Recent Advances in Insect Embryology in Japan and Poland
Edited by H. Ando and Cz. Jura
Arthropod. EmbryoL. Soc. Jpn.
(lSEBU Co. Ltd., Tsukuba) 1987

77

Embryonic Developmentof Tetrodontophora bielanensis (Collembola):
Descriptive, with Scanning Electron Micrographs

Czeslaw JURA
Anna KRZYSZTOFOWICZandElZbieta KISIEL
Synopsis
The embryonic development, from oviposition till hatching, of the collembolan species Tetrodontophora bielanensis is described. The description bases on 87 scanning electron micrographs.
The micrographs demonstrate surface and internal morphological changes.

Introduction
In this paper the whole embryonic development of Tetrodontophora bielanensis is described, utilizing informations gained from scanning electron microscopy studies.
The foundations for understanding of T. bielanensis embryogenesis come from papers by Jura (1965, 1966, 1967a, b) and Tyszkiewicz (1975); these authors examined thespecies by light microscope. Some data based on scanning electron microscope studies
were also published (Jura and Krzysztofowicz, 1978, 1982, 1986; W6jtowicz, 1978;
Krzysztofowicz, 1986b) and have provided much of the informations included in thispaper, nevertheless, new ones have been added especially those concerning the cytoarchitecture of the freshly laid egg, cleavage, dorsal organ formation as well as concerning final developmental stages.
The scanning electron micrographs were selected, to illustrate most characteristic
embryological events. The emphasis was put especially on those stages with changing

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Cz. Jura, A. Krzysztofowicz and E. Kisielsurface characteristics, but some mention is made also of concurrent internal changes
in embryo morphology. Making selection the authors intended to summarise the results
obtained until now, to make basis for further study.

Material and Methods
For observations the specimens of Tetrodontophora bielanensis were collected in thevicinity of Krak6w, especially of Bielany Hills, where they can be found in great numbers in the forest under loose bark of trees, dead leaves, on moss, or on mushrooms.
In nature oviposition occurs during the first days of November, especially after 23 days with temperature below ot. The eggs are usually laid in a group of from 1525. Every female deposits about 60 eggs. The females deposit their eggs mainly in theclefts and caves of ground under the forest litter, and sometimes between the deadleaves of the littter. In such environment, humid and with temperatures ranging between + 4 t - ot, the embryos developed during the period of about six autumnwinter-spring months.
The fertilization of eggs is internal. The males of T. bielanensis deposit spermatophores, in groups of 10 - 20 (Krzysztofowicz and Byczkowska-Smyk, 1966; Krzysztofowicz, 1967, 1980a). Before laying eggs, the females collect spermatophores and introduce them to their reproductive systems.
The animals collected at the beginning of November and kept under laboratory
conditions, in cool chamber (+ 4 t ) in glass dishes filled with fragments of decaying
wood, moss and leaves, laid eggs in mid November. The animals were fed withmushrooms collected in nature.
The developing embryos were also kept in glass dishes, filled with humid forest litter, in cool chamber at ± 4 t. Under such condition the developmental period lasts, as
in nature, about six months.
The eggs were examined daily under dissecting microscope and fixed in appropriate stage of development. Observations are especially instructive when eggs are place
under dissecting microscope in embryological hollow black slides filled with water.
Through semitransparent chorion the progress of the developmental processes, goingon inside the egg, may be easily observed.
Embryos were fixed in 2% osmium tetroxide in phosphate buffer of pH 7.3 for 19
hr and dehydrated in grade aceton. The eggs had to be pricked to facilitate penetrationof fixative into the egg interior. Best results were obtained by pricking under fixativewith a fine tungsten needle. Some procedures require removal of chorion, before or after fixation. Before fixation this was done chemically , by immersing the eggs in a 3 %solution of sodium hypochlorite for about a minute. After fixation, the eggs were dechorionated manually by using fine forceps. Manually, if required, was also removed
vitelline membrane. Some eggs were fractured manually by means of fine needle.
Fixed embryos were dried by the critical-point technique, using CO 2 , The driedembryos were coated with carbon and gold and examined in a JEOL ISN scanningelectron microscope.
The developmental age of each illustrated embryo is indicated, but all timing data
are approximate since the progress of development varies among individual eggs.

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79

Among eggs from different stocks of females, as a result of very small temperature fluctuations, the variations were rather high, even up to 20 days.

Observations
Egg
The egg, immediately after oviposition, is yellowish in colour, perfectly spherical,
and measures about 0.5 mm in diameter. Externally it is not possible to find any detailwhich would allow to determine either anterior or posterior pole, ventral or dorsal sideof the egg.

Egg envelopes
The surface of a freshly deposited egg, as viewed in scanning electron microscope,
appears smooth owing to presence of the external sticky layer. The layer envelops theunderlying chorion and is about 0.3 fL m thick. Most probably, the function of thislayer is to cement individual eggs into a bunch. The origin of the sticky layer is notknown and its elucidation would require further studies.
The chorion is about 6.6 fL m thick. Figure 2 illustrates a fragment of the egg surface with the external sticky layer partly removed, thus unveiling the surface of thechorion itself. The surface of the chorion does not show any ornament or sculpture, as
it frequently occurs in insects, only numerous pits, distributed regularly, can beobserved. The pits are probably replicas of the follicular cells. According to W6jtowicz(1978), on the average, there are about 42,000 pits per 1 mm 2 , the distance between
adjacent pits ranging from 4 to 10 fL m. The pits are about 0.6-1.0 fL m deep andtheir diameter averages 3 fL m. W6jtowicz has also described the ultrastructure of thechorion and Krzysztofowicz and Kisiel (1987) its formation during oogenesis.
The chorion of T. bielanensis does not possess such structures as micropyle, pseudomicropyle or aeropyle, which occur commonly in other insect species, and are involvedin physiological processes of vital importance to the developing embryo. It is possiblethat lack of these structures is connected with chorion rupture which occurs during themid period of development, after formation of the blastodermic cuticles by the embryo(see below).
The chorion of T. bielanensis exhibits a polarized permeability, permitting loss ofwater from egg but preventing the income of water from surrounding. This statement isbased on observations of the eggs development in natural and laboratory conditions. In
laboratory, it was observed that eggs can develop, without any damage, in water as environment, what means that chorion is fully waterproofed. On the other side, the eggs
develop only at high humidity, at dry condition they immediately degenerate. Under
natural conditions water loss is normally restricted by the particular environment of
the oviposition site selected by female. In litter, where eggs are laid, the transpirationis restricted because of very moist environment. Thus it is natural that chorion iswaterproofed but not respiratory resistant.

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- - - - - - -- ---- -80--- - - --Cz. Jura, A. Krzysztofowicz and E. KisielFig. I. Part of outermost sticky layer of freshly laid egg. Surface view. X 3,000.
Fig. 2. Part of sticky layer and of underlying chorion of the same egg as in Fig. 1. X
7,260.
ch, chorion; rf, replica of follicular cell; si, sticky layer.

Embryonic development of TetrodontophoraFig. 3. Fragment of fractured freshly laid egg. X 180.
Fig. 4. Fragment of peripheral portion of fractured egg same as in Fig. 3. X 1,080.
cy, inner cytoplasm; mv, vitelline membrane; sc, peripheral cytoplasm; y, yolk.

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Cz. Jura, A. Krzysztofowicz and E. Kisiel

The surface of the vitelline membrane, the second egg envelope underlying chorion, is completely smooth (Figs. 3, 4). The vitelline membrane is about 0.6 p m thickand, in opposite to chorion, it does Qot ruptures during the mid period of embryo development (see below).

