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A limited role in parasitism for Microplitis demolitor polydnavirus
Journal of Insect Physiology 44 (1998) 795–805
A limited role in parasitism for Microplitis demolitor polydnavirus Dominique Trudeau, M.R. Strand
*
Department of Entomology, 237 Russell Labs, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA Received 1 October 1997; accepted 23 October 1997
Abstract Spodoptera frugiperda larvae stung by Microplitis demolitor undergo physiological alterations characteristic of parasitism. However, despite these physiological modifications, parasitized S. frugiperda larvae never yield adult wasps. Our original hypothesis that unsuccessful parasitism was due to a transcriptionally inactive polydnavirus proved untrue. Microplitis demolitor polydnavirus (MdPDV) successfully infected and expressed, albeit transiently, in S. frugiperda hemocytes. MdPDV expression was most abundant in the first three days of parasitism, then sharply declined on Day 4 post-parasitization and continued to decline for the remainder of the study. During the period of MdPDV expression, S. frugiperda hemocytes were non-adherent, incapable of spreading in vitro and did not encapsulate M. demolitor eggs in vivo. Concurrent with diminishing viral expresssion, S. frugiperda hemocytes regained their ability to adhere and spread in vitro and encapsulated M. demolitor eggs in vivo. Although MdPDV disrupted S. frugiperda’s encapsulation response for the first three days post-parasitization, M. demolitor was unable to develop in this noctuid species. Failure to develop was independant of viral activity, all M. demolitor eggs oviposited in S. frugiperda larvae failed to complete embryogenesis and died within 24 hour of oviposition. S. frugiperda larvae infected with MdPDV exhibited alterations in development very similar to other lepidopterans that are permissive hosts for M. demolitor. In addition, MdPDV DNA persisted in Spodoptera frugiperda hemocytes in the absence of viral expression. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Parasitoid; Immunity; Hemocytes; Host suitability
1. Introduction Parasitoids encounter many potential hosts when foraging. Some of these species will be completely suitable for offspring development, others will be marginally suitable, and most will be unsuitable (Godfray, 1994). Many parasitoids in the families Braconidae and Ichneumonidae carry polydnaviruses which replicate in the reproductive tract of female wasps and are injected into potential hosts along with one or more eggs at oviposition (Stoltz, 1993). In suitable hosts, polydnaviruses enter a variety of tissues with transcription in the apparent absence of replication occurring over the period required for the wasp’s progeny to complete development (Strand and Pech, 1995a). Transcriptionally active polydnaviruses induce several physiological alterations in hosts that are considered important for survival of the wasp’s progeny. Most notably, polydnaviruses have
* Corresponding author. Tel.: 1 608 262 6902; Fax: 1 608 2623322; E-mail: [email protected] 0022–1910 /98 /$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 1 2 - 2
immunosuppressive and juvenilizing effects on hosts (Whitfield, 1990; Fleming, 1992). In the presence of virus, host hemocytes are unable to encapsulate parasitoid progeny, whereas in the absence of virus hemocytes rapidly encapsulate and kill the parasitoid. Likewise, by delaying or inhibiting pupation, the presence of virus appears to maintain the host in a state that facilitates proper development and successful emergence of the parasitoid. Microplitis demolitor (Hymenoptera: Braconidae) is a solitary, polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens (Lepidoptera:Noctuidae) and several other species of noctuids (Shepard et al., 1983). Expression of Microplitis demolitor polydnavirus (MdPDV) in P. includens is detectable by 4 h post-parasitization or injection and continues over the 7 day period required for completion of development by the immature wasp (Strand et al., 1992; Strand, 1994; Strand et al., 1997). Although hemocytes are the primary target of infection, MdPDV-specific mRNAs are detectable in several tissues of P. includens (Strand et al., 1992; Strand and
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Pech, 1995a). Injection of a physiological dose of MdPDV into P. includens inhibits the ability of this host to encapsulate foreign targets and prevents normal pupation (Strand and Dover, 1991; Strand and Noda, 1991). Many polydnavirus-carrying wasps appear to successfully parasitize multiple species of hosts (Strand and Obrycki, 1996). Based on our studies of M. demolitor and those conducted on other species of polydnaviruscarrying wasps, we have long assumed that the role of polydnaviruses in parasitism is to secure a suitable host environment. That is, when MdPDV infects and expresses its normal complement of transcripts in a given host species, it induces physiological alterations that are required for successful parasitoid development. Thus, hosts successfully infected by MdPDV are suitable for development of M. demolitor, whereas species unsuitable for MdPDV are not. The fall armyworm, Spodoptera frugiperda, is a noctuid species that occurs in some of the same habitats as P. includens. In the laboratory, M. demolitor readily oviposits into S. frugiperda, and these larvae exhibit developmental alterations similar to those documented in P. includens. However, no M. demolitor ever emerges from S. frugiperda. The goal of this study was to determine whether the failure of M. demolitor to develop in S. frugiperda was due to a deficiency in MdPDV transcriptional activity. Here we report that MdPDV persists in S. frugiperda for at least 7 days post-oviposition, alters growth, and disrupts encapsulation. Despite these alterations, however, all M. demolitor die because they fail to successfully complete embryogenesis. The implications of these results in defining the role of polydnaviruses in parasitism are discussed.
