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

181

Embryonic and Early Post-embryonic
Development of the Parasitic Wasp,
Trichogramma chilonis(Hymenoptera, Trichogrammatidae)

Masahiro TANAKA

Synopsis
The embryonic development, from the germ layer formation to the organogenesis, and the earlypost-embryonic development of Trichogramma chilonis were described. The central mass which occupies
the core of the embryo at the blastoderm stage was seen to stream out from the embryonic dorsum, andthen became flattened between the surface of the embryo and vitelline membrane. It leaves from theembryo at hatching and eventually degenerated in the yolk of the host egg. A longitudinal groove
appeared on the ventral surface of embryo, and cells at the surface of embryo sank and increased in
number. They differentiated into the mesodermal and endodermal cells, but most of them degenerated
the next stage of the development. The stomodaeum was formed an invagination of the ectoderm, andthe mid-gut epithelium originated from the endodermal cells which lie near the center of embryo and theproctodaeum was formed by the growing of the depression which appeared at the posterior end of theembryo. The primordial germ cells just appeared were located outside the blastoderm, and then they
moved ventrally and sank inward in the posterior pole of the embryo. The nervous system originatedfrom the ectodermal cells derived from the ventral part of the embryo, but neither ventral ganglion nor
ventral nerve cord was formed.

Introduction
In the previous paper, the author described the state of the host eggs, the number ofparasites within a single host egg, and the duration of embryonic and post-embryonic development, also the egg structure about a half hour after OViposition and the early embryonicstage; from fertilization to the completion of blastoderm, of the parasitic wasp, Trichogramma chilonis Ishii.
So far as the author is aware, no paper concerning the organogenesis of Trichogramma
has been published except those by Silvestri (1908) and Gatenby (1917).

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Tanaka, M.

In the present paper, the author intends to describe the embryonic development, especially the germ layer formation and the organogenesis, and the early post-embryonic stageof T. chilonis.

Materials and Methods
The materials and methods used in the present study are the same as described in theprevious paper (Tanaka, M., 1985).

Results
1.

Germ layer formation

At about 10 hr after oviposition the eggs of T. chilonis are elongated oval in shape, about
80 J.lm in length and 35 J.lm in width. At this time the eggs are at the blastoderm stage(Fig. 1). Size of the blastoderm cells is different from place to place, that is, the blastoderm
cells haVing a spherical large nucleus distributing in the dorsal and posterior regions of theegg are larger than those in other regions. Each of these nuclei has a single large chromatin
mass which lies closer to the egg surface. With the lapse of time the height of blastoderm
cells increases, and at the same time, the eggs become slightly shorter but remarkably
broader. At this time the number of nuclei is about 20 in the longitudinal section throughthe dorsal part of the blastoderm (dorsal blastoderm), about 22 through the ventral part(ventral blastoderm) and about 23 in a cross section through the major axis of blastoderm.
Fourth to fifty nucleolar granules are seen scattered in the core of the egg surrounded by theblastoderm, being termed as central mass hereafter, and some of them lie closer to thedepresssion occurred in the dorsal blastoderm (Fig. 3).
The primordial germ cells, spherical in shape and about 7 J.lm in diameter, are locatedoutside of the blastoderm in the posterior end of the egg (Figs. 1, 3) and clearly differ fromanother cell in their position, size and affinity for stains.
As the axial part becomes narrow by ingrowing of the blastoderm cells, the central masscontaining the nucleolar granules gradually streams out to the dorsal periphery with someblastoderm cells. The mass expands and breaks through the dorsal blastoderm at theposition between one-third and two-thirds from the anterior end of the egg (Fig. 3). Thisout-streaming of central mass the arrangement of larger blastoderm cells found in the dorsal
part of the blastoderm is disturbed.
At this stage the cells in the posterior half of the dorsal blastoderm become larger andlarger. The primordial germ cells change their position toward the ventral edge in theembryonic posterior pole and they stay there for a while. When the central mass has
streamed out and lies in the space between the blastoderm cells, the core of the egg is occupied by the ingrowing and multiplying cells. After the expulsion of the central mass theenlargement of the chromatin mass in each nucleus takes place in parallel with that ofnuclear volume, and the rapid multiplication of cells occurs, The elongated nuclei with longchromatin ttlass are found in the peripheral cell layer (Fig. 4). No mitotic division isobserved during this process. Through the growth and multiplication of cells the embryo

Embryonic development of Trichogramma

183

AP

NG

LG

NG
Figs. 1 - 6.

