An unusual virus from the parasitic wasp Cotesia melanoscela

VIROLOGY

162,311-320(1988)

An Unusual Virus from the Parasitic DONALD B. STOLTZ,**‘PETER

Wasp Cotesia melanoscela

KRELL,t DOUG COOK,* EDMUND A. MAcKINNON,+

AND

C. J. LUCAROTTlg

*Department of Microbiology, Dalhousie University. Halifax, Nova Scotia B3H 4H7; tDepartment of Microbiology, University of Guelph, Guelph, Ontario NlG 2Wl; *Department of Anatomy, Queen’s University, Kingston, Ontario K7L 3N6; and SDepartment of Biology, Mt. St. Vincent University, Halifax, Nova Scotia, Canada B3M 216 Received August

17, 1987; accepted

October

14, 1987

Certain strains of the braconid parasitoid Cofesia melanoscela carry two different viruses within their ovaries, one of which (here designated CmV2) is apparently not a polydnavirus. Virus replication occurs in the ovarian calyx and in some other tissues of both male and female parasitoids; as yet, no replication has been observed in the testis, however. In addition, CmV2 is one of only two parasitoid viruses known to replicate in host insect larvae, and we now show that this virus is also capable of replicating in vitro; the virus is nevertheless nonpathogenic for gypsy moth larvae. The virus is not transmissible per OS, either to host animals or to larvae of parasitoid strains lacking it. CmV2 is stably maintained within strains carrying it apparently by a vertical transmission mode involving the maternal line; transmission via the male germ line could not be demonstrated. While purification of the virus was not achieved, preliminary work allows us to suggest that the genome consists of a single double-stranded DNA molecule of approximately 125 kb. o 1999 Academic PWB. hc.

Stoltz et a/., 1984), and it was therefore assumed that this virus was indeed very likely a polydnavirus. When initially described, CmV2 was seen to coexist with a typical braconid polydnavirus (bpv) (Stoltz and Faulkner, 1978); this is by no means unusual, since other examples are known in which females of certain species harbor two different viruses (Stoltz and Vinson, 1979a; Hamm and Styer, 1985). Unfortunately, both our C. melanoscela colony and that from which it originated were lost in 1980. A colony subsequently established in Connecticut from field-collected material (R. M. Weseloh, Connecticut Agricultural Experiment Station, personal communication) carried only the bpv particle. An examination of material collected from various locations in the United States, Canada, Korea, and Russia failed to provide evidence for CmV2 particles in C. melanoscela populations until 1984, when it was rediscovered in wasps parasitizing satin moth larvae near Truro, Nova Scotia; more recently, a similar virus has been detected in a wasp population originating from France. It is now recognized that several “strains” of C. melanoscela exist, each of which carries a bpv variant (manuscript in preparation): however, only the Truro and French populations are known to contain both a braconid polydnavirus and CmV2. In this paper, we describe some recent studies with CmV2. In particular, we provide new information relating to the nature of the genome (apparently not polydnavirus-like), replication in vitro, and transmission within parasitoid populations.

INTRODUCTION Viruses of parasitic insects include the polydnaviruses (Stoltz er a/., 1984), a family of viruses having polydisperse DNA genomes (Krell and Stoltz, 1979, 1980; Stoltz et a/., 1981; Krell et a/., 1982) and several other as yet uncharacterized agents (e.g., Hess et al., 1980; Stoltz, 1981; Stoltz et al., 1981; Hamm and Styer, 1985). Most of the known parasitoid viruses are assumed to share a common life cycle: replication in the parasitoid ovary (in a//females of affected species), secretion into the oviduct, injection into host insects during oviposition, and entry of viral DNA into host cell nuclei (Stoltz and Vinson, 1979a). While the replication of polydnaviruses is thought to occur only within the wasp ovary, this may not be the case for other parasitoid viruses: these include an unusual virus (here designated CmV2) from the gypsy moth parasitoid Cotesia melanoscela (Stoltz and Faulkner, 1978), the subject of the present report, and an uncharacterized virus from C. marginiventris (Hamm and Styer, 1985). As has been previously shown (Stoltz and Faulkner, 1978), the CmV2 virion consists of a large quasicylindrical nucleocapsid surrounded by two unit membranes; this is, in short, the typical morphology of ichneumonid polydnaviruses (Stoltz and Vinson, 197913;

