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

273

Fine Structure of Endoderm of Embryoof Ageniaspis fuscicollis (Chalcidoidea, Hymenoptera)*

Boguslaw KOSCIELSKI
and,Maria Krystyna KOSCIELSKA
Synopsis
Ageniaspis fuscicollis is a parasItIc insect developing polyembryonically. The cells of theectodermal and endodermal layers of the embryo absorb the food materials synthesized introphamnion. In fine structure the cells of endoderm are similar to the trophamnion. During
epidermis formation stage the endoderm-trophamnion contact disappears, and then the endodermal cells take over the function of trophamnion.

Introduction
Ageniaspis fuscicollis is a parasitic hymenopteran developing polyembryonically incaterpillars of Yponomeuta millinella (Lepidoptera). Caterpillars of Yponomeuta infested byA. fuscicollis contain long, branching cords filled with numerous parasite embryos whose
number can reach 180. Marchal (1904) demonstrated that all embryos develop fromone egg as a result of its divisions. He called this type of reproduction polyembryony,
and the membrane surrounding the embryos and enabling them to absorb nutritive
materials from the haemolymph - as trophamnion.
The egg of A. fuscicollis is devoid of yolk and its segmentation is total. Prior to thebeginning of segmentation the ooplasm, constituting the proper embryo material, separates from the peripheral one, containing the chromatin of non-rejected polar bodies.
The chromatin of the polar bodies changes into giant, lobe-shaped trophamnion nuc-* Supported by the Nencki Institute of Experimental Biology of Polish Academy of Scien-ces, Warszawa.

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leus - the paranucleus, which in the course of further development divides amitotically (Silvestri, 1908; Martin, 1914; Koscielska, 1963; Koscielski, 1981). According to Ivanova-Kasas (1961, 1972) the trophamnion is an embryonic membrane (serosa) whoseformation has been shifted to early development stages. This type of membrane is characteristic of the polyembryonic Hymenoptera. As indicated by the ultrastructural studies on the polygerm of A. juscicollis, the outer surface of the trophamnion is provided
with microvilli and absorbs exogenous materials by meas of the micropinocytosis (Koscielski et al., 1978). Multivesicular bodies resulting from the fusion of the micropinocytotic vesicles are a form of yolk precursor. Thus the micropinocytotic vitellogenesis
usually taking place in the oogenesis, in the trophamnion same process is used as ameans of accumulating food reserves for the developing embryos. It can be supposedthat the disappearance of yolk and the change in the way of cleavage (from partial tototal) following it, and the use of the food accumulating mechanism originating fromthat of the egg, are two important factors responsible for the origin of the polyembryony in Hymenoptera.
Furthe, ultrastructural studies showed that the embryos of A. juscicollis absorb foodmaterials synthesized in the trophamnion, also by means of micropinocytosis. The
trophamnion cytoplasmic membrane, contacting directly with the embryos, forms
numerous folds which interlock with embryo ectodermal microvilli. In older embryos
the trophamnion contacts also with the endoderm cells through a wedge-like process
penetrating the dorsal groove of the embryos. In the terminal section of the wedge situated close to the endoderm, the cytoplasmic membrane folds are especially strongly developed. This direct contact enables also the endoderm cells to absorb micropinocytotically materials from the trophamnion (Koscielski and Koscielska, 1985).
The present paper deals with the fine structure of the endoderm of the embryo ofA. juscicollis at the dorsal groove stage.

Material and Methods
The polygerms of A. foscicollis were dissected in physiological solution from caterpillars of Y. malinella and fixed in 3 % glutaraldehyde in o. 1 M phosphate buffer, pH
7.3 for 2 hr. Then the material was rinsed in the buffer and postfixed in 1 % OS04 for
2 hr. Following the dehydration in ethanol and acetone the polygerms were embedded
in Epon 812. Ultrathin sections were contrasted according to Reynolds (1963) and examined in the electron microscope Tesla BS 613. Semithin sections were stained with
1 % methylene blue in 1 % borax.

Results
The endoderm cells in A. juscicollis, despite their position inside the embryo, contact with the trophamnion through the dorsal groove (Figs. 1, 2). The lumen of thegroove penetrates, as narrow canals, between the endoderm cells. The cell surface contacting with the canal lumen is provided with microvilli. The lumen of the canals is filled with a granular material (Fig. 3). In the apical parts of the cells micropinocytotic

Embryonic endoderm of Ageniaspis

275

Fragment of polyembryonic cord of Ageniaspis fuscicollis. Embryos at dorsal groovestage lie in trophamnion (Ta). On surface of trophamnion cyst of host cells (C).
Numerous lipid drops visible in trophamnion. Semi thin section. Scale: 50 f.L m.
Fig. 2. Embryo at dorsal groove stage. Trophamnion cytoplasm (Ta) penetrates embryo
dorsal groove as a wedge: ectoderm (Ec), endoderm (En). In endoderm numerous
lipid drops. Semi thin section. Scale: 50 f.L m.
Fig. l.

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Fig. 3.
Fig. 4.

B. Koscielski and M. K. Koscielska

Electron micrograph showing polarized endoderm cells contacting with canal lumen: microvilli (Mc), nucleus (N). In canal lumen granular material (Gm). Scale:I pm.
In polarized endoderm cell numerous pinocytotic vesicles and dense bodies visible.
Scale: I p m.