Egg cytoarchitecture
The description of general egg cytoarchitecture is based on fractured eggs (Figs. 3-5).
The. egg proper of T. bielanensis, as in other insects, contains three major components: periplasm, endoplasm with nucleus, and yolk. We shall describe these threecomponents, but neglecting the nucleus which can not be observed under scanningelectron microscope.
Periplasm and endoplasm consists of a cytoplasmic matrix that is rich in free ribosomes and encloses a multitude of organelles and other components (Komorowska,
1970; Fryc, 1971; Klaja, 1971; Malcher, 1971; Adamiec, 1975; Gancarzewicz, 1975;
Sikora, 1975; Bilinski, 1976; Klag, 1982a, b, 1983, 1984).
By means of scanning electron microscope it is not possible to distinguish any
axial polarity in T. bielanensis egg. However, Gancarzewicz (1975) and Klag (1982)
documented that one pole of freshly laid egg is rich in components containing RNA,
which according to Klag are germ-cells determinants.
Periplasm, as viewed in scanning electron microscope, consists of cytoplasmic matrix which lacks yolk particles, and is extremely thin (Figs. 3, 4). In this respect T.
bielanensis egg does not agree with corelation between oogenesis type and periplasm
thickness established for Pterygota, where egg cytoarchitecture in general reflects thecondition of oogenesis as well as the future course of embryogenesis. In Pterygota theeggs with thin periplasm are produced in result of panoistic oogenesis and have shortgerm band, whether the eggs with thick periplasm are produced in result of polytrophic
oogenesis and have long germ band (for review see Counce, 1973, and Sander, 1983).
In case of T. bielanensis the oogenesis is polytrophic (Krzysztofowicz, 1971, 1975), eggs
have extremely thin periplasm, but extremely long germ band (Jura, 1986).
The endoplasm consists of a network of strands, occupying the space between yolk
components, varying in thickness that here and there form cytoplasmic islands of different sizes (Figs. 4, 5). Peripherally, the endoplasm merges into the periplasm without
any change in the ultrastructural aspect.
The egg of T. bielanensis is very rich in storage substances. The meshes of the endoplasm are filled by storage substances that can be classified as proteid yolk bodies,lipid droplets, and glycogen granules (Fryc, 1971; Klaja, 1971; Malcher, 1971; Ksi~z­
kiewicz-Ilijeva, 1974; Bilinski, 1976). All these components are arranged concentricallywith some regularity. The components showing the largest diameters are concentrated
in the egg centre, whether the diameter of yolk components diminishes toward the egg
periphery. This concerns especially proteid yolk bodies (Figs. 3-5).
Proteid yolk bodies dominate, but the egg is also lipid-rich and this probably is
being related with the low temperature at which it develops. The lipids occur in theform of smaller globule-bodies than those of proteid-bodies. In the freshly laid egg,

Embryonic development of TetrodontophoraFig. 5.

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Fragment of central part of the storage material of the same egg as in Fig. 3. X
2,160.
Fig. 6. About 5-days old embryo, surface view. Note flattening of one of egg pole. X 80.
Fig. 7. About 6-days old embryo, surface view. Note polar body extruded on the flattened
pole. X 80.
ap, animal pole; cy, inner cytoplasm; p, polar body; y, yolk.

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Cz. Jura, A. Krzysztofowicz and E. Kisiellipid globules occur in the largest number in the vicinity of periplasm, whether the glycogen granules are distributed more or less uniformly in periplasm as well as in endoplasm.
Experimental studies showed that the egg of T. bielanensis is equiped with some extracaryotic information, elaborated during oogenesis. This information is most probably
localized in the egg periphery and is necessary for such process as cell membrane
formation during early cleavage. The stratification of such components as RNA, proteins, glycogen or lipids, in result of centrifugation, does not influence the normal
formation of the embryo (Ksiqikiewicz-Ilijeva, 1974). On the other hand, in the fertilized egg with partially injured periplasm cytokinesis does not occur (Jura, 1975, 1986).

Maturation
As we have mentioned, the freshly laid egg is perfectly spherical. After about 6days from oviposition the first changes in this egg shape occur.
During the first 5 days of development the fertilized egg smugly fills the space enclosed by the vitelline membrane. Beginning with 6th day of development the egg shortens and flattens on one pole (Fig. 6), apparently by changing volume since its chorionvitelline membrane girth remains constant. Fluid-filled pocket appears between thevitelline membrane and the flattened pole of the egg.
The egg remains flattened on one pole until the end of the first cleavage (see below). Meantime, the polar bodies emerge and occupy the pocket (Fig. 7).
Most probably, the flattening and lengthening of the egg involves the passage of a
fluid out and into the egg. Also the occupation of the pocket by the polar bodies suggests that the egg flattening may facilitate polar bodies formation.
The flattened egg portion represents the pole on which latter first cleavage furrows
will appear, and because of this, we may call it as animal egg pole (compare Figs. 7,

8).

Cleavage
Cleavage begins 6 to 8 days after oviposition, the early being total.
Prior to cleavage, zygotic nucleus undergoes two successive divisions withoutaccompanying cytokinesis. As the result of this, four daughter nuclei are formed andthe egg at this stage represents four-nuclei syncytium.
Fig. 8. About 8-days old embryo, beginning of holoblastic cleavage. Animal pole view. X
150.
Fig. 9. About 8-days old embryo, beginning of holoblastic cleavage. Vegetal pole view. X
150.
Fig. 10. About 9-days old embryo, 4-blastomere stage. Side view. X 105.
Fig. 11. About lO-days old embryo, transition from 4- to 8-blastomere stage. Animal poleview. X 105.
Fig. 12. About lO-days old embryo, 8-blastomere stage. Animal pole view. X 105.
Fig. 13. Fragments of two meridional cleavage furrows of the same embryo as in Fig. 12.
X 306.

Embryonic development of Tetrodontophora

85

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Fig. 14.
Fig. 15.
Fig. 16.
Fig. 17.
Fig. 18.

Cz. Jura, A. Krzysztofowicz and E. Kisiel

About IO-days old embryo, late 8-blastomere stage. Side view. X 105.
About 12-days old embryo, transition from 8- to 16-blastomere stage. X 105.
Fractured embryo at 16-blastomere stage. Note central yolk mass.
8-blastomere stage with "additional blastomere" (see text) . X 105.
Fragment of the surface of the embryo about 14-days old during the transItionfrom 16- to 32-blastomere stage. Note cleavage furrow formation in the lower
blastomere. X 369.
cf, cleavage furrow; cym, central yolk mass; sb, superficial blastomere.

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87

Fig. 19. About 14-days old embryo, transition from 16- to 32-blastomere stage. X 105.
Fig. 20. About 14-days old embryo, 16-blastomere stage preparing itself for cleaving. X
105.
Fig. 21. Surface view of one of the blastomere at 32-blastomere stage. X 620.
cf, cleavage furrow; cs, depression marking localization of cleavage spindle (note formationof cleavage furrow in relation to cleavage spindle).

