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

171

Early Embryonic Development of theParasitic Wasp, Trichogramma chilonis(Hymenoptera, Trichogrammatidae)

Masahiro TANAKA

Synopsis
The early embryonic development, from fertilization to completion of the blastoderm, of Trichogramma chilonis in the eggs of Papilio machaon hippocrates and Cephonodes hylas was described. In T.
chilonis the embryonic period lasted about a half day, the larval period about 2 days, and the prepupal
and pupal ones about 5.5 days in early summer and autumn. At about 50 min. after oviposition there
were found the female and the male pronucleus and the germ cell determinant in the egg. Fertilization
finished at about 1 hr after oviposition, and then cleavage occurred. The 1st to the 4th cleavage weresynchronous, and in the 5th the synchronism was lost and the formation of syncytial blastoderm began.
The. blastoderm was completed within 10 hr after oviposition, and about 6 primordial germ cells differentiated in its posterior end. Nucleolar granules were formed by multiplication of the remnant of energids
left in the core of egg and the extrusion of daughter cells or nuclei were found in the syncytial and thecellular blastoderm.

Introduction
Embryological studies on parasitic wasps have been done up to the present, but on theembryonic development of the genus Trichogramma, belonging to the superfamily of Chalcidoidea and of considerable economic importance as an egg parasite of insects, there are
only few studies, so far as the author is aware.
Silvestri (I 908) briefly described on the embryonic and post-embryonic development ofOophthora semblidis belonging to the subfamily Trichogrammatinae. Gatenby (I 917) fully
described on the embryonic development of Trichogramma evanescence, however the cleavage stage was not described by him. Krishnamurti (I938) showed photographs of somestages in the embryonic development of T. minutum,but he also did not describe them indetail.

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Tanaka, M.

The present paper deals with the description of the early stages, namely from fertilization
to the completion of blastoderm, of T. ch ilon is.

Materials and Methods
Trichogramma chilonis Ishii is an important egg-parasite of lepidopterous insects. It
attacked the eggs of Papilio machaon hippocrates C. et R. Felder and Cephonodes hylas
Linne, and many broods of the parasite appeared in early June to October in Gifu Pref.,Central Japan.
All materials collected at several parts in Gifu Pref. were used for the present study. The
parasitized eggs of Papilio machaon hippocrates and Cephonodes hylas obtained during Juneto September of 1981, 1982 and 1983 were kept at room temperature in the laboratory.
The females of T. chilonis captured in the field during July to September of 1982 and 1983
laid their eggs in the host eggs of the both species mentioned above in the laboratory. These
were also kept at room temperature. The host or parasitized eggs containing the parasite
eggs at various stages were fixed with Carnoy's or alcoholic Bouin's fluid for 20 to 60 min.
After fixing the chorion of host eggs was removed, then they were dehydrated and embedded in paraffin. They were sectioned 6 to 8 I1m in thickness and stained with Delafield'sor Heidenhain's haematoxylin and eosin.

Results
1.

Host eggs and number of parasites within single host egg.

The eggs of Papilio machaon hippocrates were approximately spherical in shape, about
1.2 mm by 0.9 mm in size and those of Cephonodes hylas were spherical, about 1.2 mm by
1.1 mm. Eggs of these species have a thick chorion. In the normal or non-parasitized eggs ofthe former species the 1st inster larvae hatched out about 7 days after oviposition, and thelatter about 3 days at early summer and autumn. In the infected host egg the size of theyolk globules changed in very tiny around the region where the parasite eggs were found.
Most of the parasitized eggs were at the early embryonic stage, from fertilization to the germ
band formation, however in rare cases the parasitized eggs or embryos developed to the stageof revolution of the embryo, in which the formation of alimentary canal had been completed. In some cases the parasite eggs were found in the developing parasite larva. The embryonic development of the host eggs was arrested by the parasitism and they were usuallyturned dark owing to the peculiar blackening of the vitelline membrane just before the parasites reached the prepupal stage. As many as 10 to 30 eggs of T. chilonis, average 19, developed in the SIngle egg of P. machaon hippocrates and a large majority of adults emerged
through. the single hole of the chorion of a host egg, while 7 to 25 parasites, average 14, grew
and flew out from each egg of C. hy/as. The maximum number of parasites within a single
host egg was 30. Generally 10 to 25 parasites were found as a group, but in rare cases there
were only 2 or 3 parasites were seen, and sometimes 2 groups of develping parasites were observed in a single host egg.

Early embryonic development of Trichogramma

173

AP

PB

FPN

OS

E

Figs.

