The eggshell of the almond wasp Eurytoma amygdali (Hymenoptera, Eurytomidae) - 1. Morphogenesis and fine structure of the eggshell layers

TISSUE AND CELL, 19Y4 26 (4) 5%.56X 0 1994 Longman Group Ltd.

DESPINA

G. MOUZAKI

and LUKAS H. MARGARITIS

THE EGGSHELL OF THE ALMOND WASP EURYTOMA AMYGDAL/ (HYMENOPTERA, EURYTOMIDAE) - 1. MORPHOGENESIS AND FINE STRUCTURE OF THE EGGSHELL LAYERS Keywords:

Almond

wasp. Hymenoptera.

eggshell.

folhcle cells. pcroxldase

ABSTRACT. The almond wasp Eu+oma urnygduli (Ilymenoptrra: Eurvtomldac) tee& ,md ovipostts cxclusivcly tn almonds and therefore is characterized as an~inscct of ccormm~c importance Its meroistic polytrophic ovaries m&de follicles with a tri-partite configuratwn The mature follicles exhibit two filaments occupying the two poles of the egg. One 15 the micropylar filament while the other might serve for resptration since it is likely that ttr flattened end layers remain outside the almond fruit. The eggshell is formed by aposltion and the lolhcle cells. which surround the follicle until the end of oogenesis. may be responsible for protein synthesis and secretion which finally lead to the assembly of the eggshell. The eggshell compnsc\ the thin vitelline membrane, possibly a ‘wax’ layer of waterproofing function. a translusccnt layer which appears amorphous even at the end of chortogenests, a granular layer, includmg large and small electron-dense granules, and finally a columnar layer very similar to layer5 found in other insect species of the same or different orders. Peroxldasc is histochemlcall) found for the first time in an eggshell of the Hymenoptera order: the tranlusccnt layer III particular is positively stained (electron-dense). Two possible roles of this peroxidatic activity are discussed. first, in comparison to other fruit-infesting insects. WCassume that elastic chorton is produced through the function of peroxidase Induced bonds (resilin-type bonds). \crk important for avoiding premature breaking, while being oviposited through a narrow ovipositor. Second. referring toothrrstudies. ihis layer can play a bactericidal role for additional embryonic protection.

Introduction

Insect follicles during the last stages of oogenesis have been shown to undergo several structural and functional changes such as the degeneration of nurse cells and the formation of the eggshell layers (for reviews see King and Buning, 198.5; Margaritis, 1985a). Oogenesis in Hymenoptera is of the meroistic polytrophic type and ovarian development in the parasitoid bragonid wasp Habrobragon juglandis has revealed the origin and the differentiation of the oocyteDivision of Biochemistry, Cell-Molecular Biology and Genetics, Department of Biology, Athens University, Panepistimiopolis 15701. Athens, Greece. Received Accepted

17 June 1993 15 February I994

nurse cell complex (Cassidy and King, 1972). Studies of eggshell formation of parasitoid species is of particular interest due to clarifying and evaluating parasitoid-host relationships (Norton and Vinson, 1982). The almond wasp Eurytoma amygdali (Hymenoptera: Eurytomidae) is a considerable fruit pest in Greece reducing the quality and quantity of edible almonds. This insect possesses several common characteristics to other fruit-infesting insects such as the dipteran tephritids: (1) Similar oviposition process inside the fruits by penetrating the seed with a very long and narrow ovipositor. (2) oogenesis in both cases is of the meroistic polytrophic type and, (3) there are similarities in the pulp of the fruits in which these eggs are oviposited. The structural similarities might reveal that these insects have