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MATERIALS

AND METHODS

Insects The parasitoid used in our studies was Cotesia (formerly Apanteles) melanoscela, several interbreeding strains of which are now recognized (Table 1). Strains CT-l and -2 were supplied by R. M. Weseloh, Connecticut Agricultural Experiment Station; Korean (KO) and French (FR) strains were provided by L. D. Rhoads, Pennsylvania Bureau of Forestry; strain TR is from material collected near Truro, Nova Scotia. Adult wasps were maintained on a diet of honey and water. Parasitoid strains were routinely reared in the laboratory from larvae of the gypsy moth, Lymantria dispar, a nondiapausing colony of which was maintained on an enriched wheat-germ diet as described (Bell et a/., 1981). Larvae of the white-marked tussock moth, Orgyia leucostigma, and the fall web-worm, Hyphantria cunea, were used in some experiments; these were reared on the same diet. Cells The IPLB-LD-652Y line of L. dispar cells was provided by E. M. Dougherty, U.S.D.A. Insect Pathology Laboratory (Beltsville, MD); this line was originally established from pupal ovaries (R. H. Goodwin, personal communication). The Estigmene acrea hemocyte cell line (BTI EAA) was obtained from R. R. Granados, Boyce Thompson Institute. Both cell lines were grown at 28” in 30 ml LUX tissue culture flasks in TC-100 medium (KC Biologicals) supplemented with 8% fetal bovine serum. In order to establish an initial in vitro infection, four female TR wasps were rinsed in 3% Nonidet-P40 for 30 set, absolute ethanol for 10 set, and finally washed in sterile distilled water. Ovaries TABLE 1

Cotesia melanoscela POPULATIONSUSED’

Origin Connecticut Connecticut Truro, Nova Scotia, Canada Russia Korea* France

Strain designation

Virus(es) carried

CT-l CT-2

bpv @raconid polydnavirus)la3 bpvlb -

TR RU KO FR

bpvlc and CmV2 bpvld bpvle bpvlf and CmV2

a For the purposes of this paper, populations are regarded as equivalent to strains. *The KO strain is regarded by one expert as possibly a different species (W. R. M. Mason, personal communication), but in our hands readily interbreeds with CT-l, CT-2, and TR (RU not done); KO might perhaps more appropriately be referred to as a subspecies.

were dissected out under aseptic conditions, and ovarian calyx fluid (crude virus) was released into 1 ml of complete tissue culture medium. This was applied to a monolayer of LD-652Y cells and left to adsorb for 3 hr with occasional agitation, after which 1.5 ml of fresh medium was added. Characterization