NG

Longitudinal and cross sections through the major axis of egg, showing
successive changes from the blastoderm stage to the germ layer formation.
1.
2.
3.
4.

s.

6.

Longitudinal section showing blastoderm stage.
Cross section showing blastoderm stage.
Longitudinal section showing out-streaming of central mass.
Longitudinal section showing germ layer formation.
Cross section showing longitudinal groove.
Longitudinal section at slightly later stage than that shown in Fig. 5.

APanterior poleDSdorsal side, GC
primordial germ cell, LG
groove, NG nucleolar granule, VSventral side. Scale: 50 J,!m.

longitudinal, 184

Tanaka, M.

broadens to form an elongated oval body.
At the stage when the egg measures about 75 ~m by 40 ~ in major axes, the cells at theegg surface retains regularity of their arrangement, whereas the inner ones are disposed irregularly. The primordial germ cells located in the postero-ventral region of embryo are enclosed by the somatic cells. Soon after, on the ventral surface which is approximately onethird from the anterior end of embryo, a logitudinal depression or groove apperars (Figs.5,
6). This groove is about 7 ~m in length, 3 ~m in width and 5 ~m in depth. Each of oval
cells arranged regularly around this groove has a large chromatin mass and is distinguishable
from other cells in their form and distribution (Figs. 5, 6). This state has a striking resembrance to the inner layer formation in other insect embryos. These cells, soon after,
gradually sink inward and increase in their number.
When the longitudinal groove on the ventral surface is formed, the extruded masscontaining nucleolar granules lies between the dorsal peripheral cells (Fig. 4). Such astagnation of the out-streaming does not last long, owing to the growth of embryo and theincrease in somatic cell number. The extruded mass is pressed out on the surface of theperipheral cell layer and flattens between the embryo and the vitelline membrane. The space
formed by the out-streming is soon closed up, and the gap once appeared in the peripheral
cell layer is repaired.
At about 15 hr after oviposition the embryo is ovoid in shape, about 75 ~m by 45 ~ inmajor axes, composed of polygonal cells with in which single, large chromatin mass situatesat their center, and several tiny granules on their inner surface. Most of the somatic cells
arranged irregularly have almost the same shape and size. It is difficult to distinguish thecells destined to form the mid-gut epithelium i. e., endodermal cells, from the mesodermal
ones by their shape and size. Now the depression on the dorsal surface of embryo has
disappeared, and neither any metameric segmentation nor a cavity is found in the embryo.
The flattened extruded mass containing the nucleolar granules and some somatic cells covers
the surface of embryo. By this time the primordial germ cells stained faintly and embracedby the somatic cells sink inward and are situated in the ventral edge of posterior pole of theembryo (Figs. 4, 6, 7).
The amnion and serosa are not found in any stages of the development.

2.

Organogenesis

Alimentary canal
At about 15 hr after oviposition the stomodaeum appears as a depression of the ectoderm
on the antero-ventral surface of embryo. Shortly after its appearance the stomodaeal invagination rapidly extends dorso-posteriorly, forming a long and narrow lumen (Fig. 8). And
then the extremity of the stomodaeal invagination impinges on the anterior end of the
mid-gut epithelium and connects with the mid-gut lumen at the region about one-third ofembryo (Fig. 9). No stomodaeal valve is formed. In the first instar larva which is about
115 ~m by 60 ~m in major axes, the stomodaeum or the fore-gut is about 45 ~m in lengthand its dorsal wall is thicker than the ventral one. Soon after the invagination of stomodaeum occurred a narrow lumen of the mid-gut appears in the center of the embryo, and it issituated slightly dorsally. This lumen is surrounded by about 9 cells in cross section andEmbryonic development of Trichogramma

185

about 11 cells in longitudinal one. With the development of mid-gut lumen the cells consisting of the mid-gut epithelium and the neighboring cells become larger than the others. On
the other hand, a lumen of the proctodaeum begins to be formed in the posterior end ofembryo and it develops rapidly (Fig. 9). At this time single-layered blind end of proctodaeum is closely in contact with that of the mid-gut epithelium. By the longitudinal sectionof this stage the proctodaeum is U-shaped, and it measures about 11 J.lm in width and 15 J.lm
in depth. The author failed to determine whether the proctodaeum is formed with the same
way as that observed in the case of stomodaeum formation or not.
Even when the body cavity has been formed, the demarcation between proctodaeum andmid-gut epithelium still remains intact (Fig. 13). The wall of the anterior half as well as theblind end of proctodaeum is thick and consists of the large cells having a striking resembrance to the mid-gut epithelial cells, while that of its posterior half or opening is extremely
thin. This state persists until the further development of the first instar larva occurs.
After the formation of stomodaeum, mid-gut and proctodaeum the endodermal cells
which do not take part in the formation of mid-gut epithelium and most of the mesodermal
cells gradually degenerate, which results in appearance of the body cavity.