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Cz. Jura, A. Krzysztofowicz and E. KisielDuring the next day of development, the nuclei migrate from the centre to the
opposite quarters of the egg and then on the flattened pole two cleavage furrows
appear (Fig. 8) perpendicular to each other. These two first furrows may be named as
meridional since they are localized on animal pole. The furrows deepen, extend to thesides, toward the opposite pole, and increasingly engirdle the whole egg. Within 10-15
hr the embryo becomes subdivided into four blastomeres (Figs. 8-10). Later a third
furrow system appears, a horizontal one, which runs parallel to the equator of the egg(Figs. 11, 12). The resulting 8-blastomere stage consists of four animal-pole blastomeres and four vegetal-pole ones, of equal size. The complete 8-cell stage is shown in
Figs. 12 and 14.
Thus, the early cleavage is holoblastic, synchronous and equal. However, this was
not the rule for all embryos studies. Within about 25 % of the embryos, the early cleavage was somewhat unequal. Moreover, some embryos showed one or two" additional"
flimsy blastomeres (Fig. 17). It was not possiblle to determine whether these additional
blastomeres were equiped with nuclei, or were purely cytoplasmic structures, since
under scanning electron microscope the nuclei could not be observed. Nevertheless,further development of such embryos was quite normal.
During described cleavage divisions, the cells of the embryo must gain new surface
areas, which are composed of new periplasm + new cell membrane portion. Jura andKrzysztofowicz (1982) have noted that when holoblastic cleavage is going on thegrooves, marking the cleavage planes, are wide but shallow (Fig. 13). The egg surface,in other planes than the grooves, remains smooth and seems to be stationary. Under
scanning electron microscope no traces of insertion of preexisting surface area, from thebottoms of the grooves into the embryo interior, or splitting of ooplasm, can beobserved. This remains in deep contrast with events accompanying the cleavage processes in a frog egg (for review see Beams and Kessel, 1976). The evidences obtained
for T. bielanensis suggest that in this species during holoblastic cleavage formation ofquite new surface areas and synthesis of new cell membranes, is necessary to complete
the separation of cells.
After reaching 8-blastomere stage, the further cleavage furrows do not involve thewhole yolk material of blastomeres. Innermost parts of blastomeres are cut off asanucleate yolk masses, in result of formation of new cell membranes of blastomeres
above previous ones. The separated yolk masses fuse into a single central yolk material,
filling the interior of the embryo, leaving the layer of pyramidal blastomeres at the egg
surface (Fig. 16). Thus, during the transition from 8- to 16-blastomere stage the superficial cleavage is initiated and the stereoblastula is formed. At the centre of stereoblastula the yolk globules are uniformly distributed, previous blastomere boundaries had
disappeared and no even traces of them could be seen.
Since 16-blastomere stage, the layer of superficial blastomeres of the embryo of T.
bielanensis is equivalent to early cellular blastoderm, very characteristic embryonicstructure of Thysanura and Pterygota species. Also beginning with 16-blastomere stage
further cleavages are asynchronous and unequal.
During the transition from 16- to 32- and than up to about 3, ODD-blastomere stage
the superficial blastomeres further diminish in size (Figs. 16 - 35), and at every division
separate off at their bases additional yolk, increasing the central yolk mass (Figs. 2628).

Embryonic development of TetrodontophoraFig. 22.

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About 16-days old embryo, 32-blastomere stage at early transition to 64blastomere stage. X 105.
Fig. 23. More advanced in development embryo than shown in Fig. 22, transition from 32to 64-blastomere stage. Note surface activity of cleaving blastomeres. X 105.
Fig. 24. More advanced in development embryo than shown in Fig. 23, transition from 32to 64-blastomere stage. Note difference in size of blastomeres on animal and vegetal poles. X 250.

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Fig. 25.

Cz. Jura, A. Krzysztofowicz and E . Kisiel

About 17-days old embryo, fragment of surface view of 64-blastomere stage. X
340.
Fig. 26. Fragment of fractured embryo during transition from 16- to 32-blastomere stage.
X 700.
sb, superficial blastomere; y, yolk.

Embryonic development of TetrodontophoraFig. 27.

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Fragment of fractured embryo during transition from 32- to 64-blastomere stage.
X 818.
Fig. 28. Fragment of fractured embryo at 64-blastomere stage. X 888.
cf, cleavage furrow; sb, superficial blastomere; y, yolk.

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Cz. Jura, A. Krzysztofowicz and E. Kisiel

From 16-blastomere stage on, another new event alters the morphological organization of the embryo. On the animal hemisphere the divisions are more frequent, theblastomeres smaller, whereas the opposite pole is occupied by the lager blastomeres.
Such condition is illustrated in Figs. 22-24, 29 and 30.
Another fact must be also noted. From 16-blastomere stage on, under scanningelectron microscope, the localization of nuclei or cleavage spindles inside the bias tomeres is clearly marked by depressions. The depressions, the postfixative effects ofshrinkage of cytoplasmic islands surrounding the nuclei, were comparatively shallow in
16-blastomere stage (Figs. 16, 19), but much deeper in the consecutive stages (Figs. 22- 24, 29 - 32). This phenomenon illustrates progressive increasing of amount of cytoplasm arround the nuclei.
Analysing the cleavage process Jura and Krzysztofowicz (1982) have suggested
that beginning with 16-blastomere stage there is marked contrast according to events
concerning cytokinesis, when compared with previous holoblastic cleavage. During partial superficial cleavage, cytokinesis involves drastic changes in cell surface, wellobservable under scanning electron microscope. Furrow formation begins as a processof splitting, which is especially well visible in fractured embryos (Fig. 27). The furrowis deep (Figs. 19, 31), its outlines are very irregular (Figs. 18, 21). The surface area of
a dividing blastomere, neighbouring the furrow, wrinkles and forms irregular folds(Fig. 18). The scanning electron micrographs also suggest, that the whole surface of acleaving superficial blastomere is not stationary. Between successive mitoses it is relatively smooth, whereas during the metaphases it is highly irregular. This is wellvisualized when a resting embryo (Fig. 30) is compared with actively cleaving one (Fig.
29).
The described observations strongly indicate that stretching and at last partial inserting of preexisting surface membrane accompanies cytokinesis in case of partial superficial cleavage. Moreover, firm evidences exist that during this type of cleavage thetime and place of appearance of furrows are determined by the mitotic events. In every
superficial blastomere, the cytokinesis begins when the process of mitosis has advanced
to the metaphase stage. The furrow starts to develop in a position perpendicular to the
middle of the spindle (compare Figs. 19, 32). This was not the case during first holoblastic cleavage. As it was described above, at this stage of development firstcaryokinesis occurs, the embryo becomes four-nuclei syncytium and only after that thecleavage furrows appear.
Another fact is of interest from the evolutionary point of view. As we remember
the yolky eggs of T. bielanensis show first holoblastic then partial superficial cleavage.
Beginning with 8-blastomere stage at every following cleavage the innermost parts of
blastomeres are cut off as yolk masses. As the superficial blastomeres became more andmore narrow, the nucleus and associated cytoplasmic island within each blastomere
moves outwards towards the exposed surface of the cell. Such process of formation of
blastoderm is connected with repetitious synthesis and loosing of cell plasma membranes. This type of blastoderm formation is energy-absorptive, biologically uneconomical, and in consequence the development is relatively slowed. In yolky eggs of Pterygota the cleaving nuclei wander directly to periplasm. Synthesis of plasma membranes
is completely omitted until the nuclei invade periplasm. Thus the cleavage process ofT. bielanensis eggs may be estimated as primitive and making link between the eggs

Embryonic development of Tetrodontophora

93

Fig. 29. Resting embryo, 64-blastomere stage. X 105.
Fig. 30. About 18-days old actively cleaving embryo, transition from 64- to 128-blastomere
stage. X 105
Fig. 31. Fragment of the surface view of the embryo shown in Fig. 30. X 517.
cf, cleavage furrow.

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Cz. Jura, A. Krzysztofowicz and E. KisielFig. 32. Fragment of the surface view of embryo at about 120-blastomere stage. X 510.
Fig. 33. Fragment offractured embryo at about 120-blastomere stage. X 517.
cs, depression marking localization of cleavage spindle; ib, inner yolk material of blastomere; n, depression marking localization of nucleus; su, surface of blastomere.

Embryonic development of TetrodontophoraFig. 34. About 20-days old embryo, 128-blastomere stage. X 295.
Fig. 35. About 22-days old embryo, 300-blastomere stage. X 105.
Fig. 36. Fractured embryo at about 300-blastomere stage. Note secondary yolk cleavage.
b, superficial blastomere; y, yolk.

