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

139

Early Embryonic Development of
Amata fortunei (Lepidoptera, Amatidae)

Masahiro TAN AKA

Synopsis
The egg structure, the early embryonic development from oviposition to serosa formation and theformation of primordial germ cells of Amata [ortunei are described in detail. In a newly laid egg the egg
surface just beneath the micro~ylar region is covered with a thin membrane. The whole egg surface isuniformly covered with numerous microvilli. The periplasm is divided into a large light-stained area
encircling almost the entire quatorial region of the egg and a dark-stained dumbell-shaped area. The
cytoplasm in the anterior pole of the egg is very thick and protrudes into the yolk forming a cone. The
yolk mass consists of three regions, the central core, the Auflosungszone and the outer zone, and the yolk
granules are grouped into four classes according to their form and affinity for dyes. Three polar bodies
resulted from maturation division soon fuse and change into a single chromatin group, and form a row
perpendicular to the surface of egg. This chromatin group persists until the energids penetrating into theperiplasm. With the lapse of time, within one hour after oviposition the microvilli shrink and become
irregularly winding microprojections. Synchronous divisions at the cleavage stage are usually maintained
until the seventh cleavage and each energid lacks a long cytoplasmic tail. The blastoderm cells are formedby the protrusion of energids beyond the initial level of the periplasm and by the lateral elongation of theplasma membrane. The partition of embryonic and extra-embryonic areas agrees precisely with that of
the surface of the egg in which a difference of stainability for Delafield's haematoxylin is found justafter oviposition. The primordial germ cells are formed by mitosis near the center of the germ band.
shortly after the germ band formation, but before the inner layer formation. The number of them is 13 to
18.

Introduction
The moth, Amata fortunei belongs to the family Amatidae. Though the embryogenesis of
the order Lepidoptera has been widely studied to compare with those of other insect orders,the family Amatidae, so far as the author is aware, has never been studied from the em-

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Tanaka, M.

bryological standpoint. The author therefore intends to study the embryogenesis of thisinsect in detail and to compare with those in the other families of the Lepidoptera.
In the present paper, the author describes the early embryonic development which covers
the egg structure, maturation, fertilization, cleavage, blastoderm formation, ventral plate andserosa formation and formation of primordial germ cells of A. fortunei.

Materials and Methods
Pregnant females of Amata fortunei de L'Orza were captured at Gifu Prefecture, in Juneor September of 1982 and 1983. The female moths laid their eggs on the leaves of various
plants in the laboratory. The eggs were kept at room temperature, and fixed with Carnoy'sor alcoholic Bouin's fluids at 50 - 60°C. They were cut into 7 to 10 ~ thichness andstained with Delafield's or Heidenhain's haematoxylin and eosin. For observation of thewhole egg, Delafield's haematoxylin was used to stain the eggs after removing the chorion.

Results
Duration of embryonic development
The duration of development from oviposition to hatching of A. fortunei was about 6.5
days at room temperature in June in Gifu.
Table 1 shows the outline of early embryonic development of A. fortunei at room
temperature.

Table 1. Time table of the early embryonic development of Amata
fortunei at room temperature (in June).
Approximate
age of egg
0- 40 min1 - 1.5 hr
2 - 2.5 hr
3 - 3.5 hr
4 - 5 hr
9 - 10 hr
12 - 13 hr
15 - 16 hr
18 - 21 hr

State of development
1st maturation division.
2nd maturation division.
Union of the male and female pronuclei.
1st cleavage.
2nd and 3rd cleavages.
Migrating energids enter the periplasm.
Blastoderm formation.
Ventral plate formation.
Serosa covers the ventral plate completely.
Differentiation of primordial germ cells.