2. Materials and methods 2.1. Insects S. frugiperda larvae were reared on the artificial diet of Greene et al. (1976). Larvae were maintained in 30 ml plastic cups with paper lids at 27 ± 1°C and a 16 h light (L):8 h dark (D) photoperiod as described by Strand (1990). S. frugiperda in our culture pupated after six instars. Moths were fed a 20% sucrose solution. M. demolitor was reared in P. includens at 27 ± 1°C and a 16L:8D photoperiod according to Strand and Wong (1991). The mixture of ovarian proteins and virus in oviducts of female wasps is referred to as calyx fluid while material from the wasp’s poison gland is called venom. Calyx fluid and venom were collected by established methods with quantities used to inject S. frugiperda larvae expressed in wasp equivalents (Strand and Wong, 1991; Strand et al., 1992). For experiments involving parasitization, M. demolitor females were allowed to
singly parasitize 8–12 h old fourth instar S. frugiperda larvae. Newly parasitized larvae were weighed and placed individually in plastic cups half filled with diet. Every day for seven days a cohort of 10 larvae was weighed, hemolymph collected and larvae dissected. At each time point, the number and developmental stage of the parasitoid progeny present per host was recorded. The number of encapsulated eggs was also recorded. Experiments were repeated three times. For convenience, S. frugiperda larvae stung by M. demolitor are referred to as parasitized. 2.2. Encapsulation assays To obtain M. demolitor eggs free of MdPDV, ovaries were carefully teased apart from female wasps into Pringle’s saline. Using sharp forceps, ovaries were opened and the eggs within collected. After rinsing to remove any calyx fluid, 5–7 wasp eggs were injected into individual fifth instar S. frugiperda larvae (12–24 h old) using a glass needle mounted on a micro manipulator. A total of 12 injected larvae were placed individually into plastic cups half filled with diet and incubated for 8 h at 27 ± 1°C prior to dissection. Following incubation, injected larvae were anaesthesized with CO2 and dissected in Pringle’s saline. The number of unencapsulated and encapsulated eggs recovered from each larva was then recorded. Eggs were scored as fully encapsulated if hemocytes covered their entire surface in multiple layers. Eggs with no or only a few adhering hemocytes were scored as unencapsulated. Encapsulation assays were also conducted using Dowex 1X-2 chromatography beads. Five S. frugiperda larvae, Day 7 post-parasitism, were injected with 7–10 beads then placed individually into plastic cups with diet and incubated at 27 ± 1°C for 22 h. Larvae were dissected as described above with the same criteria used to score encapsulation of beads. For each larva, the number of unencapsulated and encapsulated beads present was recorded. 2.3. Hemolymph collection and cell culture Parasitized larvae (Day 1 to Day 7 post-parasitism) were anaesthetized with CO2, surface sterilized with 70% ethanol, and bled from a cut proleg directly onto a piece of parafilm. From each parasitized larva, hemolymph aliquots of 0.6 l (104–105 cells) were transferred in quadruplicate to 96-well tissue culture plates containing 70 l of TC-100 medium (Sigma) plus 2 mM glutathione. The spreading behavior of hemocytes was observed by incubating one replicate plate at room temperature for 3 h. Wells containing aliquots of hemolymph from unparasitized larvae served as positive controls. Fifty cells from a randomly selected field in each well were examined by phase-contrast microscopy and the percentage of spread cells was scored as described
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by Pech et al. (1994). Briefly, granular cells were considered as spread when they became flattened, phasedark, and attained a diameter of 20–28 m. Plasmatocytes were scored as spread when they developed a flattened, phase-dark, fibroblastic morphology and were ⱖ 35 m along their longest axis (Pech et al., 1994). Cells were fixed in 10% formalin in phosphate buffer saline (PBS) for 8 min, rinsed 3 × in PBS, and resuspended in PBS containing 0.1% sodium azide. The replicate plates to be used for in situ hybridization and immunocytochemical analyses were fixed after a 15 min incubation period. Percentage and standard deviation (s.d.) are reported. 