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277

vesicles are visible as well as multivesicular bodies and dense bodies. In the cytoplasm
there are numerous, elongate mitochondria of a typical structure (Fig. 4).
The remaining endoderm cells do not form a compact arrangement; between them
there are space filled, like the lumen of the canals, with a granular material (Fig. 5).
Probably they are continuous with the canals. The cytoplasmic membranes of thesecells do not form microvilli, thus the cells are not polarized. Very rarely direct connections between the cells are observed, as cytoplasm bridges (Fig. 5). The cytoplasm, richin mitochondria and free polyribosomes, contains numerous parallel cysterns of granular cytoplasmic reticulum. Sporadically, multivesicular bodies are observed. Lipid inclusions of various size, sometimes occupying a considerable part of the cytoplasm area characteristic feature of the endoderm cells (Fig. 5) .

Fig. 5.

Electron micrograph showing endoderm cells far from dorsal groove; in cytoplasm
cysterns of granular endoplasmic reticulum, cytoplasm bridge (Cb) and lipid inclusions of various size visible, nucleus (N). Between endoderm cells spaces filled with
granular material (Gm) . Scale: 1 f-l m.

Discussion
The endoderm in A. Juscicollis results from the multipolar migration of the cells
into the blastula (Koscielska, 1977). In the further course of its development theembryo shape changes from spherical to ellyptical, and at the moment of the dorsalgroove appearance changes to kidney-like. The cells, that entered the inside of theembryo and contact with the trophamnion through the dorsal groove, show the presence of phospholipids. The phospholipids are the main energy source for the living

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processes during the polyembryonic development (Ivanova-Kasas, 1961; Koscielska,1962, 1963). The lipid-rich cells in the course of the development form the mid gut(Koscielska, 1977). There are no electron microscope studies on insect endoderm. The
existing data pertain to the definite mid gut epithelium (Smith, 1968; Krzysztofowicz etal., 1973).
Some structures contained in the endoderm cells of A. fuscicollis are similar to thoseof the trophamnion: micropinocytotic vesicles, multivesicular bodies, dense bodies andlipid inclusions. Like in the trophamnion, in the endoderm cells an intense protein
synthesis takes place which is evidenced by the strongly developed granular endoplasmic reticulum. The endoderm cells start absorbing and accumulating the reserve materials as soon as they enter in contact with the trophamnion through the dorsal groove.
In the monoembryonic development the endoderm differentiating into the mid gut encloses the egg yolk in its lumen. In A. fuscicollis the endoderm-yolk contact is retained
as a close contact with the "external yolk" which - according to Marchal (1904) - isthe trophamnion. The system of the canals distributes the materials coming from thetrophamnion to endoderm cells situated farther from the dorsal groove. After the disappearance of the dorsal groove the further development is possible due to the endoderm in which the reserve materials, mainly lipids, are accumulated. Numerous lipid
inclusions are present also in the mid gut cells. After having used the reserve materials
the mid gut gets in connection with the fore and hind guts. Then the embryo at its larval stage can feed on its own account (Koscielska, 1977).
The electron microscope studies on the polyembryony in A. fuscicollis showed, thatthe development of the parasitic Hymenoptera is not only due to the trophic membrane- trophamnion - adaptations to the absor.ption of nutritive materials from the hosthaemolymph, but also due to the adaptations of the embryo cells to the micropinocytotic uptake of the materials synthesized in the trophamnion. This adaptation is shown,at early development stages, by both the ecto- and endoderm cells. At more advanced
development stages the endoderm cells take over the trophamnion function synthesizing
materials necessary for further development of the embryos.

References
Ivanova-Kasas, O. M., 1961. Essays on the Comparative Embryology of Hymenoptera (in Russian). Leningrad Univ. Pub!.
- - - - - . , 1972. Polyembryony in insects. In S. j. Counce and C. H. Waddington (eds. ),Developmental Systems: Insects, Vol. 1,243-271. Academic Press, London, New York.
Koscielska, M. K., 1962. Investigation upon trophamnion Ageniaspis foscicollis Dalm. (Chalcidoidea, Hymenoptera). Studia Soc. Sci. Torunensis, Sec. E (Zool. ) 6: 1-9.
- - - - - , 1963. Investigations on polyembryony in Ageniaspisfuscicollis Dalm. (Chalcidoidea,Hymenoptera). Zool. Pol. 13: 255-276.
- - - - - , . 1977. Gastrulation and differentiation of endoderm in Ageniaspis fuscicollis Dalm.
(Chalcidoidea). Zool. Pol. 26: 159-173.
Koscielski, B., 1981. Early development of Ageniaspis fuscicollis Dalm. (Chalcidoidea, Hymenoptera). Zool. Pol. 28: 315-320.
- - - - - , and M. K. Koscielska, 1985. Ultrastructural studies on the polyembryony in Ageniaspis fuscicollis (Chalcidoidea, Hymenoptera). Zool. Po!. 32: 203-210.

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fuscicollis Dalm. (Chalcidoidea, Hymenoptera). Zoomorphologie 89: 279-288.
Krzysztofowicz, A., Cz. Jura and S. Bilinski, 1973. Ultrastructure of midgut epithelial cells ofTetrodontophora bielanensis (Waga) (Collembola). Acta Bioi. Crac., Ser. Zool. 16: 257-265.
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Martin, F., 1914. Zur Entwicklungsgeschichte des polyembryonalen Chalcidiers Ageniaspis (Enryrtus) fuscicollis Dalm. Z. Wiss. Zool. 110: 419-479.
Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron-opaque stain in electron
microscopy.J. Cell Bioi. 17: 208-213.
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dell'Ageniaspisfuscicollis. Ann. R. Scu. Sup. Agr. Portici 8: 1-27.
Smith, D. S., 1968. Insect Cells. Their Structure and Function. Oliver and Boyd, Edinburgh.

Authors' addresses: Prof. B. Koscielski
Department of General Zoology, Institute of Zoology, Wroolaw University,ul. Sienkiewicza 21, 50-335 Wrodaw,PolandDr. M. K. Koscielska
Department of Animal Systematics andZoogeography, Institute of Zoology,Wrodaw University