/. Embryol. exp. Morph. Vol. 24, 2, pp. 287-303, 1970
287
Printed in Great Britain
Study of the development
of the internal organs of the double malformations
of Chironomus dorsalis by fixed and sectioned
materials
By HIDEO YAJIMA 1
From the Department of Biology, Ibaraki University, Japan
SUMMARY
The development of the internal structures was investigated byfixedsections of the 'double
cephalon' and 'double abdomen' of Chironomus dorsalis.
The cell proliferation that gives rise to 'germ An/age' or embryonic rudiment begins, in the
double cephalon, along the entire convex (ventral) side of the egg and, in the double abdomen,
at both ends of the flat (dorsal) side. As a result, a single fused Anlage of the double cephalon
appears along the entire convex side of the egg and two germ Anlagen of the double abdomen
appear at both ends of the flat side.
During the formation of the germ band, both the posteriormost part of the double cephalon
which lies at the middle of the convex side of the egg and the anteriormost part of the double
abdomen which is located at the middle of the convex side, fail to differentiate and later
degenerate.
In each of the duplicated heads of double cephalon, cephalic segments anterior to the first
maxillary segment are formed, but the thoracic and abdominal segments are entirely missing.
In each half of the double abdomen, eight abdominal segments posterior to the second
abdominal segment are produced and the cephalic and thoracic segments are omitted
altogether.
The two pairs of mid-gut rudiment from both halves of the double cephalon are temporarily
united but they break apart by the end of the blastokinesis. When the two pairs of mid-gut
rudiment from both halves of the double abdomen meet, they remain fused with each other,
being surrounded by the visceral mesodermal cells in the normal way, and develop into the
mid-gut epithelium.
In the double malformations, the pole cells are contained in only one member of the duplicated structures. The pole cells of the double cephalon develop into the tetra-nucleate state
(Hasper's second step), but they fail to fuse to form tte gonad. In the double abdomen, the
gonad de\elops in the one abdomen containing the pole colls and no replacement occurs in the
sister abdomen without the pole cells.
The embryonic envelopes of the double cephalon do no* retract into the interior of the
embryo, while they do in the normal way in the double abdomen.
The double cephalon can never hatch but the double abdon^en can emerge.
1
Author's address: Department of Biology, Faculty of Sciences, Ibaraki University,
Bunkyo 2-chome, Mito, Japan.
288
H. YAJIMA
INTRODUCTION
Double malformations of Chironomid embryo, both 'double cephalon' and
'double abdomen', can be obtained by centrifugation (Yajima, 1960; Gauss &
Sander, 1966; Overton & Raab, 1967) or by partial u.v.-irradiation (Yajima,
1964; Kalthoff & Sander, 1968). The double cephalon consists of two heads
pointing in opposite directions connected at their first maxillary segments, the
thorax and abdomen being altogether absent. The double abdomen is a monster
with two abdomens joined at their bases, the head and thorax being absent. In
the present paper embryos of serial ages of both types of the double monsters
symmetrical to the equatorial plane of the egg were sectioned to study the development of their internal structures, although the treated eggs also develop
into double malformations which are asymmetrical to the equatorial plane, as
shown by Kalthoff & Sander (1968).
The present research deals chiefly with the development of the alimentary
system and the gonads where there is anomalous combination or interaction
among the cell groups.
MATERIALS AND METHODS
The eggs of Chironomus dorsalis were collected in the field.
For centrifuging, a banana-shaped egg-mass containing several hundred eggs
was sucked into a glass tube of diameter a little less than that of the egg-mass so
that most of the eggs, if not all, would lie with their long axes parallel to the
glass tube. Double cephalon and double abdomen are obtained by centrifugation along the long axis of the egg, i.e. along the length of the tube. The tube was
placed into a large centrifuge tube and spun at 4000 rev/min for 5min. After
centrifuging, the egg-mass freed from the tube was treated with sodium hypochlorite to liberate the individual eggs from the jelly material of the mass. The
incubated eggs were fixed from time to time and left in F.A.A. (formalin,
alcohol, acetic acid, 5: 15: 1) fixative for 24 h. Sections were cut at 5-7/6 and
stained with Heidenhain's haematoxylin and light-green.
RESULTS
In the present report the description of the embryonic development of double
malformations will begin from the formation of cellular blastoderm, since the
earlier stages were described elsewhere (Yajima, 1960) and no differences could
be found between double cephalon and double abdomen.
