/. Embryol. exp. Morph. Vol. 31, 2, pp. 415-422, 1974
Printed in Great Britain
415
The development of
Bithynia tentaculata (Prosobranchia, Gastropoda)
after removal of the polar lobe
By JAMES N. CATHER 1 AND NICO H. VERDONK 2
From the Department of Zoology, The University of Michigan,
and the Zoological Laboratory, The University of Utrecht
SUMMARY
The polar lobe of the egg of Bithynia, with a volume of less than 1 % of that of the egg,
was surgically removed. Lobeless embryos form no mesentoblast cell nor mesoderm bands.
They fail to establish bilateral symmetry and to form eyes, foot, operculum, shell, etc. They
always differentiate larval head cells, ganglia, larval liver and mesenchyme and sometimes
columnar epithelium, stomodeal entrance cells, endodermal tubes and muscles.
These results are discussed in relation to the structure of the lobe, the analysis of development
of Bithynia and to similar experiments in other species.
INTRODUCTION
The polar lobe, formed at the first and second cleavage in eggs of a number of
species of annelids and molluscs, has been one of the foremost examples used to
illustrate cytoplasmic control over developmental events since Crampton (1896)
removed the polar lobe from the eggs of Ilyanassa and showed that the mesoderm
bands failed to form. Since then a number of species with polar lobes have been
investigated (see Verdonk, 1968a; Cather, 1971; Clement, 1971) to determine the
role of the polar lobe in development and to determine what is present in the lobe
which causes it to exert such a profound influence on polarity and organogenesis.
In all species so far investigated the polar lobe makes up a rather large proportion
of the total egg cytoplasm, even up to a third in some cases. The polar lobe is
passed to the CD blastomere after the first cleavage, to the D blastomere after
second cleavage and then its cytoplasm becomes an integral part of the D quadrant, the major regulatory region of the embryo. Although there are some differences in the concentration of enzymes and of fine structural elements between
the cytoplasm of the polar lobe and that of the blastomeres, this can generally be
accounted for by the relative abundance of yolky and non-yolky cytoplasm.
Furthermore, the centrifugation experiments of Clement (1968) and Verdonk
1
Author's address: Department of Zoology, The University of Michigan, Ann Arbor,
Michigan 48104, U.S.A.
2
Author's address: Zoological Laboratory, Janskerkhof 3, Utrecht, The Netherlands.
416
J. N. CATHER AND N. H. VERDONK
(1968 b) present strong evidence that such elements are not of primary importance
in the regulation of development.
The general development of Bithynia was described by von Erlanger (1892) but
he failed to note the very small polar lobe of this species which was first observed
by Hess (1956). Dohmen & Verdonk (1974) have done a detailed cytological and
ultrastructural study of the polar lobe in this species. The polar lobe has a volume
of less than 1 % of that of the egg. Within the first polar lobe there is a special
body which has an unusual fine structure and a high concentration of RNA. The
vegetal body is dispersed in the CD blastomere so that both C and D apparently
receive part of its contents. In Bithynia, C and D have approximately equivalent
regulatory ability, and when either one is present, adult organs such as eyes, foot,
shell, etc., which are considered to be lobe-dependent, are formed (Verdonk &
Cather, 1973).
It appears crucial at this time to determine if the very small polar lobe of
Bithynia does in fact exert an equivalent influence on development to the larger
ones such as in Ilyanassa, or whether the behaviour of C and D is due to another
factor.
MATERIALS AND METHODS
Eggs used in these experiments were obtained from snails (Bithynia tentaculata L.) taken from the ditches near Utrecht. The snails were kept in glass
aquaria in the laboratory, where spontaneously laid egg masses were collected
from leaves of aquatic plants at regular intervals. Operations were done in small
wax-bottom dishes to which the egg mass was attached by a horseshoe-shaped
piece of tungsten wire. A small opening was made in the wall of each capsule in
the egg mass with a sharp knife. At first cleavage, a glass needle was inserted
through the opening and used to lift the polar lobe away from the blastomeres so
that the thin cytoplasmic connexion was broken. After a few hours, the opening
of the capsule wall is closed, apparently by coagulating capsule fluid. Eggs in the
opened capsules of the egg mass which were not used for operations, served as
controls. In some cases, the two blastomeres were separated and the polar lobe
was removed. Out of more than 500 attempts, 24 lobeless embryos were obtained
and 7 pairs of separated blastomeres without the lobe.
