured as dividing into two tetradiform daughter-chromo-
somes on the spindle.
Although the division of the oégonia frequently occurs in
GAMETOGENESIS OF GRANTIA COMPRESSA, 331
the flagellate chamber, the author evidently regards this as a
more or less accidental circumstance, which takes place only
if the 6ogonia have not got room enough to divide in the
mesogloea. I think myself that the migration of the odgonia
into the chambers before dividing must have a deeper signifi-
cance than this.
After their divisions are over the daughter-cells of the
oégonia wander back into the mesoglcea as young oocytes.
Here they undergo a short resting stage, and then they pass
through the prophases of what must apparently be regarded
as the mitosis belonging to the first maturation division. A
long spireme is formed which arranges itself in a characteristic
“bouquet ”’ form, and even shows a contraction phase similar
to synapsis, though this may be due to the action of reagents ;
at the same time “chromidia” appear in the cytoplasm,
probably ejected from the nucleus. A large nucleolus is like-
wise present, but this is also a characteristic feature of the
odgonia. Some of these young odcytes now degenerate,
possibly furnishing nutrient cells for the older odcytes. In
others the spireme breaks up again into chromatin granules,
and the odcyte continues its growth.
In the later stages the oOcyte increases enormously in size
and puts out long, branching pseudopodia. When the
growth of the cytoplasm has reached its completion the
nucleus enters upon the so-called “critical stage,” in which
the nucleolus has completely disappeared and the nucleus is
almost entirely devoid of chromatin. Apparently the chro-
inatin has migrated through the nuclear membrane into the
surrounding cytoplasm, where it is represented by “ chro-
midia.” Chromatin-granules and nucleolus now reappear in
the nucleus, which becomes very large and vesicular. Then
a second diminution in the amount of chromatin takes place,
this time apparently effected by solution or absorption within
the nucleus itself and not by extrusion into the cytoplasm.
The nucleus, with its diminished quantity of chromatin
arranged once more in the form of tetrads, approaches the
surface of the odcyte, which has in the meantime rounded
Bo2 ARTHUR DENDY.
itself off. The nuclear membrane now disappears, and the
eight tetradiform chromosomes, now much diminished in
size, arrange themselves on the equator of the first maturation
spindle. Each chromosome divides into two daughter
tetradiform chromosomes and the mitosis is completed in the
ordinary way. Then the first polar body, containing eight of
the daughter-chromosomes (‘tetrads’’), is twisted off from
the surface of the odcyte in a very characteristic manner. A
second maturation division now takes place and eight chro-
matin elements finally remain in the fertilised egg, eight
having passed out into the second polar body, but the forma-
tion of the second polar body was not fully observed.
It is difficult to understand completely the author’s views
as to the manner in which the reduction of the chromosomes
is carried out, and, indeed, although he discusses the problem
at some length, he does not profess to have come to any
certain conclusions: ‘ Leider ist mein Objekt zu klein und
ungiinstig, um diese wichtige Frage sicher zu entscheiden.”
At any rate he evidently considers that the somatic number
of chromosomes is sixteen and the reduced number eight, for
he finds sixteen in the segmentation nuclei of the embryo.
The most curious thing appears to be that the number is
already reduced to eight in the odgonial mitoses, but it is
suggested that this may be a pseudo-reduction. Possibly it
is connected with the tetrad formation which is supposed to
take place at this stage.
The author describes an interesting process of nutrition of
the growing odcyte by ingestion of nutrient cells. I have
observed a somewhat similar phenomenon myself in the case
of Grantia and shall discuss it at length later on.
As regards the fertilisation of the ovum and the subse-
quent segmentation stages the most interesting feature
appears to be the splitting up of the pronuclei and segmen-
tation nuclei into karyomeres, a phenomenon which I have
also observed in Grantia.
GAMETOGENESIS OF GRANTIA COMPRESSA. 333
(b) Origin and Growth of the Primary Oégonia.
Before proceeding to describe the origin and growth of the
primary odgonia as observed in Grantia compressa ib
may be well to point out the great difficulties that arise with
regard to the problem of seriation in the earlier stages of
odgenesis. These difficulties are accentuated by the absence
of definite localisation of the germ-cells and the consequent
mingling of different stages in the mesoglcea or in the chambers,
so that it is often impossible to be certain even to which
generation a particular cell belongs. We can only fit the
different observed stages together in what seems to be the
most probable order on the sometimes scanty evidence avail-
able.
In the later stages of growth of the odcyte there is less diffi-
culty, because the size of the cell not only shows that it is an
odcyte, but indicates at the same time its place in the series.
Inasmuch as amceboid cells of various sizes frequently
immigrate into the chambers and come to he between the
collared cells, the mere fact of the occurrence of a young
germ-cell in such a position affords no conclusive evidence
that it has been derived from a collared cell. The only way
in which such an origin can be demonstrated, as it seems to
me, is by finding cells which exhibit the characters of young
germ-cells while still retaining the collar or flagellum, or
both, of the collared cells. It must be admitted that it is not
often that such intermediate forms are met with, and it is
obvious that they can only be hoped for in very carefully pre-
pared sections. I have, however, seen a few such cases,
which seem to me to place my conclusions almost beyond
doubt. Figs. 10, 11, and 12 are all taken from sections of
specimen 21, stained with paracarmine and picro-indigo car-
mine. In Fig. 10 two collared cells are represented side by
side. In both the collar and flagellum are clearly visible, and
in one the body of the cell is already considerably enlarged,
apparently by the accumulation of reserve material. This
enlarged collared cell I take to bea primordial germ-cell, but
334 ARTHUR DENDY.
whether it would have turned into an o6gonium or a spermato-
gonium cannot be decided. The appearance of a vacuole
around an unusually large granule of reserve material, however,
suggests to my mind the latter.’
Figs. 11 and 12 represents a later stage, in which the body
of the cell is greatly distended with reserve granules similar to
those which occur in the ordinary collared cells, and the remains
of collar and flagellum are, unless J am mistaken in my inter-
pretation of the appearances, still visible. The nucleus has
retreated to about the middle of the cell and a distinct
nucleolus or karyosome has appeared. The whole cell pro-
jects conspicuously beyond its neighbours into the cavity
of the flagellate chamber, and there is a clear indication
of pseudopodium formation represented in fig. 12, the
pseudopodium being formed by a drawing out of the proximal
end of the cell between the adjacent collared cells. (An
apparent pseudopodium shown in fig. 11 may belong to
another cell not seen in thesection.) In fig. 11 it will be seen
that the young odgonium les actually next to the exhalant
aperture of the chamber, and the one represented in fig. 12
also lies close to an exhalant aperture, but I have not sufficient
evidence to show whether or not there in any constancy in
this position.
The primary odgonia appear to leave the layer of collared
cells very soon after their origin and migrate into the
mesogloea (fig. 13). The collar and flagellum completely dis-
appear and pseudopodia are put out (fig. 14). The nucleus
at first appears uniformly stained except for the large
nucleolus, but presently small granules of chromatin appear
scattered between the nucleolus and the nuclear membrane,
though the nucleolus appears to be surrounded by a narrow
ring free from granules (fig. 15). The reserve granules are
still abundant and easily recognisable in the cytoplasm.
The primary o6gonium now appears to round itself off more
or less completely before entering upon mitosis, as shown in
‘ Compare the account of the origin of the primary spermatogonia,
later on.
GAMETOGENESIS OF GRANTIA COMPRESSA. 335
fig. 16. In specimen 22 the mesoglcea between the chambers
is crowded with o6gonia in this condition, and their numbers
suggest that it may be a resting state. It will be seen that
the cytoplasm is only slightly granular, but may still contain
reserve material in the form of polygonal granules. The
nucieus is faintly reticulate, and there is a very large and con-
spicuous nucleolus and a thin nuclear membrane.
(c) Multiplication of the Odgonia.
Having reached the stage represented in fig. 16 the odgonia
begin to prepare for division, which is effected by mitosis.
During the progress of this mitosis they migrate through the
layer of collared cells into the flagellate chambers (fig. 21),
where the cell-division actually takes place. Although the
earlier stages of the mitosis are found in the mesoglea (figs.
17, 18), I have never seen the actual division taking place
except in the chambers themselves, and cannot therefore
agree with Jorgensen that the odgonia only migrate into the
chambers when they are short of room in the mesoglea. This
view is not in harmony with the fact that the fertilised eggs,
which are many times larger than the odgonia, manage to
find room for their development in the mesogloea by pushing
out the gastral epithelium without rupturing it. I therefore
think that there must be some special reason for the migra-
tion of the odgonia into the chambers. I would suggest that
it may enable the young odcytes to find abundant nutriment
in the chambers in the first instance, and subsequently to dis-
tribute themselves more readily throughout the sponge by
creeping along inside the walls of the chambers and re-enter-
ing the mesoglea at various points.
Jorgensen is of opinion that there are two generations of
oogonia in Sycon, and the large number of odcytes present
seems to indicate that there must be at least two odgonial
divisions in Grantia. Further evidence of this is to be found
in the small size of the youngest odcytes as compared with
the daugchter-cells formed by division of the primary odgonia
(compare figs. 29 and 26).
336 ARTHUR DENDY.
Before proceeding to describe the mitosis of the primary
odgonium it 1s necessary to say a few words with regard to
the character of the chromatin substance in the nucleus. The
growing odgonium contains, as we have seen, a large spherical
nucleolus or karyosome (fig. 14, etc.), which stains very darkly,
and in its later stages at any rate minute granules of chro-
matin may also appear in the nucleoplasm (fig. 15). During
the prophases of mitosis all the darkly staining granules of
chromatin disappear from the nucleus, while the nucleolus
may be cast forth from the odgonium altogether (figs. 17,
18). There remains behind a quantity of more lightly stain-
ing chromatin which forms the spireme, and, subsequently, the
chromosomes. It is, I think, impossible to avoid the conclusion
that there are here two totally different kinds of chromatin, for
which we may accept the terms “trophochromatin ” and
‘“jdiochromatin ” respectively, the former being concerned in
the metabolism and growth of the cell and the latter in the
processes of reproduction. The trophochromatin is represented
chiefly by the nucleolus. Small granules of chromatin are
possibly cast out into the cytoplasm as chromidia, for there is
some evidence that chromidia may be found at this stage, but
they are nothing like so conspicuous as they are in the
oécyte. In the latter the chromidia are, of course, supposed
to be concerned in yolk-formation, which has hardly com-
menced in the oégonium. The large nucleolus appears to be
bodily cast forth from the odgonium during mitosis (fig. 18).
It can, therefore, hardly be supposed to be directly concerned
in yolk-formation at this stage. It may possibly be of a
different nature from the chromidia, and represent a mass
of waste products accumulated in the nucleus during the
growth and metabolism of the odgonium. In the young
odcyte, however, as we shall see shortly, the nucleolus
definitely gives rise to chromidia (“yolk nucleus ”), while in
the maturing odcyte it appears to undergo degeneration and
absorption in the cytoplasm.
Jorgensen gives an interesting discussion on the nature and
behaviour of the chromatin in the odcytes of Sycon, to which
GAMETOGENESIS OF GRANTIA COMPRESSA. 387
I must refer the reader, but he appears to have paid very
little attention to this question so far as the odgonia are con-
cerned, and does not seem to have observed the bodily ejection
of the nucleolus.
We may also say a few words here with regard to the
formation of the chromosomes. Jérgensen figures both
spireme and chromosomes as being stained perfectly black—
as black, in fact, as the chromidia. His material, like mine,
was fixed in Flemming’s solution, and he used iron-hzema-
toxylin for staining (controlled by borax carmine and
saffranin preparations). My own preparations were for the
most part stained with iron-brazilin, but I have also used
iron-hematoxylin. There is, of course, a good deal of
variation in the results obtained by either of these methods,
but my experience is that, as a rule, the spireme thread and
chromosomes stain comparatively lightly as compared with
the nucleolus and chromidia. I have never seen the chromo-
somes so sharply defined as Jorgensen figures them, and I
have seen nothing of the so-called tetrad formation which he
describes during the odgonial mitoses and in the maturing
oocyte. The chromosomes have always appeared to me much
more like those figured by Prof. Minchin and Miss Robertson
for the dividing collared cells—small subspherical or irregular
bodies, so crowded together and ill-defined that it is impos-
sible to count them accurately. The number characteristic
of the odgonia appears to be about eight, as will be seen by
reference to figs. 19-23, and this is possibly the somatic
number (compare fig. 9).
After these preliminary observations the actual division of
the primary odgonia may be described very briefly. The
prophases of the mitosis occur while the odgonium is still
lying in the mesoglcea outside the chamber which it is about
to enter, and while it is in a more or less amceboid condition
(figs. 17, 18). Whether or not there is any casting out of
chromidia into the cytoplasm I am not certain, but as a few
small, densely staining bodies resembling chromidia sometimes
appear in the cytoplasm in later stages of this mitosis, it
338 ARTHUR DENDY.
seems not unlikely that such may occasionally be the case.
Apart from the nucleolus, however, there is very little
chromatin in the nucleus to be cast out.
The large vesicular nucleus approaches the surface of the
odgonium until it is bounded on the outside by only a very
thin layer of cytoplasm. In the meantime a spireme thread
makes its appearance, and the nucleolus also approaches the
surface (fig. 17). The nuclear membrane disappears, and
the nuclear sap merges into the cytoplasm. A very curious
phenomenon now takes place, the nucleolus being expelled,
not only from the nucleus, but from the odgonium. Fig. 18
shows it in the process of extrusion, surrounded by a drop of
nuclear sap. ‘The pear-shaped form of the nucleolus at the
moment of extrusion suggests that it must be a very soft,
perhaps a semi-fluid, body in life. I have only seen this
phenomenon exhibited very rarely in what can be considered
as at all a conclusive manner. I have frequently seen the
nucleolus apparently cast out of the odcyte, but minute
inspection shows that this is (? always) an artificial result
brought about in the act of cutting the sections. During the
process of fixation, etc., the nucleolus appears to become very
hard, and the knife then tears it bodily out of the odcyte.
In the case represented in fig. 18, however, I think there can
be no question of the normality of the process of extrusion ;
indeed, that the nucleolus must be extruded at this stage
seems to be indicated by its complete absence in later stages
of the mitosis.
It is extremely difficult to determine whether a particular
spireme stage under observation belongs to an odgonial or to
au odcyte mitosis. Jam inclined to think, however, that in
the former case there are few or no chromidia, while in the
latter the chromidia are fairly strongly developed (cf. figs.
44,45). I must admit again, however, that the sorting out
of these stages is to a large extent arbitrary.
The spireme thread now breaks up into chromosomes, a
typical spindle is formed with a minute centrosome at each
pole, and the chromosomes arrange thems:2lves in the usual
GAMETOGENESIS OF GRANTIA COMPRESSA. 339
“equatorial plate” (figs. 19, 20). The chromosomes now pre-
sumably divide, though I can hardly claim to have seen the
actual division (cf., however, fig. 21), and the two groups of
daughter-chromosomes migrate towards the two centrosomes
(figs. 22, 28). The spindle disappears, and we are left with
two closely aggregated groups of chromosomes as the founda-
tions of the two daughter-nuclei (fig. 24). Constriction of the
cytoplasm between these two daughter-nuclei now follows
(fig. 25), and finally the entire odgonium becomes divided
into two daughter-cells (fig. 26).
Apparently not until the prophases have been passed
through does the odgonium migrate through the layer of
collared cells into the adjacent flagellate chamber, as shown
in fig.21. Here it rounds itself off, usually into an oval form
(figs. 19, 20), before completing the mitosis. At this stage
the cytoplasm exhibits a fairly uniformly and densely granular
character, shown especially well in fig. 22. A few small
densely staining granules, resembling chromidia, are some-
times visible in it (fig. 19), and may even persist in the
daughter-odgonia after completion of the division (fig. 26).
It seems almost certain that a second odgonial division
takes place in the flagellate chambers very shortly after the
first one. Fig 27 represents a stage which I interpret as an
odgonium of the second generation with reconstituted nucleus,
and fig. 28 represents what I take to be such a secondary
oégonium in mitosis. Without the intervention of such a
stage it would be difficult to explain the origin of the next
series of stages (figs. 29-39), which occur very abundantly
in the chambers, and which I interpret as young oocytes.
With regard to this period of the odgenesis my conclusions
differ widely from those of Jorgensen, who makes his odgonia
of the second order larger than those of the first order, and
his young odcytes larger still, which would be very difficult
to understand in view of the repeated division, and the
apparent absence, so far as his account goes, of any process
of nutrition.
340 ARTHUR DENDY.
(d) Growth and Feeding of the Young Odcytes in
the Flagellate Chambers.
The growth of the odcytes may.be divided into two very
distinct periods, during the first of which they are found in
the flagellate chambers, while during the second they lie in
the mesogloea between the chambers.
As already indicated, it is by no means an easy matter to
sort out all the very numerous amceboid cells that occur in
the flagellate chambers, some representing stages in oégenesis
and others stages in spermatogenesis, into their proper cate-
gories. Amongst them, however, may be distinguished a type
which occurs very abundantly and exhibits certain peculiarities
by which it is more or less readily recognised. The cells in
question are small and distinctly amceboid, of irregular form,
and usually attached to the wall of the chamber by pseudo-
podia. They have a rather small nucleus, with a relatively
large nucleolus surrounded by a narrow clear space, and then
by a broad ring of minute granules extending to the nuclear
membrane. The cytoplasm usually exhibits more or less
numerous inclusions which may be surrounded by vacuoles.
Some of these inclusions, which I interpret as chromidia or
yolk-nuclei, stain nearly black, others may stain much more
lightly, and look like food-particles undergoing digestion. A
typical series of these curious cells is shown in figs. 29-39.
I interpret them as young odcytes engaged in feeding opera-
tions.
Figs. 29 and 30 show what appears to be the youngest
stage of this series, a stage which may be derived from the
mitotic division of a secondary odgonium such as is repre-
sented in fig. 27 or 28. The cytoplasm at this stage is seen
to be uniformly and rather coarsely granular and contains no
chromidia or other inclusions of any kind. The structure of
the nucleus even at this early period, with its large nucleolus
and broad zone of chromatin granules distinctly separated
from it, appears to me to be essentially that of an immature
female gamete. Presently the characteristic inclusions make
—————— =
ee
GAMETOGENESIS OF GRAN'ITTA COMPRESSA. 341
their appearance in the cytoplasm, which otherwise may come
to exhibit a more homogeneous appearance (figs. 31-39).
Some of these inclusions are comparatively lightly staining
bodies enclosed in vacuoles (figs. 31, 56, 37), and in one case
(fig. 89) a nucleated cell was distinctly recognised amongst
other bodies. I therefore believe that the lghter coloured
inclusions are food-particles undergoing digestion, captured
by the young odcytes from the stream of water that flows
through the chambers.
The nature of the intensely black stained bodies that
appear in the cytoplasm is easily interpreted. These
resemble the nucleolus in appearance, but may be much
larger (figs. 33-35). They may or may not be surrounded
by distinct vacuoles. On the other hand, they may take
the form of small granules or groups of granules (figs.
35, 36). Fig. 38 gives the clue to the manner in which
they are formed, for here the nucleolus is seen actually
discharging part of its own substance into the cytoplasm
through the nuclear membrane. I think there can be no
doubt that in these cells very active metabolism, accom-
panied by the formation of chromidia, or “ yolk-nuclei,” is
going on, and that the bodies in question are of this nature,
and are concerned in the elaboration of yolk-granules in the
cytoplasm.
Apparently feeding now ceases and the remains of food-
particles disappear from the cytoplasm. The odcyte next
migrates through the chamber wall into the mesogloa (fig.
40).
(e) GrowrH AND FrepinG oF THE OocyrEs IN THE MesocLaa.
The formation of chromidia, or “ yolk-nuclei,” by extrusion
of matter from the nucleolus, which was already commenced
within the chambers (fig. 88), may now be continued very
freely. Specimen 23 contains an immense number otf odcytes,
lying in the mesogloea between the chambers, in which this
chromidium-formation is going on (figs. 41, 42, 43), and often
giving rise to very fantastic appearances. ‘The nucleolus is
342 ARTHUR DENDY.
evidently in a liquid or semi-liquid state and appears to be
squeezed out into the cytoplasm in threads or drops, Just as
an artist’s colours may be squeezed out of their tubes. During
this process the nucleus itself may become distinctly pear-
shaped (fig. 43). The drops squeezed out into the cytoplasm
are at first enclosed in distinct vacuoles, apparently derived
from the nuclear sap. In these vacuoles they disintegrate
into granules (fig. 43), which probably became scattered
through the cytoplasm. The nuclear membrane may become
very indistinct during the process, and there are indications
that the nucleus is passing into the spireme stage to be des-
cribed next. It is doubtful whether the nucleolus is ever
completely eliminated from the nucleus at this stage. It
seems to me more probable that some of it always remains
behind as the foundation of the huge nucleolus which forms
such a conspicuous feature in later stages of the odcyte. The
irregularity in shape of the odcyte during this process of
chromidium formation indicates that it is still amoeboid, and
it seems possible that the extrusion of the chromidia may be
due to strong contraction of the cytoplasm, though it must be
admitted that the mechanism of the process is very obscure.
The whole of the series of stages representing the feeding
and growth of the young odcytes in the chambers and the re-
markable process of chromidium-formation just described
appears to have been unobserved by Jérgensen. It is true that
that observer worked upon Sycon, but it is unlikely that two
such closely related types as Sycon and Grantia should differ
in this respect, especially when they agree so closely as regards
other features of the odgenesis. JOrgensen figures the young
odcyte, supposed to be directly derived from the last o6gonial
division, as being very similar to the stage represented in my
fig. 47a (compare his fig. 32). This stage may very easily be
derived, however, through the intermediate stages represented
in figs. 41-43, from the last of the feeding stages observed
in the chambers (fig. 39).
It also seems very probable that shortly after leaving the
flagellate chambers and undergoing the process of chromidium-
GAMETOGENESIS OF GRANTIA COMPRESSA. 343
formation just described, the young odcyte exhibits the pro-
phases of a mitosis which really belongs to the first maturation
division. J6rgensen puts this incomplete mitosis, represented
by a well-marked spireme stage, immediately after the stage
which, in Sycon, probably corresponds to my fig. 47a. I
prefer to place it immediately before this stage, where it seems
to me to fit in better. Figs. 44 and 45 show two of the pro-
phases in question. ‘I'he former is evidently a leptotene phase
and the latter a pachytene. The latter always shows the
characteristic contraction of the spireme described by Jérgen-
sen, which may possibly represent a synapsis or be due simply
to the action of reagents. Both these figures show well-
developed chromidia in the cytoplasm, which, as I have already
said, inclines me to include them in the odcyte series rather
than in the odgonial series, though I do not consider that the
evidence is by any means conclusive. It is obvious from
fig. 45 that the odcyte may still be highly amceboid during
this phase, exhibiting a very irregular outline.
Jérgensen considers that some of the odcytes at this stage
undergo degeneration and may have something to do with
forming the nutrient cells for the older odcytes, but I
have obtained no good evidence of such degeneration and
my observations on the feeding of the older odcytes do not
support this view. The spireme thread now disappears
(fig. 46) and the odcyte rounds itself off, takes up a definite
position in the mesoglcea behind the layer of collared cells
(fig. 47), and enters upon its main period of growth.
Fig. 47a represents a condition of the odcyte which is very
commonly met with. Jorgensen figures a similar condition
in Sycon (fig. 52), and speaks of it as a resting condition, but,
as I have already pointed out, he regards it as the direct
product of the division of an odgonium of the second
generation. I am not quite sure, however, that Jorgensen’s
fig. 32 really represents the same stage as my fig. 47a, for he
does not show, or if so only very faintly, the chromidia in the
cytoplasm, which appear to me to be very characteristic of
this stage. It is by the abundance of these chromidia,
/
344 ARTHUR DENDY.
indeed, that this stage is chiefly distinguishable from the
primary odgonium just before mitosis, as represented in fig. 16,
taken in conjunction with the fact that the latter occurs
especially in association with the odgonial mitoses going on in
the chambers, while the former occurs especially associated
with the later stages of odcyte growth.
In Grantia the young odcyte, passing out of this “ resting
condition,” simply flattens out somewhat on one side (fig. 47b),
and puts out long branching pseudopodia by means of which
it attaches itself to the mesoglceal surface of the layer of
collared cells, as shown in fig. 48. Whether these psendo-
podia are merely “ anchoring ”’ pseudopodia, or whether they
also serve to extract nutriment for the growing odcyte
from the collared ceils with which they are in contact, must
for the present remain an open question. The appearances
represented in fig. 48, however, suggest to my mind the
latter. We are reminded of the manner in which the super-
ficial cells of the embryo of Stelospongus attach themselves to,
and evidently draw nutriment from, the large capsule-cells
by which they are surrounded, as described by me many
years ago (Dendy, 1888). It will be seen that the collared
cells contain abundant “reserve-granules,’ and _ similar
granules appear in the odcytes, while the chromidia have
entirely disappeared from the cytoplasm. The nucleus is
distinctly reticulate, with numerous darkly stained chromatin
granules and a large nucleolus, the latter surrounded by a
clear space (possibly due to shrinkage ?).
The odcyte continues to increase in size and the nucleus
grows more rapidly than the cytoplasm. Presently we reach
a stage which is very characteristic and very frequently met
with, and which I propose to call the “ contraction stage of
the odcyte.” This is represented in fig. 49. It will be seen
that the entire cell has rounded itself off again and the
pseudopodia have contracted into blunt knobs. The nucleus
is very large and pretty uniformly granular, all or nearly all
the darkly staining chromatin having evidently been expelled
into the cytoplasm in the form of chromidia (yolk-nucleus),
GAMETOGENESIS OF GRANTIA COMPRESSA. 345
which at this period are apparently not derived, at any
rate directly, from the nucleolus. ‘I'he latter is very large,
spherical, and fairly darkly staining. ‘This stage, again, does
not appear to have been observed by Jérgensen in the case
of Sycon.
It is at about this period that the feeding of the odcyte by
means of nurse-cells begins, a process which continues right
on until the odcyte has reached its maximum size or nearly
so, and which will be dealt with separately in a later
section (see, in the meantime, figs. 50, 51, 52).
During this process the odcyte increases enormously in
size, and long, root-like pseudopodia are put out again (fig. 54)
in a plane parallel to the layer of collared cells against which
the odcyte lies. The nucleus assumes the form of an enor-
mous, thin-walled vesicle, containing a reticulum of lightly
staining, flocculent material, which looks very much like a
precipitate or coagulation, and includes small, darkly staining
chromatin granules scattered through it. Most of the darkly
staining chromatin, however, appears to be expelled into the
cytoplasm in the form of chromidia, which may be extremely
numerous (fig. 53). The nucleolus is very large,and frequently
exhibits a differentiation into more and less darkly staining
spheres, which may or may not be concentric (figs. 52, 53,
54). Occasionally the nucleolus appears to be cast out into
the cytoplasm, but, as already stated, I have come to the
conclusion that this is an artificial result, due to the action of
the knife in cutting. Sometimes the cytoplasm around the
nucleus exhibits a faint radial arrangement of its granules,
as shown in figs. 52 and 54. ‘This is a feature upon which
Jérgensen has laid some stress,in the case of Sycon.
({) Maturation of the Odécytes.
The odcyte now withdraws all its pseudopodia and rounds
itself off into a compact ellipsoid body about 0:045 mm. in
maximum diameter, preparatory to maturation. The huge
vesicular nucleus disappears and its contents are apparently
VoL. 60, PART 3.—NEW SERIES. 25
346 ARTHUR DENDY.
diffused throughout the cytoplasm, which is dense, and, with
the exception of certain inclusions to be mentioned imme-
diately, uniformly and rather coarsely granular. Atno stage
have I seen any vitelline membrane. ‘The cytoplasm contains
a large number of very small chromidia, mostly aggregated
in irregular groups or clouds (fig. 55), which are probably,
in part at any rate, derived directly from the small chromatin
granules that remained in the nucleus at the close of its
career. The immense nucleolus in two cases (out of the very
few met with) was clearly discernible in the cytoplasm,
where it appeared to be undergoing absorption (fig. 55, no.).
In one case a group of small bodies that might be chromo-
somes (fig. 55, chr. ?) was observed in the cytoplasm at some
distance from the nucleolus; but the nature of these bodies
is really uncertain, as also is the nature of a protrusion of the
surface of the odcyte opposite to the supposed chromosome-
group. It is possible that we have here the beginning of the
formation of the first polar body, but I think that that is
extremely doubtful.
Ata slightly later stage, however, the chromosomes appear
unmistakably in connection with the first maturation spindle,
which is represented in fig. 56. It is obvious that this agrees
closely with the first maturation spindle as described by
Jorgensen (cf. his fig. 59), but unfortunately I have only
been able to find one really good maturation spindle in my
preparations, and I am therefore unable to give any details
with regard to the process. I am not even sure of the number
of chromosomes, but there appear to be eight or ten in each
daughter-group represented in fig. 56. Jérgensen makes the
number eight in each group, but represents each chromosome
as a sort of tetrad. From my own observations I can only
say that the chromosomes are small irregular bodies, and it
appears to me that, whenever chromosomes are concerned,
Jérgensen’s figures must be somewhat diagrammatic.
The formation of the first polar body in Grantia is repre-
sented in fig. 57, and it is evident that it takes place very
much as described by Jérgensen for Sycon. The polar body
GAMETOGENESIS OF GRANTIA COMPRESSA. 347
itself is seen to be of large size, and the remains of the spindle
are seen as a very distinct cord, slightly thickened in the
middle, connecting the group of chromosomes in the polar
body with the group that remains behind in the odcyte.
I have searched in vain for the second maturation spindle
and second polar body, but, considering the rarity with which
the first occurs in my specimens, their apparent absence has
no significance. Kven Jorgensen, however, was not able
fully to observe the formation of the second polar body,
though he tells us that apparently it also contains eight
chromosomes, while eight (dyads ?) remain in the mature
ovum.
(g) The Nurse-Cells and their Origin; Phago-
cytosis.
A process of feeding on the part of the growing oécyte at
the expense of certain nutrient cells has been described in
the case of Sycon by both Gorich (1903) and Jorgensen (1910).
Gérich says that the oocytes (“ Hizelle”’) ingest entire cells,
which are probably themselves egg-cells of smaller size. The
process is represented as taking place very much as in the
case of an Amoeba ingesting food-particles, except that no
vacuole is formed around the ingested nutrient cell, whose
protoplasm appears to mingle directly with that of the oocyte.
Hach nutrient cell appears to be asmall nucleated amcebocyte,
with a relatively large, darkly staining particle (? chromi-
dium) in the cytoplasm. Numerous very similar bodies,
evidently chromidia, are figured in the cytoplasm of the
feeding odcyte, which appears to be at about the stage figured
in my figs. 50 and 51 (compare Gorich’s figs. 1-6).
Jorgensen tells us that the ingestion of nutrient cells by
the odcyte takes place towards the end of the growth period,
and goes on right up to the formation of the maturation
spindle. ‘The ingested cells are believed to be mostly odgonia
and the ingestion is supposed to be due to chemotaxis. The
ingested cells are said to be taken into a preformed gullet in
348 ARTHUR DIENDY.
the odcyte, and not, as described by Gérich, by means of
pseudopodia. Jdérgensen has, however, observed ingestion
by means of pseudopodia in the case of Sycon setosum. He
also finds the presence of a compact chromidium in the cyto-
plasm to be characteristic of the nutrient cell. This chromidium
may be taken into the gullet of the feeding odcyte without the
nutrient cell, but usually the entire nutrient cell is taken in.
The chromidia received by the odcyte in this way are distin-
guished by their size from those cast out from the nucleus,
but both undergo like degeneration in the cytoplasm.
I have never seen anything resembling the formation of a
gullet (“Schlund’’) in Grantia, and I think that Gdrich’s
account of the taking in of the nutrient cells in Sycon seems
the more probable of the two.
In Grantia the process is complicated by the intervention
of what I propose to term “ nurse-cells,’’ which capture the
nutrient cells and bring them to the odcyte. Possibly the
supposed taking in by the odcyte of the chromidium only
from the nutrient cell really indicates something of the same
kind for Sycon.
I have observed the feeding of the odcyte by nurse-cells in
specimens 1] and 23 and I have seen many instances of it, and
I do not think there can be any doubt either as to the
observations themselves or as to their interpretation. The
peculiar arrangement shown in figs. 50 and 51 is frequently
met with in my sections. I interpret it as indicating that a
nurse-cell (n. c.) has captured a smaller cell which I regard as
a nutrient cell or food-cell (f. c.), and is passing it into the
cytoplasm of the odcyte. I shall discuss the origin of the
nurse-cell directly ; in the meantime I may point out that it
exhibits certain fairly well-marked characters of its own.
Its cytoplasm is thin-looking and stains very lightly, and the
nucleus is of moderate size, with a well-developed nuclear
membrane and reticulum and a rather small nucleolus. The
cytoplasm usually contains conspicuous inclusions which are
evidently the remains of ingested and partially disintegrated
cells, but nothing that can be identified with the “ chromidium”
GAMETOGENESIS OF GRANTIA COMPRESSA. 349
of the nutrient cells described by Gérich and Jorgensen in
Sycon ; indeed, the nurse-cell does not appear to be itself a
nutrient cell in the sense of these authors. The real nutrient
cell (f. c.) is, however, a very conspicuous object, lying in the
middle between the nurse-cell and the odcyte. It appears
to be always an oval or spherical cell with a reticulate
nucleus and rather dense, finely granular cytoplasm, and it
appears to be passed on to the odcyte by the nurse-cell before
it has undergone any disintegration, for its outlines are
perfectly definite. Judging from its small size and the absence
of chromidia in the cytoplasm I am inclined to think that the
nutrient cell is probably a rounded-off collared cell, but it is
impossible to be certain on this point, though I shall bring
forward evidence presently to show that the nurse-cells do
capture collared cells.
The process of feeding by nurse-cells does not appear to
begin until the odcyte has attained a considerable size, and it
may be fed in this way both in the contracted and in the
expanded condition, 1. e. when the pseudopodia are reduced
to short knobs and when they are fully extended. Figs. 50
and 51 represent the process of feeding by the nurse-cells
as usually observed. A somewhat different condition is
represented in fig. 52. The nurse-cell is here crushed in
between the odcyte, which is now much larger, and the layer
of collared cells against which it lies. The nutrient cell has
passed completely into the cytoplasm of the odcyte, where it
is surrounded by a small vacuole in which it appears to be
undergoing disintegration, for instead of a distinct nucleus
it exhibits two masses of chromatin—one at the middle and one
at the side. Both nurse-cell and nutrient cell appear to be
much smaller than in the cases previously described. The
whole arrangement somewhat resembles the taking in of a
chromidium from a autrient cell as described by Jorgensen,
but it is probably simply a more advanced stage of the feeding
process shown in the preceding figures.
We come now to the question of the origin of the nurse-
cells. I think there can be little doubt that they are derived
350 ARTHUR DENDY.
from small amcebocytes which occur scattered in the mesoglea,
such as is represented in fig. 58. It will be seen that we
have here a cell of about the same size as the nurse-cell shown
in fig. 51, and, except for the absence of cytoplasmic inclusions,
closely resembling it. The cytoplasm is faintly staining and
thin-looking, the nucleus is reticulate, with a small nucleolus
and a well-developed nuclear membrane, and even the some-
what angular outline of the entire cell appears to be more or
less characteristic, and is frequently met with again in the
nurse-cells while in the act of feeding the odcytes (compare
figs.50,51). Whence these amcebocytes come it is impossible
to say. They do not look like metamorphosed collared cells
or epithelial cells, and they are very likely derived direct
from embryonic amcebocytes. They probably do not all
become nurse-cells, for some, which appear to be of the same
nature, grow to a large size and may put out long filiform
pseudopodia (figs. 59-61) In this extended condition I have
found them both inside the flagellate chambers (fig. 60) and in
the mesogloea between them (fig. 61), while in a more or less
rounded-off condition they are sometimes to be seen passing
through the layer of collared cells, especially in the neighbour-
hood of the exhalant apertures of the chambers (fig. 59).
Such amceboid cells sometimes develop into very active
phagocytes, apparently entirely on their own account. I
have observed this in specimens 11, 22 and 24. Specimen 24
in particular is crowded with large and small phagocytes,
which have evidently been feeding voraciously, and apparently
chiefly on young germ-cells.
Fig. 62 represents a phagocyte from the mesogloea, which
has ingested a single relatively large cell, too large, I think,
to be a spermatogonium, and so large that the cytoplasm of
the phagocyte is only able to stretch itself around its prey in
the form of a thin envelope. In fig. 63 an actively amceboid
phagocyte, with filiform pseudopodia, is apparently in the act
of ingesting a collared cell. Fig. 64 represents a phagocyte
rounded off in the mesoglcea, with two partially disintegrated
cells in its cytoplasm. This one looks as if it mght have
GAMETOGENESIS OF GRANTIA COMPRESSA. sor
become a nurse-cell, though rather large. Fig. 65 represents
a very actively amoeboid phagocyte, while fig. 66 shows one
squeezing itself through the layer of collared cells. Fig. 67
shows one with root-like pseudopodia, half in and half out of
a chamber; this one has collected and ingested no less than
six cells, which I judge from their size and appearance to be
spermatogonia. Fig. 68 represents yet another hanging on to
the inner surface of the layer of collared cells.
It appears, then, that while some of these amcebocytes
exercise their phagocytic propensities in favour of the odcytes,
and become nurse-cells, others feed voraciously on their own
account, even entering the flagellate chambers and appa-
rently collecting the numerous young germ-cells that are found
therein. It seems not improbable that this excessive phago-
cytosis may be regarded as a perverted instinct, for the phago-
‘cytes appear to take in food far beyond their own possible
requirements, and yet I have never seen a large phagocyte
feeding an odcyte. It is perhaps worth noting that speci-
men 24, in which most of the cases of phagocytosis by large
amcebocytes were observed, had been kept in the circulation
for a week before being killed and preserved. The abnormal
conditions may have stimulated the amcebocytes to an
abnormal activity in phagocytosis.
‘he possibility also occurs to one that some of the large
phagocytes are parasitic Amoebze which do not belong to the
sponge at all. Mr. Orton (1913) has recently described
Amoebee from the gastral cavity of Sycon which certainly
seem to be quite independent organisms, though I at first
thought otherwise (Dendy, 1913), and it seems by no
means improbable that such Amcebee may enter the chambers
and feed upon the young germ-cells, or even force their way
into the mesoglea. The chief argument against this view
appears to be the impossibility of distinguishing between the
larger and smaller phagocytes, and the apparent identity of
the latter with the nurse-cells, which must certainly be
regarded as belonging to the sponge itself.
352 ARTHUR DENDY.
(h) Summary and General Remarks on Odgenesis.
It will be seen from the foregoing account of my observa-
tions on the odgenesis of Grantia, that in their main features
they agree with what has already been recorded, especially by
Jorgensen, for the closely related genus Sycon. In some
important respects, however, and especially as regards the
derivation of the odgonia from collared cells, my observations
differ from those of Jérgensen, and, while I have not been
able to obtain anything like such precise results as he claims
with regard to the mitotic phenomena, I have been able to
describe a great deal that has either escaped his notice or does
not occur in Sycon, concerning, for example, the feeding of the
young odcytes in the chambers and the subsequent formation
of chromidia by extrusion of nucleolar matter into the cyto-
plasm, the very remarkable feeding of the odcytes by means
of nurse-cells, and the process of phagocytosis in general.
The process of odgenesis in Grantia may be briefly
summarised as follows :
The primary odgonia are directly derived from collared
cells, which accumulate reserve material, enlarge, with-
draw their collars and flagella, become amceboid and wander
into the mesoglcea, re-entering the chambers before dividing
mitotically into the odgonia of the second generation. Prior
to this division the nucleolus appears to be bodily cast out of
the odgonium.
The odgonia of the second generation divide again while
in the chambers, and probably almost immediately, into small
oocytes.
The small odcytes become amoeboid, and, while still within
the chambers, attached by pseudopodia to the layer of
collared cells, take in food-particles and form conspicuous
chromidia in their cytoplasm. Having increased considerably
in size they leave the chambers and enter the mesoglea.
Here they continue to undergo a process of extensive
chromidium-formation by extrusion of chromatin from the
nucleolus into the cytoplasm.
GAMETOGENESIS OF GRANTIA COMPRESSA. 353
They also probably undergo about this time the prophases
of the first maturation division.
They now send out anchoring pseudopodia by which they
became attached to the layer of collared cells and probably
draw nutriment from them.
Presently they undergo a remarkable contraction, the
pseudopodia being almost completely withdrawn, and about
this time they begin to be fed by special nurse-cells, which
collect smaller cells and pass them into the growing oocyte.
The oécyte increases greatly in size, and long root-like
pseudopodia are again put out in a plane parallel to the layer
of collared cells against which it lies.
The nucleus becomes very large and vesicular, with a huge
spherical nucleolus, and chromidia are abundantly formed in
the cytoplasm, though apparently no longer directly derived
from the nucleolus, but formed probably by extrusion of
granules of chromatin from the nucleus. ‘he chromidia
(yolk-nucleus), whatever their source, are probably concerned
in the elaboration of the deutoplasm, with which the cyto-
plasm becomes uniformly and densely charged, though
definite individual yolk-granules can hardly be recognised.
When the odcyte has reached its full size it withdraws its
pseudopodia and rounds itself off, the nuclear membrane
disappears and the contents of the nucleus disperse themselves
through the cytoplasm. The nucleolus remains recognisable
for some time after this event, but gradually becomes
absorbed.
Chromosomes have not been recognisable since the odgonial
mitoses, when they appeared in the equatorial plate as a group
of eight or ten minute, irregularly rounded bodies, each of
which presumably divided into two. They now appear again
on the first maturation spindle, but only one really good
spindle was found, and that already in the anaphase.
The first polar body is formed in apparently a typical
manner, exactly as described by Jorgensen for Sycon, and is of
large size.
The second maturation spindle and second polar body,
304 ARTHUR DENDY.
described by Jérgensen for Sycon, were not met with, though
they probably occur.
No evidence was obtained of a reducing division, and, indeed,
the number of chromosomes could never be accurately counted.
In spite of the fact that we have no reliable information
with regard to the phenomena of meiosis in sponges, we
cannot fail to be struck with the close general agreement of
the process of odgenesis with the same process as observed in
higher animals. ‘The multiplication of oédgonia; the formation
of chromidia or yolk-nucleus by the odcyte; the early inception
of the first maturation division (if this be confirmed), inter-
rupted by the long period of growth; the co-operation of other
cells in the process of nutrition ; the character of the nucleus
and the formation of the polar bodies; are all features which the
sponges share with higher groups, and one can hardly avoid
asking the question, Does this close similarity in odgenesis
point to a nearer relationship of the sponges with the Enterozoa
than is usually admitted in this country ? The question is well
worthy of consideration, but we can hardly hope for a final
solution of it in the present state of our knowledge. We can
hardly abandon the choano-flagellate ancestry of the sponges
without much stronger evidence than we possess, and we
certainly are not justified in attributing a choano-flagellate
ancestry to other groups of Metazoa. May we, then, suppose
that all the essential processes of odgenesis already existed in
pre-choano-flagellate Protozoon ancestors common to sponges
and Enterozoa? Such a supposition would certainly be in
harmony with the view now generally held that the germ-cells
of the higher animals are really equivalent to so many
Protozoa, for if this be so then the phenomena of odgenesis
must be such as we might reasonably expect to find in
Protozoa. Recent advances in protozoology, I think, show that
such an expectation is likely to be fulfilled, for we already
know that the gametes themselves may attain as high a
degree of differentiation (into ovum and spermatozoon) in
Protozoa as in Metazoa. We also know that the female
gamete may accumulate yolk (e. g. in Coccidium), and that
GAMETOGENESIS OF GRANTIA COMPRESSA. Bis)
something that may reasonably be interpreted as a forma-
tion of polar bodies may take place (e. g. Paramcecium).
I venture to predict that a good deal of light will, in the near
tuture, be thrown upon the complex phenomena exhibited in
the life-history of many Protozoa, by comparison of the events
that take place in the gametogenesis of higher animals. Some
more satisfactory and uniform system of terminology will,
however, have to be evolved before much progress can be
made in this direction. We shall have to know, for example,
exactly what we mean by “‘chromidia.” Prof. Minchin (1912)
tells us that “in a great many Sarcodina, especially in those
belonging to the orders Amcebxa and Foraminifera, chromidia
may be present in the gamete-forming individuals as a
permanent constituent of the body-structure. In such cases
the chromidia represent, wholly or in part, the generative
chromatin, and give rise by formation of secondary nuclei to
the nuclei of the gametes.” In the present paper I have,
following Jérgensen, used the term “ chromidia” for all the
chromatin which occurs in the cytoplasm. This, I think, is
probably ali extruded from the nucleus (and nucleolus), and is
almost certainly concerned in yolk-formation, and therefore
‘trophochromatin ” and not “ idiochromatin.” The difficulty
of distinguishing between these two kinds of “chromatin”
forms perhaps the chief obstacle in the way of further
progress in the direction indicated.
(G) SPERMATOGENESIS.
(a) Historical.
I have already referred to Haeckel’s discovery of the
Sperm-morulee of calcareous sponges in the gastral epithelium
of the flagellate chambers, and to his opinion that they arise
by division of collared cells. These observations never met
with general acceptance, and are usually peuerded as having
been superseded by Poléjaeff’s well-known work, ‘ Uber das
Sperma und die Spermatogenese bei Sycandra raphanus
Haeckel’ (1882).
356 ARTHUR DENDY.
Poléjaeff lays stress upon the discrepancies between the
account of the spermatozoa given by Haeckel and that given
by Eimer. According to Eimer, the spermatozoa, if not
isolated, occur scattered through the tissues, united in millions
in oval balls; according to Haeckel, they lie between the
collared cells with their tails projecting freely into the cavity
of the flagellate chamber, and it is never possible to find them
in considerable quantities. According to Himer, again, they
are to be distinguished from the collared cells by the character
of the movements of the flagella; while, according to Haeckel,
these movements show no essential differences in the two
cases. I think it almost certain myself that, although Haeckel
saw the sperm-morule in the situation he describes, he did
not see the tails of the spermatozoa, for the sperm-morule
represent a comparatively early stage of spermatogenesis at
which no tails have yet appeared. Haeckel probably mistook
for spermatozoon tails some of the flagella of the collared
cells amongst which the sperm-morule lie. The discrepancy
as to numbers and position is not a very serious matter.
Poléjaeff himself has shown how enormously the number of
Spermatozoa produced differs in different individuals, and I
find myself that in Grantia, although the sperm-morule are
generally to be observed in the walls of the flagellate cham-
bers, or lying free in the chamber-cavities, some of the early
stages of spermatogenesis occur in the mesoglea, while later
stages are found in the inhalant canals (possibly of different
individuals), and these might well appear in sections to be
lying in the tissues.
Poléjaeff (1882) found that in Sycon raphanus, although
the sponge is hermaphrodite, the vast majority of individuals
are predominantly female, and only very occasionally a pre-
dominantly male specimen is forthcoming. In the latter,
however, the sperm-balls (‘‘ Spermaklumpen”) were so nume-
rous that their whole development could be traced in a single
section. He derives these sperm-balls from ordinary amcebo-
cytes (“Wanderzellen”’) in the mesogloea (mesoderm), similar
to those which, in his opinion, give rise to the ova. These
GAMETOGENESIS OF GRANTIA COMPRESSA. 357
cells have a diameter of 0°008-0:02 mm., and their bright,
vesicular nuclei are distinguished by their relatively large
size and their highly refractive nucleolus. Such a cell is
represented in Poléjaeff’s fig.3a as a spherical body with an
excentrically placed nucleus. ‘The nucleus now divides into
two somewhat unequal parts, which take up their positions at
opposite poles of the cell, which becomes differentiated into
two corresponding parts, a cover-cell and a sperm mother-cell
(“ Ursamenzelle”’). The cover-cell does not divide again, but
the nucleus of the sperm mother-cell divides repeatedly, and
finally gives rise to a large number of very minute, granule-
like spermatozoon-heads, enclosed within a capsule formed
by the cover-cell. Hach of these heads presently becomes
provided with a cytoplasmic tail. During this process there
is no increase in volume of the sperm-ball, and Poléjaeff
remarks upon the extraordinarily small size of the sper-
matozoa. He also points out that the spermatogenesis of
Sycon as described by him diifers in several respects from
that described for non-caleareous sponges by Schulze,
Keller and others. Thus the division of the nucleus of the
“Ursamenzelle” is not immediately followed by division of
the cytoplasm, so that there arises a multinucleate mother-
cell and not a true sperm-morula, but he remarks that this is
not a matter of any very great importance. More significant,
perhaps, is the absence of the endothelial capsule formed
around the sperm-ball by the mesoglcea cells in some of the
non-calearea. This, I think, is a matter of some importance
in connection with the problem of how the spermatozoa are
transferred from one sponge to another. This problem Polé-
jaeff does not attempt to solve, and, indeed, we are left in
doubt, after studying his paper, as to whether or not he
considers that self-fertilisation takes place in Sycon. His
fig. | shows immense quantities of what appear to be sper-
matozoon heads in the inhalant canals, the chambers and the
exhalant canals, as well as sperm-balls in the mesoglea, but
no attempt is made to decide the question whether or not all
this mass of sperm has been derived from the same sponge.
358 ARTHUR DENDY.
I think the investigation of this question would probably
have gone a long way towards reconciling the discrepancies
between the observations of Haeckel and those of Eimer, and
have shown that the cover-cells retain their wandering pro-
pensities for some time, and carry the spermatgonia from the
mesogloea into the collared-cell layer, whence they are dis-
charged into the water-stream and carried out of the sponge
altogether, possibly to find their way back again, either into
the same or into another sponge, through the inhalant canal-
system. My own observations clearly indicate that this is the
course of events, though there appear to be noteworthy
differences in details of behaviour between the two genera
Sycon and Grantia.
During the thirty-two years that have passed since the
publication of Poléjaeff’s memoir, the only contribution that
has been made to the very difficult problem of the spermato-
genesis in calcareous sponges is that contained in the paper
by Wilhelm Gorich—* Zur Kenntnis der Spermatogenese bei
den Poriferen und Coélenteraten nebst Bemerkungen tber die
Oogenese der erstern”’ (1903). This author again deals with
the process as exhibited in Sycon raphanus, and although
he describes only a few of the earlier stages in this sponge,
his results are in one respect strikingly at variance with those
of Poléjaeff. He agrees with the latter in deriving the
spermatogonia from mesogloal amoebocytes, which round
themselves off at an early stage of their growth as compared
with the odgonia. He also describes the formation of a cover-
cell, but in a totally different manner from that described by
Poléjaeff. The spermatogonium and the cover-cell, though
both derived from amcebocytes of the mesogloea, differ from
one another in certain particulars and do not arise by division
of a common mother-cell. hey only come into relation
with one another secondarily, the cover-cell spreading itself
around the spermatogonium, and finally ingesting it, in a way
which is evidently very similar to the process of phagocytosis
already described by me for Grantia. The result, however,
is a Spermatogonium enclosed in a mother-cell very much as
GAMETOGENESIS OF GRANTIA COMPRESSA. 359
described by Poléjaeff. The spermatogonium is represented
as dividing mitotically into two and then incompletely into
four parts, beyond which its history was not followed.
This is all that is known of the spermatogenesis in
Calcarea. Amongst other sponges the most frequently and
most fully investigated form is Spongilla, and it is perhaps
worth while to say a few words about what is known in this
case, which seems to be typical of the non-Calcarea, before
proceeding to describe my own observations on Grantia.
As far back as 1888, Fiedler published his memoir, “ Uber
Hi- und Spermabildung bei Spongilla fluviatilis.” He
describes the formation of cover-cell and sperm mother-cell
(““Spermatocyte”’) by division of a common mother-cell
(“Spermatogonium”) exactly as described by Poléjaeff for
Sycon. The cover-cell, however (of which more than one may
be found), not infrequently disappears before the contained
“spermatocytes ” have completed their development, and the
mass of sperm, which may have been derived from several
“spermatogonia,” becomes enclosed in a secondary follicle
formed from ordinary mesogleeal cells (“‘ Parenchymzellen”).
The original “spermatocyte” divides repeatedly by mitosis
into daughter-cells which are completely separated from one
another. The smallness of the objects, however, makes the
examination of the process very difficult and the details of
mitosis are not very satisfactorily given. The last generation
of spermatocytes, the spermatids, develop directly into the
spermatozoa. A compact chromatin-ball is formed by con-
traction of the nucleus, as previously described by Schulze
for Halisarca (= Oscarella) (1877) and Aplysilla (1878), and
the enveloping cytoplasm is drawn outinto aslender tail. In
the fully formed spermatozoon the chromatin-ball forms a
minute spherical head.
Gorich (1903) added some interesting particulars as to
Spongilla, especially with regard to the structure of the fully
formed spermatozoon, in the paper which I[ have already
quoted. He finds that the number of cover-cells taking part in
the formation of the capsule or spermatocyst varies from one
360 ARTHUR DENDY.
to about six, and maintains that these cover-cells are derived
from mesoglceal cells distinct from the spermatogonium as in
the case of Sycon. He brings forward very little evidence,
however, in support of this view. He describes the often
repeated mitotic division of the sperm-cells within the
spermatocyst very much as it was described by Fiedler. In
the fully developed spermatozoa, however, he finds a far more
complex structure than had been observed by any of his
predecessors, for in addition to the spherical head and long
slender tail he describes and figures middle piece, apical body
and centrosomes, thus bringing the structure closely into
line with that of the spermatozoon in Enterozoa, as ex-
emplified by Aurelia, which he describes and figures in the
same paper.
With regard to the explanation of the close resemblance
thus established between the spermatogenesis of sponges and
that of the Enterozoa, and its bearing upon the relationship
of the two groups, I may refer to what I have already said in
my summary on the odgenesis.
(b) Origin and Growth of the Primary Spermato-
gonia.
In returning to Haeckel’s view that the spermatozoa are
formed by division of collared cells, I must admit that it is
extremely difficult to bring forward convincing evidence that
this is really the case. Haeckel, of course, was of opinion
that the collared cells become divided up into spermatozoa
in siti, and he says nothing of the existence of the sper-
matocyst or cover-cell described by Poléjaeff and Gorich,
while both the latter hold that the spermatozoa develop from
amcebocytes of the mesoglea. I believe that the view of
each of these authors expresses part of the truth, and I hope
that my own observations may serve to account for, and to a
large extent to reconcile, the discrepancies between them.
The manner in which I have interpreted these observations
and arranged the different stages cannot even yet, however,
be regarded as more than tentative.
GAMETOGENESIS OF GRANTIA COMPRESSA. 561
The first stage in spermatogenesis, as in that of odgenesis,
appears to be the enlargement of individual collared cells in
the lining epithelium of the chambers (figs. 69,70). At the
same time the cytoplasm acquires a peculiar curdled appear-
ance (if I may use this expression for want of a better), which
looks as if it might be due to the running together of the
reserve granules. Irregular inclusions of large size may thus
be formed, around which vacuoles frequently make their
appearance. By these appearances it is possible to distin-
guish between what I believe to be the primary spermato-
gonia and the primary odgonia respectively, for in the latter
(figs. 11, 12) it will be remembered that the reserve granules
remain separate and do not run together in irregular masses.
The nucleus now becomes very distinctly reticulate as com-
pared with that of neighbouring collared cells (figs. 71, 72),
collar and flagellum are withdrawn, and the cell puts out
pseudopodia and becomes ameeboid (figs. 72-74). In this con-
dition the primary spermatogonia are to be found hanging
into the chamber from the layer of collared cells by means of
their pseudopodia. Definite inclusions disappear from the
cytoplasm.
The cell now rounds itself off and assumes a very charac-
teristic appearance (figs. 75, 76). It is readily distinguished
from the oogonia by its smaller size and by the character of
the nucleus. It is also quite different in character from the
young odcytes, which are of about the same size (figs. 29, 30),
especially as regards the nucleus, which is coarsely reticulate
and without a really well-defined nucleolus, while that of the
young oocyte has a very conspicuous nucleolus surrounded by
a clear zone and then by a zone of fine granules.
I have found the primary spermatogonia in their rounded-
off condition in the mesogloea as well as in the flagellate
chambers, so that it seems probable that while still in the
amoeboid state they may migrate through the chamber-walls
as the o6gonia and odcytes so frequently do.
It is interesting to observe that the primary spermatogonia
exhibit a good deal of variation in size, as is shown in fig.
VoL. 60, PART 3.—NEW SERIES. 26
362 ARTHUR DENDY.
75. In one case also (fig. 75a) I have observed mitosis in the
free spermatogonium, but I have no evidence that the sper-
matogonium ever actually divides until it has been provided
with a cover-cell.
(c) Formation of the Spermatocysts or Cover-
cells.
It will be remembered that according to Poléjaeff the
original amcebocyte in Sycon divides into two parts, one of
which forms the cover-cell and the other the primary sperma-
togonium, while Gérich claims that the cover-cell is formed
by an independent amcebocyte which approaches and enve-
lopes the spermatogonium in the mesogloea. My own obser-
vations strongly support the latter view, and I regard the
process of envelopment of the spermatogonium by the cover-
cell as a special case of phagocytosis. Indeed, as already
pointed out, spermatogonia are frequently ingested by the
phagocytes, and it is difficult, if not impossible, to distinguish
an ordinary case of phagocytosis in which only a single
spermatogonium has been ingested, from a case of cover-cell
formation (cf. fig. 62, in which the ingested cell, however,
seems too large to be a spermatogonium). Figs. 77, 80 and
81 represent spermatocysts, with enclosed primary spermato-
gonia, lying in the mesoglea. In these cases the cover-cell
appears to resemble closely a small phagocyte such as gives
rise to the nurse-cells (cf. fig. 58).
The majority of the spermatocysts, however, are found
lying in the walls of the flagellate chambers between the
collared cells, with the enclosed spermatogonium either still
undivided, as shown in figs. 78 and 79, or in process of
division, as shown in fig. 82, or, much more frequently,
divided up into a sperm-morula (figs. 84, 85). When in
this position the spermatocyst certainly looks very much as
if it were derived in siti from a collared cell. Sometimes,
it is true, the nucleus is distinguishable from that of adjacent
collared cells by its reticulate character (figs. 82, 85), but in
other cases (figs. 78, 84) no such distinction can be made out.
GAMETOGENESIS OF GRANTIA COMPRESSA. 363
I have already pointed out, however, that the nuclei of the
collared cells themselves vary very greatly in appearance,
and that reticulate and umiformly dark-stained nuclei may
occur in adjacent cells. Sometimes the cytoplasm of the
cover-cell may even contain reserve granules like those found
in the collared cells (fig. 85).
The history of the spermatogonia themselves, however, and
the phagocytosis observed in the mesogloea, seem to me to
indicate very clearly that the spermatocysts so often seen
lying between the collared cells have reached their position
by migration, and there is no sufficient reason for concluding
that the cover-cells are ever derived from collared cells.
As the spermatocyst lies in the collared cell layer its
nucleus is situate, usually, at any rate, towards the lumen
of the chamber, just as are the nuclei of the collared cells
themselves, and the spermatogonium or sperm-morula is
enclosed in its basal portion (figs. 78, 82, 84).
As the sperm-morula develops the cyst formed by the
cover-cell becomes extremely thin (fig. 87) and finally
ruptures, discharging the sperm-morula into the cavity
of the chamber (fig. 85). Thus the liberation of the
sperm-morula from the cover-cell takes place much earlier
than in the case of Sycon, where, according to Poléjaeff,
spermatozoa are formed while still within the cysts.
(d) Development of the Sperm-morule from the
Primary Spermatogonia.
The first division of the primary spermatogonium at least
appears to take place mitotically, as has already been
described by Gorich for Sycon. At any rate one sometimes
finds spermatogonia in which the nucleus has disappeared,
and what appear to be small, scattered chromosomes, eight or
ten in number, are scattered through the cytoplasm as shown
in figs. 81 and 82, while fig. 80 represents what may be a
spireme stage.
I have only once observed what appears to be the two-
564 ARTHUR DENDY.
celled stage of the sperm-morula (fig. 85), and I regard this
as a somewhat doubtful case; it shows, however, two distinct
spherical bodies, which I take to be secondary spermatogonia,
enclosed in what is presumably a cover-cell (cov.) lying in the
layer of collared cells.
The four-celled stage I have never been able to find, in
spite of prolonged searching. The eight-celled stage, however,
T have seen several times (figs. 84, 85, 86), and it appears
that the sperm-morula may be liberated from the sper-
matocyst as early as this (fig. 86).
A stage in which the morula consists of sixteen cells, or
thereabouts, is the most frequent in my preparations. This
stage sometimes occurs still enclosed in the cover-cell (fig. 87),
but more frequently lying free in the cavity of the flagellate
chamber (figs. 88-92). A remarkable feature of the eight-
celled and sixteen-celled stages is the extraordinary distinct-
ness with which the daughter-spermatogonia are defined, but
the exact appearance evidently depends somewhat upon the
method of preparation. Very often they appear as little
heaps of highly refractive spherical balls, like small shot,
stained black or nearly so (figs. 83, 88). At other times they
are much more lightly stained, and one or more minute, more
darkly stained granules appear within them (figs. 86, 87, 89,
90). How the division takes place I cannot say, but I have
seen no sign of mitosis after the first division of the primary
spermatogonium. Whether or not the spherical bodies
represent entire cells, or nuclei only, I am also unable to say
positively, but I conclude from their general appearance that
the former is the case. It will be noticed that there is con-
siderable difference in size between the spermatogonia in
different sperm-morule of apparently the same stage of
development (cf. figs. 87, 88), but this is only what might be
expected from the differences in size of the primary spermato-
gonia already mentioned. Although usually spherical, the
daughter-spermatogonia sometimes appear to be polygonal
from mutual pressure (figs. 89, 91).
A noteworthy feature is the appearance between them in
GAMETOGENESIS OF GRANTIA COMPRESSA. 565
the sperm-morula of a substance that looks like residual
protoplasm (figs. 86-92). As development proceeds (at the
sixteen-celled stage), this material seems to swell up so as to
separate the spermatogonia more or less from one another.
(e) The Formation of Spermatozoa; Comparison
with Sycon, etc.
As to how the spermatozoa are developed from the sperm-
morule in Grantia I have no definite information to offer.
My material does not suffice to settle this question, but I
have some reason to believe that the spermatogonia of the
sixteen-celled stage undergo further repeated subdivision. I
believe that this usually takes place after the sperm-morule
have been transferred by the water currents to the inhalant
canals of another individual. I have occasionally found, both
in the inhalant canals and flagellate chambers, small masses
of darkly stained granules (fig. 93) which appear to be
identical with the masses of granules which Poléjaeff showed
to be spermatozoon heads in Sycon. If they be of this nature,
as I think highly probable, their minute size certainly serves
to indicate a further breaking up of the spermatogonia of the
sixteen-celled stage. I have also a small amount of direct
evidence of such breaking up of the sperm-morule in the
imhalant canals, but it is not conclusive enough to bring
forward definitely.
Poléjaeff tells us that in Sycon the spermatozoa are formed
by repeated subdivision of the spermatogonia within the cover-
cell, during which process the spermatocyst does not increase
in size. The final products of these divisions are represented
as being extremely minute, and each becomes provided with
a slender flagellum.
I happen to have in my possession one of Poléjaeff’s own
preparations of Sycon, sent by him to Mr. Carter in 1883.
This preparation shows “sperm-balls”’ (sperm-morule) in
the inhalant canals, and I suppose them to have come from
another individual. The sperm-balls vary considerably in
366 ARTHUR DENDY.
size (fig. 94) and each is still enclosed in the cover-cell, whose
nucleus is very distinctly visible. In the interior a number
of ill-defined, faintly stained bodies are present, which may be
daughter-spermatogonia, or, as Poléjaeff supposes, merely the
nuclei of these. They are nothing like so definite as the
spermatogonia which I find in the sperm-morule of Grantia,
but this may be partly because they belong to a later stage
of development, and partly because of differences in the mode
of preparation. According to the information given in his
memoir Poléjaeff’s preparations were made with osmic acid
(0°01-0°05 per cent.) material stained with alum carmine,
and this probably applies to the preparation in my possession.
I give in fig. 94 drawings of two sperm-balls from this
preparation. It will be seen that they are of just about the
same size as the spermatocysts with enclosed primary sperma-
togonia in Grantia (cf. figs. 77, 78, 81), and, allowing for the
longer retention of the cover-cell and the further subdivision
of the spermatogonia after the sixteen-celled stage, I think
there is no serious discrepancy. 1 am unable to make a direct
comparison between the earlier stages in the two genera as I
have found none of these in Poléjaeff’s preparations.
That the sperm-morulz found by me in Grantia are identical
with the structures already referred to as described by
Haeckel in the lining epithelium of the flagellate chambers of
various calcareous sponges, appears to me to admit of very
little doubt, although of course I cannot agree with him in
the details of his account.
It has occurred to me as just possible that someone may
suggest that these bodies are not sperm-morulz at all, but of
quite a different nature; that they may be Sporozoa living
for a period as intracellular parasites in the collared cells and
possibly also in the amcebocytes of the sponge. Apart from
the fact, however, that sporozoan parasites have never yet
been observed in sponges, I think the developmental history
of the bodies in question is sufficient to negative this view.
No infection of the sponge-cells by small forms that might
be young Sporozoa has ever been observed, and the cell which
GAMETOGENESIS OF GRANTIA COMPRESSA. 367
is surrounded by the cover-cell, and which I believe to be the
primary spermatogonium, has nothing about it to suggest a
sporozoon.
I can only regret that I am unable to give a more satisfac-
tory account of the spermatogenesis, but I hope that what I
have said will arouse more interest in this extremely difficult
problem, and enable future workers at any rate easily to find
the structures in question, and perhaps fill up the many gaps
which I have left.
(4) FERTILISATION OF THE Ovum.
I have not been fortunate enough to observe the actual
entrance of the spermatozoon into the mature ovum. According
to Jorgensen (in Sycon), the head swells up and gives rise to
the male pronucleus, in which a nucleolus very soon makes its
appearance. ‘I'he female pronucleus has in the meantime
developed from the group of chromosomes that remained
behind in the ovum after the formation of the second polar
body. These unite together into a compact chromatin ball,
around which a vacuole, enclosed in a nuclear membrane,
makes its appearance. A nucleolusis very early differentiated
in the midst of this mass, the remainder of which is broken up
into chromatin granules, which become scattered over the
nuclear reticulum.
Sometimes a portion of one of the pronuclei (either male or
female) is represented by a small separate “ karyomere,” so
that there appear to be three nuclei in the fertilised ege.
The number of nucleoli present in the pronuclei varies.
So far as they go my own observations on Grantia are
entirely in harmony with these results. I have been able to
study four ova with well-developed male and female pronuclei.
In only one of these cases were the two pronuclei both single,
and in this case the number of nucleoli was either 4 + 1 or
3 + 2,one of them having been displaced in the cutting. In
the other three cases a karyomere was present in addition to
the principal pronuclei, and the numbers and distribution of
368 ARTHUR DENDY.
the nucleoli were 1 + 14+ 1,14 14 2,and1+414 2 re-
spectively.
Fig. 95 shows one of these cases. The position of the
degenerating polar body (p. b.) shows that the large pro-
nucleus with the single large nucleolus is evidently the
principal female pronucleus, and the small karyomere, with
single nucleolus, doubtless belongs to it. The male pro-
nucleus, on the right and below, has two nucleoli.
The formation of karyomeres is a very curious and striking
phenomenon, for further details as to which I may refer the
reader to Jérgensen’s paper. That author points out that
karyomere formation also takes place in Sycon in the process
of segmentation of the fertilised ovum, and I| find the same
to be true in the case of Grantia.
.It is perhaps worth while pointing out that the nucleoli in
the male and female pronuclei do not stain nearly so deeply as
the nucleoli of the young odcytes. The same is also true of
the nucleoli in the older odcytes. The difference may perhaps
be correlated with the fact that in the young odcytes the
nucleolus is actively engaged in the formation of chromidia or
“ yolk-nuclei,’” while probably it is not directly engaged in
this process in the older odcytes, and certainly not in the
fertilised ovum, where yolk-formation has ceased.
(x) List or LirERATURE REFERRED TO.
1854. Carter, H. J.—‘* Zoosperms in Spongilla,” ‘Ann. and Mag. Nat.
Hist.,’ vol. xiv.
“ Notes Introductory to the Study and Classification of
the Spongida,” ‘ Ann. and Mag. Nat. Hist.,’ vol. xvi.
1888. Dendy, A—‘ On the Anatomy and Histology of Stelospongus
flabelliformis, Carter; with Notes on the Development,”
‘Quart. Journ. Micr. Sci.,’ vol. 29, N.s.
1891. “On the Anatomy of Grantia labyrinthica, Carter,
and the so-called Family Teichonide,” ‘Quart. Journ. Micr.
Sci.,’ vol. 32, N.s.
1913.
“A meebocytes in Caleareous Sponges,” ‘ Nature,’ December
4th and December 25th, 1915.
1910.
1856.
1894.
1900.
1898.
1900.
1912.
OS.
1914.
1882.
1911.
1910.
GAMETOGENESIS OF GRANYTIA COMPRESSA. 369
2. Kimer, Th.—‘‘ Nesselzellen und Samen bei Seeschwiimmen,”
‘Arch. Mikr. Anat.,’ vol. vii.
. Fiedler, K.—*‘ Uber Ei- und Spermabildung bei Spongilla
fluviatilis,” ‘ Zeit. wiss. Zool.,’ vol. xlvii.