1. From the completion of the cellular blastoderm to the
formation of the germ band
Double cephalon. The cellular blastoderm of the double malformation is, in
size and appearance, not different from a normal one, although the yolk and oil
in the interior of the egg remain stratified. As soon as the cellular blastoderm is
Double malformations of Chironomus dorsalis
289
complete, there are successive cell divisions on the convex (ventral) side as well
as on the two ends of the flat (dorsal) side of the egg, which together produce the
'germ An/age' in Counce's sense (1961). The rest of the cells on the flat side do
not divide and thin out, which gives rise to an extra-embryonic portion (eep,
Fig. 3 B). As a result, the germ Anlage appears on the convex side separated
from the extra-embryonic portion on the flat side (Fig. 3C).
Soon after, the cells in the middle of the Anlage rapidly divide, giving rise to an
inward thickening (arrows, Figs. 1 A; 3 A, B). This thickening is then pushed
into the yolk by the growth and extension of the neighbouring cells and it is
finally cut off from the cells of the surface (sc, Figs. 1B-E; 3 C-E). The submerged
mass of cells forms a round ball for a while. The nuclei of the cells, however,
become pycnotic and the cell boundaries disappear by the end of blastokinesis.
The function of the sunken cell mass and its homology with a structure of the
normal embryo are not entirely clear at present. Judging from its size and location, it may correspond to the posteriormost part of the head.
After the cell mass sinks into the yolk, another group of cells of the germ
Anlage that lie along the mid-ventral line of the embryo begins to sink inward
forming a groove which gradually extends toward the ends of the embryo. This
is the ventral groove which gives rise to the inner layer (mesoderm) (mes,
Fig. 1 B, G, 2, 4; Fig. 3C). The groove is gradually closed from the middle
toward the ends of the embryo by pushing and folding of the lateral cells. As the
cells of the groove are cut off from the surface by the lateral cells, the cells of the
groove now begin to spread laterally along the inside of the embryo.
The pole cells which migrated into the yolk near one end (the posterior end in
the normal embryo) of the egg hardly move during the formation of the germ
Anlage and of the inner layer (pc, Fig. 3 A-E).
At an early stage of germ Anlage formation, the distal parts of the embryo at
the ends of the flat side of the egg begin to grow into the yolk. By further growth
of the embryo toward the centre of the egg and extension of the extra-embryonic
portion of the flat side toward the ends of the egg, two folds of the extraembryonic portion (amniotic folds) appear near the ends of the flat side (amf,
Fig. 3 B). The two folds are shifted toward the convex side of the egg by further
extension of the extra-embryonic portion, curving around the ends of the embryo
(am, amf, Figs. 1C; 3C, D). Finally the rims of the two folds unite at the middle
of the convex side (Fig. 3E). Thus, the extra-embryonic portion now becomes a
double structure; the outer part lying just beneath the chorion is the serosa (ser)
and the inner part tightly covering the germ Anlage is the amnion (am) (Fig. 3E).
There is a space between the two.
During the time when the two folds are stretching toward the middle of the
convex side, the lateral parts of the embryo at each end grow outward along the
egg surface so as almost to touch each other near the mid-dorsal line (the growth
occurs in the direction perpendicular to Fig. 3 C-E). This is the formation of the
head lobe at both ends of the germ Anlage (hi, Figs. IB, 3E).
290
H. YAJIMA
hi
eep
hi
Double malformations of Chironomus dorsalis
291
When the head Jobes are formed, the post-oral segments of the head appear
on the middle of the ventral surface of the embryo. In the normal embryo of
Chironomus, three post-oral cephalic segments are formed (mandibular, first and
second maxillary segments), but in the double cephalon only the first two
segments appear for each of the duplicated heads. The second maxillary segment of the double cephalon must belong to the submerged part of the germ
An/age. The first maxillary segments of the two heads are fused at their bases
into a large segment. In the double cephalon the thoracic and abdominal segments are entirely missing.
The stage in which the two amniotic folds completely coalesce may probably
correspond to the germ-band stage of the normal embryo. In the normal embryo
at this stage, the germ band curls along the egg-shell on the median longitudinal
plane and the head and caudal ends lie rather close together near the anterior
end of the convex side of the egg. On the contrary, the germ band of the double
cephalon at this stage is restricted to the convex side of the egg.
In contrast to continued lengthening of the normal Anlage, the germ Anlage
of the double cephalon shortens at this time, pulling back the structures that
extend over to the flat side. This, in turn, indicates that the elongation of the
normal germ Anlage prior to the germ-band stage is the result of the elongation
of the thoracic and abdominal parts and not that of the head.