MOUZAKI AND MARGARITIS

560

chosen similar physiological routes despite their distal evolutionary relationships (Kristensen, 1981). In certain cases the chorion imparts elasticity to the eggs due to covalent di- and trityrosine crosslinks between residues of the chorionic proteins (for review see Margaritis, 1985a). The process has been well-established in the case of Drosophila melanogaster to occur in the last stages of choriogenesis upon the activity of endogenous peroxidase (Giorgi and Deri, 1976; Mindrinos et al., 1980), activated by endogenous H202 (Margaritis, 1985b). Concerning other insect orders it was found in the eggshells of the tephritids Ceratitis capitata, Dacus oleae (Margaritis, 198%) and Rhagoletis cerasi (Mouzaki and Margaritis, 1991). Peroxidase exists also in the eggs of the lepidopteran Calpodes ethlius (Griffith and Lai-Fook, 1986) and might be an endogenous component of cytoplasmic inclusions in the follicle cells of a dragonfly (Kessel and Ganion, 1979). This study was undertaken in order to answer basic biological questions concerning choriogenesis in Hymenoptera and essentially in species with major economic importance like Eurytoma amygdali. Studying the formation of the eggshell of the almond wasp, we were interested to see if the eggs of E. amygdali also show positive reaction of peroxidase since all four species of economic importance share common egg oviposition mechanisms. The presence of elastic chorions are considered to prevent premature breaking of the egg during oviposition (Margaritis, 1985~). Materials and Methods Eurytoma amydgali insects were obtained in the field. The larva diapauses inside the infested almond, from February-March until November-December. Following metamorphosis the adult fly opens a hole to the hard almond pericarpium and flees to the environment. The adult flies might need a period of 3-9 days to open a round hole and exit the fruit (Plaut, 1971). Early in December mummified grey almonds were collected from the trees and kept until insects were released. They were then kept in a culture room of 22°C and fed under honey and water (Plaut, 1971). Dissection was carried in cold Ringer’s

solution and ovaries were separated into ovarioles. Several different developmental staged follicles were selected and processed for transmission and scanning electron microscopy as described elsewhere (Margaritis et al., 1980). Peroxidase was detected under the light microscope through the o-dianisidine assay (Worthington, 1972). Histochemical detection of peroxidase under the electron microscope was conducted as follows: Follicles following dissection were fixed in 8% formaldehyde in 0.1 M cacodylate buffer for 30 mins. Then, histochemical stain was prepared according to Fahimi (1970) utilizing diaminobenzidine (DAB) and H202. Samples were then processed conventionally for TEM including 1 hr osmication period. Appropriate control samples were prepared excluding either Hz02 or DAB and H202. Sections were observed unstained. ResuIts Eggshell structure Eurytoma amygdali’s ovaries belong to the

meroistic polytrophic type and each includes several ovarioles. Mature eggs show a tripartite configuration (Fig. 1). The oocyte is 0.3 mm long, the anterior end forming a short filament, and the posterior end forming a 1.425 mm filament, flattened at the end 0.25 mm (Fig. 5). The terms anterior and posterior end which are used here to characterize the egg’s polarity are refering to the way the egg is oriented in the ovipositor, i.e. the anterior pole exits first. Thin cross-sections of the main body of the egg reveal that the oocyte is surrounded by several eggshell layers: the vitelline membrane, a transluscent layer, a granular layer and a columnar layer (Fig. 3). The latter forms numerous drappings as seen under the scanning EM (Fig. 2). These are also seen underneath the follicular epithelium (Fig. 1). The basic eggshell structure is the same all around the oocyte, however, several differences occur from the main body to the edge of the egg. A major difference is found at the flattened filament edge where the granular layer is not exhibiting large granules but small, fine mesh, thickened at the basis of the layer (Fig. 6). The columnar layer of the same region shows a finer more compact formation than in the main body and the rest

EGGSHELL

MORPHOGENESIS

IN THE ALMOND

of the filament (compare tangential in Figs 4. 6).

