A Polydnavirus from the Spruce Budworm Parasitoid,Tranosema rostrale(Ichneumonidae)

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

72, 50–56 (1998)

IN984750

A Polydnavirus from the Spruce Budworm Parasitoid, Tranosema rostrale (Ichneumonidae) Michel Cusson,*,1 Christopher Lucarotti,† Don Stoltz,‡ Peter Krell,§ and Daniel Doucet¶ *Laurentian Forestry Centre, Natural Resources Canada, Canadian Forest Service, P.O. Box 3800, Sainte-Foy, Quebec G1V 4C7, Canada; †Atlantic Forestry Centre, Natural Resources Canada, Canadian Forest Service, P.O. Box 4000, Fredericton, New Brunswick E3B 5P7, Canada; ‡Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3H 4H7,Canada; §Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada; and ¶Department of Biology, Queen’s University, Kingston, Ontario K7L 3N6,Canada Received July 22, 1997; accepted December 16, 1997

cells situated between the ovarioles and lateral oviducts. The nucleocapsids acquire envelopes both before and during release into the lumen of the ovary, where mature virions form the particulate fraction of the calyx fluid (Cxf). During oviposition, Cxf is injected along with eggs into the host caterpillar, where the virus infects various tissues in which some viral genes are expressed. Polydnaviral gene products are believed to be the causal agents of symptoms such as alteration in the behavior of capsule-forming hemocytes, reduction in the number of circulating hemocytes, and inhibition of phenoloxidase (PO) activity in the hemolymph, all of which may contribute to a suppressed immune response toward parasitoid eggs. Other significant effects attributed to PVs include developmental retardation, hormonal disruption, growth reduction, and altered trehalose levels (see Stoltz, 1993). Although similar in terms of genome organization and life cycle, the PVs of braconids and ichneumonids differ significantly with respect to virion morphologies. Braconid PVs (genus Bracovirus) have cylindrical nucleocapsids of varying lengths, with virions made up of one or more nucleocapsids surrounded by a single membrane, whereas those of ichneumonids (genus Ichnovirus) have lenticular nucleocapsids of uniform size, individually surrounded by two membranes (Stoltz, 1993; Stoltz et al., 1995). The ichneumonid wasp Tranosema rostrale (Brischke) is a parasitoid of the spruce budworm, Choristoneura fumiferana (Clemens), in eastern Canada and the United States (Cusson et al., 1998). While T. rostrale Cxf induces developmental retardation in the final host instar (Doucet and Cusson, 1996a), its effects on host immunity appear to be limited. Injection of Cxf into 6th instar budworm larvae prevents the age-dependent increase in the number of hemocytes seen in control larvae and induces an inhibition of PO activity, but this does not appear to reduce the ability of hemocytes to encapsulate foreign objects other than T. rostrale eggs

The calyx epithelium of the campoplegine wasp, Tranosema rostrale, contains typical ichneumonid polydnaviruses (PVs) that display an apparently uncommon association with the egg chorion. The latter structure features fine hair-like projections, longest around the egg’s apices. In the lumen of the ovary, T. rostrale virus becomes lodged between these projections and forms a particulate coat around the egg. In the host, Choristoneura fumiferana, projections and associated virions are observed in close contact with basement membranes of fat body and muscle tissues, to which the eggs rapidly become attached following introduction into the host hemocoel. We discuss the implications of this unusual virus–chorion association in terms of immune protection, delivery of virus to specific host tissues, and the evolution of PVs. r 1998 Academic Press Key Words: Tranosema rostrale; Choristoneura fumiferana; eastern spruce budworm; polydnavirus; parasitism; chorionic projections; electron microscopy; agarose gel electrophoresis.

INTRODUCTION

Parasitic Hymenoptera have evolved various strategies to evade the immune response of their hosts. Perhaps the most intricate and fascinating is that employed by some braconid and ichneumonid wasps capable of abrogating the encapsulation reaction of their hosts through the activities of a polydnavirus (PV) injected at the time of oviposition. These viruses have a polydisperse genome composed of double-stranded circular DNAs of variable molecular mass (Stoltz et al., 1984) derived from linear copies permanently integrated within the wasp genome (Fleming and Krell, 1993). Virion assembly takes place in the nuclei of calyx

1

To whom correspondence should be addressed.

