Studies on polydnavirus transmission

155.120-131

VIROLOGY

(1986)

Studies on Polydnavirus DONALD Department

B. STOLTZ,’

of M&&io~, Received

Dalh.mAe Februaq

DAVID University,

Transmission

GUZO, Halqax,

24, 1986; accepted

DOUG

AND Nova July

Scotia,

COOK Canada

BsH

.~HT

8, 1986

Polydnaviruses are thought to replicate only in the ovaries of certain hymenopteran species. Nevertheless, in the present study, polydnaviral DNA was found to exist in males of the braconid parasitoid species Cot&a meZano.sceZa and in both male and female nonovarian tissue of an ichneumonid, Hyposotw fugitivus: preliminary results suggest that viral DNA may be present in an unintegrated form, but whether or not it is encapsidated is unknown, Using interstrain genetic crosses, we demonstrated that C. melanism& males can apparently transmit at least some viral DNA to female progeny. We suggest that polydnavirus DNAs may be present in most if not all tissues of certain parasitoid species, and are probably maintained within parasitoid populations by vertical transmission through the germ line. In parallel experiments, manually injected eggs of the ichneumonid parasitoid (H, fugitives) survived and hatched in Mahwsm americanurn larvae in the apparent absence of exogenous polydnavirus; female parasitoids reared in this manner nevertheless carried virus in their ovaries. Experiments utilizing different strains of C. nzelunoscela also suggest that per OS transmission of polydnaviruses (to parasitoid larvae) does not occur, despite the fact that inoculum viral DNA can be shown to persist for several days in the tissues of parasitized host larvae. o 1996 Academic POW. h.

the International Committee for the Taxonomy of Viruses, Sendai, 1984); no generic name has as yet been proposed for the braconid viruses. Despite the rather dissimilar morphologies of these two recognized groups of polydnaviruses, all appear to share a common life cycle, which involves replication in the wasp ovary, secretion into the oviduct, and injection into the host during oviposition (Stoltz and Vinson, 1979a). Following penetration into host tissues (Stoltz and Vinson, 1977,1979b), virus-specific transcripts can be detected (Fleming et CLL,1983; Blissard et al, 1986; our unpublished observations), in the apparent absence of viral replication. It has been recognized for some time that all females of affected parasitoid species carry virus in their ovaries; this observation is in support of recent experimental evidence to the effect that polydnaviruses are required, at least in some cases, for successful parasitism. The same observation suggests as well that parasitoid viruses must be maintained within wasp populations by a very efficient mechanism.

INTRODUCTION

The new virus family Polydnaviridae comprises a group of unusual agents which are characterized by the possession of multipartite, double-stranded, circular DNA genomes (Krell and Stoltz, 1980; Stoltz et al., 1981; Krell et aL, 1982); thus far, they are known only from certain species of parasitic hymenoptera (Stoltz et d, 1984). At present, two morphologically distinct groups of viruses are recognized as belonging to the polydnavirus family: (1) viruses found only in braconid wasps, and having cylindrical nucleocapsids of variable length surrounded either individually or as groups by a unit membrane envelope, and (2) viruses found only in ichneumonid wasps, and having fusiform nucleocapsids surrounded by two envelopes (Stoltz and Vinson, 19’79a). The ichneumonid viruses have now been assigned to the genus Polydnavirus (Fifth Report of ‘To whom dressed. 0042-6822/86 Copyright All rights

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Recent work has shown that polydnavirus DNA is present both in Campoletis scmorensis males and in nonovarian female tissue (Fleming and Summers, 1986); while these observations are suggestive of some form of hereditary transmission, the authors of that study did not specifically rule out the possibility of polydnavirus transmission by the per OSroute. In the present communication, we consider two possible scenarios for the transmission of polydnavirus genomes: vertical, with viral DNA present in the egg and/or sperm, and per OS (horizontal). The results of our experiments strongly suggest that polydnavirus transmission is effected exclusively by hereditary means. We report in addition that male parasitoids not only carry viral DNA, but can also transit it to F1 female progeny resulting from appropriate genetic crosses. MATERIALS