sections

The follicles inside the ovaries are oblong in shape and contain a constriction (canal) separating the oocyte from the nurse cells (Fig. 7). Upon vitelhne membrane formation, dramatical changes are observed in the follicles. The oocyte lenghtens and the cytoplasm near the nurse cells contains less yolk, whereas the nurse cells degenerate (Fig. 8). The vitelline membrane is very electron lucent while mtcrovilli of the follicle cells are associated with secreted material in the form of vesicles (Fig. 9). This material is more or less fibrous as can be seen under high magnifications (Fig. IO). In more advanced stages only the long filament at the posterior pole is well defined (Fig. 11). The yolk spheres appear in two forms, polygonal and elongated, depending on the configuration they occupy in space (Fig. 12). In the main body, only polygonal yolk spheres are observed (Figs 13, 14). In each pole of the oocyte the short filament and the long filament with the flattened end are observed (Fig. 15). AtOthis stage above the transparent layer 500 A granules are formed both in the main body of the oocyte (Fig. 16) and in the long filament (Fig. 17). Follicle cells at that stage contain rough

WASP

i(,l

endoplasmic reticulum (Fig. 16). The granules seem to be connected with a filamentous meshwork (Fig. 18). Histochemical detection of peroxirbse When we initially used this organism. and started dissections we observed that the mature follicles were highly elastic and moreover, they didn’t break very easily. Our results, using the o-dianisidine assay and the DAB histochemical reaction for peroxidase (see Materials and Methods), were impressive. Follicles showed positive reaction with o-dianisidine (red colour), indicating the presence of peroxidase. Red colour was also apparent in isolated eggshells. The cytochemical assay revealed, in thin sections, positive reaction of DAB in the translucent area (Fig. 19). The electron density of the reaction was somehow retained even when Hz@ was ommited from the incubation medium (used as a control) (Fig. 20). The exclusion of both DAB and H20z, in control samples showed negative reaction as was expected. Discussion The mature egg of the almond wasp includes the oocyte, the eggshell layers. and two distinct filaments: A short micropyiar filament (Zarani and Margaritis. included in the same issue of Tissue and Cell) and a long filament.

Fig. 1. Whole mount of mature follicle. The configuration is tri-partite. ma: micropylar appendage, ooc: oocyte. F: filament. A drapped structure is observed under the follicular eplthelium (asterisk). bar 100,um. Fig. 2. Higher magnification at the main body of a mature Observe the characteristic drappings. bar IOum.

follicle peeled from follicle cells

Fig. 3. Thin section at the main body of mature follicle. The vitelline membrane is very thm (1500 A). An irregular zone is observed above the vitelline membrane (arrow). Note the three choriomc layers. The translucent layer (TL) the granular layer (GL) which contains large and small electron dense granules and the columnar layer (CL). x 13.000. Fig. 4. Tangential section of the upper granular and the columnar layers. The upper part of the granular layer includes only small granules while the columnar layer is totally perforated interiorly ( t ) and exteriorly ( -1). x25.000. Fig. 5. The flattened

edge of the long filament.

being smooth

on the surfacc

bar 100 vrn

Fig. 6. Tangential section similar to that in Figure 4. This is taken at the flattened edge oi the filament. Note that the corresponding granular layer of the main eggshell, has a meshworklike appearance which is more condensed in the bottom of the layer and less near the columnar layer. Another difference concerns the latter layer. It is finer, compared to that of the mam body of the follicle, though the basic holey structure is conserved. ~40,000.

L

\ I’.’

564

According to Plaut (1971) these flies oviposit their eggs inside the nuscellar seed and prefer larger fruits (mean length of fruit 25-30 mm). The width of the fruits is approximately 10 mm whereas the length of the ovipositor is 3 mm). At this point, we faced a contradiction since, according to these measurements, the whole egg should be immersed inside the fruit (total egg length 2 mm including the filaments), however, we know that the last part of the filament remains outside the pericarp (Tzanakakis, personal communication with L. H. M.). On the other hand, a semi-immersed egg would imply that the fruits selected by the females could not have more than a width of 4-6 mm. Unfortunately, our collected flies did not oviposit under lab conditions in freshly cut almonds. It is obvious that, whether the egg is totally positioned inside the fruit or not, is