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141

Structure of newly laid egg
Abou t 100 to 150 eggs are laid as a mass in a neat pattern of diagonal rows in a single
brood, and each egg touches neighboring ones. The newly laid eggs are oval in shape, about
0.75 mm by 0.7 mm, and milky white in colour. The lower end of the egg contacting withthe surface of a leaf is the posterior pole and somewhat flattened.
The eggs are covered with a thin and nearly transparent chorion with a characteristic
polygonal network. The micropylar area is located exactly in the center of the upper side
(anterior pole) of the egg and surrounded by a rosette with 14 - 16 petal-like reticulations(Fig. 1). Five to seven micropyles are found inside a rosette. An aeropylar opening is present
at each junction of the networked chorionic ribs (Fig. 2).
The egg surface just beneath the chorion is covered with a very thin membrane
dark-stained with Heidenhain's iron alum haematoxylin, which does not adhere closely tothe egg surface, but separates by numerous microvilli on the periplasm (Fig. 5). The
microvilli average 5 f.J.ffi long and are longer in the anterior pole than in other parts of theegg.
The periplasm averages 20 J.!m in depth, except for the anterior region of the egg, and isdivided into the outer and inner layers. The former, about 5 f.J.ffi in depth through theegg surface, has higher affinity for eosin and lower for Delafield's haematoxylin than in thelatter. The inner layer is 15 f.J.ffi in average depth, but slightly thin at the anterior andposterior regions, and it contains a lot of small dot-like yolk granules dark-stained with
Heidenhain's iron alum haematoxylin. The polar cytoplasm under the micropylar region isvery thick, does not contain any yolk granules, and protrudes into the yolk forming a cone(Fig. 6). The innermost periplasm is continuous with cytoplasmic reticulum in the yolk.
In the outer layer of the periplasm two areas are distinguished from the stainability: theone is a light large area enCircling almost the entire surface of the egg except at the anterior
and posterior poles, and the other is the dark dumbell-shaped one covering a narrow dorsal
surface and both poles of the egg (Figs, 20, 21). This difference of stainability is maintained
until the migrating energids reach the periplasm.
The egg nucleus is in metaphase or anaphase of the first maturation division andembedded in a cytoplasmic island separating or expanding inward from the periplasm near
the anterior pole (Figs. 11, 22).
The yolk consists of three regions, the central core, the "Auflosungszone" termed by
Christensen (1942), and the outer zone (Figs. 3, 4). The central core consists of a mass of
irregularly shaped yolk spherules which are qomposed of light-stained small yolk granules
and a few dark-stained dot-like granules. This region connects with the anterior polar
cytoplasm. The Auflosungszone encircling the central core except at the anterior pole of theegg is tumbler-shaped, and contains more yolk substance than in the central core. This region
is composed of large yolk spherules, about 25 J.!m in maximum diameter.
The outer zone is a region between the Auflosungszone and the periplasm. This zone isdivided into the outer and inner layers; the former contains only a lot of dot-like granules,
and the latter consists of two kinds of yolk granules supported in a cytoplasmic reticulum;
the one is dark-stained dot-like granules and the other dark-stained yolk granules with asmall light-stained part inside them (Fig. 7).
A sperm showing a straight rod without a tail, about 12 f.J.ffi in length and about 1.5 f.J.ffi inwidth, lies in the polar plasm or cytoplasmic island just under the micropyle (Figs. 8, 22).

