Recent Advances in Insect Embryology in Japan
Edited by H. Ando and K. Miya.
ISEBU Co. Ltd., Tsukuba 1985

125

Blastoderm Formation in the Silkworm,Bombyx . mori (Lepidoptera, Bombycidae)

Sachiko TAKESUESynopsis
The mechanism of blastoderm formation in the silkworm, Bombyx mori, has been investigated not
only by light and electron microscopy, but also by immunofluorescence microscopy.
In B. mori eggs, blastoderm cells are formed in a mechanism different from that usually seen in manyother insect species; that is, the formation of a syncytial blastoderm and the typical cleavage furrows
cannot be observed. In the egg of B. mori, when a cleavage nucleus arrives near the periphery of the egg,
the egg surface is raised into a hillock over the nucleus. Cleavage nuclei continue to migrate further
toward the surface and protrude beyond the initial level of the egg surface. The periplasm fused with
their associated cytoplasm is partitioned. Each nucleus is separated by a laterally-invading limiting
membrane from the yolk granule-occupied layer to yield a blastoderm cell.

It has been believed that the mode of blastoderm formation is essentially similar among
many insect species (Anderson, 1962). A typical and well-studied example is found in
Drosophila, where, after the syncytial blastoderm is formed, cleavage furrows grow inward
between from the egg surface to finally yield blastderm cells (Huettner, 1923; Ede &Counce, 1956; Mahowald, 1963; Fullilove & Jacobson, 1971; Sanders, 1975; Turner &Mahowald, 1976). Iwasaki (1931) reported that in Bombyx also blastoderm cells areformed in the same manner as in Drosophila. Takesue et al. (1980) have found, however,that blastoderm cells are formed in a different way in Bombyx in this species neither typicalsyncytial blastoderm nor cleavage furrows are formed. Recently, a similar mode of blastoderm formation has been reported in Callosobruchus (Miyamoto & Van der Meer, 1982).
In this paper I will summarize our studies on the morphological aspect of blastoderm
formation in Bombyx egg and discuss the relevant structural elements. The results on
Bombyx eggs have been published (Keino & Takesue, 1982; Takesue et al., 1976, 1980,1982, 1983, 1984). The morphological features observed during blastoderm formation in126Takesue, S.

Bombyx are illustrated in Fig. 1.
Eggs up to 9 hr after oviposition
In the egg just after oviposition are seen three kinds ofyolk granules: ygl, located in the inner region of the egg and intensely stained with toluidine blue; yg2, located in the narrow peripheral region beneath the periplasm; and ygd,
small intensely-stained dot, located in the very thin periplasm. A similar dot (yg2 -d) is also
seen in yg2' Fluorescence microscopy using anti-vitellin showed that ygl, ygd and yg2 -d arevery rich in vitellin but yg2 has a negligible, if any, amount of the protein. The whole surface of the egg is covered with fine finger-like microprojections (microvilli).
When the egg is incubated at 29°C, there are seen no significant morphological changes in
the interior of the egg, except for migration of cleavage nuclei, uI).til 6 hr. On the other
hand, the egg surface changes considerably. In scanning electron microscopy the surface ofthe 4-hr egg appears covered with numerous microvilli of 3 pm long and 1.3 J.L1l1 in diameter.
In the 6-hr egg the microvilli have almost completely been replaced by thick and abundant
ruffle-like microprojections, implying intensive synthesis of the plasma membrane during
this period.
In the 8.S-hr egg many cleavage nuclei, surrounded by a little of the cytoplasm, havealready migrated near the peripheral yg2 -occupied region but still remained in the yolk-richygl -occupied region. In Drosophila the initially thin periplasm becomes gradually thickerand thicker during the period of migration of cleavage nuclei, but no such thickening of theperiplasm or the yg2 -occupied region occurs in Bombyx.
Up to 9 hr, cleavage nuclei with the associated cytoplasm have migrated into the yg2occupied region at the anterior part. This nuclear invasion of the egg periphery is later at
the posterior part. Since blastoderm formation proceeds in the same manner in the tworegions irrespective of this difference in timing, I will focus on the processes occurring at theanterior part of the egg.
Blastoderm cell formation
Blastoderm cells are formed 9-10 hr after oviposition at theanterior part. When a cleavage nucleus arrives near the periplasm, the egg surface is raised
into a hillock over the nucleus and the periplasm begins to fuse with its associated cytoplasm. Cleavage nuclei continue to further migrate toward the surface. When they protrude
beyond the initial level of the egg surface, the periplasm that has already fused with thenuclear associated cytoplasm is partitioned among and gathered around the nuclei. Theneach nucleus is separated by a membrane, which invades laterally under the nucleus, fromthe yg2 -occupied region to give a blastoderm cell. Neither syncytial blastoderm nor typicalcleavage furrow formation as seen in many insect species occurs in the Bombyx egg. The
apical portion of the surface of a newly-formed blastoderm cell is covered with ruffles, butmany interdigitating microvilli are seen in the groove region between neighboring blastoderm
cells. The apical cytoplasm is abundunt in mitochondria but poor in vacuoles, while in thecytoplasm under the nucleus there are ygds, many vacuoles of different sizes and, sometimes, some yg2 s.
At this stage the number of blastoderm cells is not enough to completely cover the wholesurface of the egg, so that the yg2 -occupied region becomes situated outermost at the surface between blastoderm cells, though there is sporadically residual periplasm there. Newly
formed blastoderm cells divide tangentially on the egg surface, which becomes completely
covered with blastoderm cells by 12 hr after oviposition. Ruffles on the apical portion of