1-10. Eggs of Trichogramma chilonis at various stages of early embryonic development.
1. About a half hr after oviposition. AP anterior pole, DS dorsal side, FPN
female pronucleus, GD germ cell determinant, PB polar body, VS ventral side
Scale: 50 J.l.m.
2. About 50 min after oviposition. MPN male pronucleus.
3. About 1 hr after oviposition. ZG zygote.
4-8. Cleavage stages. EG energid, NN naked nucleus.
9. Syncytial blastoderm stage, all nuclei show synchronous division.
10. Cellular blastoderm stage. GC germ cell.

Figs.

11-15. Cross sections through the middle portion of the egg of T. chilonis fromcleavage to blastoderm stage.
11. About 16 energids stage. EG energid.
12-13. Syncytial blastoderm stage. NG nUcleolar granule14-15. Cellular blastoderm stage.

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174

2.

Duration of embryonic and post-embryonic development of T. chilonis

The duration of development, from oviposition to emergence, of T. chilonis was about 8
to 9 days at early summer and autumn. The parasite eggs began to develop immediately
after oviposition.
The embryonic period was about a half day, the larval 2 days, and the prepupal and pupal
period about 5.S days. The adults emerged about 8 days after oviposition.
Table 1 shows the outline of embryogenesis of T. chilonis within the host egg.
Table 1. Time table of the embryonic development of Trichogramma chilonis
at room temperature (in early summer)
Approximate age of egg

State of development

0- 1 hr
1- 4 hr
4 - 10 hr

Fertilization
Cleavage
Blastoderm formation
Germ layer formation and inner layer formation
Stomodaeum, proctodaeum and mid-gut formation
Hatching

10-15hr
15-20hr
20 - 25 hr
3.

Egg structure of T. chilonis at about a half hour after oviposition

The author failed to observe the eggs just deposited. At about a half hour after oviposition the parasite eggs lay in or near the peripheral yolk of the host egg as a group or an eggmass. The long axis of each parasite egg was directed to the center of the host one, and theanterior pole of the parasite eggs was located outside of the host egg.
Parasite eggs are light ivory in color and elongate with the one-third portion from theanterior pole distinctly expanded. Both poles of them are smoothly rounded and the anterior one is somewhat broader than the posterior, and they are convex dorsally. In fixedand sectioned materials the egg measured about 75 pm by 23 pm in major axes.
The eggs are covered with the thin membrane which is closely associated with the egg
surface, but it is very difficult to distinguish the chorion from the vitelline membrane. The
spherical egg nucleus or female pronucleus of 5.5 pm in diameter is located at or near thecenter of the egg, about one-third from its anterior end (Fig. 1). A few chromatin granules
are touching on the periphery of the nucleus. Degenerating polar bodies are found at the
one-third from the anterior pole of the egg. The darkly stained large body lies near theposterior end of the egg (Fig. 1). This is so-called germ cell determinant and is oval or el1iptic in shape, about 5.5 pm by 7 pm. The homogenous ooplasm stained with hematoxylin isobserved except for in the posterior pole of the egg in which the germ cell determinant appears. Though the deutoplasmic substances or yolk granules of the host eggs are strongly
stained with eosin, the heavily stained yolk granules are not found in the parasite eggs. In
these a very thin layer of the periplasm continues with the internal protoplasmic reticulumand neither maturation of the egg nucleus nor formation of the polar body was observed.

Early embryonic development of Trichogramma
4.

175

Fertilization

In the fertilized eggs at about 50 min. after oviposition, there were found three distinct
structures, i. e., the female and the male pronucleus and the germ cell determinant (Fig. 2),
while in the unfertilized egg only the female pronucleus and the germ cell determinant wereobserved.
Soon after the female and the male pronucleus come to lie close together at the region
about one-third from the anterior pole. At this time one nucleus is larger than the other,and it is thought that the former is a male pronucleus and the latter female one (Fig. 2).
With the lapse of time they come to contact with each other, and finally fuse to form a single elliptic nucleus, i. e., zygote, about 9 p.m by 7 p.m in diameter (Fig. 3). The zygote canbe distinguished from the small female nucleus of the unfertilized egg. Thus the fertilizationis completed at about 1 hr after oviposition.

5.