Double abdomen. After the completion of the cellular blastoderm of the double
abdomen, cell proliferation that gives rise to the germ Anlage begins at two points
ABBREVIATIONS ON FIGURES
ob.c, abnormal cells containing pycnotic nuclei; am, amnion; amf, amniotic fold;
br, brain; civ, dorsal vessel; ect, ectoderm; eep, extra-embryonic portion; ent, entoderm (mid-gut rudiment);^, fore-gut; g, ganglion; go, gonad; h, hypodermis; hg,
hind-gut; ///, head lobe; Ir, labrum; me, mid-gut epithelium; mes, mesoderm; mst,
mid-gut strand; Mt, Malpighian tubule; nb, neuroblast; nc, nerve cell; pc, pole cell;
pr, proctodaeum; pri, proctodaeal invagination; sc, submerged cell mass; ser,
serosa; sg, suboesophageal ganglion; st, stomodaeum; sti, stomodaeal invagination;
vg, ventral groove; vm, visceral mesoderm; vns, ventral nervous system.
Fig. 1. Sections of the double cephalon.
(A) Sagittal section of early germ Anlage formation, showing the inward thickening
of the cells (arrow).
(B) Parasagittal section at the stage of the inner layer (mesoderm) formation.
(C) Parasagittal section at the middle stage of the germ Anlage formation, showing
amniotic folds (amf).
(D) Sagittal section at early stomodaeal invagination.
(E) Horizontal section when the temporary union of the mid-gut strands (mst) is
complete.
(F) Sagittal section at the middle of blastokinesis.
(G) 1-5, Transverse sections at the middle stage of inner layer formation. Sections
7-5 are taken at a distance of •}, f, £, f and f the body length from one end of the
embryo.
19
EMB 24
292
H. YAJIMA
ent
ent
1
Fig. 2. Sections of the double abdomen.
(A) 1-3, Transverse sections at an early stage of inner layer formation. Sections 1-3
are taken at a distance of %, \ and f- the body length from one end of the embryo.
(B) Sagittal section at the middle stage of germ-band formation, showing the
amniotic folds (amf).
(C) Transverse section at an early stage of proctodaeal invagination.
(D) Sagittal section at an early stage of blastokinesis, showing the proctodaeal
invaginations (pri).
(E) Parasagittal section at the middle of blastokinesis, showing the proctodaeum
and the mid-gut strand (mst).
(F) 1-4, Transverse sections at the middle of blastokinesis, showing the proctodaeum and the entoderm. Sections 1-4 are taken at a distance of •}, $, f and % the
body length from one end of the embryo.
Double malformations of Chironomus dorsalis
293
which are about one-tenth the length of the egg from either end (Fig. 3F). The
two rudiments then widen laterally and grow toward the middle of the flat side
along the surface of the egg (Fig. 3G, H). One of the two Anlagen carries the
pole cells with it toward the middle of the flat side {pc, Fig. 3F-J). As the ends of
the growing germ Anlagen approach the middle of the flat side, the ends, namely
caudal ends, begin to sink into the yolk in one of the following ways: in some
cases, the two growing ends pass each other at the middle of the flat side and
enter obliquely into the yolk; in the other cases, the ends press against each other
at the middle of the flat side and become bent into the yolk at right angles
to the egg surface, and almost reach the convex side. In the former cases, as
shown in Fig. 2C, the two caudal ends of the double abdomen can be observed
in transverse section.
While the cells of the germ Anlagen at the ends of the egg increase in number,
the cells of the middle of the flat side become flattened and stretched. As they do
so they become distinguished from the cells of the germ Anlagen. The cell group
gives rise to the extra-embryonic portion (eep, Fig. 3G).
Soon after the two caudal ends of the germ Anlagen sink into the yolk near
the middle of the flat side, their front ends, which lie at the ends of the egg, gradually grow towards the opposite (convex) side of the egg, curving around the
ends of the egg (Fig. 31). Finally, the growing ends meet at the middle of the
convex side and the two Anlagen unite into one (Fig. 3J).
Shortly before the tips of the growing germ Anlagen unite, nuclei of some
blastodermal cells lying in between the tips become pycnotic (ab.c, Fig. 31).
Finally, they are pushed into the yolk. The submerged cell group is gradually
cut off" from the surface of the egg and breaks down before the blastokinetic
movement of the embryo begins (sc, Figs. 2D, 3 J). Although it is again not easy
to compare this submerged part with a structure of the normal embryo, its size
and location in reference to the embryo may suggest that it represents the
anteriormost part of the abdomen.
Shortly after the appearance of the germ Anlagen, the extra-embryonic portion
of the flat side extends toward both ends of the egg and folds over the germ
Anlagen. This is the formation of two amniotic folds {amf, Fig. 3G). The folds
gradually grow toward the convex side along the surface of the embryo, curving
around the ends of the egg {am, amf, Figs. 2B, 3H, 1). Finally the rims of the
two folds meet and fuse with each other at the middle of the convex side (Fig. 3 J).
Thus, the formation of the two embryonic envelopes of the double abdomen is
complete. Since both outer serosa {ser) and inner amnion {am) of the double
abdomen are closely applied to the surface of the embryo, there is no space
between them; this is seen in the double cephalon as well as in the normal embryo.