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Cz. Jura, A. Krzysztofowicz and E. Kisielcleaving purely totally and those ones cleaving purely superficially.
The processes connected with cleavage stage of development, which could not be
observed under scanning electron microscope, like formation of yolk cells and primary
germ cells, were analysed by light. and transmission electron microscopes by Jura
(1965, 1967a, b) and Klag (1982a, b, 1983, 1984). Jura (1986) also analysed the regulative capacity of the embryo from fertilization till blastoderm stage. The morphological significance of yolk cells was studied by Jura (1966), Jura and Krzysztofowicz(1977).

Secondary yolk cleavage
During the holoblastic cleavage the storage material of the egg becomes divided
into individual blastomeres, then, this material left in the centre of the embryo, in result of superficial cleavage up to about 64-blastomere stage, represents an aggregationof yolk globules uniformly distributed within the mesh of the inner ooplasm (Figs. 16,
28). Following 64-blastomere stage the central yolk mass becomes temporarily divided
by membranes into large blocks or spherules, each containing one or more vitellophages. At about 200-blastomere stage whole central yolk is segmented into irregular
blocks of different size (Fig. 36). This organization of storage material, which may becalled as secondary yolk cleavage, lasts comparatively short period of time. Before theembryo reaches mature blastoderm stage (see below) the yolky blocks disintegrate into
separate yolk globules and storage material again assumes uniform organization.

Mature blastoderm
The layer of superficial blastomeres, formed in result of superficial cleavage, up to
about 3, OOO-blastomere stage, only roughly resembles cellular blastoderm of pterygote
insects. At early superficial cleavage phase the blastomeres are truncated, pyramidal inshape and have large amount of yolk (compare Figs. 26 - 28). As the superficial cleavage progresses, the blastomeres loose the character of yolky pyramids and become more
spherical. At the stage of about 1,500 blastomeres the organization of the embryo is asfollows.
The whole egg material is segmented, including central yolk mass. The blastome res forming the outer layer have completely lost the character of pyramids. Stilllarge and heavily yolked, the blastomeres have ball-like shape and form loose surface
layer (Figs. 35 - 38) which, because of its morphological character, can be named aspremature blastoderm. From this blastoderm, in the next step of development, arises
typical cellular blastoderm, well known for Pterygota embryo, which in turn may be
named as mature blastoderm.
The formation of mature blastoderm is preceded by the process of liberation of
blastomeres from yolk material (see Jura, 1965). After liberation from yolk, the bias tomeres continue to divide and in about 35-days old embryo the blastomeres are situatedside by side and form columnar epithelium. Thus the embryo enters upon the mature
blastoderm stage (Figs. 39 - 41), typical for purely superficially cleaving insect eggs.

Embryonic development of Tetrodontophora

97

Fig. 37. Fragment of surface view of embryo at about 300-blastomere stage. Note dropletsof lipid-like material extruded by blastomere. X 1,700.
Fig. 38. Fragment of fractured embryo at about 400-blastomere stage. X 1,440.
b, blastoderm; y, yolk.

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Cz. Jura, A. Krzysztofowicz and E. KisielFig. 39. About 35-days old embryo, 3,000-blastomere stage (premature blastoderm stage).
X 105.
Fig. 40. About 40-days old embryo, mature blastoderm stage. X 150.
Fig. 41. Fragment of fractured embryo same as shown in Fig. 40. Note tertiary yolk cleavage. X 1,287:b, blastoderm; y, yolk.

Embryonic development of TetrodontophoraFig. 42.

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Fragment of mature blastoderm, surface view. Note high secretory activity of
blastomeres. X 7,293.
Fig. 43 . Fragment of fractured embryo at mature blastoderm stage. Note highly vacuolized
cytoplasm. X 7,200.
b, blastoderm; bs, blastoderm surface; cy, cytoplasm of central yolk mass; y, yolk.

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Cz. Jura, A. Krzysztofowicz and E. KisielAs the cells of the blastoderm multiply, the epithelium itself appears narrower andmore compact (Fig. 41). A fractured embryo with a mature blastoderm is shown in
Fig. 40; when compared with the earlier stage, shown in Fig. 38, the blastoderm cells
are now about the size of a single yolk globule, but under scanning electron microscope, the cell boundaries are difficult to recognize.
The cytochemistry of mature blastoderm was analysed by Komorowska (1970),
Fryc (1971), Klaja (1971), Malcher (1971), Gancarzewicz (1975), and Micherdziiiska(1975).

Tertiary yolk cleavage
During the early stage of blastoderm differentiation into dorsal organ and material
for future germ band and embryonic membrane (see below and Jura, 1965), within thecentral storage material tertiary yolk cleavage occurs. This process in especially widely
seen at the time when blastodermal cuticles (see below) are formed. In this period ofdevelopment, the central yolk mass divides into large cubical or subspherical blocks.
The blocks are first formed close to the blastoderm (Figs. 40, 41), but ultimately extend all through yolk. This condition of central yolk material does not last long. At the
beginning of germ band formation stage the yolky blocks disintegrate into separate globules and yolk material again assumes syncytial organization.

Blastodermal cuticles
The mature blastoderm of T. bielanensis secretes two blastodermic cuticles, the protective highly resistant layers underlying the chorion. Both cuticles are illustrated inFig. 49.
Before blastodermic cuticles formation, the blastomeres of immature blastoderm
start to extrude, into the space between the body of the embryo and vitelline membrane, many lipid-like material in form of droplets (Fig. 37). As the development progresses, the droplets become more numerous and larger in size (Fig. 42). This process isaccompanied by progressing vacuolization of cytoplasm within the cells of blastoderm
as well as within the cytoplasm of central yolk mass (Fig. 43). The droplets soon dissolve and form a dense fluid, separating the vitelline membrane from the embryo,which meantime diminishes slightly its volume.
During the next developmental stage, the blastoderm matures and the vitelline
membrane again adheres to its surface. The mature blastoderm extrudes the electrondense granules which are incorporated by the vitelline membrane (Krzysztofowicz,
1986a). In result of this, the vitelline membrane is fortified with new substance and its
previous structure changes.
All above described processes are preparatory for formation of blastodermic cuticles. The first sign of formation of definite blastodermic cuticles is the appearance of
microvilli on the outer surface of the cells of mature blastoderm (Figs. 33, 47, 48). The
cytoplasm of blastodermal cells and of central yolk mass, as before, remains strongly
vacuolized (Figs. 45, 46). The microvilli secrete the material destined for the first blas-Embryonic development of TetrodontophoraFig. 44.

101

Fragment of fractured embryo at mature blastoderm stage, shortly before secrection of first blastodermic cuticle. Note appearance of microvilli on blastoderm surface. X 1.200.
Fig. 45. Fragment of fractured embryo same as shown in Fig. 45. Note high vacuolizationof cytoplasm of blastomeres. X 2.620.
b, blastoderm; y, yolk.

102Fig. 46.

Cz. Jura, A. Krzysztofowicz and E. KisielFragment of fractured embryo shortly before blastodermic cuticle formation stage.
Note high vacuolization of cytoplasm of central yolk mass. X 2,700.
Fig. 47 . Fragment of fractured embryo at mature blastoderm stage shortly before formationof blastodermic cuticles. Note numerous microvilli of blastoderm surface. X
8,140.
Fig. 48. Fragment of surface view of the same embryo as in Fig. 47. Note microvilli. X
4,400.
b, blastoderm; cb, cytoplasm of blastoderm; cy, cytoplasm of central yolk mass ; mi, microvilli; y, yolk.

Embryonic development of TetrodontophoraFig. 49.

103

About 60-days old embryo, covered by blastodermic cuticles. Both cuticles are
seen. Chorion and vitelline membrane removed. X 195.
Fig. 50. Fragment of blastoderm surface at early second blastodermic cuticle formation
stage. Note primary, regular protuberations composed of several blastodermal
cells. X 7,700.
bel, first blastodermic cuticle; bc2, second blastodermic cuticle; mv, vitelline membrane.