AND

METHODS

In the present study, it was of interest to examine the question of polydnavirus transmission using both a braconid and an ichneumonid system, for two reasons: (1) the viruses involved are quite different, at least in terms of morphology, and (2) certain types of biological experiments could only be performed with the braconid/host system at hand, and vice versa. It was anticipated that observations made using one parasitoid/host system would complement those made using the other. Insects. Hyposoter fugitivus (Hymenoptera:Ichneumonidae) was routinely reared from larvae of the forest tent caterpillar, Malacosoma disstria, and occasionally from the eastern tent caterpillar, M am&canum. Two different populations of Cotesia melanoscela (Hymenoptera:Braconidae), established from material collected originally in Connecticut (strain CT-l) and Truro, Nova Scotia (strain TR), were employed; both were reared from a laboratory colony of the gypsy moth, Lymantria dispar. Hybrids between C. melanoscela strains were obtained more or less as described (Weseloh, 1982). All adult parasitoids were maintained on a diet of honey and water. Tent caterpillars and gypsy moth larvae were reared on an artificial medium (Bell et al, 1981); in some experi-

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ments, tussock moth (Orgyia leucostigwut) larvae were also used. Vimcses. Viruses from different strains and hybrids of C. rnelanoscelu were distinguished on the basis of gel electrophoretic profiles of DNAs extracted from wasp ovaries (Fig. 1). Briefly, ovarian calyx fluid from female parasitoids was extruded into phosphate-buffered saline (without cations; pH 7.4), made to 4% sodium lauryl sarcosinate, and heated at 60” for 1 hr. Proteinase K (from a stock solution at 10 mg/ml in 1.0 MTris, pH 7.4, 0.05 MEDTA, 0.5 M NaCl) was added to a final concentration of 300 pg/ml, and digestion allowed to proceed for 2 hr at 30”; sarcosinate was maintained at 4% during this step. Nucleic acid was then extracted with hot (SO’) HzO-saturated phenol:chloroform:isoamyl alcohol (50:49:1), followed by several diethyl ether extractions. Samples were run overnight on horizontal 0.8% agarose gels in TEA buffer (TEA is 0.04 M Tris, 0.005 M sodium acetate, 0.001 M EDTA, pH 7.8). Ichneumonid polydnavirus type 3 (IPV3, from H. fugitivus; Stoltz et al., 1984) was purified by standard procedures (Krell and Stoltz, 1980). Parasitwid and host genomic DNA. The heads and thoraces of adult male braconid parasitoids were homogenized on ice in 400 ~1 of 20 mMTris, 5 mMEDTA, pH 8. Subsequently, 4 ~1 of a saturated solution of phenylthiourea (PTU) was added, and the mixture heated to 80” for 10 min. Solubilization was in 4% sarcosinate at 60” for 1.5 hr, followed by proteinase K as described above. Debris was removed by centrifugation at 12,000 g for 10 set; the supernatant was extracted five times with hot phenol, followed by one extraction with hot phenol/chloroform/isoamyl alcohol, and several extractions with diethyl ether. Total nucleic acid was precipitated overnight at -80” after bringing the solution to 0.2 MNaCl and adding 2 vol of ethanol. In the case of the ichneumonid H. fugitivus, nucleic acid extractions were as above except that the wasp abdomens (both male and female) were removed and discarded. Agarose gels were run using a BRL H4 apparatus, loaded at 100 pg total nucleic acid per lane.