of the genome

Our primary goal was to determine whether or not the genome was polydisperse, as determined by gel electrophoresis; subsequently, we wished to determine its approximate size. Unfortunately, despite repeated attempts, we were unable to purify CmV2 particles; DNA extracted from infected fat body was therefore examined using standard procedures. Briefly, fat bodies were excised into 49/o sodium lauryl sarcosinate in SSCE (SSCE is 0.15 M NaCI, 0.015 M sodium citrate, 0.01 M EDTA, pH 7.5) and heated at 60” for 1 hr; at this point, samples were often stored at -20”. Following lysis with detergent, samples were digested for 2 hr at 30” with proteinase K (300 pg/ml), extracted twice with phenol:chloroform:isoamyl alcohol (50:49:1), and finally precipitated under ethanol. Viral DNA was extracted from infected cell cultures in the same manner. DNA samples were typically analyzed by electrophoresis in 0.8% agarose gels in TEA buffer (40 mM Tris, 5 mM sodium acetate, 1 mM EDTA, pH 8.0) containing 0.5 pg/ml ethidium bromide. Transmission Our primary concerns in this regard were to determine whether TR and FR males carried CmV2 DNA and, if so, could transmit it during fertilization to strains lacking this virus. In order to carry out this portion of the study, it was deemed necessary to clone some viral DNA sequences so that appropriate hybridization probes could be utilized, but no attempt was made to generate a complete genomic library. Infected fat body DNA (from TR-parasitized larvae) was digested with a variety of restriction endonucleases and cloned into either pBR325 or pUCl9, and then propagated in Escherichia co/i strains RR1 and TBl, respectively, using standard protocols. Recombinant colonies were subjected to a double screening protocol in which duplicate filters were hybridized with 32P-labeled DNA from infected or control fat body tissue; colonies reacting only with the former probe were assumed and subsequently found to carry virus-specific sequences. After electrophoresis, male DNA was blotted bidirectionally onto nitrocellulose (Smith and Summers, 1980). Hybridization vs 32P-labeled CmV2 sequences was for 12-96 hr at 42’ in 4X SET buffer (SET is 0.15 M NaCI, 0.03 MTris, 0.001 M EDTA, pH 8.0) contain-

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ing 50% formamide, 100 pg/ml calf thymus DNA, 50 pg/ml of heparin, and 0.2% SDS. Filters were subsequently washed for 2 hr in hybridization buffer, followed by two 1-hr washes in 2x SET containing 2x Denhardt’s solution, 50 pg/ml of heparin, 0.2% SDS, and 100 pg/ml of calf thymus DNA. Filters were air-dried for 1 hr prior to autoradiography using Kodak X-Omat AR film and DuPont Cronex intensifying screens. In order to determine whether males could transmit CmV2 DNA, both TR and FR males were crossed with females belonging to strains CT-l, CT-2, KO, and RU, all of which lack CmV2; F, hybrid females from two of these crosses (using CT-l or KO as the female parent) were further back-crossed for several generations to Truro males. The presence or absence of CmV2 in hybrid females was determined simply by examining the hemocytes and fat body of parasitized larvae for the presence of viral inclusion bodies: CmV2-positive females invariably transmit infectious virus to parasitized host larvae (Stoltz and Faulkner, 1978) and it was assumed that any hybrid females carrying the virus would do likewise. In order to determine whether parasitoid larvae could be infected per OS, we injected host larvae with CmV2-containing calyx fluid and then parasitized the same larvae with either CT-l or KO females. The rationale for this was that, well before reaching maturity, parasitoid larvae would almost certainly consume some CmV2-infected hemocytes and free-floating fat body cells, as well as extracellular virus particles. Adult females reared from these hosts were allowed to parasitize other hosts, which were subsequently examined cytologically for the presence of viral inclusion bodies. RESULTS’

It was known from previous work that CmV2 replicates in the ovarian calyx (Stoltz and Faulkner, 1978) and this has been confirmed in the case of the currently available isolates from TR and FR parasitoids. We have now observed virus replication in a small number of female muscle and tracheal epithelial cells (Fig. 1); in addition, virus particles have occasionally been seen in association with what appeared to be lysed hemocytes in both male and female parasitoids (not shown). Replication was not observed in the following parasitoid tissues: epidermis, testis, and midgut; other tissues have not as yet been examined. ’ Unless otherwise indicated, results are from experiments ing only the TR population of C. melanoscela.