MOUZAKI

AND MARGARITIS

of great importance for its physiology. If part of the filament, and especially the very end, remains outside the fruit, then, respiratory gas exchan e between the environment and the oocyte Bdeveloping embryo region should be very efficient, provided that the protruding filament is structured suitably, i.e. being perforated in order to allow air diffusion (for a review on eggshell physiology see Margaritis, 1985a). Our structural evidence supports this function (see Fig. 5). The vitelline membrane is a very thin and loose layer and may enable the oocyte to absorb nutrients from the host haemolymph as has been similarly shown for other Hymenoptera such as the parasitoid Campoletis sonorensis (Norton and Vinson, 1982). Above the VM a configuration similar to a waxy or lipid layer is observed. This is very similar to some wax layers shown to exist in

Fig. 7. Whole mount follicle prior to vitelline membrane synthesis. There is a constriction (arrow) between the oocyte (ooc) and the nurse cells (nc). The yolk is less dense posteriorly, in the area where the long filament is formed. The canal of organelle and cytoplasmic material passage from the nurse cells to the oocyte is observed (arrowhead). x 120. Fig. 8. Whole mount follicle during vitelline membrane synthesis. The shape of the follicle has been changed. It has been highly elongated while the nurse cells at the posterior end have already been degenerated. Note that the yolk content is gradually decreasing from the anterior part (A) to the posterior one (P), x 140. Fig. 9. Thin section at the main body of the follicle shown in Figure 8. The vitelline membrane is loose. The follicle cells (FC) contain numerous free and membrane-bound ribosomes whereas their apical plasma membrane is full of microvilli. Secreting activity is visible (arrows) towards the granular VM. Part of the oocyte is shown which also contains microvilli. No secretion or pinocytic activity is observed. x 14,COO. Fig. 10. Higher magnification of Figure 9 at the interface between the vitelline membrane and the follicle cells. Fibrous material is deposited above the vitelline membrane (arrows). x20,000. Fig. 11. A more advanced follicle during vitelline membrane formation. The oocyte can be clearly defined as well as the long filament and the area which is going to become the flattened end of it (arrow). The filament and the latter area are separated by a barrier (arrowhead). The short filament is not visible, yet. x140. Fig. 12. Thin section at the main body of the follicle shown in Figure 11 (continuous line). The yolk shows two different structures, a polygonal and a longitudinal, depending on the configuration they occupy in the space. Between these two extremes intermediate forms can be observed (arrows). FC: follicle cells, VM: vitelline membrane. x5OCQ. Fig. 13. Cross section at the long filament of the same follicle, also shown in Figure 11 (dashed line). Mainly polygonal yolk particles are observed, which could be explained since a) these are cross-sections of the filaments b) the yolk ‘spheres’ are arranged longitudinally within the long filament because this position is the most favourable (long items within a tube). They are scattered basically on the periphery of the filament. x2450. Fig. 14. Higher magnification of the polygonal yolk particles of Figure 13. The electronlucent vesicles probably represent lipid droplets (arrows). x 14,000.

EGGSHELL

MORPHOGENESIS

IN THE ALMOND WASP

Diptera (Margaritis, 1985a); moreover ‘wax’ may also be found in Campoletis sonorensis (Norton and Vinson, 1982) and in the orthopteran Scistocerca gregaria (Kimber, 1981). Beament (1946) has shown in his studies in the heteropteran Rhodnius prolixus, that a thick hydrophobic layer in the eggshell prevents the oocyte from water loss or water uptake. A similar mechanism might prevent the oocyte of the almond wasp swelling inside the watery nuscellar seed. The transluscent layer of the eggshell does not exhibit any kind of substructure and is not stained (in sections) with uranyl acetate and lead citrate. In several other species a similar layer is found to occupy the same position in the eggshell, however, it shows a distinct periodicity, of either vertical to the vitelline membrane crossbands, as in the case of Campoletis sonorensis (Norton and Vinson, 1982) and Nemeritis canescens (Rotheram, 1973), or parallel to the vitelline membrane as in Cardiochiles nigriceps (Vinson and Scott, 1974). It is highly possible that the transluscent layer is crystallized after oviposition, or during the egg removal through the calyx or the oviduct. A similar phenomenon is described for Acheta domesticus (Furneaux and Mackay, 1976). Further discussion on the possible role and appearance of this layer is presented below. The granular layer appears structurally different in the main body of the oocyte and in the filament. In the oocyte, the larger granules at the base of the layer are formed possibly by the fusion of smaller ones pressed downwards, while the apical area includes mainly small granules. All of them seem to join together with fine fibers. In the filament the granules are generally smaller than in the main body, however the two-layered structure of larger-smaller granules &sstill maintained here. The columnar layer, found in the eggshell of E. amygdali, is very similar to the projections found in the chorion of Nemeritis (Rotheram, 1973). Campoletis (Norton and Vinson, 1982) and Cardiochiles (Vinson and Scott, 1974). All three previous species also showed a fibrillar material on and between the projections, a case not found in Eurytoma. This material is reported to function as inhibiting or suppressing the host immune system (Norton and Vinson, 1982), or even retain particulate secretions from the