0022-2011/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

50

51

POLYDNAVIRUS OF Tranosema rostrale

and larvae (Doucet and Cusson, 1996b). These observations suggest that T. rostrale eggs evade encapsulation primarily through a passive mechanism. Here we begin to explore an apparently unusual association between T. rostrale PV (TrV), T. rostrale eggs, and host tissues; in addition, we provide preliminary information regarding the nature of the TrV genome. MATERIALS AND METHODS

Insects and parasitization. Spruce budworm hosts and T. rostrale wasps were obtained and reared as described elsewhere (Doucet and Cusson, 1996a,b; Cusson et al., 1998). For parasitization, mated T. rostrale females were provided with 2-day-old last-instar C. fumiferana larvae in a Petri dish and monitored until oviposition occurred. Microscopy. Wasp and host tissues were prepared in a similar fashion except that host (parasitized) larvae were first injected with fixative (2.5% glutaraldehyde containing 0.1 M sucrose in 0.05 M sodium cacodylate buffer, pH 7.4) using a 1-cc, 27-G 1/2 tuberculin syringe. Care was taken not to puncture the gut and fixative was injected into the hemocoel until the insect became fully distended. The syringe was left in place for 30–60 s so that fixative did not leak out of the puncture wound. Each larva was then dissected in fresh fixative under a stereo microscope. Larval pieces with attached parasitoid eggs were then placed into fresh fixative and left for 3 h at 20°C. These were sequentially rinsed for 15 min each in 0.05 M sodium cacodylate buffer, pH 7.4, containing 0.1, 0.05, and 0.025 M sucrose and finally buffer alone. Material was postfixed in 1% osmium tetroxide in 0.05 M sodium cacodylate buffer, pH 7.4, for 2 h at 20°C. Larval tissues were rinsed twice in 0.05 M buffer and once in distilled water, 15 min each, and then en bloc stained overnight at 4°C in 2% aqueous uranyl acetate. Tissues were dehydrated in an acetone series and embedded in Epon–Araldite (Mollenhauer, 1964). Light-microscopy sections, 1.0 µm thick, were cut with a Diatome histoknife and were stained on glass slides in warm 0.1% toluidine blue in 2.5% aqueous sodium carbonate, pH 11.1 (Trump et al., 1961). Ultrathin sections for EM, 70–80 nm thick, were cut using a Diatome diamond knife and were stained in 0.01% lead citrate, followed by 2% aqueous uranyl acetate. Photomicrographs were taken with a Leitz Aristoplan photomicroscope on Ilford XP2 film. Electron micrographs were taken using a Philips 400 electron microscope operating at 80 kV. Staining of wasp eggs. To qualitatively assess the ability of T. rostrale eggs to adhere to internal host tissues, eggs were dissected from two female wasps and stained for 10 s in saturated aqueous methylene blue

followed by three successive 2-min rinses in Pringle’s (1938) saline. The lightly stained eggs were then transferred, in saline, to exposed C. fumiferana internal tissues using a Pasteur pipet, and observed under a stereo microscope. Agarose gel electrophoresis (AGE) of viral DNA. Ovaries from five virgin females were dissected in 50 µl of buffer (100 mM Tris, 10 mM EDTA, pH 8.0), teased open, and macerated with an insect pin. Following addition of 2 µl of proteinase K (10 mg/ml) and 10 µl of 20% sarcosyl, the mixture was incubated for 1 h at 65°C, and incubated for another 15 min following addition of a second 2-µl aliquot of proteinase K. Lastly, 10 µl of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 15% Ficoll) was added to the extract. Samples were electrophoresed at 15 V for 15 h in a 0.6% agarose gel in TAE buffer (without ethidium bromide) using a Mini-Sub Cell (Bio-Rad). A supercoiled DNA ladder (2–16 kb, Gibco) was used to estimate the molecular mass of viral genome segments. To help distinguish superhelical molecules from their relaxed circular equivalents in TrV samples, a small aliquot was boiled for 5 min to denature the open circular DNA; bands remaining after boiling represented supercoiled DNA. RESULTS

Particles in Wasp Ovary and Association with Eggs Immediately following dissection in saline, the ovaries of T. rostrale often appeared to contain little or no Cxf. However, if the eggs were released from the lateral oviducts and allowed to stand in saline for a few minutes, a cloud having the characteristic opalescence of Cxf would form around the eggs, seemingly as a result of slow diffusion of calyx particles away from the egg surface. The latter displayed hair-like projections which seemed to cover most of the egg although they were longest and most apparent around the apices (Fig. 1). Examination of thin sections of T. rostrale eggs by transmission EM revealed the presence of virus-like particles adhering to the egg surface and apparently lodged between the chorionic projections (Fig. 2). At higher magnification, the particles were seen to be lined up between the projections (Fig. 3). Electron micrographs of cross-sections through the calyx region of the ovary showed particles with features typical of ichnoviruses (Fig. 4). The lenticular particles are assembled in the nuclei of calyx cells where they acquire an initial (inner) membrane. They then migrate through the cytoplasm and bud out into the lumen of the ovary, at which time they acquire a second envelope (outer membrane). In the oviduct, the particles become associated with chorionic projections of

52

CUSSON ET AL.