104

Cz. Jura, A. Krzysztofowicz and E. Kisieltodermic cuticle.
In about two months old embryo the first blastodermic cuticle is already secreted.
This protective layer is comparatively thin and smooth (Fig. 49).
By the beginning of third month of development, within the blastoderm morphological changes occur leading to its differentiation into two different portions. On one
pole of the egg the blastodermal become higher and in consequence of this within theblastoderm two regions become distinguishable. The portion of blastoderm with higher
cells represents the material for the dorsal organ, the rest - for the future embryonicrudiment (germ band) and embryonic envelope (amnion-serosa).
By the time the dorsal organ is formed (see below), the blastoderm develops thesecond blastodermic cuticle. Every cell of the blastoderm contribute to the formation ofthis cuticle, including cells differentiating meantime into dorsal organ.
During the early period of second blastodermic cuticle formation, the pattern ofthe blastoderm, being until now slightly folded, changes into high depressions and protuberances showing regular arrangement. This process is well observable under scanning electron microscope (Fig. 50).
During the next phase of second blastodermic cuticle formation, the protuberancesarrange into rows of complicated, but parallelly running curviline (Figs. 51 - 54). In result of this, the second blastodermic cuticle, when fully secreted, shows complicatedpattern of folds equivalent to the complicated pattern of cells within the blastoderm(Fig. 55).
Within the second mature blastodermic cuticle two regions are observable showing
different pattern of foldings. Above the dorsal organ area the folds are more numerous
and less regular than above the blastoderm destined for the embryo proper andembryonic envelope (Fig. 56).
After formation, the second blastodermic cuticle separates from the blastoderm.
Loosening of the second cuticle is initiated on the pole occupying by the cells of thedorsal organ and starts when these cells begin to sink into underlying yolk (compare
Jura, 1965). At the same time the cells of the blastoderm portion, destined for germ
band and embryonic envelope, become thinner, and outlines of the individual cell of
this blastoderm portion are apt to become indistinct. By the end of third month of development the blastoderm, until now folded (Figs. 58, 59), smoothes out while the created second blastodermic cuticle remains wrinckled. Thus both blastodermic cuticles
completely separate from underlying blastoderm and situate loosely between vitelline
membrane and the surface of the embryo.
During the time when the second blastodermic cuticle is formed, the dorsal organ
matures (see below) and produces tendrils. Some of the tendrils adhere to folds of theinner surface of the second blastodermic cuticle, especially above the area of the dorsalorgan proper (Fig. 57).
After formation of both blastodermic cuticles, the chorion ruptures into two partsand from this time the outermost embryo protective layer is represented by the vitelline
membrane. This explains the changes in its ultrastructure, previously described.
The ultrastructure of second blastodermic cuticle was described in detail by Prymus-Naczynska (1978). Detailed analysis of blastodermic cuticles formation, based onlight, transmission and scanning electron microscopes, have been published by Krzysztofowicz (1986a, b).

Embryonic development of Tetrodontophora

105

Fig. 51. Fragment of surface of blastoderm at middle second blastodermic cuticle formation
stage. Note appearance of secondary foldings. X 2,964.
Fig. 52. Embryo at middle second blastodermic cuticle formation stage. Chorion and vitelline membrane removed. X 120.
Fig. 53. Fragment of surface of embryo same as shown in Fig. 52. X 450.
doa, dorsal organ region; sf, secondary folding.

106

Cz. Jura, A. Krzysztofowicz and E. KisielFig. 54. Fragment of second blastodermic cuticle at secondary folding phase. X 2,400.
Fig. 55. About gO-days old embryo covered with mature second blastodermic cuticle. Side
view. X 80.
Fig. 56. About gO-days old embryo covered with mature second blastodermic cuticle. Dorsal organ pole view. X 202.
doa, dorsal organ area.

Embryonic development of Tetrodontophora

107

Primary dorsal organ
The primary dorsal organ is a very characteristic structure of every collembolan
embryo of the species until now studied.
In T. bielanensis embryo, the dorsal organ arises through enlargement of a circular
patch of cells of blastoderm, 2 - 4 days earlier before the germ band can be distinguished.
The cells elongate and the dorsal organ gradually invaginates into the yolk, at thetime when both blastodermic cuticles become secreted. During the invagination, thecell boundaries disappear, the nuclei migrate to the deepest parts of the cells and thedorsal organ becomes mushroom-shaped with fibrillar cytoplasm in its neck region (formore details of dorsal organ formation see Jura, 1965, 1976).
Komorowska (1972) using histochemical methods and cytophotometry
documented, that the nuclei of dorsal organ contain four times more DNA when compared with the nuclei of the rest of blastoderm. Micherdzinska (1975) noted, that thecells of dorsal organ are especially rich in RNA content, but this content decreases at
the time when dorsal organ shows a secretory activity (see below).
The dorsal organ of T. bielanensis assumes its mature organization by the end of
third month of development. The mature dorsal organ has a typical glandular organization. At mature stage it secretes through its neck radiating tendrils, which fan out(Fig. 61) beneath the second blastodermic cuticle and progressively elongate to the
opposite pole of the egg (Figs. 60-67).
During the early period of dorsal organ activity the tendrils are radiating only
arround the inner edge of its neck (Fig. 63), but soon the tendrils fill the whole openingof the neck (compare Figs. 60, 62-65).
The tendrils are very numerous. A single tendril is round on cross-section, it is about 2 pm thick and has a lenght of 0.01-0.10 mm (Figs. 68-70). Within the fan,
the tendrils are cemented by a dense substance, also secreted by the dorsal organ,
which in fixed state coagulates arround tendrils in form of irregular net (Fig. 68).
At about the time when the embryo appendage rudiments become visible (see below), the tendrils flatten. The free ends of tendrils, occupying the outermost circles
within the fan, split into several subelements and send out thin threads, which densely
invest the whole embryo.
Meantime, as the above described processes advance, the space between second
blastodermic cuticle and the embryo surface becomes filled with more or less liquid
substance, dorsal organ derivate which metabolize some components of the yolk material. The substance when fixed coagulate arround the embryo in form of delicate film.
The film is especially well observable at the level of developing, meantime, the rudiments of head appendages (Fig. 76).
We have previously mentioned, that the second blastodermic cuticle shows a specialized area of a saucer-like appearance, localized above the dorsal organ (Fig. 56).
Below this area, the free ends of some tendrils, 2 - 4 in number, join together andattach to the second blastodermic cuticle (Fig. 69) and form with its folds plastron-likestructure localized above the dorsal organ. The significance of this structure is notknown.
The primary dorsal organ is a transitory embryonic organ, and degenerates at the

108

Fig. 57.

Cz. Jura, A. Krzysztofowicz and E. KisielFragment of second blastodermic cuticle of the dorsal organ area. Inner surface
view. Note tendrils attached to the inner cuticle folds . X 990.
Fig. 58. About 90-days old embryo, fragment of blastoderm surface after secretion of
second blastodermic cuticle. X 720.
Fig. 59. About 90-days old embryo after secretion of blastodermic cuticles. Both cuticles
removed. X 90.
t, tendrils.

Embryonic development of TetrodontophoraFig. 60. Dorsal organ at early stage of secretion of tendrils. Surface view. X 672.
Fig. 61. Dorsal organ at early stage of secretion of tendrils. Side view. X 220.
Fig. 62. Dorsal organ at middle stage of secretion of tendrils. Surface view. X 220.
od, dorsal organ orifice; t, tendrils.

109

110

Fig. 63.

Cz. Jura, A. Krzysztofowicz and E. KisielFragment of dorsal organ at middle stage of secretion of tendrils. Side view. X
1,419.
Fig. 64. Dorsal organ at advanced stage of tendril secretion. Note tendrils filling its orifice.
X 450.
od, dorsal organ orifice.