In an earlier report, it was noted that CmV2 also replicates in both hemocytes and fat body of host 0. leucostigma larvae, with other tissues remaining unaffected (Stoltz and Faulkner, 1978); it was initially thought that fat body cells were only occasionally infected. Observations made since then clearly suggest that the fat body is a major site of replication in all permissive hosts (see below) examined thus far (Fig. 2). Furthermore, it now seems certain that the hypertrophied “hemocytes” described previously were in fact free-floating fat body cells released upon partial disruption of fat body basement membrane. The ability of CmV2 to replicate in several additional hosts parasitized by the Truro strain of C. melanoscela was investigated. Specifically, virus replication occurs in the fat body and hemocytes of the following species: 0. leucostigma, Sfilpnotia salicis (satin moth), E. acrea (salt marsh caterpillar), L. dispar, and H. cunea. Virus replication was not observed in larvae of the tent caterpillar, Malacosoma disstria, even after manual injection of calyx fluid. Injection of calyx fluid into L. dispar larvae failed to elicit any symptoms of overt disease, even though viral inclusion bodies were invariably observed; similar observations were made using 0. leucostigma larvae (other host species were not examined). Inclusion bodies were detected in the hemocytes of several adult female gypsy moths which had been injected as larvae with calyx fluid from TR wasps; larvae hatching from eggs laid by such moths contained no inclusion bodies, and appeared to be free of virus. Materials containing CmV2 particles (calyx fluid; infected fat body or tissue culture cells) were not infectious per OS for gypsy moth larvae. The question of per OS transmission to parasitoid larvae is considered below (see Transmission). Viral replication

General observations

involv-

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in vitro

Our primary interest here was simply to determine whether CmV2 would in fact undergo replication in vitro, with the eventual aim of investigating the feasibility of plaque purification. Replication was readily observed in cell lines derived from both L. dispar and f. acrea (IPLB-LD-652Y and BTI EAA, respectively); most of our subsequent studies utilized the L. dispar line. Cytopathic effects included rounding up and hypertrophy, and the formation of nuclear inclusion bodies (Fig. 3, inset). Electron microscopic studies (Fig. 3) revealed the presence of an apparently typical viral morphogenesis, which included budding through the inner nuclear membrane, and the eventual release of extracellular virions surrounded by two unit membranes; these observations parallel those reported earlier for in viva replication (Stoltz and Faulkner,

STOLTZ ET AL.

FIG. 1. CmV2 replication in female nonovarian epithelium. mg, midgut epithelial cell cytoplasm.

tissues. (A) lntranuclear virus particles are observed in a muscle cell associated (6) Virus is present in a tracheal epithelial cell. t, lumen of a tracheole.

with midgut

UNUSUAL

FIG.2. The fat body is a site of virus replication. all three nuclei shown in the micrograph.

(A) Uninfected

PARASITOID

L. disparfat

1978). The virus could be readily passaged in vitro by adding medium from an infected culture to an uninfected one: this medium was also infectious in viva, when injected into the hemocoele of third or fourth instar L. dispar larvae. Characterization

of the genome

As mentioned above, all attempts to purify CmV2 particles from either infectious hemolymph or tissue cultures met with failure; therefore, a preliminary attempt was made to characterize viral DNA as it occurs in infected cells. On the basis of particle morphology, it was initially assumed that the genome would consist of a polydisperse population of double-stranded, circular, DNA molecules, which should show up as such following gel electrophoresis of DNA extracted from infected tissues. To the contrary, our studies revealed no perceptible differences between agarose gel electrophoretic profiles of control vs infected host fat body DNA; this suggested to us that the CmV2 genome might in fact be a large molecule, possibly comigrating with chromosomal DNA. In order to test this hypothesis, DNA samples were analyzed after digestion with several different restriction endonucleases; typical results are shown in Fig. 4. As expected, digestion of control fat body DNA resulted in a background smear with no discrete DNA bands; digestion of CmV2-infected fat body DNA, on the other hand, produced a