Cleavage

Clevage begins between 1 hr and 1.5 hr after oviposition, and the course of development
is the same in the fertilized and unfertilized eggs. Before beginning of the cleavage thezygote losts the nuclear membrane and becomes a naked nucleus or a chromatin mass. The
first cleavage is observed at the site where the conjugation of nuclei have taken place, andtwo daugther nuclei of equal size are formed (Fig. 4). The direction of the 1st cleavage isless than the right angle to the longitudinal egg axis. In most of eggs consisting single broodthe first cleavage begins at the same time, but in a few eggs start of the cleavage was delayed.
The 2nd and the 3rd cleavage are completed within 2 hr after oviposition, and 4 to 8 energids appear (Fig. 5 - 6). The direction of the 2nd cleavage is vertical to the egg long-axis.
These energids are spherical in shape, about same in size, approximately 7 p.m in diameter.
Sixteen energids, about 5.5 p.m in diameter (Fig. 7), formed by the 3rd cleavage are smaller
than those in previous cleavages. The spindle of the energid division not be observed at any
time of the cleavage stage. The division was synchronous until the 4th cleavage, and it waslost in the 5th one. These 16 energids arrange in the central zone of the egg, and soon after
they begin to migrate toward the anterior and the posterior pole of the egg (Fig. 7).
Number of the energids increased to about 30 by the time when they migrated toward
the surface of the egg, and finally entered the periplasm (Fig. 8). It does not occur that 'allenergids reach the egg periphery simultaneously. The first penetration of energid into theperiplasm occurs at the middle portion of the egg, and then at the anterior and posterior
poles. At this time there are a few energids remaining in the core of the egg.
The germ cell determinant retains its original size and position when the energids reachto the egg periphery. During the cleavage stage the eggs retain its original form and size.

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Tanaka, M.

Figs. 16-17. Longitudinal and cross sections of T. chilonis eggs atsyncytial blastoderm stage. A. surface view of the blastoderm, B. cross section through the middle portion of theblastoderm, C, D. longitudinal and cross sections of thesame embryos showed in Fig. 16.

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6.

177

Blastoderm formation

At about 4 hr after oviposition the syncytial blastoderm formation begins. At this time
the number of nuclei in the egg periphery is about 30, and each nucleus is about 5 to 6 J..lm
in diameter. The nuclei at the egg surface are almost evenly distributed, while several energids or yolk nuclei remain in the core of the egg. Before the syncytial blastoderm completion the peripheral nuclei divide three times. Though the synchrony of division has been
lost at the 5th cleavage, it occurs again in succesive divisions, which produce altogether
about 230 nuclei (Fig. 9). The syncytial blastoderm formed by the rapid mUltiplication of
nuclei in the periplasm is uniform in thickness throughout the entire periphery of the egg,
and its nuclei are spherical and about 5 J..lffi in diameter.
When the syncytial blastoderm is completed, the size of several nuclei locating at theposterior end of the egg increases gradually and the nuclei become to be distinguished fromthe other nuclei. These nuclei are the primordial germ cells, spherical in shape and 7 J..lm indiameter. Though the germ cell determinant is maintaining its original structure by thisstage, it changes into a crescent body when the germ cells are clearly formed, and then disappears gradually and finally it becomes impossible to trace visually. At this time about 10 to
20 nucleolar masses or granules are found in the core of the egg, and they are approximately same in size and shape. It was rarely observed that the division of blastoderm cells is
vertical to the egg surface (Fig. 13). The inner nucleus produced by this division moves into
the core of the egg and becomes the nucleolar granules. At the early blastoderm stage 20 to
30 of these nucleolar granules, about 1.5 J..lffi by 2 J..lm in diameter, are found in the core of
the egg.
Blastoderm formation occurs with the same way widely observed in other insect eggs,
which is about 8 J..lm in thickness when it has completed. The newly formed blastoderm cellswith single, spherical or oval nucleus are columnar except those at the posterior region of theegg.
During the blastoderm formation the size of the egg increases gradually and now measuresabout 85 J..lm by 35 J..lm along the longitudinal egg axis. At this time the egg becomes mulberry-like in the external appearance (Fig. 16), because each of the blastoderm cells makesa
slight bulge at the egg surface. The cellular blastoderm consists of about 350 cells, andhenceforth the number of the cells remains nearly constant during the blastoderm stage.
Thus the blastoderm is completed at 10 hr after oviposition.
Shortly after the completion of the blastoderm, the blastoderm cells begin to develop andthe cell differentiation takes place. In this process remarkable changes occur in the whole
blastoderm cells except for the germ cells at the posterior end of the egg; they are (1) nucleus changes its form from spherical to elliptic, (2) an nucleolus appears at the outer part ofeach nucleus facing the egg surface, and it grows larger and larger (Fig. 15). With the lapseof time the blastoderm at the ventral side of the egg becomes somewhat thicker than that at
the dorsal side. At the same time the size of blastoderm cells is also different from one
region of the egg to another, that is, the blastoderm cells in the anterior and mid-ventral regions become small and have a small and elliptic nucleus, while the cells in the rest of blastoderm become large, having a spherical large nucleus. The number of these blastoderm cells
slightly decreases, and the egg becomes broad. By this time the germ cells have been retaining original state and position, but they gradually move toward the ventral edge of blastoderm with advance of the development.

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178

When the blastoderm is completed, 40 to 50 nucleolar granules are found in the core of
the egg. The total number and size of the nucleolar granules, however, vary according to thestage of development; i. e., the number varies from 20 to 50 during the blastoderm stage,and it shows a tendency that these granules increase in number in the older blastoderm thanin the younger one.

Discussion
1.