2.4. In situ hybridization Two replicate plates were used for in situ hybridization studies. Hemocytes in each plate were probed with either the viral subgenomic clone pMd-1010 or the cDNA clone MdPi506. pMd-1010 is a 3.0 Kb Hind III MdPDV genomic fragment that hybridizes to a MdPDV DNA of 10 Kb but does not recognize any expressed sequence in parasitized S. frugiperda larvae. MdPi506 was isolated from a P. includens hemocyte cDNA library (18 h post-parasitization) and encodes 572 bp region at the 3⬘ end of a 1.5 Kb viral mRNA expressed in P. includens hemocytes from 2 h to Day 7 post-parasitism (Strand, 1994; Strand et al., 1997). The clones pMd-1010 and MdPi506 were gel purified and labeled by random priming using digoxigenin-dUTP and a commercially available kit (Genius, Boehringer-Mainnheim). The resulting probe concentrations were determined by dot blot analysis against a known standard according to the manufacturer’s instructions. In situ hybridization conditions were performed as outlined by Tautz and Pfeifle (1989) with some modifications. Modifications included incubation of cells in 50 g/ml Proteinase K for only 90 sec and hybridization with 0.1 g/ml of digoxigeninlabeled probe for 36 h at 48°C. Washed cells were incubated for 1 h at room temperature with 1:2000 antidigoxigenin conjugate in PBT(PBS plus 0.1% Tween 20) and visualized with NBT (nitro blue tetrazolium chloride) and X-phosphate solution. Controls included hemocytes from unparasitized larvae, omission of probe or conjugated antibody. Fifty cells from a randomly selected field in each well were examined using a Nikon Diaphot epifluorescence microscope with Hoffman modulation contrast optics and the percentage of positive cells was recorded. Percentages and standard deviations (s.d.) are reported. 2.5. Immunocytochemistry One replicate plate was used for immunocytochemical studies using the monoclonal antibody Mab55F2. This antibody was isolated from a panel of monoclonal anti-
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bodies generated against infected and non-infected P. includens hemocytes and recognizes virally infected hemocytes (Strand and Johnson, 1996). Briefly, fixed hemocytes were rinsed in PBS and permeabilized for 15 min in PBS plus 0.1% Triton X-100. Cells were blocked for 1 h in 3% bovine serum albumin(BSA) in PBT followed by a 1 h incubation with the monoclonal antibody Mab55F2 diluted 1:20 in PBT plus 3% BSA. After rinsing 4 × in PBT, hemocytes were incubated with FITC-conjugated goat anti-mouse secondary antibody (IgG; Kirkegaard and Perry) diluted 1:20 in PBT plus 3% BSA. Hemocytes were rinsed 4 × in PBS. Fifty cells from a randomly selected field in each well were examined as described previously and the percentage of positive cells was recorded. Percentages are reported with their standard deviation (s.d.). 2.6. RNA extraction and Northern analysis Every 24 h for seven days, cohorts of 10 parasitized larvae were anaesthetized with CO2 and bled through a proleg into 500 l of anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA and 0.041 M citric acid pH 4.5). The pooled hemolymph was then transferred to a 1.5 ml centrifuge tube and spun at 250 g for 1 min. The cell pellet was washed once in anticoagulant and total RNA isolated by homogenizing the pellet in equal volume of phenol-chloroform (1:1) and extraction buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl, 0.1% 2-mercaptoethanol and 0.1 volume of 2 M sodium acetate) as outlined by Sambrook et al. (1989). Northern blots were probed with either total MdPDV DNA, the viral genomic clone pMd1010 or the cDNA clone MdPi506. For Northern blots, total RNA (2 g/lane) from S. frugiperda hemocytes was fractionated on 6% formaldehyde, 1.0% agarose gels and transferred to nylon in 10 × SSC (Sambrook et al., 1989). Hybridization conditions followed the method outlined by Lee et al. (1992). Filters were prehybridized for 4 hours at 65°C in 1 M sodium phosphate, pH 7.2, 7.5% SDS, 1% bovine serum albumin(BSA) and 50 mM EDTA. Probes were added to filters at 0.5 × 106 cpm/ml and hybridized for 16 h at 65°C. Filters were washed under conditions of high stringency in 0.1 × SSC, 1% SDS at 65°C for 45 min.
3. Results 3.1. Development and survival of M. demolitor M. demolitor females readily parasitized 0–12 h 4th instar S. frugiperda larvae. Dissection of 30 S. frugiperda larvae immediately after oviposition revealed that female wasps laid 1–3 eggs per parasitization event with an average of 1.8 ± 0.7 eggs per larva. However, no M.