565

host haemolymph (Rotheram, 1973). On the other hand the drappings found in the pillar layer of the almond wasp might serve the same purpose, as traps for particles or bacteria found inside the fruit. This would prevent infection of the developing embryo. The tri-partite structure of the follicle is pre-determined early in oogenesis, prior to any vitelline membrane formation. This means that the follicle cells have already migrated at their final developmental sites. There is no structural difference between follicle cells found in the main body of the filament, as found in the follicles of the vetch aphid Megoura oiciae, where discrimination of two different follicle cell types suggests functional/structural specialization (Brough and Dixon, 1990). However, the follicle cells in our case can be distinguished into four different subpopulations, each of which will give rise to a specific follicle region: (1) the short micropylar filament, (2) the main body, (3) the long filament and (4) the flattened edge of the long filament. 10 subpopulations have been described in the follicles of Drosophila melanogaster which give rise to the regional complexity of the eggshell (Margaritis et al., 1980). Their final round of migration ends when the vitelline membrane has almost been synthesized (King, 1970). quite later than what we have observed for Eurytoma.

As referred to by many articles, follicle cells’ participation in vitelline membrane and chorion formation in insects is very wellestablished (King and Koch, 1963; Kimber, 1980; Margaritis, 1985a). The oocyte, however, might play an important role at least in the vitelline membrane formation (Norton and Vinson, 1982). Inside the follicle cells, one can observe all necessary structural evidence which indicates the high activity of protein synthesis occuring during choriogenesis. The rough endoplasmic reticulum, the Golgi complexes, as well as gap junctions found between neighbouring cells, are very elaborate. Gap junctions allow communication and coordination of cells to produce and release precursor material which will form the vitelline membrane and the chorion (Gain0 and Mazzini, 1990). Peroxidase as an eggshell component

Nature has facilitated insect eggshells with protective mechanisms versus predators.

EGGSHELL

MORPHOGENESIS

IN THE ALMOND

mechanical pressures, drying and flooding. One of the most important, however, least studied factor, is a stabilizing process developed in many eggs to withstand contradictory environmental conditions. This process usually occurs through different kinds of bonds, disulfide or covalent. The final result of such processes is, for example, the production of a hardened chorion as in the case of the mosquito Aedes aegypti (Sclaeger and Fuchs, 1974). Sclerotization in the mosquito eggs is a consequence of dopa-decarboxylase activity (Sclaeger and Fuchs, 1974). Another example is the eggshell of the lepidopteran Hyalophora cecropia which is stabilized through disulfite bonds (Smith et al., 1971). Finally, resilin-type covalent bonds could occur among the tyrosyl residues of proteins (Andersen, 1964), and this stabilization is producing elastic eggshells. Peroxidase catalyzes the reaction utilizing Hz02 as in the case of D. melanogaster (Margaritis, 198%). Peroxidase is found to exist in the endochorion, a structure very similar to the endochorions of D. melanogaster and of other Diptera (Margaritis et al., 1980; Margaritis. 198%; Mouzaki and Margaritis, 1991). Calpodes ethlius is reported to include all three different stabilizing procedures in the eggshell (Griffith and Lai-Fook, 1986). Nothing is known so far concerning Hymenoptera eggshell stabilization, although most