. Gorich, W.—* Zur Kenntnis der Spermatogenese bei den Pori-
feren und Colenteraten nebst Bemerkungen iiber die Odgenese
der Ersteren,”. ‘ Zeit. wiss. Zool.,’ Bd. lxxvi, Heft iv.
. Haeckel, E.—‘ Uber die sexuelle Fortpflanzung und das natiiwliche
System der Schwamme,” ‘ Jenaische Zeitschrift fiir Medicin und
Naturwissenschaft,’ Bd. vi.
‘ Die Kalkschwimme.’
. Huxley, T. H.—* Zoological Notes and Observations made on
Board H.M.S. ‘ Rattlesnake’; 2, ‘On Tethya,” ‘Ann. and Mag.
Nat. Hist.,’ vol. vii.
Jorgensen, M.—* Beitrage zur Kenntnis der Hibildung, Reifung,
Befruchtung und Furchung bei Schwammen (Syconen),’’ ‘ Archiv.
fiir Zellforschung,’ Bd. iv.
Lieberkthn, M.—‘ Beitrage zur Entwicklungsgeschichte der
Spongillen,” ‘ Muller’s Archiv.,’ 1856.
Maas, O—‘Uber die erste Differenzierung von Generations-
und Somazellen bei den Spongien,” ‘ Verhandlungen der
deutschen Zoologischen Gesellschaft, Gottingen,’ 1893.
“Die Weiterentwicklung der Syconen nach der Meta-
morphose,” ‘ Zeit. wiss. Zool.,’ Bd. Ixvi.
Minchin, E. A.—‘‘ On the Origin and Growth of the Triradiate
and Quadriradiate Spicules in the Family Clathrinide,’ ‘ Quart.
Journ. Micr. Sci.,’ vol. 40, n.s.
“ Sponges,” ‘ Lankester’s Treatise on Zoology.’
‘An Introduction to the Study of the Protozoa.’
Orton, J. H.—‘‘ On a Habitat of a Marine Ameceba,” ‘ Nature,’
November 27th, 1913.
* Preliminary Note on a Contribution to an Evaluation
of the Sea,” ‘Journal of the Marine Biological Association,’ 1914.
Poléjaeff, N.—‘* Uber das Sperma und die Spermatogenese bei
Sycandra raphanus Haeckel,” ‘Sitzb. der k. Akad. der
Wissensch. Wien,’ Bd. Ixxxvi.
Robertson, M.—‘ The Division of the Collar-cells of Calcarea
Heterocela,” ‘ Quart. Journ. Mier. Sci.,’ vol. 57.
- and Minchin, EK. A.—* The Division of the Collar-cells of
Clathrina coriacea,”’ ‘Quart. Journ. Micr. Sci.,’ vol. 55.
370 ARTHUR DENDY.
1877. Schulze, F. E.—* Untersuchungen iiber den Bau und die Entwick-
lung der Spongien. Die Gattung Halisarca,” ‘Zeit. fiir wiss.
Zool.,’ Bd. xxviii.
“Untersuchungen iiber den Bau und die Entwicklung
der Spongien. Die Familie der Aplysinide,” ‘Zeit. fiir wiss.
Zool., Bd. xxx.
(Lt) EXPLANATION OF PLATES 23 10 26,
Illustrating Prof. Arthur Dendy’s paper, ‘‘ Observations on
the Gametogenesis of Grantia compressa.
[All figures are magnified about 1650 diameters, and, with the excep-
tion of fig. 94, refer to Grantia compressa. |
PLATE 23.
Fig. 1.—Collared cell putting out pseudopodia, from teased prepara-
tion of living sponge. col. Collar. (Unstained.)
Fig. 2.—Collared cell (a), with flagellum still present, putting out
pseudopodia and retreating into the mesoglea from the gastral epithe-
lium. b., 6. Collared cells still in position in the gastral epithelium.
(Specimen 24. Borax carmine.)
Fig. 3—Ameebocyte (a) lying behind the gastral epithelium and prob-
ably derived from a collared cell. b., b. Collared cells. (Specimen 24.
Borax carmine.)
Fig. 4.—Portion of section at right angles to the gastral surface,
showing granular epithelial cell (a) in the “ flask-shaped ” condition at
the surface, and amcebocyte (b) in the mesoglea. sp. Spicules. (Speci-
men 24. Iron brazilin.)
Fig. 5.—Ameebocyte in the mesoglcea just beneath the gastral cortex.
(Specimen 24. Iron brazilin.)
Fig. 6.—Portion of section at right angles to the gastral cortex, show-
ing parts of two granular epithelial cells (a) in position on the surface,
and a group of amcebocytes (b) lying in the mesoglea and evidently
derived by immigration from the epithelial layer. p.g. A mass of
pigment-granules apparently discharged from an amcebocyte. (Speci-
men 24. Iron brazilin.)
Fig. 7.—Portion of section through inhalant canal (7. ¢.) and adjacent
mesoglea, showing transition from epithelial to ameeboid cells. (Speci-
men 24. Iron brazilin.)
GAMETOGENESIS OF GRANTIA COMPRESSA. 371
Fig. 8.—Corresponding portions of two consecutive sections taken
tangentially through the layer of collared cells, (a) through the nuclei,
(b) through the cytoplasm below the nuclei; showing variations in the
intensity of staining of the reserve granules. a, 8, y, 6, are identical
cells in the two sections. (Specimen 21. Paracarmine and _picro-
indigo carmine.)
Fig. 9.—A collared cell (?) in mitosis, showing the two groups of
daughter-chromosomes, seen end on. (Specimen 24. Borax carmine.)
Fig. 10.—Two adjacent collared cells, one in the normal condition and
the other beginning to enlarge to form a primary germ-cell. (Specimen
21. Paracarmine and picro-indigo carmine.)
Fig. 11.—Part of section through the exhalant opening (e. 0.) of a
flagellate chamber, showing two normal collared cells and the con-
version of another into a primary odgonium (p.o.). g.¢. gastral surface.
(Specimen 21. Paracarmine and picro-indigo carmine.)
Fig. 12.—Part of a similar section showing another primary odgonium
(p. 0.) with remains of collar and flagellum and a pseudopodial process
wedged in between the adjacent collared cells. (Specimen 2]. Para-
carmine and picro-indigo carmine.)
Fig. 13.—Primary odgonium (p. 0.) after complete loss of collar and
flagellum, migrating into the mesoglea from the layer of collared
cells (c. c.). (Specimen 24. Borax carmine.)
Fig. 14.—Actively ameboid primary odgonium in the mesoglea.
(Specimen 24. Borax carmine.)
Fig. 15—Primary odgonium in which small granules of chromatin
are beginning to appear in the nucleus around the nucleolus. (Speci-
men 24. Borax carmine.)
Fig. 16.—Primary odgonium rounding itself off and entering upon
resting stage before entering flagellate chamber. (Specimen 22. Iron
brazilin.)
Figs. 17, 18.—Two primary oégonia in prophase of mitosis (spireme) ;
the one on the right (fig. 18) showing the expulsion of the nucleolus.
ec. c. Collared cells. (Specimen 22. Iron brazilin.)
Fig. 19.—Primary odgonium in mitosis in flagellate chamber (late
prophase), showing equatorial plate, spindle and centrosomes, with
chromidia in cytoplasm. (Specimen 11. Iron brazilin.)
Fig. 20.—Oogonium in mitosis in chamber. Same stage as last, but
of smaller size. (Specimen 22. Iron brazilin.)
Fig. 21.—Primary oé6gonium in mitosis (metaphase) forcing its way
between the collared cells into a flagellate chamber. (Specimen 21.
Paracarmine and picro-indigo carmine.)
Ore ARTHUR DENDY.
Fig. 22.—-Primary o6gonium in mitosis in chamber (anaphase), showing
separation of the groups of daughter-chromosomes on the spindle.
(Specimen 11. Tron brazilin.)
Fig. 23—Primary ojgonium in mitosis in chamber (late anaphase).
(Specimen 21. Paracarmine.)
Fig. 24.—Odégonium in mitosis in chamber (late anaphase). (Speci-
men 22. Iron-brazilin.)
Fig.25.—Primary odgonium in mitosis in chamber (telophase), showing
cell-division ; chromidia still visible in cytoplasm. (Specimen 22. Iron
hematoxylin.)
Fig. 26—Two daughter-cells (secondary oégonia) resulting from
division of primary o6gonium, in chamber; nuclei not yet completely
reconstituted ; chromidia still visible in cytoplasm. (Specimen 22.
Iron hematoxylin.)
Fig. 27.—Secondary odgonium lying in flagellate chamber, with
reconstituted nucleus. c.c. Collared cells. (Specimen11. Iron-hema-
toxylin and picro-indigo carmine.)
Fig, 28.—Secondary odgonium in chamber, in mitosis. (Specimen 22.
Ivon brazilin.)
Fig. 29.—Young oécyte, attached to wall of flagellate chamber by
pseudopodium. c.c. Collared cells. (Specimen 22. Ivon brazilin.)
Fig. 30.—Young odcyte in chamber. (Specimen 22. Iron brazilin.)
Fig. 31—Young odcyte in chamber, with one inclusion in the cyto-
plasm. (Specimen 22. Iron brazilin.)
Fig. 82.—Young odcyte in chamber, with cytoplasmic inclusions.
(Specimen 24. Iron brazilin.)
Figs. 33-35.—Young odcytes in chambers, with very darkly stained
chromidia or ‘“ yolk-nuclei.” (Specimen 22. Iron brazilin.)
Fig. 36.—Young odcyte in chamber, with food-particles in vacuoles,
and chromidia. (Specimen 22. Tron hematoxylin.)
Fig. 37.—Young odcyte in chamber, with food-particle in vacuole.
(Specimen 22. Iron hematoxylin.)
Fig. 38.—Young odcyte in chamber, showing formation of chromidia
(yolk-nuclei) by expulsion of chromatin from nucleolus. (Specimen 23.
Iron brazilin.)
Fig.39.—Young oocyte in chamber, attached to wall at a,a, containing
chromidia or “ yolk-nuclei ” and remains of ingested cells; nw., nucleus
of oocyte; n.7.¢., nucleus of ingested cell. (Specimen 23. Iron brazilin.)
GAMETOGENESIS OF GRANTIA COMPRESSA. Sie
PLATE 24.
Fig. 40.— Young odcyte leaving a flagellate chamber after feeding, and
entering the mesoglea. c.c. Collared cells. (Specimen 11. Tron-haema-
toxylin and picro-indigo carmine.)
Figs. 41-43.—Formation of chromidia or “ yolk-nuclei”’ of the young
oocyte (in the mesoglea) by extrusion of chromatin from the nucleolus
nto the cytoplasm. (Specimen 23. Iron brazilin.)
Fig. 44.—Young odcyte in prophase of mitosis. Leptotene stage.
(Specimen 21. Paracarmine and picro-indigo carmine.)
Fig. 45.—Young odcyte in prophase of mitosis. Pachytene stage,
showing contraction of the skein. (Specimen 22. Iron hematoxylin.)
Fig. 46.—Young odcyte immediately after the prophase of mitosis,
the spireme broken up again. (Specimen 22. Iron hematoxylin.)
Fig. 47.—a. Odcyte in “resting condition” behind wall of chamber.
b. A similar odcyte flattening itself out against the layer of collared
cells (c.c.). (Specimen 11. Iron hematoxylin and picro-indigo carmine. )
Fig. 48—Two odcytes in the mesoglea, attached by “ anchoring
pseudopodia”’ to the layer of collared cells (c.c.). (Specimen 21. Para-
carmine and picro-indigo carmine.)
Fig. 49.—Odcyte in contraction stage, with pseudopodia reduced to
blunt knobs. (Specimen 11. [ron hematoxylin.)
Figs. 50, 51.—Odcytes being fed by nurse-cells (n.¢.), which have
captured food-cells (f. c.) and are passing them into the odcyte. (Speci-
men 11. Iron brazilin.)
Fig. 52.—Odcyte of nearly full size, lying behind the layer of collared
cells (¢.c.). ps. Base of pseudopodium which has been cut off. xn. c.
Nurse-cell, with food-cell lying in the cytoplasm of the odcyte beneath
it. (Specimen 11. Ivon brazilin.)
Fig. 53.—Odcyte of nearly full size, showing very strong development
of chromidia (“yolk-nuclei”) in cytoplasm. ps. Base of pseudo-
podium that has been cut off; the other pseudopodia are altogether
missed by the section. (Specimen 11. Iron brazilin.)
Fig. 54.—Odcyte of approximately full size, just before withdrawal
of the pseudopodia and rounding off. (Specimen 11. Iron brazilin.)
PLATE 25.
Fig. 55.—Odcyte after withdrawal of pseudopodia and disruption of
the nucleus. xo. Nucleolus undergoing absorption in the cytoplasm
(more distinct in next section). chr. (?) Group of chromosomes (°).
(Specimen 23. Iron brazilin.)
old ARTHUR DENDY.
Fig. 56.—Tangential section of maturing oécyte with first matura-
tion spindle in anaphase. (Specimen 11. Iron brazilin.)
Fig. 57.—Tangential section of maturing odcyte, showing formation
of first polar body (p.b.). (Specimen 11. Iron brazilin.)
Fig. 58.—Small ameebocyte of the phagocyte (nurse-cell) type from
the mesoglea between the chambers. (Specimen 11. Iron brazilin.)
Fig. 59.—Large amcebocyte of the phagocyte type; forcing its way
through the layer of collared cells (col.) near the exhalant opening of a
chamber. (Specimen 21. Paracarmine.)
Fig. 60.—Large ameebocyte of the phagocyte type, found in flagellate
chamber. (Specimen 24. Borax carmine.)
Fig. 61.—Large amebocyte of the phagocyte type, from the mesoglea
between the chambers, stretched out against the layer of collared cells
(c.c.), some only of which are shown. (Specimen 22. Tron hematoxylin.)
Fig. 62.—Small phagocyte, with single large ingested cell; from
mesoglea between chambers. (Specimen 24 Borax carmine.)
Fig. 63.—Phagocyte (ph.) engaged in ingesting the end collared cell
of a row, i. e. the one next to the exhalant opening of a chamber. . ¢.
Collared cells. ps. Pseudopodium cut off from another amebocyte.
(Specimen 24. Iron brazilin.)
Fig. 64.—Resting phagocyte (nurse-cell?) in mesoglea just outside
layer of collared cells (c. c.), with two ingested cells in process of diges-
tion. n.p. Nucleus of phagocyte. (Specimen 11. Iron hematoxylin.)
Fig. 65.—Active phagocyte in mesoglea outside chamber, attached
by pseudopodia to the outer surfaces of the collared cells (c.c.), ¢.¢.
ingested cells. n.p. Nucleus of phagocyte. (Specimen 24. Tron
brazilin.)
Fig. 66.—Phagocyte forcing its way through layer of collared cells.
Lettering as before. (Specimen 24. Borax carmine.)
Fig. 67.—Large phagocyte with six ingested cells, forcing its way
through the layer of collared cells. Lettering as before. (Specimen
24. Borax carmine.)
Fig. 68.—Large phagocyte with four ingested cells, lying in flagellate
chamber and attached by pseudopodia to the layer of collared cells.
Lettering as before. (Specimen 24. Borax carmine and picro-indigo
carmine.)
PLATE 26.
Fig. 69.—Section through parts of two flagellate chambers, adjacent
to the exhalant openings, showing one of the collared cells enlarging to
form a primary spermatogonium (p.s.). (Specimen 21. Paracarmine
and picro-indigo carmine.)
GAMETOGENESIS OF GRANTIA COMPRESSA. 375
Fig. 70.—Another example of a collared cell enlarging to form a
primary spermatogonium. (Specimen 21. Paracarmine and_picro-
indigo carmine.)
Fig. 71.—Section through lining epithelium of flagellate chamber
showing one of the collared cells enlarging to form a primary spermato-
gonium (p.s.). (Specimen 21. Paracarmine and picro-indigo carmine. )
Fig. 72.—Metamorphosis of enlarged collared cell into a primary
spermatogonium (p.s.). ¢.¢. Ordinary collared cell. (Specimen 24.
Borax carmine.)
Figs. 73, 74.—Primary spermatogonia (p.s.) in the amceboid con-
dition, attached by pseudopodia to the layer of collared cells (c. c).
(Specimen 24. Borax carmine.) (Fig. 74 rather doubtful; it has a
distinct but not very darkly staining nucleolus with a clear ring around
it, and is rather large for a spermatogonium.)
Fig. 75.—Group of primary spermatogonia rounded off; from flagellate
chamber. Fig. 75a.—Another example from an adjacent chamber, in
mitosis. (Specimen 24. Borax carmine.)
Fig. 76.—Primary spermatogonium rounded off; from mesoglcea out-
side chamber. (Specimen 24. Borax carmine.)
Fig. 77.—Primary spermatogonium (p.s.) enclosed by cover-cell (cov.),
behind layer of collared cells (c.c). (Specimen 24. Borax carmine.)
Fig. 78.—Cover-cell (cov.) and primary spermatog6nium (p.s.) lying in
layer of collared cells (c.c.). (Specimen 24. Borax carmine.)
Fig. 79.—Cover-cell (cov.) and primary spermatogonium (p.s.) lying in
layer of collared cells (c.¢.). (Specimen 24. Iron brazilin.)
Figs. 80, 81—Primary spermatogonia undergoing mitosis within
cover-cells ; from mesoglea. (Specimen 24. Iron brazilin.)
Fig. 82.—Cover-cell (cov.) containing primary spermatogonium (p. s.)
in mitosis, lying in layer of collared cells (c.¢.). (Specimen 24. Borax
carmine.)
Fig. 83.—Section through wall of chamber, showing cover-cell (cov.)
containing nucleus and two secondary spermatogonia; also 16-celled
sperm-morula (sp. m.) that has escaped from its cover-cell, the remnant
of which (?) is shown at x. c.c. Collared cells. ep. Epithelial cell of
inhalant canal. (Specimen 24. Iron brazilin.)
Fig. 84.—Section through wall of flagellate chamber and inhalant
canal, showing cover-cell (cov.) with sperm-morula of 8 cells (sp.m.), lying
in layer of collared cells (c.c.). phag. Phagocyte with included cells
lying in mesoglea. ep. Epithelial cells of inhalant canal. (Specimen
24, Borax carmine.)
«
376 ARTHUR DENDY.
Fig. 85.—Sperm-morula of probably 8 cells (sp. m.) enclosed in cover-
cell (cov.), with adjacent collared cell (c.c.), in wall of flagellate chamber;
seen en face. (Specimen 24. Borax carmine.)
Fig. 86.—Hight-celled sperm-morula lying free in cavity of chamber,
without cover-cell. (Specimen 24. Iron brazilin.)
Fig. 87.—Sperm-morula of probably 16 cells still enclosed in cover-
cell, in lining epithelium of chamber. n.cov. Nucleus of cover-cell.
(Specimen 24. Borax carmine.)
Fig. 88.—Sperm-morula of probably 16 cells, lying free in chamber
and partially broken up. (Specimen 24. Iron brazilin.)
Fig. 89.—Sperm-morula of probably 16 cells, after escape from cover-
cell; seen in optical section. (Specimen 24. Iron brazilin.)
Fig. 90.—Sperm-morula of probably 16 cells, lying free in chamber
after escape from cover-cell. (Specimen 24. Iron brazilin.)
Fig. 91—Sperm-morula of probably 16 cells, lying free in chamber
after escape fron cover-cell, showing large amount of residual proto-
plasm. (Specimen 24. Borax carmine.)
Fig. 92.—Sperm-morula of probably 16 cells, lying free in chamber
after escape from cover-cell, with swollen residual protoplasm. (Speci-
men 24. Borax carmine.)
Fig. 93.—Group of spermatozoon heads (?) from flagellate chamber.
(Specimen 21. Paracarmine.)
Fig. 94.—Two “sperm-balls” from inhalant canal of Sycon sp.
n.cov. Nucleus of cover-cell. (From one of Dr. Poléjaeff’s prepara-
tions.)
Fig. 95.—Ovum during fertilisation, with male pronucleus containing
two nucleoli, and female pronucleus represented by two karyomeres
with one nucleolus each. p.b. Remains of polar body. x... x Line
of contact of ovum with layer of collared cells. (Specimen 23. Iron
brazilin.)
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 3877
The Chromosome Complex of Culex Pipiens.
By
Monica Taylor, S.N.D., B.Sc.
With Plates 27 and 28, and 3 Text-figures.
ContENTs.
PAGE
I. Introduction and Methods . : : OG
II. The Reproductive Organs . ; : . 380
III. Spermatogenesis . : 2 Hoo
IV. Oogenesis 3 ; : . 389
V. Somatic mitosis. : ; : . 389
VI. Discussion : : Cee . 392
VII. Summary : : : : . 394
INTRODUCTION.
Miss Srrvens, in a paper entitled “The Chromosomes in
the Germ-Cells of Culex,” gave as one of her conclusions the
following :
“ Parasynapsis (parasyndesis!) occurs 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.”
Because of the importance of this discovery, especially in
the support it lends to the recent theory of parasyndesis, and
because of its bearing on the theory of the individuality of
the chromosomes, Dr. Agar, in the summer of 1912, at Tay-
1] have adopted the word “syndesis” for the conjugation of the
chromosomes, and “ synizesis” for their clumping together.
voL. 60, paRT 3.—NEW SERIES. 27
378 MONICA TAYLOR.
vallich, Loch Sween, collected and preserved material in order
to investigate the germ-cells of Culex pipiens. This he
very kindly gave to me, and in the spring and summer of
1913 a supply of egg-rafts, larvee, and pupe from Milngavie
and Skelmorlie has been used in conjunction with the original
stock.
The results obtained by a study of the Tayvallich material
showed clearly that a much more extensive investigation than
was originally intended would be necessary in order completely
to elucidate the problems that incidentally presented them-
selves. Hence many of the egg-rafts and larvee were placed in
artificial ponds in order that greater control might be exercised
over the material to be fixed, and single specimens were isolated
so that their exact age could be determined, and their periods
of ecdysis watched.
The life-history of Culex pipiens is to be found set forth
in innumerable text-books of Natural History, and much has
been written about mosquitoes in connection with malaria,
but in no case have I been able to find any adequate account
of the behaviour and fate of the imagines which hatch out
and live in captivity, nor of the time that elapses between
the emergence of the imago and the deposition of eggs.
Large numbers of pupz, developed, some under natural, others
under artificial, conditions, from eggs obtained in May or
August of 1913, were placed in small ponds which were
covered over with large cages made of mosquito-netting so
that the resulting imagines could be observed. From many
hundreds of these captive-reared creatures I have not
succeeded in obtaining any egg-rafts, although some of the
imagines have lived for four months. Nor could they be
induced to suck blood, which, according to the account of
some naturalists, is necessary, even in non-tropical forms of
gnats, for the development of the eggs.
A comparison of the spermathecz of adults which have
always been captive with those of adults taken in the open
shows that the former never contain spermatozoa, although
the latter do. Hence it would appear that captivity is not
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 379
favourable to fertilisation. The completion of this study by
the detailed investigation of the maturation and fertilisation
of the egg-cell will have to be postponed for an indefinite
time until a developmental series of imagines caught in the
open has been secured, or until the technique of artificial
rearing has been mastered.
Another interesting feature in connection with the larve
is the want of uniformity in the periods of metamorphosis.
Although the main body of any collection of larve will
complete their development in the usual time, there are always
some laggards who double or even treble the usual periods.
‘emperature and food-supply do not wholly account for this
retarded development.
The fixatives employed have been Benda’s fluid, acetic
bichromate, Gilson’s mercuro-nitric, Flemming, and Gilson-
Petrunkewitch, the two latter being most successful for the
cytology proper; the two former were useful for interpreting
cytoplasmic details.
Thionin, iron-hematoxylin (prolonged staining), as well as
Mayer’s cochineal, Ehrlich’s hematoxylin and safranin were
the stains employed. Many slides were first studied in
thionin, and then the cover-slip was removed, the thionin
washed out, the sections re-stained in iron-hematoxylin, and
comparisons made between the results of the two stains. For
certain stages after prolonged treatment with iron-hematoxylin
much extraction was necessary, for others little, so that the
same slide had often to be studied under various degrees of
extraction.
Although aceto-carmine preparations of the whole gonad
are very useful for mapping out quickly the main facts of
spermatogenesis, and although this stain has the advantage,
as Miss Stevens has pointed out, and as my experience has
confirmed, of increasing the size of the cellular elements and
of thus rendering them easier of examination, they are not
permanent, not so good for finer details, and not useful for
somatic mitosis. Hence the figures given in this paper have
been taken from sections (thickness ranging from 4 to 12 4)
and not from aceto-carmine preparations.
380 MONICA TAYLOR.
Miss Stevens worked on Culex pungens,' a form which
is very nearly allied to C. pipiens. It is probable that this
close relationship between C. pipiens and C. pungens
accounts for the great similarity which exists between her
figures of primary and secondary spermatocyte anaphases and
telophases and those that occur in C. pipiens.
Trext-FiG. 1.
amm
Outline of testis of pupa of Culex pipiens to show level at
which the various stages in spermatogenesis commonly occur.
1-4. Synizesis stage. 5-9. Preparation for spermatocyte 1.
10. Spermatocyte 1. 11,12. Spermatocyte 2. 13. Spermatids.
14, etc. Spermatozoa in different stages of differentiation.
Tur REPRODUCTIVE ORGANS.
The post-embryonic development of Culex has been worked
out by Hurst (1). The testes are paired, cylindrical in shape,
and possess no receptacule seminales, the ripe spermatozoa
being stored in the spermiducts.
The ovaries (Text-figs. 2 and 3) are paired and cylindrical,
each consisting of large numbers of ovarian tubes which all
open into a single duct. The two ducts, one from each side,
join to form a common oviduct into which three spermathece
open.
1 In a note appended to her paper, however, she expresses a fear that
two species were used for her research.
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 381
The gonad proper is contained in the third segment from
the hind end of the larva or pupa, which also is true for
C. pungens (Stevens (2)).
TEXT-FIG. 2.
A
2) BSS Po ea,
AT Oe
> ~ =
; ' : @ : cco
tt. ef. ot ear, <
ae Ace ‘2mm
- x o. t Ter OP pal Wo a
B
A. Section through ovary of Culex pipiens. c.o. Common
oviduct. e. f. Egg-follicle. o. Oviduct of right ovary. 0. t.
Ovarian tube. ¢.t. Tracheal tube. 3B. Diagram of ovarian
tube with egg-follicles.
TEXT-FIG. 3.
‘Os ™m™M
Section through ovary of Culex pipiens. e.f. Egg-follicle.
e. f.e. Egg-follicle epithelium. o. Oviduct. 0. ¢ i
tube. 7. c. Reproductive cells. ¢. ¢. Tracheal tube.
SPERMATOGENESIS.
A figure of a pupal testis drawn from a reconstruction is
given in T'ext-fig. 1. It will be seen that. the testis is divided
up into a number of small compartments by walls roughly at
right angles to the long axis of the organ. These cysts
contain cells in different stages of development, the contents
3882 MONICA TAYLOR.
of one cyst being presumably of the same age. In this par-
ticular testis the posterior cysts are full of ripe spermatozoa ;
higher up, the cysts contain spermatids; next to these are
cysts with immature spermatids, while higher up the com-
partments are full of interkinetic nuclei. Higher up still are
cells, which, by their more anterior position in the gonad, and
by their larger size, are presumably spermatocyte I, while at
the head of the gonad are nuclei in synizesis. Thus the
topographical relations of the cysts afford a means of identi-
fying the different stages in the spermatogenesis.
After consulting many sections of larve and pup, and
carefully studying the topographical relations of the cysts, it
has been possible to distinguish the usual spermatogenesis
stages, and this having been done, the recognition of the
different stages that occur in the development of each cell
generation was not a difficult matter. Although the contents
of one cyst are presumably of the same age, it is often possible
to find in it extremes of any stage. ‘Thus in a cyst charac-
terised by telophases of the first spermatocyte division there
may be a few anaphases, and some metaphases, and possibly
a few prophases. Working on this principle a cyst full of
early prophases will sometimes contain cells undergoing pre-
paration for prophases, and thus by linking up the informa-
tion gleaned by a study of these individual variations a full
series of stages can be obtained. Stress has been laid on the
topographical relations of the cysts for reasons that will be
apparent later.
A comparison of Text-fig. 1 with fig.5 in Stevens’ paper (2)
will show that in the case of Culex pungens any one gonad
contains a greater range of spermatogenesis stages than is
the case with C. pipiens. Only rarely in the latter case
are spermatogonial divisions found in the pupe and old larve,
these divisions taking place in younger larve. The usual
distribution of stages in old larvee and pupe of C. pipiens
is from synizesis to spermatozoa.
Resting nuclei in the testes of young larve resemble the
nuclei of the connective tissue (PI. 27, figs. 1, 2. and 3). In
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 9383
the densely staining nuclear sap the chromatin is more or less
peripherally arranged except fora central mass (PI. 27, fig. 1).
Resting nuclei are not found in old larve and pupe—the
synizesis stage apparently replacing the resting stage—nor are
they very common even in the young, as there is so much
active growth. In Pl. 27, fig. 3, a cluster of resting nuclei is
shown froma section of a larva stainedinthionin. The highly
staining capacity of the cytoplasm at this stage renders the
nuclei less conspicuous. Better differentiation is obtained in
iron-hematoxylin (PI. 27, fig. 2). In the more posterior parts
of the testis of young larve the cytoplasm of one cell is more
or less clearly marked off from that of its neighbours, but at
the head of the gonad the cytoplasm, densely provided with
metabolic products, forms a kind of syncytium in which large
numbers of small nuclei are indiscriminately scattered. It is
important to notice that there are no large dark-staining
bodies in the cytoplasm, which at this stage is uniform, these
dark bodies being confined to older larva and pupe.
Synizesis.—The telophases of the last spermatogonial
divisions are particularly interesting, because they show how
the synizesis nucleus has been formed, and, as the synizesis
nucleus marks the commencement of preparation for the first
meiotic divisions, the whole history of the nuclear changes
from the last spermatogonial division to the formation of the
spermatid can be traced.
A figure of one such telophase is given on Pl. 27, fig. 4.
The two daughter chromatin masses are still connected by the
remains of the spindle, and round about these masses is a
clear nuclear sap. The nuclear membrane is still present,
this persistence of the membrane being characteristic of
Culex mitosis as it is of that of many other insects. Hach
potential daughter-nucleus is possessed of a fairly thin rim
of cytoplasm in which are embedded dark-staining bodies.
The final separation of these two constituents would result in
the formation of two nuclei, each like that drawn in Pl. 27,
fig. 5—i. e. the synizesis nucleus. In this nucleus a volu-
minous and unstainable nuclear sap surrounds a “ coagulum”
384 MONICA TAYLOR.
consisting of chromatin and of a substance which is apparently
derived from the spindle apparatus.
This “coagulum”’ is frequently eccentric (Pl. 27, fig. 6).
The cytoplasm of the synizesis nucleus, as was foreshadowed
in the telophase of the mother-nucleus, is confined to a
narrow rim. Certain dark-staining bodies differentiated by
prolonged staining in iron-hematoxylin are to be found
closely apposed to the nuclear membrane, the rest of the
cytoplasm being more sparse. These dark-staining bodies
are most readily discovered in material fixed in Benda, aceto-
bichromate, and Flemming, and are only characteristic of the
cytoplasm of the later stages of spermatogenesis, that of earlier
stages being much more uniform, as has already been explained.
Pl. 27, fig. 7, shows a cell somewhat older than that given in
fig. 5, in which the dark mass of the synizesis nucleus has
increased in size, the chromatin being now arranged more or
less peripherally around a plasmosome, which stains very
palely in the thionin sections, the chromatin being difficultly
stainable except after prolonged treatment with iron-hema-
toxylin. In this latter stain the chromatin is seen to form a
fairly dense crown of closely matted fine threads around
a central space in which les the plasmosome.
The densely matted masses of chromatin around the plasmo-
some now become disentangled to a great extent and occupy
more space (Pl. 27, figs. 8 and 9). The underlying nuclear
sap does not stain so deeply, so that the chromatin is more
conspicuous. ‘The plasmosome stains more deeply in thionin
than it did in the stage represented by fig. 7. Fig. 8 re-
presents a section through, and fig. 9 an uncut nucleus of this
stage. In the latter figure the chromatin threads are shorter
and thicker than in fig. 8, in which the nucleus is slightly
younger. The cytoplasm of the cell is increasing and is
filled with small granules. It has a high staining capacity.
Synizesis has now broken up; the chromatin threads, which
have become thicker, lie against the nuclear membrane (PI.
27, figs. 10 and 11). In only a few cases is it possible to
count them, as they are so long and convoluted. Frequently
THE CHROMOSOMH COMPLEX OF CULEX PIPIENS. 385
the apices of the loops thicken and stain deeply. The
plasmosome is a highly staining and conspicuous structure.
Very often (Pl. 27, fig. 12) one chromosome thickens up
before the others, which are still long and zig-zag, and not
easily counted, or one part of the chromosome becomes locally
thickened. This stage occurs in cysts along with nuclei
containing fully formed chromosomes in late prophase. The
threads frequently show a double character.
Late Prophase (PI. 27, figs. 13 and 14).
The chromosomes have now condensed sufficiently to make
the investigation of their number easy. In every case it 1s
possible to count three chromosomes, which are sometimes
double, as is often the case in meiotic prophases. ‘The round
nucleus is surrounded by a much greater quantity of cytoplasm
than formerly. The chromosomes present great variety of
shape. They are rod-shaped, club-shaped, dumb-bell-shaped,
while crosses and rings are of frequent occurrence, these latter
being formed by the partial separation of the chromosomes
preparatory to metaphase. This preparation for metaphase
is very often evident in the twisted character of the chromo-
somes, three pairs of twisted chromosomes frequently appear-
ing (compare Stevens’ fig. 14).
Metaphase (PI. 27, figs. 15-18).
The cell itself becomes spindle-shaped. This characteristic
change of shape affords help in distinguishing meiotic from
spermatogonial divisions, the metaphases and anaphases of the
latter taking place in round-shaped cells, the shape of the cell
being in no wise affected by the formation of the spindle.
The spindle shape is well shown in figs. 15-18.
A typical anaphase is illustrated by Pip 24, se. 19), Ewe
of the chromosomes have each almost separated into daughter
halves, the two daughter halves being merely united end to
end. The third chromosome is not completely shown in the
figure, part of it having been cut away.
386 MONICA TAYLOR.
Telophase (Pl. 27, fig. 20).
The cell now becomes greatly elongated and the chromo-
somes massed together. Spaces become apparent in the
daughter chromatin masses, as shown in the left-hand mass
in fig. 20. In the next stage these clear spaces have increased
to such an extent that the central mass of the nucleus is free
from chromatin, the latter occupying a peripheral position
against the nuclear membrane (Pl. 27, fig. 21). It is inter-
esting to compare what takes place here with what happened
in the formation of the synizesis nucleus. In the latter case
(Pl. 27, fig. 4) the vacuoles appeared round the daughter-mass
of chromatin, converting this into the synizesis nucleus. In
this case, however, the vacuoles appearing in the daughter
mass, the chromatin is squeezed against the nuclear membrane
(Plo 27; fig. 21).
The cytoplasm in the cells, shown in figs. 12-20, is very
conspicuous by its bulk, and in specimens fixed in Gilson-
Petrunkewitch and Flemming (under certain conditions) it
is homogeneous and deeply stainable in thionin. In some
cells, notably those fixed in Benda, the cytoplasm is more
like that of those cells represented in figs. 49-52, where it
appears to be sharply differentiated into a more or less fluid
substance and a few darkly stainable bodies.
Second Meiotic Division.
As has already been explained, no reticular resting stage
follows the telophase of the first meiotic division. Vacuoles
appearing in the daughter chromatin masses push the chro-
matin against the nuclear membrane rendering it almost
invisible (Pl. 27, fig. 21). Next a plasmosome, most readily
demonstrated in sections stained in Ehrlich, appears, and the
chromatin thickens somewhat (fig. 22). Later (fig. 23) a
“ clock-face”’ stage similar to that which occurs in the develop-
ment of the primary spermatocyte results. The chromosomes
then thicken (fig. 24), though they are still in contact with
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 387
the nuclear membrane. Finally (fig. 25) three chromosomes
appear in the prophase. Crosses and rings are again present,
formed by the precocious longitudinal split, together with a
divergence of the daughter halves. Often the chromosomes
are so thick and close together that it is not easy to count
them (figs. 26 and 27).
In metaphase the spermatocyte II cells are again spindle-
shaped (figs. 28 and 29), but they are roughly only two thirds
the linear dimension of the primary spermatocytes, and, as
already explained, their position in the gonad, irrespective
of their size, would render their identification an easy matter.
Fig. 30 shows an anaphase of this division.
Formation of Spermatids (figs. 31 and 32).
In the round daughter-nuclei which result from the second
meiotic divisions the chromatin is again to be found against
the nuclear membrane (fig. 31). The nucleus now becomes
elongated, the chromatin, being still peripheral, gradually
diminishes in size, and finally assumes a rod shape.
Before going on to discuss the spermatogonial divisions, it
may be well, in view of the difficulty of recognising the
spermatogonial cells, to give some account of the nuclei in the
undifferentiated gonads of young larve.
In the following account whenever the sex is stated it must
be remembered that it is only tentatively given. ‘The presence
of cyst walls in the gonad has been the criterion for tentatively
assigning the sex in the case of the male larve.
The ovary is very richly provided with tracheal tubes, and
at a very early stage it is possible to identify these tubes.
The somatic cells forming the walls of the ovarian tubes can
also be distinguished from the germ cells at a very early
stage. By means of these criteria it is often possible to
identify as an ovary the gonad of a very young larva. How-
ever, in many cases it is not possible to say on which side lies
the balance of probability.
The general facts of mitosis in very young larve are illus-
388 MONICA TAYLOR.
trated by Pl. 27, figs. 33-40. Froma study of these figures it
will be seen that the number of chromosomes in undifferen-
tiated gonads, as well as in those of young male and female
larvee, is 3. :
Synizesis nuclei occur in very early stages of both male and
female (figs. 33 and 40). The two telophases illustrated in
figs. 37 and 38 do not suggest that the daughter-nuclei will
pass immediately into the synizesis nuclei (cf. fig. 4). They,
therefore, belong to early generations, spermatogonial.
The effects of aceto-bichromate fixation are illustrated in
figs. 59 and 40, where various metabolic constituents can be
discovered in the cytoplasm (cf. figs. 53, 56, and 88).
Spermatogonial Mitosis in Young Larve.
Mitotic divisions in the fully differentiated, though imma-
ture, gonad of fairly young larve will now be discussed. In
such larve the hinder ends of the gonad contain the more
advanced stages of spermatogenesis, the degree of differentia-
tion of spermatozoa, of course, depending on the age of the
larvee—but the anterior parts of the testis present a great
difference in appearance from that of old larve and pupe.
Conspicuous in such gonads is the absence of synizesis stages.
The cellular elements are much smaller—the cytoplasm form-
ing a syncytium, in which the small nuclei are embedded.
This, the “ multiplication” stage of spermatogenesis, seems
to be confined to young larvee.
A series of division stages taken from this “multiplication”
zone is given, the number of chromosomes being three (PI. 27,
figs. 41-51, and figs. 2 and 3).
The main point which emerges from a study of these nuclei
is that the diploid number of chromosomes cannot be demon-
strated in Culex pipiens. One cannot, therefore, use the
number of chromosomes to distinguish spermatogonial from
spermatocyte divisions. Still, a careful study of all the facts
seems to show that the occurrence of a synizesis stage marks
off the earlier divisions from those of spermatocyte I. The
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 389
divisions that occur in young larve are spermatogonial
divisions, although they possess the haploid number. With
regard to those synizesis stages in probable male larva, it
must be remembered that some cells in quite young speci-
mens differentiate very quickly. Hence the fact that synizesis
stages do occur in such young creatures does not argue against
the statement that the occurrence of a synizesis stage marks
off spermatogonial nuclei from spermatocyte I.
OoGENESIS.
As already stated in the introduction, a full history of the
facts of oogenesis can only be given when the necessary
material has been collected. However, for present purposes
it is only necessary to figure a few stages in order to show
that the number of chromosomes in the germ-cells is three.
Three chromosomes in prophase are shown in PI. 27, fig. 52,
taken from a young larva before the “rosettes”? are well
differentiated. The cell in which they occur clearly belongs
to an early generation.
A prophase drawn from a section of an imago, ten days old,
is shown in Pl. 27, fig. 53. The cell is from a young egg-
follicle, and as it is stained in thionin after fixation in Gilson-
Petrunkewitch, the cytoplasm is very densely stained.
A metaphase showing six chromosomes is given in Pl. 27,
fig. 54.
The drawing in PI. 27, fig. 55 is taken from an old larva.
It shows a prophase where the number of chromosomes is
three. The cytoplasm is abundant. *
Pl. 27, fig. 56, taken from an egg-follicle of a captive-reared
imago, illustrates the oldest stage obtained. The nucleus,
with its deep-staining plasmosome, and its fine, well-dis-
tributed reticulum of chromatin, forms a striking contrast to
the deep-staining, voluminous cytoplasm that surrounds it.
Somatic Mrrosts.
Although there is much indirect evidence to show that the
divisions described as spermatogonial are really divisions of
390 MONICA TAYLOR.
the earlier generations, in spite of the haploid and not diploid
number of chromosomes, yet, in view of the possibility that
they might be precocious spermatocyte I divisions, it has
been thought advisable to work out the somatic mitosis of
C. pipiens very thoroughly.
Mitosis in the Somatic Tissues of the Ovary
(Text-figs. 2 and 3).
The somatic cells of the ovarian tubes of the ovary can
be distinguished from the reproductive cells in very young
larvee by their minute size. The developing ovarian tubes
look like rosettes in sections cut from older larve, the large
central cells of the rosette being reproductive cells, the peri-
pheral cells, much smaller in size, being somatic. Later on
groups of from four to eight of these reproductive cells
become enclosed in an epithelium—the egg-follicle epithelium
—also somatic in character. The number of these egg-follicles
increases with the age of the creature, the ovary having
assumed its definitive arrangement in late pupal life. The
exceedingly thin walls of the ovarian tubes, the nuclei of
which are very small, are greatly distended by the large egg-
follicles, which are made up of one egg-cell surrounded by a
varying number of nurse-cells.
The ultimate fate of egg- and nurse-cells has still to be
worked out. The somatic tissue of the ovary—whether the
epithelium of the tubes, or the egg-follicle epithelium—is a
prolific source of mitotic figures. In all cases the number of
chromosomes is three.
A cell belonging to an egg-follicle in telophase is shown in
Pl. 27, fig.57, and near it is shown a reproductive cell (fig. 58),
which also brings out the difference in size between the
somatic and reproductive cell.
Pl. 27, figs. 59 and 60, also show three chromosomes in
typically somatic anaphase.
Innumerable figures of prophases could be drawn from the
preparations, it being characteristic of Culex pipiens that
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 391
metaphases and anaphases are comparatively difficult to find,
prophases being much more abundant.
In the case of all the cells of the ovary the number of the
chromosomes is three.
Somatic Mitosis (General).
Somatic mitoses, apart from those in the ovarian tissue, are
by no means easily found. They do not seem to be confined
to any particular period of larval or pupal life, or to take
place at any fixed hour of the day or night.
The process, as observed in nerve-cells, is illustrated by
Pl. 27, figs. 61-65, and Pl. 28, figs. 66-68.
It will be seen from these figures that there is a great
resemblance between the reproductive cells, at certain stages
of development, and the somatic cells in the nerve ganglia.
Similar likenesses between the body-wall cells and those of
the gonad could also be demonstrated. Thus a very well-
marked synizesis stage is characteristic, not only of the later
stages of spermatogenesis and of the reproductive cells of the
ovary, but also of the cells of the nervous system, and the
gradual breaking up of the synizesis resembles, in the main,
that which takes place in the gonad. Hence it would appear
that synizesis has not the significance in the spermatogenesis
of Culex that it is commonly believed to have in other
creatures, since this phenomenon is not confined to repro-
ductive cells.
An investigation of the tracheal tube-cells shows that the
number of chromosomes is three. Very frequently the
daughter halves of the split chromosome can be seen in late
prophase (PI. 28, figs. 69-71).
Evidence as to the number of chromosomes derived from a
study of the body-wall cells (illustrated by PI. 28, figs. 72-79)
confirms the results already obtained elsewhere, while in the
undifferentiated somatic cells in larvee just hatched (PI. 28,
figs. 80 and 81) the number is again three.
The alimentary canal wall splits into two layers during
392 MONICA TAYLOR.
pupal life, the inner layer undergoing disintegration (Hurst
(1)). Some of the mitoses that occur in connection with this
process are illustrated in Pl. 28, figs. 82-89. The number of
chromosomes is three, and in some cases the precocious ten-
dency of the chromosomes to divide in prophase for metaphase
is again evident.
Mitosis in Malpighian tubule cells is figured in Pl. 28, figs.
90-95, while fig. 96 shows an equatorial plate from a muscle-
forming cell. In all cases the somatic number of chromosomes
is three.
Discussion.
In C. pipiens, as has been shown, there appear to be two
maturation divisions, though no reduction of chromosomes can
be demonstrated. This is contrary to general experience, for,
as is well known, when, in any organism, the ripe germ-cell
has the same number of chromosomes as the somatic tissues,
one of the meiotic divisions is commonly omitted. It must
be remembered, however, that while two divisions undoubtedly
follow the synizesis stage in C. pipiens, the fact that they
follow synizesis is the only one which has led to their separa-
tion off from the apparently similar earlier divisions, and to
_their being described as meiotic rather than spermatogonial.
However, as nuclei closely resembling synizetic nuclei can be
found in the somatic tissues of this creature, it is, therefore,
possible that the synizesis nuclei in the testis have no real
value in diagnosing the beginning of the meiotic phase, and
that they merely represent a stage of inactivity. The rapid
divisions in the “ multiplication ”’ zone result in the formation
of large numbers of nuclei which, while they are awaiting
differentiation into spermatozoa, remain as synizesis’ nuclei.
When the time comes for this differentiation the synizesis
nucleus begins to be active, and the stages in this awakening
are analogous to the formation of spermatocyte I cells.
From the foregoing account, it will be seen that the para-
syndesis for each cell generation which Miss Stevens described
for C. pungens cannot be demonstrated for C. pipiens.
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 3893
No figures indicating the presence of six chromosomes are to
be found which cannot readily be interpreted as three chromo-
somes precociously split for metaphase. For example, con-
ditions like those Miss Stevens gives in her figs. 1 and 2
(oogonia showing three pairs of chromosomes on equatorial
plate) and in figs. 8, 9, and 10 (spermatogonial cells showing
all three pairs in late prophase), in thelight of other evidence,
must, when they occur in C. pipiens, be described as three
chromosomes already divided for metaphase. The conditions
of C. pungens, however, offer a suggestion as to how the
haploid number of chromosomes in C. pipiens may have been
derived. The permanent fusion of the paternal and maternal
members of the’ pair, i. e. the conversion of parasyndesis into
actual fusion, would result in the formation of three out of six
chromosomes.
Miss Stevens states that in C. pungens the intimate re-
lationship of the two conjugants persists from one cell genera-
tion to the next, the pairing taking place in telophase, and
persisting until the metaphase of the next mitosis: From this
it would seem that the conjugating chromosomes are only
“unfused ” in metaphase. In the case of C. pipiens the
pairsare fused throughout the whole mitosis, hence the haploid
number.
On the other hand, it is quite possible to give a different
interpretation of Miss Stevens’ figures of parasyndesis from
the one she offers. It is significant to note that she gives no
figures in support of her statement that each of the six chromo-
somes (i.e. each member of the three pairs of conjugating
chromosomes) found in the oogonial and spermatogonial
generations divides longitudinally. She merely states the fact
that they do so. Unless this division can be demonstrated, it
would seem as though the so-called conjugating chromosomes
were merely the daughter-halves of a precociously split
chromosome, as is the case in C. pipiens.
An alternative suggestion, therefore, as to the chromosome
complex of Culex pipiens and pungens is, that the somatic
number is the same as that of the mature gamete, being three
voL. 60, PART 3.—NEW SERIES. 28
394 MONICA TAYLOR.
an each case. This alternative would seem to involve the
non-participation of one of the gametic nuclei in development.
Whether this is the case, or whether the homologous
chromosomes are temporally united in each cell-generation of
C. pungens, and permanently so in C. pipiens, can only be
settled by an examination of the process of fertilisation, which
I hope to undertake in the near future.
I am greatly indebted to Dr. Agar for much valuable
‘criticism ; to many friends who have assisted me in collecting
material; to Mr. P. Jamieson for cutting the more important
sections ; and to Professor Graham Kerr for his sympathetic
encouragement.
SUMMARY.
(1) The somatic number of chromosomes is three, both-in the
male and female of Culex pipiens.
(2) The number of chromosomes in the spermatogonia as
well as in the primary and secondary spermatocytes and
‘spermatids is three.
(3) The spermatogonial cells are not characterised by a
‘synizesis stage, which latter stage marks off the spermatogonial
from the spermatocyte I stage.
(4) The nuclear membrane persists throughout mitosis.
(5) The synizesis stage represents an inactive phase of the
nucleus in spermatogenesis.
(6) A synizesis stage occurs in somatic nuclei.
LITERATURE.
1. 1890. Hurst, C. Herbert.—‘‘ Post-Embryonic Development of Gnat,”
‘Trans. Liverpool Biol. Soc.,’ vol. iv.
2. 1910. Stevens, N. M.—‘“ The Chromosomes in the Géaee cells of
Culex,” ‘ Journ, Exper. Zool.,’ viii.
THE CHROMOSOME COMPLEX OF CULEX PIPTENS. 3995
EXPLANATION OF PLATES 27 anp 28,
Illustrating Miss Monica Taylor’s paper on “ The Chromosome
Complex of Culex pipiens.”
[ All figures (except 3 and 6) were drawn with the Abbé camera under
Leitz ;, oil-immersion objective and Zeiss compensating ocular 12,
giving a magnification of 3300 diameters. This has been reduced 4,
i.e. to a magnification of 2200. Figs. 3 and 6 are drawn to a magnifi-
cation of 1900, giving a final magnification of about 1270. |
PIA 27.
Fig. 1.—Resting nucleus in connective-tissue.
Fig. 2.—Resting nucleus from gonad of young ¢ larva.
Fig. 3.—Group of nuclei drawn to same scale as fig. 6 from young
& larva; thionin stain.
Fig. 4.—Telophase of spermatogonial cell from head of testis of pupa.
Figs. 5-32 are taken from pupal testes.
Fig. 5.—A synizesis nucleus from head of testis of pupa.
Fig. 6.—A group of synizesis nuclei from a pupa fixed in Benda, and
‘deeply stained.
Fig. 7—A nucleus showing early stage in the breaking up of
synizesis.
Fig. 8.—F rom a section 4 » thick, showing later stage in the breaking
up of synizesis.
Fig. 9.—From a section 8 » thick, showing nucleus slightly older than
in fig. 8.
Fig. 10.—View of nucleus from one pole, showing the chromatin close
to the nuclear membrane.
Fig. 11.—Optical section through a nucleus which at this stage is
very characteristically like a “ clock-face.”
Fig. 12.—Nucleus showing that the thickening up of the chromo-
somes is not always uniform.
Fig. 13.—Advanced prophase.
Fig. 14.—Advanced prophase.
Fig. 15.—Very early metaphase, showing the spindle-shape of the cell
before the mitotic spindle has been organised.
396 MONICA TAYLOR.
Fig. 16.—A more elongated spindle-shaped cell. Early metaphase.
g. 17.—Metaphase, with indistinguishable chromosomes.
Fig. 18.—Metaphase in which one of the chromosomes has divided.
Fig. 19.—Later anaphase. Only one of the three dividing chromo-
somes is completely shown in the figure.
Fig. 20.—Telophase.
Fig. 21.—Interkinetiec nucleus.
Fig. 22.—Interkinetic nucleus stained in Ehrlich, showing a plasmo-
Fig. 25.—* Clock-face” stage for spermatocyte II nucleus.
Fig. 24.—A nucleus of spermatocyte II, in which chromosomes are
becoming visible.
Fig. 25.—A nucleus of spermatocyte II, showing three chromosomes.
Fig. 26.—Advanced prophase.
Fig. 27.—Later prophase; the shape of the cell is just beginning to
change.
Fig. 28.—Early metaphase.
Fig. 29.—Metaphase ; three chromosomes on equatorial plate.
Fig. 30.—Anaphase.
. 31.—Newly formed spermatid.
oe
5
Fig. 52.—Immature spermatid.
Figs. 33-40 are drawn from gonads of very young larve.
Fig. 35.—A synizesis ina ¢ (?) larva.
Fig. 34.—Prophase from a young ¢ (?) larva.
Fig. 35.—Metaphase from a young ¢ (?) larva.
Fig. 36.—Metaphase from gonad of larva.
° de
Fig. 37.—Anaphase from gonad of larva.
Fig. 38.—Anaphase from ¢ (?) larva!
Fig. 39.—Acetic bichromate and thionin preparation. 9 (?) larva.
Fig. 40.—Acetic bichromate and iron-hematoxylin preparation.
2 (?) larva.
Figs. 41, 42.—Prophases, spermatogonial; the number of chromo-
somes is three.
Fig. 45.—Equatorial plate, showing three chromosomes; a young
¢ larva, spermatogonial.
Fig. 44.—Metaphase, spermatogonial, three chromosomes.
Figs. 45, 46.—Late spermatogonial anaphases from young ¢ larva.
THE CHROMOSOME COMPLEX OF CULEX PIPIENS. 397
Figs. 47—51 are from head of testis of pupa.
Fig. 47.—Prophase, spermatogonial, from head of gonad of pupa
(cf. Stevens [fig. 10 (2) |). There are two long chromosomes which have
split longitudinally, and one smaller one not yet divided into daughter
halves.
Fig. 48 shows the threads of the spindle just beginning to form
inside the nuclear membrane, the chromosomes being too massed
together to be counted.
Fig. 49.—Metaphase; an equatorial plate where the three chromosomes
are just separating into their daughter-constituents.
Fig. 50.—Later metaphase, six chromosomes being easily counted.
Fig. 51—EHarly anaphase. Note the round-shaped nucleus, and the
somatic-like character of the V-shaped daughter-chromosomes.
Figs. 52-56 are taken from ovaries.
Fig. 52.—Prophase from young @ larva.
Fig. 53.—Prophase from 2? imago.
4.—Metaphase from ? larva.
Fig. ?
Fig. ¢
5.—Prophase from old 9 larva.
=
go
(re HSne Sn.e Lng (ne
6.—From 2 imago, two months old.
Figs. 57, 58.—Ege-follicle. Epithelium cell with nucleus in late
anaphase (57), and nurse or egg cell drawn to same scale (58). (2
imago section.)
Figs. 59, 60.—Ege-follicle epithelium cells in late anaphase, showing
three chromosomes. (From a section of a 2? Imago.)
Figs. 61-65 from nerve-ganglia.
Fig. 61—Synizesis nuclei deeply stained in Ehrlich from old
larva (ef. figs. 5, 6, and 40).
Fig. 62.—Nerve-cell showing six blocks of chromatin in the “coa-
gulum.” Old @ larva.
Figs. 63 and 64.—Nerve-cells showing the breaking up of synizesis.
Old 9 larva.
Fig. 65.—EKarly prophase from ¢ pupa (cf. fig. 10).
PLATE 28.
Fig. 66.—Early prophase of a nerve-cell from a larva just hatched.
Compare PI. 27, fig. 11.
Fig. 67.—Prophase of a nucleus of a nerve-cell from a larva just
hatched, Three chromosomes showing precocious splitting.
398 MONICA TAYLOR.
Fig. 68.—Prophase of a nucleus of anerve-cell from a young d larva
Fig. 69.—Three chromosomes in the nucleus of a tracheal tube-cell
from a young ¢ larva. Compare this with figs. 1, 9, and 10 of ‘The
Germ Cells of Culex,’ Stevens (2).
Fig. 70.—Tracheal tube-cell from young (?) larva.
Fig. 71.—Ditto.
Figs. 72-79 are drawn from body-wall cells.
Fig. 72.—Early prophase of a nucleus from a ? imago.
Fig. 73.—Three chromosomes in prophase from a $ larva.
Fig. 74.—One short and two long chromosomes. Prophase of nucleus-
from a young ¢ larva. Note indication of two daughter-halves in one
of the chromosomes.
Fig. 75.—Three chromosomes in advanced prophase from ? larva.
Fig. 76.—Equatorial plate. Side view showing three chromosomes ;
one of them is cross-shaped.
Fig. 77.—Anaphase from a @ larva.
Figs. 78 and 79.—Telophases from @ larve.
Figs. 80 and 81.—Undifferentiated cells of a larva just hatched,
showing three chromosomes in prophase and late metaphase.
Figs. 82-89 are drawn from cells in alimentary canal wall.
Figs. 82, 83, 84.—Prophases from cells in alimentary canal wall of
9 larve.
Figs. 85, 86.—Prophases from cells in alimentary canal wall of a 9
imago, ten days old. In fig. 85 one of the chromosomes has divided
longitudinally.
Fig. 87.—Prophase from alimentary canal wall of 9 larva.
Fig. 88.—Metaphase from cell of same larva.
Fig. 89.—An equatorial plate showing three chromosomes from wall
of intestine.
Figs. 90-95.—Malpighian tube-cells.
Fig. 90.—From young d larva.
Fig. 91.—From 2 larva.
Fig. 92.—From young ¢ larva.
Fig. 93.—From young ¢ larva.
Fig. 94.—From larva.
Fig. 95—From @ larva. Note cross-shaped chromosome.
Fig. 96.—Equatorial plate showing three chromosomes; two of them
ringed, from muscle-cell of young larva.
STUDIES ON AVIAN HH MOPROTOZOA, 399
Studies on Avian Hemoprotozoa: No. III.—
Observations on the Development of Try-
panosoma noctue (of the Little Owl) in
Culex pipiens; with Remarks on the Other
Parasites occurring.’
By
H. M. Woodcock, D.Sc.Lond.,
Assistant to the University Professor of Protozoology.
With Plates 29—31 and 1 Text-figure.
CONTENTS.
PAGE
PREFACE P 5 ate)
GENERAL hewamrn OF THE Senna, WorRK . . 400
DESCRIPTION OF THE PARASITES:
(i) Trypanosoma noctue . : . 406
Comparison with the cultural dev elonrent of T. frin-
gillinarum . . 409
(ii) The ookinetes of Eialthontdi aan odie and Leuco-
cytozoon ziemanni . : : . 421
(iii) The “resting Flagellates ” . ; . 423
GENERAL CONCLUSION REGARDING THE | Communes OR
OTHERWISE OF THE DIFFERENT PARASITES . . 425
PREFACE.
I propose in the present paper to conclude the account of
the work on the parasites of the Little Owl (Athene noctua)
which I carried out at Rovigno during the spring and early
‘For No. II of these Studies, “On the Trypanosome of the Little
Owl,” etc., vide ‘Quart. Journ. Mier. Sci.,’ vol. 57, 1911, p. 141.
400 H. M. WOODCOCK.
summer of 1909. I have delayed publishing up to the
present my observations on the developmental phases of the
Trypanosome in the mosquito, as I hoped to be able, before
now, to obtain the corresponding phases, and, indeed, the
transmission back again to the bird, of Trypanosoma
fringillinarum, here in England, to complete the work.
This latter research, however, progresses, unfortunately, very
slowly, so that I think it useful to publish my earlier observa-
tions without waiting longer, more especially as the only
other worker who has written anything of late on this subject,
namely, Mayer (2), in his account of the parasites of another
owl (Syrnium aluco), has upheld the view that the Flagel-
lates occurring in mosquitoes which have fed on an owl are
developed from Halteridia present in the blood of the bird.
In my “ Notes on Sporozoa, No. IV ” (14), dealing with the
nuclear structure of Halteridium and Leucocytozoon, I
think I have shown clearly that, considered from the stand-
point of these parasites, everything is against such a connec-
tion of either with a Trypanosome. And the evidence which
I have obtained from the Trypanosome side of the question is
equally negative, and does not bear out Mayer’s contention
in the slightest degree.
GENERAL ACCOUNT OF THE EXPERIMENTAL WORK.
The time at my disposal for experimental work with Culex
was only very short—about the last three weeks of June.
Earlier than this the bred-out females would not take blood,
and at the beginning of July I was unfortunately obliged to
leave Rovigno. Five owls were used, to which it will be
convenient to refer by their numbers, viz., 15, 16, 19, 22, and
23. The first two were quite free from any infection; No. 22
had only Leucocytozoon, while Nos. 19 and 23 both had
Halteridium noctue, Leucocytozoon ziemanni, and
Trypanosoma noctue. As regards Owl 19, the Trypano-
some was for some reason or other excessively scanty, and
was not, I believe, present at all in the general circulation
STUDIES ON AVIAN HAMOPROTOZOA. 401
(cf. Study No. II). The Halteridial infection of Owl 19 was
quite typical, the parasites, which, of course, were in the
usual form of male and female gametocytes, being fairly
numerous, and many of them full-grown and ripe for liberation
from the host-cells. On the other hand, the Halteridial
infection of No. 23 was not typical. In this owl nearly every
red blood-cell was infected, usually with several (four or
more) small forms, which in many cases, as they had increased
in size, had united together into a kind of common plasmodium.
This condition has been already described by me elsewhere
(13). I observed hardly any full-grown, normal gametocytes
in the blood of this bird. Owls 22 and 23 only arrived on
June 12th.
Karly in June, finding that my bred-out females would not
yet bite, I left Owl 19 one evening ina cage into which I had
put half-a-dozen or so “wild” females, caught in the same
little outhouse, used as a ‘ dark-room,” where Schaudinn
himself had caught many in time past. Before midnight two
of these had fed. I may note here that the favourite place
for feeding of the mosquitoes was the fleshy pad just above
the bird’s nostrils. One of the two females was examined
atter about thirty-six hours had elapsed, this being the period
when, according to Schaudinn, the ookinetes become trans-
formed into Trypanosomes. Its stomach contained a number
of fully-developed ookinetes, i. e. which had lost all the
pigment. he majority had the characteristic curved form,
but did not appear at all active. A few, however, showed a
certain amount of activity, which consisted in tending to
straighten out, and again recurve, the body, either slowly or
now and again spasmodically. I never observed any marked
forward progression of the ookinetes.
Besides the ookinetes certain other bodies were found to
occur, very scantily, in the preparation. These elements
were more elongated and spindle-shaped, somewhat resembling
an Indian club in form, one extremity tapering finely, the
other being rounded off. Further, some of them were very
slightly curved or crescentic. Apart from the difference in
402 H. M. WOODCOCK.
shape, the general appearance of these bodies, observed
living, was not at all unlike that of the ookinetes. They
were quite non-motile. While I was in the act of observing
one, and wondering whether it was a later stage in the
development of an ookinete, it gave one or two very slight,
jerky movements, and before I had fully assured myself that
these really represented active, voluntary motion on the part.
of the parasite, to my great surprise it had developed a
flagellum. I thought I had just an indication of the tapering
end of the body beginning to lengthen, but more than this I
did not see. One moment the parasite had no flagellum, an
instant later it had a fully-developed free flagellum, about
three-quarters as long as its own body. ‘The process must.
have been exactly comparable, in short, to that which I have
since found to occur in Leptomonas (“Crithidia”)
fasciculata (vide 15). I hurriedly brought a colleague,
Dr. Reichenow, then also working at Rovigno, to look, and
he, in turn, saw the process repeated in the case of another
individual. ‘hese were the only two instances in which we
saw the development of the flagellum, and we only found
one other flagellated form in the preparation. We both
carefully examined several of the other (non-motile) elements,
and satisfied ourselves that they had no sign whatever of
aflagellum. IfI may be permitted the personal reminiscence,
I well remember how, in the excitement of the moment,
we were both of us firmly persuaded that we had seen the
most important stage in the transformation from an ookinete
into a ‘l'rypanosome, as it had been described by Schaudinn.
This confidence did not endure, however, for many days. I
continued to watch certain ookinetes during the afternoon,
and felt very disappointed that I could not see any indication
whatever of a typical ookinete passing into one of the fusiform
bodies. After keeping the preparation under observation for
about three hours I removed the coverslip and made smears,
which were fixed and stained.
Most unfortunately, Owl 19 was taken ill and died on the
following day, and for some days I had no owl infected with
STUDIES ON AVIAN HZ MOPROUTOZOA. 403.
Trypanosomes. During this interval I fed several caught
mosquitoes on an uninfected bird (either 15 or 16), and
these were examined at periods of from thirty-six to fifty-
eight hours after being fed. In two cases, elongated, fusi-
form elements, perfectly similar to those above-mentioned,
were found; but none of them was seen to become active or
develop a flagellum. In another female, examined about
fifty-four hours after being fed, numerous active Flagellates
were observed in the stomach; these differed considerably in
appearance from the “resting Flagellates,”’ but, on the other
hand, agreed closely with the characteristic developmental
forms described below, and I have not the least doubt that
they also belonged to the life-cycle of some Avian Trypano-
some. It is important to note that no ookinetes. were seen 1n
any of these mosquitoes. These observations showed not
only that the fusiform resting Flagellates might probably have
another origin than from the ookinetes of Halteridium, but
also that it was essential to use only bred-out females, in
order to follow the course of development taken by the blood-
parasites in the Culex; hence, from this time onwards only
such individuals were used.
On June 12th Owl 23 arrived, and during the night of the
15th—16th I found Trypanosomes in the peripheral circulation ;
the parasites were of the characteristic fusiform, rather stout
type (Pl. 31, fig. a), described by Minchin and myself (I. c.).
The Trypanosomes were not at all infrequent—for Avian
Trypanosomes, it must be remembered—in the peripheral blood
at this time,! and were found also on other occasions. In both
of the two first mosquitoes which were examined after being
fed on this bird, after intervals of about thirty-four and forty
hours respectively, numerous active Flagellates were found in
the stomach. Digestion was proceeding normally and was
about half accomplished, or rather more. (I may state here
that females which had taken blood were always kept at a tem-
' In any living preparation, consisting of a small drop of blood spread
out into a thin layer under a coverslip seven-eighths of an inch square,
there would be one or two Trypanosomes.
404, H. M. WOODCOCK.
perature of from 25°-27° C., at which temperature digestion
took three to four days to be completed.) The develop-
ment of the Trypanosomes appeared to be at about the same
stage in both the females, and in both similar phases were
observed.
Ookinetes of Halteridium were also found, but in both
mosquitoes they were few in number—quite scarce, in fact,
when compared with the number present in the first female
examined, which had fed on Owl 19 (cf. above). This was
readily to be understood, bearing in mind the different con-
dition of the Halteridial infection in the two birds; in spite
of the very strong infection of Owl 23, there were nothing like
so many full-grown, ripe gametocytes asin Owl 19. A living
preparation made from one of these two stomachs was kept
under observation for some time. Three ookinetes in different
fields, all of which had lost their pigment grains, were noted
at intervals during two hours, but none of them showed any
change in form or the slightest indication of any development
into one of the fusiform bodies or into a flagellate condition.
The preparation was again looked at two hours later, with
the same result in the case of two of the ookinetes; the third
could not be found. By this time most of the Flagellates
seemed to be dead—at all events, only two or three could be
observed, and these were very sluggish.
Altogether, twenty-six female Culex pipiens which had
fed on Owl 23 were examined, after intervals varying from
about twelve to eighty hours. Flagellates were found in
twelve of these, i.e. in about 46 per cent.; sometimes they
were numerous, in other cases only few were seen. On the
other hand, out of thirty-two females fed on one of the other
owls (Nos. 15, 16, 22), none of which was infected with
Trypanosomes, the stomachs of which were carefully examined
after different intervals (thirty-six to fifty-four hours), in not
a single case were the parasites found! T'wo of these birds
were quite free from any Heemoprotozoan infection ; Owl 22,
however, had a fairly strong infection of Leucocytozoon
ziemanni. Seven of the thirty-two mosquitoes examined
STUDIES ON AVIAN HU®MOPROTOZOA. 4.05
had fed on this latter bird,! and in four of them a few of the
large ookinetes of Leucocytozoon were found. In form
and appearance these were very similar to the ookinetes of
Halteridium, but they were considerably larger. Those |
observed were quite motionless, and did not change at all in
shape.
The fifty-eight mosquitoes examined were barely half the
total number (about 120) which fed on blood. The mortality
amongst these newly bred-out imagines was high, and it
appeared to make little difference whether they took blood or
the sugar-water, banana and prune juice with which they
were supplied. More than a quarter of those which fed on
blood died during the course of digestion. During the short
time at my disposal I had, therefore, only very limited
material for transmission-experiments. During the last fort-
night between fifteen and twenty females which had fed on
Owl 23 and successfully completed digestion, were given the
opportunity of feeding on an uninfected bird (either 15 or
16). To my very great disappointment, however, not one of
these could be induced to feed again. Some of them drank a
little water, or partook of the food-supply which was placed
in the same cage for a few hours during the day-time for the
males to feed upon; nevertheless, many of them gradually
died off during this period. I was loath to sacrifice any more
for examination, hoping to the very morning of my departure
from Rovigno that one or more would bite again and give me
the chance of seeing whether one of the uninfected birds
would become infected. Unfortunately, the endeavour did
not succeed. Owls 15 and 16 accompanied me back to
England and lived for many months—much longer than a
single bird did at Rovigno—but neither of them ever showed
any parasites at all.