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More than one sperm are not found within an egg.
Maturation and fertilization
Immediately after oviposition the egg nucleus is in metaphase or early anaphase of thefirst maturation division. The direction of the spindle is perpendicular or parallel to the egg
surface. In the anaphase of the division a dark-stained equatorial disk is clearly seen, and it
seems to coincide with the deformed synaptonemal complex observed in Bombyx(Rasmussen, 1977). The first maturation division is completed within 40 min. after
oviposition.
The division of the first polar body is synchronized to the second maturation division(Figs. 12, 23). The direction of both spindles is usually perpendicular to the egg surface, butsometimes is parallel. At this time a rod-like sperm nucleus lies in a small cytoplasmic islandwith a penetrating trail at about 80 tIm from the anterior pole (Figs. 9, 23).
Within 1.5 hr after oviposition three polar bodies are formed in the cytoplasmic island(Figs. 13, 25). No difference in size is observed among them. While the egg nucleus migrates
interiorly and becomes swollen gradually to form the female pronucleus (Figs. 14,25), thesperm also becomes swollen to transform into the male pronucleus with cytoplasmic coat.
The female pronucleus is about 15 tlffi in diameter and is as large as the male pronucleus.
The union of the male and female pronuclei takes place in the yolk at the region about
one-fifth of the egg length from the anterior pole, within 2.5 hr after oviposition (Figs. 15,
16). During this process, many rod-like chromosomes are found in each pronucles, but thesynkaryon could not be observed.
During the syngamy three polar bodies gradually increase in size and the maximum size
reaches about 18 tIm in diameter (Figs. 17, 26). Though the whole surface of egg is covered
with numerous microvilli, 5 tlffi in average length, until 30 min. after oviposition. the microvilli shrink and become irregularly winding microprojections by 1 hr. During the cleavage
stage, no significant morphological changes occur in the surface of egg.
Cleavage
Up to 3 - 3.5 hr after oviposition the mitotic figure of the first cleavage is seen at theregion offertilization (Figs. 16,27). Within 4 - 5·hr the second and third cleavage occur andthe cleavage nuclei, or energids are observed near the center of anterior half of the egg. The
mitotic spindles do not show any definite direction.
As the number of energids increases, they are arranged in a sphere near the center of theegg and some of them destined to become the primary yolk cells or vitellophags remain
within this sphere, and then the centrifugal migration of the energids begins. At the 6th andthe 7th cleavage the number of remaining energids is about 8 and 20 - 30 respectively.
Energids migrating or remaining in the sphere are about 16 - 18 tIm in diameter andsurrounded by the amoeboid cytoplasm, but the migrating energids lack a long cytoplasmic
tail usually observed in other lepidopteran eggs. Synchrony of divisions is maintained until
the 6th or 7th cleavage. It may continue till the 8th division. After the 8th cleavage themigrating energids first enter the periplasm in the anterior half of the egg almost
simultaneously.
Soon after the formation of zygote, the nuclear envelope of polar bodies disappears andEarly embryonic development of Amata

143

their chromatin assembles to form a spherical mass. Subsequently this mass changes ~to arod-like structure being perpendicular to the egg surface (Figs. 19, 28). This figure persists
until the penetration of migrating energids into the periplasm at the anterior half of the egg.
Blastoderm formation
Up to 9 hr after oviposition the number of energids increases to about 150 and they enter
the periplasm in the anterior half of the egg (Fig. 30). At this stage the numerous, irregularmicroprojections are reformed on the surface of periplasm. When the energids arrive near the
periplasm, their associated cytoplasm fuses with the inner layer of periplasm. As the energids
penetrate into the periplasm, they push up the surface of the periplasm and protrude
beyond its initial level to form the hillocks (Fig. 31). Consequently, the surface of the egg
becomes mulberry-like in the external appearance. In the sections through the hillocks theassociated cytoplasm surrounding each nucleus of energids is enclosed with the outer layerof periplasm with abundant microprojections (Fig. 31). The penetration of energids into theperiplasm occurs not simultaneously at the whole egg surface, but at the anterior pole ofthe egg at first, and then proceeds posterioriy.
At about 10 hr after oviposition the entire periphery of the egg is covered with about 350
protruded nuclei or pre-blastoderm cells being scattered apart from each other. At thisstage the polar bodies disappear and about 80 energids remain in the yolk to become theprimary vitellophags or yolk cells.
Development from the pre-blastoderm to the blastoderm is performed not by theformation of the clevage furrows, but by the lateral elongation of the plasma membranes
between the adjacent pre-blastoderm cells, and the round blastoderm cells with the yolk
granules are formed (Fig. 32). However, another type of the blastoderm formation by theappearance of cleavage furrows is often observed in the equatorial zone of the egg. At about
13 hr after oviposition the egg periphery is covered with a single layer of the blastoderm cells(Fig. 33). Each blastoderm cell with a single nucleus at its outer part and a considerable
amount of yolk granules, Yg2 and Ygd, at its inner part is segregated from the inner yolk
mass, resulting in formation of a thin yolk membrane or the secondary vitelline membrane
just under the blastoderm.
The large majority of blastoderm cells again undergo synchronous division parallel to thesurface of egg, (Fig. 36). Consequently, the blastoderm consists of about 1500 cells andforms a compact cell layer, about 30 pm in thickness. In the yolk some mitotic figures also
are seen among the vitellophags. Immediately before the formation of the germ rudiment,
the nucleus of each blastoderm cell migrates to its center and two or three large nucleoli
appear in each nucleus.
Ventral plate formation and serosa formation
At the next stage the cells of the equatorial region of the egg divide actively to form thecolumnar cells, while those in the rest of the blastoderm decrease in thickness and increase inwidth without cell division. The broad equatorial zone is the embryonic area or ventral plate
and the rest is the extra-embryonic area or presumptive serosa. It is noticeable that theembryonic area coincides with the light stained region of the periplasm observed in the
newly laid eggs.