VIRUS

body. (B) Infected L. disparfat

315

body; inclusion bodies are present in

consistent pattern of bands, comprising an aggregate molecular weight of approximately 125 kb pairs. Visual inspection of such gels suggested that viral DNA may constitute 50% or more of the total nucleic acid present in infected fat body tissue. An apparent comigration of viral and fat body chromosomal DNA was subsequently confirmed by Southern blotting (Fig. 5). Submolar bands appeared in EcoRI, Pstl, and Hindlll digests, but were excluded from the molecular weight calculations; further work will be required in order to determine whether these are a reflection of genomic heterogeneity. Electrophoretic profiles of viral DNA extracted from infected cell cultures were indistinguishable from those seen with infected fat body (not shown). Transmission Observations extending over a period of several years clearly indicate that all female TR wasps carry this virus (we have only recently acquired the FR strain); this conclusion is based primarily upon the invariable presence of nuclear inclusion bodies in parasitized host larvae. It has seemed reasonable to assume, in the light of recent work on polydnaviruses (Fleming and Summers, 1986; Stoltz et al,, 1986) that CmV2 too may be transmitted vertically via the germ line. It was therefore of interest to see whether CmV2 DNA was present in male parasitoids, and if so

STOLTZ ET AL.

FIG. 3. Vir‘us replication in IPLB-LD-652Y cells. lntranuclear virus replication is shown at low magnification in (A); a cell with a prominent nuclear inck Jsion body, as seen at the light microscopic level, is shown in the inset. Virus particles bud through the inner nuclear membrane (B) and subseq uently enter the lumen of the rough endoplasmic reticulum (C). Ultimately, particles exit infected cells by budding through the plasma mer nbrane (D).

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456

FIG. 4. Visualization of the viral genome. DNA was extracted from control or virus-infected fat body, digested with restriction endonucleases, and run into a 0.8% agarose gel. Lane 1, control L. dispar fat body DNA, EcoRI; lane 4, BRL 1-kb ladder (several other gels incorporated high-molecular-weight markers to 48.5 kb); lanes 2, 3, 5, and 6, DNA extracted from infected L. dispar tissue and cut with Sstl, WI, HindIll, and EcoRI, respectively. Approximately 400 ng DNA was loaded in each lane. The indicated 4.0.kb Pstl and 3.0-kb HindIll fragments have been cloned, and are used as probes in Figs. 5 and 6, respectively.

whether males could transmit this DNA via sperm cells to diploid female progeny. In keeping with our preliminary electron microscopic observations, which strongly suggested that some virus replication occurs in males, CmV2 DNA was readily detected in dot blots of total male wasp DNA probed with cloned viral DNA sequences derived from infected fat body DNA (Fig. 6); following restriction endonuclease digestion and Southern blotting, viral DNA bands in male parasitoids were always seen to comigrate with homologous sequences from infected fat body (Fig. 6). Thus it was reasonable to consider the possibility that, as with polydnaviruses, CmV2 DNA might be transmitted within sperm cells. Since viral inclusion bodies are always present in larvae parasitized by CmV2-carrying females, we assumed that the same would apply in the case of females from other strains if they were to pick up CmV2 as a consequence of vertical transmission through male TR or FR germ plasm. In order to assess this possibility, F, female progeny from KOa x TR$ and similar crosses were allowed to parasitize host larvae, which were subsequently examined for the presence of inclusion bodies. These were never detected unless the female parent used in an F, cross was from the TR or FR strain (in

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317

which case, as expected, all females carried the virus). Similarly, no evidence for transmission of virus or viral DNA by TR males to strains lacking this virus was observed even after repeated backcrossing (four generations) to TR males. Furthermore, we were unable to demonstrate transmission of viral DNA between the CmV2-carrying TR and FR strains; this experiment was made possible by the discovery of minor differences in the restriction fragment profiles of CmV2 isolates from these two strains (Fig. 7). In a preliminary study, we attempted to look for CmV2 DNA in similar F, females (e.g., from a KO? x TR$ cross) by means of Southern blot hybridization. However, it soon became evident that even in the case of known CmV2 carriage, there was too little virus in the ovary to consistently generate a hybridization signal. In separate experiments (see Materials and Methods), we attempted to infect both CT-l and KO larvae by rearing them in host larvae previously injected with CmV2-containing calyx fluid. CmV2 transmission to host larvae could not be detected in any parasitoid females derived from such experiments (10 of each examined). Transmission of this virus to host larvae would appear to occur in nature solely as a consequence of