While restricting myself to bred-out females for the experi-
mental work, incidentally I examined a few more caught
“ wild” mosquitoes. None of them was infected with active
Flagellates (i. e. Trypanosome developmental forms), but again
1 This owl died on June 22nd, so that I had it only ten days.
406 H. M. WOODCOOK.
two or three contained the peculiar, spindle-like resting forms,
above noted; in no further instance, however, did I see one
develop a flagellum. I have since wished that I had been
able to devote more time to the study of this parasite, and to
ascertain, for example, whether it occurred in male mosquitoes
also. But I was intent on proving the origin of the Flagellates
which developed in the blood-fed females, and in transmitting
them back to the birds, if possible, and this took every minute
of my time, as I had no assistance whatever and had every-
thing to do myself.
DESCRIPTION OF THE PARASITES, AS THEY WERE FOUND IN THE
MosqQtitTors.
(i). Trypanosoma noctuae.
I pass on now to describe the developmental phases of the
Avian parasites in the female Culex pipiens, and begin
with those of Trypanosome noctunae. Considering the
Flagellates, first of all, as they were seen in life, in mosquitoes
examined about thirty-six hours after being fed on an infected
owl, the most striking form, which at once held my attention
on first seeing it, was a very long, extremely slender type,
which progressed rapidly, by means of its flagellum and the
undulating membrane along the anterior part of the body.
The membrane appeared to be very narrow along the middle
region of the body, and on account of this fact and the active
movements of these individuals, I could not determine exactly
where it ended, or whether this type was trypanomonad
(crithidial), or trypaniform. In fixed and stained prepa-
rations, however, it is seen to be distinctly and invariably
trypaniform (figs. 13-21). Nevertheless, these attenuated
trypanosomes were entirely different .in appearance from an
ordinary, slender, elongated blood-form of Trypanosome, e. g.
a piscine type such as T. granulosum. While the anterior
part of the body, where the membrane was conspicuous, was
‘sinuous and fiexible, the hinder part, as a rule, nearly half
STUDIES ON AVIAN HAMOPROTOZOA., 407
the entire length or more, was held quite stiffly and did not
appear to be actively flexible at all. Frequently it was
practically straight, but in some individuals:it was quite
curved round, like a crook (cf. figs. 19-21); this posterior
part would retain this shape, unaltered, even while the
parasite was moving rapidly forwards. From a comparison
of stained preparations it is evident that this portion of the
body represents a prolonged extension of the cytoplasm
behind (posterior to) the kinetonucleus. In view of my
observations on the living parasites, I regard this cytoplasmic
“‘tail ” as differing from the anterior half of the body in
lacking anything of the nature of myonemes, and conse-
quently any ability to bend or twist of itself. I consider
that it is only, as it were, passively flexible, and that any
curving or bending is produced mechanically, as the result of
contact with the blood-cells and other elements among which
the parasite happens to be working its way. What function
this remarkable development serves, I was not able to
ascertain.
The above type of individual constitutes a fair proportion
of the total number of flagellates present, even at this some-
what early stage of the development. ‘The other types of
parasite seen were for the most part relatively short trypano-
monad (crithidial) forms and individuals representing every
possible transition between such and the extremely attenuated
forms. As is shown by the fixed and stained preparations
made of stomachs at about this period, the majority of these
intermediate forms are really trypaniform, i.e., the kineto-
nucleus is on the aflagellar side of (posterior to) the tropho-
nucleus. The shorter, more typically trypanomonad indi-
viduals resembled the commonly occurring crithidial forms
which develop in cultures of an Avian trypanosome, such as
I have described in the case of T.fringillinarum. All
these forms had what is usually distinguished as the crithidial
type of movement. Progression was in a slightly zig-zag
manner, the flagellum and the anterior part of the body
(corresponding to the position of the undulating membrane)
408 H. M. WOODCOCK.
vibrating actively. The movement of these forms was not |
nearly as rapid as that of the long, slender individuals.
Lastly, other forms noted were short and pyriform, and
moved jerkily, not displacing themselves to any extent;
these were infrequent. Notwithstanding the great increase
in number of the parasites which must have taken place
since the blood entered the stomach, I found scarcely any
individuals actually dividing. In one or two cases I saw try-
panomonad individuals with two flagella, and in one instance
a pair of such forms still connected together; in another
instance which I noted, division was markedly unequal, a
short, pyriform individual becoming separated off from a
distinctly larger, broader, club-shaped form.
At a later period of the development the long, attenuated
forms predominate more and more, until in females examined
fifty-five hours or subsequently after being fed, the stomach
contained apparently only such forms ; and this is borne out by
the study of fixed and stained preparations made of stomachs
of this period or later.
All my observations on the parasites relate to the stomach-
phases of the life-cycle which occur between thirty-two and
about seventy-six hours after the mosquito has fed. I
examined four females from twelve to eighteen hours after
feeding on Owl 23, but I could not find any Trypanosomes at
this early period. It would be necessary to examine many
individuals to find the earliest changes in the parasites,
because, even if the development is proceeding all right, the
Trypanosomes have not yet had time to multiply and give rise
to any considerable number of parasites; and it must be
remembered that only very few Trypanosomes are taken up
by the mosquito from the blood to start with. Neither did I
find any phases of the parasites in the intestine ; but this did
not surprise me, because nearly .all the mosquitoes were
examined, at any rate, some time before the stomach was
quite empty. While digestion is still going on, the stomach
is most certainly the principal, if not the only situation in
which the Trypanosomes occur. If I had been able to
STUDIES ON AVIAN HAMOPROTOZOA. 4.09
examine a sufficient number of females after digestion had
been completed, or after they had had another meal of blood,
the parasites might have been found in the intestine ; I shall
discuss this point later on. My observations are, | am aware,
only incomplete ; but they were the fullest I was able to make
in the circumstances, and bearing in mind the chief objects
on which I concentrated my attention during the short time
at my disposal, namely, to determine whether the 'Trypano-
some-phases in the mosquito were derived from the Halteridial
parasites or not, and to bring about, if possible, the trans-
mission of the Trypanosomes back again to the owl.
The Trypanosomes in Permanent Preparations;
Comparison with the Developmental Stages found
in Cultures.—lI think, however, by comparing the various
forms which I did obtain in the mosquito, with the develop-
ment which I have found to take place in cultures, in the case
of another Avian Trypanosome, T. fringillinarum, that a
general idea can be arrived at of the main course of the natural
development in the stomach of the insectan host; as already
indicated, the chief gap and element of uncertainty relates to
the earliest changes undergone by the parasites.
I have no hesitation in making use of the cultural develop-
ment for this purpose, because practically all the different
forms occur in both cases. The reason it is helpful is because,
in the culture, the development continues over a much Jonger
period owing to the fact that there is no absorption of the
medium, such as occurs in the insect’s stomach, and therefore
the intermediate forms are met with abundantly and stages
in division are frequent, the latter being of much assistance
in determining the sequence of the developmental changes.
In the Culex, on the other hand, the course of digestion is
comparatively rapid and completed in three to three and a-
half days, by which time the stomach is empty of blood. It
is undoubtedly in relation with this fact that we find the early
and intermediate stages in the development of the parasites
passed through very quickly, which leads on to the production
of the ultimate stages found in the stomach. ‘This develop-
vou. 60, PART 3.—NEW SERIES. 29
410 H. M. WOODCOCK.
ment proceeds along two lines, the result being the formation
of two extreme types. The great difference in the relative
frequency of certain forms, on the one hand in the culture,
and on the other in the mosquito’s stomach, is entirely in
accordance with the different conditions prevailing in the
medium in the two cases.
The earliest developmental forms obtained are trypano-
monad individuals, such as are drawn in figs. 1-3, or b and ¢
of the scheme. ‘These are medium-sized, fusiform parasites,
with a distinct membrane and the two nuclei close together,
the kinetonucleus being usually just posterior to the middle
of the trophonucleus. Both nuclei are either about the
middle of the body or else slightly in the hinder part. This
type of individual corresponds exactly with a particular
crithidial form which occurs commonly in cultures, such as
is drawn in figs. fj’ of the parallel series of stages from
cultures, which I have reproduced ! for ready comparison with
the developmental forms in the mosquito. In the mosquito,
however, even in my earliest preparations (about thirty-two
hours), the number of those trypanomonad individuals is
relatively very small. This is in marked contrast to what is
the case in the cultures, where the trypanomonad type is by
far the predominating one. In the cultures many of these
forms are distinctly larger than those with which I compare
the mosquito forms just described (cf. figs. b’—-d’), and have
the undulating membrane often better developed. In a small
proportion of them, moreover, the kinetonucleus is just in
front of the trophonucleus, instead of being alongside, a
condition which I have not observed in this phase in the
mosquito.
As regards the immediate origin of these trypanomonad
forms in the stomach, I think it is most probable that they
arise by the division of rather larger, but otherwise similar
forms. As is seen very clearly from the full series of the
crithidial forms figured in my memoir (Il. c., Pls. 27, 29), the
1 All these figures of cultural forms are taken from my Memoir in
the ‘ Quart. Journ. Micr. Sci.,’ vol. 55, 1910, pl. 27, 29, or 30.
STUDIES ON AVIAN HAMOPROTOZOA. 411
division of the larger (earlier) individuals is at first by practi-
cally equal binary fission, the only inequality being that the
daughter-flagellum may be shorter than the parent one (cf.
figs. e’—g’ of the scheme, Pl]. 31). Unfortunately I have not
found any typical trypanomonad individuals in the act of the
dividing in my preparations, but, as already mentioned, I
observed an instance in life in which the parasite was just
completing division, and in a condition practically identical
with that seen in fig. 9’.
However, I have very little doubt that the division of these
trypanomonad individuals with the nuclei about the middle of
the body is almost, if not quite, finished by the time of my
earliest preparations. Because there are very few forms still
left in this phase of the development; most of the parasites
occurring are individuals which have lost the trypanomonad
condition already and become trypaniform, and are either at
some stage in the development of the elongated, attenuated
trypanosome-phase, or have, in fact, attained the latter. I
have been able to find, in stomachs of from thirty-two to
thirty-six hours, a regular series of transitional forms in this
connection (cf. PI. 29, figs.4-10). The elongated trypaniform
condition is reached by a progressive modification of the form
of the above trypanomonad individuals. I think this change
is most probably unaccompanied by further division, at any
rate, to any extent; otherwise, I ought to have found some
indication of the process, as the parasites are fairly numerous,
and all degrees in the gradual change of the type of form are
to be met with.
The earliest change is the passage of the kinetonucleus,
and, of course, of the blepharoplast and attached flagellum,
definitely behind the trophonucieus, the latter retaining its
position (figs. 4 and 5). Next, the general cytoplasm in the
posterior half begins to increase considerably in length ; in
this way the parasite becomes longer, but it does not increase
inuch in breadth. On the contrary, as the length increases,
the body becomes ultimately not only relatively, but actually
narrower and more slender. The change is most probably the
412 H. M. WOODCOCK.
result of two factors combined, namely, growth to some
extent, and a thinning-out of the general cytoplasm. The
latter factor is evident from the change in the form of the
trophonucleus. This body, in the early stage, has the shape
of a short and broad oval (figs. 4-6); as the trypaniform
condition develops it becomes elongated, and appears as a
narrow oval, compressed laterally (figs. 7-10). Meanwhile the
kinetonucleus has passed well back behind the trophonucleus,
but it never approaches anywhere near the posterior end of
the body, the extension of the cytoplasm backwards, as the
“tail”? develops, being so great (figs. 8-12). The final
position of the kinetonucleus is usually about the middle of
the body, or slightly in the posterior half. Nevertheless, the
attached flagellum and the membrane are relatively long ;
the membrane is always narrow and rarely at all wavy.
Although, as the parasite assumes its final attenuated con-
dition, the anterior part of the body also becomes thinned out
and tapering, it does not appear to increase in length to any-
thing like the extent that the posterior half does; so that the
trophonucleus lies, as a rule, distinctly in the anterior part of
the body, at times comparatively near to the anterior end
(cf. figs. 13-20).
As the final stage in the development of this form is nearly
attained, a characteristic change usually occurs in the appear-
ance of the trophonucleus. This organella, which has already
become narrowed and compressed, becomes broken up into
many oblong or rather rod-like blocks, arranged with their
length transversely to the long axis of the body of the parasite,
thus giving the whole nucleus a remarkable ladder-like
appearance (figs. 13-17). Apparently this change repre-
sents really the break-up of the original large karyosome,
which is not seen, asa rule, in the nucleus when stained by
Giemsa, into several small karyosomes arranged in a row.
This is shown by the comparison of wet-fixed preparations
stained by iron-hzmatoxylin, in which slender elongated
trypaniform individuals, approaching the final form, have,
instead of one large karyosome (as in Text-fig. 1a), four or
STUDIES ON AVIAN HH MOPROTOZOA. 413
more small karyosomatic bodies (Text-fig. 10). Probably
each rod or block in the Giemsa-stained parasites corresponds
to a karyosomatic grain of the greatly extended tropho-
nucleus. Whether this change in the nucleus is absolutely
necessary before the final form can be said to be completely
TExt-FIG. 1.
x 1600. Stages of Trypanosoma noctue in the mosquito,
showing different conditions of the trophonucleus (from wet-
fixed film, stained by iron-hematoxylin). A. Intermediate
trypaniform phase. The karyosome of the trophonucleus is
apparently budding off a small daughter karyasome. B. Ap-
proximately final (propagative) form. There are five distinct
karyosomes forming the elongated trophonucleus.
formed, I do not feel certain ; occasionally individuals, which
in other respects seem to have reached the final stage, are
found where the nucleus is not ladder-like (cf. figs. 19-21).
In my preparations of stomachs of fifty-five hours or later,
scarcely any intermediate changes are present, practically all
the individuals having attained the attenuated trypaniform
4.14. H. M. WOODCOCK.
condition. These are certainly very striking in appearance ;
their thread-like form can be judged from the fact that their
length is generally from 52 to 564, while they are rarely
more than ly broad at the widest part, and often scarcely
that! Superficially, they almost deserve the name of spiro-
chetiform Trypanosomes, were that not so very misleading.
The half of the body in front of the kinetonucleus—the
actively motile part—is frequently spirally twisted or coiled to
some extent; while the long, passive cytoplasmic “ tail”
behind the kinetonucleus may be either practically straight
(figs. 13, 14, 18 and 20) or bent up on itself in a curve
(figs. 15-17, 19 and 21).
The above-described trypaniform type also occurs in the
cultural development. I have observed individuals both in the
intermediate condition, and in what undoubtedly corresponds
to the ultimate form attained in the mosquito’s stomach (cf.
fies. k’-m’). It is important to note that the proportion of
such forms to the trypanomonad individuals in the cultures is
just the reverse from what is the case in the mosquito after
thirty-two hours. This provides a very interesting com-
mentary on the course of the cultural development as compared
with that of the natural one. Whereas, in the latter, the
enviromental conditions favour and lead on to the produc-
tion of the attentuated trypdniform type, by the modifica-
tion of the ordinary trypanomonad individuals, in the cultures
there is not the same stimulus (the approaching completion of
digestion) to the production of this form, and in consequence
it is only developed, as it were, in isolated instances; the
multiplicative (“crithidial”) form, on the other hand, per-
sists and increases in numbers, far bevond what occurs
under the natural conditions. The intermediate forms in the
cultures resemble closely the corresponding individuals from
the insect’s stomach (cf. figs. e-g). The few final forms
which I have found (fig. m’) also agree quitefobviously in their
chief features; there is the same long, cytoplasmic “ tail,”
and even the ladder-like nucleus may be developed. The
principal difference is that the individuals in the cultures are
STUDIES ON AVIAN HA MOPROTOZOA. 415
d
much broader relatively than the natural “ spirocheetiform ’
individuals ; their body has apparently more bulk. I think
this is simply an indication that these forms have grown
more in the cultural medium than they do in the stomach ;
and division having most probably ceased, the result is the
marked increase in ‘‘ girth.”
To consider next the other line of development followed by
the parasites in the mosquito, this proceeds also from the
original type of trypanomonad individual, by a modification in
form and the mode of division. Here, again, I think the
course of events can be understood from the cultural develop-
ment. In certain of the ordinary trypanomonad individuals,
the nuclei show a tendency to be in the hinder part of the
body. This may be in the first place due to a shght oblique-
ness in the direction in which the preceding nuclear division
has occurred. At any rate, in such individuals the direction
of the nuclear division is usually oblique, and we find not only
the persistence of the nuclei in the hinder part of the body,
but a distinct tendency already for the division of the body
to be slightly unequal (cf. fig. 7’). These individuals with the
nuclei definitely in the posterior half of the body pass, or
grow into the characteristic club-shaped forms,' the general
body-protoplasm tending to be concentrated in the region of
the nuclei (fig. 0’). Now, in these forms, the division is
always markedly unequal. It is important, I think, to note
this particular point, that where the nuclei are situated about
the middle of the body, the division is practically equal (as in
the ordinary trypanomonad individuals) ; where, on the other
hand, the nuclei are distinctly in the hinder part of a club-
shaped individual, the division of the cytoplasm is unequal.
This is seen in figs. p’ and q’ of my scheme and also in several
other figures in my memoir (12). I consider this mode of
division is primarily due to the drawn-back position of the
nuclei, and consequently of the blepharoplasts, and to the
fact that the new daughter-flagellum is, at any rate, largely
’ I formerly distinguished these by the cumbrous term of “ accentu-
ated trypanomonad ” individuals.
416 H. M. WOODCOCK.
formed by free, independent growth. Individuals which are
manifestly the product of a similar division are seen in figs.
7 and ¢’.
Now, what I have found in the mosquito agrees entirely
with the above description. One of the earliest stages along
this line of development is the characteristic club-shaped
trypanomonad form of fig. 22, ork. This individual corre-
sponds very closely, it will be seen, to that of fig. o’ or p’; it
is almost ready for division. As remarked in my account of
the living observations, I saw one instance of such a form in
the act of unequal division. The smaller of the daughter-
individuals about to result wasa short, pear-shaped form, with
scarcely any membrane; the larger one wasa rather club-
shaped individual, long and tapering, and probably with a
long, narrow membrane (cf. fig. q’). Unfortunately, I have
not obtained any examples of individuals just dividing in my
permanent preparations; in view, however, both of the living
observation and of the fact that numerous individuals.
belonging to both the distinct forms which are produced by
such division occur in my preparations (cf. figs. 24, 26, and
again, figs. 23, 25), I cannot doubt that such unequal division
takes place as a normal and typical phase of the development
in the mosquito. F
By further division of the larger daughter-individual
(possessing the long membrane), smaller forms are produced
(tigs. 30-32 or n and 0) ; these can at first be recognised as
corresponding to one or the other of the two types, but in the
smaller individuals the distinction between the daughter-
forms tends to be less marked. Here and there I have found
a stout pyriform individual, one of the early-developed pear-
shaped daughter-forms, just about to divide, after further
growth (fig. 27) ; and also a quite small parasite, one of the
ultimate stages, I should say, undergoing division (fig. 34).
There can be no doubt that this line of development leads
ultimately to the production of small, pear-shaped or oval
parasites, with the nuclei close together and situated about
the middle of the body, or nearer the posterior end, and with
STUDIES ON AVIAN HASMOPROTOZOA. 417
the flagellum drawn back but with practically no membrane
(figs. 29, 32 and 33). Here we have, unmistakably, the
haptomonad phase of the T'rypanosome, as I have proposed
(16). to term the so-called ‘‘ gregariniform” phase, which
serves for attachment (and coincident multiplication). If
these figures, for instance, are compared with certain of the
text-figures in my description (15) of Leptomonas fas-
ciculata, as I found this parasite in Culex pipiens, it
will be perfectly clear that they represent the corresponding
phase of the Flagellates in both cases. This agreement, I
may incidentally mention, affords an excellent example in
favour of the point I have urged, that from the haptomonad
phase alone (having regard only to the morphology) it cannot
be said with certainty whether a particular Insectan parasite
represents a Leptomonad, a Crithidia or a Trypanosome.
From the above account it is clear that the early develop-
ment of ''rypanosome noctue in the mosquito culminates
in the production of two distinct and extreme types. Whether
the above description includes all the modifications of form
which occur in the life-cycle in the Insectan host, I am not
able to say. I think, however, that it is not difficult to inter-
pret the significance of the end-stages, and if the view I
favour is correct, any further stages in the life-history repre-
sent in the main a repetition, or re-development of the above
sequence of forms, consequent on the persistence of the para-
sites in the mosquito and their response to fresh meals of
blood.
The haptomonad forms most probably—as, indeed, is
implied by thus designating them—become attached to some
part of the wall of the alimentary canal, lose practically all
the flagellum and enter upon the resting phase; ‘ resting,”
that is to say, as regards locomotion, but not in regard to
nutrition or multiplication. I think there is no reason to
doubt that the chief situation favoured by these hapto-
monads for attachment, as the digestion becomes finished, is
the anterior end of the stomach and especially the invagi-
418 H. M. WOODCOCK.
nated epithelium of the proventriculus, as was so graphically
described by Schaudinn (10) and illustrated by him in Text-
fig. 14. Just because the parasites were in this situation, I
feel sure that in regard to this point Schaudinn’s account
relates to actual developmental phases of T. noctuz and not
to a purely Insectan form (such as, possibly, Leptomonas
fasciculata). In the case of the latter, on the other hand,
the haptomonad forms are restricted to the intestine apparently.
Whether the haptomonad phase of T. noctuz also invades
the intestine to any extent, 1 am unable to say; considering
that these forms tend to mass themselves at the anterior
end of the stomach, it is quite likely that they do not—
unless they are also able to form cysts for passage to the
exterior.
Returning now to the first lime of development, what is the
further destiny of the remarkable thread-like individuals? I
am decidedly of the opinion that they represent the propaga-
tive phase of the Trypanosome in the mosquito, the form,
that is, in which the parasite is finally transmitted back again
by inoculation to the owl. Unfortunately, I have no proof of
this, but certain considerations point strongly to this being
the right explanation of the significance of the above very
characteristic type. In the’ first place, a brief comparison
with the known course of the life-history in piscine T'rypano-
somes is most suggestive. It has been recognised for some
time that a given species of fish Trypanosome shows—at all
events, in many cases—very considerable polymorphism during
that part of the cycle undergone in the blood of the Verte-
brate host; thus certain individuals of a species may be quite
small, slender forms (representing a young stage), others
large, massive forms, with all intermediate grades between
ef. T. granulosum, T. perce, Minchin [3]). Now I
have shown that the same pronounced polymorphism also
obtains in Avian Trypanosomes (vide [12] and, with Minchin
[5)). Whereas earlier writers frequently described a small
form from a particular host as one species, and a large, massive
form from the same host as another species, the true meaning
STTDIES ON AVIAN HAMOPROTOZOA. 419
of these different types is that they are different stages in the
life-history of one species. ‘Thus the large, massive, “ blue ”
Trypanosomes of the little owl, originally described under
the name T. ziemanni, and connected by Schaudinn with
Leucocytozoon ziemanni, are really only the large forms
of T. noctuze and not a separate species at all. And a
corresponding great variation in form and size is found in
T. fringillinarum, in the chaffinch. Hence there is a
strong family resemblance between piscine and avian Try-
panosomes, as regards the types met with in the Vertebrate
host ; and it seems quite clear that both belong to the same
group or division of ''rypanosomes, and stand somewhat apart
from Mammalian forms, for instance. Turning now to the life-
cycle of piscine forms in leeches, which has been fully worked
out by Miss Robertson in two or three cases (e.g. T. raix
in Pontobdella [7 and 8], T. danilewskyi in Hemi-
clepsis [9], it is found that, after a varying period of multi-
plication by the parasites in the trypanomonad (crithidial)
phase, as the digestion approaches completion—the time
occupied varying considerably according to the conditions—a
trypaniform type is developed, which becomes very elongated
and slender. ‘his passes forwards from the crop into the
proboscis-sheath, and is the propagative, inoculative form,
which transmits the infection to a fresh fish.
This type of form is essentially similar to that above
described in Culex, the significance of which I believe to be
also the same; in the leech it does not apparently attain the
same degree of tenuity and the remarkable thread-like
appearance which it does in the mosquito, but it may at times
show the same peculiar ladder-like nucleus. It may at first
be thought that from thirty-four hours onwards is too soon
for the final, propagative form in the mosquito to be already
present. But this rapid development appears quite explicable
when the habits of the mosquito are considered. Unlike most
of the Invertebrate hosts (transmissive agents) of Trypano-
somes, mosquitoes do not feed solely on blood. On the con-
trary, it is generally recognised that, so far as the species of
420 H. M. WOODCOCK.
temperate climates are concerned, meals of blood are not by
any means the rule, and in many cases only taken in con-
nection with the development of the eggs. In the case of
most females blood appears to be necessary for this purpose ;
it certainly is so in Culex pipiens, and as I have recently
shown (l. c.), two meals of blood will suffice to bring about
the growth and oviposition of the fertilised ova. After laying
one, or perhaps two, batches of eggs, most of the females
either die or go into hibernation. Hence, it is perfectly clear
that unless the propagative phase of the Trypanosome is
developed in time for inoculation at the second (or at most
the third) meal of blood, the chances of the parasite passing
back to the bird are very uncertain—an entirely different.
state of affairs from what is the case among tsetses or
leeches. For the above reasons, therefore, I think there is no
difficulty in assuming that the thread-like ‘‘ spirochetiform ”
individuals which I have described represent the inoculative
type, or in understanding their early development in the
mosquito.
While I consider it is quite likely that these forms are
inoculated again into the blood at the second time of feeding,
I do not overlook the possibility that they (or some of them)
pass first into some other organ of the mosquito (such as the
salivary glands, or cesophageal diverticula), and there await
a third meal; but this has still to be ascertained. I do think,
however, Schaudinn was mistaken in stating that the Try-
panosomes cannot be transmitted back again to the owl until
the fourth meal inclusive. I should say, if the parasites had
to wait until the mosquito took a fourth meal of blood, they
would rarely, if ever, have the opportunity of getting back
again at all, unless, perhaps, they were in a female which was
going to hibernate. It is obvious from Schaudinn’s account
that he was following chiefly the multiplication of the parasites
in the haptomonad condition (vide his description of their
attachment in vast numbers to the walls of the alimentary
canal). Schaudinn does not appear to have seen the real
propagative phase at ail! He nowhere describes the
STUDIES ON AVIAN H#MOPROTOZOA. 421
attenuated forms with the characteristic cytoplasmic “ tail”
(ef. on the other hand, Mayer’s account, referred to below).
Schaudinn’s slender, “ spirocheetiform” individuals, which
he describes under ‘ Spirocheta” (Trypanosoma
ziemanni), agree fairly well with the intermediate try-
paniform stages in the development of the final type (such as,
e.g. figs. 7 and 8); but I must add, nothing has astonished
me more than the lack of close correspondence, in the main,
between the different forms of the Trypanosome occurring in
the mosquito, as Schaudinn figured them, and as I have found
them.
Il. The Ookinetes of Halteridium noctue and
Leucocytozoon ziemanni,
A few words next concerning the ookinetes of Halteri-
dium and Leucocytozoon, as they occur in my permanent
preparations. As regards their general appearance, I have
little to add to the description previously given by Mayer
(l.c.). The ookinetes of both forms are fundamentally
similar in type, as was of course to be expected, considering
that two essentially similar parasites are concerned. In both
cases the body is very frequently more or less coiled up in the
form of a C. Practically the only difference between the two
is that the ookinetes of Leucocytozoon are much larger,
especially in regard to length, than are those of Halteri-
dium (c.f. figs. 35-40 and 41 and 42).
In my preparations of the Culex which fed on Owl 19,
made from tlurty-three to thirty-six hours after feeding, the
ookinetes of Halteridium are very numerous. In prepara-
tions made from mosquitoes which fed on Owl 25, ookinetes are
also usually to be found (up to thirty-six hours), but they are
very scanty ; this difference is due to the different conditions
of the infection in the two owls respectively (see above, pp. 400—
401). Speaking generally, all the ookinetes observed are in
the same phase of development, and with one or two exceptions
have lost all the pigment. In none of them is anything like
422 H. M. WOODCOCK.
a kinetonucleus, let alone a developing flagellum, recognisable.
In a certain number a small chromatinic area, or clump of
chromatinic grains, is present, in addition to the nucleus (figs.
35 and 36); but these stain quite similarly to the nucleus,
and in no case have the characteristic appearance of a kineto-
nucleus, as seen when stained by Giemsa (contrast the “ resting
flagellates ” referred to below). Unfortunately I am unable
to say whether the nucleus possesses a central karyosome or
not, but at any rate there is certainly no excentric or extra-
nuclear karyosome, such as occurs at certal periods both in
Halteridium and Leucocytozoon when in the blood,! and
which often simulates a*€inetonucleus in so marked a manner
that it was formerly mistaken for one, until I showed clearly
(14) what its true significance was. Frequently the ookinete
shows one or two vacuoles in the cytoplasm; I have not been
able to find any ookinetes in my preparations of mosquitoes
which were made later than fifty-four hours after feeding.
I have only come across very few ookinetes of Leuco-
cytozoon on my smears; I was generally able to find one
or two during the living examination of the stomachs of
mosquitoes which had fed on an owl infected with this
parasite, but unfortunately. in several cases, owing to their
scantiness, | have not succeeded in obtaining any on the
permanent slides made. ‘Those I have found are all practi-
cally similar in form and size (figs. 41 and 42). The nucleus
is always situated fairly near to the more rounded end of the
body, as it is also in Halteridium; it varies in size to some
1 Reichenow, in a note on Leucocytozoon ziemanni in his
account of the Hzemogregarines in ‘Handb. d. Path. Protozoen’ (6)
states that he was unable to find a karyosome in the male gametocytes.
As I showed in my “ Notes on Sporozoa,”’ published about the same
time, there is certainly such an organella present, excentric or even
extranuclear as in the female forms. It is unmistakable in prepara-
tions stained by iron hematoxylin, but it is rarely shown in Giemsa
smears. Reichenow does not say whether the figure he gives is from a
Giemsa smear or not, but from its general appearance (e. g. the hyper-
trophied host-cell nucleus stains quite differently after iron-hema-
toxylin), I should say it was.
STUDIES ON AVIAN HAZMOPROTOZOA. 423
extent. Now and again separate chromatinic granules occur
close to it (cf. fig. 42). In these ookinetes, also, one or two
vacuoles are often present. None of the ookinetes of Leuco-
cytozoon, any more than those of Halteridium, are in any
later stage of development. I have not seen the least indica-
tion of the remarkable skein-like formation so graphically
described by Schaudinn, accompanied by nuclear multiplica-
tion and the eventual development of a number of very small
Trypanosomes! I must say that I doubt very much now the
correctness of all this.
III. The “Resting Flagellates.”
As was described in the account of the living observations
(see above, pp. 401-402), in asmall number of female Culex
pipiens caught wild, which were examined at an early stage
of the experimental work before I restricted myself to the use of
bred-out mosquitoes, certain characteristic and rather peculiar
motionless bodies were sometimes present. I have mentioned
how on one occasion such resting forms were actually seeu
to develop into active flagellates, and the temporary illusion
fostered by the observation. But not only were they seen in
the first “ wild female,” which fed on Owl 19, well infected
with Halteridia, they also occurred now and again in females
which had fed on an uninfected bird, or which were examined
before being allowed to feed on a bird at all! Hence, what-
ever their origin, there is no reason for associating them with
Halteridium. For some cause or other, with the above-
noted exception, these resting forms were never observed to
become active, nor were active flagellates corresponding to
them ever found.
In my permanent preparations, practically all these para-
sites occur in the resting condition (figs. 43-45): in one or
two instances, however, there is an unmistakable wavy
border in the anterior part of the body, accompanied by a
drawn-out, tapering anterior end (figs. 46-47). Iam strongly
of the opinion that the flagellum is actually developed in both
424, H. M. WOODCOCK.
these cases, but it is difficult to be quite certain because for
some reason or other, it has not stained red in the usual
manner after Giemsa. Especially in the individual drawn in
fig. 47, however, a definite line is evident along one edge of
the anterior, tapering part, which is continued free for a short
distance, which in all probability represents a flagellum.
The general appearance of these forms, moreover, closely
resembles that of the particular individual referred to above,
just the instant before a well-marked, free flagellum became
apparent. I think no one can have any doubt that these are
really Flagellates, because they are certainly binucleate
forms; in all cases, a definite kinetonucleus is present,
usually immediately in front of the trophonucleus, which
has the characteristic staining reaction to Giemsa. It may
be pointed out that there is no question of this element being
a karyosome, because, the karyosome of the nucleus can at
times be seen, appearing, as is always the case in Giemsa-
stained smears, as a clearer area, with a distinct centriole in
the middle (cf. figs.43 and 46). The cytoplasm always stains
more darkly and much more bluish-purple in tint than any of
the developmental phases of Trypanosoma noctue, in
which, by the way, aflagellate phases appear to be entirely
lacking. In all my experience of crithidial forms, whether
as developmental phases of Trypanosomes, or “ Crithidial”
parasites, I have never observed any in which the cytoplasm
stains in this peculiar dark manner, or in which the flagellum
is so faint.
This parasite is certainly a crithidial form; this is evident
from the contiguity of the two nuclei about the middle of the
body, as well as from the distinct undulating membrane,
where an individual is in, or about to assume, the active
condition. In one respect it appears to be unique, 1.e., in
being non-flagellate, and moreover, without any trace of a
rhizoplast, and quite motionless, when still possessing the
elongated, fusiform shape, which is always associated in
other Flagellates of this kind, with the active, flagellated con-
dition; in all other cases of which I am aware, when the
STUDIES ON AVIAN HAMOPROTOZOA. 425
parasites are “resting” and non-flagellate they are in the
short, more or less oval, haptomonad phase. ‘l‘his form does
not ‘agree with any of the “ Crithidiz” hitherto described
from mosquitoes ; until more is known about it I am inclined
to regard it as a distinct parasite. At present I have
obtained the impression that this may bea purely Insectan
form, a parasite more particularly, perhaps, of the larval
Culex. Somehow, it does not look like a form accustomed
to a blood-medium ; its appearance and behaviour are so
different from all the other crithidial forms which I have had
occasion to study.
GENERAL CONCLUSION REGARDING THE QUESTION OF A CONNEC-
TION BETWEEN THE ‘l’RYPANOSOME AND THE H#MOSPORIDIAN
PARASITES OF THE LirrLeE Owt.
For the last time, I hope, that it will be necessary, I return
to this subject, more particularly in order to point out the
bearing upon it of the observations recorded above on the
developmental phases of these parasites in the mosquito. It
must be apparent, indeed, that these observations support
and further strengthen the conclusion at which I had already
arrived (14), that there is no connection whatever between
these different types of parasite; and I have found nothing
that in any way corroborates Mayer’s account (2), in which
he has upheld the opposite view.
In the first place, with regard to the mosquito which fed
on Owl 19. As was to be expected from the typical ripe
Halteridial infection of this bird, numerous fully-formed
ookinetes were found without difficulty in the stomach when
dissected. But not one of these ever showed any sign of
passing into a flagellate condition; nor were any active
Flagellates observed of the different types which I subse-
quently found frequently in mosquitoes fed on an owl known
to have Trypanosomes. This fact is very significant when it
is remembered that no Trypanosomes were ever seen in the
blood of Owl 19, either in life or in searching smears, and if
VoL. 60, PART 3,—NEW SERIES. 30
426 H. M. WOODCOCK.
they were present, must have been so rare at the time as to
be negligible.
As was discussed above, the matter was complicated just
at the outset by the use of a few “ wild ” females in which a
“resting Flagellate” occurred, which on one or two occasions
developed into the active condition. In my own opinion, how-
ever, it is perfectly clear that these resting Flagellates have
nothing to do with the ookinetes. Although there is un-
doubtedly a general resemblance in appearance between these
two bodies when observed in life, there are several important
reasons for concluding that this is only a coincidence. In
stained preparations the two types of element appear funda-
mentally distinct, for the resting Flagellates show without
exception the binucleate condition; the ookinetes, on the
other hand, never do. In not a single instance, whether of
Halteridium or of Leucocytozoon, have I been able to
find an ookinete which possesses the binucleate condition,
the first essential for it to be regarded as connected with a
Heemoflagellate. Again, the staining reaction of the general
cytoplasm in the two cases is entirely different ; and though,
knowing what I do of the dangers, no less than the advantages,
associated with the use of the Giemsa stain, I should be the
last to lay stress upon casual staining differences in different
cases ; nevertheless, where such a difference is constant and
uniform, weight may be laid upon it. Further, these resting
Flagellates occurred also in “wild” females fed on owls
totally uninfected with any of the parasites under discussion,
and in which no ookinetes, of course, were found. Finally,
these resting Flagellates have certainly nothing whatever to do
with the developmental phases of Trypanosoma noctue.
Now, in the bred-out mosquitoes which fed on Owl 28,
ookinetes were always scanty; particular individuals were
carefully watched on different occasions, but in these also no
change or development of any kind was ever seen. Never-
theless, in about 46 per cent. of these mosquitoes
examined, active Flagellates were observed in
different stages of development; and it is to be re-
STUDIES ON AVIAN HA MOPROTOZOA. 427
membered that Owl 23 was the only birdin which Try pano-
somes were found in the peripheral circulation,
and actually at this period. If the Trypanosomes had
indeed developed from ookinetes, it is difficult to understand
why they should occur frequently when the ookinetes were
scanty (and sometimes not actually noticed), and yet not at all
in the case where the ookinetes were numerous (the female
which fed on Owl 19, with the normal Halteridial infection).
All my observations point clearly to these active Flagellates
being the developmental forms of T. noctuz in the mosquito,
and derived directly from the stumpy, or stout fusiform
Trypanosome, present in the blood in the summer (as pre-
viously described (5). I have never once seen the slightest
indication of these developmental Trypanosome-stages in any
mosquito fed on a bird which did not contain this stumpy,
transmissive phase of the Trypanosome in the peripheral
circulation—whether it contained Halteridium, or Leu-
cocytozoon, or neither.
A few remarks, in concluding this discussion, about Mayer’s
account (l.c.). The most important statement of this worker
is that, in hanging drops of blood from an owl infected with
Halteridium, in which careful examination failed to reveal
any Trypanosomes after four days or so Flagellates were found
to be present; the inference is, of course, that these had
developed from the Halteridium-ookinetes. I can only
say that I feel absolutely convinced that Mayer was mistaken
in supposing no 'l'rypanosome-individuals to be present in the
drop at the beginning of the experiment. I know well from
experience how easily one of these forms can be overlooked,
especially if the blood-corpuscles are in a fairly thick layer.
Another possible explanation would be that certain very
minute or ultramicroscopic phases of the Trypanosome were
present, which later gave rise to flagellates. But this idea is
quite unnecessary when I have shown clearly that the same
particular flagellate developmental stages occur regularly,
both in cultures and in mosquitoes, as a result of inoculation
with a definite Avian 'l'rypanosome form. For my own part,
428 H. M. WOODCOCK.
I say candidly that I can see no sufficient evidence up to the
present for believing in “ cryptotrypanosomiasis 7! or in
“infective granules,” etc. In all the life-cycles of Trypano-
somes which are now known in the invertebrate host, there is no
suggestion of such a thing, and such an explanation is not, I
consider, required. In birds, and even more so in cattle and
sheep, Trypanosome individuals may be so excessively scanty in
the circulation that the greatest difficulty is entailed in
finding them ; but let a single individual succeed in passing
into a culture or into its right invertebrate host, and it will
multiply so rapidly that before many days have passed the
forms to which it gives rise are readily found. I may say
also that I am no longer inclined to think that any small
form of these Avian Trypanosomes occurs in the red blood-
corpuscles, which might perhaps be mistaken for a young
stage of Halteridium. Since very considerable doubt has
been thrown upon the occurrence of such astagein Try pano-
soma cruzi, it is becoming more and more probable that
Trypanosomes do not get into the red cells at all; at all
events I am disinclined now to postulate the occurrence of
such a phase in the Avian Trypanosomes which I have
studied until we have an authentic instance of it in some other
case. As in the case of fish-Trypanosomes, with which, as I
have shown above, Avian Trypanosomes have much in common,
I firmly believe that unless the transmissive trypanosome-
form of an Avian T'rypanosome passes into culture or the
invertebrate host, no development of Flagellates will occur.
Mayer describes and figures further certain “‘ large forms ”
which developed in mosquitoes, which he regards as the de-
velopmental stages of Leucocytozoon, in contradistinction
to Halteridinm. But these forms are obviously the same
as certain of those which I have described and figured as the
developmental stages of Trypanosoma noctue; his fig. 56,
for instance, represents an individual nearly arrived at the
final attenuated form. The stages which Mayer associates
1 By this term I understand some definite phase of the parasite,
hitherto unrecognised and possibly ultramicroscopic.
STUDIES ON AVIAN H#EMOPROTOZOA. 429
with Halteridium, equally with those which he associates
with Leucocytozoon, constitute part of a regular series, and
belong to a definite life-cycle, as I have clearly shown.
Correspondingly, it will be remembered, Minchin and I
showed also that the small forms of the Trypanosome in the
blood, associated by Schaudinn with Halteridium noctue,
form part of a regular series with the large individuals,
regarded by him as belonging to Leucocytozoon ziemanni,
and altogether represent only one species—T ry panosoma
noctue; there is no species I’. ziemanni. If, therefore,
one still held to the idea that the small Trypanosomes are
connected with Halteridium and the larger ones with
Leucocytozoon, one would be led to the impossible position
that Halteridium and Leucocytozoon are different
phases of one and the same thing.
I do not suppose that Mayer any longer considers that
an ontogenetic connection exists between any of these
different parasites, especially as in addition to the above-
inentioned paper, I have also published my account of the
cytology, in which I have shown that, after all, there is no
nuclear dimorphism in Halteridium and Leucocytozoon,
and that these are not related to the Binucleata. But as Mayer’s
account is the only one published of late years on the develop-
imental stages of these parasites of the owl in mosquitoes, I
have been obliged to point out where it is erroneous in the
light of my own observations on the whole subject.
The conclusion of the whole matter is, that the three
parasites of the little owl—Trypanosoma noctue, Halte-
ridium noctuze and Leucocytozoon ziemanni—are
entirely distinct and separate types; and the same is un-
doubtedly true for other species of these parasites in other
birds. So far as T. noctue is concerned I have been
able to outline above the main course of its development
in the mosquito (Culex pipiens), though there are, un-
fortunately, gaps still to be filled up. The development
of the Halteridium and the Leucocytozoon in the mosquito
remains to be ascertained—supposing, that is to say, that
430 H. M. WOODCOCK.
there is any development beyond the ookinete stage. Other
workers (e. g. the Sergents (11), Aragao (1) and Mayer (loc.
cit.) have obtained the development of one or both of the
parasites up to the same stage, but never any farther. As
regards Halteridium, Aragao, who has succeeded in trans-
mitting H. columbe from a Hippoboscid fly (Lynchia)
back again to the pigeon, is doubtful whether there is really
any further development in the Insectan host; it is possible,
moreover, that the so-called schizogony in the lung represents
the delayed sporogony of the ookinetes, as has been hinted at
by Minchin, in his text-book on the Protozoa (4). If that be
so, then I see no reason why the ookinetes of H. noctue
should not behave similarly ; in which case, Culex pipiens
may prove, after all, to be a true host—transmissive agent—
of this parasite also. Atany rate, for all that one can yet say
to the contrary, the ookinetes may be inoculated back again
into an owl, at the same time as the final propagative forms
of the Trypanosome. It is, perhaps, not without significance
that in every owl which was infected with the one parasite,
we found the other also to be present (cf. Minchin and Wood-
cock, loc. cit.). The .development and transmission of
Leucocytozoon are still more a matter of uncertainty. As
far as Schaudinn’s observations are concerned, if such a
remarkable nuclear multiplication and skein-development
does occur, it is very strange that neither Mayer norI myself
have seen any signs of it. One thing is, I think, practically
certain; if the ookinete does produce a number of small
elements by rapid division, these will not prove to be Try-
panosomes or other binucleate Flagellates !
Tue Lister INSTITUTE;
May 12th, 1914.
10.
19 le
12.
13.
14.
STUDIES ON AVIAN HAMOPROTOZOA, 43]
REFERENCES TO LITERATURE.
. Aragao, H. de B.—* Ueber den Entwickelungsgang und die Ueber-
tragung von Hemoproteus columbe,” ‘Arch. Protistenk.,’
xii, 1908, p. 154, pls. 11-13.
. Mayer, M.—* Ueber ein Halteridium und Leucocytozoon des
Waldkauzes und deren Weiterentwickelung in Stechmiicken,”
op. cit., xxi, 1911, p. 232, pls. 22 and 23.
. Minchin, E. A.—* Observations on the Flagellates Parasitic in the
Blood of Fresh-water Fishes,” ‘ Proc. Zool. Soc.,’ 1909, i, p. 2, pls.
1-5.
‘An Introduction to the Study of the Protozoa.’ London:
Ed. Arnold, 1912.
and Woodcock, H. M.— Observations on the Trypanosome
of the Little Owl (Athene noctua), ete.,” ‘Quart. Journ. Mier.
Sci., 57, 1911, p. 141, pls. 20 and 21.
. Reichenow, Ed.—“ Die Hemogregarinen,” in ‘Handb. d. path.
Protozoen,’ edited by Prowazek, 8. v., vol. ii, Lief. 1, p. 602, 2 pls.
. Robertson, M.—*‘ Studies on a Trypanosome found in the Alimen-
tary Canal of Pontobdella muricata,” ‘Proc. physic. Soc.
Edin.,’ 17, 1907, p. 83, pls. 4-7.
* Further Notes on a Trypanosome found in Pontobdella
muricata,” ‘Quart. Journ. Mier. Sci.,’ 54, 1909, p. 119, pl. 9.
“Transmission of Flagellates living in the Blood of certain
Fresh-water Fishes,” ‘Trans. Roy. Soc., B. 202, 1911, p. 29, pls.
1 and 2.
Schaudinn, F.—*‘ Generations- und Wirthswechsel bei Try pano-
soma und Spirocheta,”’ ‘Arb. kais. Gesundhtsa.,’ 20, 1904, p.
087, text-figs.
Sergent, E. and E.—* Etudes sur les Hématozoaires des Oiseaux,”
‘Ann. Inst. Pasteur,’ 21, 1907, p. 251, pls. 6 and 7.
Woodcock, H. M.—“Studies on Avian Hemoprotozoa: I. On
certain Parasites of the Chaffinch (Fringilla cwlebs) and the
Redpoll (Linota rufescens),” ‘Quart. Journ. Micr. Sci.,’ 55,
1910, 641, pls. 27-31.
“On an Unusual Condition observed in Halteridium,”
‘Zool, Anz.,’ 38, 1911, p. 465, text-figs.
‘ Notes on Sporozoa, Nos. II-IV,” ‘ Quart. Journ. Mier. Sci.,’
58, 1912, p. 171, pls. 9 and 10.
432 H. M. WOODCOCK.
15. Woodcock, H. Mi—* On ‘ Crithidia’ fasciculata in Hibernating
Mosquitoes (Culex pipiens), ete.,” ‘Zool. Anz.,’ 43, 1914, p. 370,
text-figs.
“ Further Remarks on the Flagellate Parasites of Culex,
etc.,” ‘Op. cit., 44, 1914, p. 26.
EXPLANATION OF PLATES 29-31.
Illustrating Dr. H.M. Woodcock’s paper on “Studies on Avian
Heemoprotozoa : No. II].—Observations on the Develop-
ment of Trypanosoma noctue (of the Little Owl) in
Culex pipiens; with Remarks on the Other Parasites
occurring.”
[All the figures on Pls. 29 and 30 are magnified 2000 times linear;
those of the scheme, PI. 31, are x 2000 ( nearly). Iam indebted to Miss
Rhodes for kindly drawing and colouring most of the figures of the
Halteridium ookinetes. |
PLATE 29.
All the figures relate to the development of T. noctuz in the
mosquito (Culex pipiens).
Figs. 1-3.—Typical trypanomonad forms.
Figs. 4-6.—Harliest stages in the development of the trypaniform
type. ;
Figs. 7-10.—Intermediate trypaniform stages.
Figs. 11, 12.—Approximation to the final inoculative form. Fig. 11
shows an intermediate stage in the change in the nuclear condition ;
three fairly large chromatinic masses (karyosomes) are present.
Figs. 13-17.—Typical attenuated thread-like forms, with ladder-like
nucleus. These are considered to be the inoculative type.
Figs. 18-21.—Individuals which agree in character with the final
forms, except for the fact that the nucleus has not become ladder-like.
Figs. 22, 23.—Club-shaped trypanomonad forms. Early stages in the
second line of development. (See text.) .
Figs. 24, 26.—Pear-shaped forms, with the kinetonucleus tending to
be in front of the trophonucleus and with hardly any membrane. These
individuals result from the smaller member of an unequal division of
STUDIES ON AVIAN HA MOPROTOZOA. 433
such a form as that of fig. 22. They represent the beginning of the
haptomonad phase.
Fig. 25.—Individual resulting from the larger half of the same or a
similar unequal division. It tends to be slightly club-shaped, and has
the nuclei well back, with a long, narrow membrane.
Fig. 27.—Pyriform individual commencing division.
Figs. 28, 29.—Other pyriform haptomonad forms.
Figs. 30, 31.—Small individuals which still show to a slight extent
the characters of the differing products of an unequal division (probably
of such a form as that of fig. 25).
Figs. 32, 33.—More haptomonad forms, or more strictly, perhaps,
forms which are going to become attached.
Fig. 34.—One of the smallest forms found undergoing division.
PLATE 50.
Figs. 35-40.—Ookinetes of Halteridium noctuex. In fig. 38 part
of the skin or pellicle of the ruptured erythrocyte is seen still attached
to the ookinete. This occurs now and again.
Figs. 41, 42—Ookinetes of Leucocytozoon ziemanni.
Figs. 43-47.—“ Resting Flagellates” found in “wild” mosquitoes. The
individuals of figs. 46 and 47 have almost certainly developed a flagellum,
marginal, if not free.
PLATE 31.
Scheme comparing the development of T. noctuz in the mosquito
with the developmental stages of T. fringillinarum as found in
cultures. (For explanation, see text.)
x
ct
s
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 439
Studies in the Experimental Analysis of Sex.
Part 11—On Stylops and Stylopisation.
By
Geofirey Smith, M.A.,
Fellow of New College, Oxford.
And
A. H. Hamm,
Assistant in the Hope Department, Oxford.
With Plates 32-35.
It has long been known that the presence of Strepsipterous
parasites on solitary bees and wasps may exert a remarkable
influence on their hosts, and the paper by Professor J. Pérez
in 1886 (1), gave a full account of the effect of Stylops
melittee on the bees Andrena, which must always be
referred to as a classical contribution to the study of parasitic
castration. Although many authors have subsequently
written on these parasites, the observations of Monsieur
Pérez remain unsurpassed for fulness of detail and interest,
and we are indebted to the veteran entomologist for
additional information which he has kindly put at our
disposal in answer to inquiries. The re-examination of the
subject, which is undertaken here, seemed desirable, not so
much for the purpose of bringing new facts to light, but for
confirming reported facts and collating them with the newly
acquired results on parasitic castration brought about by
Sacculina, since the effect of Stylops on its hymenopterous
hosts seemed to be parallel to that of Sacculina on crabs,
436 GEOFFREY SMITH AND A. H. HAMM.
and yet to differ from it in many important respects. The
Strepsipterous parasites differ from Sacculina in that they
have the sexes separate, whereas Sacculinais hermaphrodite,
so that we can test whether the sex of the parasite has any
influence on the effect excited. Moreover, it is clear that in
certain cases the female host is induced to assume certain
male characters as the result of parasitic castration by
Stylops, an effect which is never observed in the case of
Sacculina, and which requires careful examination. We
have been able to study a large number of stylopised bees of
various species, but the most fruitful material consisted of a
large colony of heavily stylopised Andrena nigrownea,
established in a grassy bank close to the University Museum,
which was kept under observation for several years and
afforded us a rich collection. The following observations on
the structure and history of the parasite are based on the
bees taken from this colony.
1. Norges oN THE PARASITE.
- Stylops melittez, like all the Strepsiptera, has the sexes
separate; the adult malJe’ being an extremely active-winged
insect, showing a possible relationship to the Coleoptera
(Pl. 33, fig. 8), while the adult female is a degenerate grub-
like creature which remains permanently inside the body of
the bee in which it. is parasitic. The male, before hatching
out as.a winged insect, also develops inside the abdomen of
the bee, undergoing its larval stages and pupation in this
situation. ‘Ihe male pupa, in fact, closely resembles the
adult female parasite, and protrudes a little cap between
the segments of the bee’s abdomen to the exterior, which
closely resembles the head of the female parasite which is
similarly protruded. When the adult-winged male emerges
from its pupa and from the bee, it pushes off the protruded
cap of the pupa and leaves the old empty pupal case inside
the abdomen of the bee where it can often be recognised as
a hollow cavity communicating with the exterior. Since the
male Stylops emerge from their pupa soon after the bees
STUDIES. IN THE EXPERIMENTAL ANALYSIS OF SEX. 437
come out of their burrows for the first time in spring, the
empty pupal case of the male Stylops is much more
frequently met with in the bee than the pupa with its cap on
containing the male itself. In the case of the female
Stylops, it is quite different, since she remains permanently
in the bee, and may be found all through the spring and early
summer with her body distended with developing eggs or
larvee.
The appearance of a bee’s abdomen with three female
Stylops in it is shown on PI. 32, fig. 1. ‘The heads of the
female Stylops are seen protruding between the segments
of the abdomen, while the rest of their bodies are hidden
inside the abdomen of the bee. If we dissect out the whole
of the female parasite from the bee (Pl. 32, fig. 2), we see
that its body consists of two chief parts, a hard yellow
chitinous portion, the cephalo-thorax, which is protruded to
the exterior, and a soft white segmented portion or abdomen,
which is buried in the bee’s abdomen, occupying a very large
proportion of the hemocoel or body cavity of the bee.
There was for a long time some doubt as to whether the
hard protruded chitinous part was really the head or the tail
end of the Stylops, but it is now quite certain that it is
really the head end, as it is possible to prove by means of a
median sagittal section, such as is shown on PI. 382, fig. 3, that
the brain (gn. 1) and the subcesophageal ganglion (gn. 2) are
situated there. We can also establish from the position of
the gangha and the ventral nerve cord (n), that the surface
of the cephalo-thorax exposed to view when the parasite is
im sit on the bee is the ventral surface.
Looking at this ventral surface of the cephalo-thorax we
can see (PI. 32, fig. 2), certain tubercles and slits which are
of importance. A median tubercle near the extreme anterior
end is placed just behind a minute pore which represents the
mouth (m). On each side of the mouth are a pair of lateral
tubercles which probably represent the atrophied man-
dibles (md). Behind these tubercles is a bow-shaped slit (0),
the opening of the brood passage, by which the larve are
438 GEOFFREY SMITH AND A. H. HAMM.
finally liberated. This slit at maturity communicates with a
superficially-placed passage running all along the ventral
surface of the body. The lateral limits of this passage are
indicated by the ridges (7) showninthe figure. The relations
of this brood passage are shown in the sections on Pl. 32,
figs. 3 and 4 (b). It will be seen that the passage is simply
a space between the chitinous integument and the body wall,
the epithelium of which (ep) is peculiarly modified by the
cells being produced into spiny processes. The function of
this curiously modified epithelium of the brood-passage has
never been suggested, but possibly its rough surface is
convenient for the young larve to travel over in their journey
to the exit at o.
The eggs complete their development in the body of the
female, which at maturity consists of a mere sack contain-
ing them, all the other organs being reduced to vestiges. An
idea of the appearance of a fully ripe female with the body
full of active larvee and embryos is given by the photograph
on Pl. 33, fig. 6. The fully-developed larvee reach the
brood passage from the body cavity of the parent by means
of five peculiar trumpet-like invaginations which lead into the
brood passage and acquire openings into the body of the
parent at the moment that the larve are ready to escape.
The five trumpets are shown attached to the epithelium of
the brood passage, the rest of the body having been re-
moved, in the photograph on Pl. 33, fig. 7, and a trumpet
with its opening into the brood passage is shown in the
sections on Pl. 32, figs. 3 and 4.
We may make some mention of the other organs of the
body of the female parasite. The skin, except where the
epithelium of the brood passage is specially modified, is ex-
ceedingly thin, and the nourishment of the body must take
place by absorption through this thin skin. There are no
special cells for seizing on or elaborating a special kind of
food either in the skin or elsewhere, so that we may suppose
that the hemolymph of the bee affords a ready-made medium
which supplies the parasite with all that is requisite. This is in
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 439
perhaps significant contrast to Sacculina, where a special
and highly-developed system of roots ramifies through the body
of the host, and is engaged in seizing on a special constituent
of the food, viz. fat.
The alimentary canal (Pl. 32, figs. 3 and 4, g), of the
Stylops is clearly recognisable, but in a quite degenerate and
useless state. There is a minute mouth opening (m), and an
equally minute anus at the hind extremity, but the lumen of
the gut through the body is obliterated. The whole apparatus
is obviously functionless. There is a peculiar mass of cells
with dark staining nuclei and eosinophilous cytoplasm (PI. 32,
fig. 3, c) situated in the ventral part of the cephalo-thorax ;
the function of these cells is unknown, but possibly they are
of the nature of supporting-cells analogous to cartilage, to
stiffen the exposed region of the body. Dorsally to the gut
the remains of the dorsal blood-vessel or heart (Pl. 32, figs.
3 and 4) can be recognised. The nervous system consists of
three ganghonic masses (Pl. 32, fig. 3, gn 1, 2, and 3), the
dorsally-situated ganglion, (gn 1), being connected by means
of a thin commissure round the cesophagus with a large
ganglion in the thoracic part of the cephalo-thorax (gn 2).
From this ganglion a thin, nervous strand (n), representing
the ventral nerve cord, passes toa third ganglion (gv 3) in the
abdomen, and from this a thin filament passes away, but no
other ganglia could be found in the adult female.
Of great importance for the economy of the parasite is the
tracheal system. ‘There are two main tracheal trunks which
open on conspicuous tubercles (Pl. 32, fig. 2, tr.) on each side
of the cephalo-thorax. These two trachez (see Pl. 32 and
33, figs. 4 and 7, tr) pass right through the body, giving off
numerous branches, which ramify among the developing eggs
and supply them with oxyyen from the outside, quite inde-
pendently of the bee. We may note again a contrast between
Stylops and Sacculina here, since the developing Saccu-
lina roots have to obtain their oxygen from the blood of the
crab.
The above account of the structure of the female parasite
4.4.0 GEOFFREY SMITH AND A. H. HAMM.
is sufficient to give an idea of its mode of life and nourish-
ment; for further details the reader may refer to the papers
of vy. Siebold (2), Pierce (11) Nassonow (10) and Brues (8).
There are certain points relative to the life-history of the
parasite which remain obscure but suggest features of great
interest. It is known that the larve, which are called
Triungulins and have the form shown in ventral view on
Pl. 32, fig. 5, leave the body of the parent by means of the
opening of the brood passage, and: find their way on to
flowers visited by the bee. They then clamber on to another
bee which visits the flower, and clinging on to its hairs are
carried back to the burrow, where they ultimately enter the
cells and infect the next generation. At exactly what stage
they enter the young bee larve is not known, but it is
presumably at an early stage of development.
The really obscure part of the life-history concerns the
mode of development of the eggs, and the question, how the
female parasite is fertilised by the male, if, indeed, fertilisa-
tion ever takes place.
In our account of the structure of the adult female parasite
it was shown that the portion of the body protruded to the
exterior is the head, and not the tail, as certain authors have
supposed. Apart from the anomaly of fertilisation taking
place through the head end of an insect, we have been unable
to find any opening or organ such as a spermatheca for the
entrance or reception of the spermatozoa of the male, either
on the protruded cephalo-thorax or on the rest of the body
inside the bee. It has been suggested by some authors (16)
that fertilisation may take place through the opening of the
brood passage, but this appears to us improbable, as there is
no means of entrance from the brood passage into the body of
the parasite, the trumpet-like invaginations being completely
closed until shortly before the larve are ready to emerge.
It has also been suggested that fertilisation might take place
through the mouth and alimentary canal, but this rather
extravagant suggestion is not supported by the actual state
of the alimentary canal, which appears in sections as a con-
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 441
tinuous narrow tube passing through the body without any
outlet into the body cavity where the eggs are contained.
It has already been suggested that the eggs of Strepsiptera
may develop parthenogenetically in certain cases. Thus Brues
(8), describing the oogenesis in Acrochismus wheeleri
(Pierce), a Xenid parasite on the wasps Polistes, contends
that the eggs develop parthenogenetically, the second polar
body re-entering the egg and fusing with the egg nucleus.
Since Brues, however, did not follow polar-body formation his
evidence is incomplete.
Dr. R. C. L. Perkins (5) has expressed the view that the
parasites of the bee Halictus must be parthenogenetic, at
least in certain cases, as when the eggs begin to develop it is
impossible that a male could have fertilised the female. Dr.
Perkins also has kindly informed us that out of 500-1000
specimens of this parasite seen by him only one or two were
males, so that evidently the males are on the point of dis-
appearance. ‘Taking these facts in conjunction with the
practical impossibility, as it seems to us, of the spermatozoa
in Stylops ever entering the body of the female at the time
the eggs begin to develop, we are led to the conclusion that
development is always parthenogenetic in the Stylopide. If
this is correct, it follows that the active winged males are
useless for the propagation of the species, a conclusion which
few would accept without misgiving.
Various observers have attempted to observe the act of
copulation in the Strepsiptera, but mostly with no or very
equivocal success. Pierce (11), in his valuable revision of the
Strepsiptera, remarks—‘‘ That the female must be fertilised
can hardly be doubted, and yet the nature of the act and the.
fact itself has been but slightly proven.’ Observations on
the behaviour of the male by Saunders and Crawford (11)
show that he is actively attracted by the female Stylops, or,
at any rate, by the bee on which a female Stylops is situated,
and that he runs about on the body of the bee, evincing the
greatest excitement. F. Muir (17) has made similar observa-
tions, but is uncertain how copulation takes place.
vou. 60, PART 3.—NEW SERIES. 31
4.49 GEOFFREY SMITH AND A. H. HAMM,
The following account, compiled by one of us (A. H.) from
notes on the habits of the male, may be given:
At the end of April and beginning of May, 1912, the male
Stylops was not uncommon in the vicinity of the colony of
Andrena nigroznea, being seen on the wing at mid-day in
sunshiny weather. The singular flight of the male Stylops has
often been seen and commented upon by many observers. When
once recognised they can never be forgotten, the peculiar
fight and milky-white wings at once distinguishing them
from all other insects. None were observed actually flying
over the burrows of the bee, and nearly all, when first seen,
were some 10 or 15 feet from the ground, sufficiently high to
prevent some of them being captured. All the specimens
caught were boxed alive for further observation or experi-
ment. On three occasions a male Stylops was, immediately
after capture, introduced into a large glass-bottomed box
containing a freshly caught bee, infected with one or more
female Stylops. In each case the behaviour of the male was
identically the same. The male Stylops, directly it was intro-
duced into the box, fluttered on to the bee, and quickly ran
over its body to where the head of the female Stylops was
everted between the bee’s abdominal segments. At this time
the male is rapidly vibrating its wings and protruding its
last two or three apical segments, which are long and tapering
like an ovipositor. The insect is thus quite unlike a dried
specimen in which these segments are invariably telescoped
into the body. Actual pairing did not occur on any of the
three occasions. The bee, which had been resting quietly in
the box, became extremely restless as soon as the male Stylops
flew on to it, and kept repeatedly climbing to the top of the
box and then suddenly dropping, as if in the endeavour to
rid itself of its unwelcome rider. After about ten or fifteen
minutes of ceaseless running to and fro over the bee, the
male Stylops voluntarily quitted the Andrena, but still
continued to run and vibrate its wings for about two hours
longer, after which time it dropped apparently exhausted,
and died shortly afterwards. The other males which had
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX, 443
been boxed also continued to flutter and to vibrate their
wings ceaselessly, until, in about two hours, all movement
came to an end, and they were apparently dead. It will be
seen from this account that the male Stylops, besides retain-
ing its structure and activity unimpaired, also possesses the
instinct for attaching itself to the bee, but in no case has
actual copulation been observed.
The copulatory apparatus of the male Stylops (fig. 8, a
and B) consists of a hollow chitinous penis, shaped rather like
a pick-axe, which can be everted on a hinge, but when with-
drawn is covered by a grooved sheath. Although the penis
is a slender organ it has a sharp point, and might be used
for hypodermic injection of spermatozoa into the body of
the female. It is, however, very difficult to see how the
- Injection of spermatozoa into the body of the female would
result in fertilisation, because the eggs never quit the ovary,
and are always completely surrounded by follicular epi-
thelium, which would prevent the access of spermatozoa
casually injected into the body-cavity. ‘lhe male does not
show any ‘trace of degeneracy in its internal reproductive
organs, the vesicule seminales being crowded with active
spermatozoa.
In several females the eggs have been found in an early
stage of development, the features of which strongly confirm
our suspicion that development is parthenogenetic. In these
cases all the developing eggs are at approximately the same
stage of development, exhibiting two, or, in some cases, more
segmentation nuclei (Pl. 32, fig. 4 a,b/.), while at the periphery
of the egg a mitotic spindle is observed (p. 1), which invari-
ably exhibits a single large chromosome and three, or four
smaller ones, often in process of division. Hach egg is com-
pletely invested by the follicular epithelium (/).
Now, it is quite clear that the mitotic spindle inust represent
the first polar body in process of division. There is, however,
no trace of a second polar body, which there certainly ought
to be if a second polar body was given off and fertilisation
effected in the usual way.
VoL. 60, PART 3.—NEW SERIES. B1§
444, GEOFFREY SMITH AND A. H. HAMM.
It is possible to explain the appearances in these eggs on
three suppositions: (1) That no second polar body is formed
and that the female pronucleus develops parthenogenetically,
(2) that a second polar body is formed but fuses with the
female pronucleus, which then develops parthogenetically, or
(3) that a second polar body is formed but disappears and
leaves no trace, although the first polar body is still only im
the metaphase of its mitosis, and that fertilisation is effected
by a spermatozoon.
It must be admitted that the third supposition is exceed-
ingly improbable from what we know of the development of
any other egg, the entire disappearance of the second polar
body before the completion of the mitosis of the first polar
body being altogether unknown.
During the present year (1914) the burrows of the colony -
of A. nigrownea were carefully watched at the beginning
of the season, and the first bee carrying a female Stylops
was captured, and the parasite preserved and its eggs
examined by serial sections. These eggs were found to be
already in a fairly late segmentation stage, which is strong
presumptive evidence that development had already begun
before the bee had left its burrow, and before the Stylops
would have had a chance of being fertilised.
It will therefore be seen that the cumulative evidences in
favour of the parthenogenetic development of the eggs of
Stylops are exceedingly strong, consisting in the following
main heads: (1) There is no opening or apparatus in the
female adapted for conveying the spermatozoa to the eggs;
(2) the eggs remain throughout their development encased in
the follicular epithelium of the ovary, so that access to them
by spermatozoa which had entered the body cavity is very
difficult to imagine; (3) parthenogenesis must occur as a
normal rule in the parasites of Halictus; (4) the known
stages in the polar-body formation of Stylops are inconsistent
with the view that fertilisation by a spermatozoon has been
effected; (5) actual copulation by the male has never been
adequately observed.
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 445
We may finally note that in a large number of colonies of
infected Andrena it would appear that the male parasite is
very much scarcer than the female, and in certain cases may
have almost entirely died out. This rule is, however, not
invariable, and in the case of Xenos, Wheeler (12) records
an actual preponderance of males over females.
The most difficult thing to explain, on the assumption that
the males are now useless, is their marked instinct for
clambering on the body of the bee when infected by a female
Stylops. This surely indicates that at some time pairing
took place in this situation. In explanation of this it must
be remembered that under present conditions the female
Stylops never develops beyond what is really a larval or rather
pupal stage, and that at some previous period in the history
of the parasite it is certain that the female developed further
and probably issued from the bee as a fully-formed and pos-
sibly winged imago. It may well have been that when this
was the case the males waited for the emergence of the
females and paired with them directly they issued from the
bee, and that they still retain the instinct of attaching them-
selves to the infected bees and waiting for the appearance of
the female imago, an appearance which now is never realised.
For an explanation we are forced to fall back upon some such
hypothesis as this, since no transitional forms are known to
exist in nature which might show the intermediate steps by
which the endo-parasitic habit and arrested development of
the female parasite have been acquired.
If we are correct in supposing that the males of the
Stylopide are useless and that development is invariably
parthenogenetic, it may be pointed out that such a condition
of affairs is not altogether without parallel according to the
results of recent researches. In the Rhizocephala (18)
degenerate Cirripedes parasitic on various Decapod crustacea,
we find that in certain genera, e.g. Sacculina and
Peltogaster, the parasites are hermaphrodites which propa-
gate themselves by a continuous round of self-fertilization.
Nevertheless, degenerate larval males are found, often in
4.4.6 GEOFFREY SMITH AND A. H. HAMM.
numbers up to twenty or thirty, fixed round the mantle
opening of about 80 per cent. of young Sacculine, and
these larval males are entirely degenerate and useless in the re-
production of the species. Other genera of the Rhizocephala,
e.g. Sylon, are purely female, and reproduce entirely by
parthenogenesis, and have thus got rid of the marked dis-
harmony (to borrow Metchnikoff’s term) which characterises
the sexual economy of Sacculina and Peltogaster.