Extruded nucleolar granules
In the embryo which has finished the germ layer formation, the extruded mass flattens
between the periphery of embryo and the vitelline membrane, and covers the dorsal surfaceof the embryo as if it is an embryonic envelope. When the alimentary canal has been
completed and the body cavity begins to be formed, the beginning of hatching occurs. As
the chorion is torn and cast off posteriorly, the flattened extruded mass leaves from theembryo. Then it becomes a spherical body containing nucleolar granules and several somatic
nuclei or cells, and is found by the dorsal body wall of the embryo (Fig. 13). This bodyramains intact for a while, but eventually it degenerates in the yolk of the host egg.

Primordial germ cells
Just before the proctodaeum invagination, about seven primordial germ cells lost anaffinity for staining and surrounded by the somatic cells are observed in the ventral edge ofposterior end of the embryo. After the proctodaeum formation they are embraced by theflattened cells between the ventral proctodaeal wall and body wall of embryo (Figs. 9,12,
13).

Mesoderm
After the alimentary canal formation a large number of cells sink inward from theperiphery of the embryo and increase in number during the germ layer formation. They,
however, degenerate except the cells which have taken part in the formation of alimentary
canal and nervous system. Consequently a small number of loose spherical cells with achromatin body and protoplasmic network is found in the newly formed body cavity (Fig.

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Tanaka, M.

AP

Figs. 7 - 9.
Longitudinal sections through the major axis of egg, showing successive formation of alimentary canal.
Fig. 10.

Horizontal section through the major axis of egg at the stage little later than thatshown in Fig. 9.

Fig. 11.

Cross section through the major axis of egg at the same stage shown in Fig. 10.

Fig. 12.

Cross section through the proctodaeum at the same stage shown in Fig. 11.

Fig. 13.

Longitudinal section of a first instar larva and a extruded mass.

Apanterior pole, BR
brain, ECTectodermal cell, END
endodermal cell,
mesodermal cell,MGE
mid-gut epithelium,GC
primordial germ cell, MES
neuroblast, NEM
nerve cell mass;
NG nucleolar
MGL
mid-gut lumen,
NB
proctodaeum, ST
stomodaeum, VSventral side. Scale : 50 p.m.
granule, PREmbryonic development of Trichogramma

18713). These cells are probably mesodermal cells. Neither the aggregation of these cells nor
the formation of mesoblastic somite was observed at any stages of the development.

Nervous system
When the formation of mid-gut lumen begins, the ventral two-cell layer and dorsal monocell layer are recognizable between the rudimental mid-gut epithelium and the peripheral
layer of embryo in cross section (Fig. 11). The cells consisting of the ventral outer layer
probably correspond with the neuroblasts in other insect embryos, and the inner and thedorsal layer are the mesoderm, but most of which are destined to degenerate during thefollowing development. As the development proceeds, nearly all outer cells also degenerate.
In the oldest embryo or the newly hatched first instar larva large masses of the nerve cells,
namely the brain and suboesophageal ganglion, may be obserbed.
The brain develops over the antero-dorsal side of the fore-gut, and the sub oesophageal
ganglion is formed beneath the fore-gut wall (Fig. 13). The former is elliptic, about 25 JJ1Il
in length and 15 Ilm in width, and the latter is smaller than the former. Though there a few
number of cells are found on the inner surface of the ventral body wall, neither theganglion nor the nerve cord differentiates.

Discussion
1.

Primordial germ cells

According to Silvestri's (1908) illustrations, the primordial germ cells in the eggs ofOophthora semblidis are situated in the posterior part of the blastoderm, and when thestomodaeum begins to invaginate, the germ cells are located in the postero-central region ofembryo, being sandwitched by the peripheral layer and inner somatic cells.
In T. chilonis, the primordial germ cells differentiated outside the blastoderm at theposterior pole of the egg, and they remained intact for a while. After the expulsion of
nucleolar granules they changed their position to the ventral edge in the embryonic posteriorpole and sank inward during the formation of germ layer. These observations agree withGatenby's (1917) descriptions on T. evanescens.

2.

