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

207

Early Embryonic Developmentof Tritneptis diprionis(Chalcidoidea, Hymenoptera) *,Maria Krystyna KOSCIELSKA
and,

Boguslaw KOSCIELSKI
Synopsis
Early embryonic stages of Tritneptis diprionis, an ectoparasite of Diprionidae, are described.
A freshly laid egg contains the oosome. The chromatin of the nuclei of the blastoderm cells
shows polarity. The germ cells enter the embryo interior partly through blastoderm. The primary
endoderm arises from primary vitellophages. The definitive mid gut epithelium is formed by thecells lying at the bottoms of stomodaeum and proctodaeum. The gastrulation is accomplished by
the sinking of the middle plate of germ band.

Introduction
The genus Tritnepsis is one of .the rarer ectoparasites of Diprionidae. In Polishfauna the genus is represented by two species: T. klugii and T. diprionis. So far, theembryonic development of any species of this genus was studied.
The present paper is a continuation of series of works on the embryonic development of hymenopterans - parasites of diprionid larvae (Koscielska, 1980, 1981, 1985).

* Supported by Nencki Institute of Experimental Biology of Polish Academy of Sciences,Warszawa.

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M. K. Koscielska and B. KoscielskiLongitudinal section through the egg of Tritneptis diprionis. Posterior pole of the egg
contains oosome. Scale: 25 pm.
Fig. 2. Formation of the syncytial blastoderm. Cleavage .nuclei and oosome (0). Scale: 25pm.
Fig. 3. Posterior pole of the egg. Oosome fragments visible. Scale: 25 pm.
Fig. 4. Germ cells. Scale: 25 pm.
Fig. 1.

Early embryonic development of Tritneptis

2096Fig. 5.

Penetration of germ cells through the blastoderm. Chromatin polarization In thenuclei of the blastoderm cells. Scale: 25 f.L m .
Fig. 6. atin granule underneath the blastoderm cells. Scale: 25 f.L m
Fig. 7. Irregular accumulations of the vitellophages. Chromatin polarization in the nucleiof the blastoderm cells visible. Scale: 25 f.L m .
Figs. 8, 9. Formation of the primary epithelium of the mid gut. Longitudinal sections. pen,primary endoderm; v, vitellophages. Scale: 25 f.L m .

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M. K. Koscielska and B. Koscielski

Material and Methods
The material used in the study ,was taken from a laboratory culture in which larvae (in cocoons) of Diprion pini and D. frutetorum were used as hosts of parasite.
Larvae in cocoons had been collected in pine woods. This made it possible to obtainany number of Tritneptis diprionis eggs in various growth stages. After puncturing thechorion with a tungsten needle sharpened in molten sodium nitrite, the eggs were fixedfor lO - 20 min in Carnoy's fluid, and then embedded in paraffin. The sections, 5 ft mthick, were stained in Delafield's haematoxylin and eosin.

Results
A freshly laid egg contains fine yolk granules evenly scattered in the ooplasm. Inthe posterior part of the egg there is an oval oosome (Fig. I). During the cleavage theoosome migrates to the peripheral region of the posterior egg pole and there it undergoes fragmentation (Figs. 2, 3). Finally the material of the oosome gets into the germcells. The germ cells get out of the embryo even before the definitive blastoderm isformed (Fig. 4). These processes take place within the first few hours after the egglaying. Between the 10th and 20th hour of the embryonic development single germ
cells penetrate through the blastoderm inside the embryo(Fig. 5). The chromatin of theblastoderm nuclei shows a polarization, being accumulated mostly in the apical portions of the nuclei (Fig. 5). Immediately below the blastoderm, most often on the posterior pole of the embryo, chromatin granules may occur, coming from the blastoderm
nuclei (Fig. 6). At the stage of blastula a larger accumulation of yolk is localized in theanterior egg portion. In course of the further development the yolk distribution again
becomes even. In an initial period of the blastoderm formation yolk nuclei (vitellophages) arrange in two rows parallel to the egg long axis. At the stage of the definitive blastoderm the vitellophages divide intensively and at the same time their linear
arrangement becomes disturbed: irregular accumulations of several and sometimes
more than ten vittelophages form (Fig. 7). Initially the division of the vitellophages ismitotic, then probably amitotic. After numerous divisions the primary vitellophages
assume again their linear arrangement. A part of the vitellophages (ca. 30th hour of thedevelopment) get to the surface of the yolk to form the primary mid gut epithelium(Figs. 8, 9). In the posterior part of the primary and the definitive mid gut a few nuclei
can be observed originating probably from the germ cells (Fig. 12). After the definitive
mid gut epithelium has been formed, the primary vitellophages undergo a gradual degeneration.
Between the 20th and the 30th hour of the development gastrulation takes place,effected by the sinking of the middle plate (Figs. 10, 11). The process begins in the midpart of the germinal band and from there it proceeds to the posterior and anterior partof the embryo. The course of this process is rapid and is completed within a few hours.
During the gastrulation also the only embryonic membrane - the serosa - forms. At
about the 40th hour of the development, in the anterior part of the embryo on its ventral side salivary glands arise from the ectoderm, characterized by large, polyploid nuclei (Fig. 13). At that time also the definitive mid gut epithelium forms fro m a group ofEarly embryonic development of Tritneptis

211. mer

13
Figs. 10, 11. Sequence of formation of the mesoderm (mes). Cross sections. Scales: 25 fl m.
Fig. 12.
Fig. 13.

Longitudinal section through the embryo. In the posterior part of the definitive
mid gut the nuclei derived from the germ cells can be seen (arrow). Scale: 25 flm.
Formation of the salivary gland. Longitudinal section. sgd, salivary gland duct.
Scale: 25 fl m .