In a similar 'double abdomen' monster of Smittaparthenogenetica, which was
produced by partial u.v.-irradiation of the egg, although the extra-embryonic
portion differentiated from the embryonic part, it did not develop enough to
cover the entire embryonic area (Kalthoff & Sander, 1968). This may partly be
19-2
294
H. YAJIMA
due to insufficiency of material of the embryonic envelopes in the Smitta
malformation.
Shortly before the growth of the germ Anlagen toward the convex side begins,
the cells of each Anlage along the mid-ventral line become invaginated to form
H
pc
mes
ect
Fig. 3. Semi-diagrammatic sagittal sections of the two types of double malformation
at successive stages from early germ Anlage to germ band. A-E, Double cephalon;
F-J, double abdomen.
the ventral groove. Imagination first takes place in the middle of the ventral
surface of each Anlage and then is gradually closed from the middle toward the
ends of the Anlage by the growth of the lateral cells on the border of the groove
toward the mid-ventral line and is finally cut off from the surface, which gives
Double malformations of Chironomus dorsalis
295
rise to mesoderm (mes, Fig. 3H, J). Fig. 2 A, 1-3, shows transverse sections at
the early stage of the formation of mesoderm roughly corresponding to Fig. 3H.
Both nos. 1 and 3 are taken respectively at a distance of one-quarter of the length
of the egg from either end, and contain areas of the mesoderm formation of the
two opposite sides. No. 2 is a section cut through the middle of the egg, which
has no trace of cell proliferation. From these three sections it is clear that the
formation of the two rudiments of the embryo and of the inner layer occurs
independently at the two ends of the egg.
When the two germ Anlagen of the double abdomen meet at the middle of the
convex side of the egg, the two abdomens cover the entire median line of the
dorsal and ventral surfaces of the egg with the two caudal ends at the centre of
the egg (Fig. 3 J). The stage may correspond to the germ-band stage of the
normal embryo.
A little later, segmentation of the germ band begins almost simultaneously
along the entire germ band. In most cases, fifteen segments can be seen externally
in the double abdomen. In the middle of the convex side a large segment can be
seen which contains two masses of nerve cells internally (Fig. 5G-J). The serial
sections show that the true number of the segments in the double abdomen is
sixteen. Since there are nine abdominal segments in the normal embryo, the
large segment at the middle of the double abdomen may represent the second
abdominal segment. The first abdominal segment must belong to the submerged
part. There are no traces of head and thoracic segments in this malformation.
2. Stomodaeal and proctodaeal imaginations and
formation of mid-gut strand (entoderm)
Double cephalon. Soon after the formation of the post-oral segments, two
shallow depressions appear a little proximal to each mandibular segment. These
are the stomodaeal invaginations (sti, Figs. 1 B, D; 5 A, B). As the cells increase
in number, the depressions are deepened, extending toward the centre of the
egg, and giving rise to stomodaeum (st, Figs. 1 F, 5C). At this time, by extension
of the stomodaeum, the pole cells which have lain near one end of the embryo
(pc, Fig. 5 A, B) are shifted either to the circum-labral space (pc, Fig. 5C-E) or a
point near the tip of the stomodaeum.
At an early stage of stomodaeal invagination, a few cells at the tips of the
stomodaea begin to take up stain much more heavily than others (ent, Figs. 1 B,
D; 5 A, B). They are entodermal cells, which are destined to form the mid-gut
strand. These cells multiply rapidly and form a mass along the middle of the
inner side of each cephalon. Then the tip of the mass gradually separates laterally
into two arms. The mid-gut rudiments are further pushed inward by the elongation of the stomodaeum as well as vigorous division of the cells in the rudiment.
Eventually the two arms of the mid-gut rudiment from each cephalic end meet
and fuse each side at the centre of the egg, establishing a temporary union
(mst, Figs. 1 E, 5C).
296
H. YAJIMA
In the double cephalon, this gut strand after the union is a little more slender
than those of the normal embryo. The number of cells appearing in the transverse section of the strand is less than in the normal one (mst, Fig. 4 A, 2, 3).
The strands in the oily centripetal half of the egg are circular in transverse
section (mst, Fig. 4A, 2), while the strands in the yolky centrifugal half are
flattened (mst, Fig. 4A, 3).
st
-
far
ent
br
br
dv
Fig. 4. Sections of later stages for both types of double malformation.
(A) 1-4, Transverse sections of d.c. (double cephalon) when the union of the mid-gut
strands is almost complete. Sections 1-4 are taken at a distance of i, £, f and -f the
body length from one end of the embryo.
(B) Parasagittal section of d.c. at late blastokinesis, showing the accumulation of
entodermal cells.