FIG. 1. Photomicrographs of T. rostrale eggs immediately following dissection of an ovary in saline. (a) Single egg. (b) Group of eggs. Note the presence of chorionic hair-like projections on the egg surface, most apparent around the apices (arrows), and the cloud (i.e., virions; arrowhead) diffusing away from the eggs. Bars 5 0.1 mm. FIG. 2. Electron micrograph showing T. rostrale chorion and chorionic hair-like projections. Note how virions (arrowheads) coat the surface of the egg. Bar 5 2.5 µm. FIG. 3. Electron micrograph showing positioning of T. rostrale virions (arrowheads) between chorionic hair-like projections (arrow). Bar 5 0.5 µm. FIG. 4. Electron micrograph showing T. rostrale virions (small arrowheads) in calyx cell nucleus (n), in the cytoplasm (c), and lodged between chorionic hair-like projections (arrowhead) of an egg (e) after budding out into the lumen of the ovary (arrow). Bar 5 1 µm. FIG. 5. Photomicrograph of a cross-section through a T. rostrale ovary showing calyx epithelium with nucleus (n) and three eggs (e) in the lumen. Note chorionic hair-like projections (arrowhead). Bar 5 25 µm.

53

POLYDNAVIRUS OF Tranosema rostrale

nearby eggs; this association may be facilitated by an apparent proximity of eggs to the calyx epithelium (Figs. 4 and 5). Extraction and Electrophoretic Analysis of DNA from Calyx Fluid Extracts of T. rostrale Cxf contained two sets of eight clearly identifiable DNA bands which, by comparison with published PV AGE profiles, are assumed to represent open circular and supercoiled DNA species ranging in size between ca. 5 and 10 kb (Fig. 6). Confirmation that bands A to H are supercoiled species was provided by AGE analysis of boiled DNA samples, in which only eight bands remained; these bands could be clearly aligned with bands A to H (data not shown). This analysis, along with the morphological characteristics described above, indicates that T. rostrale particles are true ichneumonid PVs, with a genome comprising at least eight DNA segments; as with other ichnoviruses, several submolar genome segments are expected to be present. Eggs and PVs in the Lepidopteran Host In C. fumiferana larvae, T. rostrale eggs were always found in close association with host tissues, and chori-

FIG. 6. Agarose gel electrophoresis of TrV DNA. Lanes 1 and 2 were loaded with ca. 5 and 1 female equivalents of TrV, respectively. Lane M contains supercoiled molecular weight markers (kb). Bands labeled with lower case and upper case letters have been identified as open circular and supercoiled species, respectively. See text for details on electrophoresis conditions.

onic projections usually extended right to these tissues (typically fat body, muscle, and midgut; Fig. 7). Approximately 1 h after parasitization, virions adhering to the egg surface were seen in direct contact with host basal lamina (Fig. 8). Viral nucleocapsids were also observed to gain entry to the cytoplasm (Figs. 9–12) and nuclei (Fig. 13) of host cells as described for other ichnoviruses (e.g., Stoltz and Vinson, 1979). Because T. rostrale eggs were never found freefloating in the host’s hemocoel, we tested the hypothesis that the eggs’ surface properties are such that they adhere strongly to host tissues, possibly to facilitate delivery of PVs to host cells. In order to improve detection of the eggs inside the host, we first stained them with aqueous methylene blue. The eggs were then transferred with a Pasteur pipette to exposed host tissues such as fat body and midgut where they were observed to immediately become attached (within 5 s). These egg–tissue associations were made particularly apparent by the peristaltic movement of the gut in the freshly decapitated larva, as the attached eggs moved in synchrony with the tissue. DISCUSSION