After the operations, the egg masses were left in the operating dishes overnight
and then transferred to small covered dishes (Boveri type) with copper-free tapwater. In most cases 10 mg of streptomycin sulfate and 10 mg of sulphanilamide
were added to each liter of water but some embryos were raised without antibiotics to be sure that operated embryos were not more sensitive to them than the
controls. Embryos, which were maintained at 25 °C, were inspected and changed
to clean water daily. Egg masses were cleaned with pieces of paper tissue if
mycelia were adhering to them.
Control embryos hatch after 12-14 days at 25 °C. Lobeless embryos were
Lobeless embryos o/Bithynia
417
Fig. 1. Photograph of living lobeless embryo, 11 days old, in the egg capsule. The
balloon-like ectodermal vesicle includes most of the endoderm but there is a small
projection to the exterior, cw, Capsule wall; ec, ectoderm; en, endoderm. x 95.
reared for various periods through 13 days. Embryos were prepared with silver
nitrate (Verdonk, 1965) or were fixed in Zenker's fluid and sectioned and stained
using standard histological procedures.
RESULTS
Lobeless eggs of Bithynia undergo typical cleavage, with normal cleavage
rates, through the formation of the third quartet of micromeres, except that the
second polar lobe is not formed. Four lobeless embryos were stained with silver
after the formation of the mesentoblast, 4d, in the control embryos to determine
whether the mesentoblast forms at the same time in lobeless embryos as in the
controls or whether the divisions of the D quadrant of lobeless embryos lose their
unique features to become similar to the other quadrants. There is no mesento-
418
J. N. CATHER AND N. H. VERDONK
~ Ihc
\ Ihc
Fig. 2. Drawing from silver-stained embryo of 13 days which shows the size and
distribution of ectodermal cells. The embryo was balloon-like in life but collapsed
somewhat in preparation. Ihc, Larval head cells; see, small-celled ectoderm, x 300.
blast formed in lobeless embryos and the cells at the vegetal pole at this stage
retain the same radial symmetry as in earlier stages.
Gastrulation in lobeless embryos occurs in the same way as in the controls,
after which the lobeless embryos swell into balloon-like structures with the
endodermal mass lying against the wall of the transparent ectoderm cells, or in
some cases projecting through to the exterior (Fig. 1).
Four lobeless embryos were sectioned during the third and fourth day of
development to determine if mesoderm bands were present. A few cells, apparently the ectomesenchyme, were found between the ectoderm and endoderm in
the general region of the blastopore but no mesoderm bands were formed in the
lobeless embryos. Mesoderm bands are very conspicuous in control embryos
throughout this period. Both controls and lobeless embryos have distinctive
prototrochal cells at this stage.
Three of the balloon-like embryos of 6 or 7 days were fixed and sectioned. They
FIGURES
3-6
Fig. 3. Section of 13-day-old lobeless embryo. Note the extrusion of the liver and
the invagination of columnar epithelium which could represent early shell gland.
cle, Columnar epithelium; g, ganglion; /, liver; m, mesenchyme; n, nerve, x 350.
Fig. 4. Section of 9-day-old lobeless embryo through the stomodaeum entrance
cells. Other structures such as liver were not in the plane of section, g, Ganglion;
sec, stomodaeum entrance cells, x 300.
Figs. 5, 6. Twin embryos from the separated blastomeres of a delobed egg. cle,
Columnar epithelial cells; ent, endodermal tube;#, ganglion; Ihc, larval head cells;
y, yolk, x 350.
Lobeless embryos o/Bithynia
419
420
J. N. CATHER AND N. H. VERDONK
had small-celled ectoderm, larval head cells and ganglia. The endoderm was
differentiated into characteristic larval liver and there were sparsely scattered
mesenchyme cells throughout the fluid-filled vesicle.
Thirteen lobeless embryos were allowed to develop to full maturity between
9 and 13 days. All had small-celled ectoderm, larval head cells (Fig. 2), ganglia
with nerve outgrowths and well-formed larval liver (Fig. 3). The majority of the
ectoderm is made up of small cells but there are patches of the large larval head
cells, though these do not appear to be in any consistent pattern. The liver is
usually made up of several lobes. In three cases an endodermal tube connected
to the liver but ended blindly within the embryo. These tubes appear to be either
midgut or anterior hindgut but the distinguishing characteristics are absent.
Very large, distinctive ciliated stomodaeum entrance cells were present in one
embryo (Fig. 4) and they were probably present in one of the young embryos
where they were not well differentiated. An invagination of columnar epithelium
was present in two embryos (Fig. 3) and flattened columnar epithelial patches
were usually present. These may represent shell gland but the cells never complete their differentiation to become characteristic of shell gland, and shell was
never observed.