Embryonic development of TetrodontophoraFig. 65.

111

Dorsal organ at more advanced stage of secretion of tendrils than that shown in
Fig. 64. X 774.
Fig. 66. About lOO-days old embryo at advanced stage of secretion of tendrils by dorsalorgan. Note papilla-like evaginations of rudiments of appendages. X 132.
Fig. 67. About l20-days old embryo wholly invested by tendrils. Dorsal organ pole view.
X 206.
t, tendrils.

112

Cz. Jura, A. Krzysztofowicz and E. KisielFig. 68. Fragments of tendrils with attached dense substance. X 5,400.
Fig. 69. Fragments of two tendrils connected by ends and attached to mner surface of
second blastodermic cuticle of dorsal organ area. X 6,820.
bc, blastodermic cuticle; t, tendril.

Embryonic development of Tetrodontophora

113

Fig. 70. Fragments of tendrils before embryo invagination into the yolk. X 11,650.
Fig. 71. About ISO-days old embryo after invagination into the yolk. Dorsal organ poleview. Note involution of dorsal organ. X 150.
Fig. 72. Fragment of embryo at dorsal-closure stage. Dorsal organ pole view. Xl, 600.
od, dorsal organ orifice.

114

Cz. Jura, A. Krzysztofowicz and E. Kisiel

end of embryogenesis. Already after invagination of the embryo into the yolk, the tendrils dissolve, and at the time when dorsal closure begins, this is when the ectodermal
cells of the embryo start to replace the amnio-serosal cells, the tendrils are no longer
observable (Figs. 71, 72). However, during some time a narrow opening is visible leading to the degenerating dorsal organ (Fig. 72).
The fluid originating from dissolved tendrils, as well as from dense substance surrounding the tendrils, fills the space created beneath the embryo invaginating into theyolk (Fig. 85; compare also Fig. 1, Jura, 1967b).
After invagination, the embryo flattens on both sides (Fig. 71) and elongates inanterio-posterior direction. The fluid fills also the space between the embryo sides andthe second blastodermic cuticle. The increasing of the volume of the fluid as well as its
pressure leads to the cracking of the chorion, but the second blastodermic cuticle
stretches without breaking because of presence of folds which smooth out. The vitelline
membrane and first blastodermic cuticle also do not break, most probably, because of
their high extensibility.
The function of the dorsal organ is widely discussed in insect embryological literature. It is postulated that this structure functions as organ for gas exchange, or asorgan for absorbing the water from environment (for review see Tamarelle, 1975).
The dorsal organ in T. bielanensis reaches its maximum activity, in fluid and tendrils secretion, when invagination of the embryo into the yolk is taking place. The
swelling of the second blastodermic cuticle and the chorion rupture accompanies invagination. It seems clear, therefore, that the dorsal organ plays a part in the change ofthe shape of the embryo, essential for its invagination and elongation. It is also reasonable to suggest, that the radiating tendrils act in strengthening of the second blastodermic cuticle, which stretches to accommodate the shape of the embryo. Jura
(1967b) published experimental evidences, that in absence of functional dorsal organ
the embryo of T. bielanensis fails to undergo invagination. In consequence of this, thebody segments and appendages develop flatly on over the surface of the yolk material,
further morphogenetic processes are disturbed and larva does not hatch.

Body segments and appendages
The period in which the part of blastoderm, destined for the embryo proper andFig. 73. About 95-days old embryo, first phase of head appendage formation. Dorsal organ
pole view. X 150.
Fig. 74. Antennal rudiment formation stage. Dorsal organ pole view. X 150.
Fig. 75. More advanced antennal rudiment formation stage than shown in Fig. 74. Side
view. X 150.
Fig. 76. Rudiment of antenna. X 750.
Fig. 77. Embryo with rudiments of head, thoracic, and tubus ventralis appendages. Side
view. X 150.
Fig. 78. Embryo with vestigial rudiments of appendages of premandibular segment. Ventral view. X 150.
a, rudiment of antenna; d, dorsal organ; f, substance produced by dorsal organ and condensed in result of fixation; i, rudiment of appendage of premandibular segment; m, rudimentof mandible.

Embryonic development of Tetrodontophora

115

116

Cz. Jura, A. Krzysztofowicz and E. KisielEmbryonic development of Tetrodontophora117

the embryonic envelope, is uniformly thick does not last long. The blastodermic cutides and dorsal organ formation phase is accomplished with differentiation of uniform
blastoderm into germ band and amnio-serosa.
The germ band appears in form of a band (Jura, 1965), on the opposite pole in relation to developing dorsal organ. The cells of germ band gradually become columnar,
between them distinct boundaries appear, and in result of many cell divisions and cell
migrations the germ band becomes first two-layered, then many-layered.
The cells of the blastoderm, which occupy the sides of the embryo between thedorsal organ and germ band, become much flattened and thinner and remain onelayered. This thin walled part of blastoderm represents amnio-serosa.
At the mature dorsal organ stage, the head lobes within the germ band become
already evident and this is the first indication of germ band differentiation. The headlobes represent the region bearing labrum, mouth and antennae.
At the head lobe stage, the germ band is very long its head and tail ends nearlymeeting at the dorsal organ. The formation of appendage rudiments precedes the sequestration of germ band into definite body segments. In case of T. bielanensis it ispossible to distinguish three phases in development of the rudiments of appendages.
The rudiments of head appendages appear first, are followed by thoracic, and a little
later by abdominal ones. All rudiments of appendages appear as papilla-like evaginations of germ band (Figs. 73 -77).
Of the head appendages as first appear rudiments of maxillae. The rudiments ofthe first thoracic appendages appear almost simultaneously with rudiments of maxillae.
These are most conspicuous rudiments during early period of appendage formation(Fif. 73). Subsequently: rudiments of antennae, mandibles, intercalary appendages, andsecond and third thoracic appendages appear (Figs. 73 - 78). As last appear rudimentsof abdominal appendages (Fig. 77).
The antennal, intercalary, mandibular, maxillary I and 11 appendage rudiments
mark dearly five head segments of T. bielanensis embryo. But, because between theantennal segment and the most frontal part of the germ band incisions appear (Figs.
82, 83), we postulate that the head is make up of six segments:I) The preantennal segment, deveoid of appendages.
2) The antennary segment.
3) The intercalary segment (premandibular), with vestigial appendages.
4) The mandibular segment.
5) The maxillary I segment.
6) The maxillary 11 segment.
Fig. 79. Orifice of stomodaeum. Xl, 280.
Fig. 80. Embryo more advanced in development than shown in Fig. 71. Ventral view. X
84.
Fig. 81. Advanced appendage rudiment stage. Dorsal organ pole view. X 180.
Fig. 82. Advanced appendage rudiment formation stage. Beginning of involution of rudiments of premandibular segment. X 246.
a, rudiment of antenna; d, dorsal organ; i, rudiment of appendage of premandibular segment; in, incisions marking border between preantennal and antennal segments; m, rudiment of mandible; s, orifice of stomodaeum; tv, rudiment of tubus ventralis; xl, rudiment of
first maxilla; x2, rudiment of second maxilla.

118

Cz. Jura, A. Krzysztofowicz and E. KisielFig. 83. Embryo shortly before invagination into yolk. Ventral view. X 246.
Fig. 84. Embryo shortly before invagination into the yolk. Side view. X 90.
Fig. 85. Embryo during invagination into the yolk, covered with blastodermic cuticles andvitelline membrane. Ventral view. X 90.
a, antenna; as, antennal segment; d, dorsal organ; e, embryo proper; in, incision marking
border between preantennal and antennal segments; I, labrum; m, mandible; xl, first maxilla; t, tendrils.