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Briefly, BPV-la DNA was digested to comNucleic acids were extracted from either control or H. fugitivus-parasitized larvae pletion with EcoRI, ligated into pUC 19, in the same manner, except that in the case and subsequently propagated in Escheof M. disstria larvae the heads, posterior richia coli strain TBl. Recombinant DNAs body segments, and guts were first re- were screened by colony hybridization, using probes prepared by nick translation of moved; for each time point post oviposition, nucleic acids were from five to seven pooled specific DNA bands extracted from agarose larvae. M. disstria were used as 4th instar gels by the freeze/squeeze method (Thurlarvae; control and C melanoscela-parasiing et al, 1975). We were particularly intized 0. leucostigma and L. dispar larvae terested in cloning sequences derived from (1st or 2nd instar) were used intact. band D (see Fig. l), and to this end idenDNA blotting/spotting. After electrotified colonies reacting with pooled DNA from CT-l bands C-E; these colonies were phoresis, DNA was blotted onto nitrocellulose as described (Southern, 1979). After subsequently probed with pooled DNA drying and baking at 80” in vacua, filters from bands C and E from strain TR, with nonreacting colonies assumed to contain were prehybridized for 18 hr in 50% deionized formamide, 0.02% each of Ficoll, bo- band D inserts. As it happened, TR bands vine serum albumen, and polyvinylpyrolC and E were ultimately found not to be lidone (Denhardt’s solution), 4X SET (SET entirely homologous with those from strain CT-l, so that it became necessary to probe is 0.15 M NaCI, 0.03 M Tris, 0.001 M EDTA, pH 8), 50 pg/ml of heparin (Singh and the viral genome with a number of putative Jones, 1984), 0.2% sodium dodecyl sulfate inserts from band D in order to identify (SDS), and 125 rg/ml of calf thymus DNA. sequences which in fact would hybridize Probe DNAs were labeled with 32P by nick specifically with that DNA band. Plasmids translation to specific activities of approx containing appropriate inserts were di1.0 X lo8 cpm/pg of DNA. Hybridization gested with EcoRI and run into an agarose was for 48-72 hr at 42”, in the same buffer gel; virus-specific DNA was then extracted used for prehybridization. Posthybridizaand used to probe blots containing male tion treatments consisted of a 2-hr wash parasitoid DNA. in hybridization buffer, followed by two lRecombinant E. coli (strain RRl) conhr washes in 2X SET, 2X Denhardt’s so- taining ichneumonid polydnavirus (IPV-3) lution, 50 pg/ml of heparin, 0.2% SDS, and sequences ligated after EcoRI digestion 125 pg/ml of calf thymus DNA. Filters into pBR325 plasmids were generously were air-dried for 1 hr prior to autoraprovided by Peter Krell. In our studies, we diography using Kodak X-OMAT AR film used IPV-3 sequences from two different and DuPont Cronex intensifying screens. plasmids, pIPV-3-1.9 and -4.9; these seHost larval nucleic acids were either un- quences are nonhomologous and hybridize treated or else digested with nucleases ac- to different (but as yet unidentified) viral cording to the protocols of Brandsma and DNA bands. Miller (1980). Nucleic acids were spotted Transmission per 0s. This question was onto nitrocellulose filters using a BRL addressed in two ways: (1) by determining HybriDot apparatus and methods outlined whether parasitoids developing in the abby Kafatos et al. (1979). Prehybridization, sence of homologous polydnavirus nevhybridization and posthybridization ertheless carried that virus, and (2) by treatments were as described for Southern providing opportunities for developing blotting. parasitoid larvae to consume a polydnaDNA cloning and h&%dization probes. virus carrying a readily identifiable genetic For braconid polydnavirus probes, DNA marker. In order to carry out these experpurified from braconid polydnavirus (BPV) iments, it was necessary to make use of type la (carried by wasp strain CT-l) was two different biological systems, since neiused, primarily because this particle pos- ther alone proved suitable for all of the apsesses at least two circular DNA species proaches adopted in this study. not observed in some others (see Fig. 1). The first approach involved use of

POLYDNAVIRUS

the ichneumonid parasitoid, H. fbgitivus, washed eggs of which can, in the apparent absence of homologous polydnavirus (IPV3), develop to maturity either in larvae of the eastern tent caterpillar, M. urn&canum (Stoltz and Vinson, 1979a) or in tussock moth larvae which have been previously immunosuppressed by a braconid polydnavirus (from C. mdanoscela; Guzo and Stoltz, 1985). Resultant female H. fugitivus progeny were examined for the presence of homologous polydnavirus by agarose gel electrophoresis of calyx fluid DNA. The second approach involved use of the braconid parasitoid, C. melunoscela, different populations of which carry polydnaviruses which can be readily distinguished from each other by means of gel electrophoresis (see Fig. 1). Of particular interest was a DNA band, D, which is observed in virus carried by the CT-1 but not the TR wasp population. In appropriate injection and double-parasitization experiments, we simply provided an opportunity for developing TR larvae to consume virus from CT1. For example, in one experiment, TR eggs were washed free of homologous virus and then injected along with BPV-la (from C. melunoscela strain CT-l). In another experiment, host larvae were injected with BPV-la and then parasitized 1 hr later by a TR female (TR wasps lack viral DNA band D, which is present only in BPV-la); TR females emerging from host larvae were examined for the presence of band D DNA by agarose gel electrophoresis. Alternatively, larvae were parasitized by both CT-1 and TR females; we reasoned that if a productive infection could be achieved by the per OS route, then all female wasps emerging from doubly parasitized hosts should carry band D. Before conducting any such experiments, we determined that polydnavirus DNA could in fact persist in host larvae for several days postinjection, or after oviposition; this information was obtained by dot/blot hybridization, as described above. RESULTS