(C) Sagittal section of d.c. at about the time of completion of dorsal closure.
(D) Parasagittal section of d.a. (double abdomen) at about the time of completion of
dorsal closure.
(E) Parasagittal section of d.a. at the same stage as D, showing the gonad (go).
Double malformations o/Chironomus dorsalis
297
While the stomodaeum and the mid-gut strand are developing in the double
cephalon, the germ band shortens toward the middle of the convex side until its
length is reduced by about 20 % (Fig. 5C, D). This stage may correspond to the
blastokinesis of the normal embryo. In the normal embryo, since the trunk
regions contract more than the head region during blastokinesis, the degree of
contraction of the cephalic segments alone cannot be denned, while in the double
cephalon, since the decrease is doubled, the shrinkage is quite obvious.
During this contraction the stomodaea are bent at right angles at about the
middle of their lengths (st, Figs. 4 B, 5D), and an irregular bulge is formed at the
middle of each mid-gut strand (Fig. 5D). All these may be the results of the
contraction of the embryo.
In the normal embryo, after the union of the paired arms of the anterior and
posterior mid-gut rudiments, the mid-gut strands are gradually surrounded by
mesodermal cells which are the rudiments of visceral musculature, while the
mid-gut strand grows laterally to form a mid-gut tube. In the double cephalon,
since mesodermal cells do not surround the mid-gut strands, the strands, once
joined, gradually break off by the end of blastokinesis. Broken halves shorten
and draw themselves back toward the stomodaea (ent, Figs. 4B, 5E).
The absence of the visceral mesoderm around the mid-gut strand in the double
cephalon may result in the failure of maintenance of this union.
Double abdomen. The proctodaeal invaginations of the double abdomen begin
to appear at the caudal ends when the ends are still in the yolk. A little later,
entodermal cells, which are the rudiment of the mid-gut, appear at the tip of each
proctodaeal invagination (ent, Figs. 2D, 5F). The entodermal cells gradually
increase in number and grow into a mass, and then the mass separates laterally
into two arms which elongate along the venter of the embryo toward the ends of
the egg.
Immediately after the appearance of the proctodaeal invagination, the shortening or the blastokinesis of the germ band of the double abdomen begins.
The submerged caudal ends of the embryo are drawn out from the yolk until the
ends contact each other at the mid-ventral surface (Fig. 5F). During this movement, the proctodaeal invaginations grow deeper and longer (the first stretching)
until their free tips reach about the level of the second segment from the caudal
end, where they remain until after blastokinetic movement (pr,pri, Figs. 2D, E;
5G-I).
A further shortening of the entire embryo causes the two caudal ends of the
double abdomen to be pulled away from each other and the tips of the growing
mid-gut rudiments to reach the ends of the egg {ent, Fig. 5G). After bending
around the ends of the egg, the rudiments continue to elongate toward the middle
of the convex side of the egg, where eventually the paired arms of the rudiment
from each abdomen meet and fuse on each side (mst, Figs. 2E, 5H). At this
time, the caudal ends of the embryo are located at about one-third the length of
the embryo from the ends of the egg on the flat side.
298
H. YAJIMA
As soon as the union is complete the mid-gut strands are gradually covered
with visceral mesoderm cells (vm, Fig. 51). The strands stretch laterally to enclose
the yolk eventually, and differentiate into the mid-gut epithelium.
ent
sti ent
mes
nc
sti
nb ent sti
sc
ent
nc
mes
sc
ent pc
nc
ncent
pri
pri
ent
sti
nc
Ir
H
st
mst nc
br
mes
st
br
Fig. 5. Semi-diagrammatic sagittal sections of double malformations from early segmentation to completion of blastokinesis. A-E, Double cephalon; F-J, double
abdomen.
After blastokinesis, the proctodaea of the double abdomen stretch for the
second time through the distance of three segments (the second stretching). This
is the final location of the proctodaeum, where it develops into the hind-gut
(hg, Fig. 5J).
Double malformations of Chironomus dorsalis
299
3. Development of the pole cells in the double malformations
Since the double cephalon lacks the thorax and abdomen (see section 1), the
pole cells cannot help lying in an atypical position, in the cephalic part. In many
cases the pole cells are carried by stomodaeal invagination into the circumlabral space (pc, Figs. 1B, 5C), or in a few cases remain near the tip of the stomodaeum. The pole cells are gradually surrounded by mesodermal cells which
give rise to a sheath-like structure.
In the double abdomen, since the pole cells are found in a normal location,
they are shifted toward the middle of the embryo by the growth of the germ
Anlage (pc, Figs. 2B, 3F-J). When the caudal ends are bent into the yolk, the
pole cells are located inside one of the caudal ends (pc, Fig. 3J). As the proctodaea grow longer, the pole cells are separated into two lateral groups (pc,
Fig. 2F, 7) and are carried toward one end of the egg.