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demolitor eggs hatched. Embryogenesis proceeded up to germband extension but thereafter development ceased between 12 and 24 h (Fig. 1). Out of 210 parasitized S. frugiperda larvae dissected in the course of this study, not a single first instar parasitoid larva was observed. Unhatched eggs remained in circulation in the host’s hemocoel for several days. From Day 1 to Day 3 postparasitism, eggs were free of adhering hemocytes. By Day 4 post-oviposition, however, 50 ± 10% of parasitized S. frugiperda larvae contained encapsulated eggs. The proportion of larvae that contained encapsulated eggs increased to 80 ± 10% on Day 5 and reached 93.3 ± 5.8% by Day 7 post-parasitism (Fig. 2). To determine whether M. demolitor eggs were encapsulated in S. frugiperda in the absence of MdPDV, twelve 5th instar larvae were injected with 5–7 parasitoid eggs in the absence of virus. Eight hour post-injection, an average of 3.8 ± 1.5 encapsulated M. demolitor eggs were recovered per larva. An unencapsulated parasitoid egg was only detected in one S. frugiperda larva. No melanization was found associated with capsules of M. demolitor eggs. Whether parasitized S. frugiperda larvae could encapsulate targets other then M. demolitor eggs was investigated using Dowex 1X-2 beads. Seven to ten Dowex
Fig. 2. Mean percentage ( ± s.d.) of parasitised S. frugiperda larvae containing encapsulated parasitoid eggs. A total of ten larvae were dissected at each time point and the number of hosts that contained an encapsulated M. demolitor egg was recorded.
Fig. 1. Fate of M. demolitor eggs oviposited in S. frugiperda. (a) 12 h post-oviposition, the parasitoid egg has completed germ band extension. (b) Day 1 post-oviposition the embryo is dead and tissue histolysis has begun. (c) Day 2 post-oviposition, the egg is necrotic. (d) Day 4 postoviposition, the egg (E) is encapsulated by multiple layers of hemocytes (arrow). Bar = 145 m.
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1X-2 beads were injected into each of five 5th instar S. frugiperda larvae Day 7 post-parasitism. Twenty-two hours post-injection, an average of 5.3 ± 1.6 encapsulated beads and 1.6 ± 1.1 unencapsulated beads were recovered per larva. Out of a total of 33 beads recovered from injected S. frugiperda larvae, 75.8% were fully encapsulated whereas 24.2% were free of adhering hemocytes. Unlike M. demolitor eggs, all recovered beads were melanized whether they were encapsulated or unencapsulated.
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burrowed into the artificial diet in preparation for pupation and pupated by Day 10. In contrast, the majority of parasitized larvae molted to the 5th instar on Day 4 and reached the 6th instar on Day 7. From Day 7 on, the course of development of parasitized S. frugiperda larvae varied greatly. Out of a total of sixty parasitized S. frugiperda larvae, fifty eight percent died as either larvae or larval-pupal intermediates by Day 17 post-parasitization. The remaining larvae successfully pupated between Day 11 and 13 and gave rise to adult moths.
3.2. Development of parasitized S. frugiperda larvae 3.3. Northern analysis From Day 1 to Day 7 post-parasitism, S. frugiperda larvae fed minimally and gained little weight (Fig. 3). Parasitized larvae did not exhibit weight gain in the first two days of parasitization but showed a modest increase in weight between Day 3 and Day 7. The mean larval weight at the time of parasitization was 13.73 ± 2.59 mg and by Day 7 post-parasitization was 105.49 ± 67.65 mg. Control larvae more than tripled in weight in the first two days of the experiment and reached a maximum mean weight on Day 6 of 432.88 ± 72.71 mg. Control larvae molted to the 5th instar on Day 2 and reached the 6th instar on Day 4. By Day 7, unparasitized larvae
To determine whether expression of MdPDV transcripts could be detected in S. frugiperda hemocytes, total hemocyte RNA was isolated from parasitized fourth instar larvae, at 24 h intervals, for 7 days. Northern blots of this time course were prepared and then hybridized with selected probes. Northern analysis using 32P-labeled MdPDV DNA revealed the presence of several viral transcripts in infected hemocytes. Viral messages ranged in size from 400 bp to 3.3 Kb with a 1.5 and 3.3 Kb transcripts being the most abundant (Fig. 4a). Viral expression was strongest Day 1 to 3 post-parasitism, then sharply declined on Day 4 and continued to decline for the remainder of the sampling period. The expression profile of MdPDV was unique on Day 2 post-parasitism in that two abundant transcripts, a 2.0 Kb and a 3.3 Kb transcripts, were not present. To determine whether the cDNA clone MdPi506 was a reliable and specific marker for monitoring the expression of the 1.5 Kb transcript in infected hemocytes, a time course Northern analysis was performed with this clone. 32P-labeled MdPi506 gave a single hybridization signal that corresponded with the 1.5 Kb mRNA. The temporal expression of the 1.5 Kb mRNA followed that of the other viral transcripts detected using 32 P labeled-MdPDV DNA (Fig. 4b). Briefly, the 1.5 Kb message was most abundant Day 1 to Day 3 post-parasitization then declined on Day 4 and was barely detectable by Day 7 post-parasitization. Total RNA isolated from unparasitized larvae did not give a hybridization signal. 3.4. Hemocyte spreading behavior post-parasitism
Fig. 3. Mean weight ( ± s.d.) of parasitized (open circles) and nonparasitized (closed circles) S. frugiperda larvae Day 0–7 post-parasitism. M. demolitor females were allowed to singly parasitize seventy 4th instar (0–12 h) S. frugiperda larvae. Larvae were weighed immediately following parasitization, then placed in plastic cups with diet (one individual per cup) and maintained at 27°C ± 1°C. Every 24 h for seven days, larval weight and instar were recorded. Controls consisted of a cohort of seventy unparasitized 0–12 h 4th instar larvae placed individually in plastic cups and maintained at 27°C ± 1°C. Unparasitized larvae were weighed and staged every 24 h for seven days. Each datum point represents the mean weight of 10 larvae.
