Study of the development of the internal organs of the double malformations of Chironomus dorsalis by fixed and sectioned materials

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.

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 5 min. 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 μ and stained with Heidenhain’s haematoxylin and light-green.

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.

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 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 Anlage’ 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. 1 B–E;,3C–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. 3A–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 extra-embryonic 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. 3C–E). This is the formation of the head lobe at both ends of the germ Anlage (hl, Figs. 1B, 3E).

When the head lobes 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 Anlage. 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.

After the completion of the cellular blastoderm of the double abdomen, cell proliferation that gives rise to the germ Anlage begins at two points 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. 3I). Finally, the growing ends meet at the middle of the convex side and the two Anlagen unite into one (Fig. 3 J).

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. 3I). 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 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 the ventral groove. Invagination 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 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.

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. IB, 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).

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, 5). 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, 5).

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 defined, 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.

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–1).

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.

As soon as the union is complete the mid-gut strands are gradually covered with visceral mesoderm cells (vm, Fig. 5I). The strands stretch laterally to enclose the yolk eventually, and differentiate into the mid-gut epithelium.

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).

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 circum-labral space (pc, Figs. 1 B, 5C), or in a few cases remain near the tip of the stomo-daeum. 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. 3 J). As the procto-daea 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).

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.

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.

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.

The double cephalon can never hatch, in spite of the fact that the body colour turns yellowish brown, as in the normal embryo near 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.

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 μ 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.

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.

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).

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 melano-gaster, 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.

Le développement des structures internes a été étudié sur des coupes fixées de Chironomus dorsalis chez ‘double cephalon’ d’une part et chez ‘double abdomen’ d’autre part.

La prolifération cellulaire qui est à l’origine de l’ébauche germinale, se présente dans ‘double cephalon’, tout au long du côté convexe (ventral) de l’œuf et dans ‘double abdomen ‘, aux deux extrémités du côté aplati (dorsal).

Il en résulte chez ‘double cephalon’ une ébauche fusionnée, simple, tout au long du côté convexe de l’œuf, et chez ‘double abdomen’, deux ébauches germinales, aux deux extrémités du côté aplati.

Pendant la formation de la bande germinale, chez ‘double cephalon’, la partie la plus postérieure, située au milieu du côté convexe de l’œuf, et chez ‘double abdomen’ la partie la plus antérieure, localisée au milieu du côté convexe, ne se différencient pas et dégénèrent.

Dans chacune des têtes rédupliquées de ‘double cephalon’, les segments céphaliques, antérieurs au premier segment maxillaire, sont formés, mais les segments thoraciques et abdominaux sont entièrement absents.

Chez ‘double abdomen’, huit segments abdominaux, postérieurs au second segment abdominal, sont formés et les segments céphaliques et thoraciques sont absents.

Les deux paires du rudiment de l’intestin moyen, chez ‘double cephalon’ sont unies temporairement mais se séparent à la fin de la blastocinèse.

Quand les deux paires de rudiment d’intestin moyen chez ‘double abdomen’ se rencontrent, elles restent fusionnées, entourées de cellules mésodermiques viscérales, de façon normale, et se développent en épithélium intestinal.

Dans ces malformations doubles, les cellules polaires sont contenues dans une seule des structures rédupliquées. Les cellules polaires de ‘double cephalon’ présentent l’état tétra-nucléé (2e stade de Hasper) mais elles ne fusionnent pas pour former la gonade. Dans ‘double abdomen’ la gonade se différencie dans la structure qui contient les cellules polaires, mais non dans l’autre.

Les enveloppes embryonnaires, chez ‘double cephalon’ ne se rétractent pas à l’intérieur de l’embryon bien qu’elles le fassent de manière normale chez ‘double abdomen’.

Un individu ‘double cephalon’ ne peut jamais éclore, ce qui peut être le 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.