WASP

iI17

of the parasitoid species follow the same pattern of oviposition through a narrow ovipositor in order to lay eggs inside the host caterpillars (i.e. Nasonia oitripennis, Ritchards. 1969). A similar way of oviposition has been observed in insects of different orders such as the dipteran fruit Aies. Our observations in the eggshell of E. amvgdali showed that the translucent layer is positively stained for peroxidase and therefore can be of proteinaceous composition. Since we were even able to detect peroxidase in the absence of exogenous H703 the following can be suggested: Peroxidase either functions through an alternative mechanism other than that proposed so far for D. melanogaster uia WY.01 utilization (Margaritis, 198Sb, Keramaris et al., 1991), or, if it does, then the HZ02 might be concentrated in high amounts, so that the reaction is accomplished without ttie addition of any exogenous H202. The role of peroxidase in this eggshell is quite clear and can be summarized as follows: (1) Elasticity of the particular eggshell is of utmost importance since the egg to be oviposited is stretched inside the ovipositor for a period of 9-34 min (Plaut, 1971). Any other insect eggshell, devoid of resilin-type bonds, would not have withstood such pressure and would have collapsed. The very long and relatively narrow shape of the egg also helps in that direction. (2) While already heing oviposited, this peroxidase layer would have

Fig. 15. Whole mount of a follicle undergoing choriogenesis. filament at the posterior pole(P). The short micropylar filament (A). x140.

Note the flattened end of the is located at the opposite pole

Fig. 16. Thin section at the main body of the follicle shown in Figure 15. The transparent layer is highly electron lucent (TL) while granules are seen above. They will give. in a later stage, the granular layer. The follicle cells show a very high content of rough cndoplasmic reticulum (RER). ~12,000. Fig. 17. Thin section at the long filament of the same follicle. temporally with that of the main body. x 15,000.

Chorion

formatloo

coincides

Flp. 18. High magnification of the newly secreted granules. Their mean dmmeter while very thin filaments seem to withhold the granules (arrowheads). ~SO.009

is 51)OA

Fig. 19. Histochemtcal assay for the detection of peroxidase using DAB + HzOz m an early choriogenic stage. At that penod the transparent layer is formed and appears to he electrondense. Ohserve also the electron-dense vesicles inside the follicle cells (arrows). % 14.000. Fig 20. A mature folhcle after the peroxidase transparent layer appears to be electron-dense.

DAB assay without any exogenous x 14.000.

H,O:. The

MOUZAKI

568

bactericidal properties to protect the developing embryo. A similar process has been reported for the fish Tribolodon hokonensis (Kudo et al., 1988). To function in such a

AND MARGARITIS

way, peroxidase should remain active after oviposition, a hypothesis which cannot fully be supported, yet.