The calyx epithelium of female T. rostrale contains and releases particles whose morphology and DNA electrophoretic profile are similar to those of previously described ichnoviruses (Stoltz et al., 1995), with genomic segments in approximately the same size range as reported for other ichneumonid PVs (Stoltz et al., 1981). However, once in the lumen of the ovary, TrV becomes associated with the egg chorion in a fashion that has not been documented before for true PVs. Although the eggs of other well-known PV-carrying wasps feature chorionic projections, these are usually much shorter than the ones seen on the eggs of T. rostrale, and virions do not become bound to the egg surface (e.g., Campoletis sonorensis, Norton and Vinson, 1977; Cardiochiles nigriceps, Vinson and Scott, 1974). The PV–chorion association described here for T. rostrale most closely resembles the association observed between the chorion and the virus-like particles (VLPs) of the ichneumonid Venturia canescens (Rotheram, 1967, 1973a), with the exception that VLPs do not contain DNA and cannot, therefore, be considered PVs (Bedwin, 1979). In both T. rostrale and V. canescens, the chorionic projections are short over the sides of the eggs but long around the ends, and particles become lodged between the projections to form a coat on the egg surface (see Rotheram, 1973a). In V. canescens, chorion-bound VLPs have been shown to confer passive protection to the egg against encapsulation. Apparently, some VLP structural proteins share antigenic determinants with a protein from the host Ephestia kuehniella, thus preventing the eggs from

54

CUSSON ET AL.

FIG. 7. Photomicrograph of T. rostrale egg (e) in hemocoel of C. fumiferana host ca.1 h after parasitization. Chorionic hair-like projections (arrowhead) extend toward host tissues. Bar 5 50 µm. FIG. 8. Electron micrograph showing portion of T. rostrale chorion (c) and chorionic hair-like projections (cp) with virions (arrowheads) extending toward C. fumiferana host tissue (h) (1 h after parasitization). Bar 5 1 µm. FIG. 9. Electron micrograph of T. rostrale virions (arrowheads) passing through the basal lamina of a C. fumiferana tracheole epithelial cell. Bar 5 0.5 µm. FIG. 10. Electron micrograph of T. rostrale virion entering C. fumiferana host cell. Note virion ‘‘tail’’ (arrowhead). Bar 5 0.2 µm. FIG. 11. Electron micrograph of T. rostrale virion entering C. fumiferana host cell. Note the basal lamina. Bar 5 0.2 µm. FIG. 12. Electron micrograph showing two T. rostrale virions within a C. fumiferana host cell. Note the plasma-membrane-derived envelope (arrowheads) around the virions. Bar 5 0.2 µm. FIG. 13. Electron micrograph of a T. rostrale nucleocapsid (arrow) within a C. fumiferana cell nucleus (n); c: cytoplasm. Bar 5 0.2 µm.

being recognized as foreign by the host (Theopold et al., 1994). It is possible that the PV coating of T. rostrale eggs plays a similar protective role since TrV infection in C. fumiferana does not prevent encapsulation of foreign objects other than T. rostrale eggs and larvae; notably, hemocyte behavior does not appear to be affected by TrV infection, although the host immune system is compromised in some other ways (e.g., inhibition of PO activity and inhibition of the rise in total hemocyte counts normally observed in healthy larvae; Doucet and Cusson, 1996b). The projections alone may also be involved in confer-

ring some level of immune protection inasmuch as ‘‘fibrous layers’’ on the surface of other parasitoid eggs have been shown to provide temporary protection against encapsulation, possibly during the interval between oviposition and the beginning of active immune suppression by putative polydnaviral gene products or other factors transmitted by the parasitoid to the host (Davies and Vinson, 1986; Asgari and Schmidt, 1994). In this context, it would be of interest to monitor the fate of both chorionic projections and associated PVs during the 3-day interval between parasitization and hatching of T. rostrale eggs in C. fumiferana larvae.