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Soon after formation of the ventral plate, the presumptive serosal cells lying along themargin of the ventral plate begin to grow over the ventral plate to form a complete covering,
while the ventral plate sinks slightly into the yolk. At beginning of the serosa formation thepresumptive serosal cells undergo one or two amitotic divisions without the formation of thecell membrane, and the cells of the ventral plate or germ band continue to divide
tangentaliy. Consequently, the serosal cells are large in size and are two- or four-nucleate,
while the cells of the ventral plate are rectangular in shape and mono-nucleate. At this stage
aminion is not yet formed.
Primor~al germ cells

In A. fortune; there is neither the polar granules at the posterior pole in the newly laid
egg, nor the pole cells among the blastoderm cells. Further, during the formation of theserosa, no specialized cells -£.an be observed among the cells of the ventral plate.
After the completion of the serosa several mitotic figures are seen at the mid-line near thecenter of the germ band (Fig. 38). They show the first appearance of primordial germ cells.
These mitotic figures are clearly distinguishable from those of somatic cells by their position
and the direction of spindles. The mitotic figures of somatic cells are always parallel to thesurface of the germ band and locate at its outer part (Fig. 37). The primordial germ cells
formed through this process are remarkably different from the somatic cells in their shapeand size (Fig. 39). As the development progresses, the primordial germ cells aggregate to
make a cluster near the center of the germ band (Fig. 40). At this time they are polygonal,
and their nucleus is round, about 10 Il1Il in diameter and has several nucleoli. Then thecluster of primordial germ cells is pushed out from the germ band into the yolk in which
they scattered in the limited area (Fig. 41). Now, the number of primordial germ cells is 13
to 18.
Shortly before the differentiation of protocephalon and protocorm the primordial germ
cells reenter separately among the somatic cells near the original area, and representing thesimilar characteristic feature to that in other species of the Lepidoptera. At this time they
are spherical in shape, about 20 Jllll in diameter and have a round, large nucleus with one or
two large nucleoli and light-stained cytoplasm (Fig. 42).

Discussion
Yolk
In the A. fortune; eggs the yolk consists of three regions, namely the central core, theAuflosungszone, and the outer zone. Similar structures were also described by Gross andHowland (1940) in Prodenia eridania and Presser and Rutschky (1957) in Heliothis zea.
In regard of the yolk granules Takesue et al. (1971, 1976, 1982) described that the newly
laid eggs of Bombyx mori have three kinds of yolk granules, i. e., Yg1, Yg2 and Yg3 or Ygd.
In the A. fortunei eggs the yolk granules were grouped into four classes according to theirform and affinity for dyes. The large yolk spherules located in the Auflosungszone, thelight-stained yolk granules with a small dark-stained granule located in the outer zone, andthe dark-stained dot-like granules located in the outer zone and the periplasm are similar toEarly embryonic development of Amata

145

Figs. 1 - 7. Structure of newly laid egg.
1. Micropylar region of anterior pole of egg. Scale: 100 J.1m. 2. Surface structureof chorion. Scale: 50 J.1m. 3. Longitudinal section of egg. Scale : 200 J.1m. 4. Cross
section of egg. Scale: 200 J.1m. 5. Longitudinal section through ventral side of egg,
showing vitelline membrane, microvilli and periplasm. Scale: 100 J.1m. 6.
Longitudinal section of anterior pole plasm. Scale: 200 J.1m. 7. Longitudinal
section through anterior region of egg, showing variant yolk granules. Scale: 30
J.1m.