Apparent purasitoids.

viral transmission by male In preliminary studies, we

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found that it was possible to identify several different strains of the braconid parasitoid, C. mehnoscela, two of which (CT1 and TR) are central to the present study; identification was based on relatively minor but consistent differences in their respective braconid polydnavirus DNA profiles (Fig. 1). In this regard, strain CT-1 was of particular interest since it carried

l

2

3

FIG. 1. Gel electrophoretic profiles of viral DNAs from three different strains of the braconid parasitoid C.melanoscela;eachlanecontainsDNAextractedfrom the calyx fluid of a single female wasp. Lane 1, strain CT-l; lane 2, strain RU, lane 3, strain TR. Some of the superhelical DNA bands are indicated on the left. Two of these, bands D and H, are unique to strain CT-l wasps. Band D is of particular relevance to the present study. A detailed examination of genome organization in the CT-l virus BPV-la will be presented separately (D. Guzo and D. B. Stoltz, in preparation).

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a virus (BPV-la) which possessed at least one unique DNA band (D; 27.4 kbp) not present in some other strains (lane 1, Fig. 1). Having determined as well that all of our C melunoscela populations were capable of successfully interbreeding to produce fertile F1 hybrids, we were provided with an opportunity to address the question of whether males could contribute viral DNA to progeny females (no such system has as yet been identified for ichneumonid polydnaviruses). Genetic crosses were set up between males of the CT-l strain and females belonging to the TR strain, which lacks the 2’7.4-kbp DNA species (i.e., band D). In such crosses, we consistently observed an apparent transmission of CT-l-specific viral DNA to progeny Fi females; transmission of band D DNA was readily detected either by ethidium bromide staining of agarose gels, or by Southern blot hybridization (Fig. 2). Presence of viral DNA in male parasitoids. While the results of genetic hybridization experiments seemed to indicate that males could transmit polydnavirus DNA, it was necessary to prove that they in fact carried the viral genome. In particular, it was important to determine whether CT-l males carried DNA band D, which is apparently not carried by some other strains; otherwise, it becomes reasonable to entertain the possibility of band D being induced in, rather than transmitted to, F1 hybrids. In order to carry out this portion of the study, it was necessary to clone DNA sequences from band D, for use as specific hybridization probes. Genomic DNAs were extracted from males belonging to different strains, and probed with virus-specific DNA from plasmids pBPV-la-14.9 and -4.2; these plasmids contain nonhomologous BPV-la inserts of 14.9 and 4.2 kbp, respectively, hybridizing to different circular viral DNA bands (Fig. 3). Results obtained thus far indicate that 17. melanoscela males appear to contain the same spectrum of viral DNAs as do females of the same strain, As is true for CT-1 females, for example, the pBPV-la-14.9 probe hybridizes with two species of circular viral DNA (i.e., bands D and G), whereas only one is detected in males of strains lacking