Whether parasitism affected the function of the major hemocyte morphotypes was investigated by observing the in vitro behavior of granular cells and plasmatocytes. Typically, granular cells and plasmatocytes from noctuids like S. frugiperda first adhere to the bottom of the tissue culture vessel and then spread in a morphotypespecific fashion as previously described (Strand and Pech, 1995a; Gillespie et al., 1997). To assess whether MdPDV affected the spreading behavior of S. frugiperda hemocytes, mixed hemocyte populations were collected
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Fig. 5. (a) Mean percentage ( ± s.d.) of S. frugiperda hemocytes spread Day 0–7 post-parasitism by M. demolitor. (b) Spread hemocytes from an unparasitized S. frugiperda larva. (c) Hemocytes from a parasitized larva 24 h post-parasitism. (d) Hemocytes from a parasitized larva seven days post-parasitism. Hemocytes from individual larvae were placed into individual wells of a 96 well culture plate. After 3 h, a total of 50 hemocytes was counted per well and the proportion of hemocytes that had spread was recorded. Each datum point represents the mean proportion of spread cells from 10 larvae. Bar = 40 m.
Fig. 4. (a) Hybridization of MdPDV DNA to RNA from hemocytes of parasitized S. frugiperda larvae. 32P-labelled MdPDV DNA was hybridized to Northern blots of total RNA (2 g per lane) from hemocytes from unparasitized larvae (C) and larvae Day 1 to 7 post-paratism. Size markers are shown at the left in kilobases. Blots were washed under conditions of high stringency and exposed to autoradiograph film using an intensifying screen at −80°C for 24 h. (b) Hybridization of MdPi506 to RNA from hemocytes of S. frugiperda larvae Day 1 to Day 7 post-parasitism. Insert from MdPi506 was gel purified, radiolabeled, and hybridized to Northern blots of total hemocyte RNA (2 m per lane). RNA samples and hybridization conditions were as stated above.
from nonparasitized larvae and at selected intervals Day 1 to Day 7 post-parasitism. Eighty-three percent of hemocytes from nonparasitized larvae were spread 3 h after placement in culture, whereas only 21.5 ± 8.9% of hemocytes from Day 1 parasitized larvae were spread (Fig. 5a,Fig. 5b). Neither granular cells nor plasmatocytes from Day 1 parasitized larvae were able to adhere to the bottom of the tissue culture plate and many membrane bound vesicles, suggestive of apoptosis, were present (Fig. 5c). The number of spread hemocytes in parasitized individuals steadily increased over the seven day obser-
vation period. By Day 4 post-parasitism, 53.8 ± 17.4% of hemocytes were able to adhere and spread on the foreign surface and this proportion reached near control levels seven days post-parasitism (Fig. 5d). 3.5. Viral expression in hemocytes To determine whether hemocyte dysfunction correlated with viral expression, in situ hybridization experiments were conducted using the cDNA clone MdPi506. The percentage of hemocytes positive for the 1.5 Kb mRNA was highest from Day 1 to Day 3 post-parasitism and then declined by Day 4 post-parasitization. This decline persisted over time so that by Day 7 post-parasitism only 27.5 ± 18.0% of hemocytes were labeled when probed with MdPi506 (Fig. 6a). A nuclear and cytoplasmic signal was detected in hemocytes positive for MdPi506 (Fig. 6b). This distribution is consistent with MdPi506 recognizing the 1.5 Kb transcript. No hybridization signal was detected when hemocytes from nonparasitized individuals were used or when probe or antibody were omitted (data not shown). Viral expression in hemocytes was also characterized using the monoclonal antibody Mab55F2 (Fig. 6c). The spatial and temporal pattern of viral expression obtained using Mab55F2 closely
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3.6. Persistence of viral DNA To determine whether viral DNA persisted in infected hemocytes in the absence of viral expression, in situ hybridization experiments were performed using the MdPDV genomic clone pMd-1010. In situ experiments with pMd-1010 revealed that greater than 80% of hemocytes were positive for MdPDV DNA throughout the course of parasitization (Fig. 7a). Thus unlike viral expression which declined 4 days post-parasitization, viral DNA persisted in hemocytes. Consistent with pMd1010 being a non-expressed viral sequence, only a nuclear signal was detected in hemocytes (Fig. 7b). No hybridization signal was detected in hemocytes from nonparasitized larvae or when either probe or antibody were omitted (data not shown).