Another instance of sexual disharmony has been described by
Maupas in certain free-living Nematodes of the genus
Rhabditis, where again the majority of the individuals are
self-fertilizing hermaphrodites, but a certain number of males
are still produced which are useless for reproduction. In
other species these males have been entirely eliminated.
These instances of undoubted disharmony, or imperfection
of adaptation in sexual economy, should make us pause
before we assume that the males of the Stylopide must still
be functional in those species in which they occur in
considerable numbers.
2. Tur EFFECT oF THE PARASITES ON THEIR Hosts.
We will consider first the effect of the parasites on the
internal reproductive organs of the hosts.
In the case of twenty female Andrena nigrownea, of
which four carried male Stylops puparia and sixteen
female Stylops, it was found in every case that the ovary
was very greatly reduced in size and was incapable of pro- |
ducing mature ova. The appearance of such reduced ovaries,
as compared with that of normal ovaries, is shown on
Pl. 33, figs. 9 and 10.
No marked difference was observed in cases where male
parasites were present from those in which female parasites
were concerned.
It was found, therefore, without exception, that stylopisa-
tion brought about a reduction in size of the ovary and
complete sterility.
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 447
This result is in agreement with the observations of
Pérez (1).
In the case of fifteen male A. nigroznea, of which four
carried male puparia, ten female Stylops, and one had a
male and a female parasite, it could not be observed that the
presence of the parasites in any case had exerted any effect
on the development of the testes or their ducts. The figures
given on Pl. 33, figs. 11 and 12, show the male reproduc-
tive apparatus in normal and stylopised males, and it will be
seen that there is no reduction in size in the stylopised
individual. In order to test whether the testes of the
stylopised males produce ripe spermatozoa, it was found
necessary to examine bees early in the year soon after their
emergence from the burrows, since both normal and stylopised
individuals later in the year were generally found with the
vesicules empty of spermatozoa.
If, however, stylopised males are taken early in the year,
it is possible to show that their testes and ducts are in the
same condition as normal males, and that abundance of ripe
spermatozoa are present in the large vesicles which lead from
the three testicular tubes on each side into the vas deferens.
The section on PI, 33, fig. 18, through the three testicular
tubes and the vesicle of one side of a stylopised bee, shows
the presence of abundant spermatozoa in the vesicle. ‘The
testicular tubes in this section are more or less empty with a
rather ragged degenerate epithelium, but this appearance is
due to the fact that spermatogenesis is over, and is equally
to be noticed in normal males.
This absence of any effect of the stylopisation on the male
internal organs is on the whole in agreement with what other
authors have found, though Pérez records some cases of a
one-sided damage being inflicted on the testes by the
parasite, and Theobald (7) is inclined to believe that the
damage may be considerable. Perkins (5), on the other
hand, both for males and females, tends to minimise the
effect of the parasites on the internal organs, and records the
fact that stylopised males have been taken in copula with
448 GEOFFREY SMITH AND A. H. HAMM.
stylopised females, showing that the sexual instincts may still
persist in stylopised individuals.
There can be no doubt from our own observations, and
from the general consensus of opinion, that the female bees
suffer far more serious reduction in their ovaries than the
male bees experience in the case of their internal reproductive
organs.
The reason for this difference in effect appears to us fairly
obvious. The testes of the male bee are exceedingly minute
structures, about a hundredth part of the ovaries in size, and
they, therefore, require a small fraction of the nutriment
which is demanded by the ovaries. The presence of the
parasite, therefore, while cutting off a large part of the
necessary nutriment from the ovaries, does not succeed in
depriving the minute testes of the small amount of nourishment
which they require, and hence they are able to attain their
normal development, though the ovaries are seriously
starved.
We may now proceed to the effect on the external
characters.
The males and females of A. nigroznea differ, firstly
from one another in their external genital armature which
consists of a complicated copulatory apparatus in the males
and of an ovipositor in the female. After examining a long
series of stylopised males and females, we are unable to find
any reduction or abnormality in these structures as the result
of stylopisation. In this respect we are not in complete
agreement with Pérez (1), who reports a marked reduction in
the development of the genital armature as the result of
stylopisation in several cases. We do not doubt that this is
correct, but the effect in any case is a comparatively slight
one, and there is never the slightest difficulty in at once
recognising the male and female stylopised individuals by the
genital armature which is always typically developed, though
it may be in certain cases somewhat reduced in size.
Another important secondary sexual character, affecting
the hard chitinous structure of the bee is found in the
,
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 449
antennz, which are 13-jointed in the males and 12-jointed in
the females, of all Andrena (see Pl. 35, figs. 14-15).
This character in our experience and in the experience of
all other observers is quite unaffected by stylopisation, the
infected individuals always having the number of joints
typical of their sex. Pérez again is of opinion that in certain
cases the relative length of the joints in infected individuals
is slightly altered, but here the effect is admittedly very
slight indeed, and the figures given to illustrate the effect do
not appear to us to bear out the contention.
A marked distinction between the sexes of all Andrena
is found in the structure of the femur and tibia of the hind
legs, which are thin and not markedly hairy in the male, but
in the female are greatly enlarged to form the scopa or pollen-
collecting apparatus. The condition of the normal male
(A. nigroenea) is shown on Pl. 34, fig. 16, of the infected
wale, in Pl. 34, fig. 17, of the normal female in figs. 18 and
19, and of two stylopised females in figs. 20 and 21.
These figures bring out the fact, which we have found
invariably in A. nigrownea, that as the result of stylopisa-
tion the male does not acquire in any degree the scopa of the
female, while the scopa of the female is always to some extent
reduced in size by the action of stylopisation.
We also find that stylopised females never carry any pollen
on their scope, in marked distinction from the normal
females, the majority of which are found with their scope
plastered with pollen as shown in fig. 18. The stylopised
females have evidently entirely lost the instinct for collecting
pollen, though they still continue to visit the burrows. Of
the hundred or so stylopised females examined not a single
individual had pollen on it, but we are not in a position to say
that such a thing cannot ever occur, as there is certainly a
great degree of variation in the intensity of the effects of
stylopisation in different individuals and species.
We have found that stylopisation affects the punctuation of
the chitin of the abdomen to a certain extent, though the
effect can only be appreciated by examining a good series of
4.50 GEOFFREY SMITH AND A. H. HAMM.
normal and infected individuals together. If we look at a
series of normal males and females together, we shall notice
that the males reflect the light more brightly than the females,
owing chiefly to the less degree of punctuation and hairiness
of the abdomen. }
The stylopised males, on the other hand, tend to have the
abdomen dull, very much as in the female, and this appears
to be due to the deeper and more frequent punctuation on
the abdomen, and not to a greater hairiness. The stylopised
females do not appear to be affected either in punctuation or
hairiness.
We have now dealt with the most important secondary
sexual characters which concern the structure of the hard
chitinous parts, and it will be recognised that the effect
exerted by stylopisation is small and consists in a reduction
of certain sexual characters, and never in a real assumption of
characters proper to the opposite sex. The most constant
and striking effect is the reduction of the scopa in the female
and the loss of the instinct for collecting pollen. Comparing
these effects with the effect of Sacculina on the secondary
sexual characters of Inachus (14), it will be admitted that
the complete inversion suffered by the males of Inachus has
no parallel in the bees modified by stylopisation, so far as
structure is concerned.
‘here remains for consideration, however, a very important
character which may undergo a very complete inversion as
the result of stylopisation. In certain species of Andrena,
e.g. A. chrysosceles and A. labialis, the female has the
ordinary black clypeus, but the male has a yellow or white
one (see Pl. 35, figs. 22, 23, 25 and 27). Pérez discovered
that as the result of stylopisation the female might assume
completely or in part the coloured clypeus of the male (see
Pl. 35, fig. 26), while the male might undergo considerable
retrogression and lose a great part of the yellow colouration
(fig. 28). Pérez makes it clear and has personally informed
us that this remarkable effect is by no means invariable and
that very frequently stylopised males and females of A.
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 451
labialis may exhibit their proper clypeus colouration with-
out any modification. It is therefore necessary in this case
to be able to examine a long series of infected individuals, and
it would appear that other observers have not been fortunate
in securing such aseries because no confirmation of Monsieur
Pérez’s discovery has hitherto been published. We are
fortunately in a position to produce confirmatory evidence in
the case of A. chrysosceles. PI. 35, fig. 22, depicts the
head of a normal female specimen, while fig. 23 shows the
head of a normal male with the coloured clypeus. Fig. 24
shows the head of a stylopised female, taken at Sandford
near Oxford by one of us (A. H. H.), which has developed
the coloured clypeus of the male in a typical manner. ‘This
specimen was parasitised by a male Stylops. It may be stated
that long series of normal A. labialis and chrysosceles
have been examined, and that in no case has any assumption
of the coloured clypeus by the female or vice versd been
observed apart from the effects of stylopisation.
This acquisition of clypeus colouration by the female is by
far the most striking alteration brought about: by stylopisa-
tion, because it really amounts to a true acquisition of a
positive character belonging to the opposite sex, and not to a
mere negative suppression of characters that should normally
be developed. ‘lhe alterations in the hard parts and the
blackening of the clypeus in the males, can all be interpreted
as mere negative suppressions, but the acquisition of the
yellow clypeus by the stylopised females is in a different
category. In the majority of Andrena the clypeus of both
sexes is black, so that the loss of the yellow colour in the
stylopised males of A. labialis may be considered a mere
repression, but not so the acquisition of the yellow colour by
the female.
Before discussing these results there are two points
which merit attention. Since the Stylops parasites are
of separate sexes, it appeared possible that the sex of the
parasite might have an important imfluence on the effect
exerted upon the bee. For instance it might be found that
452 GEOFFREY SMITH AND A. H. HAMM.
only females stylopised by male Stylops could develop the
white clypeus characteristic of the normal male bee. Of
course if such a contention could be proved it would have a
most important bearing on the theoretical interpretation of
how the effect is brought about. It would suggest, in fact,
that the male Stylops exerted a specific male influence on
the bee, and the female Stylops a specific female influence.
In answer to our inquiries Monsieur J. Pérez has kindly
sent us some of his specimens of A. labialis, which satisfac-
torily settle this point. Among these specimens there are
two female A. labialis parasitised by female Stylops
which show a considerable amount of white colour on the
clypeus, and there is also a male A. labialis parasitised by
a male Stylops, which shows avery marked reduction of the
white colour on the face.
Dr. R. C. L. Perkins has also sent us two valuable instances
bearing on this question, viz., two females of A. labialis,
with their faces coloured as in the male, both of which are
parasitised by a single female Stylops. These instances are
abundantly sufficient to demolish the view that the sex of
the parasite has any determining influence on the effect pro-
duced on the secondary sexual characters. It is probably
true that the presence of a male Stylops has a more generally
damaging effect on the bee, but there is no evidence of the
male parasite exciting a specifically male effect and of the
female exciting a female effect upon the host.
The theoretical importance of this fact will be given its
due weight in a later paragraph.
The second point to which attention may be called is the
great amount of variation exhibited by Andrena and other
insects in their reaction to strepsipterous parasites. This
variation does not only subsist as between different species of
hosts, but also as between different individuals of the same
species of host. Wheeler (12), who has made a most detailed
and exhaustive examination of the effect of Xenos on the
wasps Polistes came to the conclusion that the parasite
had no definite effect on the secondary sexual characters of the
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 4038
host, and it is clear that his conclusion is perfectly correct.
The same may probably be said of the effect of Eleuchus
on the Homoptera.
We have already seen that no other observer has apparently
described the effect of Stylops on the clypeus colouration of
certain Andrena, noticed by Pérez, until we came across
the case of A. chrysosceles published here. These appa-
rently contradictory results have led to much confusion, but
to anyone familiar with the facts of parasite castration in
other branches of the animal kingdom they will occasion no
surprise. In cases where the effects of the parasite lead to
the most startling and complete inversions, as in Sacculina
on Inachus and Peltogaster on Eupagurus (14), there
is always a certain small percentage of individuals which
remain almost completely unaffected by the presence of the
parasite, while in other cases, such as Sacculina on
Carcinus, the effect of the secondary characters is often
nil, and never consists in more than a slight approximation
of the male to the female type. Such variations then,
whether due to differences in individual or specific suscepti-
bility or to some casual event in the history of the disease,
are to be expected, and should warn the observer not to draw
conclusions without examining a long series of infected
individuals.
(5) Discusston or Resvtrs.
If we compare the effects of stylopisation with those of
Sacculina on Inachus, we shall recognise that they are
much slighter and less radical in the former than in the
latter case. Thus in the internal reproductive organs stylopi-
sation only causes a reduction in size of the ovary, and
preveuts ripe ova from being produced, while the testes of
the male are practically unaffected. ‘The presence of Saccu-
lina, on the other hand (18), may occasion the complete
destruction of all the internal genital organs, with the excep-
tion of some remnants of, germinal epithelium, while infected
A454, GEOFFREY SMITH AND A. H. HAMM.
male crabs may be induced by the Sacculina to produce
ripe ova in their testes.
Correspondingly the effects of stylopisation on the second-
ary sexual characters are comparatively slight, and amount
to no more than a reduction of certain characters, such as
the scopa of the female, while in the case of Sacculina the
whole morphological structure of the male crab may be
eutirely converted to the female state.
In one case, however, that of the colour of the clypeus, the
female bee when stylopised may assume the positive male
character.
It is clear, therefore, that the reaction of the bee to the
Stylops does not go so far as that of the crab to Sacculina,
either internally or externally, and whereas in sacculinisation
we are forced to the conclusion that the Sacculina exerts
an active feminising influence on both sexes of infected crabs,
in the case of stylopisation it is sufficient to hold that the
action here consists merely in an arrest of development inci-
dent on the cutting off of a certain amount of nutriment from
the ovaries, and to a less extent from the testes. In sacculi-
nisation we have argued (18) that the Sacculina roots, by
demanding a certain type of nutriment, viz. fat, stimulate a
certain type of metabolism in the crab, which is characteristic
of the adult female when maturing its ovaries, and that this
internal change of metabolism brings in its train all the
deep-seated changes in the internal and external genital
structures. he Stylops, on the other hand, does not,
initiate such wide-spreading changes; it stops short at
abstracting a certain amount of nutriment from the blood,
and so causes a merely quantitative alteration in the develop-
ment of the internal and external genital organs. It has
been pointed out that the Stylops parasite does not appear to
be taking up any special nutriment from the blood of the
host, but rather to receive the nutriment from the blood,
ready-made, and thus it would not be expected to stimulate
any special line of metabolic changes. Further, the Stylops
always receives its oxygen from the outside air, while the
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 455
Sacculina roots are living amerobically, and must split
off their oxygen from the blood of the host. This implies a
more intimate relation between the metabolism of host and
parasite in the case of Sacculina.
We thus see that the effects of stylopisation may be inter-
preted as due toa merely quantitative abstraction of nutriment
normally destined for the reproductive glands, and that this
abstraction brings in its train a reduction in size of these
glands, especially the ovaries, and a corresponding reduction
in the development of some of the secondary sexual
characters.
It may be urged, however, that this explanation does not
apply to the assumption of the yellow clypeus by the female,
as this is a positive male character. We have an excellent
analogy for this case in sterile female birds (15) which, either
as the result of operative ovariotomy or else of ovarian
disease and atrophy, may assume male plumage to a very
marked extent. The assumption of the yellow clypeus by
stylopised female bees with reduced ovaries seems to us
exactly parallel to the assumption of cock’s plumage by
female birds with ovaries either atrophied or removed by
operation. It seems that in both cases the mere atrophy or
suppression of the ovary is sufficient in both cases to induce
the development of certain male characters in the colouration.
It is not necessary, therefore, to ascribe a special mascu-
linising influence to the Stylops parasite in the same sense as
one must ascribe an active feminising influence to the
Sacculina roots. The masculinising influence resides in
the female bee itself, just as in the female bird, and is
called into activity by the mere suppression of the ovarian
function.
In this manner we may look upon the acquisition of the
yellow clypeus by the female as due to the same cause as the
other alterations brought about by stylopisation, viz. to
the mere quantitative cutting off of nutriment from the ovary,
and not to any specific or qualitative action of the parasite,
as in the case of Sacculina on Inachus.
456 GEOFFREY SMITH AND A. H. HAMM.
It appeared possible to us at one time that a qualitative
action might account for the assumption by the female of the
male clypeus in the following manner: It might be possible
that this assumption by the female only followed when she was
parasitised by a male Stylops, which might exert a specific
male influence on the host. This supposition is not confirmed
by the facts, as the presence of a female Stylops can equally
bring about the assumption of the male clypeus by the
female.
The fact that the sex of the parasite has no influence on
the effect exerted on the host is in reality a strong confuta-
tion of the idea that the effects of parasitic castration are due
to a specific internal secretion produced by the parasite.
For if such a secretion were produced by the parasite, we
should certainly expect that the female parasite would
produce a female internal secretion and the male a male one,
whereas we find that the parasites of both sex exert a similar
effect. This is perfectly intelligible if we suppose that the
parasites of both sexes act on the host merely by cutting off a
certain amount of nutriment from the gonad, a process which
reacts more profoundly on the female than on the male, owing
to the larger size of the ovaries and the larger demand made
by them on the nutriment in the body.
The peculiarity of the case of Sacculina consists in the
fact that the roots of the parasite happen to demand an
excessive supply of the same sort of nutriment which the
ovary of a normal female crab requires, and so bring
about a series of profound metabolic changes leading to the
feminisation of the host.
The result of all the above considerations is to show that a
parasite may act on the sexual characters of its host in two
ways. Firstly, it may simply take up a certain amount of
nutriment from the blood so as to deprive the gonad of its
proper supply and lead to its partial atrophy, but without
bringing about any deep-seated alteration in the metabolism
or stimulating any special set of metabolic changes. ‘The
abstraction of this nutriment, by depriving the blood of its
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 457
proper supply, may lead to the atrophy of the gonad and to
the reduction of the secondary sexual characters, and even to
an inversion of certain secondary sexual characters, e. g. the
colour of the clypeus in Andrena. Secondly, as in the
ease of Inachus parasitised by Sacculina and of
Eupagurus parasitised by Peltogaster, the parasite in
obtaining its food from the blood of the host, may set going
a special set of metabolic changes in the host, and these
changes may result in diverting the metabolism of the host
to the female state, so that the host assumes female
characters throughout and the infected male may even be
induced to produce ova in its testes. This second type is not
a mere passive inhibition like the first, but an active reaction
explicable, as has been shown, on the basis of an immunity
reaction (14).
Another point which emerges from this study is that the
bee, Andrena, belongs to the same category as many birds
(Pheasants, Fowls, Ducks, Ostriches, etc.), (15) in that a mere
atrophy of the ovary is followed by an assumption of a posi-
tive male secondary sexual character. In all these animals it
would appear that the normal ovary exerts an inhibitory
action preventing certain male characters from emerging, and
that when the ovarian influence is removed or interfered
with the stimulus is given for the development of these
characters.
SUMMARY.
(1) From a study of the anatomy and life history of
Stylops, it appears that despite the existence of active
winged males, fertilisation cannot occur and development is
always parthenogenetic.
(2) The parasite obtains its oxygen from the outside air by
means of tracheal openings on the cephalo-thorax, and it does
not possess any special absorptive organs for taking up a
special kind of food from the host. Nutrition appears to
take place by simple filtration from the host’s blood through
the very thin skin of the parasite.
458 GEOFFREY SMITH AND A. H. HAMM.
(3) The effect of the parasite on the internal genital organs
is slight, as compared with the effect of Sacculina on
Inachus, and leads to a reduction in the size of the ovaries
to about quarter the normal size, while the testes are usually
unaffected. The ovaries of stylopised bees never produce
ripe ova, but the testes generally produce normal ripe
spermatozoa.
(4) The effect on the secondary sexual characters is again
slight as compared with that of Sacculina on Inachus.
The external gonapophyses are usually unaltered, or they may
be slightly reduced in size ; the antenne are unaltered. The
scopa of the parasitised female is generally reduced in size,
and she never or very rarely collects any pollen. The
punctuation on the abdomen of the male may be increased.
(5) The most striking effect occurs in certain species (e. g.
A. labialis and chrysosceles) in which the male normally
has a yellow clypeus and the female a black one. Stylopisa-
tion in those cases may lead to the female assuming a yellow
clypeus as in the male, while the male may lose the yellow ~
and acquire a partially black clypeus.
This acquisition of the yellow clypeus by the female is the
only change which can undoubtedly be interpreted as a
positive acquisition of a secondary sexual character proper to
the opposite sex. ;
(6) This effect may be brought about by male or female
Stylops indifferently, the sex of the parasite having nothing
to do with the nature of the effect exerted.
(7) The effects of stylopisation may be ascribed to a merely
quantitative abstraction of nutriment from the gonad, leading
to its partial atrophy, and not to a qualitative alteration of
the metabolism such as is brought about by Sacculina.
This also applies to the assumption of the yellow clypeus by
stylopised females, on the analogy of the assumption of male
plumage by many female birds as the result of simple
ovariotomy or ovarian atrophy.
10.
11.
12.
13.
14.
15.
16.
17.
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 459
LITERATURE.
. Pérez, J—* Des effets du Parasitisme des Stylops sur les Apiaries
du Genre Andrena,” ‘ Actes Soc. Linn. Bordeaux,’ vol. xl, 1886.
. Siebold, Th. v.—‘ Uber Strepsiptera,” ‘Archiv. f. Naturg., Jahrg. 9.
. Dale, C. W.—* Stylopide,” ‘Entom. Monthly Mag.,’ p. 50, 1892.
. Eaton, A. E.—‘‘ Notes on Elenchus tenuicornis,” loc. cit.,
p- 250.
. Perkins, R. C. L.—* Stylopised Bees,” loc. cit., p. 7.
. Saunders, E.—‘ Elenchus tenuicorius,” loc. cit., p. 249.
. Theobald, F. V.—“ Stylopised Bees,” loc. cit., p. 40.
. Brues, C. T.—* A Contribution to our Knowledge of the Stylopide,”
‘Zool. Jahrb. Abth. f. Anat.,’ xviii, p. 241, 1903.
. Perkins, R. C. L.—‘ Report of Work of the Experimental Station
of the Hawaiian Sugar Planters’ Association,’ ‘‘ Leaf. Hoppers
and their Natural Enemies,” Part iii, “Stylopide,” Honolulu,
1905.
Nassonow, N. V.— Xenos rossii, seine Anatomie und Entwick-
lungsgeschichte,” ‘ Bull. Univ. Warsaw,’ 1892. ‘On the Meta-
morphosis of the Strepsiptera,” ‘Warsaw University News,’
No. 7, 1892. “On the Morphology of Stylops melitte,”
‘ Warsaw University News,’ Nos. 8 and 9, 1893.
Pierce.—* A Revision of the Strepsiptera,” ‘Harvard Museum Bull.,’
1906.
Wheeler, W. M.—<‘ The Effects of Castration in Insects,” ‘ Journ.
of Exp. Zool.,’ vol. viii.
Smith, G.—*The Rhizocephala,” ‘Naples Monographs, No. 29,
1906.
“Studies in the Experimental Analysis of Sex,’ Parts
7 and 10, ‘ Quart. Journ. Mier. Sci.,’ vols. 57 and 59.
and Mrs. Haig Thomas.—* Sterile and Hybrid Pheasants,”
‘ Journ. of Genetics,’ vol. iii, 1913. :
Meinert, F.—“ Contribution a l’histoire naturelle des Strepsiptéres,”
‘Bull. de ’Acad. Royale des Sciences de Danemark,’ November,
1896.
Muir, F.—“ Notes on Some Fijian Insects,” ‘Report of Work of the
Experimental Station of Hawaiian Sugar Planters’ Association,’
Bulletin No. 2, 1906.
4.60 GEOFFREY SMITH AND A. H. HAMM.
EXPLANATION OF PLATES 32-35,
Illustrating Mr. Geoffrey Smith’s and Mr. A. H. Hamm’s
“Studies in the Experimental Analysis of Sex.” Part
II.—* On Stylops and Stylopisation.”
LETTERING.
b. Brood-passage. 6. v. Dorsal blood-vessel. bu. Bulb on vas
deferens. c. Supporting cells. ch. Chitinous investment. ep. Modi-
fied epithelium of brood-passage. g. Gut. gn.1. Brain. gn. 2. Sub-
cesophageal ganglion. gn. 3. Abdominal ganglion. m. Mouth. md.
Mandible. n. Ventral nerve. nu. Nurse-cells. o. Opening of brood-
passage. ov. Ova. te. Testes tubes. tr. Tracheal tube. vd. Vas
deferens. v.e. Vasa efferentia. ves. Vesicula seminalis.
Fig. 1—Abdomen of Andrena nigrow#nea ? with heads of three
Stylops melittze 2, protruding in natural position.
Fig. 2—Stylops melittw 2, from ventral surface, removed from
bee.
Fig. 3.—Stylops melitte 2, in sagittal section.
Fig. 4.—Ditto, transverse section through middle of body.
Fig. 44.—Section through developing egg with first polar body (p. 1),
blastomeres (b/.), and follicular epithelium (f/f).
Fig. 5.—Triungulin larve, from brood-passage, ventral view.
Fig. 6.—Stylops 2, adult, with body full of triungulins ready to
hatch.
Fig. 7.—Ditto, triungulins removed, showing five trumpets attached
to epithelium of brood-passage. Two trachex spread out laterally.
Fig. 8—Stylops melittx, ¢, dorsal view; abdomen telescoped.
Fig. 84.—Copulatory apparatus of S. melittez, lateral view. B.
Penis.
Fig. 9.—Ovary of normal Andrena nigroenea 2°.
Fig. 10.—Ovary of Stylopised A. nigro#nea ¢.
Fig. 11.—Testes and ducts of normal A. nigroxnea @.
Fig. 12.—Testes and ducts of Stylopised A. nigroxnea J.
Fig. 13.—Section through testes, ducts, and vesicula of one side of
Stylopised A. nigroznea, showing ripe sperm in vesicula.
Fig. 14.—Antenna of normal A. nigroenea 2°.
Fig. 15.—Antenna of normal A. nigroewnea d.
STUDIES IN THE EXPERIMENTAL ANALYSIS OF SEX. 461
Fig. 16.—A. nigrownea, normal ¢.
Fig. 17.—Ditto, stylopised ¢, showing unaltered legs and gonapo-
physes.
Fig. 18.—Ditto, normal ?, carrying pollen on scope.
Fig. 19.—Ditto, normal ? , showing scope without pollen.
Fig. 20.—Ditto, stylopised 9, showing reduced scopw, otherwise
unaltered.
Fig. 21.—Ditto, stylopised 2, showing reduced scope, otherwise
unaltered.
Fig. 22.—Andrena chrysosceles, head of normal 9.
Fig. 23.—Ditto, head of normal ¢.
Fig. 24.—Ditto, head of stylopised 9.
Fig. 25.—Andrena labialis, head of normal 9, after J. Pérez.
ig. 26.—Ditto, head of stylopised ?, after J. Pérez.
Fig. 27.—Ditto, head of normal ¢, after J. Pérez.
ig. 28.—Ditto, head of stylopised ¢, after J. Pérez.
voL. 60, PART 3.—NEW SERIES. 32
-
THE RAT-TRYPANOSOME, I'RYPANOSOMA LEWISI.
4.63
The Rat-Trypanosome, Trypanosoma lewisi,
in its Relation to the Rat-Flea, Cerato-
phyllus fasciatus.
By
E. A. Minchin
and
J. D. Thomson.
With Plates 36—45 and 24 Text-figures.
ConTEN's.
Part I—IntTRODUCTORY .
(1) Personal Narrative .
(2) Notes on the Flea, Cer Roca IheL ae fageratae
(a) Anatomy, Methods of Dissection ;
(b) Notes on the Parasites of the Flea p
(c) Notes on the Histological Structure of the Suonmedh
of the Flea é :
(3) Technique
Part I].—TuHE DEVELOPMENT OF T. LEWISI IN THE FLEA.
(1) General Introduction
(2) The Developmental Series
A. The Stomach-Phase
(a) The Extracellular rey pRnGsGrien
(b) The Intracellular Multiplication of the Tr ypano-
somes ‘
Appendices to ne Siomdck Phase,
(i) The Occurrence of the Intracellular
Multiplication
(ii) The Type of Cell Attacked by the
Trypanosome 5 ;
voL. 60, PART 4.—NEW SERIES. 33
545
547
4.64 E. A. MINCHIN AND J. D. THOMSON.
PAGE
iii) The Relation of the Trypanosomes to
the Cells : 548
(iv) The Effects of the Tey parioseaiee on
the Epithelial Cells . : 549
(v) The Relation of the Ty panoeoraes
Infection as a whole to the Stomach 551
B. The Migration to the Rectum : ; . 568
c. The Rectal Phase ; - oD
(a) The Transition to the Crithidial For . 570
(b) The Established Rectal Phase : ei eis.
(3) The Degenerative Series. : . 592
Appendices to the Meyelopinent:
(i) Previous Investigations on the Deve-
lopment of Trypanosoma lewisi 597
(ii) On the Possibility of the Occurrence
of Sexual Phenomena in T. lewisi. 603
Part JII.—ExXxpPEeRIMENTAL STUDY OF THE PROBLEMS OF
TRANSMISSION AND DEVELOPMENT . = 605:
(1) Introduction : : : : . 605
(2) General Problems . : : . 609
(3) Problems of Special Nate ‘ ; . 659
BIBLIOGRAPHICAL REFERENCES . : : Be OS:
DESCRIPTION OF PLATES. E ‘ 3 211682
PART I. INTRODUCTORY.
(1) Personat NarRATIVE.
In this memoir we give a detailed account of investiga-
tions which have occupied us intermittently, with many
interruptions, during the past five years. When we
undertook this task our object in view was to work out as
fully as possible the life-history and mode of transmission of
a trypanosome, so that in at least one species of these
important parasites its relation to the invertebrate. host
might be as thoroughly known as, for instance, the relation
of the malarial parasite to the mosquito, thus furnishing a
standard with which the life-histories of other species of
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 465
trypanosomes might be compared and contrasted as they
become known. How far we have succeeded in our task
must be left to our readers to judge.
The species which we selected as the subject of our in-
vestigations was Trypanosoma lewisi, the common
parasite of rats which is apparently of world-wide distribu-
tion. ‘This species offers many advantages for such a study.
It is common in London and easily procured when required ;
its vertebrate host, the rat, is a mammal of small size, the
domesticated variety of which lives well and breeds rapidly
in confinement, is inoffensive, and is easily handled; its
invertebrate host, the rat-flea, is also easy to keep in
captivity and is extremely prolific, and it is of a size which,
though it increases to some extent the difficulties of manipu-
lation, has a great advantage that the material to be searched
and studied microscopically is confined within a _ small
compass!; and finally, by no means the least of the
advantages of working with T. lewisiis the fact that it is
non-pathogenic to its natural host and cannot live at all in
human blood.
Since there is no difficulty whatever in obtaining the
vertebrate host in abundance, either in the clean (i. e. non-
infected) or infected condition, our first care was to obtain a
stock of the invertebrate host, the flea. This we succeeded
in doing from rats trapped in the open near Mr. Gurney’s
Laboratory at Sutton Broad, Norfolk. Fifty specimens of
Ceratophyllus fasciatus were obtained in this way in
the autumn of 1908, and were kindly identified for us by the
Hon. N. C. Rothschild, and with these fleas a breeding-cage
was stocked and a flea-farm started. The cages used were of
the type used by the Plague Commission, as figured in the
‘ Journal of Hygiene,’ vol. vi, pl. iv. A rat was kept in the
cage to feed the fleas, and they were left to themselves.
Karly in 1909 one of us (H. A. M.) went to Rovigno for some
1 An advantage which those will appreciate who have had practical
experience of searching for trypanosomes through many centimeters of
the digestive tract of the tsetse-fly, for instance.
466 E. A. MINOHIN AND J. D. THOMSON.
three months, during which time the fleas were left to breed
under the care of an assistant, whose duties consisted of
attending to the rat and replacing it if it fell ill or died.
When the cage was examined after Haster it was found to be
swarming with fleas, and onr work began in May, 1909. We
have worked ever since then with the fertile progeny of the
original fifty fleas from Norfolk, and have never added
further to our stock from without. The fleas breed so fast
that it is often necessary to keep their numbers down, other- ,
wise they take too much blood from the rat and affect its
health. Fresh breeding-cages have also been started, and
during the greater part of the time that we have been at
work we have kept two cages constantly going, one in which
the fleas are fed always on a clean healthy rat, and another
in which an infected rat is always kept. We shall refer to
these two cages as the non-infected and the infected breed-
ing-cages respectively. As will be shown below, the stock of
fleas with which we have worked all along was fortunately
quite free from any natural infection with leptomonad or
other flagellate parasites. Thus we have been saved from a
fertile source of confusion and error, since we can be quite
certain that any flagellates found in our fleas are stages of
T. lewisi and nothing else.
Although we cannot claim that in our work we have solved
completely every problem presented by the transmission of
the trypanosome and its development in the flea—a result
which probably no man could achieve in a life-time—we
think it now fitting that we should publish such results as we
have obtained, after having done as much as we were able to
do in the time and under the circumstances. We claim
at least that we have not jumped to our conclusions; our
note-books contain not only the records of many experiments,
but also of the dissection and examination of over 1,600 fleas,
and we have over 700 drawings of stages of the development
of the trypanosome, from which those given in this memoir
are a selection. It would, indeed, have been easier for us to
have written a plausible and apparently complete account of
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 467
the development of T. lewisi, full of positive statements,
after one year of our work than it is after five years, during
which we have been forced by the logic of facts to abandon
or modify many of our earlier conclusions or beliefs.
It is our pleasant duty at this point to express our thanks
to those of our friends and colleagues to whom we are
indebted for assistance. To Dr. Woodcock and Miss
Robertson we are grateful for much advice, friendly criticism,
and valuable suggestions. Our work could not have been
carried out, certainly not in the time at all events, without
the assistance of Miss Rhodes, who has not only drawn all
the illustrations with a skill to which it is quite unnecessary
for us to draw the reader’s attention (since the figures speak
for themselves), but has also relieved us of a large part of
the wearisome drudgery of searching through the microscopic
preparations. Mr. George Kauffmann has been most helpful
in every part of the investigation, not only assisting in
making preparations, examining rats, and other similar
duties, but more especially in carrying out intelligently and
enthusiastically all the details of the experiments, in which
his extraordinary skill and resourcefulness in controlling the
wayward flea were invaluable. Dr. D. J. Reid has given us
the benefit of his skill and experience in microphotography.
From our colleagues of the Lister Institute, Dr. C. J. Martin,
the Director, and Mr. Bacot, who have been themselves
engaged in studying the transmission of plague by fleas, we
have had many valuable hints and help in various ways. ‘To
each and all of these we desire to express our cordial thanks
and gratitude.
(2) Notes on THE FLEA, CERATOPHYLLUS FASCIATUS.
(a) Anatomy. Methods of Dissection.
The fleas collected for dissection and examination were
thrown, or allowed to hop, on to the surface of a small
468 E. A. MINCHIN AND J. D. THOMSON.
quantity of salt-citrate solution’ placed in a suitable glass
capsule. The fleas are quite helpless on the surface of the
liquid, and each flea that it is required to dissect can be
picked off the surface of the liquid and transferred to a
small drop of the same solution on a slide for further
operation.
The examinations of the fleas were usually conducted by
both of us acting in concert. One of us worked with the
dissecting - microscope, extracted the parts of the flea
required, placed them on slides, covered them with glass
slips, and handed them to the other, who proceeded to search
- them carefully through under a microscope, using dry lenses
of fairly high magnification (Zeiss D or apochromatic 4 mm.).
In some cases one of us worked entirely alone, but it is
difficult for one person to carry out satisfactorily both the
dissection and examination of the flea; the various parts of
the digestive tract often require prolonged and careful
searching to find the flagellates, and if the operator be
working single-handed, one preparation may dry up while
he is searching through another.
For the dissection of the flea? the following apparatus
was used: A pair of fine needles mounted in wooden
handles, a fine pair of forceps, and a dissecting-microscope,
besides slides and coverslips. The needles used were
sharpened on a hone, one to a sharp point, the other to a
flat, chisel-like edge with rounded corners. ‘he pointed
needle was the more useful for holding, the flat-edged needle
1 Made up as recommended by Laveran and Mesnil, namely: 1 grm.
of sodium citrate and 1 grm. of sodium chloride dissolved in 200 ce. of
distilled water. This mixture appears to be most favourable for the
examination of living trypanosomes.
2 If an operation can be properly called dissection which consists
in treating the flea as the Thracian women are said to have treated
Orpheus: “Discerptum latos Juvenem sparsere per agros”
(i. e. fields of the microscope, in this case). It need hardly be said
that our object was not to study the anatomy of the flea, but to extract
from its body those organs which might possibly harbour developmental
stages of the trypanosome.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 469
for cutting. The dissecting-microscope used was a Zeiss
binocular, with No. 4 eyepieces and the paired objectives
F*, It is also necessary for the dissector to have at hand an
ordinary microscope armed with a low power, since it is
often difficult to distinguish the minute’ organs of the flea
under the dissecting-microscope ; the intestine, for instance,
if severed from its connections might easily be confounded
with a portion of a Malpighian tubule.
In the following paragraphs is described the method of
procedure for making what may be considered an exhaustive
examination of a flea for trypanosomes; it was not always
necessary, however, to attempt so much, nor is it claimed
that the entire operation was always successfully carried out,
since both our knowledge of the flea’s anatomy, and our skill
in extracting the organs required, advanced considerably
during the progress of our investigations.
The flea, as stated above, is picked up with a fine pair of
forceps, holding it by its head, and placed on a slide (slide 1)
in a drop of salt-citrate solution. The first operation is to
cut off the head, which is not always easy if the flea be a
lively one, in which case it is best to asphyxiate or drown the
flea partially by holding it under water with the forceps for
a short time. ‘lo decapitate the flea, hold it still by pressing
the pointed needle across the thorax, and with the flat-edged
needle cut across the head in the region of the eyes. ‘The
severed head may then be removed to another slide (slide 2),
covered with a cover-glass, and the contents of the pro-
boscis examined ; but as the proboscis was never found to
contain trypanosomes we ceased to trouble about it in our
later studies.
It is frequently the case that the flea has its rectum filled
with faeces or with partially digested blood, and when this is
so it happens commonly that the rectum empties itself by a
violent contraction at the instant that the head is severed
(sometimes also eggs are extruded) ; or if the evacuation does
not take place at this point in the proceedings, it is very
difficult to avoid squeezing out the contents of the distended
470 E. A. MINCHIN AND J. D. THOMSON.
Trxt-Fic. 1.
Digestive tract of a female flea, dis-
sected out and drawn with the
M Eq. camera lucida at a magnification
"of 60, reduced in the reproduction
to 40. Theanterior part of the dis-
section is seen in ventral view ; the
rectum and its surroundings in side
view. cs. @sophagus. prov. Pro-
ventriculus. St. Stomach. Mt. a.
Malpighian tubule of the anterior
pair; that on the left side of the
stomach is shown in its normal -
position, that on the right has its
distal limb pulled out and away
from the stomach. Mt. p. Malpig-
hian tubule of the posterior pair.
int. Intestine. 7. p. The six rectal
papille. R. The rectum. ¢.s. Ter-
minal segments. an. The anus.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 471
rectum during the subsequent operation of opening the
abdomen. In cases where feces are thus extruded the body
of the flea is removed at once to another slide (slide 5), and
the feeces left on slide 1 are covered with a slip and
examined.
Through the integument of the flea the stomach can be
seen lying ventrally in the anterior ? of the abdomen, and
often the rectum can be seen at the hinder end in the
dorsal region. The ventral posterior and dorsal anterior
part of the abdomen is seen to be occupied by a whitish
mass, most conspicuous in the female, and consisting chiefly
of the reproductive organs.
The next stage in the proceedings is to open the body of
the flea. This is done near the hinder end, at about the level
of the fourth or fifth tergite of the abdomen. The body is
held still with the pointed needle, with which the thoracic
region is pressed down or speared, and with the flat-edged
needle the body-wall is cut through dorsally and ventrally in
the region indicated, and the hindermost segments of the
abdomen gently detached in such a way as to separate the
integumental portions without rupturing or tearing the
internal organs. It is especially important, if it be desired
to examine the contents of the body-cavity, that the
digestive tract should not be in any way torn or punctured.
By holding the anterior part of the body and pulling gently
on the detached hinder part, the gut can be stretched out
and seen in nearly its full length; the stomach, usually
containing a greater or less amount of more or less digested
blood, is seen projecting from the anterior part of the body,
the rectum is contained in the detached hinder part, and
stretching between the two is the intestine like a delicate
white filament, exposed in its whole length, but more or less
obscured by the fat-body, Malpighian tubules, and generative
organs, especially by the large ovaries in the female; these
organs render the female flea much more difficult to dissect,
in spite of its larger size, than the less-encumbered male.
The generative organs and as much as possible of the fat-
472 E. A. MINCHIN AND J. D. THOMSON.
body are now pulled out on to the slide and cut off from the
body, care being taken not to injure the gut. The carcase of
the flea, with the hinder part hanging on by the still intact
intestine, is now removed to another slide (slide 4), and the
extracted contents of the body-cavity on slide 3 can be
covered with a slip and passed on for examination ; but so far
as stages of T. lewisi are concerned, it is superfluous to do
So, since they are never found in the body-cavity unless the
gut has been punctured or ruptured.
The next step is to divide the digestive tract into two
parts, thereby severing completely the hinder part of the
body from the fore-part. This is done at the point at which
the Malpighian tubules are given off at the junction of the
stomach and intestine, the region which represents the
transition from the mid-gut, lined by endoderm, to the hind-
gut or proctodzeum, lined by ectoderm. The Malpighian
tubules are four in number in the flea; two of them run
forward a short way on the wall of the stomach right and
left, attached to it by fine tracheal tubes, and then turn
backwards again with a sharp, elbow-like bend towards the
dorsal side of the body ; the other two tubules run backwards
parallel to the intestine and alongside of it towards the hinder
end of the body. The posterior pair of the tubules are also
bent on themselves towards their distal extremities, but not
so regularly as the anterior pair. The gut is cut across with
the flat-edged needle at the point of origin of the tubules, and
if this be performed accurately one pair of tubules (the
anterior pair) remains attached to the stomach, the other pair
to the intestine ; sometimes, however, all four tubules remain
attached to one or other of these organs. The hinder part
of the body, with the intestine and rectum, is now removed
to another slide (slide 5). The stomach is then pulled back-
wards out of the anterior part of the body on slide 4, and
with it come out also, continuous with its anterior termina-
tion, the proventriculus and the cesophagus, these two parts
representing the embryonic stomodeeum, lined by ectoderm,
while the stomach represents the whole of the embryonic mid-
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 473
gut. The proventriculus is lined by a thick chitinous cuticle
prolonged into stiff, curved, pointed spines, densely planted
and forming, apparently, a straining apparatus ; it is approxi-
mately globular in form and usually contains blood. The
cesophagus is a delicate tube, its walls composed of the
chitinous cuticle internally and a delicate network of
muscles externally ; it generally performs active move-
ments, twisting from side to side, when freshly extracted.
The two pairs of salivary glands are situated in the anterior
region of the abdomen right and left of the stomach. Hach
gland has the form of a simple oval pouch, the wall of which
is composed of a single layer of large cells with very large
nuclei. From each gland comes off a duct, which, after
running a short distance, unites with the similar duct of the
other gland of the same pair. (In one instance we have seen
the two glands of one side of the body fused into one, but
with their ducts quite separate; on the other side of the
body there was a pair of distinct glands in their normal
relations). ‘The common duct of each pair of glands then
passes forwards alongside the gut through the thorax into
the head, where it meets and joins with the corresponding
duct from the other side of the body. The common salivary
duct then runs a short distance and opens into the proboscis,
doubtless on the hypopharynx as in other insects. The
salivary ducts are recognisable at once under the microscope
by their trachea-like structure, being lined by a thick cuticle
which has ring-like thickenings; the rings are, however, some-
what irregular and easily distinguishable from the very even
and regular spiral thickening of the wall of a tracheal tubule.
Externally to the cuticular lining the tubule is covered by an
investing layer of protoplasm, of uneven thickness in different
parts and containing fairly large nuclei at irregular intervals.
The ring-like thickenings of the cuticular lining become less
marked as the ducts approach their point of junction, and
cease altogether before they unite ; the cuticular lining being
quite smooth in the common duct and for short distances in
the paired ducts.
474 E. A. MINCHIN AND J. D. THOMSON.
Not infrequently the salivary glands come out with the
stomach when it is pulled out; more usually, however, they
do not do so, but remain in situ. In such cases the anterior
part of the body is removed to another slide (slide 6), and
the stomach, left on slide 4, is teased up, covered, and handed
on for examination.
Now the dissection of the hinder part of the body, on
slide 5, is proceeded with, in order to extract and separate
the intestine and rectum. The rectum, situated dorsally to
the accessory reproductive apparatus, penis or receptaculum
seminis, is a fairly large pear-shaped organ, the stalk of the
pear terminating in the anus. The slender intestine joins
the rectum at its broad end, and in this region are situated
the six conspicuous rectal papille, remarkable and very
characteristic structures, the presence of a single one of which
makes it easy to recognise even a small fragment of the
rectum. Behind the papille the rectum has a thin wall, to
which the crithidial stage of the trypanosome, when present,
is usually found attached, sometimes in vast numbers. In its
anterior part, the region of the papilla, the rectum has only
circular muscle bands, between which are wide interspaces.
In the hinder region, behind the papille, there are both
circular and longitudinal muscle-bands; the latter can be
traced forward to just behind the papille, at which point
each band becomes rapidly narrowed to a tendon-like fibre,
and at the same time the striations of the muscle disappear.
The tendinous continuations can be traced forwards, in the
living condition, for some distance, but we have not made out
the exact points of their insertion.
The intestine is characterised by a continuous coat of ring-
like muscle-bands, with interspaces, arranged very regularly
external to the epithelium. When the edge of the intestine is
focussed under the microscope, the layer of circularly-disposed
muscle-fibres is seen in optical transverse section like a string
of beads. ‘I'he intestine is frequently seen to be performing
active peristaltic movements, and it may be thicker in some
parts than in others, owing to the contraction of the muscles.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 479
The rectum must be dissected carefully out of the hinder
part of the body, so that it remains on the slide, free from all
the adjacent organs or chitinous plates of the integument.
The easiest way to do this is to make an obliquely longitudinal
cut with the flat-edged needle so as to sever the ventral-
anterior half of the hindmost segments, together with the
genitalia, from the dorsal-posterior half containing the rectum
and anus. The genitalia can then be removed and the rectum
extracted without much difficulty. It requires some care to
separate it from the anus without injuring it. When this has
been accomplished, all unnecessary débris is cleared away.
If it be desired to make separate examinations of the intestine
and rectum, the intestine is cut off as close as possible to its
junction with the rectum. ‘To effect this it is best to spear
the rectum with the pointed needle and make the cut with
the flat-edged needle; or the operation of cutting off the
intestine may be performed before the rectum has been dis-
sected out from the hinder part of the body. In either case,
the intestine is removed to another slide (slide 7), and both
rectum and intestine, on their respective slides (5 and 7) are
teased up, covered, and passed on for examination. It is
not difficult to tear the rectum into several pieces with the
needles, but it is not so easy to tease up the intestine; it is
too slender to make sure of splitting it lengthways, except by
good luck and more or less accidentally, and itis necessary as
arule to content oneself with cutting it transversely into two
or three short pieces, the contents of which are generally
squeezed out during the process.
Finally there remain the salivary glands, on slide 6, in the
portion of the carcase consisting of the thorax and fore-part
of the abdomen from which the gut has been extracted. The
salivary glands, as has been stated above, are lodged in the
fore-part of the abdomen beside the stomach, and it is
generally by no means difficult to extract them when the
stomach has been removed. To do this it is best first to
spear the thorax with the pointed needle, then insert the flat-
edged needle into the abdominal cavity from behind, and rake
4.76 E. A. MINCHIN AND J. D. THOMSON.
out gently the contents of theabdomen. The salivary glands
sometimes come out fairly clean, but more often they are
embedded in fat-body, trachez, etc., from which they must
be carefully freed as much as possible. In such cases they
are sometimes a little difficult to detect under the dissecting
microscope, but their position may be traced by their long,
thread-like ducts. They are much smaller in the male flea than
in the female. Another method which sometimes succeeds
better in extracting the glands is to pull on the integument
of the thorax with one needle and on that of the abdomen with
the other. The body-wall then often tears across at the
junction of the thorax and abdomen, and the salivary ducts
are seen at once stretched out between thetwo. By continuing
to pull the thorax forwards, the glands may be pulled out of
the abdominal cavity and are seen hanging on to the back of
the thorax, from which it is not difficult to detach them. By
this method the glands may often be obtained very clean and
free from encumbering fat and other tissue. When the
glands have been extracted, other débris is cleared away
and the coverslip is put on. The glands are very soft and
are crushed immediately by the weight of a coverslip if
there is no other tissue under it; but for examination of
their contents this is not a disadvantage.
In the foregoing paragraphs we have given a detailed
account of a full examination of the flea, such as we practised
in the earlier periods of our investigation. But when it
became evident to us that the trypanosome, during its
development in the flea, never strays beyond the limits of
the digestive tract proper, we were able greatly to curtail
the ritual of the examination and to omit entirely the proboscis,
body-cavity, and salivary glands. It is also unnecessary, as
a rule, to separate the intestine and rectum in the dissection.
Consequently, our later examinations were reduced to (1) the
excluded feces, if any, on the slide on which the flea was
decapitated ; (2) the stomach, on a second slide; and (3) the
rectum and intestine, on a third.
Tt was no part of our task to make a special and detailed study of the
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 477
anatomy of the flea, but a few points observed by us incidentally in our
dissections may be noted briefly here.
The nervous system, of which some beautiful dissections were made
in this laboratory by Major Christophers, I.M.S., consists, as in insects
generally, of (1) the brain or supra-csophageal ganglion-complex,
sending off the peri-cesophageal connectives which pass on either side of _
the esophagus to connect with the foremost of (2) the three large
thoracic ganglia, joined by connectives to form a series which passes on
into (3) the abdominal chain of ganglia. It isa very difficult operation
to dissect out the brain and the first two thoracic ganglia, but it happens
very frequently that in the ordinary dissections of the flea the third
thoracic ganglion and the abdominal chain of ganglia are exposed entire
and in continuity. It is then seen that the abdominal chain consists
of a series of small ganglia terminated posteriorly by a larger ganglion ;
and further that in the male there are seven smaller ganglia, in the
female only six, in the abdominal chain. The larger hindmost ganglion,
from which nerves are sent off to the genitalia and rectum, evidently
represents a fusion of several ganglia equivalent to the more anterior
smaller ganglia. Consequently it is seen that the concentration and
fusion of ganglia at the hinder extremity of the ventral chain has
proceeded a step further in the female than in the male.
The genitalia consist, in the male, of a conspicuous pair of testes,
situated dorsally in the abdomen, and a pair of filamentous glands
(prostates?) not unlike Malpighian tubules at first sight, but of slightly
smaller calibre, and differing entirely in histological structure. There
is no separate seminal vesicle, but each testis is a tightly convoluted
tubule, the lower end of which is dilated to contain the ripe spermatozoa.
Ducts from the testes and prostates unite to form a median Ductus
ejaculatorius, which opens into a large penis of very complicated
structure. In the female the two ovaries occupy practically the same
position as the testes, but take up much more space and extend forwards
to the most anterior limits of theabdomen. Each ovary consists usually
of four egg-tubes or ovarioles, but in one specimen that we have
mounted as a permanent preparation there are five ovarioles on each
side. The ducts of the ovarioles unite to form the paired oviducts, and
these unite in their turn to form the common oviduct. Ventral to the
common oviduct lies the unpaired receptaculum seminis, consisting of a
brown, chitinous capsule of a peculiar shape. The main body of the
capsule is spherical, but gives off a curved, horn-shaped diverticulum,
ending blindly. The horn-shaped portion has its concave curve turned
towards, and connected by striped muscles with, the spherical part of
the capsule. Aslender duct of great length, and much convoluted near
its origin, arises from the spherical part of the capsule, and runs back
to open probably into the distal extremity of the oviduct or into the
478 E. A. MINCHIN AND J. D. THOMSON.
genital vestibule. The spherical part of the capsule and duct of the
receptaculum are surrounded with unicellular glands, thickly clustered
round the capsule and the convoluted portion of the duct, but thinning
out and becoming smaller towards the distal end of the duct. The
receptaculum, dissected out, stained and mounted for the microscope,
is a singularly beautiful object. It usually contains a dense mass of
spermatozoa.
The heart is frequently seen in dissections at the hinder end of the
body as a delicate filament, which by its own contractions twists and
lashes itself about. Under the microscope it appears a delicate tube,
beset towards the hinder end by the pericardial cells which are attached
to it oneither side, right and left, and are crowded together towards the
hinder end, but occur more sparingly towards the middle region and are
wanting in the anterior third of the heart. The ostia appear to be con-
fined to the posterior region of the heart, but we have not made out their
exact number or arrangement. For the pericardial cells, see Minchin
(1910).
(b) Notes on the Parasites of the Fleas.
In a former publication one of us (E. A. M., 1910) has described some
parasites found in our stock of fleas. The most important was a form
to which the name Malpighiella refringens was given, occurring,
as the generic name implies, in the Malpighian tubules of the flea.
Since that time this infection seems to have died out entirely inour fleas,
and we have not seen any Malpighiella in the fleas dissected by us
for the last three years or more. Why this parasite should have died
outin our fleas it is impossible to say, but it may be remarked that no
conditions could possibly be more favourable for contaminative infection
from flea to flea (whether from adult to adult, or larva to larva, or adult
to larva, or vice versa) than those in our breeding cages, where vast
numbers of fleas in all stages of development are herded together in a
confined space. Consequently the disappearance of Malpighiella in
our cages rather indicates that the fleas do not acquire infection with
this parasite by the contaminative method.
In the publication referred to, numerous yeast-like bodies were
described and figured from the digestive tract of the flea. Since then
we have found organisms of this kind abundantly in smears of the
salivary glands (text-fig. 24, p. 642).
In the larve of fleas that we have dissected and examined from our
cages we have found the gregarine Agrippina bona (Strickland,
1912).
The cysticercoids of tapeworms are found not infrequently in the
fleas. Nicoll and Minchin (1911) described two species of cysticercoids
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 479
from our fleas, representing Hymenolepis diminuta and another
species of the same genus. We have found the same two species
frequently, and also have in our possession specimens of a third species
notidentified. The cysticercoids appear sporadically, and are sometimes
quite common for a period, and then are not found again for a long
time. This uncertainty in their occurrence is quite intelligible, since
their appearance must be caused by the introduction into the cage of a
rat infected with tape-worms, which doubtless infects a large number of
the larve that later become adult fleas.
The point upon which we wish to lay special stress is the
absence in our stock of fleas of any flagellate parasites, and
more especially of the leptomonad described by Swingle (1911)
under the name Herpetomonas (Leptomonas) pattoni.
We have been at great pains to convince ourselves upon this
point. In the first place we dissected at various times about
eighty! fleas from the non-infected breeding-cage without
finding any flagellates of any kind in them, while flagellate
parasites occur in a very large percentage of those known to
have fed upon infected rats, though not in all, since the
trypanosome often fails to establish itself in the flea, and
even when the insect has been fed on a rat with trypanosomes
swarming in the blood, they often disappear completely from
the digestive tract of the flea within twenty-four hours of its.
having fed.
We give here three tables (A, B (1), and B (2)) showing the
results of dissections of fleas from our stock which had been
put upon infected rats, and so had had the chance of
acquiring an infection of T.lewisi. Fleas do not always
feed, however, when given the opportunity to do so,
especially in cold weather,’ and if the fleas are dissected and
examined within tweuty-four hours after having been put ou
the rat (the fleas in all cases having been kept hungry for
1 As a matter of fact we dissected far more than this, all with nega-
tive results, but we have not kept exact records of more than seventy-
nine.
? Note especially the twenty-one fleas of November 11th, 1911, in
Table A, of which eighteen did not feed. This was due to a sudden
cold snap, the first breath, so to speak, of winter.
vot. 60, PART 4.—NEW SERIES. B4
A480 E. A. MINCHIN AND J. D. THOMSON.
two or three days previously to being put on), it is quite easy
to distinguish those which have been fed from those which
have not availed themselves of the opportunity of doing so. In
this way useful controls are obtained for determining whether
the fleas contained any flagellate infection before being used
for putting on the infected rat.
Taste A.—Fleas Examined within Twenty-four
Hours after being put on an Infected Rat, to
show the Numbers that had or had not Fed, and
the Numbers of those that had Fed but in
which no Flagellates were Found.
Number of fleas | Number of fleas which ap-
Date onwhich| Time since#| Number of | apparently not peared to have fedand
the fleas were] fleas put on | fleas put on |fed and contain- which contained :
examined. | infected rat. the rat. ing no flagel- | (a) Stages of (b) No
lates. T.lewisi. flagellates.
5:vii:’710| 6 hours 3 i Z 0
(Oe wai TE) 12) ,, 10 2 8 0
Bene is) 15 .., 14 3 a! 0
5.3 Yen eat Eel ces 4 1 3 0
D6cwe 11 18: © ,, 12 il 7 4
Pordic cls 8, i, ad 3 5 1
S0ea-da | 18 ;, 5 at 4 0
Gea lio| 1S' Vie) 3 5 0
4:71:°13) 20 ,, 14 113, 2 0
10: vi:10} 24 ,, 4 2 2 0
6:vii:710| 24 ,, 3 2 1 0
Viens | 24 2, 5 A: 3 1
15:vi:11| 24 ,, itl 6 4 1
262ix<11| 24 ° ,, 7 a 8 2
30:ix:711| 24 _,, 22 2 18 2
Seed Used is 13 il 12 0
6:x:711 | 24 ,, 14 9 1 1
exue 11) 24 45, 1) 2 11 4
Uae | a 21 18 2 1
7 xed 24. |, 1a? 5 10 2
24:xi:711| 24 ,, 20 2 12 6
Hepaneg il Bh 9 12 3 7 2
IDPS cba l| Gis 6 1 5 0
2:vil:°12| 24 ,, 14 8 6 0
20:vi:"11| 24 ,, 14 3 8 3
Total . F = uzeo 92 167 30
Percentage . 7 3100 31°83 57°79 10°38
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 481
From Table A it is seen that of 289 fleas which were put
on infected rats, 92 (31°83 per cent.) had not fed and con-
tained no flagellates, 167 (57°79 per cent.) had fed and
contained T’. lewisi, and 30 (10°38 per cent.) had fed but
contained no flagellates.
In addition to these negative data we have had the
opportunity of comparing our stock of fleas with another
stock which was actually infected with Leptomonas
pattoni. When one of us (H.A.M.) was in Paris in
January, 1913, he was very kindly presented by Dr.
E. Chatton, of the Pasteur Institute, with some living fleas
(Ceratophyllus fasciatus) from a stock infected with
Leptomonas pattoni. These fleas were brought back to
London and a fresh breeding-cage colonized with them. The
fleas were left to breed for a year, during which time the rat
in the breeding-cage was changed frequently, but none of
the rats put in acquired any trypanosome-infection. When
the fleas were examined at the beginning of 1914, they had
multiplied enormously, and were found to contain Lepto-
monas-infections. We did not keep any exact records of
our dissections of the Leptomonas-fleas, but, roughly
speaking, about 50 per cent. of the fleas contained teeming
infections. The leptomonads appear to establish themselves
in the fleas as readily as does T. lewisi, perhaps more so,
since, as will be seen from Table B (1), barely more than
14 per cent. of our stock of fleas contained swarming infec-
tions when exposed permanently to infection with T. lewisi
in the infected breeding-cage, and Table B (2), if we count
only those known to have fed on an infected rat, not less than
six, not more than fourteen days previously, gives but a
slightly higher percentage (21°19).
We may conclude, therefore, from a comparison of our
stock of fleas with those bred from Dr. Chatton’s stock
infected with the leptomonad, that, had our stock also been
infected with leptomonads, we should not have failed to find
fleas containing leptomonads in those fed on clean rats in
the first place, and secondly, that the percentage of fleas
482 E. A. MINCHIN AND J. D. THOMSON.
Taste B.—Summary of the Condition of Fleas known
to have Fed on Infected Rats. (1) Fleas taken
at Random from the Infected Breeding Cage.
Trypanosomes. -
Caleee He 2.2 ree | eames of the
mescee: ) f
| alee: None. Scanty. Swarming.
18 ie MOTs oe |e 1 1() Infection produced by 5 fleas
of which 3 were dissected
| | (Table J).
23:11:710} 4) 1) 2 1 (s)
Sornr= tO Ga! S13 2 (s)
Deives LO | 6 0|3 3 (Lr, 2s)
8:iv:710} 2 0 | 1 (si) 1 (si)
10:iv:’710| 2 taal: | 0
22:iv:710| 4 33 |) | 1(s)
3:v: 10 4A. 4/0 0
95:vii:710| 5 ales 0
27:vii:’10| 3 12 1 0
5:vii: 10) 5 ee || e33 0 One positive (Table K).
6 :ix:°10 3 33, || | 0
voix: 10 | 4 ae 0
4-1x3°10| 3 0/2 | 1(s)
tocix 103) 3 () |} 33 | 0
paca: LOi) 6S bale 1 (s) One positive (Table K).
mois LO) 1; 0 0 Negative (Table K).
Q4:ix:"10)| 1 120 | O
96:ix:°10| 2 110 | 1 (r)
act AO) 3 Sao 0
2ecam< 10) 9S 3/0 0
29 :ix:710| 3 210 1 (ir)
3:x: 10 2 2:1 0 im
6:x:’10 2 Ot 1 (r)
10:x:710 | 4 IL |) 1 (r)
Usese sO) |p as 33. |) 0) 0 All negative (Table K).
WAR Sst E OM SS |e 3) "0 0 One positive (Table K).
h 6 hear Ve ae ibe 1 (x) All negative (Table K).
LS ace OS Spe i 0 iS (Table K).
24:x:710| 3 210 ma) e (Table K).
Bos ee LOU see | 0 | 0 One positive (Table K).
Ave xa SAO | es 2)1 | 0 (Table K).
8:xi:710 | 3 Pee: 0 All negative (Table K).
AS xa) es PAN 1 (x) One positive (Table K).
16:xi:‘10| 3 a | 1(@) All negative (Table K).
i Ms cra 2/0 | O Both negative (Table K).
22:xi:°10| 2 2) 0 0 =: (Table K).
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 483
Trypanosomes.
(fens | Experimental infectivity of the |
dissected.) | flea. |
Fleas..None.| Scanty. Swarming.
z meal ne |e
2.08) 5 ered C0) Pil Ee at EC) 0
sues 10) 2) 2) 0 1 (r)
earce lO.) 2 OFT 1 (s)
b:xi: 10) 5 1 | 4 0 Injection of sal. gl.negative.|
gexue 10) 29) 0 | 2 i)
foxa= 109) - 2° | ~ 0) 2 0
Sexans 105) 72) 227) 0 0 Both negative (Table K). |
isoxas tO} 3) 21 0 All negative (Table K). |
|14:xii:710| 3 Pee ie a 0
115:xii:°10} 6 5 | 1 (sr) 0 Fleas infected rat239, stom-
| achs rat 257 (Table I). |
pea eom 9:5) IO 0 Fleas positive (Table 1).
hae it} 6 4 | 2 (r) 0 Stomachs injected, positive
(Table I).
260: 11} 6 5 | 1 (ir) 0 Negative (Table I).
Zor bls! -6* 601-0 0 Fleas infected rat 251,|
| stomachs rat 261 (Table
| 10;
eae uh | or! 5 10 i) Fleas positive (Table I). |
eae LE | 10°) 21 6 2 Stomachs injected, 2 posi- |
tive.
Zone I 10 | 7 | 2 1 (r) Stomachs positive, recta |
negative. |
(cont: 13 | 8 8 | 0 0)
13:10:7138| 4 0 | 1 (sr) 3 (2s, Isr) |
14:ii1:713| 3 0 | 0 3 (1s, 2sr) |
Peat 13 | 73 S320 0 |
28:11:13). 9 5 | 4 (3s, Isr)| 0 | |
Sin 1S || 7) 76 0 | |
A:iv:713 | 9 4 | 0 1 (sr)
7:iv:713 | 6 0 | 3 (sr) 3 (s)
10:iv:713| 6 1 | 4(1s, 3sr)} 1 (sr)
14:iv:°13) 2 | 0| 1(s) (al
Total .|249 | 144 |70 35
Percentage 100 o 78328711 14-06
The batches marked * were fleas of which the stomachs and other
organs were kept for injection into rats, and were therefore examined
hastily and imperfectly.
s = stomach; r=rectum; i= intestine.
484
E. A. MINCHIN AND J. D. THOMSON.
Taste B.—Summary of the Condition of Fleas known
to have fed on Infected Rats.
Definite Periods.
(2) Fleas fed at
Age of infection
in flea (approxi-
mately).
6 hours
Date.
No.of
fleas.
None.
:710
: 10
Paid
ie ld
2711
Sekt
2711
alo
3° 1183
13
eels
oak
2710
el.
ra
eal
lull
:711
palit!
Soll
riggs!
rg
reee th
ree 8
Sal
ay lik
eZ
ape IP
a3
el a
: 710
:710
: 710
All
eral
2D,
2 2,
5 lit
LD,
: 09
: 09
: 09
bo
OT OTS He DO OU CaF HB CO SO Ot
7
© Or Ot DH
ot
WokhAaLOOD
eS oe
LO DODO COR HDD OTH OD ODN HD HESIOD DOM NNN OSOHMBHMOMOS ©
Fleas containing Trypanosomes.
Scanty.
1 (si)
0
6 (4s, 2sr)
10 (6s, 4sr)
4 (1s, Isr, 2r)
9 (7s, 2sr)
8 (5s, 2sr, lr)
4 (1s, 2sr, 1r)
3 (sr)
3 (r)
1 (sr)
12 (1s, 3sr, 8r)
2 (1s, 1r)
1 (s)
0
Swarming.
3 (2s, Isr)
1 (s)
3 (Is, 2sr)
6 (5sr, lr)
3 (1s, 2sr)
2 (sr)
4 (1s, 3sr)
3 (2s, Isr)
2 (1s, Isr)
8 (6s, Isr, 1r)
8 (4s, 4 sr)
4 (3s, 1sr)
0
6 (1s, 5sr)
5 (1s, 4sr)
5 (sr)
5 (4s, Isr)
0
1 (s)
6 (5sr, 1s)
3 (1s, 2r)
0
0
0
(
0
2 (1s, Isr)
0
0
1 (s)
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI.
Age of infection
in flea (approxi-
mately).
3 days
bole
10
ll
12
14
33
33
“
vy
e
3 ss
Fleas containing Trypanosomes.
Grand total
Percentage
Total of six days and over
Percentage
No.of
fleas.
None. Scanty. Swarming.
on nei 2 (sr) 2 (1s, Ir)
By |S 0 0
4] 4 0 0
10 | 6 | 4 (2s, Isr, 1r) 0
10; 1 | 5 (1s, 2sr, 2r) 4 (1s, 3sr)
12! 6 | 5 (1s, Isr, 3r) L@)
16 | 4 8 (1s, 7r) 4 (1s, 3r)
Gola 4 (sr) 1 (sr)
8| 4 2 (sr) 2 (sr)
3] 0 1 (sr) 2 (1s, Isr)
10| 5 5 (1s, 4sr) 0
12 9 i3 (sr) 0)
12] 8 3 (r) 1 (v)
2} 2 0 0
Za O 0 2 (sr)
3 | 2 1 (i) 0
10} 6 1 (s) 3 (r)
1h 27) 5 (x) 0
403 1 (s) 0
6] 2 2 (r) 2 (Isr, Ir)
2 2 0 0
5| 3 1 (r) i)
4| 0 1 (sr) 3 (Isr, 2r)
10) 3 2 (Is, Ir) 5 (2sr, 3r)
6 5 1 (sr) 0
6| 3 0 3 (r)
(Auta 3 (sr, 2r) 3 (sr)
6| 2 2 (Isr, Ir) 2 (sr)
G3 1 (s) 2 (sr)
oie 1 (sr) 1 (s)
6 6 0 0
6| 6 0 0
Gr 2 3 (1s, 2r) 1 (sr)
4| 2 2 (r) 0
6] 3 2 (1s, lr) E(x)
San 7 0 1 (7)
2) 1 1 (@) 0
Te ell 0 0
2 2 0 0
. 609 |230 293 156
. {100 |37-77 36°62 25°61
. (118 | 65 28 25
23°73 21:19
F bi 55°08
* Stomachs only examined.
485
486 E. A. MINCHIN AND J. D. THOMSON.
containing flagellates would have been far higher than is
shown by our tables, in fleas exposed to infection by T.
lewisi.!
(c) Notes on the Histological Structure of the
Stomach of the Flea.
We shall have occasion, when describing the developmental cycle of
the trypanosome in Part II below, to relate how the trypanosome pene-
trates into the epithelial cells of the stomach of the flea and goes through
a process of multiplication within them. It is a necessary preliminary,
therefore, to understanding the effects of the parasites that we should
preface our description of their development by some remarks upon the
structure and contents of the flea’s stomach; and in the following
section we give an account of our observations upon these matters,
without claiming to have added anything to the scientific knowledge of
insect histology.