Beament. J. W. L. 1946. The water proofing process in eggs of Rhodnius prolixus (Stahl.). Proc. R. Sot. B. 133, 407418. Brough, C. N. and Dixon, A. F. G. 1990. Ultrastructural features of egg development in oviparae of the vetch aphid Megoura oiciae Buckton. Tissue Cell. 22(l), 51-63. Cassidy, J. D. and King, R. C. 1972. Ovarian development in b’abrobragon jugkmdti (Ashmead) (Hymenoptera: Braconidae). I. The origin and differentiation of the oocyte-nurse cells complex. Biol. Bull.. 143(3), 483-505. Fahimi, H. D. 1970. The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver. .I. Ce[L BioL, 47. 247-262. Furneaux. P. J. S. and Mackay, A. L. 1976. The composition, structure and formation of the chorion and the vitelline membrane in the insect eggshell. In The Insect Znregumenr (ed. H. R. Hepburn), pp. 157-176. Elsevier, Amsterdam. Gaino, E. and Mazzini, M. 1990. Follicle cell activity in the ovarioles of Habroph/ebia eldae (Ephemeroptera: Leptophlebidae). Trans. Am. Microsc. Sot., 109, 300-310. Giorgi. F. and Deri, P. 1976. Cytochemistry of late ovarian chambers of Drosophila melanogasrer. Histochemisrry. 52, 105-117. Griffith, C. M. and Lai-Fook, J. 1986. Structure and formation of the chorion in the butterfly, Calpodes. Trssue Cell. 18(4). 589-601. Kessel. R. G. and Ganion, L. R. 1979. Localization of horseradish peroxidase in the panoistic dragonfly ovary. J. Submicrosc. Cytol., ll(3). 313-324. Kimber, S. J. 1980. The secretion of the eggshell of Scistocerca gregaria: Ultrastructure of the follicle cells during the termination and eggshell secretion. J. Cell. Sci.. 46, 455-477. King. R. C. 1970. Ovarian Development in Drosophila melanogaster. Academic Press, London. N. York. King, R. C. and Buning, J. 1985. The origin and functioning of insect oocytes and nurse cells. In Comprehensive insect Biochemistry, Physiology and Pharmacology (eds. L. I. Gilbert and G. A. Kerkut). Vol. 1. pp. 37-82. Pergamon Press, Oxford, N. York. King, R. C. and Koch. E. A. 1963. Studies on the ovarian follicle cells of Drosophila. Quarr. J. Micr. Sci.. 104, 2Y7320.

Kristensen, N. P. 1981. Phylogeny of insect orders. Annu. Reo. Enromol.. 26, 135-157. Kudo. S.. Sate, A. and Inoue, M. 1988. Chorionic peroxidase activity in the eggs of the fish Tribolodon hokonmcs J. Exp. Zool. 245, 63-70.

Margaritis, L. H. 1985a. Structure and physiology of the eggshell. In Comprehensioe Insecf Biochemisfry, Physiolog! and Pharmacology (eds. L. 1. Gilbert and G. A. Kerkut), Vol. 1. pp X3-230. Pergamon Press. Oxford. N. York. Margaritis, L. H. 1985b. The eggshell of Drosophila melanogaster. III. Covalent crosslinking of the chorion proteins involves endogenous hydrogen peroxide. Tissue CelI, 17, 553-560. Margaritis, L. H. 198%. Comparative study of the eggshell of the fruit flies Dacus oleae and CerorirrJ, capitatu. C‘un J. Zool., 63(9). 21962206. Margaritis, L. H., Kafatos. F. C. and Petri, W. H. 1980. The eggshell of Drosophila melanogasrer. I. Fine structure of the layers and regions of the wild type eggshell. J. Cell Sci., 43, 1-35. Mindrinos, M. N., Petri, W. H.. Galanopoulos, V. G., Lombard, M. F. and Margaritis. L. H. 1980. Crosslinking of the Drosophila chorion involves a peroxidase. Roux’s Arch. Deu. Biol., 189, 187-196. Mouzaki, D. G. and Margaritis, L. H. 1991. The eggshell of the cherry fly Rhagoletis cerasi. Tissue Cell. 23(5), 745754.

Norton, W. N. and Vinson, S. B. 1982. Synthesis of the vitelline membrane and chorionic membranes of an ichneumomc parasitoid. J. Morphol. 174, 185-195. Plaut, H. N. 1971. On the biology of the adult of the almond wasp. Euryroma amygdali End. (Hym.. Eurytomldae), in Israel. Bull. Em. Res., 61, 275-281. Rotheram, S. 1973. The surface of the egg of a parasitic insect. I. The surface of the egg and first-instar larva of Nemeritis. Proc. R. Sot. Land. B., 183, 179194. Smith, D. S., Telfer, W. H. and Neville, A. C. 1971. Fine structure of the chorion of a moth, Hyalophora cecropia. Tissue Cell, 3, 477-498. Vinson, S. B. and Scott, J. R. 1974. Parasitoid eggshell changes in a suitable and unsuitable host. J. Ulrrasrruct. Res.. 47, l-15. Worthington enzyme manual. 1972. Worthington Biochemical Corporation, Freehold. NJ.