Blastoderm formation in the silkwormFig. 1.

127

Summary of morphological features observed during blastoderm
formation in the Bombyx egg.
ac associated cytoplasm, blc blastoderm cell, cn cleavage nucleus, mp
microprojection, ppl periplasm, ygl and yg2 inner yolk granules, ygd
small yolk granules, ysm yolk-sac membrane.

blastoderm cells become flattened, possibly owing to their proximity to the vitellin membrane.
Interestingly, the yg2 -occupied region remains almost the same in width at least up tothis stage. Vitellin has been thought to serve as a main source of nutrients for the embryonicdevelopment. As described above, however, blastoderm cells develop on the periphery thatis separated from the extremely vitellin-rich ygl -occupied interior region by the very vitellin-poor yg2 -occupied region, suggesting an important role of yg2 s in early embryogenesis(Takesue et al., 1983).

Possible involvement of cytoskeletons in blastodenn fonnation

Blastoderm formation
involves migration of cleavage nuclei and remarkable changes of the egg surface. These
processes are possibly controlled by cytoskeletal systems, because cellular movement isgenerally believed to be regulated by these systems. The results as described below support
this possibility.

128

Takesue, S.

In the associated cytoplasm of a cleavage nucleus which has migrated near the periplasm
are found many long microtubules extending to the periphery of the associated cytoplasm
from the perinuclear region, where the micro tubules appear associated with the nuclear
membrane. In a cleavage nucleus which is about to protrude from the egg surface the microtubules radially extend from each of the centrioles located above and under the nucleus. Ina newly-formed blastoderm cell the microtubules are found only under the nucleus. A similar distribution of microtubules has been reported in the gall midge, Wachtliella persicariae(Wolf, 1980). No microtubules are seen outside the nuclear associated cytoplasm on electron microscopic sections. In indirect immunofluorescence microscopy, however, antibodies
against ~ubulin stain the perivitellin space between the egg surface and chorion in addition tonuclear associated cytoplasm.
No micro filaments are also detected even in the microprojections covering the egg surface
on electron microscopic sections. In indirect immunofluorescence microscopy antibodies
against actin stain the perivitellin space, the periplasm, yg2 s and the gap regions among yg 1 S,
but not cleavage nuclei in the 8.5-hr egg, indicating that actin exists in the internal andperipheral regions of the egg.
When the 8.S-hr egg is exposed to colchicine for 3 hr, nuclear division arrests and cleavage
nuclei remain in the ygl -occupied region as in the 8.S-hr egg, but the microprojections onthe egg surface become wider and larger as in the normally developing egg. After 7-hr exposure, many blastoderm-cell-like but nucleus-lacking structures are found on the egg surface. These results suggest that microtubules are involved in migration of cleavage nuclei but
not in changes of the microprojections during blastoderm formation and, furthermore, thatthe changes of the egg surface is independent of the nuclear migration.
Cyto~halasin B inhibits the morphological change of the microprojections and decreases
the migration rate of cleavage nuclei. When the 8.5-hr egg is exposed to the drug for S hr,cleavllge nuclei have arrived at the egg surface but the microprojections have disappeared.
The same results have been obtained with cytochalasin D. These results suggest that themorphological changes of the egg surface during blastoderm formation are controlled bymicrofilaments. The micro filaments are also probably involved in nuclear migration at leastnear the egg surface.

References
Anderson, D. T., 1962. The embryology of Dacus tryoni (Frogg.) [Diptera, Trypeptidae(=Tephritidae)], the Queensland fruit-fly. J. Embryol. expo Morph. 10: 248-292.
Ede, D. A. & S. J. Counce, 1956. A cinematographic study of the embryology of Drosophila melanogaster. Wilhelm Roux's Arch. 158: 402-415.
Fullilove, S. L. & A. G. Jacobson, 1971. Nuclear elongation and cytokinesis in Drosophila montana. Dev. Bioi. 26: 560-577.
Huettner, A. F., 1923. The origin of the germ cells in Drosophila melanogaster. J. Morph.
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129

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Author's Address:

Dr. Sachiko Takesue,
Biological Institute,
Faculty of Science,
N agoya University,
Furo-cho, Chikusa-ku,
Nagoya, 464 Japan