1

2

3

4

5

6

1

2

3

45

6

FIG. 5. Apparent comigration of viral and fat body chromosomal DNA. (A) DNAs are shown after gel electrophoresis and ethidium bromide staining. (B) A blot of this gel is shown probed with the indicated CmV2 fstl fragment (4.0 kb). Note that the 4.0-kb probe develops a hybridization signal only at the position of host chromosomal DNA in lane 1. Lane 1, DNA from infected L. dispar fat body; 2, polydisperse DNA from TR calyx fluid. It should be kept in mind that TR calyx fluid consists almost entirely of braconid polydnavirus, containing only a few CmV2 virions; 3, DNA from infected L. dispar fat body cut with Psrl; 4, calyx fluid DNA cut with fstl; 5, DNA from uninfected L. d/spar fat body; 6, as in 5. but cut with fstl.

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1

2

1

2

FIG. 6. Viral DNA in male parasitoids. (A) Serial twofold dilutions of a 3.0-kb CmV2 HindIll fragment (column 1) and TR male total nucleic acid (column 2) were spotted onto nitrocellulose; amounts spotted start at 10 ng of DNA in column 1, and total nucleic acid from two males in column 2. (6) An autoradiogram showing comigration of the 3.0-kb HindIll fragment in CmV2-infected fat body (lane 1) and TR male parasitoid (lane 2) DNA. Approximately 3 rg total nucleic acid was applied to lane 1, and 25 gg to lane 2; the latter is equivalent to the total amount of nucleic acid in 25 male parasitoids. The 3.0-kb HindIll fragment (see Fig. 4) was used as probe in both (A) and (B).

parasitism; we have observed replication

have genomes consisting of a population of circular molecules of different sizes. In terms of both morphology and morphogenesis, the CmV2 particle closely resembles typical ichneumonid polydnaviruses (Stoltz and Faulkner, 1978), although it has been isolated from a braconid parasitoid. Nevertheless the present study clearly indicates that the CmV2 genome is almost certainly not polydisperse, but rather would appear to consist of a single large molecule (of as yet unknown topology), and we therefore conclude that this virus does not belong to the polydnavirus group. In support of this argument is the observation (unpublished) that CmV2 nucleocapsids do not possess the obvious surface substructure (Stoltz and Vinson, 1979b) associated with those of ichneumonid polydnaviruses. The only other known group of viruses with an at least superficially similar morphology are the “ascovigeruses,” which also possess a nonpolydisperse nome (Federici, 1983). However, ascoviruses have a characteristic and very obvious membrane surface structure, which we have never observed on negatively stained CmV2 virions; in addition, it is as yet by no means clear that ascoviruses have two envelopes.

in parasitized

L. dispar, 0. leucostigma, H. cunea, S. salicis, and, more rarely, E. acrea larvae. In the laboratory, infection of host larvae could also be achieved by intrahemocoelic injection of calyx fluid, but all attempts to passage the virus by the per OS route met with failure.

DISCUSSION CmV2 is not a polydnavirus Virtually all of the known parasitoid viruses can be assigned to one or the other of two reasonably welldefined groups, both of which are presently included within the new virus family, Polydnaviridae (Stoltz et a/., 1984; Fifth Report of the International Commission for the Taxonomy of Viruses, Sendai, 1984): (1) viruses in the genus folydnavirus. These viruses are characterized by the possession of two envelopes surrounding a quasicylindrical nucleocapsid; thus far, they are known only from the hymenopteran family, Ichneumonidae, and (2) viruses known only from the Braconidae, and having cylindrical nucleocapsids of variable length enclosed within a single unit membrane envelope (no generic name has been proposed for this group of viruses). Unlike other double-stranded DNA viruses, those belonging to the family Polydnaviridae

1

2

3

FIG. 7. Apparent absence of genetic transmission of CmV2 DNA between wasp populations carrying different virus strains. Viral DNA was extracted from infected host fat body tissue, digested to completion with EcoRI, and examined following agarose gel electrophoresis. DNA samples are from larvae parasitized by TR, (FR? X TR6) F,, and FR females (lanes 1-3, respectively). The indicated TR bands (arrows opposite lane 1) do not appear to have been transmitted to female F, progeny (lane 2).