The histology of the digestive tract of insects has been the subject of
1 Noller (1912), discussing the question of the leptomonas-infection of
the fleas, remarks, p. 398, that since the larva of the flea acquires the
infection, adult fleas bred in a cage can be infected, and that conse-
quently “the arrangement of the experiments (‘ Versuchsanordnung’) of
Minchin and Thomson, who used fleas bred in a rat-cage, does not
correspond to the requirements (‘ Anforderungen’).” We are at a loss
to understand to what this criticism applies or what are the ‘ Anforde-
rungen”’ to which Noller refers. At the time Noller wrote we had
published only our three preliminary reports. The first two of these
(1910, 1911, 1) refer only to the transmission of T. lewisi by fleas,
and it is sufficiently obvious that the presence of leptomonads in the
fleas could not affect in any way the value or significance of positive
results obtained in experiments on the transmission of the trypano-
somes, since, ex hypothesi, the trypanosome and the leptomonad
parasite are in no way connected. Our third report (1911, 2) gave an
account of the intracellular multiplication of T. lewisi in the flea’s
stomach, a discovery which Ndller himself has confirmed, and which
also would be quite unaffected by the presence of leptomonads in the
fleas. Ndller’s criticism appears to us, therefore, both premature and
superfluous ; premature, because our stock of fleas was not, as a
matter of fact, infected with leptomonads; and superfluous, because,
even if the fleas had been infected with leptomonads, it would have
made no difference to the experiments and observations which Noller
criticises.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 487
numerous memoirs, and its general characteristics are very well known.
It would be beyond the scope of this memoir to attempt to discuss this
subject in detail or to cite the very copious literature dealing with it;
but of recent works we may refer more especially to the very excellent
monograph of Léger and Dubosegq (1902), who have studied the intestinal |
epithelium of Tracheata from the same point of view as ourselves, that
is to say, with the object of describing the changes produced in the
epithelium by parasites (gregarines) attacking the cells, None of the
insects studied by Léger and Duboseq, however, were of blood-sucking
habit and the stomach-epithelium of the flea differs in a number of
points from any of the epithelia described by the French authors.
The wall of the stomach consists of the following principal layers,
counted from within outwards (PI. 39, fig. 126): (1) the lining epithe-
lium, (2) a layer of circular muscle fibres, and (3) a layer of longitu-
dinal muscle fibres. In addition to these layers, which are very easily
seen, there are to be found also, though by no means in every section,
flattened epithelial cells external to, and in contact with, the lining
epithelium, between it and the circular muscle-layer; they occur
sparingly and far apart, but appear to represent an integral and perhaps
primitive constituent of the wall of the mid-gut. Similar flattened cells
are found here and there on the Malpighian tubules, the wall of which
is similar in histological composition to the stomach-wall if the latter be
imagined as reduced to the lining epithelium and the flattened cells
alone, without the muscle-layers.
We are concerned here only with the lining epithelium of the
stomach, but it may be mentioned in passing that the circular muscle-
fibres occur as bands or separate rings with considerable intervals
between them, and consequently do not appear in every transverse
section of the stomach. The longitudinal muscles are also separated
from one another by intervals, a fact at once apparent in the transverse
section, in which the muscles are seen cut across and in which it can
further be seen in a well-preserved section, that each longitudinal
muscle-fibre is connected to its neighbours by a delicate membrane,
appearing asa fine line running between each adjacent pair of muscle-
bands and forming a delicate sheath or investment round the whole
stomach. The circular muscle-layer is continued on into the intestine,
where it forms a continuous investment without intervals between the
bands; the longitudinal muscles end at the pylorus posteriorly and
start anteriorly behind the proventriculus, which has its own system of
musculature, running for the most part in oblique bands arranged sym-
metrically right and left. Hach muscle-band in the stomach-wall is a
single, transversely-striated fibre, in which an occasional elongated
nucleus is seen, embedded in a small quantity of protoplasm.
The following account of the epithelium and contents of the stomach
488 E. A. MINCHIN AND J. D. THOMSON.
applies, unless otherwise stated, to sections of the stomach fixed with
Flemming’s fluid and stained with iron-hematoxylin followed Eby
TrxT-FIG. 2.
Diagrammatic representations of sections of the epithelium of
the flea’s stomach, to show the various conditions: a, to show
a section through an epithelial crypt from which clear,
regenerated epithelium is arising on all sides, while on one
side three black, degenerated cells are seen (compare fig. 316,
Pl. 44) ; b, to show the manner in which the border of the cells
arises in relation to the gradually-developed separation of the
cells from one another; c, to show the transition from the ordi-
nary, columnar type of epithelium to the flattened type.
Lichtgriin-picric (see the following section dealing with technique). If
such a section through a number of stomachs—all taken from a batch
THE RA'T-TRYPANOSOME, TRYPANOSOMA LEWISI. 489
of fleas dissected at the same sitting and at the same interval of time
after having fed on the infected rat, all preserved in the same way, all
stuck on the same slice of liver, cut and stained simultaneously—be
examined even inthe most cursory manner, very considerable differences
are seen between the stomachs in one and the same microtome-section.
These differences affect both the epithelium and the contents. The
epithelium varies, in the first place, in the form of the cells, from
flattened to columnar, and secondly, in the staining reactions of the
cells. The contents of the stomach, that is to say the blood-débris,
vary greatly in colour, staining in some cases deep opaque black, or less
deeply in various shades of grey, in other cases, however, bright yellow.
The variations in the form of the epithelial cells are to be ascribed to
the differences in the degree to which the stomach is dilated by the
ingested blood. In a gorged flea the distension of the stomach stretches
the epithelium until the cells become thin and flattened ; but when the
flea is hungry, or has taken ina small quantity of blood, or when the
quantity ingested has become reduced by digestion and absorption,
the epithelium resumes what may be considered its normal columnar
form. Every gradation between the flattened and columnar conditions
can be found in different sections or in different parts of one and the
same section.
The variations in the staining reactions of the epithelial cells depend,
in the first place, on the age or senescence of the cells. It is a matter
of common knowledge that the lining epithelium of the mid-gut of
insects is continually being thrown off and regenerated. The ordinary
epithelial cells do not multiply and no mitoses are ever found in them ;
the centres of regeneration are the so-called epithelial crypts, each
representing morphologically a small diverticulum of the epithelium in
which the approximation of the cells usually obliterates the cavity and
produces a solid, bud-like mass of cells (Text-fig. 2, a and PI. 44, tigs.
314, 316). Whena flea’s stomach, containing a certain amount of ingested
blood, is plunged into a fixative, the epithelial crypts are very easily
seen with a hand-lens or with the naked eye as little opaque white spots
in the semi-transparent stomach-wall, very conspicuous against the
reddish-brown background of the stomach-contents. In the sections it
is common to find mitoses in such a crypt, especially towards its fundus
(Text-fig. 2, a). As the cells multiply they are pushed upwards to the
general level of the epithelium and outwards from the crypt to replace
the old epithelial cells which, having degenerated, are cast off from the
wall into the lumen of the stomach, and are digested there.
The young, freshly-regenerated epithelial cells have the cytoplasm
clear, staining light-grey, and are relatively poor in granulations; the
older cells, on the contrary, have the cytoplasm full of granules that
stain very deeply, until finally the whole cell, including its nucleus
490 E. A. MINCHIN AND J. D. THOMSON.
becomes black and opaque. Consequently, the epithelia of different
stomachs show very varied appearances. A recently regenerated
stomach will show clear epithelium all round, and, according to the
time that has elapsed since regeneration, there may be no detached
cells in the lumen of the stomach, or there may be a certain number of
detached black cells, or there may be still, here and there, isolated
black cells or patches of such cells in situ in the epithelium or in pro-
cess of being cast off from it. On the other hand, a stomach which is
about to be regenerated shows very dark epithelium all round, and in
places this may bein process of rejection and replacement from the
crypts, in which the cells have clear cytoplasm. The condition of the
epithelium may vary in different parts of the same stomach, and from
what we have observed we have gained the impression that the regenera-
tion proceeds usually from before backwards, so that the anterior part
of the stomach is further advanced in degeneration or regeneration, as
the case may be, than the posterior region. We find in our preparations
all possible conditions of the epithelium in different stomachs in one
and the same microtome-section, and we have not been able to establish
any definite relation between the feeds of the flea and the regeneration of
the epithelium, but we have not paid sufficient attention to this point to
be able to state positively that no such relation exists; the differences
seen in the epithelia of flea-stomachs examined at the same interval of
time after feeding may be due to inequalities in the rate at which
digestion proceeds.
In addition to what appears to be the normal process of senile decay,
in which the cells take up the iron-hematoxylin stain very deeply and
become black and opaque, we have observed a second mode of degenera-
tion, which we are inclined to ascribe to the action of the trypanosomes,
since in all cases where it occurs in our preparations there are trypano-
somes to be found in the stomach, and frequently in the degenerated
cells themselves. In this second type of degeneration the black-staining
granules in the cell diminish in quantity, without, however, disappearing
entirely, while the cytoplasm of the cell stains yellow (Pl. 39, fig. 133;
Pl. 40, fig. 140); hence we have generally referred to this condition in
our notes as * yellow necrosis.” Inall the stomachs in which we have
found it the blood-débris is also stained yellow, and it is often very
difficult to make out the precise boundary of the necrosed cell-body, or
to distinguish the cells from the débris when they lie free in it (Pl. 39,
fig. 125), except by the presence of the nucleus and of a certain number of
black granules in the cytoplasm of the necrosed cel]. Indeed, our first
impression was that the yellow colouring matter of the blood had in
some way penetrated into the cell and stained its cytoplasm, but there
can be no doubt that this idea is an illusion and that the yellow colour,
both of the blood-débris and of the necrosed cells, is due to the picric
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISE. 491
acid in the Lichtgriin-picric staining combination, though it is, of
course, possible that the substance, whatever it may be, which stains
yellow in the blood-débris may have become infiltrated into the dead
cells and given them their peculiar staining properties.
The variations in the staining reactions of the contents of the
stomach are more difficult to explain. There appear to be two types of
staining after the use of the iron-hematoxylin-Lichtgriin-picric com-
bination, one in which the contents stain in various shades of grey up to
black, with a greenish tinge, and one in which they stain a bright
lemon-yellow. It is not possible to bring these two types of staining
into one series, as there is no transition between them; the grey-black
and the yellow types occur side by side in different stomachs in one and
the same microtome-section, and each stomach shows either the one or
the other condition through the whole series of sections. So far as our
observations permit us to generalise, the grey-black series represent
the normal stages of the digestion of the blood; the yellow reaction
appears to be due to some abnormal condition.
The blood ingested by the flea is very soon affected by the digestive
action of the stomach, and the red corpuscles cease to be recognisable
within a few hours after digestion. In the middle period of digestion,
that is twenty-four hours, or thereabouts, after feeding, the blood has
become thick, viscous and brick-red in colour, and contains immense
numbers of irregularly shaped grains of all sizes, but for the most
part coarse and large. Towards the end of digestion, forty-eight
hours or so after feeding, the stomach contents are fluid and watery,
dark brownish-black in colour, and the grains are much diminished in
number and in size.
In the sections, taking first the grey-black series, the blood in the
earlier phases of digestion (eighteen to twenty-four hours) usually con-
sists of densely-packed grains and spherules, varying in size from
very coarse to very fine, and staining intensely black. Between the
grains there is visible a coagulated albuminous matrix, stained
greenish with the Lichtgriin-picric combination. The stomach-contents
fill the whole section and adhere closely to the epithelial cells,
penetrating down between them when they have the columnar form,
but in the centre of the section there is generally a clear patch,
circular in outline, which is seen to owe its clear appearance to the
fact that coarse grains are absent, and it consists only of the albuminous
matrix with finer granules. Hence the digestion, or more probably the
passage backwards towards the rectum of the indigestible remnants, of
the blood-débris appears to proceed from the centre of the section—
that is to say, from the axial region of the stomach—towards the
periphery.
As the digestion proceeds, the grains in the débris become smaller
492 E. A. MINCHIN AND J. D. THOMSON.
and stain less deeply ; consequently the stomach-contents stain grey, in
varying shades of darkness, while the matrix still shows the greenish
hue. In stomachs thirty-six hours after feeding the contents of the
stomach are generally greatly diminished in quantity, and are absorbed
in the centre of the lumen, leaving a clear space of variable form, while
round the periphery the greenish-grey débris adheres close to the
epithelium. Leucocytes, especially the polymorphonuclear forms, can
be recognised in the blood twenty-four hours after feeding, but at
thirty-six hours we have not found them. Owing to the lighter tints of
the stomach-contents at thirty-six hours, the trypanosomes free in the
blood-débris can be seen more easily, in contrast with the earlier state
of affairs.
In the yellow stomachs the contents appear at first almost uniform, but
on close examination they are seen to consist of closely-packed granular
substance, all of which, both granules and matrix, is coloured by the
picric acid in the staining combination used. The first point that
strikes one immediately is that the contents in such stomachs are large
in quantity and fill the whole stomach, or show but a slight amount of
absorption towards the centre of the section, even at thirty-six hours,
when the contents of, the grey-black stomachs are considerably
diminished. The epithelium of the yellow stomachs may vary from
the flattened to the columnar form, but the normal cells stain grey or
black, in sharp contrast with the yellow contents.
It seems obvious from these data that the yellow stomachs represent
an abnormal condition; we have endeavoured, not very successfully, to
find a relation between this condition and either the presence of
trypanosomes, on the one hand, or the state of the epithelium on
the other.
In the yellow-staining stomachs which we have studied we have found
trypanosomes to be present in the stomach in every case except one, and
in that case there were attached clumps of crithidial forms imme-
diately behind the pylorus, showing that the stomach-phase was over.
But on the other hand, we have found the grey-black condition of the
contents in well-infected stomachs also, showing at least that, if the
yellow condition is in any way due to the parasites, they do not always
produce that effect. On the other hand, in those cases in which we
have found no trypanosomes at all, either in the stomach or outside it,
the contents are always in the grey-black condition. A significant
circumstance is, perhaps, the fact that we have only found the “yellow
necrosis ’ of the cells in stomachs with yellow-stained contents.
As regards the condition of the epithelium, we have found the yellow
condition of the contents associated (1) with epithelium black all round
and in process of being cast off, or (2) with epithelium mostly clear, but
with black patches of cells in situ or detached ; in one such stomach the
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 493
first condition is found in the anterior half, the second in the posterior.
We can state, therefore, that in our experience the yellow-stained
contents occur only in stomachs about to be regenerated, or in process
of regeneration, or very recently regenerated. But, again, we have
found the grey-black condition in stomachs that appeared also to have
undergone regeneration very recently, which makes it difficult to
correlate this condition with the process of regeneration. The question
of the significance and cause of the yellow-staining condition of the
stomach-contents must be left an open question at present; the data to
hand do not suffice for drawing decisive conclusions, and it would lead
us too far to attempt further investigations upon this problem. On the
whole, however, it seems at least probable that we are dealing with an
abnormal state of the digestive processes towards which the trypano-
somes are a contributory cause, if not the sole one.
As already stated, the different conditions of the stomach-contents
described above are those seen after staining with iron-hematoxylin and
Lichtgriin-picric. After the use of Giemsa’s stain the colour of the
contents differs considerably in different cases.
Most of our sections stained with Giemsa were fixed with Maier. In
those in which the trypanosomes were best stained and show the flagella
clearly and sharply the grains and spherules of the débris are coloured
for the most part orange-pink, especially in those stomachs in which
the digestion of the blood is further advanced (figs. 109, 113, Pl. 38) ; in
the earlier stages of digestion many of the larger grains and masses in
the débris are stained deep purple, making the contents of the stomach
more opaque. In one of our series preserved in Flemming, consisting
in all of seven slides, the first six were stained with the iron-hematoxy-
lin-Lichtgriin-picri¢ combination, the seventh with Giemsa’s stain; on
this seventh slide there are sections of four stomachs, two of which, on
the other six slides, show grey-black contents, while the remaining two
have the contents yellow in colour. In the Giemsa-stained slide the
blood-débris shows a coloration very different after Flemming to that
which it shows after Maier, being stained a bluish-green tint. The
stomachs of the yellow type are slightly more blue in tint than those of
the grey-black series, but otherwise the difference between them is but
slightly marked.
Having now described the chief variations in the conditions of the
stomachs and their contents, or at least those differences which are
obvious upon the most cursory inspection of the sections, it remains
to give a more detailed account of the epithelial cell. In any given
stomach the cells show great individual variation in form and structure,
but, nevertheless, it is not possible to divide them into distinct classes.
There are no special glandular or secreting cells, as described by Léger
and Duboscq in other insects, and all the cells of the general epithelium
494, E. A. MINCHIN AND J. D. THOMSON.
of the stomach of the flea are to be regarded as equipotential, the
differences visible between them being merely the expression of varying
physiological conditions in relation to their changing environment,
on the one hand, or to their constitutional vigour or senescence, on the
other. Hence it is possible to give a generalised description of thé
cells, beginning first with the normal, healthy cell and dealing afterwards
with the changes it undergoes in the process of degeneration.
The epithelial cells are produced, as already stated, in the “ crypts of
regeneration,’ which have been described in various insects by Léger
and Duboseq. In the flea these structures appear usually as solid, bud-
like cell-masses that often project outwards from the wall of the stomach
to a considerable extent (PI. 44, figs. 514, 316) beyond the level of the
muscle-layers, which pass on either side of them. Internally the crypts
do not rise up beyond the general level of the epithelium. The closely-
packed cells of the crypts show distinct limits, and do not form a
syncytial mass of protoplasm, as described by Léger and Duboseq
(1. ¢., p.410) in the larva of Anthrenus verbasci, for example. At
the fundus of the crypt mitoses are often found, sometimes in two cells
simultaneously in the same crypt; in other cases a]l the nuclei are in
the resting state. Doubtless the crypts have periods of active multipli-
cation, alternating with periods of repose, as in other insects. The
crypts are often seen to be marked off from the general epithelium by
slender dark cells, the “cellules de recouvrement” described by
Léger and Dubosegq (1. c., Pl. II, fig. 2, ¢.7.; p.388). The crypts appear
to have the monopoly of cell-production in the stomach of the flea. We
have not found basal cells, “cellules de remplacement,”’ in the
general epithelium.
By multiplication and increase in their numbers the cells are pushed
outwards on all sides from the crypt to take their place in the general
epithelium (PI. 44, fig. 316, and Text-fig. 2a). The young epithelial cells
seen in the immediate neighbourhood of the crypts are columnar
cells, roughly rectangular in form, and generally about twice as high
as they are broad. The lateral boundaries of the cell are approxi-
mately parallel, and each cell is in contact with its adjacent neighbours
for its whole length. The free, apical surface of the cell is convex, and
on this side is developed a very distinct, thick border, at first covering
only the upper surface of the cell, which projects like a dome towards
the lumen of the stomach.
The further development in the form of the cell consists in an
extension of the upper free surface, brought about by the cells
becoming free and separated from one another at their sides, first at
their apices and then downwards along almost the whole length of
the side of the cell, till finally each cell is connected with the adjacent
cells only by a narrow isthmus at its base (Text-fig. 2b). As the cell
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 495
becomes free the border develops also on the exposed surface, so that,
instead of being confined to the apex of the cell, it extends down the
vertical sides also (Pl. 40, fig. 156). This process of separation between
the cells has an obvious significance in connection with the process of
flattening which they undergo when the stomach is dilated after
feeding ; it can be regarded as an adaptation to the blood-sucking habit.
When the flea gorges itself each cell is so stretched that its tallest part
in the vertical direction is scarcely thicker than the nucleus, which
bulges out the middle part of the cell in an even curve towards the
lumen of the stomach, while towards the periphery the verticle height
of the cell diminishes to the isthmus connecting it with its neighbours
(Text-fig. 2c). As the cell resumes the columnar form the nucleus
remains at or near the base, as a rule, and the cytoplasm of the cell is
heaped up over it. In the extreme columnar form the apex of the
cell is generally slightly expanded, the middle region more narrowed,
so that spaces are left between adjacent cells, into which a con-
siderable quantity of blood-débris penetrates (Pl. 39, fig. 126). The
nucleus is usually situated at the base of the cell, but occasionally
towards the apex (PI. 40, fig. 136). The border clothes the whole free
surface of the cell, whether flattened or columnar, and is of considerable
thickness over the apex and the sides, becoming thinner as it approaches
the isthmus, but in the columnar form of the cell, when its apical region
is expanded, the border may be thinner, as if stretched, at the apex of
the cell (Pl. 40, fig. 144).
The blood-débris has a great tendency to adhere closely to the border,
so much so that the border is often more sharply marked off from the
cell-contents within than fromthe blood-débris without, in the sections,
but places can be found occasionally where the blood-débris has split
away from the epithelium, leaving the border distinct and sharp. The
border appears usually homogeneous and refringent, though in some
preparations indications are seen of a vertical striation, as if it were
composed of little darkly-stained rods, placed at right angles to its two
limiting surfaces, and separated by intervening substance of lighter
colour (Pl. 39, fig. 129, and Pl. 40, fig. 142). After sublimate-fixation
the border is colourless, but when stained with iron-hematoxylin the
blood-débris adhering to it often hinders the extraction of the stain
and at these spots it remains black ; when the hematoxylin is extracted
it tends to take up the green from the Lichtgriin-picric mixture (Pl. 40,
fig. 147). With Giemsa after sublimate-fixation it stains a pinkish-
yellow. After Flemming-fixation the border is yellowish, as if tinged
by the chromic acid in the mixture, and when this fixation is followed
by the Giemsa-stain the border is coloured green (PI. 38, figs. 99-103),
There is no “ bordure en brosse,” or palisade of stiff rod-like cilia,
external to the border, as in many insects. The condition in the flea
voL. 60, PART 4.—NEW SERIES. 35
496 E. A. MINCHIN AND J. D. THOMSON.
more resembles that figured by Léger and Duboseq for Scolopendra
(1. ¢., pl. vi).
The border is evidently a fairly tough structure since in teased up
stomachs examined fresh, the borders of cells are often seen quite empty,
but retaining their shape, like shells.
The nucleus of the epithelial cell calls for no special comment: as
can be seen in our figures, it is rounded or oval, with the typical
structure seen in tissue-cells, namely, a distinct membrane, a reticulum
containing chromatin-grains of various sizes, and one or more nucleoli
which stain black, like the chromatin, after iron-hematoxylin. Mitoses
of the usual type are found commonly in the crypts of regeneration, but
we have never seen the slightest evidence of nuclear division in cells
forming part of the general epithelium outside the crypts.
The cytoplasm of the epithelial cell varies at different ages. In the
youngest cells bordering the crypts the cytoplasm appears more or less
homogeneous and finely granular in all parts of the cell; it stains light
purplish-grey or grey-black after iron-hematoxylin, bluish-purple after
Giemsa, and no coarse granulations are to be seen. In the fully
developed cell the cytoplasm has undergone local differentiation ; round
the nucleus, in the basal half of the cell, it has a denser texture, but
above the nucleus, in the apical region, it has become of looser con-
sistence, more spongy, so to speak, in appearance, with irregular spaces
(Pl. 39, figs. 126, 127, Pl. 40, figs. 136, 144), containing fluid in the living
condition, and transversed by strands of protoplasm disposed irregularly.
The more the apical part of the cells is expanded the more watery its
contents appear. Sometimes the apical region appears almost empty
in the sections, with only a few traces of cytoplasm close under the
border and at the sides. It is in this region in which the stages of the
trypanosomes are most often found, and into which the parasites first
penetrate.
In addition to these changes in the cytoplasm, numerous grains and
enclosures of various kinds make their appearance in it. A detailed
study of these granulations would require a lengthy investigation, an
expenditure of time and trouble, that would go beyond the scope
and objects of this work. We must confine ourselves to a brief
summary of the appearances seen in our sections, without attempting
to give physiological explanations of the various conditions seen. Itis
obvious that the bare observation that a granule is stained black by
iron-hematoxylin or red by Giemsa’s stain does not permit very far-
reaching conclusions as to its nature or function in the cell; bodies of
most diverse properties might agree to this extent in their reactions.
The first granulations to appear are minute grains which, whatever
the fixation, Flemming or sublimate, stain black after iron-hematoxylin
and red after Giemsa. They are seen at first chiefly at the sides of the
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 497
nucleus, between it and the cell-wall and extend up the sides of the cell
close under the border. Scanty at first in the apical spongy part of the
cell; they are soon deposited in this region also, appearing often in con-
siderable numbers and varying in size from small granules to conspicuous
_ grains, and even large masses (PI. 40, figs. 136, 137.) The larger grains
are seldom homogeneous, but appear as rings, black or red, as the case
may be, with clear centre, apparently hollow (Pl. 38, fig. 99). Those
of still larger size show, especially after Giemsa, darker and lighter
parts disposed in various ways; inside the peripheral deeply-stained
shell there may be darker grains or patches. After iron-hematoxylin,
however, the whole mass may be opaque black, but usually shows lighter
inner portions. The extent to which these granulations are developed
varies in different stomachs, doubtless in relation to their secretive or
absorptive activity at the moment of preservation. When a number of
stomachs are cut in the same block, one stomach all through the series
may show the cells clear and very free from granulations, while another
stomach shows nearly every epithelial cell loaded with coarse grains in
its apical region.
The red grains, as they may be termed from their distinctive reaction
to Giemsa’s stain, appear to be always present in greater or less quantity
in the fully-developed cells of every stomach. In addition there are
often found, lodged in the apical spongy region of the cell, masses of
relatively large size which do not retain the iron-hematoxylin stain
firmly throughout their substance, and consequently appear for the
most part light grey in colour after this stain (Pl. 40, figs. 145, 146);
after Giemsa they are either scarcely stained at all, appearing a sort of
neutral tint, or they are coloured bluish-purple in various shades, some-
times very deeply, with streaks and blotches more reddish in tint
(P1. 38, fig. 102). These masses vary considerably in size and contour,
and show differentiation of their substance into lighter and darker parts.
With superficial examination they often simulate the intracellular
stages of the trypanosomes to a remarkable degree, especially in the
living condition, when they are often very conspicuous ; for a long time
we confused them with the spheres in the freshly teased-up stomachs,
and spent much time watching them in the expectation, never of course
fulfilled, of seeing them perform the characteristic movements. After
we had made smears of stomachs in which these bodies were abundant,
without finding any intracellular stages of the trypanosomes in such
preparations, we came to the conclusion that these motionless spheres
(as they appeared to be) were merely cell-products, and referred to them
in our notes as “ pseudospheres.” Even in sections the pseudospheres
often mimic the true spheres and might be confused with them at first
sight, but only by an inexperienced observer who had never seen the
actual intracellular stages of the trypanosome in the epithelium. The
498 E. A. MINCHIN AND J. D. THOMSON,
idea occurred to us at one time that some of the pseundospheres might
possibly be degenerated stages of the trypanosomes, destroyed, and in
process of absorption, within the epithelial cells into which they had
penetrated; but we have found no decisive evidence for this. It is
most probable that the pseudospherés represent secretion-masses
formed by the cell itself.
In some of the stomachs. especially in those preserved about twenty-
four hours after feeding, there are to be seen dense and very conspicuous
accumulations of coarse grains in the epithelial cells immediately below
the border (PI. 38, fig. 98, Pl. 40, fig. 147). The grains in question are
especially distinct after fixation with Maier’s fluid; they are more
difficult to make out in the stomachs fixed with Flemming. The grains
resemble very closely those of the blood-débris adherent to the border
external to the cell, so much so that the first impression gained is that
the débris has been absorbed into the cell through the border. It is
easy to imagine this after iron-hematoxylin, which stains both these
granules and the débris very black after sublimate-fixation (fig. 147);
but the Giemsa-stain colours the grains within the cell differently from
the débris (fig. 98), and when the digestion of the blood has gone
beyond a certain point the grains inside the cell may be stained much
darker with iron-hematoxylin than the grains in the blood-débris. It
is improbable that the coarse grains of the débris would pass bodily .
through the border, which is to all appearances a dense, tough
structure ; but it is probable that these grains are formed in the cell in
direct relationship with the process of absorption of nutriment from
the blood.
Amongst the enclosures of the epithelial cell must be mentioned
finally peculiar yellow grains which occur with great frequency in some
stomachs, not at all in others. Their presence or absence is in no way
connected with that of the trypanosomes, and they occur both in
normal as well as in degenerating cells, though perhaps more
abundantly in the latter. In the Flemming-iron-hematoxylin sections
these grains have a brownish-yellow tint, often with a darker shell
(Pl. 40, fig. 141). They vary in size from small granules up to the large
grains reaching as much as 13 in diameter (fig. 142). Their tint also
varies in depth, being usually much lighter in the larger grains. With
Giemsa, after Flemming, they are stained bright green (Pl. 38, fig. 103),
probably as the result of a blue stain (azure) imposed upon their original
yellow tint.!. These yellow bodies are very similar to, probaby identical
1 A similar result is seen in the chitinous spines of the proventriculus
in sections stained with Giemsa; the cuticle at the base of the spine is
stained red, but that of the spine itself, from near the base to the tip, is
coloured emerald green. The unstained spine is yellow in tint. Com-
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 499
in nature with, the enclosures characteristic of the pericardial cells. As
one of us has described elsewhere (E.A.M., 1910), the pericardial cells of
the flea may beso crammed with yellowish-brown grains and spheres that
the cell becomes visible with the naked eye as an opaque black spot
through the integument of the living flea. In some of our stomach-
sections there are also casual sections of pericardial cells which have
been pulled out of the flea together with the stomach, so that we have
had the opportunity of making a direct comparison between the yellow
grains in the epithelial and pericardial cells. The yellow grains are
probably an excretory product, eliminated by the flea under certain
physiological but apparently normal conditions, and elaborated either
in the epithelial cells, to be cast out into the lumen of the stomach, or
in the body-cavity, to be taken up by the pericardial cells.
We come now to the process of cell-degeneration which occurs in the
effete, senile epithelial cells. This process is very different in the flea’s
stomach from that described by Léger and Duboscq in various insects,
none of them of blood-sucking habit. It is described by these authors
asa‘ Dégénérescence mucoide,” an infiltration of the cells with
mucoid substance. In the flea’s stomach the process appears to be more
of the nature of a fatty degeneration, combined perhaps with a mucoid
infiltration.
In our sections fixed with Flemming’s fluid and stained with iron-
hematoxylin the intensely black, often perfectly opaque, degenerated
cells, which are seen frequently detached completely or in process of
detachment from the epithelium, are very distinct from the clear,
lightly-stained cells originating from the crypts of regeneration and
taking the place of the degenerated cells (Pl. 44, fig. 516). In some
of our sections the stain has been over-extracted: the trypanosomes
have become ghosts, faintly visible only to the practised eye, the nuclei
of the epithelial cells are pale, and even the blood-débris has had its
usually intense black stain reduced to a shade of brown; but the black
grains and masses in the epithelial cells remain as black as ever, showing
that they do not owe their colour to the stain but to the fixation, that
is to say, to the osmic acid in the Flemming’s fluid. Such preparations,
spoilt for other purposes, are very useful for showing the gradual
process of deposition of the blackened grains. First they appear as fine
granules round the nucleus, near the base of the cell (Pl. 40, figs. 143,
144). Next, other, and for the most part larger masses, are deposited in
the cytoplasm above the nucleus. The cell then becomes gradually
filled up with black grains from below towards the apex; often an
pare also the green stain of the border, mentioned above, after Flemming
and Giemsa, evidently due also to the super-position of a blue dye upon
a yellow ground.
500 E. A. MINCHIN AND J. D. THOMSON.
empty space is seen at the apex, immediately below the border (PI. 40,
fig. 138), but finally this, too, is filled up and the whole cell becomes an
opaque black mass (fig. 139).
Very instructive is one of our series preserved in Flemming, in which
there is one stomach in which nearly all the epithelium is degenerate.
The sections of this stomach are spread over seven slides, six of which
were stained with iron-hematoxylin, while the seventh, on which are
sections through the hindmost region of the stomach, was stained with
Giemsa. On this slide the degeneration is not so far advanced as in
the more anterior region of the stomach, and in different parts even of
the same section the following conditions are to be found: (1) Cells of
normal type, with clear cytoplasm containing a few red granules
(Pl. 38, fig. 99) ; (2) cells with cytoplasm of a darker bluish-purple tint,
with many more red granules and amongst them a few coarser grains
intensely black in colour (PI. 58, fig. 100) ; (3) cells in which both the
red and the black grains, but especially the latter, are greatly increased
in number, leading up to (4) opaque black cells in which nothing can
be focussed clearly. The black grains, it is obvious, can only owe their
colour to the action of the osmic acid in the fixation, and must there-
fore be of a fatty nature. On the other hand there is also a marked
increase of the red grains in the degenerating cells, indicating, perhaps,
that in addition to deposition of fat, there is also a tendency to mucoid
infiltration, as described by Léger and Duboseq. The darker tint of
the cytoplasm, in so far as this is not an optical effect due to crowding
of the grains, indicates that it becomes impregnated with the substances
produced in the process of degeneration.
The deposition of the fat round the nucleus in the first instance indi-
cates that the nucleus takes an active share in the process, and this is
borne out by the fact that the nuclei themselves become very dark in
the degenerating cells and are sometimes quite opaque.!
In sections of stomachs fixed with sublimate mixtures the blackening
of the degenerating cells seen in the Flemming-fixed sections is con-
spicuously absent, so that at the first glance it is difficult to pick out
the senile portions of the epithelium. More careful study of the subli-
mate sections shows that here the degenerated epithelium is dis-
tinguished from the regenerated by its pale, empty appearance, owing
to the fat-grains having entirely disappeared, leaving empty spaces to
mark their former position. This is best seen in stomachs fixed in
sublimate-acetic, since, after sublimate-aleohol mixtures (Maier’s and
Schaudinn’s) the cells are often much deformed and shrunk. In a
favourable spot it is seen that the young cells, freshly produced from
Léger and Duboseq have noted also that the mucoid substance is
deposited first in the nucleus.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 501
the crypts, have denser cytoplasm filling the cell throughout, except in
the apical expanded portion of the cell; the cytoplasm stains deep
grey or neutral tint after iron-hematoxylin and shows relatively few en-
closures. The senile cells, on the contrary, are full of cavities, so that the
cytoplasm has a spongy appearance throughout the cell and not merely
in its apical region; and scattered through the spongy cytoplasm are
grains, fine or but moderately coarse, which are stained black after iron-
hematoxylin, red after Giemsa.
The difference between the senile cells after the two methods of
fixation is easily explained if the grains deposited in them are principally
fat. In all the sections alike the fat has been dissolved away during the
process of imbedding in paraffin. In the Flemming-fixed sections, how-
ever, each fat-grain has reduced the OsO, to metallic osmium, and
consequently is represented in the sections by a black mass, a model of
the fat-globule in metallic osmium. In the sublimate-fixed sections no
such reduction takes place, and the fat-globule is represented by an
empty space; only the mucoid grains (if we are right in calling them
so) remain in the cytoplasm, stained red or black according to the
stain used.
It should be mentioned finally that after sublimate-fixations the
blood-débris is stained very much blacker by iron-hematoxylin, and
holds the stain much more tenaciously than after Flemming-fixation.
This is especially true of that part of the débris which penetrates down
between adjacent epithelial cells, and which often remains jet-black
after all the rest of the débris has become pale in tint. In consequence
the cells of the columnar epithelium in sublimate-fixed sections are
often seen to be separated by black masses, which careless observation
might confuse with the black stain of the degenerated cells after
Flemming-fixation, especially when, as often happens in such sections,
the main mass of the débris has shrunk away from the epithelium into
the centre of the stomach-lumen. Such a mistake could only be made,
however, with powers too low to discern that the black masses are
between the cells and not in them.
The degenerated cells are thrown off bodily into the lumen of the
stomach, which often contains great numbers of them in the blood-
débris. There they are doubtless digested and absorbed along with the
other contents of the stomach. Légerand Duboscq described a process
of mucoid degeneration in which the entire cell, having a remarkable
and deceptive resemblance to a gregarine, is engulphed by a basal cell ;
ultimately the latter also degenerates, and is thrown off with the cell
it has taken in (1. ¢., p. 451). We have seen nothing of this sort in the
flea, in which basal cells do not occur in the epithelium of the stomach.
502 E. A. MINCHIN AND J. D. THOMSON.
(5) TECHNIQUE.
We have already described above our methods of dissecting
the flea and extracting from it the organs which it is required
to examine for the presence of stages of T. lewisi. Here
we propose to describe the methods by which the trypano-
somes, when found, were preserved as permanent preparations
for microscopic study.
The organs of the flea, extracted in the manner described
above, are at once examined carefully under the microscope
for the presence of trypanosomes in their various phases of
development. When trypanosomes were found in any of the
internal organs, after note had been taken, or sketches made,
of their forms, position, and other points of interest, we pro-
ceeded to make permanent preparations of them. For this
purpose the coverslip is carefully raised up, by means of the
pair of fine needles that were used in the dissection of the flea,
lifted off, and dropped at once with wet surface downwards
into a suitable fixative. The slide is then handed to the
collaborator or to an assistant, who places it bodily into a tube
containing a small quantity of four per cent. solution of osmic
acid. In the tube the slide remains about ten to fifteen
seconds, tightly corked up, in order to fix the trypanosomes
with the vapour of osmic acid. Subsequently the slide is
fixed with absolute alcohol for about fifteen minutes and
stained with Giemsa’s stain in the usual manner.
For the fixation of the coverslip-films we used, in the earlier
periods of our investigation, either Schaudinn’s fluid (corro-
sive sublimate, saturated solution in distilled water, 100 c.c.;
absolute alcohol, 50 c.c.; glacial acetic, a few drops) or
sublimate-acetic (Hg Cl, saturated in H,O, 95 volumes; glacial
acetic, 5 volumes). Both these fixatives gave results about
equally good; it is difficult to choose between them. Latterly,
however, we used only Maier’s modification of Schaudinn’s
fluid (distilled water, 200 c.c.; absolute alcohol, 100 c.c.;
sodium chloride, 1:2 grm.; HgCl,, 10 grm.), since this
appeared tous to give better preservation, and, in particular,
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 503
less shrinkage of the bodies of the trypanosomes, than the
others. The fluid being put into a large watch-glass, the
coverslip is dropped into it with the film downwards. ‘The
coverslip usually sinks in the fluid and then rests on its
corners on the rounded bottom of the watch-glass, so that
the film itself escapes any friction or injury. The coverslips
are left in the fixative from ten minutes to half-an-hour or
longer (the exact time appears to be immaterial), and are
then passed through 50 and 70 into 90 per cent. alcohol,
where they can be kept until it is convenient to stain them.
The coverslip films were stained almost invariably with
Heidenhain’s iron-hzmatoxylin, using 35 per cent. iron-alum
solution and } per cent. hematoxylin-solution, both in dis-
tilled water. ‘he film, after having been brought down
through graded strengths of alcohol (80, 70, . . . 10
per cent.) to water was left about twenty-four hours in the
iron-alum, then as long in the hematoxylin. Immediately
before using the hematoxylin-solution a few drops of a
saturated watery solution of lithium carbonate was added
to it, drop by drop, until the solution, when shaken up,
was a bright claret-red colour. After the film had been
twenty-four hours in the hematoxylin-solution the differ-
entiation of the stain was carried out under control by the
microscope in a weak (light brown) watery solution of iron-
alum. When differentiation was complete the film was washed
in a current of tap-water for at least twenty minutes, then
rinsed in distilled water and brought up through graded
strengths of alcohol to absolute alcohol. At this stage the
coverslip was usually dipped for a moment into Lichtgriin-
picric solution (Lichtgrin, 1 grm.; picric acid, 4 grm.;
absolute alcohol, 100 e.c.), then washed again in absolute
alcohol, passed through xylol, and mounted in pure xylol-
balsam on aslide. The Lichtgriin stain must be used very
rapidly, as it stains intensely.
In this way two preparations were obtained of the contents
of each organ—one on the coverslip, the other on the slide—
and as a rule trypanosomes were found more or less
504. E. A. MINCHIN AND J. D. THOMSON.
abundantly on both of them, so that it was possible to
compare corresponding phases of the development prepared
by distinct methods of technique. It is very important,
however, that the operation of removing the coverslip and
fixing the films should be performed very rapidly and
expeditiously, in order to avoid any drying taking place.
The coverslip is particularly liable to dry, since the film of
liquid that adheres to it is very thin; the slide, on the
contrary, does not dry so quickly. A coverslip that has
dried before fixation is quite useless for staining by the iron-
hematoxylin method; the trypanosomes acquireacharacteristic
shiny appearance, as if they had been glazed, and when the
stain is extracted in order to differentiate the preparation, it
does not come out of the cytoplasm evenly, but gives a
blotchy appearance, with no sharp differentiation of the
nucleus or flagellum. It sometimes happens that a coverslip-
fim may be otherwise satisfactory, but may have dried
slightly at or near the edges, thus affording opportunities for
comparing the effects of desiccation on the trypanosomes
with the condition of others that have never been dried. It
is then seen that, in addition to the defective staining
already described, the trypanosomes are flattened and
distorted in various ways.
The fragments of tissue in the dissection adhere, for the
most part, to the coverslip ; it is not possible, however, to
make out anything of trypanosomes which remain within the
organs in film-preparations, and it is therefore necessary to
tease up the organs well, after dissecting them out, in order
to set free the trypanosomes. In the case of those phases
which are attached to the gut-wall many remain so attached
even when the wall is teased up, but a certain number are
usually set free. When such forms are seen in the fresh film
they should be dislodged, as far as possible, by tapping
gently on the coverslip with a needle.
In some cases a coverslip-film which had been stained with
iron-hematoxylin was unmounted by dissolving the Canada
balsam in xylol, after the trypanosomes on it had been
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 505
studied and drawn, and the hematoxylin-stain completely
extracted by placing it for twenty-four hours in a 3} per
cent. solution of iron-alum. The coverslip was then washed
for an hour in a current of tap-water, and could then be re-
stained by some other method—for example, Twort’s stain.
Trypanosomes that had been already drawn after the
hematoxylin-staining could then be drawn again after bemg
stained in a different manner. This double staining did not
seem to injure the trypanosomes in any way, but it is note-
worthy that after re-staining with Twort’s stain they always
came out a little smaller, when re-drawn with the camera lucida,
than they had done previously after the hematoxylin-stain
(compare figs. 260-63, Pl. 42, with figs. 260a—263a, PI. 38).
When, as sometimes happened, the trypanosomes were so
scanty on the coverslip as to require prolonged searching to
find them, it was often very difficult to judge the right
amount of extraction of the hematoxylin in the process of
differentiation by means of iron-alum. Morever, a degree
of differentiation which is sufficient for trypanosomes in
the thinner parts of the film is insufficient for the thicker
parts. Hence it was often necessary to unmount the pre-
parations and differentiate them further, perhaps two or
three times, before the right degree was attained. It is
difficult to judge of the required differentiation by the
fragments of tissue in the films, since the minute bodies of
the flagellates give up the stain much more quickly than the
relatively thick tissue-cells, and in a preparation in which the
latter are satisfactorily differentiated the trypanosomes
become mere ghosts, requiring to be re-staimed altogether.
The counter-stain with the Lichtgriin-picric mixture was
found to show up the cytoplasm and flagellum of the
trypanosomes more clearly.
However carefully the preparations have been made, it is
often difficult to make out clearly and with certainty the
structural details of some of the minuter phases of the life-
cycle, and for this purpose the best optical apparatus was
required, both as regards the objectives and the illumination
506 E. A. MINCHIN AND J. D. THOMSON.
used. All trypanosomes in the permanent preparations were
drawn by Miss Rhodes, under our supervision, with the
camera lucida at a constant scale of magnification which was
as nearly as possible 3000 diameters in the case of the film-
preparations, 2000 diameters in the case of sections.
Our study of the development of T. lewisi in the flea
was based principally upon the examination of films, made as
described above, but it was found necessary also to cut
sections both of the stomach and rectum of the flea. The
following is an account of the technique employed by us in
preparing sections of the stomach; the same applies to
sections of the rectum, the only difference being that the
stomachs were cut transversely, the recta longitudinally.
The stomachs of which sections were cut were taken from
fleas fed eighteen, twenty-four, or thirty-six hours previously
on an infected rat; the fleas themselves had been collected
from the non-infected breeding-cage and kept hungry for
about three days before being put on the infected rat. The
stomach in each case was carefully dissected out from the
flea, if possible without puncturing or injuring the stomach,
in a drop of salt-solution on a slide, and then plunged into
the fixative by inverting the slide in such a way that the
stomach alone, all other parts of the flea having been
removed, was in a hanging drop. If the stomach was
ruptured or punctured in the process of extraction it was
not, as a rule, preserved, except perhaps as a smear after
teasing it up.
A number of different fixatives were tried, but the best
results were obtained with Flemming’s fluid! and Maier’s
modification of Schaudinn’s fluid, and especially with the
former. After Flemming the histology of the stomach is
extremely good in all details; the blood fills the whole section
1 The strong solution, made up as follows: a gramme tube of osmic
acid is broken into a clean bottle, and to it is added distilled water,
50 ¢.c.; 1 per cent. solution of chromic acid in water, about 187°5 c.c. ;
and glacial acetic about 12°5 ¢.c.; the whole allowed to mix and
dissolve.
THE RA'T-TRYPANOSOME, I'RYPANOSOMA LEWIST. 507
andis not shrunk away from the wall, and the trypanosomes,
both free and intracellular, are well-preserved both in
structure and form, and they stain well either with iron-
hematoxylin or Giemsa, especially the former. After Maier’s
fluid the histology of the stomach-tissue is ndt so good; the
cells are shrunk and the minute structure of the nuclei is
deformed. It is evident from a careful study of the prepara-
tions that the defects of Maier’s fluid are due to unequal or
differential penetration of its constituents ; the alcohol evi-
dently diffuses into the tissues first and produces the shrinkage
and deformation of the nuclei; the sublimate does not get to
the various tissue-elements until they have already been fixed
in a defective manner by the alcohol. The blood-débris is
also much shrunk after the Maier; while the greater part,
sometimes the whole of it, contracts to form a central mass in
the section, a certain amount remains usually adherent to the
epithelium at the periphery, leaving an irregular empty ring-
shaped space between the central and peripheral zones of the
blood-débris. But to compensate for these disadvantages,
the trypanosomes are extremely well-preserved and stain
admirably with Giemsa’s stain; some of our stomach-sections
prepared in this way are as clear and demonstrative, so far as
the trypanosomes are concerned, as any smear or film-
preparation ; in fact more so in the case of the large
“spheres,” which do not suffer so much from the tendency
to opacity which is so disagreeable a feature in the smears.
One is here confronted with the extraordinary difference,
familiar to everyone who has worked at trypanosomes,
between the reaction of these parasites, and that of tissue-
cells, to the ordinary fixatives and stains used in cytological
technique.
Whatever the fixative used, it was allowed to act for about
an hour. ‘The stomachs preserved in Flemming were well
washed in tap-water and then brought up through a series of
alcohols of gradually increasing strength ; those preserved in
Maier were transferred from it direct to 50 per cent. alcohol.
In either case the objects were brought up to 90 per cent.
508 E. A. MINCHIN AND J. D. THOMSON.
alcohol and there fixed on liver preparatory to being
imbedded for section-cutting. Amyloid human liver was
used. A moderately thin slice of a block of liver preserved
in alcohol was cut by hand with a razor wetted with alcohol,
and floated into a shallow glass vessel with a flat bottom,
placed on the stage of the dissecting-microscope, and contain-
ing 90 per cent. alcohol to the depth of about a centimeter.
The stomachs, taken up in a pipette of suitably coarse calibre,
were placed on the slice of liver and carefully arranged side-
by-side, their axes parallel to one another and similarly
orientated, with their proventriculi all at the same level
and all pointing in one direction, their pylori in the oppo-
site direction. Then a tiny drop of glycerine and albumin
solution, such as is used commonly for sticking sections
on slides, was taken up on the point of a needle and
caused to touch the surface of the alcohol immediately above
the stomachs. The dense albumin-solution falls at once
through the alcohol and spreads out over the stomachs on the
liver; at the same time the glycerine is extracted and the
albumin coagulated by the alcohol, with the result that
the stomachs are stuck to the slice of liver. From six to
nine stomachs were thus attached side-by-side on a slice
of liver. As the stomachs, before being stuck on, are very
liable to roll about or become shifted in position with the
slightest disturbance or touch of the microscope, it was found
best in practice to put them on not more than three at a
time; that is to say, three stomachs having been arranged
and fixed upon the liver, three more are then put on beside
them. When the required number of stomachs have been
stuck on, the slice of liver is trimmed with a scalpel into a
rectangular form, in such a way that the longitudinal axes of
the stomachs are parallel to the shorter sides of the rectangle;
so that by cutting sections of the liver parallel to the longer
sides of the rectangle the stomachs are all cut transversely at
the same time.
We have thought it worth while to describe the method
of fixing the stomachs on liver, although no novelty is
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 509
claimed for it, in some detail, as it may not be familiar
to some investigators working on similar objects, and
because it is a procedure which saves much time and
trouble. In the first place, it is much easier to imbed a
relatively large block of tissue than a number of separate
tiny little stomachs, and the orientation of the objects can
be made much more accurate. In the second place, a great
economy of labour in the section-cutting and of space in
the slides and preparations is effected. To have a number
of stomachs cut in the same section diminishes the labour
of looking through the preparations under the microscope,
and the presence in the section of the slice of liver makes it
much easier to go from one section to the next under the high
power. Thirdly, with a little experience the liver itself
furnishes useful guidance in staining the sections, especially
by the iron-hematoxylin method; one soon learns what
degree of extraction of the stain from the liver-cells gives
the best results for the trypanosomes, so that the process of
differentiation can be carried out under low powers of the
microscope—a great advantage. And finally, since it may be
assumed that all the stomach-sections contained in one and
the same microtome-section have received exactly the same
treatment, it is legitimate to ascribe the very considerable
differences seen in different stomachs in the same section to
constitutional or functional differences in the stomachs them-
selves and not to varying local effects of the stain.
The stomachs, after being fixed to the liver in 90 per cent.
alcohol, were imbedded in the usual way in paraffin, with a
melting point of about 54°C. Methods of celloidin-imbedding
were tried, but yielded no advantages to compensate for the
extra trouble, especially that of extracting the celloidin from
the sections—an indispensable preliminary to staining them.
The best thickness for the sections of stomachs was found
to be 6m; with less than that the trypanosomes are too
* One of us (EK. A. M.) first became acquainted with this method in
1891 from fellow-workers in the Zoological Station at Naples, and has
practised it constantly ever since.
510 E. A. MINCHIN AND J. D. THOMSON.
fragmentary. The recta may with advantage be cut thinner
than 6, since the crithidial forms are very minute.
Various methods of staining were tried on the sections,
but the results of the trials were that we kept finally in
practice to two methods only, namely, iron-hematoxylin
(Heidenhain), followed by Lichtgriin-picric in absolute alcohol
as a counter-stain, and Giemsa’s method. For the iron-
hematoxylin method the sections were treated first as has
been described above for the coverslip-films. The Lichtgriin-
picric, which stains very rapidly, was merely washed over the
sections for a moment and then washed off again with
absolute alcohol. Giemsa’s stain was used, according to the
published prescription, as follows: The sections have their
paraffin removed, and are brought down to water in the
ordinary way. They are then washed in tap-water and put
into dilute Lugol’s solution (1 ¢.c. of Lugol to 25 e.c. of
distilled water) for ten minutes. After this they are rinsed
quickly in tap-water and put into a 0°5 per cent. watery
solution of hyposulphite of soda for ten minutes. Next they
are washed in a current of tap-water for five minutes or
longer, and then put into the stain. The distilled water used
to dilute the Giemsa-stain has to be neutralised in the way
prescribed by Giemsa.t The sections were first placed in
fairly strong stain—say, 1 drop of Giemsa to 1 c.c. of
neutralised distilled-water—for about an hour, and then were
left overnight in a weaker stain—1 drop of Giemsa to 4 or 5 c.c.
of neutral distilled-water. The excess of stain is removed
by rinsing in water, and after the excess of water has been
drained off differentiation of the stain is carried out with
1 A measured volume of the distilled water to be neutralised is taken,
and to it are added a few drops of hematoxylin-solution (5 per cent. in
distilled water), sufficient to tint it. Then a very weak solution (1 per
cent. in distilled water) of potassium carbonate is added drop by drop,
the water being well shaken after each drop has been added, and left
for a minute or two, until the colour of the tinted water changes from
yellowish-red to reddish-purple. In this way the number of drops of
the carbonate-solution required for neutralising a given volume of the
distilled water is known.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 511
different strengths of acetone mixed with xylol, beginning
with 95 per cent. acetone used for a very short time, in order
to dehydrate the sections and extract the stain, and ending
with pure xylol, after which the slides are mounted in dammar
or Canada-balsam.
Of the two staining methods principally used, iron-hema-
toxylin gave admirable results after Flemming, especially for
the intracellular stages; for the extracellular trypanosomes
this stain is not so satisfactory, owing to the fact that the
blood-débris, especially in the earlier stages of digestion,
stains very intensely with it and refuses to give up the stain—
at any rate not until after it has been all extracted from
the cells and parasites. Consequently trypanosomes free in
the blood may be entirely obscured by the opaque, deeply-
stained débris, and hence quite invisible. The black stain of
the blood-débris is even more intense after Maier than after
Flemming ; sections of stomachs fixed in Maier less than
thirty-six hours after feeding are hopeless for the iron-
hematoxylin staiu, so far as the free trypanosomes are con-
cerned, and those fixed in Flemming are not much better.
By Giemsa’s method, on the other hand, the trypanosomes in
blood-débris are sharply differentiated and admirably shown;
in the cells they are also good, better, perhaps, as “ show ”
preparations, but not so precise in minute cytological details
as by the iron-hematoxylin method.
To sum up the results of our experience in the technique
of stomach-sections, we recommend: (1) Flemming’s fluid,
followed by iron-hematoxylin and Lichtgriin-picric ; and (2)
Maier’s fluid, followed by Giemsa’s stain. These two methods,
supplementing each other, may be relied upon to reveal all
essential details of the intimate life of the trypanosome and
of the disturbances produced by it in the tissues of the host.
vou, 60, PART 4,—NEW SERIES.% 36
512 E. A. MINCHIN AND J. D. THOMSON.
PART II.—THE DEVELOPMENT OF TRYPANOSOMA
LEWISI IN THE FLEA.
(1) Generat Inrropuction.
From the results of experiments, described further below,
it is shown that fleas fed on rats infected with Trypanosoma
lewisi do not become infective to rats again until a period of
at least five or six days has elapsed from the time that the
fleas first ingested blood containing trypanosomes. From these
experimental data it may be inferred that the developmental
cycle of T. lewisi in the flea requires a minimum of five days
for its complete course. The conclusions drawn from the
experiments are confirmed by direct observation, since it is
found, as will be described presently, that the little stumpy
trypanosome which is the final form of the development in
the flea, makes its first appearance in the rectum of the flea
about five days after the development begins.
During the entire course of its development the trypano-
some is confined to the alimentary canal proper of the flea,
and is found in the stomach, intestine, and rectum; it is
never found in the body-cavity (heemoccele), and by a series.
of observations and experiments, which in our opinion are
exhaustive (see below), we have convinced ourselves that the
trypanosome does not penetrate into the salivary glands. It
may, however, occur in the Malpighian tubules exceptionally,
as the small crithidial form, characteristic of the rectal phase,
attached to the wall of the tubes at or near their proximal
opening into the proctodzeum.
The developmental cycle can be divided conveniently into
phases characteristic of the parts of the gut in which the try-
panosomes are found, and we can thus distinguish a stomach-
phase and a rectal phase. These distinctions are useful and
natural, but their sharpness is blurred by not infrequent
variations in the course of events; thus forms belonging
normally to the rectal phase may sometimes be found in the
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 413
pyloric region of the stomach, though the converse case of
the typical stomach-phase occurring in the rectum is not
found. We may consider these phases first in their normal
and typical modes of occurrence, and deal with the variations
subsequently.
The stomach-phase (Pls. 36-39) is the first period of the
development and is characterised by a peculiar mode of multi-
plication on the part of the trypanosomes, which penetrate
into the epithelial cells lining the stomach and there repro-
duce themselves by a process of multiple fission. Hence in
this period of the development free and intracellular forms
can be distinguished. The stomach-phase is of short.
duration, perhaps in some cases lasting not more than
twenty-four hours, in others two or three days, in rare cases
four or even five days, but probably always terminated by the
second feed of the flea, counting as the first feed that by
which the flea became infected.
In the intestine the trypanosomes find, as a rule, no resting
place, but merely pass through it on their way to the rectum.
Hence, the forms found in the intestine are usually active,
migratory forms which have completed the stomach-phase and
are on their way to the rectum to initiate the rectal phase.
Occasionally, however, forms similar to those characteristic
of the rectal phase may be found attached to the wall of the
intestine, especially near the pyloric opening.
The rectal phase (Pls. 41 and 42) consists chiefly of small,
often minute individuals, which are crithidial in structure
and are attached by the tip of the flagellum to the wall of the
rectum, where they keep up a continual multiplication by
binary fission. The crithidial form of the development takes
origin in the rectum and is first established there, but may
migrate forward to the pyloric region of the stomach later on.
When once established in the flea, the crithidial phase endures,
probably, as long as the flea lives, and thus constitutes a
permanent stock of the parasite, enabling the infectivity of
the flea to be maintained without renewal of the infection.
From the crithidial phase arise by modification of individual
514 E. A. MINCHIN AND J. ‘D. THOMSON.
crithidial forms the small trypanosome-forms by which the
infection of the rat is brought about, and which are the final
forms of the developmental cycle in the flea.
By no means all the trypanosomes, however, which are
taken up from the rat by the flea undergo the course of
development sketched out briefly in the foregoing paragraphs.
By experiment it is found that only a relatively small number
of the fleas fed on infected rats become infective, apparently
not more than one flea in four, on an average (see below) ;
and these results are confirmed by direct observation. If a
number of fleas are fed on a well-infected rat, trypanosomes
will be found in the gut of all the fleas dissected and examined
a short time after feeding; but the longer the interval between
the feeding and the examination of the fleas the larger the
proportion of the fleas in which the trypanosomes have dis-
appeared or become very scanty, until finally trypanosomes
will be found in but few (see Tables A and B (2) above). Pro-
bably the percentage of fleas in which the trypanosome
succeeds in establishing itself permanently may be taken, on
the average as about 25 per cent. (see p. 663 below). It follows
that in about 75 per cent. of the fleas which digest blood con-
taining T’. lewisi the parasites die out altogether, and it is
probable that in all the fleas a certain number of the ingested
trypanosomes die off, since fleas that have been fed on a rat
with trypanosomes swarming in the blood may exhibit a very
scanty infection of the gut at any subsequent period.
From these data it is to be expected that together with
developmental forms of the trypanosomes, various stages in
their degeneration would also be found in the fleas, at least
during the first few days after the parasites were ingested
by them, and this expectation is fully realised. Itis necessary,
therefore, to recognise a degenerative series of forms (PI. 43)
as well as a developmental series in the gut of the flea, and to
distinguish carefully the two series from one another. Any
particular flea, when dissected and examined, may present an
extraordinary medley of different forms of the trypanosome.
To distinguish between the different forms and to refer each
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 515
form to its proper position and sequence in the series, whether
developmental or degenerative, is our task, and it is no light
one. When we were at an earlier stage in our investigations
we did not recognise sufficiently the importance of the
degenerative series, and consequently tried to interpolate
degenerative forms into the developmental series, greatly to
our own confusion. On the other hand it is necessary to steer
very clear of a tendency to explain any form as degenerative,
of which the developmental position is not immediately clear ;
thus we were at first inclined to regard the peculiar recurved
forms in the stomach as degenerative, until we discovered the
intracellular multiplication and were thereby enabled to refer
the recurved forms to their true position.
In the problem of piecing together and reconstructing
the sequence of the two series, developmental or degenerative,
there is, to begin with, a known and fixed starting point for
each, namely, the ordinary form of T. lewisi as it occurs in
the blood of the rat. Further clues are obtained. by linking
together, through gradual transitions, the forms seen in the
fleas, but more especially by the study of “‘time-fed” fleas,
that is to say, fleas dissected and examined at known periods
of time after they have been fed on the infected rat. The
part of the gut in which a given form occurs is a further
guide as to its significance; and all data and conclusions
obtained from observation are controlled and checked by
the results of experiment, especially useful in determining
the final form of thedevelopment. Guided by these various
considerations we have arrived at the conception, set forth
below in fuller detail, of the changes undergone by the
trypanosome in the flea (see especially Pl. 45 and description).
In our account we describe separately the two series which
we regard as developmental and degenerative respectively ;
but it must be pointed out that while these two series are
very distinct and easily recognisable as a whole, certain
forms or stages of the one series are sometimes very difficult
to distinguish decisively from very similar forms belonging in
reality to the other series. Consequently it is impossible to
516 E. A. MINCHIN AND J. D. THOMSON.
be free from doubt, occasionally, with regard to the place
to be assigned to a particular specimen or type of indi-
vidual.
It remains only to be stated at this point that we adhere to
the following nomenclature for the parts of the body of the
trypanosome or crithidia: Blepharoplast for the basal granule
of the flagellum; kinetonucleus for the smaller, tropho-
nucleus for the larger, of the two nuclei. In order to save
space we shall, however, use for the kinetonucleus the
symbol » (plural nz) and for the trophonucleus the symbol
N (plural NN).
(2) Tue DEVELOPMENTAL SERIES.
(4) The Stomach-Phase.
The blood ingested by the flea passes in the first instance
into the stomach, that portion of the digestive tract which is
derived from the embryonic mid-gut or mesenteron, and
which is lined by a layer of epithelium representing the true
hypoblast or endoderm of the embryo. In the post-embryonic
stages of the insect, this part of the gut is characterised by
the absence of the chitinous cuticular lining secreted by the
ectodermal! epithelium of the parts anterior or posterior to if,
namely, the stomodzeum, comprising the pharynx, cesophagus,
and proventriculus, and the proctodeum, comprising the
intestine and rectum. The boundary between mid-gut and
hind-gut is further indicated by the origin at this point of
the Malpighian tubules.
in what may be called a normal feed, the flea fills the
stomach and proventriculus alone. It is not an infrequent
occurrence, however, for some fleas to gorge themselves to
such an extent that the freshly ingested blood not only fills
the stomach completely, but overflows beyond it into the
intestine and rectum ; we have observed this to happen most
frequently in the case of female fleas, rarely in the case of
males. In such cases some of the ingested trypanosomes
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 517
may be carried on at once into the proctodzal regions of the
gut, but all such trypanosomes degenerate, and need not be
reckoned with in the developmental series, the first phases of
which take place always in the stomach alone.
(a) The Extracellular Trypanosomes.
The trypanosomes introduced into the stomach very soon
begin to undergo changes (PI. 36, figs. 1 and 2; Pl. 37, figs.
47 and 48). The first change is probably purely physio-
logical, since long before any alteration is observable in form
or structure these ingested trypanosomes seem to have lost
their power to infect when injected subcutaneously into clean,
susceptible rats (see below, p. 634). The next change
observed in these ingested trypanosomes may be seen on
examining microscopically the contents of a flea’s stomach
four to six hours after the first feed on an infected rat. A
certain number of trypanosomes will then be seen to pass
rapidly in a straight course across the field of the microscope
with their flagella directed anteriorly. ‘lhe posterior third
ot the body is held more or less straight and appears more
rigid, as it does not share in the rapid undulations of the
anterior end of the body. The movements of these trypano-
somes thus contrast strongly with the sinuous, serpentine and
wriggling rather than progressive movements characteristic
of the trypanosomes in the blood. When not actively pro-
eressing, the trypanosomes in the stomach have a tendency to
attach themselves by the tips of their flagella to pieces of
débris, to the wall of the stomach, or to the surface of any
other firm body. The stiffening of the trypanosome-body is
probably due to increased tension of the cytoplasmic contents
produced by absorption of fluid from the ingested blood as it
undergoes alteration in the process of digestion. As a result
of absorption or imbibition of fluid, the body of the parasite,
previously more or less distinctly flattened, acquires a cylin-
drical and more rigid contour. If this explanation be correct,
it follows that the first stimulus to developmental change is
518 E. A. MINCHIN AND J. D. THOMSON.
to be ascribed to differences of osmotic tension in the fluid
medium, as has been shown experimentally by Miss Robertson
(1911) to be the case in the development of the trypanosomes
of fishes.
In stained preparations most of the ingested trypanosomes
show at first little modification from. the ordinary blood-try-
panosomes. In rare instances the nuclei may be approximated
(Pl. 36, fig. 2). Some show a darker staining-reaction of the
posterior third of the body, which appears, from the back-
ward position of 7 in such forms, to bea sign of degeneration
beginning to set in (compare PI. 43, figs. 289, 290).
In their free active state the trypanosomes in the stomach
are never found to be undergoing multiplication by any form
of fission, and it is doubtful if they undergo any develop-
mental changes further than those described above, until
after they have multiplied within the cells of the lining
epithelium of the stomach. The multiplication of the stomach-
phase takes place solely within these cells, and although, in
strict chronological order, we should now describe the intra-
cellular stages of multiplication, it is more convenient for
purposes of description to divide the stomach-phase into
“ free’ and “intracellular” stages, and to describe all the
free developmental forms before describing the intracellular
multiplication.
It can be established by direct observation that the process
of intracellular multiplication produces a long, free type of
trypanosome which may be characterised by the term
“ crithidiomorphic,” because while externally similar in form
and movements to a large crithidial type of flagellate, it
lacks, as a rule, the diagnostic structural feature of a true
crithidial form, since only exceptionally is » found actually
beside or in front of N (Pl. 36, figs. 3-11, Pl. 57, figs. 49-57).
We shall now proceed to describe in more detail this late,
free form of trypanosome. It must, of course, be understood
that as several generations of the intracellular stage may
follow each other in succession (see below), free and intra-
cellular forms in all stages of development can be found
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 519
together in the same stomach. We believe, however, that
the typical crithidiomorphic type of trypanosome always
follows an intracellular stage, that in its less developed form
it is the direct product of intracellular multiplication, and
that though in this form it may again enter an epithelial cell
and multiply, it is, in the more advanced form, the highest
developmental type of the stomach-phase and is destined to
pass down the intestine into the rectum where, after under-
going further modification, it initiates the characteristic
crithidial rectal phase to be described below.
In the living condition the crithidiomorphic form pro-
gresses at a great pace in a straight line with the flagelluia
directed anteriorly (“mouvement en fléche”’), in much the
same manner as does the early stomach-form above described.
It is, however, considerably longer and the posterior end is
more rigid and swollen, often distinctly clubbed. Owing to
the rapid motion and imperfectly straight body, the clubbed
appearance is exaggerated in the living condition, but stained
preparations also show that some of the trypanosomes are
distinctly clubbed in shape. Like the early stomach-form,
when not actively progressing the crithidiomorphic type has
a strong tendency to attach itself by the tip of its flagellum
to cells or débris, etc. Apart from its size the distinguishing
characteristic between this and the earlier form is the marked
approximation of the two nuclei, best seen in stained
preparations (Pl. 36, figs. 7, 8, 10; Pl. 37, figs. 50, 51).
It has been mentioned that while, as a rule, the long, free stomach-
trypanosomes have n behind N, it is found in a few cases that they have
the typical crithidial structure with » in front N. We have observed
altogether but three instances in which such forms occurred in sufficient
abundance to make them worthy of special note. The first and most
striking was the case of a flea taken from a bell-jar in which a number
of fleas had been kept for some time with an infected rat, so that the
length of time since the flea had ingested the parasites was not known.
The body-cavity of the flea contained a cysticercoid of Hymenolepis
diminuta (vide Nicoll and Minchin, 1911). The intestine of this flea
showed a peculiar malformation in the form of a globular pouch-like
appendix, distended with red fluid, and due apparently to an obstruc-
520 E. A. MINCHIN AND J. D. THOMSON.
tion or strangulation of the intestine. The stomach, examined fresh,
was seen to contain a great number of active trypanosomes, some of
which were adhering together in couples, and in the intestine a clump
of attached forms was seen near the origin of the Malpighian tubules.
In the preparations of the stomach of this flea a great number of try-
panosomes were found showing every possible gradation of structure,
from forms similar to the ordinary blood-trypanosomes to a long
crithidial type with n far in front of N (Pl. 36, fig. 11 and PI. 37,
figs. 60-66). Many of these were found closely adherent in couples, just
as had been seen in the fresh state, each such couple being composed of
two crithidial forms in most cases, but sometimes of two ordinary
forms (PI. 36, fig. 12, and Pl. 37, figs. 67, 68). In every couple seen the
two individuals appeared quite distinct and showed no signs of actual
fusion; one couple was found attached téte béche (as in PI. 43,
fig. 310). In the preparation of the rectum and intestine (preserved
together) a few similar large trypaniform or crithidial individuals were
seen, and also a fair number of dwarfed, degenerative forms, but no
couples.
Special mention has been made of this flea because we were at first,
and remained for some time, under the impression that the couples seen
represented a true sexual fusion, and that we had discovered the sexual
phase of the trypanosome. We have been quite unable, however, to
confirm this notion or to find a similar state of things in any other flea
of all those examined by us, and we now regard the state of things
found by us in this particular flea as exceptional and abnormal, in rela-
tion probably to the malformations noted by us in the flea itself. It is
possible that the malformed condition of the intestine prevented, to some
extent, the passage onwards of the trypanosomes from the stomach, and
so caused an arrest of development in the parasites, in which the
tendency towards the crithidial type of structure became realised to its
fullest extent. The coupling of the trypanosomes must then be regarded
as agglomeration due to abnormal and unfavourable conditions, though
in no case were more than two trypanosomes seen adhering together.