55

POLYDNAVIRUS OF Tranosema rostrale

Our observations on the rapid adherence of T. rostrale eggs to host tissues following their introduction into the host’s hemocoel suggest another function for the virus–chorion association: delivery of PVs to specific host tissues. Electron micrographs of T. rostrale eggs in the hemocoel of C. fumiferana have indeed shown virions at the egg–basement membrane interface (Fig. 8), and TrVs have been observed to pass through basement membranes and to gain entry into fat body and muscle cells (Figs. 9–13). The eggs of the braconid parasitoid Asobara tabida, which attacks larvae of several Drosophila species, have also been observed to stick to host tissues (Mollema, 1988). In this case, however, the ‘‘stickiness’’ apparently helps the egg avoid complete encapsulation by protecting surfaces embedded in host tissue from circulating hemocytes (Kraaijeveld and van Alphen, 1994). Whether a similar mechanism is instrumental in protecting T. rostrale eggs from encapsulation remains to be determined. In most other host–parasitoid systems examined to date, however, the eggs and PVs are free-floating in the host hemocoel, a condition which may be necessary for rapid infection of capsule-forming hemocytes (Strand, 1994). In the case of T. rostrale, infection of hemocytes may not be vital for avoidance of encapsulation (see above), but the implication of T. rostrale Cxf in host developmental disruption (Doucet and Cusson, 1996a) may require that virions infect specific tissues such as fat body, to which most eggs were found to be closely associated during dissection of host larvae. Given that the VLPs of V. canescens display many ichnovirus-like features, including particle morphology and a common site of particle genesis and assembly (Rotheram, 1973a,b; Schmidt and SchuchmannFeddersen, 1989), a detailed comparison of these particles with those of T. rostrale may provide new clues as to how PVs have evolved. Speculations on the origin of PVs have generated a few hypotheses, one of which— perhaps the most attractive—suggests that they have evolved from preexisting viruses (Stoltz and Whitfield, 1992; Whitfield, 1990). In this context, the similarities between V. canescens and T. rostrale, in terms of their calyx particles and how these are associated with the eggs, lend some support to the hypothesis that the VLPs of V. canescens are former PVs that have ‘‘lost’’ their genetic material (Schmidt and SchuchmannFeddersen, 1989), with the latter no longer being required for successful parasitization. Presumably, the immune protection provided by the particulate coat on the egg is all that the parasitoid needs to successfully develop in its habitual hosts. In the case of T. rostrale, although the encapsulation avoidance strategy may be related to that of V. canescens, the parasitoid apparently needs to alter host physiology in some additional ways (e.g., induce developmental arrest) in order to

successfully parasitize its host. In this respect, V. canescens should perhaps be considered a ‘‘conformer’’ (Harvey and Thompson, 1995; Harvey, 1996) while T. rostrale would be best described as a ‘‘regulator,’’ hence the necessity for T. rostrale to maintain a set of polydnaviral genes to effect other alterations of host physiology. It would now be of value to examine other wasp species that are very closely related to either T. rostrale or V. canescens [these two species may, themselves, be closely related to one another as suggested by their earlier assignment to a common genus (Campoplex); Cusson et al., 1998; Salt, 1976] to establish whether other wasps share their characteristic PV/VLP–chorion association and whether or not the particles contain DNA. In addition, efforts should be made to determine if V. canescens VLPs and T. rostrale virions share antigenic determinants. Such an approach may shed new light on the evolution of PVs. ACKNOWLEDGMENTS We thank D. Trudel and M. Bolduc for their assistance in rearing the insects. This research was supported by the ‘‘Green Plan’’ Program (Government of Canada). REFERENCES Asgari, S., and Schmidt, O. 1994. Passive protection of eggs from the parasitoid, Cotesia rubecula, in the host, Pieris rapae. J. Insect Physiol. 40, 789–795. Bedwin, O. 1979. An insect glycoprotein: A study of the particles responsible for the resistance of a parasitoid’s egg to the defense reactions of its insect host. Proc. R. Soc. Lond. B. 205, 271–286. Cusson, M., Barron, J. R., Goulet, H., Re´gnie`re, J., and Doucet, D. 1998. Biology and status of Tranosema rostrale (Hymenoptera: Ichneumonidae), a parasitoid of the eastern spruce budworm (Lepidoptera: Tortricidae). Ann. Entomol. Soc. Am. 91, 87–93. Davies, D. H., and Vinson, S. B. 1986. Passive evasion by eggs of braconid parasitoid Cardiochiles nigriceps of encapsulation in vitro by haemocytes of host Heliothis virescens. Possible role for fibrous layer in immunity. J. Insect Physiol. 32, 1003–1010. Doucet, D., and Cusson, M. 1996a. Alteration of developmental rate and growth of Choristoneura fumiferana parasitized by Tranosema rostrale: role of the calyx fluid. Entomol. Exp. Appl. 81, 21–30. Doucet, D., and Cusson, M. 1996b. Role of calyx fluid in alterations of immunity in Choristoneura fumiferana larvae parasitized by Tranosema rostrale. Comp. Biochem. Physiol. 114A, 311–317. Fleming, J.-A. G. W., and Krell, P. J. 1993. The polydnaviruses: Multipartite DNA viruses from parasitic Hymenoptera. In ‘‘Viruses of Invertebrates’’ (E. Kurstak, Ed.), pp. 141–177. Marcel Dekker, New York. Harvey, J. A. 1996. Venturia canescens parasitizing Galleria mellonella and Anagasta kuehniella: Is the parasitoid a conformer or a regulator? J. Insect Physiol. 42, 1017–1025. Harvey, J. A., and Thompson, D. J. 1995. Developmental interactions between the solitary endoparasitoid Venturia canescens (Hymenoptera: Ichneumonidae), and two of its hosts, Plodia interpunctella and Corcyra cephalonica (Lepidoptera: Pyralidae). Eur. J. Entomol. 92, 427–435. Kraaijeveld, A. R., and van Alphen, J. J. M. 1994. Geographical variation in resistance of the parasitoid Asobara tabida against encapsulation by Drosophila melanogaster larvae: the mechanism explored. Physiol. Entomol. 19, 9–14.