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Finally, the cytopathology of ascoviruses and CmV2, both of which replicate in fat body tissue, is very different. Nevertheless, given the nonpolydisperse nature of the ascovirus genome (Federici, 1983), it may be of interest in future studies to look for possible sequence homologies between CmV2 and the ascoviruses. Function

and transmission

Parasitoid viruses are invariably found in the ovaries of all females of all affected species (Stoltz and Vinson, 1979a). This observation has led to the development of two working hypotheses: (1) that these viruses may per-form some function of benefit to the parasitoid; and (2) that transmission of such viruses within parasitoid populations may be achieved by vertical means. In the case of polydnaviruses, there is now ample evidence in support of the former: these viruses appear to promote successful parasitism by suppressing host immunity to parasitoid eggs and/or larvae (Edson et al., 1981; Guzo and Stoltz, 1985; Stoltz and Guzo, 1986). On the other hand, it seems unlikely that CmV2 plays any functional role in successful parasitism since not all C. melanoscela strains carry it, and those that do also carry polydnavirus; nevertheless, this question will remain unresolved until purified CmV2 is available, thus permitting the appropriate biological experimentation to be carried out. Until recently, the subject of parasitoid virus transmission has received little attention. In theory, three possible scenarios can be invoked: (1) per OS transmission (virus injected during oviposition infects developing parasitoid larvae, eventually reaching and infecting germ line tissue; i.e., horizontal transmission); (2) maternal transmission, as observed in the case of mitochondrial genomes (e.g., Dawid and Blackler, 1972); and (3) vertical transmission via the germ line of either sex. In our view, the results of this and a previous study (Stoltz et a/., 1986) would appear to have effectively ruled out per OS transmission of parasitoid viruses in general. On the other hand, while polydnaviruses can apparently be transmitted via the germ line of either sex (Fleming and Summers, 1986; Stoltz et al., 1986) this appears not to be the case for CmV2, for which only maternal transmission has been demonstrated (present study). Thus, our observations tend to suggest that not all parasitoid viruses are necessarily transmitted in precisely the same manner, and support the view that more than one family of viruses may reside in parasitoid ovaries. Pathogenicity While CmV2 shares many features of a typical polydnavirus life cycle, it does differ in one important re-

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spect: whereas no known polydnavirus replicates in host tissue, CmV2 does. It is therefore of interest to consider whether this agent has any potential for biocontrol, particularly with reference to the perennial gypsy moth problem. Our studies in this area would tend to suggest that CmV2 is not sufficiently pathogenic to warrant serious consideration as a microbial pesticide; gypsy moth larvae injected with either CmV2-containing calyx fluid or with infected LD-652Y cells did become infected, but invariably survived to produce normal progeny. Furthermore, the virus was not infectious per OS, and can apparently be transmitted only as a consequence of oviposition by the parasitoid, an event which in any case serves to destroy the host. Since CmV2 apparently replicates in several tissues of both male and female parasitoids, the question arises as to whether it is pathogenic for the parasitoid itself. In response, it should be noted that only a few cells appear to be infected, even in the ovary (Stoltz and Faulkner, 1978); in addition, we have no evidence to suggest that strains TR and FR differ from other parasitoid strains in terms of either longevity or fecundity. ACKNOWLEDGMENTS This study would not have been possible without the generous assistance of personnel associated with the Canadian Forestry Service, the Pennsylvania Department of Environmental Resources (Dr. L. D. Rhoads), and the U.S. Department of Agriculture. We are particularly indebted to Dr. T. D. Smith. Nova Scotia Department of Lands and Forests, for directing our attention to the satin moth infestation near Truro. We thank Carolyn Murphy and Marie Richard for typing the manuscript. This research was supported by grants from the Medical Research Council and by the Canadian Forestry Service to D.B.S., and from the Natural Sciences and Engineering Research Council to P.K. and C.J.L.

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