4. Discussion
Fig. 5. Continued.
followed that of the in situ experiments with 70–75% of hemocytes positive in the first three days following parasitization by M. demolitor but then declining Day 4 to Day 7 post-parasitization (Fig. 6a). No labeling was observed among cells from nonparasitized individuals or when either the primary or secondary antibodies were omitted (data not shown).
We have long assumed that the developmental and immunological alterations observed in P. includens larvae, as a result of MdPDV infection, to be necessary and sufficient for parasitoid survival and development. Previous studies have shown that M. demolitor eggs, by themselves, are readily encapsulated when injected into a permissive host, like P. includens, in the absence of any wasp derived factors (Strand and Noda, 1991). However, in parasitized P. includens or P. includens injected with MdPDV, M. demolitor eggs are never encapsulated (Strand and Noda, 1991). The inability to encapsulate the parasitoid is due to the inability of plasmatocytes to spread and the death of granular cells via apoptosis. Both of these alterations in hemocyte function are due to direct infection of hemocytes by MdPDV and subsequent transcription of MdPDV specific genes (Strand et al., 1992; Strand and Pech, 1995b; Strand et al., 1997). Concurrent with immunosuppression, infected P. includens larvae exhibit a severe reduction in weight gain, temporal delays in larval to larval molts and inhibition of normal pupation (Strand and Dover, 1991; Dover et al., 1995; Balgopal et al., 1996). Host juvenilization is believed to contribute to successful parasitism by providing an adequate time frame for completion of parasitoid development whereas immunosuppression ensures wasp survival by inhibiting parasitoid encapsulation (Strand and Obrycki, 1996). As documented in P. includens, MdPDV also induced significant changes in the cellular immune system of S. frugiperda larvae. Viral transcripts detected in hemocytes Day 1 to Day 4 post-parasitism were identical in size and, with the exception of Day 2 post-parasitism, in abundance to those reported for P. includens. Consistent with viral expression, S. frugiperda hemocytes became nonadherent, incapable of spreading and did not encapsulate M. demolitor eggs in vivo. Since viral tran-
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Fig. 6. (a) Mean percentage ( ± s.d.) of S. frugiperda hemocytes positive for the monoclonal antibody Mab55F2 and hybridizing with the digoxigenin-labelled MdPi506 Day 0–7 post-parasitism. Hemocytes from individual larvae were placed into individual wells of a 96 well tissue culture plate. Following in situ hybridization or immunocytochemistry (performed as described in Materials and Methods), a total of 50 hemocytes were examined per well and the proportion of hemocytes that had a positive signal was recorded. Each datum point is the mean percentage of cells exhibiting a positive signal from 10 different larvae. (b) In situ hybridization of S. frugiperda hemocytes infected in vivo with MdPDV and hybridized with the dioxigenin-labeled MdPi506. Note that the hybridization signal is both nuclear and cytoplasmic (arrow). Uninfected cells are not labeled (asterisk) (c) S. frugiperda hemocytes infected in vivo with MdPDV and labeled with the monoclonal antibody, Mab55F2. Bar = 40 m.
scription is required for the induction of immunosuppression in many polydnavirus-host systems (Davies et al., 1987; Edson et al., 1981; Guzo and Stoltz, 1985, 1987; Stoltz and Guzo, 1986; Strand and Noda, 1991; Strand and Pech, 1995a; Asgari et al., 1996), we suggest that the alterations observed in S. frugiperda hemocytes are also likely due to MdPDV transcription. Supporting this conclusion, we found that disruption of hemocyte spreading is correlated with high levels of viral expression whereas the subsequent ability of hemocytes to spread at Day 7 post-parasitism is correlated with cessation of viral expression. In Lepidoptera, granular cells and plasmatocytes are the principal hemocyte morphotypes involved in capsule formation (reviewed by Gillespie et al., 1997). Normally, these cells circulate freely in the hemocoel in a nonadhesive state. However, upon recognition of foreigness, granular cells and plasmatocytes become activated whereby they attach to and spread on foreign surfaces such as tissue culture plates (Davies et al., 1988). In P. includens, granular cells are responsible for both the initiation and termination of encapsulation whereas plasmatocytes make up the bulk of capsules by forming concentric layers of flattened cells around the target (Pech and Strand, 1996). Although the role of individual hemocyte morphotypes in encapsulation may vary between noctuid species, all encapsulation reactions require hemocytes to change from non-adhesive cells to adhesive, flattened cells that strongly adhere to the foreign target and one another (Pech and Strand, 1995). In MdPDVinfected hemocytes, the loss of adhesive capability directly interferes with the ability of granular cells and plasmatocytes to participate in encapsulation of foreign targets, assuring survival of the M. demolitor egg or larva. Permissive hosts have traditionally been defined as those that fulfill all the developmental requirements of parasitoids. The present study suggests that, contrary to our original assumption, the ability of MdPDV to infect and express in S. frugiperda did not make this host suitable for M. demolitor development. That is, host suitability is not defined solely by the transcriptional activity
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Fig. 7. (a) Mean percentage ( ± s.d.) of S. frugiperda hemocytes hybridizing with digoxigenin-labelled pMd-1010 Day 0–7 post-parasitism. Hemocytes were collected from individual larvae at selected intervals post-parasitism (Day 1 to Day 7) and placed into individual wells of a 96 well tissue culture plate. Following in situ hybridization (performed as outlined in Materials and Methods), a total of 50 hemocytes per well were examined and the proportion of cells exhibiting a positive signal was recorded. Each datum point is the mean percentage of positive cells from 10 different larvae. (b) In situ hybridization of digoxigenin-labelled pMd-1010 to hemocytes from parasitized S. frugiperda larvae. Note that the hybridization signal is restricted to the nucleus (arrow). Uninfected cells are not labeled (asterisk). Bar = 40 m.