During blastokinesis, the pole cells are gradually surrounded by mesodermal
cells which will develop into the gonadal sheath. After blastokinesis, their final
position is in the sixth abdominal segment as in the normal embryo (go, Fig. 5 J).
Nuclear condition of the pole cells
As was stated by Hasper (1911), the pole cells of the Chironomus embryo
develop through three steps, each involving a change in the number of nuclei
in the cell. The last nuclear division takes place in the posterior perivitelline
space, producing eight bi-nucleate cells (step 1).
The bi-nucleate cells then migrate into the interior of the egg. They are
separated into two lateral groups by proctodaeal invagination and are surrounded
by mesodermal cells. The mesodermal cells come close together, surrounding the
pole cells two by two, four groups in all. As the surrounded pairs fuse, four
tetra-nucleate cells result, two on either side of the proctodaeum (step 2).
The tetra-nucleate cells are shifted toward the posterior end by the shortening
of the embryo and two on either side approach each other and adhere (step 3).
But in this case the boundary wall between the cells is retained in which state
the pole cells remain during early larval development.
In the double cephalon, although the pole cells advance up to the tetranucleate condition (Hasper's second step), they fail to fuse. This may mainly be
due to the atypical environment in which the pole cells of the double cephalon
are placed.
In the double abdomen the pole cells which are found in only one of the two
abdomens reach their final level of the differentiation.
In some cases centrifuged embryos develop otherwise 'normally' but lack the
pole cells in the interior of the embryo. No trace of gonadal structure is seen in
these embryos.
300
H. YAJIMA
4. Retraction of the embryonic envelopes, dorsal closure and
the differentiation of the internal organs
Double cephalon. As the blastokinesis of the double cephalon nears completion, the serosa, which has been lying just beneath the chorion, gradually
separates from the chorion. The amnion, which has been covering the embryo,
also begins to separate from the surface of the embryo. The compound envelope
is then retracted toward the middle of the dorsal surface of the embryo, but
before it is fully retracted, it begins to break down. Eventually, broken pieces
of the envelopes are left in spaces at both ends of the egg.
Although the process of the dorsal closure of the lateral walls of the double
cephalon cannot clearly be traced, the time of its completion can be judged to
some extent. Fig. 4C is a sagittal section of the double cephalon at about the
time of completion of dorsal closure. The two brains (br) and two suboesophageal
ganglions (sg) can be seen. The two fore-guts (fg) form proximally to the brain
and heavily stained masses of entodermal cells (ent) can be found around the
far ends of the fore-guts. In the double cephalon, the inner surface of the labrum
remains exposed at either end of the embryo and the labral lobe does not move
to cover the mouth opening. Just above the masses of entodermal cells, yolk
granules are lying free. There is no trace of the submerged cell mass which had
been maintained during earlier stages. A hypodermis (h) covers the entire
surface of the embryo.
Double abdomen. The retraction of the embryonic envelopes in the double
abdomen proceeds in a similar way to the normal embryo. At late blastokinesis,
the compound envelope separates from the embryo and is retracted toward the
middle of the dorsal surface of the embryo, where it is absorbed into the yolk.
Concerning the dorsal closure, as soon as blastokinesis is complete, the lateral
walls of the double abdomen expand latero-dorsally until they meet and fuse at
the mid-dorsal line. Fig. 4D and E show sagittal sections at the stage just after
the dorsal closure. As seen in D, the dorsal vessel (dv) is well differentiated in
each abdomen. In the centre, the mid-gut enclosing heavily stained yolk granules
lies between the two hind-guts (hg). In E, the bundle of nerve fibres from both
halves runs through the well-differentiated ventral nervous system (vns). The
gonad (go) is present near the left side of the embryo, while it is entirely lacking
on the right side. A little above the gonad, Malpighian tubule (Mt) is seen.
As can be judged from Fig. 4C-E, the internal organs of the two types of
double malformations are differentiated as well as the external structures.
Furthermore, the sizes of these organs are almost equal to those of the normal
ones.
5. Hatching
Double cephalon. The double cephalon can never hatch, in spite of the fact
that the body colour turns yellowish brown, as in the normal embryo near
Double malformations 0/Chironomus dorsalis
301
hatching time. However, as the natural hatching time approaches, the chorion
becomes softer and sometimes stretches longitudinally. Unpublished data
suggest that the hatching enzyme is being secreted by the double cephalon.
The failure of hatching in the double cephalon may partly be due to a greater
distance intervening between the chorion and the tips of the heads, but may
chiefly be due to its lack of motility.