In the seven cases where the cells were separated and the polar lobe removed at
first cleavage, nearly identical twins were formed (Figs. 5, 6). They gastrulate,
become swollen, and then become more compact as they are filled with mesenchyme, so that they appear like AB halves (Verdonk & Cather, 1973). They form
small-celled ectoderm, larval head cells and some muscle. In one case an endodermal tube (Fig. 5), such as was described above, was present but no other adult
structures were seen.
In all cases muscle cell development was restricted to the older embryos.
These muscle cells were never organized into the band-like structures seen in the
lobeless embryos of Ilyanassa. Most of the mesenchymal cells retain their typical
branching character.
DISCUSSION
In Crampton's (1896) original work with Ilyanassa, he found that neither the
mesentoblast nor the mesoderm bands were formed in lobeless embryos. Clement
(1952) showed that the many unique features of cleavage in the D quadrant - the
quadrant which receives the polar lobe in Ilyanassa - were lost in lobeless eggs.
The cells of the D quadrant were similar in size and time of formation to the other
quadrants. Since the precocious division of 3D to form the mesentoblast does
not occur in lobeless embryos, they retain their radial symmetry.
The development of lobeless embryos in Bithynia is consistent with previous
results. In normal embryos a second polar lobe is formed though it remains
broadly connected to the CD-cell. When the polar lobe is removed at first
cleavage the second lobe is not formed. In normal development the mesentoblast,
4d, is formed and divides twice before 3A-3C divide to form 4a-4c. In lobeless
Lobeless embryos of Bithynia
421
embryos the cell 3D does not divide ahead of the other macromeres and no
mesoderm bands are formed.
Clement (1952) developed methods for rearing the partial embryos of Ilyanassa
through the stages of organogenesis. He found that the monstrous lobeless
embryos formed endodermal vesicles, ganglia, muscles and radially symmetrical
velar lobes, but no shell, foot, operculum, eyes, etc., were formed. In later (1962)
experiments he found internal shell masses and noted that the posterior protrusion was the everted stomodaeum. Atkinson (1971) made a detailed histological
analysis of normal and lobeless embryos of Ilyanassa and found in addition to
confirming and extending Clement's observations that endoderm could differentiate into digestive gland, style sac, stomach and esophagus and that
glandular epithelium resembling pedal or mantle tissue was formed.
The influence of the polar lobe on organogenesis is to a great extent similar in
Bithynia and Ilyanassa. The eyes, foot, external shell, operculum and statocysts
are lobe-dependent structures, and are never found in the monstrous lobeless
embryos of either species. However, the development of digestive tract structures
in lobeless embryos of Bithynia is far less extensive than in Ilyanassa. A recognizable stomodaeum was never present and only one or two lobeless embryos had
stomodaeum entrance cells. The endoderm develops into larval liver, sometimes
paired and at times attached to an endodermal tube, apparently mid- or hindgut.
Further differentiation of the endoderm does not occur and some of the older
embryos showed degeneration of the liver.
Hess (1956) described shell gland in isolated half-embryos of Bithynia. In
lobeless embryos, columnar epithelium was often present and could represent
early anlage of a shell gland but shell material is never secreted by it, nor were
internal shell masses, typical in lobeless embryos of Ilyanassa, ever observed.
The only adult structures which differentiate apparently independently of the
polar lobe in both prosobranchs are ganglia. These are well developed and send
out nerves, but no obvious pattern in the position of ganglia was observed.
In general terms, then, removal of the polar lobe in Bithynia has the same
result as removal of the polar lobe in other species. This is remarkable when the
small size of the polar lobe of Bithynia is compared to that of other spiralian
species which have been studied. The large polar lobes of Ilyanassa (PucciMinafra, Minafra & Collier, 1969; Geuskens & de Jonge d'Ardoye, 1971),
Mytilus (Humphreys, 1964) and Dentalium (Reverberi, 1970) are filled with the
usual cytoplasmic organelles of eggs such as yolk granules, lipid droplets and
mitochondria as well as various vesicles, none of which are unique to the lobe.
In Bithynia the small lobe is nearly free of yolk but it contains a dense body
consisting of vesicles filled with electron-dense particles (Dohmen & Verdonk,
1974). A further analysis of this vegetal body now is of major interest since it
appears that it may be responsible for the morphogenetic influence of the lobe.
422
J. N. CATHER AND N. H. VERDONK
The authors wish to thank Professor Dr Chr. P. Raven for his encouragement during the
course of this work and for critically reading the manuscript. Thefirstauthor wishes to express
his appreciation to Professor Raven for the hospitality of his laboratory. We sincerely
appreciate the excellent technical assistance of Mr Gideon Zwaan.
The work of J. N. C. was supported in part by institutional Research Grant IN-40M to the
University of Michigan from the American Cancer Society.
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ATKINSON,
{Received 27 June 1973)