The second case in which the long crithidial forms were prominent
was in a flea of a batch which had been fed onan infected rat three days
before being examined and dissected. The fleas had been kept in an
incubator at a temperature of 25° C. after the infective feed, and had not
been fed again. In one of the fleas long crithidial forms with m in front
of N, were fairly numerous, together with intracellular multiplicative
stages, in the stomach (PI. 37, figs. 56,57); in the rectum one active
trypanosome of the long stomach-type and a clump of degenerative
forms were seen in the fresh state.
The third case to be noted was in a flea of a batch which had been fed
twenty-four hours previously on an infected rat. There was nothing
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 521
special to note about this flea; the stomach contained many long active
trypanosemes, with x and N closely approximated, or with nm in front of
N (PI. 36, figs. 9,10), and also some dwarfed degenerative forms, but no
multiplicative stages. In the rectum a few clumps of degenerative
appearance and some developmental forms were seen.
Besides these three cases which have come under our observation, in
which the long crithidial type was conspicuously abundant in the
stomach, we have noted the occasional occurrence of this type at
various ages—twenty-four hours, forty-eight hours, and sixty hours—
after the flea had fed on an infected rat. It is evident that it must be
regarded as exceptional for the trypanosomes to reach the complete
crithidial condition in the stomach, and that no special significance can
be attributed to the crithidial form in this part of the life-cycle,
although in rare instances and under special circumstances it may be
abundant.
It will be clear from the foregoing remarks that we are quite unable
to agree with the statements of Swellengrebel and Strickland (1910):
who, having examined two fleas one day after feeding on the infected
rat and five others two days after the infected feed, describe the trans-
formation of the long crithidiomorphie type of trypanosome into the
long crithidial type as the normal and usual method of development in
the stomach. On the strength of somewhat more extended experience,
we consider the long crithidial form to be of highly exceptional occur-
rence, both in the stomach and elsewhere, at so early a period of the
development, as already stated; we can only explain the results of
Swellengrebel and Strickland on the supposition that they were so
unfortunate as to have chanced upon abnormal fleas, similar to the
three cases described by us above, or that they may have regarded as
crithidial forms the very commonly-occurring recurved forms, which
they do not describe at all.
In view of what is known with regard to both the later
development of T. lewisi in the flea and the life-cycle of
other trypanosomes in their invertebrate hosts, it is evident
that the crithidial type of form and structure is the principal
and most characteristic phase of the development, and that
there is a pronounced tendency for the trypanosome to assume
crithidial characters when taken up by the flea—a tendency
which asserts itself more strongly after the trypanosomes
have undergone multiplication in the cells of the lming
epithelium of the stomach. So long, however, as the trypa-
nosome remains in the stomach the atavistic tendency towards
522 E. A. MINCHIN AND J. D. THOMSON.
the assumption of the crithidial form (+) does not normally
(or, at all events, usually) get beyond the crithidiomorphic
form (=). Occasionally, nevertheless, the crithidial form
n
asserts itself, as it were, even during the stomach-phase ; more
especially, perhaps, under the influence of any circumstances
which tend to retard the development of the trypanosome and
retain it in the stomach after it is ripe for passage into the
proctodeum, but not infrequently even under conditions
which cannot be asserted to be in any way abnormal.
(6) The Intracellular Multiplication of the
Trypanosome.
As already stated, the multiplication never takes place in
the free, active condition of the trypanosome, but only after
it has penetrated into one of the large epithelial cells lining
the stomach, within which it goes through a process of
multiple fission to produce a number of daughter-individuals.
which escape from the cell and pass back into the lumen of
the stomach as free trypanosomes again. The whole pro-
cess of intracellular multiplication, so far as it could be made
out by observation of living trypanosomes in the stomachs of
freshly-dissected fleas, was described by us in our preliminary
report (1911) ; we had not then had sufficient time or oppor-
tunity to make detailed studies, which present peculiar diffi-
culties, of the multiplication in preserved and stained material.
The ordimary smear-methods seldom permit any finer details
to be made out of the trypanosomes within the cells, on
account of the large size and thickness of the cells and con-
sequent opacity of the preparation. It is only possible in
smears to study the stages of multiplication set free by the
rupture of the cells; but even of such specimens it is difficult
to get perfectly satisfactory preparation for microscopic study
of detail. With the method of fixation by vapour of osmic
acid and subsequent coloration with Giemsa’s stain or other
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 523
modification of the Romanowsky method of staining we have
obtained occasionally very clear preparations of the later
stages of the multiplication, but as a rule, the “ spheres ”
with many nuclei take up the stain with such intensity that
they become opaque masses showing nothing of the internal
structure, although in the same preparations the free trypano-
somes may be stained to perfection. If in such preparations
the stain be cautiously extracted by means of acetone or other
suitable media, it is possible to obtain specimens showing the
nuclei satisfactorily, but then, as a rule, the flagella are
invisible, having lost the stain completely, while the free
trypanosomes or early stages of multiplication on the same
slide have become mere ghosts or have vanished altogether,
beyond the power of visual resuscitation by the most delicate
and refined methods of microscopic illumination. Very often
in such preparations only the kinetonuclei can be seen, the tro-
phonuclei having disappeared. Inthe study of Romanowsky-
stained preparations it was generally found necessary to begin
by drawing all that could be seen, general outline, project-
ing flagella, in the opaque, untouched preparations of the
spheres, and then to perform a number of successive opera-
tions of cautious extraction of the stain, examining the
preparations after each such operation and adding to the
drawing any fresh details of structure brought to hight. It
was difficult, however, to control the extraction of so sensitive
a stain with sufficient exactness to avoid losing the whole of
it in an instant. The last state of the preparation was
generally one which left it useless for purposes of demonstra-
tion; always a disappointment to the microscopist and his
friends. We have never succeeded in re-staining satisfac-
torily preparations in which the Romanowsky stain has been
over-extracted.
By far the best and most instructive preparations of the
intracellular multiplication were obtained in the coverslip
preparations fixed in Schaudinn’s or Maier’s fluid and subse-
quently stained by the iron-hematoxylin method, as described
above. Only in such preparations was it found possible to
524 E. A. MINCHIN AND J. D. THOMSON.
control the stain so that in the largest spheres both nuclei
and flagella were visible; even then, however, the tropho-
nuclei were sometimes faint and difficult to make out clearly
when the flagella were still sharp and distinct. Of one film
in which the smear was thickly crowded with spheres of
various sizes, some free, others still in the tissue, a very
satisfactory preparation was obtained by staining with Mann’s
hematoxylin, carried out with the friendly help of the inventor
of the stain himself. The result was a very good “ show ”
preparation of the multinucleate spheres, sharp and clear,
even in the thick parts of the smear, and especially suitable
for moderate magnification; the flagella, however, could not
be made out.
While the ordinary smear-methods presented special diffi-
culties, very convincing and beautiful preparations of the
intra-cellular phase. were obtained ir sections of fleas’
stomachs extracted carefully from the body and preserved in
various ways; a full account of the technique employed is
given above. Such preparations have the immense advan-
tage of exhibiting the exact relations of the trypanosomes to
the cells ; it is possible to look through every section of each
series, to note every trypanosome, free or intracellular,
occurring in each stomach, and to observe what each parasite
was doing at the moment the stomach was preserved. On the
other hand, for the study of the stages of multiplication,
sections have the disadvantage that the parasites themselves
are often halved or mutilated, so that any given specimen
may be only a part or fragment of the whole body. Both
smears and sections are therefore indispensable, and supple-
ment each other in obtaining a complete picture of the course
of events.
So much for methods and technique; we proceed now to
give an account of our observations.
From Noller’s investigations on the development of T.
lewisi in Ctenocephalus canis, it appears that! the
intracellular multiplication begins about six hours after the
ingestion of the trypanosomes by the flea. In our prepara-
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 525
tions of a batch of fleas, of which the infection could not have
been more than nine and a half or less than seven and a half
hours old, we have found the recurved forms fairly commonly
and also some of the rolled-up forms characteristic of the
early intracellular stages (Text-fig. 23, p. 635). We have
no other records of intracellular multiplication in our fleas
earlier than twelve hours. The stages of the intracellular
multiplication are to be found in all parts of the epithelium of
the stomach, from close behind the proventriculus to the
pylorus.
Our investigations upon the intracellular multiplication
contain, unfortunately, one gap which we have been unable
to fill; we have not succeeded in observing the actual pene-
tration of the epithelial cell by the trypanosome. Noller,
however, has been so fortunate as to observe the process, and
gives the following account of it. In a dog-flea which had
sucked infected blood five hours and fifty-five minutes
previously, he saw “a trypanosome, of which the pointed
hinder end had already penetrated into an epithelial cell.
The flagellum-bearing anterior end beat violently and
incessantly, whereby the trypanosome penetrated further
and further into the cell. After I had watched this
spectacle for about five minutes the trypanosome, which
had so far penetrated into the cell as far as the middle of
its body, shot suddenly into the cell and stirred up the
granular cell-contents by its lively movements. Since, how-
ever, the cell was torn on the opposite side, the trypano-
some soon shot out of the cell again.” Néller thus confirms
the suggestions we made in our preliminary report (1911)
with regard to the probable method in which the penetration
of the cell is affected.
We have frequently seen trypanosomes, not distinguish-
able in the living state from the ordinary type, singly within
cells; the first time we ever discovered the trypanosomes
within the cells was just such a case, a single trypanosome of
quite ordinary appearance, wriggling and squirming actively
in the cytoplasm of an epithelial cell, in a flea which had
526 BE. A. MINCHIN AND J. D. THOMSON.
been fed twelve hours previously on an infected rat. Careful
examination of the cell, at different foci of the microscope,
convinced us, greatly to our astonishment, that the parasite
was really within the cell and not above or below it. ‘This,
and other observations, repeated subsequently upon trypano-
somes of ordinary appearance, contained singly within epithe-
lial cells, suggest that the trypanosomes in each such case
had but recently penetrated into the cell; but the observation
might also be interpreted to mean that the trypanosome seen
was the last of a batch produced by multiple fission within
the cell from which its sister-trypanosomes had already
escaped. In the latter case, however, the trypanosome
would probably be within a vacnole, as will be described
presently. ;
Observation of the free trypanosomes in the living state
shows that, as already stated, they are extremely active, but
have a great tendency to attach themselves by the tip of the
flagellum to firm objects; to the wall of the stomach, to
pieces of débris, even to the glass surface of the slide or
coverslip when under observation. The study of sections of
the stomach confirms this observation in an unmistakable
manner; many trypanosomes of the long, stiff type are seen
in the sections attached to the epithelial cells by their
flagella. The attachment is not, as a rule, to the outer pro-
jecting ends of the cells, but to their sides; the trypanosomes
put their long flagella down between the epithelial cells and
often adhere to the cell close to its base; it would appear as
if the side of the cell, at least in its columnar form, is its
vulnerable region. A still more striking point is that many
of these trypanosomes attached to, but still quite outside the
cell, have already assumed the recurved form. These obser-
vations make it very probable that in some cases the trypano-
some may first attach itself to the cell by its flagellum and
then bore its way into the cytoplasm in some way.
Several trypanosomes may penetrate independently into
one and the same cell. We have frequently observed
numbers of the parasites, from five or six up to a dozen or
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 527
more, in different stages of multiplication, side by side in a
cell both in the living condition and in sections (Pl. 38,
figs. 112, 118; Pl. 39, fig. 130). The parasites lie in the
cytoplasm usually in a distinct vacuole, produced, apparently,
by the liquefactive action of the parasite on the cytoplasm of
the host-cell. The trypanosomes are nearly always in a state
of movement, a point to which we shall return again. It is
not infrequent to observe a number of trypanosomes in the
same vacuole, wriggling actively one over the other.
The infected cell may become reduced simply to a bag
containing fluid in which large numbers of trypanosomes,
generally with their multiplication completed or far advanced,
move actively. Such cells are found commonly in sections
(Text-tig. 3); they are generally thrown off from the epi-
thelium and lie quite free in the blood-débris, sometimes
even in the centre of the lumen of the stomach; nothing
remains of the cell-contents except a thin superficial layer
of cytoplasm, under the cell-membrane, and the nucleus,
adherent usually to the wall at some point. This condition
obviously represents the last stages of the exhaustion and
death of the cell, from which the trypanosomes will escape
either by their own activity or by disintegration of the cell.
We shall consider the effects produced by the parasites on
the cells in more detail subsequently ; at present it will be
more convenient to confine our attention to the development
of the trypanosomes themselves.
The study of the trypanosomes in the living cells, checked
by the examination of preserved material, permits readily
enough of the recognition of a number of well-marked stages
in the process of multiplication :
(1) Trypanosomes of quite ordinary appearance, which
have apparently but recently penetrated into the cell, as
already described.
(2) Pear-shaped forms, with the flagellum continuing the
stalk of the pear; the body of the pear is distinctly flattened,
and therefore presents a contour which differs according as
it is seen from the edge or from the flattened surface ; as the
voL. 60, PART 4,—NEW SERIES. 37
528 E. A. MINCHIN AND J. D. THOMSON.
parasite is in constant motion within the cell it presents con-
tinually different views to the observer. The body of the
parasite also shows in life incessant “metabolic” changes of
form, movements of an active protoplasmic body imperfectly
restrained by the thin, vielding envelope or periplast. It is
very easy to seein the living condition that these pear-shaped
forms are produced by the body of the trypanosome being
doubled upon itself, an interpretation confirmed by the
examination of preserved specimens. When the stomach is
teased up and examined fresh, many of the recurved pear-
shaped forms are found swimming freely, with the flagellum
forward, in the salt-citrate solution used in the dissection.
If such a form be watched attentively, it is often seen to
uncurl itself, straightening out the body and thus passing
from the pear-shaped form to that of an ordinary trypano-
some. In some cases a trypanosome which had been seen to
unbend itself in this manner can be observed to curl up
again, while swimming freely, and thus to assume or to lose
the pear-shaped form several times in succession.
In the fixed and stained preparations it is seen that the |
trypanosome is bent upon itself in such a way that the
posterior part of the body, containing the kinetonucleus, is
closely applied to the anterior half of the body. ‘Thus a pear- |
shaped body results in which N is lodged in the thickest part
of the pear, at (Pl. 36, fig. 15) or near (fig. 13) the blunt end
of the body; while is usually well in front of N, that is to
say, nearer to the pointed end of the body (PI. 37, fig. 70).
In some cases, however, 1 is close beside N (figs. 13-17), or
even, exceptionally, behind WN (fig. 16). The variations in
the positions of 7 and N are easily explained by their
variability in this respect, already described, in the free
trypanosomes, on the one hand, and on the other by varia-
tions in the exact region of the body at which the bending
takes place. As a general rule, the body appears to be bent
between 7 and N, so that N lies a little way from the extreme
posterior end and m in front of it (figs. 13, 17); but the point
of greatest curvature may be in the region of N, which is
THE RAT-TRYPANOSOME, I'RYPANOSOMA ILEWISI. 5929
then at the hindermost extremity of the body (fig. 15), or
even in the region of n, which is then at the extreme blunt
end (fig. 16). In exceptional cases the bending takes place
in front of N. In all cases the flagellum runs backwards
along one side of the body, round the blunt posterior end, and
forward, for a variable distance, to the basal granule or true
blepharoplast situated close beside ». Thus the course of
the flagellum, as a whole, may be compared to the letter U
modified by making one arm of the letter much longer than
the other.
In some cases the distinction between the two limbs of the
recurved body can be seen plainly in the fixed specimens
(Pl. 36, fig. 15; Pl. 37, fig. 70), but in other cases no line of
demarcation can be made out, and the applied portions of the
body appear to have formed completely into a compact pear-
shaped mass, leading on to the stage next to be described.
The recurved forms differ remarkably in size, and from
a comparison of these forms with one another, with the free
trypanosomes, and with later stages of the intracellular
development, there can be no doubt that the initial stages of
the lite of the trypanosomes within the cells is accompanied
by a pronounced diminution in the size of the flagellates (see
especially Pl. 36, figs. 13-17, and compare them with figs. 1-10
of free trypanosomes, and figs. 21-34 of later stages, on the
same plate). How this shrinkage takes place it is difficult to
say; probably the cytoplasm of the flagellate gives up a
large amount of watery fluid and so diminishes in bulk,
while becoming at the same time correspondingly denser in
texture, a change which would account for the intensity with
which the intracellular forms take up the stain and the
consequent opacity which they acquire, as already noted. It
is necessary, however, to exercise caution in estimating the
size of the forms in preparations, since there is no doubt that
they vary owing to differences in fixation. Thus, Pl. 36,
fig. 14 shows a specimen from the same slide as fig. 13, but
the former is from a part of the film which appeared to have
dried before it was-exposed to the action of the osmic vapour ;
530 E. A. MINCHIN AND J. D. THOMSON.
its large size, light colour and the elongation of N are all
indications that the soft body had become flattened out by
being dried before fixation. ‘The specimens may also become
deformed in other ways; Pl. 37, fig. 72 is probably to be
explained as representing a recurved form in which the
flagellum has become torn away from the side of the body
and so projects freely from the rounded posterior end.
We were at first under the impression that the pear-shaped,
recurved trypanosomes found free in teased-up stomachs
examined fresh were forms that had been originally intra-
cellular and had been set free by rupture of their host-cells.
As stated above, however, examination of sections proved
that these recurved forms may be extracellular in occurrence,
attached to the epithelial cells or even free from them. It is
evident, therefore, that the recurved form is not simply an
adaptation to life within the confined limits of the cell.
(3) Forms with rounded or oval body, derived from the
pear-shaped recurved forms by a further contraction and roll-
ing up of the body ; these are the forms which we described
in our preliminary account as ‘ block-like” since the body
often shows during life irregular contours, changing con-
tinually owing to the active metabolic movement. ‘The
flagellum, which runs in a U-shaped course in the recurved
forms, acquires now an additional bend (compare especially
Text-fig. 23, p. 655, hand 7; it usually rans round the outercon-
tour of the rounded body and protrudes from it to a variable
extent. In some cases a very considerable length of the
flagellum is free (Pl. 37, figs. 77, 78), in other cases a very little
(Pl. 36, fig. 28; Pl. 37, fig. 82), while in other cases again the
flagellum is simply wrapped closely round the body (Pl. 36,
figs. 29, 30, 35; Pl. 37, figs. 81, 84). The extreme length of
the free flagellum seen in Pl. 37, fig. 76 is possibly due in
part toits having become artificially detached from the body
in the process of making the smear.
The data in the foregoing paragraph have been obtained
chiefly from the study of preserved specimens. In the living
condition this stage appears as a small, rounded or oval body
HE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI, 531
within the cell, usually in motion and showing a distinct
flagellum. Sometimes, however, these bodies are quite
motionless with no flagellum visible. In our preliminary
communication (1911), we were unable to decide whether
a flagellum was always present, and were prepared to admit
that in some cases this stage might be a non-flagellated
leishmania-like form. We had observed in one case that a
body which had been for some time quiet and motionless
within the host-cell became suddenly active, showing a dis-
tinct flagellum. In all our permanent preparations, however,
whenever flagella can be made out in the other stages or in
the free trypanosomes, they can be seen to be invariably
present at this stage also, and there can be no doubt that the
motionless forms are those in which the flagellum is wrapped
round the body. We are now convinced that non-flagellated
leishmanial forms do not occur. We have the impression
that the rolled-up trypanosome can wrap its flagellum round
the body and pass into a resting, quiescent condition for
a time, after which it can become active again by un-
curling and setting free its flagellum, or at least a certain
length of it, probably never quite the whole length.
The body in this stage, as in the last, is actively metabolic,
with constantly changing contours in life. When the rounded
forms are set free by rupture of the host-cell they swim
actively in the liquid, progressing with the flagellum directed
forwards; they then resemble ordinary flagellate monads,
and the observer might easily have the impression that he
was watching some intruding flagellate derived from contami-
nation of the salt-solution or from some extraneous source.
In a few rare instances the rolled-up forms have been
found in preparations to exhibit a central perforation or
fenestration (Pl. 36, figs. 24, 30, 34), evidently produced by
the trypanosome curling itself round so as to leave a central
space. ‘his condition, when it occurs, is probably quite
transitory, the plastic cell-body of the trypanosome fusing
into a compact lump sooner or later.
It seems probable that some of the rolled-up forms degene-
532 E. A. MINCHIN AND J. D. THOMSON.
rate at this stage and are absorbed; that is, at least, the
only explanation we are able to offer for such minute forms
as those shown in Pl. 36, figs. 43-45, which appear to be
undergoing degeneration.
The smallest of the rounded forms have each a single kine-
tonucleus, trophonucleus and flagellum. Now they begin to
grow in size, with concomitant multiplication of their nuclei
and formation of daughter-flagella. The division of these
various parts appears to go on much as in other trypano-
somes, independently, but more or less synchronously. The
division of » may be slightly in advance of that of N, or
slightly after it: thus, stages are found in which m and N
appear to be both in the same stage of division (PI. 37, fig. 79) ;
or in which N appears to be in advance of m (PI. 36, figs. 28
and 37) ; or with two distinct nv and N still in division (PI. 37,
figs. 80, 82) ; or finally with n and N both completely divided
(Pl. 36, figs. 33, 36). The division of 1 is dependent on, or
connected with, that of the basal granule or true blepharo-
plast, which may be regarded as representing the centriole
or division-centre for x. The original flagellum does not
divide, however, but remains attached to one of the daughter-
blepharoplasts, from which it arises in close proximity to one
of the daughter nn; and from the other blepharoplast a new
flagellum grows out, at first a very fine and delicate structure
and consequently very difficult to make out clearly or with
certainty in the opaque body. In many cases in which 7 is
divided completely no second flagellum can be seen, but it
would not be safe, in view of the difficulties of technique
presented by these objects, to conclude in all such cases that
the formation of the new flagellum had not begun, since in
other cases a very delicate line can be seen plainly growing
out from the daughter-blepharoplast—that is to say, from
the blepharoplast other than that from which the original
flagellum arises (P]. 37, figs. 79-81). As a rule the daughter-
flagella can be made out in the preparations stained with
iron-hematoxylin, but not in those stained by Giemsa’s
method ; in the latter case, as already mentioned, the body
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 533
usually stains so intensely as to obscure completely the
delicate daughter-flagella, which are imbedded in the mass
of the cytoplasm, while the original flagellum runs for the
most part on the exterior of the body; and if the stain be
extracted sufficiently to make the body clear, it comes out of
the growing flagella and leaves them invisible.
We may infer, therefore, that as the division of the nuclei
proceeds the formation and growth of new flagella follow
hard upon the division of the blepharoplast and kinetonucleus,
and that a new flagellum grows out from each blepharoplast,
in close proximity to 7, quite independently of the original or
parent flagellum, which remains unaltered.
As a general rule the multiplication of the nuclei begins at
the rolled-up stage, but this rule is by no means invariable.
Sometimes the nuclei are found to have multiplied even
before the trypanosome has taken on the recurved form
(Pl. 36, figs. 18-20). Such forms might possibly be specimens
which, after becoming recurved, have straightened themselves
out again, but their appearance is that of trypanosomes in
which nuclear multiplication has begun before change of
body-form. Attention must also be drawn to the peculiar
elongated forms with 2 mn and 2 NN, such as Pl. 36, figs.
21-23; Pl. 37, fig. 74. At one time we were inclined to sus-
pect that these forms, and also the unaltered trypanosomes
with 2 nn and 2 NN, might be examples of fusion instead of
multiplication (see below, p. 604) ; but we could find no definite
evidence of there being fusions of two trypanosomes, and the
fact that forms occur with 3 nn and 3 NN (fig. 18, pl. 36)
makes it more probable that they are early stages of multi-
plication. On the other hand the possibility cannot be
excluded that in some cases accidental and purely plasto-
gamic (non-sexual) fusion of intracellular stages may occur,
and that such fusions may explain the enormous size of some
of the spheres and later stages of multiplication (Pl. 36,
fig. 42).
The multiplication of the nuclei proceeds apace, and the
duplication of 7 and N is followed by a stage in which the
534 EK. A. MINCHIN AND J. D. THOMSON.
body contains 3 nn and 3 NN (PI. 36, fig.40!; Pl. 37, fig. 84).
This stage, which is of common occurrence, indicates that
after the first division of 2 and N one of the daughter-nuclei
in each case remains undivided, while the other divides again.
This interpretation is supported by fig. 39, showing a specimen
containing 3 nn and 2 NN, one of the latter being in process
of division. roe
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5658 E. A. MINCHIN AND J. D. THOMSON.
(15) From a batch preserved in sublimate-acetic. A large stomach
which passes through 241 sections without quite reaching pylorus.
About 10 extracellular trypanosomes, most of them attached to the
epithelium, are found in the hindermost sections.
No intracellular trypanosomes are to be found.
Interpretation.—A stomach in which the intracellular multi-
plication is either completed or almost inhibited.
(8) The Migration to the Rectum.
As stated above, the trypanosomes occurring in the intestine
are usually in transit from the stomach to the rectum, and only
exceptionally attach themselves to the intestinal wall or
undergo further development there. We have been able on
more than one occasion to observe the actual passage of the
trypanosomes along the intestine. In a flea which had fed
on an infected rat about twenty-four hours previously, the
stomach, with a considerable length of the intestine attached,
was dissected out on a slide partly teased up and covered
with a glass slip; the posterior part of the stomach, however,
with the intestine attached, was left intact. Close to the
pylorus the stomach contained fluid in which a great number
of brown granules were suspended, coarse granules of fecal
appearance evidently representing indigestible residue of the
last meal. These granules could be seen to be in a state of
violent commotion, more violent than could be explained
merely by Brownian movement, since they were being churned
and stirred in every direction; but although there could be
little doubt but that the movements were due to the activity
of trypanosomes, the flagellates themselves could not be
seen clearly in the opaque fluid through the stomach-wall.
Soon, however, a bolus of fluid passed through the pylorus
into the intestine and passed down it by peristaltic action,
pushed onwards like a bead, until it reached the cut end of
the intestine and was extruded from it. It was then seen at
once that the fluid contained, in addition to the fecal granules,
a swarm of excessively active trypanosomes, long and relatively
slender forms with great powers of rapid forward progression.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 569
They began at once to spread in all directions in the fluid ;
but at this moment the coverslip was picked off with needles
and dropped instantly into Schaudinn’s fluid; after it had
been stained and mounted it was found that, by a piece of
good luck, the stomach and intestine had remained sticking
to the coverslip, and that round the cut end of the intestine
were several trypanosomes of the long stomach-type (Pl. 42,
figs. 203, 204), evidently some of those that had been seen to
pass down the intestine. In two other fleas, fed respectively
twenty-four and eighteen hours previously on infected rats,
we were able to confirm our observations on the passage of
the trypanosomes down the intestine and to obtain prepara-
tions of them (Pl. 37, fig. 50).
It is unnecessary to give a detailed description of the
migratory trypanosomes, since itis evident from the figures that
they are simply of the long stomach-type already described.
They are very active, and in form crithidiomorphic, with the
hinder part of the body stiff and straight, sometimes slightly
or even markedly clubbed and swollen. The two nuclei are
more or less closely approximated, but 71s almost always well
behind N. It is seen from this that the trypanosomes result-
ing from the intracellular multiplication in the stomach may
do one of two things ; they may penetrate again into epithelial
cells and go through another generation of multiplication ;
or they may collect in the pyloric region and be carried down
the intestine (compare Pl. 45). The migration may begin as
has been seen, as early as eighteen hours, but this appears
to be rather exceptionally early, to judge from our observa-
tions on the rectal phase; probably it does not usually begin
till twenty-four or thirty-six hours. It continues, doubtless, as
long as the stomach-phase lasts, and as already stated, we have
found intracellular stages as late as five days in fleas not fed
a second time; we may suppose, therefore, that the produc-
tion of the migratory forms and their passage down to the
rectum, may be going on continually, in some cases, until the
second feed of the flea. In other cases, however, the multi-
plication in the stomach probably comes to an end of itself,
570 E. A. MINCHIN AND J. D. THOMSON.
before the second feed, judging by the many observed
instances in which, in fleas not fed a second time, the stomach
may contain many long free trypanosomes, or the rectal
phase may be weli established, without any intracellular
multiplication occurring in the stomach.
(c) The Rectal Phase.
The starting point of the rectal phase is the long, active
“‘ crithidiomorphic” type of trypanosomes already described,
which migrates down the intestine. During its passage down
the intestine the changes of form and structure which may
have begun already in the stomach continue, so that by the
time it reaches the rectum its posterior end is generally
distinctly club-shaped. Arrived in the rectum, it very soon
undergoes changes in form, habits and structure, and multi-
plies by binary fission, giving rise ultimately to the typical
forms of the established rectal phase, forms which, apart from
other characters, are of much smaller size and bulk than
those of the stomach-phase. We will discuss first (a) the
transition from the initial to the established forms of the
rectal phase, and then (b) the various types of modifications
of the latter, culminating in the little trypanosome-form,
which is the final stage of the development of the flea.
(a) The Transition to the Crithidial Form.
If the various processes of change in the initial stages of
the rectal phase be analyzed, after study of both living and
preserved material, and by comparing the starting point of
this part of the development with its final result, we may note
the following tendencies in the organism.
In the first place it loses its intense activity and becomes
more sluggish in movement, with a great tendency to attach
itself by the tip of the flagellum ; under natural conditions
the flagellate attaches itself to the wall of the hind-gut, but
when under microscopic examination it can be seen to adhere
THE RAL-TRYPANOSOME, TRYPANOSOMA LEWISI. 571
firmly to pieces of débris of any kind, or to the surface of
the glass slide or coverslip. When not attached in this way
it progresses slowly with the flagellum directed forwards.
Secondly, the body shortens and changes in form by the
cytoplasm becoming concentrated towards the posterior end
of the body.
Thirdly, the flagellum becomes progressively shortened.
Fourthly, the two nuclei, if still in their original positions,
become transposed into the typical crithidial arrangement,
with 2 close beside or in front of N,
Fifthly, the nuclei, the flagellum, and finally the whole
body are multiplied by division or reduplication.
The order in which these different 'processes of change
have just been stated is in no way to be taken as indicating
their chronological order of succession in the development,
since they take place more or less independently and, as it
were, at different rates of acceleration in different indi-
viduals ; the result is consequently the production of a great
number of forms which at first are rather bewildering and diffi-
cult to arrange in a series. The difficulty of tracing in detail
the transition from the initial stage of the rectal phase to the
established crithidial condition is increased by the fact that
the early transitional stages are extraordinarily rare and diffi-
cult to find in the permanent preparations; a fact which
indicates that the transition takes place very rapidly and is
completed very quickly. One explanation of this rapid
change may perhaps be found in the great diminution in size
which is brought about in this part of the development.
Leaving out of consideration for the moment any structural
or other changes, it is at least quite clear that the
large individuals which come down from the stomach
initiate a series of generations of multiplication by fission
culminating in forms perhaps not more than a tenth the size
of the initial forms from which they are derived. Conse-
quently it is probable that the successive divisions of the
body follow one another at first with extreme rapidity and
without intervening pauses to allow the daughter-individuals.
572 E. A. MINCHIN AND J. D. THOMSON.
to grow to the size of the parent, as would happen in the
normal multiplication such as takes place in the established
crithidial stage. Another explanation for the rarity of the
transition-stages may be the possibility that of the long
trypanosome-forms which come down from the stomach to the
rectum, only a small number may go through their metamor-
phosis into the typical rectal phase and the greater number
may degenerate or be carried out of the flea. Whether this be
true or not, it is not necessary to suppose that all those which
pass down from the stomach do so in a single swarm; it is
more probable that they dribble down from the stomach, so
to speak, in larger or smaller bands or singly, a supposition
which would also account for the small number found at any
one time in the rectum undergoing their metamorphosis.
A further difficulty which may arise in distinguishing the
forms of the transition from trypanosome to erithidia, and in
assigning them to their proper position in the series, is the
fact that the body may become artificially broadened in pre-
parations which have been allowed inadvertently to dry up
the least bit before fixation. Deformed specimens of this kind
can berecognized by their flattened appearance and consequent
even staining of the body, which in a properly preserved
specimen should be thicker and more opaque in the axial
region than towards the edges of the body; the trophonucleus
in flattened specimens is often transversely elongated ; and,
further, the process of drying seldom affects a single
specimen, unless it is very isolated or near the edge of the
film, but, if it has taken place at all, the effects of desiccation
are apparent over at least a considerable area of the slide or
coverslip. It is, therefore, not difficult with a little practice
to detect the specimens which have become broadened arti-
ficially ; and in any case the process of drying does not affect
the length of the body or flagellum to any appreciable extent.
It is a result, doubtless, of the rapidity with which the
transition is effected that we have not been able to come to a
perfect agreement of opinion between ourselves as to certain
points of the development during this transitional period,
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 573
namely, the exact stage in the process of change of form at
which the first division of the initial rectal form (that is to
say, the long club-shaped forms that. come down from the
stomach) takes place, and consequently the type of binary
division, whether equal or unequal, which initiates the
whole series of generations in the rectal phase. Before
we discuss this doubtful point, we may first classify the
various types seen in the initial transitional stages of the
rectal phase, for which it is simplest to take the types of
body-form as the basis of classification. Bearing in mind the
considerations of technique that have been discussed already,
and being careful, therefore, to eliminate all cases where
there is reason to suspect artificial deformation of the
specimens, we can recognise the following series of forms,
each of which is a stage in the progressive shortening and
broadening of the body by concentration of the cytoplasmic
substance in the posterior third of the original slender
trypanosome :
(a) Slender forms, differing but little from the long
stomach type, and evidently but recently arrived in the rectum.
The flagellum is still long, and N is usually in front of n
(Pl. 42, fig. 208) ; sometimes, however, the reverse is the case
(PI. 42, fig. 209).
(b) Forms in which the hinder region of the body, contain-
ing the two nuclei, become swollen and club-shaped (Pl. 42,
fig. 206), and the anterior part correspondingly attenuated,
until the body as a whole becomes more or less tadpole-
shaped (Pl. 41, fig. 150; Pl. 42, figs. 205, 207). The flagellum
at this stage is usually long, but sometimes distinctly
shortened (PI. 41, fig. 158; Pl. 42, fig. 210).! The nuclei are
usually still in their original relative positions, but are some-
times crithidial in arrangement (PI. 42, fig. 211).
(c) Forms in which the concentration of the body-substance
' How the shortening of the flagellum takes place is not clear, but it
is worthy of note that in smears there are often found broken pieces of
flagella near the specimens, as if the flagellum had become brittle and
had broken off (PI. 41, figs. 158, 160).
574 EK. A. MINCHIN AND J. D. THOMSON.
in the posterior half or third of the body has proceeded so far
that the body is nearly half as broad as long, and the anterior
prolongation which forms the undulating membrane is much
reduced in length and very slender. Examples of transitions
from the last stage to this are seen in Pl. 41, figs. 151, 152;
TExt-FIG. 13.
Diagram to show the possible modes of transition from the
stomach-phase to the rectal (crithidial) phase: a, slender form
newly arrived from the stomach (compare PI. 42, fig. 208, ete.) ;
b, early club-shaped form (compare PI. 42, fig. 205, etc.) ; ¢, later
(more swollen) club-shaped form (compare PI. 41, fig. 149) ;
ec, unequal division of ¢ (hypothetical); d, large pear-shaped
form (compare PI. 41, figs. 154, 155, etc.) ; dd, division of d (com-
pare Pl. 41, fig. 179); e, forms resulting from the initial division
of the rectal phase (compare PI. 41, figs. 153, 157, 164, ete.). (x
about 2000.)
the complete realisation of this stage is seen in PI. 41, figs. 149,
154,162; Pl. 42, fig. 212. From a comparison of the figures
it is seen that the position of the nuclei varies considerably ;
n may still be at or near the hinder end (PI. 41, fig. 162), or
may be close beside N (PI. 41, figs. 149, 154; Pl. 42, fio. 212).
The hinder end may be pointed, or quite round. The
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 575
flagellum may still be a fair length, or quite short (Pl. 41,
fig. 162).
(d) Forms in which the body is contracted into a pear-
shaped mass, nearly as broad as long. The stalk of the pear
is formed by the flagellum together with a slight projection
of the body representing the anterior prolongation, now
usually reduced to its smallest limits (Pl. 41, figs. 155, 156,
161; Pl. 42, fig. 215). The nuclei are usually transposed, 1
being beside or in front of N. The length of the flagellum
varies within wide limits represented by PI. 41, figs. 155 and
156.
(e) Forms which are distinguished from the preceding stage
chiefly by their smaller size. Here again, however, caution
is necessary in referring a given specimen to its place in the
series, since the apparent size in permanent preparations may
be affected considerably by technique. In the preparations
fixed on the slide with osmic vapour and stained by Giemsa’s
method the trypanosomes always appear considerably larger
than those on the coverslips fixed wet with sublimate
solutions, stained with hematoxylin and mounted without
drying in canada balsam (vide Minchin, 1909). Conse-
quently, different standards of sizes are required for inter-
preting preparations made by these two different methods of
procedure, and preparations made by the one method must
not be compared directly, without making due allowance for
the differences in result, with those made out by the other
method. Nevertheless, after giving due weight to these
considerations, it is not difficult to distinguish forms which
are about half the bulk of the stages already described
(Pl. 41, figs. 153, 159, 160, 164; Pl. 42, fig. 218). Such forms
are, without doubt, individuals derived from at least one,
possibly more than one, division of the initial form, a conclu-
sion supported by the occurrence of such forms in pairs,
possibly as the result of division recently completed (PI. 41,
fig. 157; Pl. 42, fig. 216a).
The question which must remain open at present is, in
which of the four stages of changes of form (a), (b), (c), or
vou. 60, PART 4, —NEW SERIES. 40
576 E.' A. MINCHIN AND J. D. THOMSON.
(d) (see Text-fig. 15), does the initial division of the rectal
phase take place?
If, as is possible, and as one of us (J. D. T.) thinks probable,
the first division takes place in the club-shaped form as soon
as the concentration of the protoplasm is completed and
when (as in Text-fig. 15, c) the two nuclei lie close together,
it may well be the case that the products of division would
then be markedly unequal in appearance owing to the reten-
tion of the old flagellum by one of the two daughter-indi-
viduals; that is to say, the division would be of the type
found in similar club-shaped forms in the trypanosome of the
gold-fish both in cultures and in the leech, and in bird-
trypanosomes both in cultures and in the mosquito, and which
has also been seen in an early culture of I’. lewisi itself.
On this view the swollen, pear-shaped forms of stage d would
have to be interpreted as forms subsequent to, and the pro-
ducts of, the initial division. In spite of much searching,
however, through our preparations, we have been unable to
find actual examples of club-shaped forms showing division
markedly unequal in appearance.
If, on the other hand, as one of us (EH. A. M.) believes, it is
the most usual state of things for the organism to continue
the process of contraction and broadening of the body until
it has reached the condition of stage d, it is probable that
the flagellate would then divide by the type of binary fission
characteristic of the subsequent generations of the crithidial
phase, that is to say, producing two daughter-individuals that
are equal, or not markedly unequal in bulk, and differing
only in that one of them has the flagellum temporarily shorter
than that of the other. Pl. 41, figs. 179, 180, and Pl. 42,
fig. 216, may then be interpreted as examples of the initial
division, and Pl. 41, fig. 157 and Pl. 42, fig. 2164, as pairs
resulting from the initial division recently completed.
- The problem of the initial division of the trypanosomes in the rectum
is one which involves more than the question as to the type of form,
club-shaped or pear-shaped, in which the division takes place, or the
question whether the products of division differ in visible characters of
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 577
bulk, structure, or appearance; it raises a much deeper and more
fundamental problem, namely, whether the division which initiates the
rectal phase is an equating division which gives rise to two equi-
potential daughter-individuals or a differentiating division which pro-
duces inequipotential forms. If the division-products are equipotential,
then visible differences between them of any kind would be merely
temporary and of no significance for the future behaviour and destiny
of the sister-individuals, which would be true twins ; all such differences,
however pronounced, would be immaterial for the development as a
whole, and the same would apply to the parent individual, whether club-
shaped or pear-shaped. If, on the other hand, the daughter-individuals
are inequipotential, in the sense that the smaller of the two division-
products becomes the starting point of an indefinite number of genera-
tions of small crithidial individuals, while the larger is merely a “ parent ”
which, though it may divide in the same manner to produce small
crithidial forms several times in succession, does not ultimately develop
further, but drops out, as it were, of the direct line of the development
when its powers of reproduction are exhausted; if they are inequi-
potential in this sense, it follows that the observed difference between the
two daughter-individuals would have an important significance and that
they would not then be true twins, and further, that the club-shaped form,
assuming that this is the form in which division takes place, would then
be a developmental form of special and peculiar significance in the life-
cycle and not merely a stage in the change of form leading to the pear-
shaped stage. We must be content, unfortunately, with enunciating
these possibilities, without being in a position to decide between them ;
owing to the fact, to which reference has been made above, that the
extreme rarity of transitional forms in our preparations has supplied us
with insufficient material for a decisive judgment.
The type of the initial division and the exact point at which
it occurs in the series of progressive form-changes of the
rectal phase must be left at present an open question, unfor-
tunately ; but this much may be stated positively about the
process of division in the rectal phase in all cases, whether in
the initial or subsequent stages. No multiple division occurs
henceforth in the developmental cycle, but the parasites settle
down to a course of simple binary fission continued indefi-
nitely, and always taking place in the lumen of the gut, never
within cells. The process of fission is initiated by division of
the blepharoplast or basal granule of the flagellum, but the
flagellum itself does not divide; the original or parent
578 E. A. MINCHIN AND J. D. THOMSON.
flagellum remains attached to one of the two daughter-
blepharoplasts and a new flagellum grows out from the other
blepharoplast. The division of » follows hard upon that of
the blepharoplast, then N starts its division, and finally the
whole body divides ; of the two daughter-flagellates produced,
one has the original flagellum, the other has to grow a new
one, which it may not do, in some cases, until after complete
separation from its twin sister. In any case, one of the two
products of division has a much shorter flagellum than the
other, irrespective of any difference in bulk between the two.
As already stated, while these changes of form and pro-
cesses of multiplication are going on the flagellate is also
undergoing a change which, though a very small thing in
itself, produces nevertheless the characteristic morphological
distinction between the trypaniform and crithidial types;
namely, the approximation and final transposition of the two
nuclei, so that 2 comes to lie beside, or even well in front of,
N. The approximation of the two nuclei begins with the
first alteration and changes of form, but the exact point in
the development at which the change of position of the two
nuclei is completed is subject to great variation. Very
exceptionally, as has been described above, the transposition
of the nuclei may be complete in the stomach itself, but more
usually, it may be said normally, the change does not take
place until the flagellate reaches the rectum. Any of the
forms distinguished above as a, b, c, d or e, may be found
with the nuclei, but slightly or completely approximated, and
finally in the clumps of developmental forms such as that
represented in Pl. 41, figs. 182-184, evidently consisting of
forms which have completed several, at least two or three
generations of fission since arriving in the rectum, all stages
in the process of transposition of » or N are to be observed.
Since the small forms of the established rectal phase show
invariably the typical crithidial arrangement of the nuclei, all
that can be said is that the change in position of the nuclei
is effected earlier or later, but without fail, in the transition
from the stomach-phase to the rectal phase.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 579
The successive generations of the transitional rectal phase
cannot be distinguished with precision. ‘The size to which
the individuals are diminished after a given number of
generations is determined by two variable factors during
those generations, namely, the rate of individual growth and
the frequency with which multiplication takes place. All
that can be said is that the rectal forms diminish in size by
repeated division until they reach a minimum size which
is attained when growth and multiplication balance each other
more or less evenly; and that in the rectal infections of
recent origin the average size of the individual flagellates is
slightly larger, as a rule, than in the old-established infections.
(b) The Established Rectal Phase.
In its fully-established and definitive condition the rectal
phase consists chiefly of small crithidial individuals which
multiply by binary fission, attached to the wall of the gut
by their flagella or by the anterior (flagellar) extremity of
the body. The crithidial stage appears normally, if not
- invariably, to take origin in the rectum in the manner described
in the preceding section, and this part of the digestive
tract is its most usual and characteristic habitat ; consequently
we have made use of the term “rectal phase” for this part
of the development, in spite of the fact, presently to be dis-
cussed in more detail, that the crithidial and other forms of
this phase may be found not uncommonly in other parts of
the gut, more especially in the intestine close behind the
pylorus, but also in the stomach itself.
In one flea we have found the rectal phase well-established so
early as eighteen hours after the infective feed (Text-fig. 14),
and in another flea we found a few typical examples of this
phase at twenty-four hours, but both these examples are
abnormally early cases of the fully-developed crithidial phase,
which, in our experience, is seldom completely established
before thirty-six or forty-eight hours after the infective
feed.
580 E. A. MINCHIN AND J. D. THOMSON:
The extent to which the crithidial infection is developed in
different fleas varies greatly, from a swarming infection cover-
ing the wall of the rectum like a pile-carpet, especially in the
region behind the rectal papilla, to a condition in which a
few scanty flagellates are to be found only by careful search-
ing; in either case, however, the crithidial stage represents
Text-Fic. 14.
@)
saa
©
Mac
Uo.
Various forms from the rectum of a flea eighteen hours after the
infective feed, a, b, c and d are probably degenerative forms,
the rest developmental; e and f, early rectal forms, g—/, hapto-
monads (x 2000).
the permanent stock of the parasites in the flea, multiplying
continually and indefinitely and maintaining itself, in all
probability, as long as the flea lives. We have succeeded in
keeping a single flea alive for nearly three months, and during
that time seven rats were infected by it (see p. 640 below).
This permanent infectivity can only be explained by the
establishment of the crithidial stock in the rectum and its
continued multiplication. Each crithidial individual may do
one of two things : it may multiply by binary fission to produce
two crithidial forms (multiplicative phase); or it maybe
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 581
transformed gradually into small stumpy trypaniform indi-
viduals (final propagative phase). Hence the flagellates ot
the rectal stock may be classified, broadly-speaking, into
crithidial, transitional and trypaniform individuals.
The forms that occur in a well-established rectal infection
are very varied, and it is a somewhat difficult task to classity
them and to assign to each and every form its due place in
the developmental series. ‘l'o obtain a general notion of the
various types met with, it is best to examine fleas some eight
or nine days after they have fed once on an infected rat and
subsequently on clean rats, so as to obtain the rectal phase in
its typical condition, free from admixture of earlier develop-
mental or degenerative forms. Such fleas may have no
flagellates at all in their rectum or may present every grada-
tion between an extremely scanty and a swarming infection.
If a rectum containing numerous flagellates be examined
fresh after having been dissected out in salt-citrate solution,
opened up by tearing it with fine needles, and then covered
with a coverslip, the majority of the flagellates are seen
attached to the wall of the rectum, chiefly in the region
behind the rectal papilla. Many of them will have become
detached as the result of the dissection, and will be seen
floating about singly or adhering together in larger or smaller
clumps. If the preparation be sealed up carefully and
watched for some time many of those that lie about singly
will attach themselves to the glass of the slide or coverslip,
and it can be seen very clearly in all cases, whether in the
forms still attached to the rectal wall or in those set free in
clumps or singly, that the attachment is by the tip of the
flagellum or by the flagellar extremity of the body in those
that have no free flagellum. The flagellates may be attached
to the rectal wall in such numbers and so closely crowded
together that they resemble a furry lining or pile on it. Seen
in profile they appear in serried ranks, each in contact with
its neighbours; seen in surface view they present the appear-
ance of a honeycomb, each flagellate in optical transverse
section, showing outlines nearly polygonal as the result of
mutual pressure. In the living condition the attached
082 EK. A. MINCHIN AND J. D. THOMSON.
crithidias are motionless for the most part, but occasionally a
given individual performs a kind of jerky nutating move-
ment ; the body, remaining attached, sways rapidly from one
side to the other, with a slight curvature of the axis. After
bending, first to one side and then to the other, in this way
the animal remains quiescent for a time, but when there are
a large number of the flagellates, there is scarcely a moment
in which one or another, or several at once here and there,
are not performing these movements.
In addition to the attached forms, with the flagellum for
the most part very short or wanting altogether, there are
usually a certain number of free forms. Some of these are
crithidial in form, with the undulating membrane feebly
developed and scarcely, if at all, recognisable, and with a
distinct free flagellum, often quite long. The function of
these flagellated forms appears to be that of migrating in
order to colonize other parts of the wall of the gut. When
a clump of attached forms has been multiplying actively
at one spot it doubtless tends to become overcrowded, at
least in its central part. Then probably one of two things
may happen; some of the crithidias may become transformed
into the final trypanosome-form and detached from the clump;
or a certain number will develop flagella, remaining
crithidial in type, migrate to another part, and attach them-
selves again.!
Besides the crithidial forms with long flagellum there can
be seen free forms, also with a flagellum of variable length,
but with a distinct undulating membrane running along the
whole or greater part of the length of the body, which
appears more flexible and performs sinuous undulatory
movements more or less distinctly. These forms are the
ttle trypanosomes, or transitional stages in their develop-
ment, the forms which constitute the final infective stage of
‘One of us has seen in the living rectum a crithidia with a long
flagellum become detached from the rectal wall, swim actively across the
rectal cavity, with its flagellum directed forwards, and attach itself
again to the wall on the side opposite to its former attachment.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 583
the cycle in the flea (so-called ‘‘ metacyclical trypanosomes ”’
of Brumpt).
We can thus distinguish in the rectum of the flea three
principal types of individuals, each of which varies con-
siderably in size and details of form, and between which are
to be found every possible transition, namely :
(1) The attached or haptomonad! form, which is_ the
multiplicative stage of the rectal development.
(2) The free or nectomonad! form.
(3) The final trypanosome-form.
The many variations of these three types and the transi-
tions between them cause the rectal phase of the develop-
ment to present a variety of form which is at first very
bewildering, but which becomes easily intelligible if the
classification into the three types given above be used as
the key. It may be noted here that it is very common,
in the development of other trypanosomes, for the crithidial
phase to exhibit great variation in size, form, and structure.
Compare the works of Chagas on the development of
T. cruzi in the bug, and of Miss Robertson (1911, etc.) on the
development of the trypanosomes of fishes in leeches.
We will now proceed to describe the variations of these
types and the transitions between them in more detail.
(1) ‘he haptomonad or attached type is the phase in which
multiplication by binary fission takes place ; consequently its
variations of form are related mainly to the function of
multiplication. Leaving out the forms which are actually in
process of division, we find that chief variations in form are
seen: first, with regard to the shape of the body, whether
relatively slender, with the hinder end sharply or bluntly
' Woodcock (1914) has proposed the useful term “ haptomonad” for
the attached phase of a Crithidia or Leptomonas, the so-called
“ gregariniform”’ individuals of Léger, but of which the resemblance
to a gregarine is not very striking, not at least in the developmental
phases of Trypanosoma lewisi. To denote the locomotor crithidias
with long flagella we propose the term ‘‘nectomonad,” i. e. swimming
monad, as a correlative to haptomonad, fixed monad.
584. E. A. MINCHIN AND J. D. THOMSON.
pointed, or stouter, with rounded hinder end, or finally ovoid
or even globular in form ; secondly, with regard to the extent
to which the flagellum is developed.
The typical haptomonad, when not preparing for division,
has the body spindle-shaped or pear-shaped with the thickest
part anteriorly in front of the nuclei, and the hinder end
more or less sharply pointed (PI. 41, figs. 174, 176,193; Pl. 42,
fig. 252). The two nuclei are usually close together, situated
either about the middle of the body or nearer to the hinder
end; 2 is either just in front of N or close beside it. The
cytoplasm has a great tendency to stain very dark by any
method, especially towards the hinder end. After Giemsa
the body has a purplish-blue tinge ; after Twort’s combination
of neutral red and Lichtgriin it is seen to be full of very fine
granules, stained red and scattered irregularly (Pl. 38, figs.
260a-263a) ; from these reactions it is evident that the
opacity of the body is due to the deposition in the cytoplasm
of very fine “ chromatoid ” grains, probably of the nature of
volutin. The cytoplasm is usually free from coarse granula-
tions, which are, however, present occasionally.
In preparation for division the hinder end of the body
begins to swell and to become rounded at the hinder end,
while the nuclei are shifted more posteriorly. In consequence
the body becomes pear-shaped, but in the opposite manner to
that previously described, since now the thickest part of the
pear is the posterior end, while the anterior extremity of the
body is narrowed, with the flagellum representing, as it were,
the stalk of the pear (PI. 41, figs. 168, 188; Pl. 42, figs. 231,
247-249). In other cases the body becomes evenly ovoid or
even globular in form (PI. 42, figs. 243-247).
The process of division calls for no special remark (see
Pl. 42, figs. 218, 214, 226-2380, 249, 253, 266). The
blepharoplast or basal granule of the flagellum divides first,
one of the daughter-blepharoplasts retaining the old flagellum
attached to it, while the root of the new flagellum begins to
grow out from the other daughter-blepharoplast (PI. 42, fig.
:
o
5
252, lowest specimen). Following the blepharoplast, n divides
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 585
next and after that N. Next the body is constricted into
two, beginning from the anterior or flagellar end (Pl. 42,
fig. 253). We have never seen any but binary fission of the
crithidial forms ; multiple fission does not occur in any form
at this stage. We have also sought without success for
multiplication by endogenous budding (the so-called “infec-
tive granules” of Balfour and others). In Leptomonas
pattoni of the flea we have found very clear instances of
apparent endogenous budding (PI. 42, figs. 281, 282-284), and
Pl. 42, figs. 268, 269 are rather suggestive of a similar process
occurring in the haptomonads of T’. lewisi, but further proof
of it is lacking.
The flagellum in haptomonad forms is extremely short, and
as a rule is reduced practically to its intra-cellular root or
rhizoplast, the projection beyond the limits of the cell-body
being very slight or quite imperceptible. At the point where
the rhizoplast comes to the surface at the anterior apex of the
body there is usually a fairly large and distinct, but often ill-
defined patch of substance which stains like the flagellum,
that is to say, red, after Giemsa’s stain, black or greyish-
black after iron-hematoxylin, and green with 'l'wort’s stain,
and which appears to represent a secretion produced by the
flagellate, a sort of cement by which the animal adheres to
the wall of the rectum (PI. 41, figs. 174, 178; Pl. 42, figs. 223,
226, 243-245, 260, 262, etc.).
In many cases the rhizoplast fails to reach the surface of
the body, or may even, very exceptionally, be absent altogether
(Pl. 42, figs. 241-243, 267). ‘he body is then always ovoid or
globular in form. In such cases we have true leishmanial
forms, which appear to owe their origin to very rapid multipli-
cation of the ordinary haptomonad type; multiplication is so
rapid that one of the two daughter-individuals resulting from
binary fission has no time to form completely its new flagel-
lum or even the rhizoplast, before being split off from its
twin sister and beginning to divide again. This view receives
support from the extremely small size which is commonly a
feature of these leishmanial forms. In no case have we seen
586 E. A. MINCHIN AND J. D. THOMSON.
anything that could be interpreted as encystation or encapsu-
lation of the leishmanial forms; they appear to represent a
purely trophic and multiplicative phase.
The haptomonad forms occur usually, as has been said,
attached to the cuticle lining the rectum; in surface views of
the rectal wall, living or preserved, they may be seen attached
singly, in clumps, or in a continuous carpet-like layer, and
exactly the same is found in microtome-sections of preserved
recta (Pl. 44, fig. 317; Pl. 42, fig. 277). In sections of a well-
infected rectum the haptomonad forms are seen in a long,
continuous line, like soldiers on parade. But, both in living
and preserved recta, in film preparations or in sections, free
clumps of haptomonads are also found, in which the indi-
viduals all have their flagella directed pomand a certain point
(Pl. 42, fig. 274). In spite of careful scrutiny it is not possible
to detect any body or particle of débris at the centre to
which the monads are attached ; they appear to adhere simply
to one another by their flagellar extremities. The question
at once arises whether these free clumps are a natural or an
artificial condition. If they were only seen in teased-up recta
one could have hesitation in ascribing their detachment to the
manipulation, but they are found also in sections of recta.
It is, of course, impossible to dissect out and preserve a rectum
without subjecting it to great stresses and strains which might
detach the monads, but it is remarkable how tenaciously they
adhere to the wall. In one of our series of sections of a
rectum it can be seen that it has been badly torn in getting
it out ; part of the torn wall has curled right back and turned
inside out. ‘The tear goes right through an attached carpet
of crithidias which have nevertheless remained adherent to
the wall, even on the part that has curled back, giving at a
first glance the erroneous impression that the monads are
attached to the exterior of the rectum. They must, therefore,
be attached very firmly to the wall, which is intelligible when
it is recognised that the crithidial forms are not the ripe,
propagative stages of the cycle and that if they were carried
to the exterior with the faces they would be lost. For this
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 587
reason alone it seems highly probable that the free clumps of
crithidial forms represent either clumps artificially detached
by manipulation, or an abnormal condition of the flagellates
detrimental to their future welfare.
As transitions between the nectomonad and haptomonad
phase we would expect to find both stages of the development
of the nectomonad into the haptomonad, and stages of the
development in the reverse direction. It is of course almost
impossible to say, by simple inspection of a transitional form
in a permanent preparation, in which direction it is develop-
ing. We are inclined, however, to interpret as transitions
from the nectomonad to the haptomonad the more slender
forms, with short flagella and hinder ends pointed or but
slightly blunted, such as Pl. 41, figs. 175,176; Pl. 42, figs.
220, 252; and as transitions in the opposite directions the
rounded or broad pear-shaped forms with flagella of various
lengths, such as PI. 41, fig. 166, Pl. 42, figs. 231-234, 251, 265.
In some forms of the latter type the distal ends of the flagella
are very thin, much thinner and more delicate than the proxi-
mal portions (PI. 42, figs. 246, 250, 268, 269), and we interpret
this appearance as indicating that the flagellum is in process
of growth rather than of regression in length, for the reason
that a similar condition ‘is seen in the flagella of forms
transitional to the final trypanosome-type (PI. 42, figs. 255-
257), in which the flagellum is not likely to be in process of
shortening.
In the series which we interpret as transitional from the
haptomonad to the nectomonad type we find globular forms
with flagella of considerable length (Pl. 41, fig. 166; Pl. 42,
fig. 234), and, since we have not observed such forms
swimming freely in the rectum, we conclude that the hapto-
monad, while attached, first develops its flagellum to a
considerable length, and then acquires the elongated form
of body, before becoming detached fromthe wall and set free.
‘ Comparable, for example, to the sponge-larve, which attach them-
selves to the surface-film of the water instead of becoming fixed to a
firm object, and which in consequence perish inevitably—E. A. M,
588 E. A. MINCHIN AND J. D. THOMSON.
(2) The nectomonad or free type of crithidial flagellate has
a more slender body, in its fully-developed form about five
times or more as long as its greatest breadth, but with great
variations in its relative proportions (Pl. 41, figs. 190, 194-197 ;
Pl. 42, figs. 217, 235, 236, 254). The body is usually spindle-
shaped, pointed at both ends, its thickest part at the level of
or slightly behind, the middle point of its length, and 1m is
usually well in front of N. When it swims the flagellum,
directed forwards, is thrown into even sinuous undulations
which begin at the tip and run backwards, in contrast to the
type of movement so often seen in free-living flagellates, in
which the proximal two-thirds, or so, of the flagellum is held
stiff and straight, while the distal third performs lashing
movements which drag the body forward. We have not
found the fully-developed nectomonad type undergoing
multiplication by fission, unless Pl. 42, figs. 253 and 266 are
to be so interpreted.
(3) The final trypanosome-form appears to rise in most
cases from the haptomonad type, with which it is usually
found closely associated in preparations; compare Pl. 41,
fig. 202, of a section through the intestine close behind the
pylorus ; the trypanosomes are seen with their posterior ends
projecting above the level of the serried ranks of the hapto-
monad crithidias, as if they were pushed upwards by the
development and growth of their flagella. It is possible,
however, that the final forms may sometimes arise from the
nectomonad type, and that such an origin explains the
occurrence of the slender forms of the trypanosomes, the
stout forms being derived from the haptomonads. Forms
such as Pl. 42, fig. 287, are perhaps to be interpreted
as transitional from the nectomonad type to the final
trypanosome-form.
The essential feature in the origin of the final form from the
erithidial form, of whatever type, is the transposition of the
two nuclei, x, and N. Both nuclei move backwards usually, —
but N only for a short distance, while » passes N and goes to
the posterior extremity of the body (Pl. 42, figs, 238, 239,
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 9589
255-259, 270). In some cases, especially in the slender
forms, 2 stops short of the extreme posterior end of the
body (fig. 259), but in the stumpy forms » becomes quite
terminal in position, as a rule (Pl. 41, figs. 199, 200; Pl. 42,
figs, 259, 271). Further characteristic of the final stage is
the relatively large size of both n and N, and the faint stain
that N usually takes in the permanent preparations. In
many cases N appears distinctly elongated in the longitudinal
direction (figs. 199, 259). With the displacement backwards
of w and of the attachment and origin of the flagellum, the
undulating membrane becomes correspondingly extended and
lengthened.
The occurrence of stout and slender forms of the final
trypanosomes has been mentioned already, and was pointed
out by Swellengrebel and Strickland (1910) ; but it is a fact
somewhat difficult to explain. It may be, as already sug-
gested, that it is simply due to difference of origin, the
slender forms arising from the nectomonads, the stout forms
from the haptomonads. On the other hand, it may be that
the trypanosomes, when ingested by the rat, become exceed-
ingly active in order to find their way from the digestive
tract into the blood, and that the slender forms in the flea
represent merely the precocious assumption of a type of
structure which belongs strictly to a later period of the life-
cycle. These are the only suggestions we can offer at present
in explanation of the two forms.
We have never in any case seen the final trypanosome-
form dividing, but it is stated to do so by Swellengrebel and
Strickland (1910), who, after having examined one batch of
thirty-seven infected fleas, have been able to figure no less
than three examples of a process of division that we have
never been able to find in all the many hundreds of infected
fleas we have dissected and examined. For our part we
agree with Brumpt (1915), that these “ metacyclical trypano-
somes,” as he proposes to call them, are “ phases d’attente ”
which do not multiply further in the flea.
- With the development of the final trypanosome-form the
590 E. A. MINCHIN AND J. D. THOMSON.
cycle of T. lewisi inthe fleais ended. It only remains to say
a few words with regard to the occurrence of the rectal phase
in regions of the gut situated further forwards than the
rectum. It is by no means an infrequent occurrence to find
clumps and carpets of various forms characteristic of the
rectal phase attached in the intestine and even in the stomach.!
In the intestine they occur most frequently at the upper end,
close behind the pylorus. When they occur in the stomach
they are probably always attached towards its hinder end,
near the pylorus. Hence the two chief situations of the
crithidial forms, when occurring outside the rectum, may be
designated briefly ‘‘ pre-pyloric ” and ‘ post-pyloric.”
Two possibilities present themselves at once to the mind
with reference to these extra-rectal crithidial infections ; first,
that the infection of the stomach or intestine is a direct one,
brought about by forms which have attached themselves
there immediately after completing their stomach-phase,
without having ever travelled further back in the digestive
tract; secondly, that the infection has been brought about in
an indirect manner by forms which have migrated forwards
from the rectum.
So far as post-pyloric intestinal infections are concerned,
we have some evidence that the infection may be sometimes a
direct one; in one of our series of sections of the stomach of
a flea that had fed thirty-six hours previously to being pre-
served, there are two large clumps of crithidial forms attached
close behind the pylorus. Probably in such cases those
attached in the intestine represent but a small numerical
proportion of those that migrated backwards from the
stomach, the majority having passed on down to the rectum,
while a few have stuck, as it were, higher up.
With regard, however,to the pre-pyloric crithidial infections,
we have no evidence of direct infection taking place, but all
our data indicate that such infections of the stomach are
1 Since a certain length of intestine was usually cut off with the
stomach it is possible that many of the crithidial forms found by us in
our stomach-films were really post-pyloric in situation.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 591
brought about indirectly, and the same is probably true, in
most cases, of the post-pyloric infections of the intestine. In
the first place we have no record of the occurrence of crithi-
dial infections in the stomach (pre-pyloric) earlier than seven
days after the first infective feed of the fleas; but at later
periods than this we have so many records of such infections
in the stomach that, had they been in all cases brought about
directly, we should have expected to have found crithidial
forms in the stomach during the period when such forms are
being established, that is to say, from about thirty-six hours
and five days or so, which we have never done. Secondly,
the evidence furnished by experiment 39 (see below, p. 634),
indicates very strongly that the final infective forms of the
life-cycle were first produced in the rectum on the fifth day
and were there also on the seventh day, but had migrated
forwards to the stomach on the tenth day.
It seems, therefore, most probable that in the majority of
cases at least, the pre-pyloric and even the post-pyloric
infections are the secondary results of a migration forward
from the rectum of crithidial forms previously established
there ; and since neither the haptomonads nor the final
trypanosome-forms appear capable of undertaking such
migrations, it must be the nectomonads, which are obviously
active locomotor forms, that are responsible for such migra-
tion. We have performed some experiments from which it is
clear that the migration forwards is dependent on conditions
of nutrition in the flea and that starvation favours a forward
migration of the nectomonads towards the stomach.
In many cases the forwards migration of the flagellates
leads to the rectum being quite deserted by them. ‘This is
well shown by the following instance, by no means an isolated
one of its kind in our experience, but very typical. A flea
was taken from the infected breeding-cage and put by itself
on a clean rat for three days, from the 19th to the 22nd of
September; 1t was then recovered and dissected. The
stomach-preparations were found to contain a considerable
infection of the typical rectal phase (T'ext-fig. 17, p. 627),
vou. 60, PART 4,—NEW SERIES. Al
592 E. A. MINCHIN AND J. D. THOMSON.
but no flagellates of any kind were found inthe rectum. The
rat became infected, and first showed trypanosomes in its
blood on September 28th. The age of the infection of the
flea was not known, but the crithidial stock seems in this case
to have died out in the rectum and to have established itself
exclusively in the pyloric region.
We have also, though rarely, seen the attached crithidial
form in the proximal portions of the Malpighian tubules.
On the other hand we have never seen in our rat-fleas
(Ceratophyllus fasciatus) infections such as are
described by Noller (1912), and Wenyon (1913), in the
dog-flea, where both the rectum and the intestine are
described as being carpeted along their whole extent with
the crithidial phase; though we have seen such infections
in fleas harbouring the Leptomonas. It is a fact which
seems at first strange, but is probably very significant, that,
as we have pointed out elsewhere (p. 610), the rat-flea is not
so efficient a host for the rat-trypanosome as other species of
fleas which do not usually or of choice feed upon rats; from
which circumstance it would appear as if the rat-flea has
acquired a certain degree of natural immunity to the trypano-
some of the rat which other fleas do not possess.
For a general summary of the development of Trypano-
soma lewisi in the flea, see Plate 45, and the description
of it (p. 691).
(8) Tue DEGENERATIVE SERIES.
Trypanosomes undergoing degenerative changes may be
found in either the stomach or rectum during the first few
days after the flea has fed for the first time on an infected
rat. They are most abundant in batches of fleas examined
during the first twenty-four hours after feeding. After this
period trypanosomes may have disappeared altogether from
the gut of the flea, and after thirty-six hours degenerative
forms are of infrequent occurrence. In some cases, however,
degenerative forms may be found in the rectum much later
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 593
than the first day, namely, up to three, four, or even five days
after the infective feed. The degenerative forms of late
occurrence are probably to be interpreted as individuals
which have become degenerative after having developed
in a normal manner for a longer or shorter period. The
trypanosomes which begin to degenerate immediately after
being ingested by the flea probably do not last long beyond
twenty-four or thirty-six hours, usually not so long. Fleas
that have fed on an infected rat whose blood is swarming
with trypanosomes often show no trace of the parasites in
any part of the gut by twenty-four hours. The majority of
the degenerative trypanosomes that are found in the fleas
are those that begin to degenerate immediately after being
taken up from the rat.
There is no essential difference between the degenerative
forms found in the stomach and the rectum. We may, there-
fore, give a general description of the forms of the degenera-
tive series without taking special note of their provenance.
In direct contrast to the changes undergone by the develop-
mental forms in the stomach, the principal sign of degenera-
tion is a progressive diminution in size, more especially in the
length of the body. The trypanosome gradually dwindles
and wastes away, beginning at the flagellar end, during
which process the flagellum becomes converted progressively
into a fluffy mass, which frequently shows a tendency to stain
blue or bluish with the Giemsa stain, instead of the normal
red (Pl. 48, figs. 294-296, 308). Meanwhile N is pushed
backwards towards . The displacement of N does not
appear to be due to any active migration on its part, but to
be the purely passive consequence of the dwindling of the
anterior part of the body, whereby it is forced backwards. On
the other hand n shows no tendency whatever to move for-
wards, but may do one of two things: it may remain where it
is, or be shifted backwards only to a slight extent, in which
case the hinder end of the body retains the sharp point
characteristic of the trypanosome in the blood (PI. 43, fig. 301);
or it may pass back towards the extreme posterior end, and
594 E. A. MINCHIN AND J. D. THOMSON.
even become terminal in position, in which case the hinder
end becomes bluntly pointed or even rounded (Figs. 290, 291,
308). If at the same time the body becomes broadened out
posteriorly, as sometimes happens, the result is a form which
may mimic very exactly the small stumpy trypanosome which
is the final form of the development (Figs. 299, 306, 307).