AO aeropylar opening, AP anterior pole, APP anterior polar plasm, AD
Auflosungszone of yolk, CH chorion, CO central core of yolk, MP micropyle,
MV microvilli, OL outer layer of yolk, PE periplasm, PP posterior pole, VM
vitelline membrane, YG, YG 2 , YGd 3 types of yolk granules.

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Figs. 8 -Tanaka, M.

16. Maturation, fertilization and early cleavage. Scales: 20J.Lm.
8. Sperm incytoplasmic island, about 5 min after oviposition. 9. Transformed sperm
surrounded with radial cytoplasm, about 1 hr after oviposition. 10. Male
pronucleus, about 2 hr after oviposition. 11. Anaphase of 1st maturation division,
about 10 min after oviposition. 12. Metaphase of 2nd maturation division,
about 1 hr after oviposition. 13. 1st and 2nd polar bodies in cytoplasmic island,
about 2 hr after oviposition. 14. Female pronucleus, about 2 hr after oviposition.
15. Conjugated nuclei, about 2.5 hr after oviposition. 16. Metaphase of 1stcleavage, about 3 hr after oviposition.
CRR chromosome, CP conjugated nuclei, FP female pronucleus, MP malepronucleus, PB 1 , PB z 1st and 2nd polar bodies, SP sperm, TS transformed
sperm.

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Figs. 17 - 19. Successive changes of polar bodies. Scale: 20 p.m. 17. Polar bodies before
union, about 2.5 hr after oviposition. 18. United nuclei of polar bodies, about 3
hr after oviposition. 19. Rod-like chromatin mass resulted from union of polar
bodies, about 4.5 hr after oviposition.
CHR chromatin mass, PE periplasm.

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20

Tanaka, M.

AP

pp

21

VP

DP

Figs. 20 - 21. Surface structure of newly laid egg stained with Delafield's haematoxylin,
schematic diagrams. 20. Lateral view. 21. Apical view.

Figs. 22 - 28. Successive changes of anterior region of egg from fertilization to cleavage
stage, schematic diagrams. 22. Anaphase of 1st maturation division and sperm,
about 1 hr after oviposition. 23. Metaphase of 2nd maturation division andtransformed sperm, about 1 hr after oviposition. 24. Four daughter nuclei in ornear cytoplasmic island and transformed sperm, about 1.5 hr after oviposition. 25.
Three polar bodies, female and male pronuclei, about 2 hr after oviposition. 26.
Conjugation of male and female pronuclei, about 2.5 hr after oviposition. 27.
Fusion polar bodies and mitotic figure of 1st cleavage, about 3 hr after
oviposition. 28. Chromatin mass resulted from fusion of polar bodies and mitotic
figures of cleavage nuclei, about 4.5 hr after oviposition.
AP anterior pole of egg, CRR chromosome, CM chromatin mass of polar bodies,
CN cleavage nucleus, CON conjugating nuclei, DS dorsal side of egg, FP female
pronucleus, MP male pronucleus, MV microvilli, PB 1 , PB 2 1st and 2nd polar
bodies, PE periplasm, PP posterior pole of egg, SP sperm, UP united polar
bodies, VM vitelline membrane, VS ventral side of egg.

Early embryonic development of Amata

149

Figs. 29 - 34. Longitudinal sections through egg periphery, showing process of blastoderm
formation, 9 - 12 hr after oviposition. Scale: 40 J.l.m.
Figs. 35 - 36. Tangential sections through blastoderm. Scale: 40 lAm. 35. Blastoderm cells. at same stage shown in Fig. 32. 36. Mitotic figures of blastoderm cells at samestage shown in Fig. 33.
AC associated cytoplasm, BC blastoderm cell, IPE inner periplasm, ME
migrating energid, OPE outer periplasm, PE periplasm.