56

CUSSON ET AL.

Mollema, C. 1988. ‘‘Genetical Aspects of Resistance in a HostParasitoid Interaction.’’ Ph.D. thesis, University of Leiden, The Netherlands. Mollenhauer, H. R. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111–114. Norton, W. N., and Vinson, S. B. 1977. Encapsulation of a parasitoid egg within its habitual host: An ultrastructural investigation. J. Invertebr. Pathol. 30, 55–67. Pringle, J. W. S. 1938. Proprioception in insects. J. Exp. Biol. 15, 101–103. Rotheram, S. 1967. Immune surface of egg of a parasitic insect. Nature 214, 700. Rotheram, S. 1973a. The surface of the egg of a parasitic insect. I. The surface of the egg and first-instar larva of Nemeritis. Proc. R. Soc. Lond. B. 183, 179–194. Rotheram, S. 1973b. The surface of the egg of a parasitic insect. II. The ultrastructure of the particulate coat on the egg of Nemeritis. Proc. R. Soc. Lond. B. 183, 195–204. Salt, G. 1976. The hosts of Nemeritis canescens, a problem in the host specificity of insect parasitoids. Ecol. Entomol. 1, 63–67. Schmidt, O., and Schuchmann-Feddersen, I. 1989. Role of virus-like particles in parasitoid-host interaction of insects. In ‘‘Subcellular Biochemistry,’’ Vol. 15 (J. R. Harris, Ed.), pp. 91–119. Plenum Press, New York. Stoltz, D. B. 1993. The polydnavirus life cycle. In ‘‘Parasites and Pathogens of Insects,’’ Vol. 1, ‘‘Parasites’’ (N. E. Beckage, S. N. Thompson, and B. A. Federici, Eds.), pp. 167–187. Academic Press, New York. Stoltz, D. B., Beckage, N. E., Blissard, G. W., Fleming, J. G. W., Krell, P. J., Theilmann, D. A., Summers, M. D., and Webb, B. A. 1995.

Family Polydnaviridae. In ‘‘Virus Taxonomy’’ (F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo, and M. D. Summers, Eds.), pp. 143–147. Springer-Verlag, New York. Stoltz, D. B., Krell, P. J., and Vinson, S. B. 1981. Polydisperse viral DNA’s in ichneumonid ovaries: A survey. Can. J. Microbiol. 27, 123–130. Stoltz, D. B., and Vinson, S. B. 1979. Penetration into caterpillar cells of virus-like particles injected during oviposition by parasitoid ichneumonid wasps. Can. J. Microbiol. 25, 207–216. Stoltz, D. B., and Whitfield, J. B. 1992. Viruses and virus-like entities in the parasitic Hymenoptera. J. Hym. Res. 1, 125–139. Stoltz, D. B., Krell, P. J., Summers, M. D., and Vinson, S. B. 1984. Polynaviridae—A proposed family of insect viruses with segmented, double-stranded, circular DNA genomes. Intervirology 21, 1–4. Strand, M. R. 1994. Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes. J. Gen. Virol. 75, 3007–3020. Theopold, U., Krause, E., and Schmidt, O. 1994. Cloning of a VLP-protein coding gene from a parasitoid wasp Venturia canescens. Arch. Insect Biochem. Physiol. 26, 137–145. Trump, B. F., Smucker, E. A., Edward, A., and Benditt, E. P. 1961. A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5, 343–348. Vinson, S. B., and Scott, J. R. 1974. Parasitoid egg shell changes in a suitable and unsuitable host. J. Ultrastruct. Res. 47, 1–15. Whitfield, J. B. 1990. Parasitoids, polydnaviruses and endosymbiosis. Parasitol. Today 6, 381–384.