AND

COOK

DNA band D. Only the open circular form of viral DNA was observed in male DNA extracts, whereas their superhelical equivalents are readily detected in calyx fluids (crude virus) taken from wasp ovaries. An approximate comigration of male parasitoid and viral DNAs was routinely observed. Visible hybridization signals were in each case removed from the location of chromosomal DNA. In parallel studies, we examined both male and nonovarian female tissues from an ichneumonid parasitoid, H. fugitivus, for the presence of polydnavirus DNA. Typical results are shown in Fig. 4; while only open circular forms of viral DNA were observed in males, extracts from females always contained a small amount of superhelical in addition to relaxed circular DNA. Again, hybridization signals in uncut male and nonovarian tissues were situated well below the position of chromosomal DNA. Persistence of viral DNA in host insects. In preliminary experiments, we determined that inoculum viral DNA could persist in host larvae, and might therefore be available for transmission per OS. Results for ichneumonid polydnavirus type 3 are presented in Fig. 5. In dot/blots, viral DNA in host larvae was detectable for at least 5 days following parasitization; interestingly, viral DNA was also observed to persist in host species (L d&par and 0. leucostigma) in which this parasitoid cannot successfully develop. It was established by means of Southern blot hybridization that a seemingly complete spectrum of polydisperse viral DNA was present in parasitized host larvae. Similar results were obtained for the braconid polydnavirus, BPV-la. In this case, it was of particular interest (for the experiments described below) to determine whether sequences homologous to pBPVla-14.9 persisted in host larvae for an appreciable period post oviposition. That this occurs is shown in Fig. 6, in which it can be seen that such sequences persist for at least 5 days PO. Interestingly, this blot revealed the existence of some unsuspected heterogeneity in the CT-l colony, i.e., pBPV-la-14.9 hybridized to three EcoRI

POLYDNAVIRUS

fragments, rather than the two usually observed (not shown). We are now establishing isogenic colonies of all our parasitoid strains. The observation does not affect the validity of certain conclusions arrived at during the course of the present study since band D was always present in CT-1 females, and never in TR females; similarly, while pBPV-la-14.9 hybridized to either

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two or three EcoRI fragments in virus from CT-l, it never hybridized to more than one fragment (derived from band G) in the virus from the TR strain. Lack of transmission per OS. Working with H. fugitivus, one of us (D.B.S.) had previously determined that larvae of the eastern tent caterpillar, M. americanurn, would support parasitoid development in

A

Gs Ds

123456789 FIG. 2. Apparent either by ethidium viral DNAs from 6, (TR9 X CT-lb)Fi also seen in hybrid from an Fi hybrid 1, strain TR; lane signals developed

transmission of viral DNA to Fi progeny by male parasitoids can be demonstrated bromide staining (A) or filter hybridization (B). The gel shown in (A) includes several individual female parasitoids, as follows: lanes l-3, strain CT-l; lanes 4hybrids; lanes 7-9, strain TR. The position of band D, unique to strain CT-l, but progeny, is indicated. In (9) calyx fluid DNAs from the two parental strains and female have been probed with vector-free =P-labeled pBPV-la-14.9 DNA. Lane 2, (TR9 X CT-1B)Fi hybrid; lane 3, strain CT-l. The positions of hybridization at bands D and G are indicated; s = superhelical DNA.

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1

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GUZO,

4

AND

COOK

1

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FIG. 3. Viral DNA in C. melanoscela males. Uncut DNAs from male parasitoids are compared with DNA profiles from calyx fluid taken from females. In the case of males, 100 pg of total nucleic acid (approximately 60 males) was run in each lane; for females, approx 0.5 gg of viral DNA (calyx fluid from the equivalent of 0.1 parasitoid) was run. The great disparity in amount of DNA loaded into the gels accounts for the observation that male DNA routinely migrates slightly in advance of corresponding viral sequences. q-Labeled probes were prepared from plasmids pBPV-la-14.9 and -4.2 as described under Materials and Methods. Blot (A) was probed with the 14.9-kbp insert from DNA band D. Lane 1, uncut CT-l male wasp DNA; lane 2, uncut CT-1 viral DNA; lane 3, uncut TR viral DNA; lane 4, uncut TR male wasp DNA. The probe hybridizes with two circular DNA species, bands D and G, in CT-l parasitoids, but only with one (band G) in strain TR; s and r denote superhelical and relaxed circular forms, respectively, of these DNAs. Blot (B) was probed with the 4.2-kbp insert from band A, the position of which is indicated in Fig. 1; note that no hybridization signal is developed at the position of uncut male chromosomal DNA, which is indicated by an arrow. Lanes 1 and 3, uncut CT-l viral DNA; lane 2, uncut male wasp DNA. The lettering is as shown in blot (A).