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Tanaka, M.

Figs. 37 - 42. Longitudinal and cross sections through central region of germ band. Scale:
40 J-Im. 37. Mitotic figures of somatic cells, longitudinal section, about 18 hr after
oviposition. 39 - 40. Group of primordial germ cells, cross sections, about 22- 23 hr after oviposition. 41. Invagination of germ band and migrating
primordial germ cells, cross section, about 25 hr after oviposition. 42. Primordial
germ cells located in germ band, longitudinal section, about 32 hr after
ovip'osition.
IN invagination of germ band, MF mitotic figure of somatic cells, PGC
primordial germ cell, SC serosal cell, SOC somatic cell.

Early embryonic development of Amata

151

Ygl, Yg2 and Yg3 or Ygd by Takesue et al. (1980) respectively. However the fourth class ofyolk granules composed of light-stained small granules and a few of dark-stained dot-likegranules located in the central core are observed in the egg of A. fortunei.
Changes of the surface of egg
The microvilli or microprojections on the egg surface are seldom mentioned in
lepidopteran insects, except for the silkworm, B. mori (Miya, 1978, 1980; Takei andNagashima, 1975; Takesue et al., 1980; Keino and Takesue, 1982). Takei and Nagashimadescribed that the numerous microvilli were seen in the eggs from 1 to 3 hr after oviposition,but ~hey disappeared 2 hr after oviposition. Takesue et al. and Keino and Takesue statedthat the whole surface of the egg was covered with numerous microvilli up to 4 hr after
oviposition, then they disappeared gradually and were replaced with ruffle-like microprojections at 6 hr after oviposition.
In A. fortunei, eggs immediately after oviposition the surface of egg was uniformly
covered with numerous microvilli. And then the microvilli shrink and become irregularly
winding microprojections by 1 hr after oviposition. Since the change of surface structure of
the egg takes place very earlier in this species than in B. mori, it seems not to correspondwith the stages of syngamy and cleavage.
Periplasm

In the newly laid egg the outer layer of the periplasm was divided into two areas; a large
area encircling almost the entire equatorial periphery of the egg and a dumbell-shaped one
covering a narrow dorsal surface and both poles of the egg. The former was somewhat
thinner than the latter and contains a lot of tiny dot-like yolk granules dark-stained with
Heidenhain's haematoxylin. These partitions are maintained until the migrating energids
reach the periplasm, and the large area and the rest may correspond to the presumptive
embryonic area and the presumptive extra-embryonic area respectively. This fact seems toindicate morphologically the predetermination of the embryonic and the extra-embryonicareas.
Polar bodies
Considerable works have been done on the formation and fate of the polar bodies in thevarious lepidopterous insects.
Eastham (1927, Pieris), 10hannsen (1929, Diacrisia), Presser and Rutschky (1957,
Heliothis) and Glavanakova and Dryanovska (1969, Sitotroga) described that two polar
bodies resulted from the maturation divisions, while Hiue (1918, Eudemis), Sato et al.
(1927, Bombyx), Bataillon and Su (1933, Bombyx), Gross and Howland (1940, Prodenia), Rempel (1951, Mamestra), Okada (1960, Chilo), Kobayashi and Ando (1982,
Neomicropteryx) and etc. stated that three polar bodies occurredJ>y subsequent divisions
in the egg maturation.
Almost all students reported that the polar bodies fused to form a single vesicle and thendisappeared in the early cleavage stage, but few ones described as to the fate of the fusedvesicle (Gross and Howland, 1940; Sato et al. 1927; Bataillon and Su, 1933). In P. eridania

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Tanaka, M.