Double abdomen. The double abdomen can 'hatch', although the frequency
of occurrence is very low. As soon as dorsal closure is complete, the body of the
double abdomen begins to stretch longitudinally and move frequently. Because
of the limited space within the chorion, the body becomes bent and coiled.
Sooner or later, the larva bursts through one end of the chorion. The hatching
of the larva in this way is apparently rather difficult. Indeed the time between
the beginning of the pre-hatching movement and the hatching itself may be
twice that for the normal larva. This may be due to the failure of the chorion to
be softened owing to the lack of hatching enzyme in the double abdomen.
The freshly hatched double abdomen is 760/t long, which is twice the length
of the malformation at the completion of dorsal closure. The freshly hatched
larva is also one and a half times longer than the normal larva.
DISCUSSION
Only a few outstanding features of the development of the double malformations of Chironomus dorsalis will be pointed out and briefly considered in this
section.
(a) Differentiation of the mid-gut strand
As shown in section 2, in the double cephalon the union of the mid-gut
strands from each half of the duplicated structures is only temporary, and after
severance the strands stop differentiating into mid-gut epithelium, unlike the
joined mid-gut strand of the double abdomen. Why does the mid-gut strand
in the double cephalon break off and fail to differentiate into mid-gut epithelium ? Obviously, the difference in the state of the mid-gut strands between
the two double malformations is the presence or absence of the visceral mesodermal cells around the strands. In the double cephalon, although the mesodermal elements do occur, they develop into cephalic mesodermal structures and
do not envelop the mid-gut strands. The anterior and posterior mid-gut strands
of the normal embryo as well as the strands of the double abdomen are surrounded by trunk mesodermal cells. It can be inferred from the above facts that
the cephalic mesodermal cells are not effective in the morphogenesis of the
mid-gut.
The significance of the visceral musculature cells (mesoderm) in the mid-gut
formation may be that (1) the muscle cells provide a firm support for the midgut strand to join the strands when they come into contact, and (2) the muscle
cells may also induce the entodermal strand cells to differentiate into mid-gut
epithelium, as has been shown in Leptinotarsa by Haget (1953).
302
H. YAJIMA
(b) The pole cell and the differentiation of the gonadal sheath
From early in this century, many studies have been performed to clarify the
relationship between the pole cell and the differentiation of the mesodermal
gonadal sheath. Hegner (Chrysomelid beetle, 1908), Geigy {Drosophila melanogaster, 1931), Aboim (D. melanogaster, 1945), Haget (Leptinotarsa, 1953),
Hathaway & Selman (D. melanogaster, 1961), Oelhafen (Culex pipiens, 1961)
and Jura (D. virilis, 1964) showed that the mesodermal gonadal elements can
develop without the pole cells (or germinal elements), while Counce & Selman
(D. melanogaster, 1955) and Poulson & Waterhouse (D. melanogaster, 1960)
suggested that the mesodermal cells fail to differentiate into the gonadal sheath
when the pole cells are absent.
The double abdomen of Chironomus is ideal material for studying the differentiation of the mesodermal gonadal sheath, because the malformation has
two abdomens—one containing the pole cells and the other without them. The
present results clearly indicate that the gonadal sheath will not develop unless
the pole cells are present (section 3). A similar relationship but in the opposite
sense is found in the double cephalon.
The differentiation capacity of the pole cells themselves may be analysed
by comparing the development of the pole cells in the two types of double
malformations. As shown in section 3, the pole cells in the double cephalon
fail to differentiate into their final forms probably owing to the atypical
surroundings, while the cells in the double abdomen reach their final level of
differentiation much as in the normal embryo.
RESUME
Etude sur la developpement des structures internes des malformations doubles
de Chironomus dorsalis par des coupes fixees
Le developpement des structures internes a ete etudie sur des coupesfixeesde Chironomus
dorsalis chez 'double cephalon' d'une part et chez 'double abdomen' d'autre part.
La proliferation cellulaire qui est a l'origine de Pebauche germinale, se presente dans
'double cephalon', tout au long du cote convexe (ventral) de l'ceuf et dans 'double abdomen',
aux deux extremites du cote aplati (dorsal).
II en resulte chez 'double cephalon' une ebauche fusionnee, simple, tout au long du cote
convexe de Pceuf, et chez 'double abdomen', deux ebauches germinales, aux deux extremites
du cote aplati.
Pendant la formation de la bande germinale, chez 'double cephalon', la partie la plus
posterieure, situee au milieu du cote convexe de l'ceuf, et chez 'double abdomen' la partie la
plus anterieure, localisee au milieu du cote convexe, ne se differencient pas et degenerent.
Dans chacune des tetes redupliquees de 'double cephalon', les segments cephaliques,
anterieurs au premier segment maxillaire, sont formes, mais les segments thoraciques et
abdominaux sont entierement absents.