Host age and parasitism

Effect of the age of host eggs on the parasitism by Trichogramma has been studied indetail by Peterson (1930), Lewis and Redlinger (1939), Klomp and Teerink (1967), Marston
and Ertle (1969), and Hiehata et al. (1976) etc.. It should be noted that the parasite eggs
were rarely found in the old host eggs in which the embryos attained to the revolution stage
and their alimentary canal had been formed. However the parasite eggs observed in thefrashly infested host eggs collected in the field were at the earlier stage of embryogenesis
ranging from fertilization to germ band formation. From these evidences the author believes
that the age of host eggs suitable for the parasitism by T. chilonis is almost limited within
one day after oviposition, and the host eggs older than one day are hardly parasitized by T.
chilonis. This fact agrees with the conclusion by Hiehata et al. (1976) in three species ofTrichogramma.
2.

Developmental delay observed among some eggs in the same brood

Delay of beginning of the cleavage and the development in some of the parasite eggs
among the same brood were found; in some instances several eggs were still at the earlycleavage stage even though majority of the brood reach the blastoderm stage. The numberof parasite which were behind the schedule of their development agreed with that of emerged male adults. Judging from the above facts it is probably true that the start of development of unfertilized eggs is later than that of fertilized one.
3.

The nucleolar granules

In T. evanescens. Gatenby (1917) examined the formation of extruded nucleoli or the
nucleolar granules, and he stated that a number of nucleolar granules were the extrusions
from the blastoderm nuclei and no dividing nuclei were found in the sections. He said,moreover, that in the younger blastoderm there were found fewer extruded nucleoli, andthat a shortening of the egg length always occurred with a decrease of th~ nuclear number inthe growing blastoderm.
As to the ori'gin of chromatin or nucleolar granules, however, the author obtained a different observations. In T. chilonis. when the energids reached to the egg periphery some ofthem remained in the cent er of the egg. The latter became chromatin or nucleolar granules.
In the syncytial blastoderm stage the naked nuclei or two daughter nuclei just produced by
the division were found between the nuclei in these blastoderm cells. Though it is not clear
whether both daughter nuclei become the nucleolar granules or not, at least the inner one

Early embryonic development of Trichogramma179

was originated from the latter. In the early blastoderm stage the naked nuclei were observed
between the blastoderm cells. In the present study, the author, however, can not determine
whether these nuclei contribute to re-form the blastoderm or they are cast out into thecenter of the egg to form the nucleolar granules.

Acknowledgments
I wish to express my sincere thanks to Prof. Hiroshi Ando, Sugadaira Montane ResearchCenter, University of Tsukuba, and Prof. Keiichiro Miya, Faculty of Agriculture, Iwate University, for their kind advice and critical reading of the manuscript. Thanks are also due toProf. Yoshimi Hirose, Division of Insect Natural Enemies, Institute of Biological Control,
Faculty of Agriculture, Kyusyu University, for his identification of the material in thisstudy.

References
Gatenby, J. B. 1917. The embryonic development of Trichogramma evanescens Westw.,
monembryonic egg parasite of Donacia simplex. Quart. I. Micr. Sci. 62: 149-187.
Hiehata, K., Hirose, Y. and H. Kimoto 1976. The effect of the host age on the parasitism
by three species of Trichogramma (Hymenoptera: Trichogrammatidae), egg parasitoids
of Papilio xuthus Linne (Lepidoptera: Papilionidae). lap. I. appl. Ent. Zool. 20: 3136. (In Japanese with English summary).
Klomp, H. and B. J. Teerink 1967. The significance of oviposition rates in the egg parasite,
Trichogramma embryophagum Htg. Archs. Neerl. Zool. 17: 350-375.
Krishnamurti, B. 1938. A microscopical study of the development of Trichogramma
minutum Riley (the egg-parasite of the sugarcane borers in Mysore) and its parasitisation of the eggs of Corcyra cephalonica Staint (the flour-moth employed in the mass
production of Trichogramma). Proc. Indian Acd. Sci., (B) 7: 36-40.
Lewis, W. J. and L. M. Redlinger 1969. Suitability of eggs of the almond moth, Cadra
Ann. ent. Soc ..
cautella. of various ages for parasitism by Trichogramma evanescens.
Am. 62: 1482-1484.
Marston, N. and L. R. Ertle 1969. Host age and parasitism by Trichogramma minutum(Hymenoptera: Trichogrammatidae). Ann. ent. Soc. Am. 62: 1476-1482.
Peterson, A. 1930. A biological study of Trichogramma minutum Riley as an egg parasiteof the oriental fruit moth. Tech. Bull. U. S. Dep. Agric. 215: 1-21.
Silvestri, F. 1908. Contribuzioni ana 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. Boll. del Lab.
di Zool. gen. e agr. della R. Scuola sup. d Agricoltura in Portici., 3: 29-85.

Author's address:

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