The trypanosomes that undergo this process of degenera-
tion show a great tendency to adhere together in clumps
attaching themselves to one another by the tips of their
flagella (Pl. 45, figs. 304, 308; Pl. 44, fig. 311). The adherence
in this way of the degenerative forms must be distinguished
clearly from the process of agglomeration which T. lewisi
undergoes so readily when placed in unfavourable circum-
stances. Agglomeration takes place by the hinder ends of
the trypanosomes and more especially by their nn, as Laveran
and Mesnil have shown, and as a result of it the trypanosomes
tend to form rosette-like clusters, in which the flagella radiate
outwards. True agglomeration of this kind can also occur in
the flea under special circumstances, as will be described
presently. But in the degenerative clusters the conditions
are precisely the opposite to agglomeration, since the flagella
are directed towards the centre of the cluster, while the hinder
ends of the trypanosomes radiate outwards. The tendency
of the degenerative forms to adhere in clumps must be inter-
preted as an expression of the general tendency (perhaps it
might be termed instinct) of the trypanosome to attach itself
by the tip of the flagellum to firm surfaces when in the body of
the flea, a tendency very pronounced in all developmental
forms, excluding the final stage of the cycle.
Clumps and masses of very considerable size are formed by
the degenerative forms adhering together in the manner
described. Towards the centre the clumps often show a
cement-like substance, which stains pinkish-red with Giemsa.
The final stages of the degeneration are small forms, which
1 Manteuffel (1909) has already drawn attention to the distinction
between rosettes, with flagella directed inwards, and true agglomera-
tion.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 595
represent simply the hinder ends of the original trypano-
somes. ‘I‘hey are usually sharply or bluntly pointed (PI. 45,
figs. 302-304), or may be rounded off (fig. 306). The large
clumps of these little degenerative forms in the rectum are
often very difficult to distinguish in the living state from
the clumps of developmental crithidias. The degenerative
clumps, however, generally occur loose in the cavity, while
the true crithidias are attached to the wall of the rectum,
though in the process of dissection the latter often become torn
away from the wall. Whena loose clump of this kind consists
entirely of forms with pointed hinder ends it is probably
degenerative. The true crithidial clumps always have a con-
siderable number of forms with rounded hinder ends,
especially in the early periods of the establishment of the
rectal phase, when the hinder ends of the crithidial forms
are almost alwaysrounded. The degenerative forms, carefully
examined, show a certain extent of undulating membrane
running down the side of the body to n, which is situated
behind N, while in the typical haptomonad phase the
flagellum is reduced to the rhizoplast which comes off close
to » and terminates at the surface of the body, n being
situated beside orin front of N. Finally, it should be noted
that the degenerative forms never multiply by division at any
time. Nevertheless, in spite of all these distinctions, which
are more easily perceived in permanent preparations than in
the living state, it is sometimes difficult to pronounce decisively
as to the nature of a given individual, whether degenerative or
developmental, in preparations of the rectum; but as a rule
there is no difficulty at all.
A remarkable fact is the occurrence of recurved forms
amongst the degenerative forms in the rectum, of a type
essentially similar to the recurved trypanosomes occurring in
the normal developmental series in the stomach (Pl. 43,
figs. 297, 298). The recurved forms are often seen in the
clumps of degenerative trypanosomes. ‘The occurrence of
such forms in the rectum may perhaps be interpreted as an
abortive effort on the part of the trypanosomes that have
596 E. A. MINCHIN AND J. D. THOMSON.
passed on prematurely into the rectum to go through a
development similar to that which they undergo normally in
the stomach, but which, in all probability, would be impossible
in the rectum, where the cuticular lining would doubtless be
an effective bar to the penetration of the epithelial cells by
the trypanosome. It is possible that some of the recurved
forms in the stomach may also degenerate without ever
succeeding in penetrating the cells; the curious forms such as
PI. 43, fig. 305, are very probably to be explained as recurved
forms in process of degeneration. It would be difficult, how-
ever, as a rule, to distinguish between degenerative and
developmental trypanosomes in the recurved condition in the
stomach ; but in the rectum all such recurved forms must be
regarded as abortive and destined to degeneration.
As has also been mentioned above, trypanosomes of de-
generative type are found in the rectum on the third and
fourth days after infection. Such forms may, in some cases,
differ but little from ordinary blood-trypanosomes, and are
then to be interpreted, probably, as trypanosomes ingested at
a later feed, which have passed on to the rectum; but in
other cases they may be forms which are undergoing de-
generation after having developed normally in the stomach.
They are found, not infrequently, mixed with true develop-
mental forms in clumps, into which they have probably
intruded themselves (Pl. 41, fig. 183). It is necessary to be
careful not to confuse them with early forms of the rectal
phase in which n is still behind N; such forms can be
distinguished by their greater stoutness and bulk, and by the
fact that 1 has generally migrated forwards to some extent
(Pl. 41, figs. 181-187).
True agglomeration very rarely occurs in the flea, but we
have found it in its most typical form (Pl. 43, figs. 309, 310)
in fleas of a batch, the record of which was as follows: The
fleas, twelve in number, had been fed once on an infected rat
in the usual way, and three days later they were fed again on
a rat, the object being to test the influence of a second feed
of clean blood on the persistence of the stomach-phase
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 597
(see p. 664 below). By mistake, however, the fleas were fed
again on an infected rat instead of a clean rat. The next
day (four days after the first infective feed) the fleas were
dissected and examined. In every flea the trypanosomes of
the second feed could be recognised, quite unaltered from the
blood-form, and in most cases agglomerating in pairs, threes,
or rosettes composed of many individuals; they were found
in the stomach in every flea and in a few in the rectum also,
where in one case degenerative forms were noted; in some
fleas these trypanosomes were very numerous ; in others they
were scanty and had evidently undergone reduction in
number. ‘lhe trypanosomes of the first feed had disappeared
in nine out of the twelve fleas, while in the remaining three
they were represented by developmental forms of the usual
type in the rectum. Agglomerating trypanosomes of the
second feed were found both in fleas in which those of the
first feed had persisted and in fleas in which they had
disappeared.
From this observation it would appear that when a flea
has once had an infective feed, its digestive tract, and more
especially its stomach, acquires properties which cause the
agelomeration and probably also the degeneration of try-
panosomes taken in at later feeds, alike whether those
ingested at the first feed have succeeded in establishing
themselves in the, flea or not.
Reference has already been made above (pp. 531-532) to
intracellular forms which appear to be undergoing degenera-
tion after having penetrated into an epithelial cell (Pl. 36,
figs. 45-45).
APPENDICES TO THE DEVELOPMENT.
(1) Previous Investigations on the Development of
Trypanosoma lewisi.
The first who attempted to follow out the development of T. lewisi
in its invertebrate host was Prowazek (1905), who studied the develop-
ment in the rat-louse, Hematopinus spinulosus, and since those
598 E. A. MINCHIN AND J. D. THOMSON.
who followed immediately after him in similar investigations also made
use of the louse, it is simplest to deal first with all those works in which
the development in the louse is studied. Since we have not ourselves
studied the development of this insect, we are not in a position to con-
trovert the statements made, but it is legitimate for us to compare the
forms and stages described with those which we have found in the flea,
and, on the ground of such comparisons, to criticise the interpretations
given by the authors.
According to Prowazek, the general course of the development in the
louse is as follows: The flagellates are first to be found in the stomach,
where they do not collect at particular spots, but swim freely everywhere
in the ingested blood. In the stomach the processes of maturation and
fertilization take place. At the second feed of the louse the parasites
are forced down to the end of the mid-gut and finally come to rest in
the hind-gut, for the most part near the Malpighian tubules, but also in
other parts. Resting stages are to be found on or between the cells of
the mid-gut, and more especially at the beginning of the hind-gut.
From the fact that the parasites disappear from the hind-gut, it is
inferred that they can pass through the epithelium of the hind-gut.
[This conclusion is by no means warranted by the observation on which
it is founded; it is also, in our opinion, extremely improbable that the
flagellates could penetrate through the chitinous cuticle lining the hind-
gut.| In this way the parasites are stated to pass into the blood-stream,
then into the larynx [ sic], and so finally back into the vertebrate host
when the louse feeds. [It is not clear whether this statement is founded
on observation, or simply on the analogy of the statements made by
Schaudinn with regard to Trypanosoma noctuex; the author
never succeeded in obtaining an infection of the rat by means of the
louse.| No trypanosomes or their resting stages were found in freshly-
deposited feces.
The author describes in great detail, with figures, various appearances
interpreted by himas maturation, fertilisation, and even parthenogenesis.
As all this part of the work is in the highest degree unconvincing and
appears to consist of forced theoretical interpretations of degenerating
forms which were occasionally seen to undergo agglomeration, it is not
necessary to do more than refer to the figures given by Prowazek. Pl. II,
fig, 32, purports to show the “ fertilisation ” in the living, and PI. III,
figs. 38 and 39, in the stained condition, while fig. 40 represents the
“ ookinete,” a non-flagellated form with a single nucleus. From the
ookinete a crithidial form is stated to arise in the manner described by
Schaudinn (PI. ITT, figs. 41-43 and 45). An active multiplication follows
[but Pl. III, fig. 55, which is given as an example of the division, is simply
an unaltered blood-trypanosome which shows the commonly-occurring
abnormality of possessing two NN; compare our Text-fig. 15}.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. o99
The author appears to regard most of the crithidias as involution-
forms, which form “agglomeration-stars.” Such a star is shown in PI. IIT
fig. 54 [which represents a typical clump of normal developmental
crithidias, similar to our PI. 41, figs. 182-184]. Inaddition to the crithidial
involution-forms there are found other much smaller forms, wedged in
between the cells and with the flagella completely absorbed. The
crithidial involution-forms may also degenerate into small non-flagellated
forms (fig. 50). No special inoculative or final form of the development
is described.
Baldrey (1909) professes to have confirmed the development described
by Prowazek, including even the process of maturation and fertilisation.
Taxr-ries 15,
Trypanosome with two VN from the stomach of a flea eighteen
hours after the infective feed. Such forms with two and even
three NN are quite common in the blood of the rat and in the
gut of the flea at early stages of the development; compare
Minchin (1909), p. 803, Pl. 21, fig. 6, Pl. 22, fig. 74, and Pl, 23,
fig. 84; they haye nothing to do with reproduction of the try-
panosome by fission. (x 2000.)
He gives two text-figures, one showing “male” and “female’’ forms
and * copulation,” the other showing “ ookinetes ”’ and crithidial forms
of the typical nectomonad type. He states that the ookinete recon-
structs a flagellar apparatus and divides rapidly to produce crithidial
forms, which, by repeated division become smaller and smaller, pass
into the body-cavity, thence to the suctorial mouth-apparatus and so
infect the rat. The complete cycle takes from eight to ten days.
Rodenwaldt (1909) studied the development of T. lewisi in the
louse in order to meet the criticisms of Patton, and succeeded inci-
dentally in proving that Patton’s “Crithidia hematopini” is a
mythical and non-existent species. On the first day of the development
he found in the louse both unaltered forms of T.1lewisi and long forms,
600 E. A. MINCHIN AND J.. D. THOMSON.
which he described as “ Lanzettformen.”’ [The latter, from the figures
given (PI. 1, figs. 7-12) are clearly the same as our long “ crithidiomor-
phic” stomach-forms.] He also found trypanosomes alleged to be
dividing (figs. 5, 6, and 12) [but these, again, are simply forms with two
or three NN, such as occur frequently in the blood of the rat; see
above]. On the second and third days he found the same forms, but
a larger proportion of the Lanzettformen. In one louse, however, he
found crithidial forms developed on the third day.. On the fourth day
he found forms with a short flagellum or none at all (as shown in his
figs. 13, 14), which he compares with the ookinetes of Prowazek and
Baldrey; they are stated to bend their bodies without changing their
place. On the fifth day long crithidial forms appear (compare his figs.
15-21), and on the sixth and following days smaller crithidial forms in
rosettes, and also, but more rarely, leptomonad forms (figs. 23, 27).
From the tenth day there were found (1) small non-flagellated forms
(figs. 31-34) ; (2) stout flagellated forms (figs. 35-39); (3) a few stout,
non-flagellated forms, “ookinetes”’ (figs. 40-47); and also forms regarded
as representing copulation of gametes (figs. 48,49). After twenty days
slender, sporozoite-like forms were found (figs. 52-58) in the gut, never
in the body-cavity. Rodenwaldt did not succeed in producing infection
by means of lice.
Breinl and Hindle (1909) describe the development in the louse mainly
as follows. The ingested trypanosomes first show characteristic
changes in the nucleus, of which the karyosome divides and the division
products move to opposite ends of the nucleus. N and then become
approximated and a division takes place. Some of the trypanosomes
show about this stage a reduction of the cytoplasm, producing tadpole-
like forms with a swollen head and the rest of‘the body reduced to a
long flagellum (see their figs. 5-9); at this stage the two nuclei take
on the crithidial (“Herpetomonas-like”) arrangement. The crithidie
multiply by division and become “agglomerated in great clusters with
the flagellum always directed inwardly” (fig. 18). [These clusters
appear to be simply developmental clumps of crithidial forms; as
pointed out above, clumps with the flagella directed inwards, whether
of degenerative or developmental forms, are not instances of true
agglomeration.] Round forms [haptomonads ?] are also found (figs.
32-36). The alleged conjugation was not confirmed.
[We cannot help remarking that all the stages of the trypanosome
figured by Breinl and Hindle, even the crithidial clumps, present an
extraordinarily sickly and degenerative appearance; we venture to
think that anyone who compares their figures with ours will agree to
this statement. |
Swellengrebel and Strickland (1910), have had the advantage over
previous authors that they were able to compare the stages in the louse
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 601
with those occurring in the flea; their memoir is illustrated by numerous
figures, for which, however, they appear not to claim great exactness,
since they refer to them as “diagrams.” They find that ‘the develop-
ment in the louse is a very irregular one and is not to be compared with
that which takes place in the flea,’ The first changes are that n wanders
in the direction of N, producing finally a crithidial form, slender or
club-shaped (diagrams xv and xvi). From the crithidial forms arise
large “ovals,” some of them without flagella and representing the
“ookinetes” of former authors. Later ovals [haptomonads] and
crithidiz [nectomonads] are found singly or in clumps (diagrams xvii
and xviii). Degenerative forms were also seen, but no flagellates were
found which could be identified with this small, final trypanosome-forms
of the development in the flea. No conjugation was observed.
From all these various works, only one positive fact emerges clearly,
namely, that T. lewisi can develop in the louse into its typical crithi-
dial phase. with both nectomonads and haptomonads. On the other
hand, no intracellular multiplication has been observed, nor has it
been proved as yet that the development can proceed so far as to
produce the small trypanosomes which end the cycle in the flea. One
form, apparently degenerative, occurs in the louse which we have not
found in the flea, namely, a large oval form without a flagellum, the
zygote or “ ookinete” of Prowazek and others.
The first published works on the development of T. lewisi in the
flea were those of Swellengrebel and Strickland (1910). We have
already noticed above their statements with regard to the development
of the stomach-phase and stated that we are quite unable to agree with
the account given by them. On the third day of the development they
find both long crithidial forms and large “ ovals” [stout crithidias of
the haptomonad type] in the mid-gut (diagram vy). On the fourth day
they state that the flagellates had all passed out of the stomach into the
intestine. On the fifth day they found only round forms [hapto-
monads| in the rectum (diagram vii). On subsequent days they found
the various forms of the rectal phase, and on the eighth day they found
the small trypanosome-forms, the final stage of the development, which
the authors were the first to discover. In their diagram xvi, they give a
summary of the development showing the following sequence of forms:
(1) the normal blood-trypanosomes ; (2) a long crithidial form ; (3) a
stumpy crithidial form with short flagellum ; (4) a haptomonad form ;
(5) the same in process of division; (6) a form transitional to—;
(7) a nectomonad; (8) a form transitional to—; (9) the final trypano-
some-form. [In view, however, of the great differences seen in the
development of the trypanosomes in different fleas, especially prior to
the establishment of the rectal phase, it was somewhat rash to attempt
to fix the order of events in so few as eighty-three fleas, and it may be
602 E. A. MINCHIN AND J. D. THOMSON.
remarked that the entire stomach-phase has practically been omitted
from the cycle as summarised by the authors. |
Swingle (1911) gave the following description of the cycle in the flea.
He states that the trypanosomes remain but a short time in the stomach,
but migrate to the intestine where important changes take place. The
first change to be seen is a diminution in size, and at the same time N
moves towards the posterior end of the body. Occasionally such forms
degenerate ; in those that do not x moves forwards till it is close beside
or in front of N, thus producing a true crithidialform. The individuals
which do not change into the crithidial type curl upon themselves to
form an oval rounded mass (figs. 15, 16) [apparently representing
recurved forms]. Development of the crithidial forms may proceed
along two separate lines which come to the same end; (1) they may
“agglutinate” by the anterior ends forming rosettes (figs. 20, 21
[representing typical early crithidial clumps]); or (2) they may form
solitary cysts (figs. 22-30) [apparently representing typical examples of
the degenerative series]. Other forms [apparently degenerative] are
also described; but the haptomonad and other forms of the rectal
phase are all referred: by the author to the form-series of the lepto-
monad described by him as Herpetomonas pattoni; aconclusion
which Swellengrebel and Strickland (1911-12), justly criticise, though
they go too far in the opposite direction in suggesting that H. pattoni
is a stage in the development of T. lewisi.
Noller (1912), studying the development of T. lewisi in the dog-flea
(Ctenocephalus canis), confirmed our discovery of the intracellular
multiplication in the stomach and added some further details; he
observed the penetration of a trypanosome into a cell five hours and
fifty-five minutes after the ingested blood had been ingested by the flea
and states that the trypanosomes go through at least two generations,
probably more, of intracellular multiplication. Whether or not the
trypanosomes establish a normal infection in the flea depends, in
NOller’s opinion, upon whether they succeed in fixing themselves in the
intestine or rectum, or not. As regards the multiplication of the
attached forms in the end-gut, Noller finds that they can always be
distinguished from the leptomonads by the possession of a typical
undulating membrane and by undergoing a process of multiple fission ;
neither of these statements accord in the least with our experience of
the development of T. lewisi in Ceratophyllus fasciatus.
It remains to mention that Swellengrebel and Strickland (1910) made
some observations on the development of T. lewisi in Ornithodoros
moubata and Cimex lectularius. In the former they got no
development of crithidial forms; in the latter they found large
crithidias but no development in the hinder part of the mid-gut.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 603
(2) On the Possibility of the Occurrence of Sexual
Phenomena in T. lewisi.
It has been seen from the foregoing summary of previous investiga-
tions on the development of T. lewisi that Prowazek first, and after
him Baldrey, Rodenwaldt, and Gonder, asserted that the development
of T. lewisi in the louse begins witha process of fertilisation, of which
the main features are stated to be as follows: Slender male and stout
female forms of the trypanosome are differentiated; their nuclei go
through a process of maturation and reduction, after which a fusion of
the gametes takes place. The zygoteis described as an ‘ ookinete” of
elongated, oval form, with no flagellar apparatus and with a single
nucleus (synkaryon). The nucleus is then stated to divide into two by
a heteropolar mitosis to produce the two nuclei of a trypanosome
nand N, and then the locomotor apparatus, flagellum and undulating
membrane, are formed. The result is a flagellate of crithidial structure,
which proceeds to multiply actively by binary fission.
It must be remarked here that Prowazek’s account of the “ ookinete ”
and its development in T. lewisi was modelled in every essential
detail on the account given by Schaudinn for Trypanosoma noctue.
The fertilisation observed by Schaudinn, however, was not that of a
trypanosome, but of Hemoproteus (Halteridium). It is a process
of true fertilisation, which was first observed in vitro by Macallum,
and its occurrence is not open to doubt. Schaudinn differed from all
previous investigators in asserting that the ookinete (zygote) of
Hemoproteus became converted into a crithidial flagellate, a state-
ment which has never been confirmed, and seems never likely to be.
There can be little doubt at the present time that in linking together
the development of Hemoproteus noctue and of Trypanosoma
noctuz into a single life-cycle Schaudinn fell into error. Prowazek,
on the other hand, derived his “ookinete’’ in T. lewisi from the
sexual union and fusion of two trypanosomes, so that in its alleged
origin the ookinete of T. lewisi is of quite different nature from that
of Hemoproteus noctuze. Prowazek is, therefore, the first
investigator who claims to have seen sexual conjugation of trypanosomes
in the invertebrate host.
_ Later investigators of the development of T. lewisi in the louse
have not confirmed Prowazek’s statements as regards the sexual phase,
nor has anything similar been found in the flea. Those who have
investigated the development of other trypanosomes in their invertebrate
hosts have also failed altogether to observe sexual phases or sexual
behaviour, in spite of much careful searching for phenomena to which
their attention has been strongly directed. As stated above, Prowazek’s
account of the sexual processes is most unconvincing, and the data he
604 E. A. MINCHIN AND J. D. THOMSON.
brings forward are quite inadequate to support the superstructure
of theoretical interpretation built upon them. In short, the question
of sexuality in trypanosomes may be summed up in the words of
Miss Robertson (1912, p. 247): “ There is at present no sound evidence
of conjugation in any trypanosome life-cycle so far worked out.”
We have ourselves searched most carefully, but in vain, for sexual
phases and syngamy in the development of T. lewisi. As stated
above (p. 519), we found in one flea long crithidial forms adhering in
couples in a manner very suggestive of true sexual behaviour, and
believed that we had observed true syngamy. We were never able,
however. to confirm this observation or carry it any further, and we
are now convinced that the phenomena observed on that occasion
were simply processes of agglomeration of abnormal forms of the
trypanosomes in a malformed flea. When we discovered the stomach-
phase we thought it very probable that the sexual processes might take
place in this part of the developmental cycle, and we were inclined to
interpret as evidence of sexual union some of those stages with two
nn and two NN, such as PI. 36, figs. 19-23, which are certainly at first
sight very suggestive of the fusion of two trypanosomes. We have no
evidence, however, of any subsequent fusion of the nuclei, nor of any
antecedent processes of nuclear reduction such as should be the pre-
liminary to the process of syngamy. We are not able to arrange the
figures of these stages in any series which would suggest a sexual
process. In short we are not able to interpret these forms as anything
but early stages of the multiplication of the trypanosome.
On the other hand, it has been shown convincingly that the cycle in
the invertebrate host effects a marked change in the properties or
idiosyncrasies of the trypanosomes that have undergone it. Gonder
showed that an arsenic-resistant strain of T. lewisi remained arsenic-
resistant so long as it was transmitted from rat to rat by direct
inoculation, but lost that property when transmitted by the. louse.
Miss Robertson (1912) also found that strains of T. gambiense
became changed in character when transmitted through the tsetse-fly,
and remarks: “It seems clear that the cycle in the fly asa whole,
whether conjugation actually occurs or not, has much of the biological
significance of the process.”
Those who believe that trypanosomes pass through sexual phases in
their invertebrate host will be inclined to ascribe the changes in the
properties of the parasite to the effects of the sexual process. At the
present time it is not possible either to affirm or to deny, with certainty,
that sexual processes occur. All that can be said with any approach
to verisimilitude is that the change appears to be connected in some
way with the metamorphosis of the trypanosome and its passage through
a crithidial stage ; but proof is lacking that the crithidial stage follows
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 605
upon, and is the product of, a sexual process. Attention may be drawn
here to another possibility already indicated above, namely, that the
crithidial phase may be initiated by a differentiating division into two
inequipotential products, one of which is destined to be eliminated
sooner or later from the direct line of the life-cycle. If this supposition
is correct a possible explanation might be afforded for the renovating
effects of the invertebrate cycle. In the present state of knowledge:
however, such an explanation must remain hypothetical, and lacking
objective foundation.
PART IIT.—_ EXPERIMENTAL STUDY OF THE PROBLEMS
OF THE TRANSMISSION AND DEVELOPMENT.
(1) Lyrropucrion.
THROUGHOUT our investigation of the relations of Try-
panosoma lewisi to the flea, we have endeavoured, as far
as possible, to make experiment and observation go hand in
hand, employing the one method to check or throw light
upon the results obtained by the other. In the following
pages we set forth our results in a number of sections which
arrange themselves naturally into two groups. One group
(sections i-xv) embraces problems that deal with the complete
cycle (including the passage of the propagative forms back
into the rat) and with the establishment of T. lewisi in the
flea, raising questions that are of general interest in the study
of trypanosomiasis. The other group (sections xvi-xix)
deals with some further problems that are of interest more
especially in relation to the flea Ceratophyllus fasciatus
and to ‘I’. le wisi itself under more or less special conditions in
the flea. Hach section is headed by a proposition which it is the
object of the experiments cited to establish. If we consider
that the proposition is proved satisfactorily by our experi-
ments, it is put in the form of a positive or negative state-
ment; if, on the other hand, the problem stands in need of
further proof, the heading of the section is expressed in
interrogative form.
The details of each experiment are given when it is cited,
606 E. A. MINCHIN AND J. D. THOMSON.
but a few general remarks upon our methods may be made
conveniently at this point. We kept going two breeding-
cages of the type used by the Plague Commission (see
‘Journal of Hygiene,’ vi, Pl. iv). In one cage a clean rat
was always kept to feed the fleas, in the other an infected
rat ; these two cages are designated, in the account of our
experiments, the non-infected and the infected breeding-cage
respectively. From the former we could always obtain a
plentiful stock of clean fleas when required, while the latter
furnished infective fleas. The rats used were almost always
white rats bred in captivity ; we found them as a rule docile
and good-tempered so long as they were handled with the
hands and not with forceps, and the operation of pricking
their tails to obtain drops of blood, when required, did not
arouse their resentment in the slightest. They live well in
captivity, and weré none the worse for being exposed to the
fleas in the breeding-cages, provided the number of fleas was
not allowed to become too great. Many of them suffered,
however, from a troublesome itch, caused by a minute
Acarine, which is very difficult to get rid of. One of our
breeding-cages became over-run by rat-mites, rendering it
necessary to destroy it and start a fresh one.
For our actual experiments we used in many cases,
especially for experiments with small numbers of fleas, cages
of special design in the form of a cylindrical tin-canister with
the bottom closed in with tin, the top provided with a lid
with a tightly-fitting rim. The canister was 10 in. high and
6 in. in diameter. The top of the lid was made of strong
wire gauze, to prevent the rat jumping out, and over that
muslin-gauze was pasted to prevent escape of fleas. After
these cages had been in use for some time, however, they
tended to become rusty on the inside and then the fleas could
climb up the tin easily. Consequently, it was found more
suitable to use inverted bell-jars, each about 154 in. in height
and 7in. in diameter. The bell-jars were supported each on
a wooden block, or several together in a wooden crate. The
open upper end of the bell-jar had a zine wire cover to
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 607
prevent the rat jumping out, but it was not necessary to take
precautions against the fleas escaping, because they are
unable either to jump so high or to crawl up the smooth glass
if kept clean. The bell-jars were cleaned out thoroughly
once a week.
The beil-jars were especially suited for experiments with
single fleas or a small number of fleas. First of all clean
saw-dust is put in the bell-jar to a depth of about 3 in., then
the rat and the flea or fleas are put in. Since Cerato-
phyllus fasciatus is a flea which does not live perma-
nently on the rat but only goes on to it for food, and lives
naturally in rat-burrows, the fleas were generally to be found
without difficulty in the saw-dust, when it was necessary to
recover them, but sometimes they were on the rat itself. In
the latter case the rat was held over a deep bowl of
enamelled iron and the flea disturbed. by blowing on to the
fur of the rat, which has the effect of soon making the flea
come to the surface of the fur. It was then captured, as a
rule, by seizing it gently byfinger and thumb, an operation
which must be performed rapidly and deftly, otherwise it
burrows down into the fur and must be dislodged again.
Sometimes the flea drops off the rat and falls into the
bowl, where it can be recaptured easily. Our assistant,
Mr. George Kauffmann, became exceedingly expert at this
job, and if a flea could not be found by him it was safe to
assume that it had died or been eaten. In our earlier experi-
ments we used chloroform for recovering the fleas, but later
we abandoned this method, often fatal to the rats.
In some cases it was required to expose a large number of
fleas—200 or so—to infection on an infected rat for a night
or a day. For this purpose the bell-jar was also handy, but
it was often found that the rat ate a great many of the fleas,
sometimes as many as 100 or more in a single night. To
prevent this a cylinder of wire gauze was made, of sufficient
length to fit into the bell-jar in such a way that its two ends
were closed by the glass wall of the jar, and of such a calibre
as to allow the rat to walk forwards or backwards along it,
vot. 60 PART 4,—NEW SERIES. 42
608 E. A. MINCHIN AND J. D. THOMSON.
but not wide enough to permit the rat to turn round or use
its paws freely, and consequently hindering it from catching
and eating the fleas.
For the purpose of collecting large numbers of fleas from
the breeding-cage the following method was found to be the
simplest: Two glass capsules were used, each provided with
a well-fitting lid, the one smaller, about 2} in. in diameter
and 1{in. in height; the other larger, about 6in. in diameter
and 3 in. in height. First of all, débris from the breeding-
cage containing fleas in all stages of their development is
scooped up with the small capsule and the lid at once clapped
on. ‘Then the small capsule is placed in the large one; the
lid of the small capsule is removed with one hand, and the lid
of the large one put on with the other. The adult fleas in
the small capsule then begin at once to jump out of it in
every direction, and so fall into the enclosing large capsule,
in which they soon collect on the side furthest from the light.
When the fleas have swarmed out in this way the small
capsule is removed and the débris contained in itis returned
to the breeding-cage. The fleas in the large capsule can then
be emptied through a glass funnel into a suitable receptacle,
such as an Erlenmeyer flask. Or, if the large capsule be left
to stand until all the fleas have congregated on the side furthest
from the light, then by suddenly turning the capsule round
through about 180°, so that the side which was furthest from
the light is now the most illuminated, the fleas begin at once
to move towards the opposite side ; and if then the small cap-
sule be held in their way they can be made to jump into it of
their own accord, and they can thus very easily be counted
and disposed of as required. Ceratophyllus fasciatus is
not a very good jumper and its trajectory is low.
The fleas collected can be kept, if required, for a considerable
time ; we found the best method was to put a little clean white
sand, moistened with two or three drops of water, at the
bottom of a flask. The fleas burrow down into the sand and
appear to live comfortably. If they are to be kept any
length of time the sand must be moistened again every
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 609
two or three days. Like most blood-suckers, the flea can
stand a prolonged fast.
(2) GunrraL PRrosiems.
(i) Trypanosoma lewisi is transmitted from Rat to
Rat by the Rat-flea, Ceratophyllus fasciatus.
It is not necessary that we should cite experiments
specially to prove this proposition, since it is established by
the experiments brought forward under the headings that
follow, and it has been proved beyond all possibility of reason-
able doubt by experiments already published by others as
well as by ourselves.
The agency of fleas in the transmission of T. le wisi was first demon-
strated by Rabinowitsch and Kempner (1899), who succeeded in infect-
ing clean rats by intra-peritoneal injection of teased-up fleas (species
not stated) which had previously been fed on infected rats. In these
experiments the trypanosomes appeared in the blood of the rats in six
to eight days after the injection. The authors state that they were not
able to find any stages of the trypanosome in the flea-débris which
was injected. They also obtained positive results by placing fleas,
previously fed on infected rats, upon clean rats; the trypanosomes made
their appearance in the blood of the rats after two to three weeks.
Their experiments with lice gave negative results.
In spite of the experiments of Rabinowitsch and Kempner, the work
of Prowazek (1905) on the development of T. lewisi in the rat-louse,
Hxematopinus spinulosus, led to this insect being regarded as the
true host of the rat-trypanosome, and no more experiments with fleas
appear to have been undertaken until those of Nuttall (1908), who
obtained positive infections of rats with fleas, using both Cerato-
phyllus fasciatus and Ctenophthalmus agyrtes. Two years
later we published accounts of a number of experiments, since when the
rOle of the flea has been established beyond the necessity of further
experiment upon the subject.
We have confined our experiments throughout solely to the common
English rat-flea, Ceratophyllus fasciatus, but it has been shown
by Noller (1912) and Wenyon (1913) that the transmission can be
effected by other species of fleas, namely, the dog-flea, Ctenoce-
phalus canis; the human flea, Pulex irritans ; and the Indian
rat-flea, Xenopsylla cheopis. It is indeed noteworthy that other
species of fleas appear to be more efficient as true hosts of the rat-
trypanosome than the species which in this country occurs habitually
610 E. A. MINCHIN AND J. D. ''HOMSON.
in association with rats, since Dr. Wenyon has informed us that in the
fleas with which he experimented, the trypanosomes never failed to
establish themselves and to go through their complete developmental
cycle, while in Ceratophyllus fasciatus we found that only a small
percentage of the fleas became infective (see below), and examination of
the fleas showed that the trypanosomes establish themselves in a corre-
spondingly small percentage (see p. 659). It would appear, therefore,
that the flea which, more than any other species, is exposed in this
country to infection by T. lewisi, has developed a certain degree of
natural immunity to the parasite. From the experiments published by
the authors cited it is probable that T. lewisi would undergo its
development in any species of flea,and would be transmitted by it, pro-
vided that the flea could be induced to suck the blood of an infected
rat. The natural efficacy of any given species of flea in transmitting
T. lewisi depends probably on the habits and tastes of the flea, and
not on any specific ability to harbour the trypanosome. Brumpt (1913)
has pointed out that all the trypanosomes of small rodents seem to be
able to develop in fleas.
A number of experiments have been performed by several investi-
gators on the transmission of T. lewisi by means of the rat-louse,
Hematopinus spinulosus. The first experiment with rat-lice
(species not stated) was carried out by MacNeal (1904), who transferred
“several” lice from an infected to a clean rat ; trypanosomes appeared in
the latter after fourteen days. Positive results in experiments of this
kind with rat-lice are reported by Nuttall (1908), Baldrey (1909), Breinl
and Hindle (1909), Manteuffel (1909), and Gonder (1911). To judge,
however, from the published accounts of these transmission-experi-
ments, positive results were by no means frequent and were obtained in
some cases at least with difficulty and by the exercise of great patience
and perseverance, or by using large numbers of lice. Nuttall obtained
one positive result in an experiment in which sixty lice were used ; two
‘other experiments, in which fewer lice were used, were negative.
Baldrey reports two experiments in which infection was obtained by
means of lice; in the first, 100 lice were used, and the result is regarded
by him as a case of direct mechanical infection, but for what reason is
not at all clear; the second, in which ten lice were used, is interpreted
as demonstrating a developmental cycle in the louse. Breinl and
Hindle report three successful transmissions by means of lice, after
carrying on numerous experiments for over a year. Manteuffel seems
to have been more successful than most other experimenters in this
field, though he does not record the actual number of his experiments
or the proportion of those which were positive in result, but he states
that infections with lice were “prompt and frequent ”; his method was
to put infected rats, with lice on them, in the same cage with clean
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 611
rats, and from his results he concludes that lice do not transmit the
trypanosome longer than from three to five days after being removed
from the infected rat, and that the transmission is effected by the act of
blood-sucking ; if the first of these two conclusions be true, it would
appear that the trypanosome does not succeed in establishing itself in
the louse in the way it does in the flea. Gonder reports that after many
fruitless attempts to transmit T. lewisi with definite numbers (80-
100) of lice, he obtained six positive results in a series of fifty experi-
ments, and eight positive results in another series of fifty, using greater
numbers (gréssere Mengen) of lice; and he also brought about six
infections by making emulsions of lice taken directly from an infected
rat, the lice having been left on the infected rat for five, nine, eleven,
thirteen, sixteen, and twenty-one days respectively, in these six experi-
ments. On the other hand, Prowazek, who first described developmental]
stages in the louse and claimed that this insect was the true host of T.
lewisi, was unable to obtain experimental transmission ; Rodenwaldt
obtained no positive results with numerous transmission-experiments ;
and we also have obtained only negative results in any attempts that
we have made to transmit T. lewisi by means of the rat-louse.
It is evident from the results summarised briefly in the foregoing
paragraph that transmission of T. lewisi can be effected by the rat-
louse, but only with difficulty, and in a small percentage of cases.
This is a great contrast to the ease and comparative certainty with
which the trypanosome can be transmitted by fleas. We have always
used our flea-cages as the simplest and easiest method of obtaining
infected rats when required by ourselves or by our colleagues or friends,
and not only have we infected rats with single fleas on many occasions,
but we have even succeeded in infecting several rats successively with
one and the same flea. We have no hesitation, therefore, in regarding
fleas as the usual agency whereby T. lewisi is transmitted from rat
to rat in Nature, a result brought about by the louse rarely and
exceptionally.
It should be noted that Brumpt (1913) has succeeded in infecting a
rat with T. lewisi by inoculating it with the rectal contents of a bug,
Cimex lectularius, fed on an infected rat thirty-eight and again six
days previously. There is no evidence, however, that this insect
transmits the infection naturally.
(i) [he Transmission takes place by the Cyclical
Method. Transmission by the Direct Method
has not been proved to occur.
These are among the conclusions drawn from experiments
described in full detail in our preliminary report (1910). It
612 E. A. MINCHIN AND J. D. THUMSON.
is sufficient here to state that experiments “A” (20) and
“B” (21) in our report were devised chiefly to separate
“ direct” from “cyclical”? infection, a matter of primary
importance at the time that these investigations were begun,
and they show that in the individual cases cited (A; and B,)
transmission was effected when all possibility of the direct
method was excluded. Experiments ‘‘C” (22) and “ D ” (28)
multiply such cases many times, and show further that fleas
once infective retain the infection so as to infect a series of
rats without themselves being exposed to fresh infection.
Since then a number of experiments have been carried out
by us, many of which are enumerated under the different
headings which follow, and the sum-total of these experiments
not only supports the conclusions in our preliminary report,
but establishes beyond doubt that the rat-flea isa true inter-
mediate host, that it can transmit the infection to other clean
rats only after the developmental cycle has been completed
within itself; that, in short, the infection takes place by the
cyclical method; and that there is no evidence whatever to
show that the rat-flea is capable of carrying the infection
from one rat to another by whatis called the “direct” or
“ mechanical” method.
The term method in the phrase * method of transmission,” if used
without qualification, should include comprehensively all that happens
in the transmission of infection from one vertebrate toanother. In the
transmission of trypanosomes the natural transmitting agent, when
such is known, is a blood-sucking invertebrate of some kind. When
the method is said to be “ contaminative” or “inoculative,’ trans-
mission is viewed from the side of the invertebrate in its relation to the
vertebrate, and the problems involved are particular, that is to say
such as deal with modifications due to special cireumstances in those
relationships, and are concerned at most with special groups of
trypanosomiases rather than with trypanosomiasis in general. On the
other hand when the method of transmission is said to be “ cyclical”
or “ direct,” transmission is viewed from the side of the trypanosome in
its relation to the invertebrate, and the problem becomes a general one,
dealing with that phase of the transmission which is concerned with the
life-history of trypanosomes as a group of parasites, and with the wider
question of their double relationship to vertebrate and invertebrate,
THE RA'T-TRYPANOSOME, TRYPANOSOMA LEWISI. 613
bringing them into line with other known relationships among parasitic
Protozoa in this respect.
A great impetus was given to the study of trypanosomes by economic
and other considerations arising out of the prevalence of tsetse-fly
disease and sleeping sickness in Africa. It was long known that these
diseases could be transferred artificially by direct inoculation of blood
from a diseased to a healthy subject by means of a hypodermic syringe.
Naturally, therefore, before much work had been done in this direction
tsetse-flies known to be associated with the spread of these diseases
were supposed to transmit them in this direct way. Bruce and others,
experimenting with bred-out flies, proved the possibility of this taking
place under certain conditions which, in the case of sleeping sickness
at all events, were very unlikely ever to be fulfilled in Nature. Only
with a swarming infection and by interrupted feeding could the
disease be passed on directly from an infected to a clean animal
with any approach to certainty, and even under the most favourable
conditions in other respects, the longer the interval between inter-
rupting the feed on the infected animal and continuing it on a clean
’ animal, the less the chance of the clean animal becoming infected,
until, with the lapse of about half-an-hour, it was just as certain
that the infection would not take place. Moreover, however short
the interruption between the feeds, an interposed partial feed on a
clean animal rendered the fiy non-infective to a second clean animal.
Later experiments showed that the contents of the stomachs of flies
that had fed on an infected animal, if injected into a clean animal,
could produce infection only up to about two days after the infective
feed. The fly itself, however, could not be shown to act in any way
resembling a hypodermic syringe, and the idea of ‘‘ delayed mechanical
transmission’ never found support from feeding experiments. The
conclusion to be drawn from all the earlier experiments on direct
transmission seemed to be that when infection was obtained it was
with ‘“‘fouled proboscis” before the blood in its lumen distal to the
entrance of the salivary duct, and perhaps also on its external surface,
had had time to dry, and that the conditions under which it was
shown to be possible were never likely to be fulfilled in Nature, in
the case of sleeping sickness, and in the case of tsetse-fly disease of
cattle, far too seldom to account for the spread of the disease, while
in no case could such a method of transmission account for the
existence of fly-belts through which healthy domestic stock cannot
pass. Other things being equal, the efficiency of an invertebrate as
a transmitter of trypanosomes would be enormously increased if the
invertebrate were a true intermediate host and not merely a‘ porter ”
of the parasites from an infected to a clean subject, and to demon-
strate beyond doubt that trypanosomes underwent an alternation of
614 E. A. MINCHIN AND J. -D. THOMSON.
generations was of primary importance in connection with the general
trypanosome problem at the time that we undertook this investiga-
tion, when it was being maintained by Patton and others that no
trypanosomes went through a developmental cycle in the invertebrate,
that all transmission of trypanosomes was direct, and that the crithidial
forms found in blood-sucking invertebrates were all of them independent
parasites of the invertebrate, having no connection with the trypano-
somes or other parasites of the vertebrate. It was known that rat-fleas
could transmit T. lewisi from infected to clean rats, and although
transmission by fouled proboscides seemed quite out of the question, it
was necessary to demonstrate beyond doubt that the rat-flea is a true
intermediate host of T. lewisi, that it can transmit the infection to
other rats only after the developmental cycle has been completed within
itself, and that once infected it remains infective for a considerable
time, so as to be able to infect a series of clean rats without itself being
exposed again to infection. These points, which we believe concern the
transmission of trypanosomes in general, and which may be taken as
typical of the relations which trypanosomes as a group bear to their
invertebrate hosts, as well as other points of more special interest (con-
fined, it may be, to T. lewisi alone or to the lewisi group), are
dealt with under different headings in what follows later. Although
the practical cannot properly be separated from the scientific, the most
interesting problem of the transmission from the scientific point of
view is perhaps the way in which the trypanosome becomes established.
There are considerable variations in the details of the cycles of
different species or groups of trypanosomes in their natural hosts due
to special conditions, but arising out of the very meaning of a cycle,
and therefore common to all is the fact that until the cycle is com-
pleted the invertebrate, though infected, is not infective. This may
be of direct practical importance in special cases, and where that
is so it is important to ascertain the length of time required for the
completion of the cycle in each case. Of more general practical
importance in questions connected with the spread of infection is the
fact that the trypanosome does establish itself in such a way that the
invertebrate remains infective for a long time without requiring to be
exposed again to infection.
(iii) The Trypanosomes make their Appearance in
the Blood of the Rat Five to Seven Days after
Infection; the Multiplication of the Trypano-
somes in the Blood of the Rat come to an End
‘Eleven to Thirteen Days after Infection.
In order to establish with exactness the length of the
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 615
incubation-period and the multiplication-period in the rat
after infection, it is necessary that the rat should have been
exposed to infection by the fleas for a short time; long
exposure leaves too wide a margin between the possible
maximum and minimum deducible from the actual data
furnished by the experiment for the mean to be of any value
im reckoning the length of the two periods in question.
When the rat is removed from contact with the infected
fleas it is further very necessary that all fleas should be
removed from its skin. The rat is then kept in a flea-proof
cage and its blood is examined daily in fresh, living films
until trypanosomes are first detected in it, in order to deter-
mine the duration of the incubation-period ; then smears of
the blood are made and preserved daily and examined until
the multiphication-period is found to be past and ended. So
long as the trypanosomes are multiplying in the rat’s blood,
they are of various sizes, some of the ordinary, normal size,
others very small, and others again much above the normal
size. Marked variation in the size of the trypanosomes is a
sure sign that multiplication is proceeding, even when actual
division-stages are so scarce in the preparation that pro-
longed search is necessary in order to find them. As soon as
the multiplication is ended the trypanosomes are all of one
type and size, allowing for slight individual variations that
are not perceptible without careful measurement; to such
trypanosomes; the normal form of T. lewisi and the sole
form occurring in the blood when once the multiplication is at
end, we shall refer always as “ ordinary.”
We cite here a few examples from our series of experiments,
choosing first (Table C), those in which the rats were exposed
to infection for one day only, so that the periods of incubation
and multiplication can be determined within a margin of one
day. In our second table (D), we quote those instances in
which the rats were exposed to infection for two days, so that
a wider margin of possible error must be allowed for in
calculating the two periods. In a third table (E), we shall
give some results obtained with rats which were infected
A. MINCHIN AND J. D. THOMSON.
E.
616
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THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 619
each by a single flea, in order to show that in many cases, at
least, the maximum periods of incubation and multiplication
which can be deduced from experiments under these con-
ditions are not greater than those indicated by the experi-
ments in which many fleas were used to obtain infection. In
a fourth table (F) we give for comparison, the results obtained
by inoculating rats with the stomachs or recta of fleas; in
such cases the length of the periods of incubation and multi-
plication can be determined with exactness, the moment of
infection being known.
The determination of the length of the multiplication-period in an
infected rat is of practical importance for interpreting other experi-
ments, since, when it has been determined, it furnishes a datum from
which approximately accurate conclusions can be drawn as to the time
at which the rats become infected, when the point is shown definitely by
the details of the experiment. As regards the first appearance of the
trypanosomes in the blood, they appear at first in such scanty numbers
that it is very easy to overlook them, and they may often be reported
absent when a more prolonged search would have detected their
presence. Similarly, the trypanosomes at the end of the multiplica-
tion-period may sometimes have been reported as “all ordinary” in a
smear in which more careful searching might have led to the discovery
of a few individuals above or below the normal size. Consequently, the
errors of observation are such as tend to over-estimate the length of
the incubation-period, and to under-estimate that of the multiplication-
period, from the serutiny of the blood-films. On the whole, however,
the results obtained in our experiments are very uniform and indicate
an incubation-period of about six days, a multiplication-period of about
twelve days. It is interesting to note that these results agree with
those obtained in the case of rats infected artificially by inoculation,
intra-peritoneal or otherwise, with blood from an infected rat. Since a
syringe would inoculate far more trypanosomes than the rat would
obtain from even a large number of fleas, it might have been expected
that the rat would, so to speak, fill up quicker when infected by means
of a syringe, and that consequently the multiplication-period would be
correspondingly shorter. In our experience, however, the length of the
multiplication-period remains approximately constant in all cases,
whether the infection is effected by a syringe, by a large number of
fleas, by a few fleas, or even by a single flea ; a fact which indicates that
the length of time during which the trypanosome multiplies in the rat
620 E. A..MINCHIN AND J. D. THOMSON.
is not determined by the number of trypanosomes put into the rat, but
by the mutual interaction of host and parasite.
It may be noted here that some rats appear to possess a certain
degree of natural immunity to infection with T. lewisi. A single
instance which came under our experience will suffice to demonstrate
this point. A rat was exposed to infection on June 9th and its blood
was examined daily; on June 25th a few trypanosomes were first seen
in the blood in scanty numbers, just as they are usually seen at their
first appearance between the fifth and seventh days of the infection.
The rat was then removed from contact with the fleas and kept apart ;
but neither on the next day nor on any subsequent day were any try-
panosomes to be found in its blood. This rat, therefore, contracted
only a transitory infection which was late in its appearance and dis-
appeared after one day ; had the trypanosomes been overlooked on that
day the experiment would have been returned wrongly as negative in
result.
(iv) The Cycle of Development in the Flea requires
a Minimum-of Five Days for its Completion.
This point was dealt with in our preliminary communication
(1910), in which we came to the conclusion that the incuba-
tion in the flea was six or seven days. Our method of deter-
mining this was, first of all to expose non-infected fleas to
infection, by putting them on a well-infected rat, for but a
single day, so that if the fleas afterwards produced an infection,
the time at which they themselves became infected could be
determined within a narrow margin, twenty-two hours in our
actual experiment. The fleas were then placed in contact for
three days with clean rat (1) which did not become infected ;
after that for three days with clean rat (2), which also did not
become infected; and then for two days on clean rat (3),
which showed trypanosomes in its blood six days after being
removed from contact with the infected fleas. Clean rat (3)
was, therefore, infected by the fleas in the interval between
the sixth and eighth day after the fleas themselves had
acquired the infection ; consequently the infection in the fleas
could not have been more than eight days old.
Subsequent experiments performed by us have indicated a
possible minimum of five days for the flea-cycle of the try-
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 621
panosome. In experiment 39 (see below, p. 630) it is proved
that the rectum of the flea, injected into the rat, can produce
an infection as early as the fifth day, and in such fleas the
examination of films shows the presence of the small trypano-
somes which are the final form of the development in the flea.
In experiments 26 and 28, undertaken in order to ascertain
whether a rat, in which the trypanosomes are still in the
multiplication-period, is capable of infecting fleas (see below,
p. 657), the results obtained indicated a short incubation-
period in the fleas. Thus in experiment 26, 127 fleas, after
being three days (from 8: ii: 10 to 11: ii: 710) on the in-
fected rat were put on rat 187 for another three days (from
11 :ii to 14: ii). Rat 187 showed trypanosomes in its blood
after five days (19 : ii), and the multiplication-period ended
five days later (24: i1). Consequently the incubation-period
in the fleas could not have been more than six days (8 : 11 to
14: ii). In experiment 28, 137 fleas were put first on the
infected rat for four days (15: ix: 710 to 19: ix: 710), and
then were put for one day (19 : 1x to 20: ix) on rat 209 ; after
this they were put on rat 215 and left on it. Rat 209 had
shown no infection when it died nine days later (29 : 1x) ;
rat 215 first showed trypanosomes on 30: ix, and the multiplhi-
cation was ended 3: x. This result indicates that rat 215
was infected about 21 : ix, in which case the incubation-period
in the flea could not have been more than six days (15 : ix to
91: ix). Since rat 209 showed no trypanosomes for at least
nine days after being exposed to infection it was probably
not infected ; so that the infection in the fleas was probably
not ripe for at least five days (15 : ix to 20 : 1x).
On the other hand we have instances, as already mentioned in our
preliminary communication (1910), of an incubation-period in the flea
apparently much longer than six days. Thus in experiment 19 a cage
(J) was stocked with seventy-two fleas from the non-infected breeding-
cage and an infected rat (No. 81, a wild black rat, naturally infected)
was put with them for three days (20:ix :’09 to 23:ix:’09). Rat 81
was then removed and rat 82, a clean, tame rat, was put in its place
(23: ix) and left in the cage. Rat 82 first showed trypanosomes in its
blood 29:x; the multiplication-period was ended about 4:xi, indi-
622 E. A. MINCHIN AND J. D. THOMSON.
cating that the actual infection of rat 82 took place about 23:x. In
this case, therefore, the fleas did not produce an infection in the clean
rat for at least a calendar month after their contact with the infected
rat was interrupted. Such a result, however, permits of no conclusion
whatever as to the length of the incubation-period in the flea; it merely
demonstrates a point proved also by other experiments, namely that in_
fective fleas often fail to infect. We have put forward already (1910)
one possible explanation for this, that a rat, which is comparatively
immune to begin with, may resist infection for a long time, but its re-
sistance may be overcome at last. Another possible explanation may
be given by the method in which infection of the rat by the flea is now
known to take place, namely by the rat licking off the moist feces of
infective fleas that are deposited on its skin (see below, p. 648). It is
evident that if the rat fails to lick off the feces while still moist, or if
the infective flea does not defecate on the rat, no infection is brought
about. A negative result of this kind is most likely to be attained when
the number of infective fleas on the rat is very small, as seen in the
large number of negative and small number of positive results in our
series of experiments in which single fleas were used (see below, p. 661).
That infective fleas in Cage J were rare is shown by the fact that
between 23 :ix and 19: x thirty fleas from this cage were dissected and
examined without finding a single one infected. On the other hand,
when fleas are sufficiently numerous and have been well infected, posi-
tive results are fairly certain (Experiment “C” of our preliminary
report).
(v) Transmission is never effected until the Deve-
lopmental Cycle is completed; that is to say,
until at least Five Days have elapsed since the
First Exposure of the Fleas to Infection.
We have found, as already stated, by direct observation,
that the final form of the developmental cycle appears in the
gut of the flea five days after the infective feed (see below,
p. 630). We bring forward here a few instances to show that
at least five days must elapse before the flea becomes infective,
after having ingested trypanosomes from an infected rat.
(1) Experiment 20.—A cage colonised with forty-four fleas that
had been exposed to infection from 4: x : 09 to8: x: 09.
Rat 93 put into the cage from 8:x tol2:x, i.e. during a period
in which the age of the infection in the fleas could not have been less
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 623
than two days old at the beginning nor more than eight days old at the
end. Result negative.
(The next rat used in this experiment died, but subsequent rats used
showed that the fleas had become infective.)
(2) Experiment 21.—A cage colonised with 157 fleas that had been
exposed to infection from 11: x : 09 to 15: x: ’09.
Rat 97 put in from 15 : x to 19: x, i. e. during a period in which the
age of the infection in the fleas could not have been less than a few
hours at the beginning nor more than eight days at the end. Result
negative.
(The next rat put in became infected, apparently about 27: x; age of
infection in the fleas then between twelve and sixteen days.)
(3) Experiment 22.—A cage colonised with 160 fleas exposed to
infection from 24 : xi: 09 to 27: xi: ‘09.
Rat 116 put in from 27 : xi to 30: xi, i. e. during a period in which
the age of the infection in the fleas could not have been less than a few
hours at the beginning nor more than six days at the end. Result
negative.
(The next rat put in became infected, apparently about 3 : xii; infec-
tion of the fleas then six to nine days old.)
(4) Experiment 23.—A cage colonised with 162 fleas exposed to
infection from 7 : xii: 09 to 8: xii: ’09.
Rat 125 putin from 8: xii to 11: xii, i.e. during a period in which
the age of the infection in the fleas could not have been less than a few
hours at the beginning nor more than four days at the end. Result
negative.
Rat 133 put in from 11 : xii to 18: xii, i. e. during a period in which
the age of the infection in the fleas could not have been less than four
days at the beginning nor more than six days at theend. Result nega-
tive.
(The next rat put in became infected, apparently about 15 : xii; infec-
tion of the fleas then seven to eight days old.)
(5) Experiment 24.—A cage colonised with fifty fleas exposed to
infection from 7 : xii: 09 to 8: xii: ‘09.
Rat 126 put in from 8: xii to it: xii, i.e. during a period in which
the age of the infection in the fleas could not have been less than a few
hours at the beginning nor more than four days at the end. Result
negative.
(The next rat put in became infected, apparently about 3:1:710; age
of the infection of the fleas then between twenty-six and twenty-seven
days.)
(6) Experiment 25.—A cage colonised with seventy fleas exposed
to infection from 31: xii: 09 to3:1: 10.
Rat 152 put in from 3:ito6:i,i.e. during a period in which the
vot. 60, part 4.—NEW SERIES. 45
624. BE. A. MINCHIN AND J. D. THOMSON.
age of the infection in the fleas was not less than a few hours at the
beginning nor more than six days at the end. Result negative.
(The next rat putin became infected, apparently about 6 or 7:1; the
age of the infection at 7 : 1 was from four to seven days.)
(7) Experiment 45, Batch C (see below).—Bell-jar colonised
with thirty fleas exposed to infection from 22 : vii : 13 to 23: vii : 713.
No infection produced in rat 370 put in fora period of two days,
during which the infection in the fleas could not have been less than
five days oldat the beginning nor more than seven days old at the end.
No infection produced in rat 370a, put in for a period of one day,
during which the infection in the fleas could not have been less than
eight days old at the beginning nor more than ten days old at the end.
(The next rat put in became infected.)
In the previous section it has also been pointed out that in Experi-
ment 28, rat 209 escaped infection when exposed to infection by 138 fleas
during a period of one day, at the beginning of which the age of infec-
tion in the fleas could not have been less than a few hours nor more than
five days at the end. Rat 215 became infected by the fleas a day later,
when the age of the infection in the fleas could not have been less than
two or more than six days.
Putting together the results of this and the last section, it
is seen that fleas in which there is a possibility, from the data
of the experiment, of the infection being more than five days
old, may fail to produce infection, although the subsequent
history of those fleas shows them to have been infected
effectively, but no infections have been obtained in any
experiment of which the data are incompatible with the infec-
tion being at least six days old in the fleas that produced the
infection.
(vi) The Infection of the Rat is brought about by
the Small Trypanosome-form which is the Final
Form of the Development.
This point is scarcely capable of direct proof, since it is
impossible to be absolutely certain that when an infection
has been produced no other forms of the developmental cycle
in the flea were introduced into the rat except the trypano-
some-forms. It can, however, be demonstrated in experi-
ments planned for that purpose, that the trypanosome-forms
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 625
are present when an infection is produced. Ndéller (1912)
and Wenyon (1913), have shown that the trypanosome-forms
were present in all cases in the feeces with which they infected
rats per OSs.
The following experimental results indicate that the try-
panosome-form is the effective agent in infection. In Experi-
ment 35 B twelve fleas were taken at hazard from the infected
breeding-cage and put on clean rat 243 for five days (23 : ii:
711 to 28:11: 711; Rat 243 was found to be infected on 3:
ii:711). Ten of the fleas recovered were then dissected (the
other two lost); of each flea the stomach was placed on one
shde in a drop of salt-citrate solution, the rectum on another
slide in another drop. Each stomach and each rectum were
then teased up and examined microscopically in order to see
if trypanosomes were present in any form; but since this
examination had to be performed very rapidly and cursorily
and without putting a coverslip over the drop, trypanosomes
may have been often overlooked, when they were not present
in abundance. Whether trypanosomes could be seen in the
fresh specimen or not, each teased-up stomach was inoculated
by means of a syringe into a separate clean rat; the rectum
was only inoculated if trypanosomes were seen in it.! After
the drop containing the teased-up stomach or rectum had
been drawn up into the injecting syringe the film of moisture
left on the slide was fixed with osmic vapour, stained with
Giemsa’s stain, and carefully searched for trypanosomes. The
following are the results obtained with each flea.
Flea (1).—Nothing seen in the fresh stomach or rectum. Stomach
inoculated into Rat 279. No infection produced. Nothing found in
the preserved film.
Flea (2)—As last, stomach inoculated in rat 280, no infection,
nothing found in the films.
1 The reason for the differential treatment of the stomach and rectum
was because we believed, at the time, that infection was brought about
by regurgitation of infective trypanosomes through the proboscis from
the stomach, and also because the presence of trypanosomes in the
rectum is not so easily overlooked as in the stomach.
626 E. A. MINCHIN AND J. D. THOMSON.
Flea (3).—As last, stomach inoculated into rat 281, no infection,
nothing found in the films.
Flea (4).—One sluggish stumpy form, which may have been erithi-
dial or trypaniform, was seen in the fresh teased-up stomach; nothing
seen in the fresh rectum. Stomach inoculated into rat 283, result
negative. Nothing found in the preserved film of the stomach.
Flea (5).—Nothing seen in the fresh stomach, numerous trypano-
somes seen in the rectum. Stomach inoculated into rat 285, result
positive. Rectum inoculated into rat 248, result negative. One try-
panosome-form and one transitional form found in the preserved film of
the stomach (Text-fig. 16, b and c). Nothing found in the preserved
film of the rectum.
Flea (6).—Nothing seen in the fresh stomach; a few forms, some
TrExt-Fic. 16.
S2C%
Small trypanosome-forms from the stomach-films of fleas 5 and 7
in Experiment 35 B, and flea 5 in Experiment 27 (see text).
(x 2000.)
stout and of crithidial appearance and some slender, apparently try-
paniform, seen in the rectum. Stomach inoculated into rat 286, result,
negative; rectum not inoculated. No films preserved.
Flea (7).—Nothing seen in the fresh stomach or rectum. Stomach
inoculated into rat 288, result positive. One trypanosome (Text-fig.
16, a) found in the preserved film of the stomach.
Fleas (8), (9), (10)—In each case nothing was seen in the fresh
stomach or rectum. The stomachs were inoculated into rats 289, 262,
263 respectively, results in each case negative. Nothing found in the
preserved films.
Summary.—In the case of two fleas out of the ten used,
the stomachs, when inoculated into clean rats, produced an
infection. The final trypanosome-stage was found in both
the stomachs that produced infections, but in none of the
remaining eight stomachs that produced no infection.
La J
Experiments 27, 29 and 32 were conducted in a different
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 627
manner. Fleas taken from the infected breeding-cage were
put each on a separate rat and left on it for three or four
days. The flea was then recovered (if it could be found),
dissected and examined.
Experiment 27.—Flea (5), placed on rat 207 for three days
(2: viii: 10 to 5 viii : °10) produced infection (see Table E). The flea
dissected (5 : viii), and one large transitional form found in the slide of
the stomach (Fig. 16, d).
TExtT-FiG. 17.
Various forms (haptomonad, a—d, nectomonad, e-h, transitional,
il, and trypaniform, m and n), from the stomach-film of flea
3, Experiment 29 (see text). (x 2000.)
Experiment 29.—Flea (3) placed on rat 212 for three days,
(19 : ix : 710 to 22 : ix : 10), recovered and dissected 22 : ix °10 (see Table
E). Large clumps of attached forms were seen in the stomach and also
free forms; nothing was seen in the intestine, rectum, salivary glands
or proboscis. The preparations of the stomach showed crithidial,
transitional and trypaniform types in abundance (Text-fig. 17). Rat
212 became infected and first showed trypanosomes in the blood on
28: ix. Four other fleas in the same experiment failed to infect their
rats; in two of these fleas nothing was found, in the third a small
628 E. A. MINCHIN AND J. D. THOMSON.
Trxt-Fic. 18.
4
>
mM.
h. J:
Various forms (haptomonad, a, b, nectomonad, ¢, d, transitional,
e-h, and trypaniform, 7—m), from the stomach-film of flea 3,
Experiment 32 (g) (see text). (x 2000.)
TExtT-FIG 19.
Various forms (haptomonads, a—d, nectomonad, e, transitional,
f~, and trypaniform, j-m) from the rectum-film of flea 3,
Experiment 32 (h) (see text). (x 2000.)
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 629
attached clump was seen in the intestine, nothing in any other part of
the flea. The remaining flea was not recovered.
Experiment 32 (g).—Flea (3) placed on rat 241 for four days (5X1:
10 to 7: xi: 10) produced no infection. In the stomach of the flea,
dissected 8: xi, crithidial, transitional and trypaniform types were
found (Fig. 18).
Experiment 32 (h).—Flea(3) placed onrat 244 for four days (11 :xi-710
to 15:xi 10) produced an infection (see Table E). The flea was
dissected 15 : xi; nothing was found in the stomach, intestine or salivary
glands ; the rectum showed a typical swarming “ pile-carpet ” infection
with all the usual types of form (Text-fig. 19).
TExt-FIG. 20.
og ade
Flea3, Experiment 32(i). a, haptomonad, and b, trypanosome-form
from the rectum-film; cand d, haptomonads, e-g, nectomonads,
from the film of the intestine (see text). (x 2000.)
Can a
Experiment 32 (i).—Flea (3), placed on rat 247 for three days
(12: xi: 710 to 15: xi: °10), produced no infection. The flea, dissected
and examined (16: xi), showed no trypanosomes in the stomach, but
in the intestine were clumps attached behind the pylorus (Text-fig. 20, c),
and the rectum contained a teeming infection (Text-fig. 20, a and b) of
the usual types.
The cases cited show that when infections were produced
the final trypanosome-form was found either in the stomach
or rectum; but it should also be mentioned that we have
three instances in which the rat became infected under similar
circumstances without our having been able to discover an
infection of the flea, which must have been so scanty as to
escape detection in our films. On the other hand the experi-
ments also show that the infective form may be present in the
630 E. A. MINCHIN AND J. D. THOMSON,
flea without any infection resulting when the flea is on the rat
for not more than four days. The failure of the flea to infect
in such cases must be correlated with the casual nature of the
contaminative method of infection by the fleas, evidently not
so sure a method as that of inoculation. It will be shown
further (Experiment 39, below) that the period at which the
flea becomes infective coincides with the first appearance of
the small trypanosomes in the rectum.
(vii) The Final Infective Form of the Cycle is deve-
loped first in the Rectum on the Fifth Day of
the Developmental Cycle, but may appear later
in the Stomach.
In order to ascertain how soon the trypanosomes, ingested
by the flea, attain to maturity in the different parts of the
digestive tract of the flea an experiment (Experiment 39) was
carried out in the following manner. A number of fleas
(about one hundred) were collected from the non-infected
breeding-cage, put into test-tubes, with clean sand, slightly
damp, and kept there for four days (20:iv:711 to 24:iv:71]),
in order that they should be properly hungry and ready to
feed. The fleas were then (24: iv) put into a special flea-
proof tin cage with a well-infected rat (No. 259).’
At regular intervals batches, each of ten fleas, were
recovered from rat 259, kept in the test-tubes on sand, and
dissected on the following day (to ensure that the fleas had
not ingested blood containing trypanosomes for at least a day
previous to being dissected). In the dissection of the fleas,
the flea was first placed on a slide in a drop of salt-
citrate solution and the proboscis removed by cutting through
the head in the region of the eyes; the proboscis was then
placed in a separate capsule in a small quantity of salt-
citrate solution. Very often feces were extruded when the
+ Rat 2459 was put in with a single flea on 22 : iii: “11; trypanosomes
were first seen in the blood on 30: iii; multiplication was ended on
4: iv; see Table E, p. 617 above.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 631
head was cut through. When this occurred the carcase of
the flea was at once removed to another slide, and the
extruded faeces examined microscopically. If trypanosomes
were found in sufficient abundance the slide and coverslip
were fixed and stained. The carcase of the flea was then
opened in the hinder end of the abdomen, and the junction
of stomach and intestine cut through behind the Malpighian
tubes, after which the portion of the carcase containing the
stomach was transferred to another slide. ‘Then the stomach
and Malpighian tubules, together with the proventriculus and
cesophagus, were removed together and transferred to a
second capsule, and the proctodzum (intestine with rectum)
to a third. In making these dissections some of the con-
tents of the stomach or rectum escaped on to the slide (the
organs being purposely punctured to allow some contents
to escape, when necessary). The escaped contents were
examined microscopically, and if trypanosomes were found in
them in sufficient numbers they were preserved.
After all the 10 fleas of each batch had been dissected in
this way the 10 proboscides were inoculated into one clean
rat, the 10 stomachs into another, and the 10 recta into a
third. In each case the whole of the salt-solution in the
capsule was injected also. he stomachs and recta were
teased up as fine as possible, and the proboscides crushed up,
before injecting them.
The following are the actual injections performed; in the results
stated, 0 signifies that the rat inoculated acquired no infection, +
that it became infected.
26: iv :°11.—The 10 fleas recovered on the previous day were dis-
sected ; trypanosomes were seen in the stomach, rectum, and extruded
feces of several fleas. All trypanosomes seen appeared to be of quite
ordinary type:
10 proboscides injected into rat 289: result 0
10 stomachs ,F of 299) e eee 0)
10 recta i - BUG oe 0
97: iv :’11—The 10 fleas recovered on the previous day were dis-
sected; trypanosomes were seen in the stomach of one, the stomach and
extruded feeces of another, in the stomach and rectum of a third, and in
632 E. A. MINCHIN AND J. D. THOMSON.
the rectum and extruded feces, very abundantly, of a fourth; the
rectal forms appeared pear-shaped or club-shaped in the living state
(Text-fig. 21), but no post-crithidial trypanosome-forms were present :
TEXT-FIG. 21.
Various forms from the rectum and feces of a flea of the batch
of 27:iv:’ll. Note that no final trypanosome-forms are
present (see text). (x 2000.)
10 proboscides injected into rat 301: result 0
10 stomachs 2 eo eens pA arr AU)
10 recta “ Se OUD a same
28: iv:’11—The 10 fleas recovered on the previous day were dis-
sected ; trypanosomes were seen in the stomachs of three, and in the
stomachs and extruded feces of two others. The feces were preserved
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 633
in one case where they were seen, but no trypanosomes were found in
the preparations :
10 proboscides injected into rat 304: result 0
10 stomachs . eee U5 en me 0
10 recta % ae iy ose f:.2 0
29:iv:°11—The 10 fleas recovered on the previous day were dis-
sected; trypanosomes were seen in the extruded feces of one, in the
rectum and feces of another (abundantly) and the rectum of a third
(abundantly) ; of the last two, preparations were made of the rectal con-
tents, and there were found pear-shaped crithidial, transitional, and
post-crithidiai trypaniform individuals (Text-fig. 22).
TEXT-FIG. 22.
boon (AL
bark OC)
Various forms from the rectum and feces of two fleas of the
batch of 29:iv:’11, Experiment 39 (see text). Note the trypano-
some-forms (last 4 figs. to the right, second row). (x 2000.)
10 proboscides injected into rat 307: result 0
10 stomachs oa ‘S ty Talis ~ o5.8 4)
10 recta 5 3 pes Neh Mgt Se
1:yv:’l1l.—The fleas recovered 29:iv were dissected; in one of
them, a male, minute crithidial individuals were seen in the stomach
contents, but not in the rectal contents; no preparation was made.