Gross and Howland described that the large, irregular chromatic strands formed by thefusion of polar bodies seemed to indicate an unsuccessful division within the vesicle andpersisted until the cleavage nuclei migrated just beneath the periplasm. Bataillon and Su andSato et al. observed the mitotic figures of the polar bodies in the eggs of B. mori.
In A. fortunei three distinct polar bodies were formed and fused to form a single
chromatin group after the conjugation of pronuclei. At the stage of eight energids (after thethird cleavage stage) the chromatin group was arranged in a row perpendicular to the surfaceof the egg and the spindle-like structure was rarely formed around the row. This structure
resembled the metaphase of the mitotic division and remained unchanged until the migratingenergids penetrating into the periplasm. Hence, the formation and fate of the polar bodies in
A. fortunei are similar to those in P. eridania.
Blastoderm formation
In regard to the mode of the blastoderm formation several different results were reported.
The first type is the formation of the blastoderm by the occurrence of cleavage furrows
between the energids migrated into the periplasm, and is observed in the most species of the
Lepidoptera (Neomicropteryx, Kobayashi and Ando, 1982; Prodenia, Gross and Howland,
1940; Mamestra, Rempel, 1951; Bombyx, Iwasaki, 1931, Miya, 1958, 1980; Endoclita,
Tanaka, 1980; etc.). The second type is the formation of the blastoderm by the protrusionof energids beyond the initial level of the periplasm and is reported in B. mori (Takesue etal., 1980; Takesue and Keino, 1981). In the third type the protrusion ofenergids occurrs
beyond the initial level of the periplasm at first, but the boundaries between the energids areformed by cleavage furrows, and this type is observed in several species of lepidopterous
insects (Eudemis, HUie, 1918: Pieris, Eastham, 1927; Chilo, Okada, 1960; Papilio, Vaidya,
1968; A ncylolomia, Tanaka, 1970).
In the A. fortunei egg, a mechanism of the blastoderm formation is similar to that
described by Takesue et al. and Takesue and Keino in B. mori. Such diffe rence in theformation of the blastoderm may be closely connected with the strength of nuclear
protrusion.
Primordial germ cells
Though many morphological observations concerning the time and the process of theprimordial germ cell differentiation in the Lepidoptera have been done, the results obtained
differ remarkably among the different species or authors. Those differences are probably
caused by the difficulty in the distinction between the primordial germ cells and somatic
cells just after differentiation. Generally, the primordial germ cells appear as a group of cells
at some definite region of the germ band after the germ band formation, but before theinner layer formation (Ephestia, Sehi, 1931; Solenobia, l..autenschiager, 1932; Bombyx,
Kawaguchi and Miya, 1943, Miya, 1952, 1958; Heliothis, Presser and Rutschky, 1957;
Epiphyas, Anderson and Wood, 1968, Endoclita, Ando and Tanaka, 1979).
Miya (1950,1952,1958) described in B. mori that the germ cells were formed throughthe process of invagination at the ventral side of germ band when the blastoderm
differentiated to the germ band and the extra-embryonic area, and the first multiplication ofprimordial germ cells occurred probably during the blastoderm formation and ceased at theEarly embryonic development of Amata

153

time of their appearance. Moreover, he (1950, 1957) presumed by the cauterization
experiment that the cells destined to be differentiated to the germ cells were predeterminedamong blastoderm cells.
In A. fortunei, the primordial germ cells also differentiate at the definite region of thegerm band at the similar period as in most species of the Lepidoptera. However unlike theresult in B. mori the primordial germ cells of A. fortunei increase in number at the time of
their first appearance as in Solenobia triquetrella (Lautenschlarber, 1932). Further it issuggested that the primordial germ cells in A. fortunei do not originate from one stem cell,because two or more mitotic figures are seen at their first appearance.

Acknowledgements
The author wishes to express his sincere thanks to ,Prof. Dr. Hiroshi Ando, SugadairaMontane Research Center, University of Tsukuba and Prof. Dr. Keiichiro Miya, Faculty ofAgriculture, Iwate University, for their kind advices and critical reading of his manuscript.

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Author's Address:

Dr. M. Tanaka,
Kano High School,
Nanyo-cho, Kano, Gifu 500, Japan