Embryonic development of Tetrodontophora

119

The antennae develop from the posterior boundaries of the procephalic lobes,
more lateral than the other paired appendage fundaments, and postorally. Afterappearance, they grow very fast, more .rapidly than the rudiments of other appendages(Figs. 80-83). Before hatching of the larva they assume preoral position.
The intercalary appendages are vestigial, but clearly observable (Fig. 78). They
appear nearest to the median plane of the head part of germ band than the other rudiments. The intercalary appendages disappear shortly before invagination of the embryo
into the yolk.
The labrum arises as an evagination anterior and distal from the bases of antennae. At no period of development it gives evidence of paired origin (Figs. 82, 83).
At the same time as the labral anlage appears, but behind it, invagination of thestomodaeum occurs, assuming a form of a crescent-like groove (Figs. 79, 81, 82). Before the invagination of the embryo into the yolk, the labral anlage already covers the
opening of the stomodaeum (Fig. 83).
The rudiments of the mandibles appear nearer to the median plane of the headpart of germ band than the maxillae, and show no trace of lobation (Figs. 82, 83).
The rudiments of maxillae I and 11 show early bifurcation into the anlagen oflobus externus and lobus internus (Fig. 82).
The metamerism of the body of the embryo becomes apparent after invagination,
in form of the transverse grooves formed in ectoderm. The development of the bodysegments in T. bielanensis offers nothing unusual, when compared with other collembolan species.

Invagination
The invagination of the embryo into the yolk occurs when all rudiments of appendages are already well differentiated. In case of T. bielanensis it is rather a simple process.
During the early period of invagination the ventral surface of the embryo, between
the maxillary segments and first abdominal segment, becomes somewhat flattened (Fig.
83) and then begins to push into the yolk (Fig. 85). At first, the thoracic segments sinkand then the head and abdomen segments also sink into the yolk.
.
Prior to invagination, the embryo lies on the surface of the yolk material with its
ventral part turned outwards (Fig. 84). As the result of invagination the embryo reverses its position, bringing the dorsal and posterior parts of the embryo body come tolie nearly parallel, and the embryo becomes restricted to less than one half of the circumference of the egg. The embryo retains the bending position until hatching (Figs.
86,87).

General remarks
Any comparative embryology of insects must begin with Collembola. In this groupthe eggs are rich in yolk, have thin periplasm, and early cleavage is total. All thesecharacteristics may be estimated as phylogenetically primitive. The study of embryonic

120

Cz. Jura, A. Krzysztofowicz and E. KisielFig. 86. Embryo shortly before hatching. Side view. X 175.
Fig. 87. Embryo shortly before hatching. Ventral view. X 165.
a, antenna.

Embryonic development of Tetrodontophora121

development of T. bielanensis, by means of scanning electron microscope, has fully confirmed this presupposition.
T. bielanensis shows many myriapod-like modes in its early embryonic development.
First of all, it has common with myriapods the type of the egg organization. The
embryos of T. bielanensis, like embryo of Myriapoda, cleavage totally, produce blastodermic cuticles, primary dorsal organ, primitive embryonic envelope, and combine
the extreme lengthenic of germ band with prolonged embryonic development. In
Myriapoda and the species studied, the invagination of the embryo into the yolk material is a simple process, the mid-gut epithelium has its origin in vitellophages.
The embryos of T. bielanensis develop the cellular blastoderm around the yolk
mass, as pterygote insects, but the mode of blastoderm formation in this species isprimitive, energy absorptive, biologically uneconomical, when compared with intralecithal cleavage. But beginning with germ band stage, the development of the external form of the embryos of T. bielanensis is similar to that of the embryos of pterygote
insects.
Although the functional configuration of the mouth-parts differs in T. bielanensis
and Pterygota (Collembola are entognathous), the segmental composition and themode of the basic components of the head are identical. The head lobes of the embryosstudied and of Pterygota are sharply rounded in outline, project in a posterio-Iateral
direction and develop antennae at their posterio-median corners. The end of the postantenna 1 region, behind the head lobes, is the premandibular segment, which develops
transient limb buds recently well observed in some Pterygota (for review see
Tamarelle, 1984). The premandibular segment is followed by mandibular, maxillary,labial, and three thoracic segments all with limb buds. Only abdomen follows in thespecies studied more specialized course, which is involved in specialized jumping
locomotion. In other respects, the development is similar. Such processes as invagination of stomodaeum, overgrowth of the labrum and forward migration of the antennal
rudiments to a preoral position, all take place both in the species studied and in Pterygota. There is no direct evidence of the preantennal segment in T. bielanensis, as inPterygota, but in the case of the species studied the appearance of incisions, during
appendage formation stage, between the antenna 1 segment and the most frontal part of
the germ band, suggests such possibility.
We are just led to the conclusion that the mode of embryonic development of T.
bielanensis makes link between Myriapoda and Pterygota.

References
Adamiec, ]., 1975. Ultrastructure of nurse cells in the male gonad of Tetrodontophora bieianensis(Waga) (Collembola). Acta Bioi. Crac., Ser. Zooi. 18: 85-90.
Beams, H. W. and R. G. Kessel, 1976. Cytokinesis: a comparative study of cytoplasmic divisionin animal cells. Ann. Sci. 64: 279-290.
Bilinski, S., 1976. Ultrastructural studies on the vitellogenesis of Tetrodontophora bieianensis (Waga)(Collembola). Cell Tiss. Res. 168: 399-410.
Counce, S. ]., 1973. The causal analysis of insect embryogenesis. In S. ]. Counce and C. H.
Wadding ton (eds. ), Developmental Systems: Insects, Vol. 2, 1-156. Academic Press, London, New York.