the apparent absence of polydnavirus (Stoltz and Vinson, 19’79a).2 This observation was confirmed for the purposes of the present study; in addition, however, we determined that IPV-3 was present in female parasitoids reared from washed eggs which had been manually injected into M amer‘M. americnnum will also support the intrahemocelic replication of injected yeast cells (unpublished observations), suggesting the presence of a generalized immune deficiency. The related species, M. disstti, encapsulates H. ji&tivua eggs in the absence of virus, and efficiently destroys yeast cells (Stoltz and Guso, 1986).

icanum larvae. Similarly, H. jkgitivw eggs will develop in 0. kucostigma larvae if these have been previously parasitized by C. melunoscela (Guzo and Stoltz, 1935); again, females reared in this manner, in the apparent absence of IPV-3 in the host, nevertheless carried this virus (Fig. 7). No such system was available for study in the case of any known braconid polydnavirus. However, as described above, C. melanoscela wasps belonging to strain CT1 carry a specific viral DNA band not found in some other strains of this parasitoid; this provided an opportunity to determine whether this “marker” DNA (band D) and,

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fined by electrophoresis of viral DNAs) emerged in approximately equivalent numbers, again suggestive of a lack of per 0s infection.

DISCUSSION

m

v

vff A

m * 6

V

V

C

f D

FIG. 4. Viral DNA from males and nonovarian female tissues of the ichneumonid parasitoid, H. fugitivus, is compared with viral DNA sequences from female calyx fluid; all are uncut. m, Male parasitoid DNA; f, DNA from nonovarian female tissues from two separate extractions; v, viral DNA (from ovarian calyx fluid); s and r are as given in Fig. 3. Blots (A) and (B) were probed with a 4.9-kbp viral DNA insert from plasmid pIPV-3-4.9; (C) and (D) were probed with a 1.9-kbp insert from pIPV-3-1.9. Male and nonovarian female samples were loaded at 100 pg total nucleic acid per lane; virus was at 0.1 pg per lane. The position of faint hybridization signals corresponding to superhelical DNA in female nonovarian tissue is indicated by long arrows. The position of uncut chromosomal DNA is indicated by a short arrow.

presumably, the BPV-la virus as well, could be transmitted per OS to other strains. To this end, we injected host larvae with a combination of eggs from strain TR, which lacks DNA band D, and virus from strain CT-l. In complementary experiments, host larvae were injected intrahemocelically with BPV-la and subsequently parasitized with TR wasps, or else larvae parasitized by both CT-l and TR were used; whether injected manually (not shown) or during oviposition by the parasitoid (Fig. 6), viral DNA could be shown to persist for several days in host animals. In all cases, female TR progeny emerging from injected hosts invariably lacked the unique viral DNA band typical of CT-l parasitoids; in the case of doubly parasitized hosts, CT-l and TR females (as de-

The life cycle of certain parasitic insects requires, as an integral component, the participation of unusual viruses, now classified as polydnaviruses; at least in some case, such viruses appear to promote successful parasitism, possibly by virtue of an immunosuppressive activity directed against host hemocytes (Edson et ak, 1981; Guzo and Stoltz, 1985; Stoltz and GUZO, 1986). The association existing between virus and parasitoid is invariable, and so cannot be considered to represent an infection; indeed, in a very real sense, the virus is a part of the parasitoid. The foregoing should suggest that an unusually effective method of transmission must operate in order to ensure that all females carry their respective polydnavirus. A plausible mechanism for accomplishing this would be one in which virus or viral DNA was present in every egg and/or sperm cell; in other words, information for the replication of polydnaviruses could in some manner be carried as part of the parasitoid genome. Alternatively, it is possible that polydnaviruses are transmitted per OS as a result of larval feeding in the “infected” host. In the present study, we define some biological systems which have allowed us to experimentally address these two possibilities. VimLs in male pcwasitoids. In a recently published study, Fleming and Summers (1986) have conclusively demonstrated the presence of CsV polydnavirus DNA in both male and female nonovarian tissues of the ichneumonid parasitoid, Campoktis scme ren.s&. Furthermore, these authors provide evidence suggesting that viral DNA can in this case apparently exist in both integrated and nonintegrated forms, as determined by comparative restriction endonuclease analyses of DNAs extracted from parasitoid tissues and purified virus par-