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M. K. Koscielska ap.d B. Koscielski

cells at the bottom of the stomo- and proctodaeum.

Discussion
In the embryonic development of Tritneptis diprionis an array of features occurs
characteristic of the development of hymenopterans, especially the parasitic ones. The
newly laid egg containing few and fine yolk granules is similar to that of Monodontomerus
dentipes (Koscielska, 1981). During the cleavage the oosome fragments, like in other
Hymenoptera, determine the germ cells (Beams and Kessel, 1974). Similarly, thepenetration of the germ cells through the blastoderm to the insid-e of the embryo wasobserved in many parasitic hymenopterans (Bronskill, 1959, 1964; Ivanova-Kasas,1950, 1952, 1958, 1960, 1961; Koscielska, 1980, 1985). Some of the germ cells penetrate
into the posterior part of the primary mid gut and fo~m there a small aggregation.
Their role has not been explained. Most authors (see Bronskill, 1959, 1964) regard
them as tertiary vitellophages which take part in digestion. Maybe in the case of T. diprionis these cells actually digest the remnants of the primary mid gut epithelium, like in
Pleolophus basi<.onus (Koscielska, 1980). In M. dentipes, however, in which the primaryvitellophages disappear early, these cells probably take part in the digestion of yolk
(Koscielska, 1981).
A polarization of the chromatin in the blastoderm cells, resembling that of T. diprionis, was observed in Dahlnominus fuscipennis (Koscielska, 1985) and in Eurytoma aciculata (Ivanova-Kasas, 1958). The significance of this phenomenon has not been explained.
The process of the vitellophage divisions , at the stage of definitive blastoderm,
was observed not only in Hymenoptera (Koscielska, 1980, 1985) but also in Diptera(Anderson" 1962). The greatest number of the vitellophages in the studied species
occurs during the gastrulation, like in D. fuscipennis (Koscielska, 1985). The vitellophage division, initially mitotic and then probably amitotic was described also in thedevelopment of P. basi<.onus (Koscielska, 1980) and D. fuscipennis (Koscielska, 1985). InT. diprionis, like in D. fuscipennis the vitellophages are still present at the stage of theprimary mid gut, in other species e. g. M. dentipes they disappear at earlier development
stages (Koscielska, 1981). The primary mid gut epithelium in T. diprionis forms fromthe primary vitellophages, like in some other hymenopterans, both parasitic and nonparasitic (Carriere and Burger, 1897; Strindberg, 1915; Bledowski and Krai6ska, 1926;
Dondua, 1953; Bronskill, 1959, 1964; Ivanova-Kasas, 1960; Koscielska, 1980, 1985).
The gastrulation in T. diprionis resembles this process in other studied species ofhymenopterans parasitizing larvae of diprionids, i. e., it is accomplished by sinking of
the middle plate. Until not long ago it was supposed that (see Ivanova-Kasas, 1961)
this type of gastrulation is characteristic of Aculeata and a few species of parasitic
Hymenoptera (Bronskill, 1959, 1964; Ivanova-Kasas, 1961). It follows from the present
paper and from the earlier studies (Koscielska, 1980, 1981, 1985) that the gastru1ation
effected by sinking of the middle plate is not a rare phenomenon in the development of
the parasitic hymenopterans.
The chromatin granules observed below the blastoderm in T. diprionis were described also in other Hymenoptera. The problem was discussed in detail in the paper

Early embryonic development of Tritneptis

213

on the development of P. basizonus (KoScielska, 1980). More recently granules of nuclear origin at the blastoderm stage were found by Tanaka (1985) in Trichogramma chilonis.

The salivary glands in T. diprionis are ectodermal origin, like in other parasitic
hymenopterans (Bronskill, 1959, 1964; Koscielska, 1981, 1985). Also the definitive midgut epithelium is formed, like in most insects, from the cells situated at the bottom ofthe stomo- and proctodaeum (Anderson, 1972).
In the development of T. diprionis there are some features characteristic of otherhymenopterans parasitizing diprionids, e. g. formation of the primary and the definitive
mid gut, and the type of gastrulation. On the other hand, these characters are typicalof the development of other Hymenoptera, especially Aculeata. Thus it is another proofof the phylogenetic affinity of the parasitic hymenopterans and Aculeata.

References
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M. K. Koscielska and B. Koscielski
Authors' addresses: Dr. M. K. Koscielska
Department of Animal Systematics andZoogeography, Institute of Zoology,Wroclaw University, ul. Sienkiewicza
21, 50-335 Wroclaw, Poland

Prof. B. Koscielski
Department of General Zoology, Institute of Zoology, Wroclaw University