Alimentary canal

In T. chilonis, before the formation of proctodaeum and mid-gut the stomodaeum began
to invaginate as a depression of the ectoderm, and then it extruded dorso-posteriorly. Shortly afterward the end of the stomodaeum impinged on the anterior end of the developing
mid-gut, and the stomodaeal lumen became continuous with the mid-gut lumen.
Silvestri (1908) illustrated the similar feature of the stomodaeal invagination of O.
semblidis. Gatenby (1917), however, stated that, although the stomodaeum was normallyformed, the cell mass situated between the stomodaeal and mesenteron cells remained
unbroken till the formation of body cavity occurs.

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188

As to the origin of the mid-gut epithelium Gatenby (I917) described that a longitudinalgroove regarded as a early blastopore appeared on the dorsal surface of the embryo and it
became deeper to form the mid-gut.
In T. chilonis this groove invaginated at the ventral surface, and the cells arranged aroundit sank inward and increased in number. Although the fate of these cells is not yet definitely
known, it is possible to say that these cells take part in the formation of the fore- or mid-gut
epithelium.
Regarding the proctodaeum formation Gatenby (I917) noted that, in T. evenescens it
was difficult to ascertain the occurrence of invagination as observed by the time of stomodaeum formation, and the demarcation between the mid-gut and proctodaeum was quite
indistinct. Though Silvestri (I908) has given an illustration of the sagittal section of O.
semblidis embryo in which the alimentary canal has been formed, he did not describe theearlier stages of proctodaeum formation.
In T. chilonis, in \vhich the mid-gut lumen was formed, the proctodaeum appeared at theposterior end of the embryo, and it grew rapidly. The invagination of proctodaeum was notconfirmed by the present author. When the proctodaeum became clear after the occurrenceof large proctodaeal cavity the single cell layer which forms proctodaeal blind end wasclosely in contact with the posterior mid-gut epithelium. These epithelial cells in the extremity of the newly formed proctodaeum had a striking resemblance to those in the midgut. In this species the contacted area of proctodaeal end and the end of the mid-gut is kept
intact and opening which may join two lumens may not appear at this stage, and the demarcation between the proctodaeal and mid-gut epithelium was indistinct for a while, but theformer became distinguishable from the latter in the stage of the body cavity formation.
From the above-mentioned facts it is conceivable that the proctodaeum may be formedby the growing of the lumen which appears at the posterior end of the embryo, and notformed by the general invagination of the ectoderm. The epithelial cells in the extremity ofproctodaeum probably originates from the endodermal cells.

3.

Embryonic Envelope

In T. chilonis neither amnion nor serosa is formed at any stages of the development.
Silvestri (I908) described that a 'pseudoserosa' was formed by the delamination of theembryonic cells in the eggs of O. semblidis and indicated that it covered the embryo.
Gatenby (I917) denied Silvestri's explanation on the pseudoserosa and stated that theembryonic membrane did not exist in Trichogramma. However, it could be observed in T.
chilonis that when the chorion was torn and cast off, the embryo floated free in the yolk
and changed its form into a round body as observed in T. evanescens (Gatenby, 1917).
This fact shows that that the flattened, extruded cells observed over these embryos are nottrue embryonic envelope.
Therefore the author agrees with Gatenby's conclusion above-mentioned.

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189

Acknowledgement
I wish to express my sincere thanks to Prof. Hiroshi Ando, Sugadaira Montane ResearchCenter, University of Tsukuba, for his kind advaice and critical reading of the manuscript.
Thanks are also due to Prof. Yoshimi Hirose, Division of Insect Natural Enemies, Instituteof Biological Control, Faculty of Agriculture, Kyusyu University, for identification of thematerial in this study.

References
Gatenby, 1. B. 1917. The embryonic development of Trichogramma evanscensWestw.,
monembryonic egg parasite of Donacia simplex. Quart. J. Micr. Sci. 62: 149-187.
Silvestri, F.
1908. Contribuzioni alla conoscenza biologica degli Imenotteri parasiti.
11. Sviluppo dell'Ageniaspis fuscicollis (Dalm.) e note biografiche. Ill. Sviluppo dell'Encyrtus aphidivorus Mayer. IV. Sviluppo dell'Oophthora semblidis Aur. Ball. delLab. di zool. gen. e agr. della R. Scuola sup. d Agricoltura in Portici. 3: 29-85.
Tanaka, M. 1984. Early embryonic development of parasitic wasp, Trichogramma chilonis(Hymenoptera, Trichogrammatidae) In Ando & Miya (ed.) Recent Advances ofInsect Embryology in Japan: 171-179. ISEBU Co. Ltd. Tsukuba, Japan.

Author's address:

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