STOLTZ,

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b

a Ld

Md

b

b

FIG. 5. Presence and persistence of H. fugitives polydnavirus DNA in host larvae; q-labeled viral DNA was used as probe. In dot/blots (A), persistence of viral DNA is shown in larvae of three different host species. Md, M. disstria; 01, 0, leucostigma; Ld, L dispar. a, Total nucleic acid spotted (7 pg per spot); b, same, but digested with DNase; c, same, digested with RNase (some contaminating DNase activity is apparent); from left to right, the dots are of larval DNA extracted at 15 min, 3 hr, 1 day, 3 days, and 5 days following parasitism. In (B), undigested DNA extracted from parasitized host larvae is compared with viral DNA. 01, Ld, and Md are as given above (at 30 pg nucleic acid per lane); Hf, H. fugitivus viral DNA (0.05 pg).

titles. As these authors suggest, integration of the CsV genome into parasitoid chromosomal DNA could provide a mechanism for the vertical transmission of polydnaviruses through the parasitoid germ line. In the present study, we have carried out similar but less extensive studies using two different parasitoids. One of these, H. fugit&us, is an ichneumonid species carrying a polydnavirus (IPV-3) which shares only minimal genetic homology with CsV (Krell and Stoltz, unpublished data); the other, C. melanoscelu, is a braconid parasitoid. We have now shown that polydnavirus DNA exists in males of both parasitoid species and, in the case of H. fhgitiwus, in nono-

varian female tissues as well. However, in contrast to the earlier study on CsV cited above, in which the majority of male parasitoid DNA may exist in an integrated form, we were unable to find evidence either for integrated viral DNA sequences or putative replicative intermediates in parasitoid tissues. It remains to be seen whether some as yet unrecognized technical parameter might account for the very different observations which have now been recorded from two different laboratories. Alternatively, it is possible that if integration indeed occurs, it may nevertheless not represent a general phenomenon; rather, it may be restricted to only some parasitoid/virus systems. It is also

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(Krell and Stoltz, 1979), at least 50% of encapsidated ichneumonid polydnavirus DNA appears to be supercoiled (Krell and Stoltz, 1980; Krell et al, 1982). We can at present offer no suitable explanation for these disparate observations.

7

FIG. 6. Persistence of braconid polydnavirus (BPVla) DNA in 2nd instar L dkpar larvae parasitized by CT-l wasps; nucleic acids were digested with EcoRI and probed with a 14.9-kbp insert from band D. Lane 1, viral DNA (0.5 pg); lane 2, nucleic acid from nonparasitized larvae (50 rg); lanes 3-7, nucleic acid from parasitized larvae at 3, 6, 24, 48 and 96 hr postoviposition, respectively (50 pg each).

possible that different viral DNAs may exist in different physical states in male wasps; for example, CsV DNA band Q could not be detected in episomal form in male parasitoids, unlikeband B (see Fig. 3 of Fleming and Summers, 1986). Interestingly, only relaxed circular DNA was detected in our genomic blots of male parasitoid DNA (female nonovarian tissue routinely contained a very small amount of supercoiled DNA), even though the superhelical form could readily be detected in purified virus. This observation seems similar to that described for male C. sonoren.sis wasps probed with DNA from CsV band B (Fleming and Summers, 1986), in which the SH form is clearly underrepresented. While such results might be expected in the case of C. melanoscelu viral DNA, most of which is in the relaxed form

FIG. ‘7. Lane 1, agarose gel electrophoretic pattern of viral DNAs extracted from the ovary of an H. fw it&s female reared from a permissive host (AZ. disstria) larva; such larvae are injected with viral DNA as a normal consequence of parasitism, and this DNA persists for several days in the host (see Fig. 5). Lane 2, viral DNAs from the ovary of an H. J%&&US female reared from an H. fugitivus egg which had been washed and then manually injected into an 0. ti stigma larva parasitized 30 min earlier by C. melono.sceZo (see Guzo and Stoltz, 1985, for rationale); in this system, H.J%&VUS viral DNA cannot be detected in the host animal by dot blotting (not shown).