10 proboscides injected into rat 310 : result 0
10 stomachs . = alll Be se (O)
10 recta Se Fe oli tes)
4:v:'11.—The 10 fleas recovered on the previous day were dissecte d,
trypanosomes were seen in the rectum of one, in the extruded feces of
another; no preparation made.
10 proboscides injected into rat 313: result 0
10 stomachs re = SeolAr .s0 i
10 recta By - olor: pee O
634 E, A. MINCHIN AND J. D. ''HOMSON.
Summary of Experiment 39.—None of the rats inocu-
lated with organs of the fleas which had been exposed to
infection two, three, or four days previously became infected.
On the fifth and seventh days inoculation of the recta pro-
duced infections, while the inoculations of the stomachs were
negative. On the tenth day, on the other hand, inoculation
of the stomachs gave a positive, that of the recta a negative
result. It is seen, therefore, (1) that the fleas first became
infective on the fifth day, when also the post-crithidial try-
panosomes were first found in preparations of the rectum ;
(2) that the developmental forms which produce infection
were present in the rectum, but not in the stomach, on the
fifth and seventh days; and in the stomach, but not in the
rectum, on the tenth day.
(vii) The Developmental Forms of the Trypano-
somes in the Flea are not infective when
inoculated into the Rat during a period ex-
tending from a short time (half an hour?)
after being taken up by the Flea until the
Developmental Cycle is complete.
After we had shown, in Experiment 39 (see above), that
the trypanosomes in the flea are not infective to the rat after
they have been in the flea for two days, and that they do
not acquire infectivity for five days after being ingested,
we instituted a number of experiments with a view of dis-
covering how soon the ingested trypanosomes lose their power
of intecting.
In our first experiment a number of fleas collected from our
non-infected breeding-cage and kept hungry for three days
(7: x1:711 to 10: xi:711), were put on a well-infected rat at
6 am. (10:xi:’11) and collected two hours later. They
were then dissected in batches, the stomachs of each batch
being placed together on the same slide, teased up in a drop
of salt-solution, drawn up intoan injection-syringe and inocu-
lated into a clean rat. After the drop had been drawn up
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 635
into the syringe the film of moisture left on the slide was fixed
with osmic vapour and stained with Giemsa’s stain, in order
to get an idea of the modification, if any, which the trypano-
somes had undergone.
The following were the batches dissected and injected :
Trxt-FIG. 23.
cv.
=r
Trypanosomes from the stomachs of fleas of the batches of
10: xi: 11 (see text).
(1) Four fleas, injected into rat 323 at 11.55 a.m. The preserved film
showed trypanosomes of the ordinary blood-type.
(2) Four fleas, injected into rat 324at 12.55 p.m. The preserved film
showed trypanosomes of the ordinary blood-type (Text-fig. 23, a, b).
(3) Four fleas, injected into rat 325 at 1.30 p.m. The preserved film
showed trypanosomes for the most part unmodified, with » approxi-
mated to N, others dwarfed slightly as if beginning to degenerate
(Text-fig. 23, ¢, d).
(4) Four fleas, injected at 2.35 p.m. into rat 326. The preserved film
showed trypanosomes of the ordinary blood-type (Text-fig. 23, e, f).
(5) Four fleas, injected at 3.30 p.m. into rat 327. The preserved film
636 E. A. MINCHIN AND J. D. THOMSON.
showed trypanosomes mostly modified, some rather lengthened out,
others recurved (Text-fig. 23, g, h, z).
(6) Three fleas, injected at 4.35 p.m. into rat 327. The preserved film
showed trypanosomes mostly unmodified,some rather long (Text-fig. 23,
j, k).
All the results were negative, since none of the rats
became infected. It is seen from the times of feeding and
injecting the fleas that none of the trypanosomes had been
in the fleas more than ten and half hours (6 a.m. to 4.35 p.m.),
or less than three and half hours (8 a.m. to 11.30 a.m.).
After obtaining this result we made a number of other ex-
periments, modifying slightly our method of procedure. Fleas
taken from the non-infected breeding-cage were fed under
observation on a well-infected rat and the time of feeding
noted. The flea was recovered, dissected, and its stomach
injected into a clean rat after being teased up in a drop of
salt-citrate solution. As before, the film of moisture left on
the slide was preserved in some cases.
In some cases also some blood from the infected rat was
inoculated subcutaneously into a control clean rat. The
controls were not always positive, however, since the sub-
cutaneous method of injection is notoriously less efficient
than the intra-peritoneal method, when a small quantity of
blood is taken direct from the rat.
27: xi: ‘11.—Fleas fed under observation on an infected rat after
having been kept hungry for three days.
Fleas (1) and (2) fed at 11.35, injected into rat 329 at 12.10 (35
minutes). The preserved film showed trypanosomes quite unmodified.
Flea (5) fed at 11.45, injected into rat 329 at 12.20 (35 minutes). The
preserved film showed trypanosomes quite unmodified.
Flea (4) fed at 11.30, injected into rat 330 at 12.30 (1 hour). Try-
panosomes in preserved film quite unmodified.
Flea (5) fed at 11.50, injected into rat 330 at 12.50 (1 hour). Try-
panosomes seen in the fresh stomach, but film not preserved.
Flea (6) fed at 11.50, injected into rat 330 at 12.50 (1 hour). Film
badly preserved.
Flea (7) fed at 10.45, injected into rat 331 at 12.45 (2 hours). Try-
panosomes in preserved film quite unmodified.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 637
Summary.
Rat 329 was injected with 3 fleas 35 minutes after feeding.
99 = .
880) iy = oe a) al hour Ay ry
ess: . 1 flea 2 hours after feeding.
The results in all three cases were negative. No control
rat was injected.
8:xii: 711.—Fleas fed under observation on an infected rat (322).
Fleas (1) and (2) fed at 11.40, injected into clean rat 324 at 12.26
(36 minutes). Active trypanosomes seen in the fresh stomachs.
Fleas (3) and (4) fed at 11°53, injected into rat 324 at 12.37 (44
minutes). Trypanosomes seen in the fresh stomachs.
Fleas (5), (6), and (7) fed at 12.4, injected into rat 324 at 12.42
(38 minutes). Trypanosomes seen in the fresh stomachs.
Fleas (8), (9), and (10) fed at 12.10, injected into rat 324 at 12.47
(37 minutes). Trypanosomes seen in the fresh stomachs.
Flea (11) fed at 12.20, injected into rat 324 at 12.55 (385 minutes).
Summary.—Rat 324 inoculated with the stomachs of
eleven fleas fed on rat 322 between 35 and 44 minutes pre-
viously. Result negative.
Control rat 323 inoculated at the same time with a small
drop of citrated blood from rat 322. Result positive (found
to be infected on 15 : xi).
24:i:°11—Hight fleas, kept hungry for three days previously, were
fed under observation on an infected rat (No. 392); the stomachs of six
inoculated into clean rat 411.
Flea (1) fed 10 a.m, injected 11 a.m. (1 hom).
Flea (2) ., 10.10 a.m., injected 11.10 a.m. (1 hour).
Flea () ,, LO0:I5 . a 11.14 ,, (59 minutes).
Flea (4) ,, 10.20 ,, un EO) Gvhour):
Flea (5) ,, 10:25 _., ae 2S 3
Bieai(G) - 2029) 5 5 20)
Fleas (7) and (8), fed 10.33 a.m. and 10.38 a.m. respectively, were
dissected and fixed preparations made of them; numerous trypano-
somes, quite unmodified in appearance, were found in them.
Summary.—Rat 411 inoculated with the stomachs of six
fleas, each of which had fed on infected rat 392 an hour
previously. Result negative.
638 E. A. MINCHIN AND J. D. THOMSON.
Control rat 412 inoculated with a small drop of citrated
blood from rat 392 at 12 noon. Result negative. (N.B.—
Rat 412 put into the infected breeding-cage on 15:11: 713
contracted an infection in due course and was therefore not
naturally immune.)
11: ii: ’°13.—Seven fleas fed on infected rat 402 under observation.
The stomachs inoculated into clean rat 414.
Flea (1) fed 10. 9 a.m., injected 11. 7 a.m.
Flea) 1012, ap, dlalOees
Fleas). .,10A7 ~,, Jer oleh yeas
Pilea (4) ,, 10.25 ., au Ob yeep
Hlea (5) ,,.1028 4, dee leo
Eiea6) 1097 ., i HESS
Hiea(7) ..1058- 5, . ss
(In all cases the time of feeding was reckoned from the moment the
flea withdrew its proboscis.)
Some of the blood-was allowed to escape from the stomach of flea (7)
and permanent preparations made; numerous trypanosomes quite
unmodified in appearance were found.
Summary.—Rat 414 acquired no infection; control rat
415, inoculated with one drop of blood from rat 402, became
infected (trypanosomes first seen, 21 : 11: 713).
25 : ii :’138.—One flea was fed under observation at 10.45 a.m. on
infected rat 415; the stomach was inoculated into clean rat 419 at 11.45.
Results negative. (The other fleas refused to feed.)
27:i1:°13.—Hight fleas fed under observation on infected rat 415;
the stomachs inoculated into clean rat 419,in each case 59 minutes or an
hour after feeding. Result negative.
Control rat 414 inoculated with a drop of blood from rat 415. Result
negative (rat 414 was later infected by being put into the infected
breeding-cage.)
It will be seen from the foregoing accounts that all experi-
ments, in which infected blood ingested by a flea was mocu-
lated into a clean rat from about half an hour onwards after
ingestion by the flea, gave uniformly negative results. ‘The
controls were sometimes negative, sometimes positive.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 639
(ix) The Flea, when once it has become infective,
remains so for a considerable Length of Time.
This point was dealt with in our preliminary account (1910),
when it was shown in two experiments (“C” and “D 7’) that
cages of fleas when once rendered infective continued to pro-
duce infections for some time without being re-infected.
Experiment 22 (‘‘C’’) was continued for some time after the
publication of our paper, but on 28:1:710 an infected rat
was unfortunately introduced into the cage, an oversight
which vitiated all subsequent results, and the experiment was
discontinued. During the period prior to this accident, how-
ever, the experiment was not open to objection, and produced
a result which may be summarised as follows. A cage
colonised with 160 clean fleas, into which an infected rat was
introduced for three days (24 to 27 : xi: ’09), and which
produced the first infection between 30 : xi: ’09 and 3: xii: 09,
continued to produce infections without being re-infected up
to 24:1:710, a period of approximately 55 days. It is,
therefore, a safe conclusion to aflirm that the infectivity
persisted in some fleas, or at least one flea, for that period
of time.
Later a more exact experiment (Experiment 37) was carried
out, in which single fleas were used. ‘To begin with, 10 fleas
were taken from the infected breeding-cage and put each on
a clean rat for four days; at the end of that time the 10 fleas
were recovered and each flea put by itself in a separate test-
tube in damp sand for six days. Meanwhile the 10 rats
were examined daily, and in due course two of them
developed infection, the remaining eight being negative.
The eight fleas that gave negative results were returned to
the infected breeding-cage.
The two fleas that had been proved experimentally to be
infective—henceforth known as Flea “A” and Flea “B”?—
were then placed singly on a succession of clean rats. That
is to say, each flea was placed by itself on a clean rat for so
many days, then was recovered and placed on another clean
rat for so many days, and so on. ‘I'he experiments with each
vou, 60, PART 4,—NEW SERIES. 44
640
E. A.
MINCHIN AND J. D. THOMSON.
flea were continued until the flea disappeared: that is to say,
until the flea could not be recovered when sought for.
The details and results of Experiment 37 have been sum-
marised in tabular form in our preliminary communication
(1911), but since we have frequently had occasion to refer
to the experiment in the present memoir, we think it worth
while to reproduce the table already published.
Taste G.-Summary of Experiment 37.
Flea “A.”
Trypanosomes.
Flea on the rat. Result. |— =
First seen in blood. Multiplication ended.
14-18:1:711 — Q4:11:°11 Py san 91 (4L
24: ii- il iil: iT: 0
1-6: i117 711 + 15:11: 711 (Rat died,
16: iii: 11)
6-13 : 11:11 0
13- hg :ii: Ll 0
yee sir 211i _ Dif = Ws AL 31:i1:°11
23-— see 0)
yf eriell aie 3 lit + Div Ul 9:iv:’1l1
Hes sree A 0
5-10 av. Lt 0
10- Deve al 0
52.0 ave on 22 :iv:711 95 :iv: 711
D0=Dbiiv eel + iboyeo- 1 Dewi wl
95-29 :iv:711 i esyeevlal! 6:v:71l
29 :iv-4:v:711 0
4-9: v:°11 ? \(Rat died, 9:iv:"11)
9215): vi: 1 0
(l5:v ot ‘flea not recov ered. )
RVieane tae
14-18 :i1: 711 = 94:1: "11 28:11:11
94:ii-1: ii: 711 0)
1-6 :11:711 0
6-13 : 111: 711 0
13=07 21 271 0
pee oii 1 0
23-202 Te + 30:10: 711 4’:iv : 711
97 :ii-L:iv:’Ll + Siawiemlal 1 .Avieoiel:
Neate 971i! 0
5-10:iv: 711
(LO cance*
0
11 flea not recovered.)
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 641
From the tabular summary it is seen that flea “A”
remained infective from about 15: 11:’11 to about 26: iv: 71]
—that is to say, for a period of 70 days—and flea “ B” from
about 16: i1: 711 to about 27 : 11: 711—a period of 40 days.
We have no data for determining the maximum period of
time during which a flea can remain infective. It is, perhaps,
not improbable that a flea, once rendered infective, may
remain so as long as it lives, but we have no knowledge with
regard to the average longevity of the flea. Flea “A” (?)
lived under our care from 14:11: 711 to 9: v :’?11—a period
of 84 days; but we have no clue as to its age when it was
first taken from the breeding-cage.
(x) The Trypanosome does not penetrate into the
Salivary Glands of the Flea, but is confined,
during its whole Development, to the Diges-
tive Tract.
To prove this point we began by dissecting out salivary
glands of fleas taken from the infected breeding-cage and
examining the glands both in the fresh condition, by teasing
them up or crushing them with a coverslip, in a drop of salt-
citrate solution, and also by means of fixed permanent smears
of the glands. In a large number of glands examined very
carefully in this way many yeast-like organisms and other
similar bodies were found, but never anything that appeared
in the least like any possible stage of a trypanosome.
Examination of fluid from the body-cavity gave negative
results also.
Thinking that we might have failed in these examinations
to obtain an infective flea, we took fleas from the infected
breeding-cage, put them singly on clean rats for three days
or so, and then recovered and dissected them. The organs of
the fleas were examined carefully in the fresh condition, and
in some cases permanent preparations were also made of
them and laidaside. If arat became infected subsequently,
and so proved the flea put on it to have been infective,
the preparations of that flea were searched very caretully.
642 KE. A. MINCHIN AND J. D. THOMSON.
In this way we were able to examine the salivary glands
and other organs of fleas which had been proved experi-
mentally to be infective. The following are the details of the
experiments and observations; the sign + signifies that
trypanosomes were found in the organs mentioned, while 0
means that none were found:
(1) Flea ($) put on rat 212 for three days (19 : ix : °10 to 22: ix 710,
Experiment 29). Rat 212 found to be infected 28 : ix; multiplication
ended 30: ix. Flea dissected 22: ix; proboscis 0, salivary glands 0,
stomach + (Fig. 17), rectum 0, intestines 0.
(2) Flea (¢) put on rat 223 for four days (10: x: 710 to 14: x:’10,
Experiment 32a). Rat 223 found to be infected 17: x ; multiplication
TEXT-FIG 24.
Yeast-like bodies of various kinds from the salivary glands of a
flea. They are shown in groups, as they were found in the
preparation. (x 2000.)
ended 22: x. Flea dissected and examined 14: x; no trypanosomes were
seen in the proboscis, body-cavity, stomach, intestine, rectum or salivary
glands ; but unfortunately no permanent preparations were made.
(3) Flea (2) put on rat 233 for five days (20: x: 710 to 25: x: 710,
Experiment 32e). Rat 233 found to be infected 28 : x; multiplication
ended 31: x; scanty infection, with few trypanosomes. Flea dissected
and examined 25 : x; no trypanosomes seen in proboscis, body-cavity,
stomach, intestine, rectum or salivary glands; permanent preparations
made of salivary glands, 0. (N.B.—Many yeast-like bodies in the
salivary glands, see Text-fig. 24.)
(4) Flea (¢) put on rat 238 for three days (1 : xi: °10 to 4: xi: “10.
Experiment 32f). Rat 238 found to be infected 14: xi; multiplication
ended 16: xi. Flea dissected and examined 4: xi; no trypanosomes
were seen in the proboscis, stomach, intestine, rectum or salivary
glands; permanent preparations made of the stomach and salivary
glands, both 0.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 643
From the foregoing experiments it is seen that nothing
which could be recognised as a trypanosome was found in the
salivary glands of four fleas known to have been infective.
In one of the four fleas the salivary glands were examined
only in the fresh condition, but in the other three fleas the
salivary glands were examined both in the fresh condition
and in the permanent preparations.
It will be remarked, however, that no trypanosomes were
found in any part of the digestive tract, when examined fresh,
in three out of the four fleas, although there can scarcely be
any doubt that trypanosomes must have been present. Asa
matter of fact, a scanty infection of the crithidial or final try-
panosome-forms, small in size and sluggish in movement, is
easily overlooked in the fresh preparations of the teased-up
digestive tract, especially in an organ relatively so large as
the stomach, which may be gorged with blood-débris greatly
hindering and obscuring the examination. It has been our
experience not infrequently that the smaller forms of the
cycle have been found scantily in permanent preparations of
stomachs in which nothing was seen in the fresh examination.
This scarcely applies, however, to organs so minute as the
salivary glands, the contents of which can be scanned com:
prehensively in one field of the microscope. We have, there+
fore, cited the second flea in spite of the fact that no per-
manent preparations of the salivary glands were examined.
After having made the negative observations recorded
above, the idea occurred to us that any form of the trypano-
some found in the salivary glands would probably be a final
stage of the cycle, destined to be inoculated by the flea
through the proboscis into the rat; and that consequently the
examination of fleas which had produced an infection recently
would be inconclusive, since in such fleas the salivary glands
might be purged of their infection, temporarily at least.
We therefore carried out some experiments in which the
object was to determine which organs of the flea contained
the infective stages of the trypanosome, by dissecting fleas
taken from the infected breeding-cage and injecting their
644. E. A. MINCHIN AND J. D. THOMSON.
organs separately into clean rats. Thus a batch of fleas, at
least five in number, was taken from the infected breeding-
cage, and all the fleas in the batch were dissected at the same
sitting. The stomachs of all the fleas were put together in
one watch-glass and the salivary glands in another. Each
flea has four salivary glands (two on each side of the body),
but we did not succeed in every case in dissecting out all
four of these minute organs; sometimes only two or three
were obtained, or even one only of the four (on foggy morn-
ings) ; but in every case at least one of the four glands was
obtained. In the case of the recta (cut off behind the pylorus
and therefore including the greater part of the intestine
as well), each was examined microscopically and only kept
for injection if seen to contain trypanosomes.
In our earlier experiment (Experiment 33) only the salivary
glands were used for injection, with the following results :
Taste H.—Experiment 33.
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670 E. A. MINCHIN AND J. D. ‘THOMSON.
answer is in the negative, it cannot be regarded as certain,
but only as possessing a greater or less degree of probability.
In order to test this point we fed batches of fleas on infected
rats and then divided each such batch usually into two batches,
Aand B. Batch A in each such case was kept starved until
it was examined; batch B was fed again before being
examined. In cases where fleas of batch B were found on
examination not to have availed themselves of the chance of
feeding, they were reckoned in batch A. Sometimes the
original batch was not divided, but treated as a whole either
as an “A” (not re-fed) or “B” (re-fed) batch.
The results of these experiments are tabulated in Table M,
which seems at first sight decidedly in favour of the conclu-
sion that the trypanosomes cannot persist beyond the second
feed of the flea. It isseen that in forty-nine fleas not fed
again after the infective feed, trypanosomes were present in
twenty cases; in thirteen of the cases the trypanosomes were
of the long stomach type and in eight cases intracellular forms
were seen. On the other hand, in thirty-two fleas examined
after having been fed a second time, the typical multiplicative
stomach-phase was not present in a single instance. Unfor-
tunately, the force of these figures is rather weakened by the
fact that, of the fleas fed a second time it can only be asserted
positively in seven cases that the rectum contained true
developmental crithidial forms and that the developmental
cycle was in these cases a “ going concern,” so to speak.
While the figures make it probable, to a certain degree, that
the stomach-phase, if it persists up to the time of the second
feed, must come to an end then, this conclusion cannot be
considered established and must remain a point for further
ce
investigation.
If it be true that the stomach-phase cannot persist beyond
the second feed, we may enquire how such a result is brought
about. It is intelligible that a fresh meal of blood might
sweep on all free, extracellular trypanosomes from the
stomach towards the rectum, but this would not account for
the disappearance of the intracellular forms. It has been
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 671
mentioned above that in some insects the epithelium of
the mid-gut is regenerated completely after each meal, and
we stated further that we were not in a position either to
affirm or to deny that, in the case of the flea, the regeneration
of the stomach-epithelium takes place in regular correlation
with the feeding. If it were so, however, it would become
quite intelligible why a second feed should put an end to the
intracellular multiplication in the stomach.
(xvill) Starvation of the Flea during the Incuba-
tion Period of the Cycle does not inhibit, nor
does it necessarily retard, the Developmental
Cycle of the Trypanosome in the Flea.
Experiment 45.—A batch of about 250 clean fleas, having been
collected from the non-infected breeding-cage and kept hungry forthree
days, were put (22: vii:°13) on a well-infected rat, for about twenty-four
hours. The next day 150 of the fleas were recovered and kept in a flask
containing some damp sand.
Two days later (25: vil), thirty of these 150 fleas in the flask were put
into a freshly-prepared bell-jar A with aclean rat 368. The next day
(26: vii) rat 568 was removed from bell-jar A, all the fleas on it being
cleaned off carefully and put back into the bell-jar. Rat 368, examined
regularly up to 26: viii, did not become infected.
Two days later (28 : vii), clean rat 368a was put into bell-jar A, con-
taining the fleas that had been in contact with rat 368. On the same
day. thirty more fleas from the flask were put into bell-jar B with clean
rat 369; another thirty in bell-jar C with clean rat 370; and another
thirty in bell-jar D with clean rat 371. It will be remembered that the
fleas in the flask had been exposed to infection for one day (22-23: vii),
and kept without food since then; consequently, bell-jars B, C, and D
were colonised each with thirty fleas that had been exposed to infection
between five and six days previously and starved since then.
The next day (29: vii), rat 368a was removed from bell-jar A and all
fleas recovered from it put back into the bell-jar. Rat 368a did not
acquire infection. The same day rats 369, 370, and 371 were removed
from bell-jars B, C, and D, all fleas being recovered from them and put
back into the respective bell-jars. Rats 369 and 370 did not become
infected ; rat 371, on the other hand, became infected in due course (see
Table C, p. 616).
Two days later (31: vii), rat 368b was put into bell-jar A, rat 369a
into bell-jar B, rat 370a into bell-jar C, and rat 371a into bell-jar D.
VoL. 60, PART 4.—NEW SERIES. 46
672 BE. A. MINCHIN AND J. D. THOMSON.
The next day (1: viii), rats 369a, 370a and 37la were removed from the
bell-jars B, C, and D, and the fleas on them carefully recovered and put
back into their respective bell-jars. Rat 368b was left in bell-jar A. In
the sequel rats 368b, 369a, and 370a did not become infected; rat 371a
showed infection in due course.
Two days later (3: viii), clean rats 369b, 370b and 371b were placed in
bell-jars B, C, and D, and left in till they should become infected. Rat
369b did not become infected ; rats 370b and 371b became infected in
due course.
The results of the experiment may be summarised in the
following manner. We start with four batches (A, B, C, D),
each of thirty fleas, which had been exposed to infection on
the same rat for one day (22: vii to 23: vii).
(1) Batch A (bell-jar A) :
Put on rat 368 from 25: vii to 26: vii (about three days
after exposure to infection); result negative.
Put on rat 368a from 28 : vii to 29 : vii (about six days after
exposure to infection) ; result negative.
Put on rat 368b, 31 : vii (about nine days after exposure to
infection), and left onthe rat ; result negative.
This batch therefore did not become infective at all.
(2) Batch B (bell-jar B) :
Starved for five days (23: vii to 28: vii), then put on rat
369 from 28 : vii to 29 : vii (about six days after exposure to:
infection) ; result negative.
~ Put on rat 369a from 31: vil to 1: vii (about nine days
after exposure to infection) ; result negative.
Left in with rat 369b on 3: viii; result negative.
This batch therefore did not become infective at all.
(8) Batch C (bell-jar C) :
Starved for five days, then put on rat 370 from 28 : vii to
29 : vii (about six days after exposure to infection) ; result
negative.
Put on rat 370a from 31 : vii to 1: viii (about nine days after
exposure to infection) ; result negative.
Left in with rat 370b on 3 : viii; result positive (the
examinations of the rat indicate that infection took place
between 4: viii and 7: viii).
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 673
This batch therefore became infevtive.
(4) Batch D (bell-jar D) :
Starved for five days, then put on rat 371 from 28 : vii to
29: vii (about six days after exposure to infection) ; result
positive.
Put on rat 37la from 31 : vii to 1: viii (about nine days
after exposure to infection) ; result positive.
Left in with rat 37la on 3 : viii; result positive.
This batch evidently became strongly infective.
Remarks.—From the above summary it is seen that
batch A (not starved) and batch B (starved) failed to become
infective, while batches C and D (both starved) became
infective.
Batch © did not produce its first infection before 3 : vill;
that is to say not until eleven or twelve days, at least, after
exposure of the fleas to infection. It is not legitimate, how-
ever, to conclude from this that the developmental cycle of
the trypanosome was retarded, since it has been shown above
that infective fleas often fail to infect. The fleas may very
well have been infective when placed in contact with rats 370
and 370a, but they were in contact with these rats for only
about twenty-four hours. The most probable explanation for
the two failures to infect is that only a small number of fleas
in this batch were infective.
Batch D produced its first infection between 28: vii and
29 : vii, and since it was exposed to infection between 22: vil
and 23 : vii it follows from these figures that the incubation-
period—that is to say, the length of time taken by the
developmental cycle of the trypanosomes—must have been
between five and seven days. We are justified therefore in
concluding that the trypanosomes in this batch went through
a cycle of perfectly normal duration. The further fact that
this batch never failed to produce infection during the time
the experiment was carried on, indicates that the trypanosomes
went through their cycle and established themselves success-
fully in a relatively large number of the fleas.
To conclude: Batches C and D show that starvation of the
674 E. A. MINCHIN AND J. D. THOMSON.
fleas during the incubation-period does not inhibit the develop-
ment of the trypanosomes; and batch D shows further that
the development is not necessarily retarded by starvation.
(xix) Starvation of the Flea following immediately
on an Infective Feed favours the Establish-
ment of the Haptomonad Phase in the Rectum,
while Starvation begun after the Incubation-
Period in the Flea is over favours Migration
to the Post-Pyloric End of the Intestine and
the Establishment of the Haptomonad Phase
there.
Experiments 49 and 50 were carried out with the object of
ascertaining what effect, ifany, varying food-conditions might
have on the incidence and location of the established hapto-
monad phase in the flea’s gut.
Experiment 49.—21: iii: °14—A number of fleas collected from
the non-infected breeding-cage two days previously were put into a
bell-jar with a well-infected rat at eight a.m., and were recovered again
at twelve noon. They were then divided into two batches. Batch A,
consisting of fifteen fleas, was put into a flask with moist sand at
the bottom. Batch B (about forty fleas) was put into a bell-jar with
a clean rat (rat 477).
26 : iii : °14.—Five fleas of batch A and four of batch B were dissected
and examined.
Of batch A four were positive, one was negative. Of the four positive
three showed developing forms of the trypanosomes in both the
stomach and the rectum. Two of the three showed large numbers in
both stomach and rectum, and in one of the stomachs intracellular
forms were found. The fourth positive showed a haptomonad infection
in the rectum.
Of batch B only one of the four dissected showed trypanosomes, and
these were found free in the stomach-slide.
27: iii: "14.—Hight fleas of batch A and eight of batch B were
dissected and examined.
Of batch A seven were positive; one was negative. Of the seven
positive one showed long active forms in the stomach and haptomonads
in the rectum, while six showed developing forms in the rectum only—
three scanty and three in fair numbers attached mostly round the
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 675
rectal surface of the projecting intestine, but in other parts as well.
Of batch B only one was found infected, and it showed haptomonads
attached about the middle region of the rectum.
The remaining fleas of batch B were now divided into two batches—
batch Al and batch Bi. Batch A1, consisting of fifteen fleas, was put
into a flask with moist sand at the bottom, and batch B1 was left in the
bell-jar with rat 477.
4: iv :’°14.—Rat 477 was removed from the bell-jar and clean rat 478
was put in its place with batch B1.
Four fleas of batch Al and four of batch Bl were dissected and
examined.
Of batch Al three were positive and one was negative. Of the three
positive two showed trypanosomes in abundance in the post-pyloric
region of the intestine and nowhere else. In these the trypanosomes
were long and slender, and some were club-shaped; while in a third,
which showed one or two in the rectum also, there was a swarming in-
fection of haptomonads, as well as long, slender and club-shaped forms
in the post-pyloric region. The fourth flea was negative.
Of batch B1 all were negative.
The remaining fleas of batch Al were now allowed to feed on a clean
rat for a short time,
9: iv: 14.—Five fleas of batch Al and four of batch Bl were dis-
sected and examined.
Of batch Al only one flea was positive, and it showed a fair number
of slender trypanosomes in the post-pyloric region and nowhere else.
Of batch BI all were negative.
11 : iv: ‘14.—The remaining fleas of batch Al were allowed to feed on
a clean rat for a short time.
16: iv : “14.—Five fleas of batch Al and five of batch B1 were dis-
sected and examined.
Of batch Al two were positive and three were negative. The two
positives showed large numbers of trypanosomes, some free, long and
active, some club-shaped, and others were small, round and pear-shaped,
in clumps and attached so as to form a lining to the wall of the gut.
All were inthe post-pyloric region and nowhere else. Of batch Bl
all five were negative.
Rat 477 became infected in due course. Rat 478 never became
infected. This agrees with results of the examinations.
Experiment 50.—20: v : 14.—A number of fleas collected from the
non-infected breeding-cage two days previously were put into a bell-jar
with a well-infected rat late in the evening, were left overnight, and
were recovered next morning. They were then divided into two
batches. Batch A was kept in a flask with moist sand at the bottom.
676 E. A. MINCHIN AND J. D. THOMSON.
Batch B was put into a freshly-prepared bell-jar with a clean rat (rat
496).
26:v: °14.—Five fleas of batch A and five of batch B were dissected
and examined.
Of batch A three were positive and two were negative. Of the three
positive two showed haptomonads in the rectum, one being a swarming
infection, and the third showed many long, active forms in the post-
pyloric region only.
Of batch B all were negative.
28:v:°14.—Seven fleas of batch A and seven of batch B were
dissected and examined.
Of batch A five were positive and two were negative. Of the five
positive four showed developing forms in the rectum only, and of these
two were swarming infections. The fifth showed haptomonad infection
of the rectum, and also free active forms in the stomach.
Of batch B only one of the seven was positive, and it showed a
scanty infection of the rectum only.
The remaining fleas of batch B were now divided into two batches—
batch Al and batch Bl. Batch Al was put into a flask with moist
sand in the bottom. Batch B1 was put into a bell-jar with a clean rat
(rat 497).
vi: °14.—Fourteen fleas of batch Al and fourteen of batch Bl
were dissected and examined.
Of batch Al four were positive and ten were negative. Of the four
positives one showed haptomonads on the rectal surface of the project-
ing intestine and three showed trypanosomes (one Seu aS in the
post-pyloric region and nowhere else.
Of batch B1 five were positive and nine were negative. Of the five
positives one which had its stomach full of red blood showed a scanty
infection in the rectum only. The other four showed infection in the
post-pyloric regions only. Of these two (females) had small ova and
their stomachs were empty. The remaining two had a fair quantity of
bro wnish-coloured blood-débris in their stomachs. Rats 496 and 497
became infected in due course.
Summary.
A.—Fleas starved from immediately after the infective feed, dissected
and examined five and six days after the infective feed.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI.
677
|
| post-pyloric only, 1
~ Number of!
No. of ex- Number | a: : .
. fleas exa- | ; | Site of infection. Remarks.
periment. | “ined. | infected. | |
|
j — rt |
49 13 1 Stomach and Intracellular forms
rectum, 4; in one stomach |
rectum only, 7 | 3 scanty, 4 swarming
i—infections mostly in
| upper part of rectum |
| 50 12 | 8 Stomachand | |
|
rectum, 1;
rectum only, 6; 3 scanty and 5 swarm-
| ing
|
B.—Fleas that were put into bell-jar immediately after the infective
feed, along with clean rat, on which they could feed at any time.
sected and examined five and six days after the infective feed.
Dis-
= Number of} ~ |
| No. of ex- |- Number | .
| periment. Pens oe Saerarede | Site of infection,
49 12 «| 2 Stomach only, 1
y
Rectum only, 1
50 12 1 | Rectum only, 1
Remarks.
~Haptomonad_infec- |
tion in middle
| region.
|
Al.—Fleas in which starvation was begun six days after the infective
feed. During the six days the fleas had been fed on clean rats.
Those
of Experiment 49 were dissected and examined fourteen, nineteen, and
twenty-six days after the infective feed. Those of Experiment 50 were
dissected and examined thirteen days after the infective feed.
Remarks.
nad infection in 2.
Pile carpet infection
upper part; swarm-
ing haptomonad in--
|
es _ |Number of} 1
eee fleas exa- anes Site of infection,
mined,
|
, 49 14 6 — Post-pyloric only,5;| Swarming haptomo-
post-pyloric and
rectum, 1
| 50 14 4 Rectum only, 1;
post-pyloric only,
3
fection in 1.
G73.) E. A. MINCHIN AND J. D. THOMSON.
B1.—Fleas kept with clean rats in bell-jar ever since the infective
feed. Dissected and examined as in Al.
7 Number of} »
No. of ex- Number . ; ‘ i
F fleas exa- | . Site of infection. Remarks, |
periment. | “shined. infected.
49 13 0 — — |
50 14. 5) Rectum only, 1;| Stomach full of red |
|
post-pyloric only,| blood; !stomach |
4 empty and ova)
small in 2; stomach
contained fair quan- .
tity of blood-débris |
in 2. |
These results seem to throw light on the important function
that the nectomonad forms described as occurring in the
established rectal-phase may have in maintaining the infec-
tion in the flea. The optimum food-conditions for the
establishment of the haptomonad stage seem to lie some-
where between abundance and poverty, and between partial
and complete digestion of the blood-supply. More extended
1 Although the fleas of batches B and B1 in both experiments had
the chance of feeding on clean rats at any time from immediately after
the infective feed onwards, all may not have equally availed themselves
of the opportunity. Itis certain, in fact, from the condition of the ova
and of the stomachs of two females of batch Bl that showed post-
pyloric infection, that they, for some reason not ascertained, had
starved in the midst of plenty; and these should really be transferred
to batch Al. The remaining two fleas of batch B1 that showed post-
pyloric infection had evidently fed, but not quite recently. The other
infected flea of batch B1 had its stomach distended with red blood, and
in it the infection was in the rectum only.
In order to test the infectivity of batches Al and B1 in Experiment 50,
twelve fleas of each batch were put on clean rats, two fleas to each clean
rat. The result was that two of the six rats belonging to batch Al became
infected, while none of the six belonging to batch Bl became infected,
This may indicate that a period of starvation heightens the infectivity
of infected fleas, perhaps by inducing increased production of the final
propagative forms of the cycle; but further experiments would be
required to justify such a deduction.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 679
and varied observations are required, but so far as these
experiments go they show that the incidence, location, and
continued existence of the haptomonad stage in the flea’s gut
depend to a large extent on the food-supply. When, under
conditions of partial starvation, a sufficient supply of nourish-
ment cannot be obtained in the rectum, the haptomonad stage,
if established there, would die out, and the flea would lose its
infection were it not that the nectomonads produced in the
rectum migrate forwards and re-establish this stage nearer to
the food-supply. In like manner it may be assumed that
when the food-supply in the post-pyloric end of the
intestine becomes continuously too rich and abundant, the
nectomonads produced there migrate backwards to the rectum
and so the balance is maintained and the infection in the flea
is kept up.
LIstER INSTITUTE,
June, 1914.
BIBLIOGRAPHICAL REFERENCES.
Baldrey, F.S. H.—‘ Versuche und Beobachtungen iiber die Entwicklung
von Trypanosoma lewisi in der Rattenlaus Hematopinus
spinulosus,” ‘Arch. Protistenkunde,’ xv, 1909, pp. 826-332, 2
text-figs.
Breinl, A., and Hindle, E.—‘ Observations on the Life History of
Trypanosoma lewisi in the Rat Louse (Hematopinus
spinulosus),” ‘Ann. Trop. Med. Parasitol.,’ iii, 1909, pp. 553-
564, pls. i and ii,
Brumpt, E.—‘‘Evolution de Trypanosoma lewisi, etc., chez les
Puces et les Punaises,” ‘Bull. Soc. Pathol. Exot.,’ vi, 1913, pp.
167-171.
Chatton, E., and Delanoe, P.—* Leptomonas Pattoni (Swingle) et
Tr. Lewisi (Kent) chez l’adulte et la larve de Ceratophyllus
fasciatus,’ ‘C. R. Soc. Biol. Paris,’ Ixxiii, 1912, pp. 291-293,
17 text-figs.
and Léger, M.—* Sur un mode particulier d’agglutination et de
cytolyse simulant un enkystement chez les Leptomonas des
drosophiles.” ibid., Ixxii, 1912, pp. 171-173, 15 text-figs.
680 E. A. MINCHIN AND J. D. THOMSON.
| Gonder, R.—‘‘ Untersuchungen iiber arzneifeste Mikroorganismen: I,
Trypanosoma lewisi,”’ ‘C. B. Bakt. Parasitenkunde’ (I, Abt.
Orig.), lxi, 1911, pp. 102-113.
| Léger, L., and Duboseq, O.—* Les Grégarines et l’epithélium intestinal
chez les Trachéates,” ‘ Arch. Parasitol.,’ vi, 1902, pp. 377-473, pls.
li-vii, 8 text-figs.
- MacNeal, W. J.—* The Life-History of Trypanosoma lewisi and
Trypanosoma brucei,” ‘ Journ. Infect. Diseases,’ 1, 1904, pp.
517-543, pls. xi-xvi.
Manteufel.—‘Studien iiber die Trypanosomiasis der Ratten, etc.,”
‘Arbeiten k. Gesundheitsamte,’ xxxiii, 1909, pp. 46-83.
~~ Minchin, E. A.—‘The Structure of Trypanosoma lewisi in Rela-
tion to Microscopical Technique,” *Quart. Journ. Micr. Sci.,’ 53,
1909, pp. 755-808, pls. xxi-xxiil.
“\»— “On some Parasites observed in the Rat-flea (Ceratophyllus
fasciatus),” ‘Hertwig’s Festschrift,’ i, 1910, pp. 289-302, pl. xxiii.
Ww and Thomson, J. D.—‘ The Transmission of Trypanosoma
lewisi by the Rat-flea (Ceratophyllus fasciatus),” ‘ Proc.
Roy. Soe.’ (B), Ixxxii, 1910, pp. 273-285.
“The Transmission of Trypanosoma lewisi by the
Rat-flea (Ceratophyllus fasciatus),” ‘Brit. Med. Journ.,’ i,
1911, pp. 1809, 1310.
“On the Occurrence of an Intracellular Stage in the
Development of Trypanosoma lewisi in the Rat-flea,” ibid.,
ii, 1911, pp. 361-364,
\onicoll, W.. and Minchin, E. A.— Two Species of Cysticercoids from
the Rat-flea (Ceratophyllus fasciatus),” ‘Proc. Zool. Soce.,’
1911, pp. 9-13, 2 text-figs.
Noller, W.—‘ Die Ubertragungsweise der Rattentrypanosomen durch
Fléhe,” ‘Arch. f. Protistenkunde,’ xxv, 1912, pp. 386-424, 5 text-
figs.
SS
Nuttall, G. H. F—‘The Transmission of Trypanosoma lewisi by
Fleas and Lice,” ‘ Parasitology,’ i, 1908, pp. 296-801.
Prowazek, S. v.—‘ Studien iiber Saugetiertrypanosomen,” ‘Arbeiten k.
Gesundheitsamte,’ xxii, 1905, pp. 351-395, pls. i-vi.
Rabinowitsch, L., and Kempner, W.— Beitrag zur Kenntnis der Blut-
parasiten, speciell der Rattentrypanosomen,” ‘ Zeitschr. f. Hyg.
Robertson, M.—* Transmission of Flagellates living in the Blood of
certain Freshwater Fishes,” ‘Phil. Trans.’ (B), ccii, 1911, pp. 29-
50, pls. i and ii, 4 text-figs.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 681
Robertson, M.—‘ Notes on certain Aspects of the Development of
Trypanosoma gambiense in Glossina palpalis,” ‘Proc.
Roy. Soe.’ (B), lxxxv, 1912, pp. 241-248.
Rodenwaldt, E—‘Trypanosoma lewisi in Hematopinus
spinulosus,” ‘C. B. Bakt. Parasitenkunde’ (Abt. I, Orig.), li,
1909, pp. 30-42, pls. i-il.
Strickland, C.—* The Mechanism of Transmission of Trypanosoma
lewisi from Rat to Rat by the Rat-flea,” ‘ Brit. Med. Journ.,’
i, 1911. p. 1049.
— “Agrippina bona nov. gen. et nov. sp. representing a new
family of Gregarines.” ‘ Parasitology,’ v, 1912, pp. 99-108, pl. iv,
oo text-figs.
and Swellengrebel, N. H.—* The Development of Trypano-
soma lewisi in the Rat-flea (Ceratophyllus fasciatus),”
‘Proc. Cambridge Phil. Soe.,’ xv, 1910, pp. 531-533.
“Notes on Trypanosoma lewisi and its Relation
to certain Arthropoda,” ‘ Parasitology, iii, 1910, pp. 456-454,
1 text-fig.
Swellengrebel, N. H., and Strickland, C—‘The Development of
Trypanosoma lewisi outside the Vertebrate Host,” ibid., ii,
1910, pp. 360-369, 21 diagrams.
“Some Remarks on Dr. Swingle’s paper, ‘The Trans-
mission of Trypanosoma lewisi by Rat-fleas ete,” ibid., iv,
1911, pp. 105-108.
Swingle, L. D.—‘* The Transmission of Trypanosoma lewisi by
Rat-fleas (Ceratophyllus sp. and Pulex sp.) with Short
Descriptions of three new Herpetomonads,” ‘ Journ. of Infect.
Dis., viii, 1911, pp. 125-146, pls. i-iv.
Thomson, J. D.—*‘ Cultivation of the Trypanosome found in the Blood
of the Gold-fish,” ‘Journ. Hygiene,’ viil, 1908, pp. 75-82, pl. iii.
Wenyon, C. M.—* Experiments on the Transmission of Trypano-
soma lewisi by means of ‘Fleas,’ ‘Journ. London School
Trop. Med.,’ ii, 1913, pp. 119-123.
682 E. A. MINCHIN AND J. D. THOMSON.
DESCRIPTION OF PLATES 36 to 45,
Illustrating Mr. E. A. Minchin and Dr. J. D. Thomson’s
paper on “The Rat-Trypanosome, Trypanosoma
lewisi, in its Relation to the Rat-Flea, Cerato-
phyllus fasciatus.”
(In all cases where the age of a trypanosome is stated, it is to be
understood as reckoned from the first infective feed of the flea, or, in
cases where the fleas were left on the infected rat for some days, from
the time when the fleas were first given the chance of feeding on it.)
PLATE 36.
[Various stages of the stomach-phase in film-preparations fixed with
osmic vapour and stained with Giemsa’s stain; drawn with the camera
lucida at a magnification of 3000. |
Figs. 1-12.—F ree (extracellular) trypanosomes.
Figs. 1 and 2.—Six hours; fig. 1, practically unmodified; fig. 2,
slightly modified in structure.
Figs. 3 and 4.—Twelve hours.
Figs. 5 and 6.—Highteen hours.
Figs. 7-10.—Twenty-four hours.
Figs. 11 and 12.—Forms of crithidial structure from an abnormal
flea (see p. 519); fig. 11, a free specimen; fig. 12, a couple adhering
together.
Figs. 13-17.—Recurved forms.
Figs. 13, 14.—Twelve hours ; both from the same preparation, but 14
s flattened out by having dried up before fixation, while 13 is normal.
Figs. 15-17.—T wenty-four hours.
Figs. 18-20.—Forms in which the multiplication of the nuclei have
begun without the typanosomes having assumed the recurved form (or
perhaps forms which have straightened themselves out again second-
arily); twenty-four hours, all from the same preparation. In fig. 19
the trypanosome is enclosed in the remains of the host-cell.
Figs. 21-23.—Rolled-up forms with 2n and 2 NN each; from the
same preparation as the preceding.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 683
Figs. 94 99—Rolled-up forms with » and N still single, in 27 begin-
ning to divide. Figs. 24 and 25 are from the same preparation as the
preceding ; figs. 26-29 are also twenty-four hours; the specimen in
fig. 26 is evidently enlarged artificially by having dried before fixation.
Figs. 30-37,—Multiplication of nuclei to 2 or 3 nn and 2 NN.
Fig. 38.—Small sphere with 4 nn and 4 NN.
Fig. 39.—Stage with 3 nn and2 NN. N.B.—Figs. 35-39 are all from
the same preparation, showing the intense staining and consequent
opacity of the body characteristic of the intracellular stages; only the
principal flagella can be made out clearly, the daughter-flagella being
scarcely, or not at all, visible. Twenty-four hours.
Fig. 40.—Sphere with 3 nn and 3 NN, from the same preparation as
fig. 26; evidently enlarged by flattening due to drying. Twenty-four
hours.
Fig. 41—A large sphere containing 13 nn and 13 NN, and two
others ; the stain is over-extracted and the flagella are not seen. These
specimens are from the same stomach as figs. 88-94 on pl. 37, in all of
which flagella are plainly seen. Thirty-six hours.
Fig. 42.—Two very large tailed spheres from the same preparation as
figs. 18-25, 30, 33, 34: the flagella cannot be made out. Twenty-four
hours.
Figs. 43-46.—Small rolled-up forms, possibly in process of degenera-
tion, from the same preparation as the last.
PLATE 37.
[Various stages of the stomach-phase from films preserved in sub-
limate mixtures—sublimate-acetic, Schaudinn’s fluid, or Maier’s fluid—
and stained with iron-hematoxylin, drawn with the camera lucida at
a magnification of 3000. |
Figs. 47-68.— Free (extracellular) trypanosomes, except 58 and 59.
Figs. 47, 48.—Six hours; fig. 47, quite unmodified; fig. 47, slightly
modified.
Fig. 49.—Highteen hours.
Figs. 50-55.—T wenty-four hours.
Figs. 56-59.—Three days, all from the same preparation. Figs. 58
and 59 are evidently early stages of the intracellular multiplication.
Fig. 60.—Four days.
Figs. 61-68.—From an abnormal flea taken from the infected breed-
ing cage (see p. 519). Every possible grade of transition occurs in the
preparation from quite ordinary forms, such as fig. 61, to large erithidial
684 BE. A. MINCHIN AND J. D. THOMSON.
forms, such as fig. 66, and of the latter some occur in couples adhering
together, as in figs. 67 and 68.
Figs. 69-73.—Recurved forms, twenty-four hours; in the specimen
sbown in fig. 72 the flagellum appears to have become torn away from
the body.
Fig. 74.—First division of n in a rolled-up form.
Fig. 75.—Recurved form with 2 nn and 2 NN; forty-eight hours.
Figs. 76.—A rolled-up form in which the flagellum has become un-
twisted from the body; from the same preparation as the last.
Figs. 77, 78.—Two rolled-up forms from the same preparation,
twenty-four hours.
Figs. 79-81.—Twenty-four hours, all from the same preparation ;
fig. 79, n divided, daughter-flagellum arismg, N beginning to divide ;
figs. 80 and 81, similar stages.
Figs. 82-86.—Twenty-four hours, all from the same preparation ;
figs. 82, 83, each 2 nn, 1 N; fig. 85, 2 nn, 2 NN; fig. 84, 3 nn, 2 NN;
fig. 86, tailed sphere with 8 nn, 8 NN.
Fig. 87.—Recurved form, twenty-four hours.
Figs. 88-94.—Various stages, all from the same preparation and from
the same flea as fig. 41 on Pl. 36. Thirty-six hours.
Fig. 95.—Small sphere with 3 nn and 3 NN; twenty-four hours.
Fig. 96.—Large, nearly ripe sphere containing about ten trypano-
somes, shown also in the photograph on Pl. 44, fig. 318; forty-eight
hours.
PLATE 38.
[Epithelial cells of the stomach and stages of the stomach-phase
from sections of the flea’s stomach, stained with Giemsa’s stain (with
the exception of figs. 260a, 26la, 262a, 263a, and 264, for which see
description of Pl. 42). Figs. 99-103 were fixed with Flemming’s fluid,
but all the rest were fixed with Maier’s fluid. Drawn with the camera
lucida at a magnification of 2000. |
Figs. 97, 98.—Epithelial cells; fig. 97 shows a cell in the columnar
form ; fig. 98 a flattened cell with, just beneath the border, a layer of
granules staining quite differently from the blood-débris externally.
Figs. 99-103.—Various details of the cells after Flemming-fixation ;
fig. 99, red-staining grains in the upper end of the cell; fig. 100, red-
staining grains mixed with osmic-blackened (fatty) grains ; fig. 101, cell
comparatively free from granules ; fig. 102, pseudosphere in the upper
end of a cell; fig. 103, two ‘ yellow bodies”’ in the upper end of a cell.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 685
Fig. 104.—Portion of the epithelium close to a erypt of regeneration,
showing a recurved trypanosome attached to an epithelial cell and a
trypanosome of ordinary type free in the débris close by. Twenty-four
hours.
Fig. 105.—Section shaving the side of an epithelial cell obliquely,
showing a trypanosome of ordinary appearance attached to it. Twenty-
four hours.
Fig. 106.—Stout form of trypanosome free in the débris. Twenty-
four hours.
Fig. 107.—Recurved trypanosome within a cell. Twenty-four hours.
Fig. 108.—Tailed sphere within a cell, also two smaller spheres and
a portion of a trypanosome cut across; the nucleus of the cell does not
come into the section. ‘Twenty-four hours.
Fig. 109.—Portion. of the epithelium showing a very large tailed
sphere, three smaller ones and a nucleus (N); the cells are much
exhausted. Twenty-four hours.
Fig. 110.—Section of a cell (not passing through the nucleus) showing
a large tailed sphere and fragments of three smaller ones. Twenty-
four hours.
Fig. 111.—A large tailed sphere and a slice of a smaller one, in a cell
near the nucleus (NV). Twenty-four hours.
Fig. 112.—Numerous multiplication-stages in a partly broken-down
cell of flattened type, and a cell nucleus (N). Twenty-four hours.
Fig. 113.—Six spheres in a broken-down cell cast off into the blood-
débris. Twenty-four hours.
Figs. 114-116.—Three figures drawn from the same slide. Fig. 114,
exhausted cell filled with trypanosomes; fig. 115, a large sphere ripe
for breaking up into a mass of trypanosomes as in the last; fig. 116,
large sphere. The preparation was over-extracted and does not show
the flagella. Twenty-four hours.
PLATE 39.
[Stages of the stomach-phase, all from the same series of sections
through nine stomachs preserved thirty-six hours after the infective
feed in Flemming’s fluid and stained with iron-hematoxylin and Licht-
grin-picric. Drawn with the camera lucida at a magnification of 2000. |
Figs. 117-129.—Extracellular trypanosomes.
Fig. 117.—Slightly club-shaped form, near the epithelium but not
attached.
Figs. 118, 119.—Recurved forms, not attached.
Figs. 120-123.—Attached recurved forms,
686 E. A. MINCHIN AND J. D. THOMSON.
Fig. 124.—A small bunch of trypanosomes attached in the interspace
between two cells. Structure of the trypanosome difficult to make out
clearly.
Fig. 125.—Trypanosomes in or attached to the débris of a necrosed
cell. Two of them are recurved forms, the third possibly crithidial in type.
Fig. 126.—A slightly hypertrophied epithelial cell containing three
spheres of different sizes, with portions of the cells on either side of it.
N, the nuclei of the cells; l.m., longitudinal muscles of the stomach in
transverse section ; ¢.m., one of the circular muscles of the stomach.
Fig. 127.—From the next section of the series, showing the same cell
in which appears one of the same three spheres lodged in a distinct
vacuole.
Fig. 128.—Large hypertrophied cell with five nuclei in process of
being thrown off from the epithelium ; it contains two spheres (sph.)
and numerous yellow bodies. The same cell is shown in the photograph
in Pl. I, fig. 3138.
Fig. 129.—Flattened cell showing a sphere lodged in a vacuole beside
the nucleus (NV).
Fig. 130.—Cell containing four spheres close to the nucleus (NV).
Fig. 131.—Young epithelial cell close to a crypt containing an early
multiplication-stage just external to the nucleus (NV).
Fig. 132.—Degenerated and cast-off cell, containing a sphere, the
flagellum of which is sticking out from the remains of the cell.
Fig. 133.— Recurved trypanosome within a cell.
Fig. 1384.—Normal columnar epithelial cell containing spheres both
above and below the nucleus (NV).
Fig. 135.—Degenerated and cast-off epithelial cell, full of fatty
deposits and yellow bodies, containing two spheres.
PLATE 40.
(Epithelial cells of the flea from sections of stomachs, all, except
fig. 147, fixed with Flemming’s fluid and stained with iron-hematoxylin
followed by Lichtgriin-picric. Figs. 136-140 are drawn at a magnifica-
tion of 1000; all the rest at 2000. }
Fig. 136.—Four young cells showing the partially developed border
and the different positions of the nucleus in the cell.
Fig. 137.—A cell showing the more granular condition.
Figs. 138, 139.—Two stages of the fatty degeneration of the cell. In
138 the nucleus, though obscured, is still faintly visible ; in 189 the cell
has become an opaque black mass.
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 687
Fig. 140.—T wo contiguous cells, of which the one to the left shows
the beginning of the “ yellow necrosis.”
Figs. 141, 142—The upper ends of cells containing “ yellow bodies ” ;
in fig. 141 several such bodies, in fig. 142 one large one.
Figs. 143, 144.—To show the deposition of osmic-blackened fatty
grains in cells beginning to degenerate; fig. 145 in a flattened cell,
fig. 144 in a columnar cell.
Figs. 145, 146.—Cells containing pseudospheres (ps.) in their upper
portions.
Fig. 147.—To show the deposition of deeply-staining grains below
the border of the epithelial cell. External to the cell are seen the
coarse black grains of the blood-débris. Maier’s fluid, iron-hema-
toxylin, Lichtgriin-picric.
Fig. 148.—Outer end of a cell containing a sphere (sph.) which is
seen to contain a darkly-staining mass (chromatin ? or fatty deposit ?).
PLATE 41.
[Various stages of the rectal-phase. Figs. 201, 202 are from sections
fixed with Flemming’s fluid, stained with Giemsa’s stain and drawn at
a magnification of 2000; all the other figures are from smear-prepara-
tions fixed with osmic vapour, stained with Giemsa’s stain, and drawn
at a magnification of 5000. |
Figs. 149-152.—Early “tadpole” forms from the rectum, twenty-
four hours; fig. 152 is perhaps an early stage of division.
Figs. 153, 154.—Early stages from the rectum, twenty-four hours.
Fig. 155.—Rectum, thirty-six hours.
Fig. 156.—Rectum, twenty-four hours.
Figs. 157, 158.—Rectum, thirty-six hours.
Figs. 159-161.—From the rectum of another flea, thirty-six hours.
Fig. 162.—Rectum, sixty hours.
Figs. 163, 164.—rectum, three days.
Figs. 165-170.—From a swarming rectal infection, three days.
Figs. 171-176.—From a swarming rectal infection, about three and a
half days old.
Figs. 177, 178.—F rom the rectum, forty-eight hours.
Figs. 179-182.—From the rectum about three and a half days.
Figs. 183-187.—Early stages from the rectum, four days. In the
clump shown in fig. 183 two trypanosomes of quite ordinary type are
seen; these are probably forms which have come in with a later feed
and have attached themselves secondarily to the clump.
vot. 60, PART 4.—NEW SERIES. A7
688 E, A. MINCHIN AND J. D. THOMSON.
Figs. 188-190.—From the rectum, five days.
Fig. 191—From the rectum of another flea, five days.
Figs. 192-200.—From the recta of fleas taken from the infective
breeding-cage, age uncertain. Fig. {192, early form from the rectum
figs. 193-196, from stomach-preparations (post-pyloric?); fig. 197,
rectum ; fig. 198, rectum; fig. 199, stomach (post-pyloric) ; fig. 200,
rectum.
Fig. 201.—F ree clump from a section of a rectum.
Fig. 202.—From a section of the intestine close behind the pylorus,
showing a number of haptomonads attached to the cuticle, and between
them, projecting up above the level of the haptomonads and distin-
guished from them by their lighter stain and terminal mn are numerous
examples of the final trypanosome-forms (T).
PLATE 42.
[Various stages of the rectal-phase. Figs. 273-277 are from sections
of recta fixed with Flemming’s fluid, stained with iron-hematoxylin,
and drawn at a magnification of 2000; all the other figures are from
smear-preparations fixed with sublimate mixtures, stained with iron-
hematoxylin and drawn at a magnification of 3000.]
Figs. 203, 204.—Forms from the intestine in process of migration to
the rectum, twenty-four hours (see p. 568).
Figs. 205-212.—Early “tadpole” forms from the rectum, twenty-
four hours.
Figs. 215, 214.—Early division-forms, rectum, forty-eight hours.
Figs. 215, 216.—Early forms, one dividing, rectum, three days.
Fig. 216 a.—Couple produced by an early division ?
Figs. 217-220.—F rom a well-infected rectum, about three and a half
days.
Figs. 221-240.—Various forms from a swarming rectal infection of a
flea that had been on an infected rat for five days; figs. 221-225, hapto-
monads; figs. 226-230, division-forms; figs. 231-234, growth of flagel-
lum figs. 235, 236, nectomonads ; figs. 237, 238, transitional forms ;
figs. 239, 240, slender and stout trypanosome-forms.
Figs. 241, 259.—Various forms from a swarming rectal infection,
eight days; figs. 241-245, rounded haptomonads without free flagella ;
figs. 246, 250, 251, flagella growing out from rounded haptomonads ;
figs. 247, 248, 249, pear-shaped haptomonads without free flagella ;
fig. 252, clump of pear-shaped haptomonads, some with well-developed
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 689
flagella ; fig. 253, division-stage; fig. 254, nectomonad ; figs. 255-258
transitional forms; figs. 259, final trypanosome-form.
Figs. 260-264.—Various forms from a swarming rectal infection,
taken from the infected breeding-cage, age unknown; figs. 260 a-263a,
the same specimens as 260-263, restained in Twort’s stain ; fig. 264, a
nectomonad stained with Twort’s stain. (N.B.—Figs. 260 a-263a and
264 have been transferred to Pl. 38.)
Figs. 265-272.—Various forms from a swarming rectal infection, age
unknown; fig. 265, early form ? fig. 266, division-form ; fig. 267, rounded
haptomonad, without flagellum, beginning to divide; figs. 268, 269,
rounded haptomonads with free flagellum beginning to grow out ;
fiz, 270, transitional form ; figs. 271, 272, final trypanosome-forms.
Fig. 273.—Clump of crithidial forms attached in the intestine just
behind the pylorus, in sections of a stomach of a flea preserved thirty-
six hours after the infective feed. (x 2000.)
Figs. 274-276.—From sections through the rectum of a flea preserved
eight days after the infective feed. Fig. 274, free clump ; fig. 275, clamp
attached to the cuticle of the rectum ; fig. 276, forms attached singly to
the cuticle. (x 2000.)
Fig. 277.—Haptomonads attached to the cuticle of the rectum, from
sections through the rectum of a flea, preserved eleven days after the
infective feed. (x 2000.) (This drawing is from the same section as that
photographed in Pl. 44, fig. 317; the patch drawn lies just to the right
hand of the middle of the three pointers that start from ¢ in fig. 317.)
Figs. 278-288.—Leptomonas pattoni, various forms from the
rectum of the flea. Figs. 278-284 a are from preparations fixed with
Maier’s fluid and stained with iron-hematoxylin; figs. 285-288 are from
preparations fixed with osmic vapour and stained with Giemsa. In the
specimens drawn in figs. 281, 282, note small bodies marked # which
appear to be endogenous buds (“ infective granules’’); in fig. 283 a
similar body is shown free, and figs. 284, 284 a appear to be stages in
the development of the bud into the leptomonad. Fig. 285 is evidently
a nectomonad form, and fig. 286a stage in the development of such a
form; they differ from the nectomonads of T. lewisi in the great
prolongation of the body behind NV.
PLATE 48.
[Degenerative forms, fixed with osmic vapour and stained with
Giemsa, drawn with the camera lucida to a magnification of 3000. |
Figs. 289, 290.—Six hours, rectum.
Figs. 291-296,—Twelve hours, rectum.
690 E. A. MINCHIN AND J. D. THOMSON.
Figs. 297, 298.—Recurved forms from the rectum, twelve hours.
9
Figs. 299-301.—From the rectum of another flea, twelve hours.
Figs, 302-304.—Rectum, thirty-six hours.
Fig. 305.—Stomach, forty-eight hours.
Fig. 506.— Rectum, twenty-four hours.
Fig. 307.—Rectum, sixty hours.
Fig. 308.—Clump of degenerative forms, rectum, eighteen hours.
Figs. 509, 310.—Agglomerating trypanosomes from a flea fed for the
second time on an infected rat the day previously.
PLATE 44.
Fig 311.—Clump of degenerative forms (compare fig. 308). Photo.
(x 1500.)
Fig. 312.—Clump of developmental crithidial forms; at 7’ is seen a
final trypanosome-form attached to the clump. From the same pre-
paration as figs. 241-259. Photo. (x 2000.)
Fig. 313.—Infected patch of stomach-epithelium in which the cells
are breaking down and being thrown off. The Jarge multinucleate cell
marked C is the cell, part of which is drawn in PI. 39, fig. 128. Photo.
(x 600.)
Fig. 314.—Section through an epithelial crypt of regeneration. Close
beside it is seen a black, degenerated epithelial cell. Photo. (x 800.)
Fig. 315.—Section of an infected patch of the stomach-epithelium
showing the cells being thrown off; the cells contain coarse black
(fatty) granules and stages of intracellular multiplication, which are
not clearly seen. Photo. (x 600.)
Fig. 316.—Section of the stomach-epithelium showing an epithelial
crypt towards the middle ; to the left of the crypt the epithelium is old
and degenerate and full of blackened fat; to the right of the crypt is
new, clear epithelium budded off from it. Compare Text-fig. 2. Photo.
x 300.
Fig. 317.—Section of the wall of a rectum showing a swarming
crithidial infection of the “ pile-carpet”’ type. Fig. 277 is drawn from
this section, from the part between the middle and the right-hand
pointers which start from ¢ to indicate the serried ranks of the crithidial
forms. Photo. (x 500.)
Figs. 318.—A sphere (the same that is drawn in fig. 96) and near it
one of the long free trypanosomes of the stomach-type, from a smear,
Photo, (x 1000.)
THE RAT-TRYPANOSOME, TRYPANOSOMA LEWISI. 691
Fig. 319.—Section passing through the opening of the pylorus into
the intestine. Photo. (x 300.)
PLATE 45.
[General diagram of the entire life-cycle of Trypanosoma lewisi
in the flea, combined from the observations and experiments set forth
in this memoir in order to give a summarised idea of the complete
course of events. The stomach-phase is represented on the upper side
of the diagram, the rectal phase on the lower side and to the left; in the
right-hand lower corner is shown the secondary infection of the pylorus.
Magnification 2000. |
1. Trypanosome as taken up by the flea from the rat.
2. Trypanosome slightly modified after a few hours in the flea’s
stomach.
3-12. Cells of the epithelium of the stomach containing the various
phases of the intracellular multiplication.
3. Cell with two trypanosomes attached to it, one of ordinary, the
other of recurved, type.
4. Penetration of an epithelial cell by a trypanosome, drawn from
Ndller’s description of the process.
Recurved form in the cell.
Or
. Rolled-up form in the cell.
. Early multiplication-form.
_ Later multiplication ; 8 nn, 8 NN.
. Large tailed sphere.
Oo © aI SD
10. Large ripe sphere without tail.
11. Daughter-trypanosomes free in the exhausted cell after bursting
of the sphere.
12. Daughter-trypanosomes escaping from the cell. After being set
free, the trypanosomes may do one of two things as shown by the
arrows. They may each penetrate another epithelial cell and repeat
the process of multiplication by which they were produced. Or they
may pass through the pylorus and down the intestine to the rectum to
give rise there to the rectal (crithidial) phase.
13-18. To show the two possible ways in which the established rectal
phase may arise from the stomach-trypanosomes :
(a) 13, 14, 15, 16. Four successive stages of the contraction of a
stomach-trypanosome into a pear-shaped form which at 17 divides by
692 E. A. MINCHIN AND J. D. THOMSON.
equal or subequal binary fission to produce two equipotential daughter-
products, similar to 18, which by further-repeated binary fission produce
the ordinary crithidial phase. .
(b) 14a, 14b. Two successive stages of the contraction of a stomach-
trypanosome into a club-shaped form, which at 17a divides by unequal
binary fission to produce two inequipotential products, the larger
parent-form which does not develop further and the smaller daughter-
form, similar to 18, which by repeated binary fission produces the
ordinary crithidial phase.
19. Established rectal infection, showing the various forms of the
rectal phase. h.h. Haptomonads, some of them dividing, some of them
transitional to the other types. u.n. Nectomonads. ¢r. tr. Forms
transitional from the crithidial type to T.T., the final trypanosome-form.
The final trypanosome-forms, as shown by the arrows, pass finally
out of the flea with the feces.
The nectomonads can, under certain conditions of food-supply,
migrate back along the intestine and fix themselves close behind the
pylorus in order to give rise to—
20. Secondary infection of the pyloric region of the intestine: the
letters have the same significance as before. Final trypanosome-forms
(7.T.) arise and pass down the intestine into the rectum and so out with
the feces ; nectomonads (nn) also arise and may migrate back again to
the rectum and re-establish the crithidial phase there ; these migrations
are indicated by the arrows.
INDEX FO, VOL: -60;
NEW SERIES.
Acrossota liposclera, a new
genus and species of Aleyonarians,
by Gilbert C. Bourne, 261
Ageregatide, sporogony of, by Helen
Pixell-Goodrich, 159
Aleyonarians, a new genus of, by
Bourne, 261
Beaver, blastocyst and placenta of,
by Arthur Willey, 175
Bourne on A crossota liposclera,
a new genus and species of Alcyo-
narians, 261
Centrifuged egg of the Frog, by
Jenkinson, 61
Ceratophyllus fasciatus, the
Rat-flea, and its relation to the
Rat-trypanosome, 463
Chromosome complex of Culex
pipiens, by Monica Taylor, 377
Conus, anatomy of, by H. O. N.
Shaw, 1
Culex pipiens, chromosome com-
plex of, by Monica Taylor, 377
Culex pipiens, trypanosome from,
399
Dendy on the gametogenesis of
Grantia compressa, 313
Egg of the Frog, centrifuged, by
Jenkinson, 61
Flea of the Rat and the Rat-
trypanosome, 463
Frog, centrifuged egg of, by Jenkin-
son, 61
Gametogenesis of Grantia com-
pressa, by Dendy, 313
Grantia compressa, gameto-
genesis of, by Dendy, 313
Hexmoprotozoa, by Woodcock, 399
Hamm on Stylops and Stylopisation,
435
Jenkinson on the development of the
centrifuged egg of the Frog, 61
Minchin, E. A., and J. D. Thomson
on the Rat-trypanosome, Try-
panosoma lewisi, inits relation
to the Rat-flea, Ceratophyllus
fasciatus, 463
Nemertines, proboscidean system of,
by Wynhoff, 273
Owl, Little, development of its try-
panosome, 399
Pixell-Goodrich on the sporogony
and systematic position of the
Ageoregatide, 159
Placenta and blastocyst of
Beaver, by Willey, 175
the
694.
Rat-trypanosome and its relation to
the Rat-flea, by E. A. Minchin and
J. D. Thomson, 463
Sex, experimental analysis of,
Part 11, by Geoffrey Smith,
435
Shaw on the anatomy of Conus,
1
Smith, Geoffrey, on Stylops and
Stylopisation, 435
Stylops and Stylopisation, by
Geoffrey Smith and A. H. Hamm,
435 ;
|
INDEX.
Taylor, Monica, on the chromosome
complex of Culex pipiens, 377
Thomson, J. D., see Minchin.
Trypanosoma lewisi and
relation to the Rat-flea, 463
Trypanosoma noctue in Culex
pipiens, by Woodcock, 399
its
Willey on the blastocyst and placenta
of the Beaver, 175
Woodcock on Avian Hemoprotozoa,
399
Wynhoff on the proboscidean system
of Nemertines, 273
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QH Quarterly journal of micro-
201 scopical science
Q2
D's
v.60
cop.2
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