122

Cz. Jura, A. Krzysztofowicz and E. Kisiel

Fryc, W., 1971. Histochemical analysis of early developmental stages of Tetrodontophora bielanensis(Waga) (Collembola). Lipids. Zeu;. Nauk. U}, 2001. 17: 39-46.
Gancarzewicz, B., 1975. Histochemical analysis of RNA in early stages of embryonic development of Tetrodontophora bielanensis (Waga) (Collembola). Zeu;. Nauk. U}, Zool. 21: 85-94.
jura, Cz., 1965. Embryonic development of Tetrodontophora bielanensis (Waga) (Collembola) from
oviposition till germ band formation stage. Acta Bio1. Grac., Ser. Zooz. 8: 141-157.
- - - - - , 1966. Origin of the endoderm and embryogenesis of the alimentary system in Tetrodontophora bielanensis (Waga) (Collembola). Acta BioI. Grac., Ser. Zool. 9: 93-102.
- - - - - , 1967a. Origin of germ cells and gonads formation in embryogenesis of Tetrodont
ophora bie1anensis (Waga) (Collembola). Acta Bio1. Grac., Ser. Zool. 10: 97-103.
- - - - - , 1967b. The significance and function of the primary dorsal organ in embryonicdevelopment of Tetrodontophora bie1anensis (Waga) (Collembola). Acta Bio1. Grac., Ser. Zool. 10:
301-311.
- - - - - , 1972. The development of apterygote insects. In S. j. Counce and C. H. Waddington (eds. ), Developmental Systems: Insects, Vol. I, 49-94. Academic Press, London,
New York.
- - - - - , 1975. Cleavage without cytokinesis of the eggs of Tetrodontophora bie1anensis (Waga)(Collembola) after periplasm damage with thermocauter. Acta Bioi. Grac., Ser. Zool. 18: 97102.
- - - - - , 1986. Effects of cautheryzation of Tetrodontophora bielanensis (Collembola) eggs from
oviposition till premature blastoderm stage. Instructive events in early development. Proc.
Arthropod. Embryol. Soc.}pn. 21: 1-12.
- - - - - , and A. Krzysztofowicz, 1977. Ultrastructural changes in embryonic midgut cells
devel9ping into larval midgut epithelium of Tetrodontophora bielanensis (Waga) (Collembola).
Rev. Ecoz. Bioi. Sol 14: 10-15.
- - - - - , and, 1978. Scanning electron microscopy of Tetrodontophora.bielanensis (Waga) (Collembola). The structure of the dorsal organ. 1st Int. Sem. Apterygota,
Accad. delle Se. Detta Da-Fisiocritiei, 105-113.
- - - - - , and, 1982. Scanning electron microscopy of Tetrodontophorabielanensis (Waga) (Collembola) embryogenesis. Fertilized egg to 64-blastomere stage. Acta
Bio1. Aud. Sci. Hung. 33: 39-48.
- - - - - , and, 1986. Scanning electron microscopy of Tetrodontophorabielanensis (Waga) (Collembola) embryogenesis. Segmentation and appendage formation ofhead. Acta Bioi. Grac., Ser. 2001. 28: 7-11.
Klag, j., 1982a. Germ line of Tetrodontophora bielanensis (Insecta, Collembola). Ultrastructural
study on the origin of primordial germ cells. j. Embryo1. Exp. Morpho1. 72: 183-195.
- - - - - , 1982b. Germ line of Tetrodontophora bielanensis (Insecta, Collembola). 2. Ultrastructural differentiation of primordial germ cells during early embryogenesis. Gytobios 33:
173-182.
- - - - - , 1983. Germ line of Tetrodontophora bielanensis (Insecta, Collembola). 3. Migration ofprimordial germ cells. Gytobios 38: 38-53.
- - - - - , 1984. Germ line of Tetrodontophora bielanensis (Insecta, Collembola). 4. Nucleoluslike bodies extruded in toto from nuclei of primordial germ cells become part of the "nuage" .
Gytobios 40: 7-20.
Klaja, E., 1971. Histochemical analysis of early developmental stages of Tetrodontophora bielanensis(Waga) (Collembola). PAS-positive substances. Zesz. Nauk. U}, Zool. 17: 47-57.
Komorowska, B., 1970. Alkaline phosphatase in early stages of embryonic development of Tetrodontophora bielanensis (Waga) (Collembola). Histochemical analysis. Zesz. Nauk. U}, 2001. 16:
93-105.
- - - - - - . , 1972. Cytophotometric analysis of nuclear DNA in the cells of primary dorsalorgan and germ band of Tetrodontophora bielanensis (Waga) (Collembola). Zesz. Nauk. U}, Zool.
18: 15-25.

Embryonic development of Tetrodontophora

123

Krzysztofowicz, A., 1967. Spermatogeneza u Tetrodontophorabielanensis (Waga) (Collembola). Zesz.
Nauk. Vj, Zool. 13: 27-69.
- - - - - , 1971. Histochemical and autoradiographic analysis of RNA synthesis in trophic
cells of the female gonad in Tetrodontophora bielanensis (Waga) (Collembola). Acta Biol. Crac.,
Ser. Zool. 14: 299-305.
- - - - - , 1975. Histochemical and ultrastructural analysis of blocking nurse cells in thefemale gonad of Tetrodontophora bielanensis (Waga) (Collembola). Acta Biol. Crac., Ser. Zool. 18:
45-53.
- - - - - , 1980a. Ultrastructure of the spermatophores in Tetrodontophora bielanensis (Waga)(Collembola). Folia Biol., Krak6w 28: 79-82.
- - - - - , 1980b. The ultrastructure of spermatogonia and germ cells of germarium in Tetrodontophora bielanensis (Waga) (Collembola). Folia Biol., KrakOw 28: 363-369.
- - - - - , 1986a. Ultrastructural studies on embryonic development of Tetrodontophorabielanensis (Waga) (Collembola). Formation of the 1st and 2nd blastodermal cuticles. Part I.
TEM studies. Acta Biol. Crac., Ser. Zool. 28: 19-26.
- - - - - , 1986b. Ultrastructural studies on embryonic development of Tetrodontophorabielanensis (Waga) (Collembola). Formation of the 1st and 2nd blastodermal cuticles. Part 11.
SEM studies. Acta Biol. Crac., Ser. Zool. 28: 27-31.
- - - - - , and W. Byczkowska-Smyk, 1966. Preliminary investigations on the fine structureof spermatozoa of Tetrodontophora bielanensis (Waga) (Collembola). Acta Biol. Crac., Ser. Zool. 9:
97-103.
- - - - - , and, 1968. Studies on the fine structure of spermatids in the Tetrodontophora bielanensis (Waga) (Collembola). Part 11. Acta Biol. Crac., Ser. Zool. 11: 29-36.
- - - - - , and, 1972. Fine structure of first spermatocytes of Tetrodontophorabielanensis (Waga) (Collembola). Ill. Zesz. Nauk. Vj, Zool. 18: 7-13.
- - - - - , and E. Kisiel, 1987. Oogenesis of Tetrodontophora bielanensis (Waga) (Collembola).
Preliminary ultrastructural studies of the first egg envelope formation. In H. Ando and Cz.
Jura (eds. ), Recent Advances in Insect Embryology in Japan and Poland, 37-49. Arthropod. Embryo!. Soc. Jpn. (ISEBU Co. Ltd., Tsukuba).
Ksi~zkiewicz-Ilijeva, M., 1974. Development of the eggs of Tetrodontophora bielanensis (Waga) (Collembola) when centrifuged before cleavage. Acta Biol. Crac., Ser. Zool. 17: 70-75.
Malcher, B., 1971. Glycogen .in the early developmental stages of Tetrodontophora bielanensis(Waga) (Collembola). Zesz. Nauk. Vj, Zool. 17: 59-67.
Micherdzinska, E., 1975. RNA of the late blastodermal cells and of the primary dorsal organ of
Tetrodontophora bielanensis (Waga) (Collembola). Histochemical analysis. Zesz. Nauk. Vj, Zool.
21: 95-103.
Prymus-Naczynska, A., 1978. Ultrastructure of the blastodermal cuticle of Tetrodontophorabielanensis (Waga) (Collembola). Folia Biol., Krak6w 26: 87-93.
Sander, K., 1983. Extracaryotic determinants, a link between oogenesis and embryonic patternformation in insects. Proc. Arthropod. Embryol. Soc.jpn. 19: 1-12.
Sikora, A., 1975. Ultrastructure of the nurse cells in the female gonad of Tetrodontophora bielanensis(Waga) (Collembola). Acta Biol. Crac., Ser. Zool. 18: 79-84.
Tamarelle, M., 1975. Nouvelles observations sur ultrastructure de l'organe dorsal des Collemboles. Mise un evidence d'un type de differenciation "en cocarde" chez Proisotoma minuta. C.
R. Acad. Sci. Paris 280: 897-899.
- - - - - , 1984. Transient rudiments of second antennae on the "intercalary segment" ofembryos of Anurida maritima Guer. (Collembola: Arthropleona) and Hyphantria cunea Drury(Lepidoptera: Arctiidae). Int. j. Insect Morphol. Embryol. 13: 331-336.
Tyszkiewicz, K., 1976. The embryogenesis of the central nervous system of Tetrodontophorabielanensis (Waga) (Collembola). Acta Biol. Crac., Ser. Zool. 19: 1-20.
W6jtowicz, A., 1978. Fine structure of the egg chorion of Tetrodontophora bielanensis (Waga) (Collembola). Folia Biol., Krak6w 26: 323-327.

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Cz. Jura, A. Krzysztofowicz and E. Kisiel
Authors' address: Prof. Cz. Jura, Prof. A. Krzysztofowicz
and MSc. E. Kisiel Department of Systematic Zoology, InstItute of Zoology,
Jagiellonian University, ul. Karasia 6,30-060 Krak6w, Poland