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In unpublished studies, we have as yet been unable to detect virus particles in testes from a limited number of either C. melunoscela or H. fugitivus males. In this context, it should be noted that Fleming and Summers (1986) have tentatively concluded that their results provide no support for viral replication in male tissue, although they do not rule it out. The question of whether polydnavirus replication may occur in male wasps or in female nonovarian tissue nevertheless remains unresolved, and will be difficult to answer without a thorough ultrastructural investigation. Virus

transmission

by male parasitoids.

Using C. mekcnoscelu populations, we have been able to demonstrate that males not only carry viral DNA, but are in addition apparently capable of transmitting it to progeny individuals resulting from appropriate genetic crosses; such experiments were made possible by the fortuitous discovery of several different C melanoscela populations, which can be distinguished from each other by agarose gel electrophoresis of their viral DNAs. Since all of these populations can interbreed successfully, we simply looked for one having a distinct DNA band not present in the others, and used this as a genetic marker in experiments designed to determine whether males could transmit such DNA. The results obtained from such experiments clearly suggest that viral DNA can be carried within sperm cells and transmitted to progeny females as a consequence of egg fertilization. The significance of male-directed viral DNA transfer is not immediately apparent, although it could of course be seen as a mechanism for introducing genetic variability into virus (and parasitoid) populations. Nevertheless, it seems unlikely that maintenance of polydnavirus DNA within parasitoid populations requires the participation of males, since some species carrying virus can reproduce parthenogenetically, producing only females which, nevertheless, still carry polydnavirus (e.g., Cot&a schaeferi; D. B. Stoltz, unpublished data). Where present, males are in any case haploid and must therefore have received their complement of viral DNA through the female germ line.

AND

COOK

Lack of transmission per OS.It is now well known that female parasitoids inject viruses into host larvae during oviposition (Stoltz and Vinson, 1979a). As is shown here, inoculum viral DNA can persist for several days following oviposition, and might presumably be available for consumption by the developing parasitoid larva; conceivably, then, such DNA could eventually reach and become inserted into germ line tissue. We have always considered this scenario to be extremely unlikely, if only because the degree of transmission efficiency required (100% ) would not likely be achieved by horizontal transfer; unique opportunities to investigate this question were nevertheless afforded by the parasitoid/host systems at hand, and warranted critical evaluation. Using several different experimental approaches, however, we could find no evidence for per OS transmission, and must conclude that it does not occur. Conclusion. Preliminary evidence would now seem to suggest that each polydnavirus is carried by both sexes of the parasitoid species with which it is associated (Fleming and Summers, 1986; the present study); furthermore, it can reasonably be suggested (although proof is lacking) that polydnavirus DNA is present in every tissue as well as in each individual parasitoid. We can therefore speculate that, in whatever physical form it exists, polydnavirus DNA may represent a unique component of the genome of certain parasitoid species. In other words, any distinction made between parasitoid genomic and viral DNA can, at least to some extent, be considered artificial. Polydnaviruses are nevertheless viruses, in the sense that they are very much like other viruses in terms of biochemical composition, morphogenesis, and structure; in other ways, however, they differ significantly from typical viruses, and can just as legitimately be regarded as nuclear secretions taking a viral form, and having as a unique functional role the genetic colonization of the parasitoid’s host. ACKNOWLEDGMENTS This Medical

research was supported by a grant from the Research Council of Canada. We thank Peter

POLYDNAVIRUS Krell for providing ichneumonid clones.

polydnavirus

type 3

REFERENCES BELL, R. A., OWENS, C. D., SHAPIRO, M., and TARDIF, J. F. R. (1981). Mass rearing and virus production. In “The Gypsy Moth: Research toward Integrated Pest Management” (C. D. Doane and M. L. McManus, eds.), pp. 599-655. USDA Tech. Bull. 1584. BLISSARD, G. W., VINSON, S. B., and SUMMERS, M.D. (1986). Identification, mapping and in vitro translation of Campoktis sonorensti virus mRNAs from parasitized Heliothis virescens larvae. J. V&l 57, 318-327.

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