Chez 'double abdomen', huit segments abdominaux, posterieurs au second segment
abdominal, sont formes et les segments cephaliques et thoraciques sont absents.
Les deux paires du rudiment de I'intestin mover, chez 'double cephalon' sont unies temporairement mais se separent a la fin de la blastocinese.
Double malformations of Chironomus dorsalis
303
Quand les deux paires de rudiment d'intestin moyen chez 'double abdomen' se rencontrent, elles restent fusionnees, entourees de cellules mesodermiques viscerales, de facon
normale, et se developpent en epithelium intestinal.
Dans ces malformations doubles, les cellules polaires sont contenues dans une seule des
structures redupliquees. Les cellules polaires de 'double cephalon' presentent l'etat tetranuclee (2e stade de Hasper) mais elles ne fusionnent pas pour former la gonade. Dans 'double
abdomen' la gonade se differencie dans la structure qui contient les cellules polaires, mais non
dans I'autre.
Les enveloppes embryonnaires, chez 'double cephalon' ne se retractent pas a l'interieur de
I'embryon bien qu'elles Ie fassent de maniere normale chez 'double abdomen'.
Un individu' double cephalon' ne peut jamais eclore, ce qui peut etre Ie cas pour un individu
'double abdomen'.
The author wishes to thank Dr Katsuma Dan, Tokyo Metropolitan University, for his
kind advice and encouragement. Thanks are also due to Dr Rodger D. Mitchell, Department
of Zoology, University of Florida, for reading the manuscript.
REFERENCES
A. N. (1945). Developpement embryonnaire et post-embryonnaire des gonades
normales et agametiques de Drosophila melanogaster. Revue suisse Zool. 52, 53-154.
COUNCE, S. J. (1961). The analysis of insect embryogenesis. A. Rev. Ent. 6, 295-312.
COUNCE, S. J. & SELMAN, G. G. (1955). The effects of ultrasonic treatment on embryonic
development of Drosophila melanogaster. J. Embryol. exp. Morph. 3, 121-141.
GAUSS, U. & SANDER, K. (1966). Stadienabhangigkeit embryonaler Doppelbildungen von
Chironomus thummi. Naturwissenschaften 53, 182-183.
GEIGY, R. (1931). Action de l'ultra-violet sur Ie pole germinal dans l'oeuf de Drosophila
melanogaster. Revue suisse Zool. 38, 187-288.
HAGET, A. (1953). Analyse experimentale des facteurs de la morphogenese embryonnaire
chez Ie coleoptere Leptinotarsa. Bull. biol. Fr. Belg. 87, 123-217.
HASPER, M. (1911). Zur Entwicklung der Geschlechtsorgane von Chironomus. Zool. Jb.
(Anat.) 31, 543-612.
HATHAWAY, D. S. & SELMAN, G. G. (1961). Certain aspects of cell lineage and morphogenesis
studied in embryos of Drosophila melanogaster. J. Embryol. exp. Morph. 9, 310-325.
HEGNER, R. W. (1908). The effects of removing the germ cell determinants from the eggs of
some chrysomelid beetles. Biol. Bull. mar. biol. Lab., Woods Hole 16, .19-26.
JURA, C. (1964). Cytological and experimental observations on the origin and fate of the pole
cells in Drosophila virilis Sturt. IT. Experimental analysis. Acta biol. cracov. (Ser. zool.) 7,
89-103.
KALTHOFF, K. & SANDER, K. (1968). Der Entwicklungsgang der Missbildung' Doppelabdomen'
im partiell UV-bestrahlten Ei von Smitta parthenogenetica (Dipt., Chironomidae). Wilhelm
Roux' Arch. EntwMech. Org. 161, 129-146.
OELHAFEN, F. (1961). Zur Embryogenesis von Culex pipiens. Markierung und Extirpationen
mit UV-Strahlenstich. Wilhelm Roux' Arch. EntwMech. Org. 153, 120-157.
OVERTON, J. & RAAB, M. (1967). The development and fine structure of centrifuged eggs of
Chironomus thummi. Devi Biol. 15, 271-287.
POULSON, D. F. & WATERHOUSE, D. F. (1960). Experimental studies on pole cells and midgut differentiation. Aust. J. biol. Sci. 13, 541-567.
YAJIMA, H. (1960). Studies on embryonic determination of the harlequin-fly, Chironomus
dorsalis. I. Effects of centrifugation and of its combination with constriction and puncturing.
J. Embryol. exp. Morph. 8, 198-215.
YAJIMA, H. (1964). Studies on embryonic determination of the harlequin-fly, Chironomus
dorsalis. II. Effects of partial irradiation of the egg by ultra-violet light. J. Embryol. exp.
Morph. 12, 89-100.
ABOIM,
(Manuscript received 10 September 1969)