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and persistence of MdPDV. Studies with H. fugitivus, H. rivalis and selected polydnaviruses in L. dispar also support this finding (Guzo and Stoltz, 1985; Stoltz et al., 1986). Although MdPDV alone does not make a host suitable for M. demolitor, MdPDV gene products are still critical to successful parasitism. Several studies, indicate that polydnaviruses play an essential role in protecting their associated parasitoid from encapsulation. Successful development of Cotesia kariyai in parasitized Pseudaletia separata but not in parasitized Spodoptera litura correlated with differential infectivity of Cotesia kariyai virus for each host (Hayakawa et al., 1994). With Campoletis sonorensis, host suitability correlated with the presence of a polydnavirus gene product in the host’s hemolymph. Host species in which viral gene products could not be detected almost always encapsulated wasp progeny (Cook et al., 1984). Furthermore, Lymantria dispar which normally encapsulates Hyposoter rivalis eggs was rendered immunologically permissive with prior parasitization by an habitual parasite of this insect, the polydnavirus-carrying wasp, Cotesia melanoscela (Guzo and Stoltz, 1985). Even transient polydnavirus expression has been shown to contribute to successful parasitism. The braconid wasp, Cotesia rubecola, evades its host encapsulation response by combining passive protection and polydnavirus activity (Asgari et al., 1996). As for MdPDV, since neither the egg nor larval stages of M. demolitor appear capable of passive immune evasion, survival of wasp progeny in the absence of viral expression is unlikely (Strand and Noda, 1991, Trudeau and Strand, unpublished). Parasitized S. frugiperda larvae also exhibited symptoms characteristic of developmental arrest. A similar response to parasitism has previously been reported for P. includens and Heliothis virescens, both suitable hosts for M. demolitor (Strand and Dover, 1991; Strand et al., 1988). Developmental arrest, like immunosuppression, has been found to correlate with the transcriptional activity of several polydnaviruses including MdPDV (Beckage et al., 1987, 1994; Doucet and Cusson, 1996; Dover et al., 1988a, b; Hayakawa et al., 1994; Soller and Lanzrein, 1996; Strand and Dover, 1991; Strand and Wong, 1991; Vinson et al., 1979). Parasitized S. frugiperda larvae remained developmentally arrested even after viral expression had ceased in hemocytes. Perhaps continuous viral expression takes place in tissues or cells other than hemocytes (Strand et al., 1992; Asgari et al., 1996). Most importantly though, the effect of parasitism on S. frugiperda’s development persisted for the seven days required for wasp development. In summary, the present study suggests that successful infection of a host by a polydnavirus does not assure that the host will also be suitable for successful development of the associated parasitoid. Studies are currently underway to determine whether host species permissive
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for M. demolitor development are reciprocally always permissive to infection by MdPDV. Acknowledgements We thank J. Johnson for assistance with figures and A. Witherell for screening of an expression library with Mab55F2. This research was supported in part by the National Institute of Health grant AI32617. D. T. was a recipient of postgraduate fellowships from the Natural Sciences and Engineering Research Council of Canada (NSERC) and from Fonds pour la formation de Chercheurs et l’Aide a la Recherche (FCAR) (Canada). References Asgari, S., Hellers, M., Schmidt, O., 1996. Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2352–2662. Balgopal, M.M., Dover, B.A., Goodman, W.G., Strand, M.R., 1996. Parasitism by Microplitis demolitor induces alterations in the juvenile hormone titers and juvenile hormone esterase activity of its host Pseudoplusia includens. Journal of Insect Physiology 42, 337–345. Beckage, N.E., Templeton, T.J., Nielsen, B.D., Cook, D.I., Stoltz, D.B., 1987. Parasitism-induced hemolymph polypeptides in Manduca sexta (L.) larvae parasitized by the braconid wasp Cotesia congregata (Say). Insect Biochemistry 17, 439–455. Beckage, N.E., Tan, F.F., Schleifer, K.W., Lane, R.D., Cherubin, L.L., 1994. Characterization and biological effects of Cotesia congregata polydnavirus on host larvae of the tobacco hornworm, Manduca sexta. Archives of Insect Biochemistry and Physiology 26, 165– 195. Cook, D.I., Stoltz, D.B., Vinson, S.B., 1984. Induction of a new haemolymph glycoprotein in larvae of permissive hosts parasitized by Campoletis sonorensis. Insect Biochemistry 14, 45–50. Davies, D.H., Strand, M.R., Vinson, S.B., 1987. Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. Journal of Insect Physiology 33, 143–153. Davies, D.H., Hayes, T.K., Vinson, S.B., 1988. Preliminary characterization of in vitro encapsulation promoting factor: a peptide that mediates insect haemocyte adhesion. Developmental and Comparative Immunology 12, 241–253. Doucet, D., Cusson, M., 1996. Alteration of developmental rate and growth of Choristoneura fumiferana parasitized by Tranosema rostrale: role of calyx fluid. Entomologia experimentalis et applicata 81, 12–30. Dover, B.A., Davies, D.H., Vinson, S.B., 1988a. Degeneration of last instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus. Journal of Invertebrate Pathology 51, 80–91. Dover, B.A., Davies, D.H., Vinson, S.B., 1988b. Dose-dependent influence of Campoletis sonorensis polydnavirus on the development and ecdysteroid titers of last-instar Heliothis virescens larvae. Archives of Insect Biochemistry and Physiology 8, 113–126. Dover, B.A., Menon, A., Brown, R.C., Strand, M.R., 1995. Suppression of juvenile hormone esterase in Heliothis virescens by Microplitis demolitor calyx fluid. Journal of Insect Physiology 41, 809–817. Edson, K.M., Vinson, S.B., Stoltz, D.B., Summers, M.D., 1981. Virus in a parasitoid wasp: suppression of the cellular immune response in the parasitoid’s host. Science 211, 582–583.
Fleming, J.G.W., 1992. Polydnaviruses: mutualists and pathogens. Annual Review of Entomology 37, 401–425. Gillespie, J., Kanost, M.R., Trenczek, T., 1997. Biological mediators of insect immunity. Annual Review of Entomology 42, 611–643. Godfray, H.C.J., 1994. Parasitoids: Behavioral and Evolutionary Ecology. Princeton University Press, Princeton. 464 pp. Greene, F.L., Leppla, N.C., Dickerson, W.A., 1976. Velvetbean caterpillar: a rearing procedure and artificial medium. Journal of Economic Entomology 69, 487–488. Guzo, D., Stoltz, D.B., 1985. Obligatory multiparasitism in the tussock moth Orgyia leucostigma. Parasitology 90, 1–10. Guzo, D., Stoltz, D.B., 1987. Observations on cellular immunity and parasitism in the tussock moth. Journal of Insect Physiology 1, 19–31. Hayakawa, Y., Yazaki, K., Yamanaka, A., Tanaka, T., 1994. Expression of polydnavirus genes from the parasitoid wasp Cotesia kariyai in two noctuid hosts. Insect Molecular Biology 3, 97–103. Lee, F.-J., Moss, J., Lin, L.-W., 1992. A simplified procedure for hybridization of RNA blots. Biotechniques 13, 210–211. Pech, L.L., Strand, M.R., 1995. Encapsulation of foreign targets by hemocytes of the moth Pseudoplusia includens (Lepidoptera: Noctuidae) involves an RGD- dependent cell adhesion mechanism. Journal of Insect Physiology 41, 481–488. Pech, L.L., Strand, M.R., 1996. Granular cells are required for encapsulation of foreign targets by insect haemocytes. Journal of Cell Science 109, 2053–2060. Pech, L.L., Trudeau, D., Strand, M.R., 1994. Separation and behavior in vitro of hemocytes from the moth Pseudoplusia includens. Cell and Tissue Research 227, 159–167. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York. Shepard, M., Powell, J.E., Jones, W.A., 1983. Biology of Microplitis demolitor (Hymenoptera: Braconidae), an imported parasitoid of Heliothis (Lepidoptera: Noctuidae) spp. and the soybean looper Pseudoplusia includens (Lepidoptera: Noctuidae). Environmental Entomology 12, 641–645. Soller, M., Lanzrein, B., 1996. Polydnavirus and venom of the egglarval parasitoid Chelonus inanitus (Braconidae) induce developmental arrest in the prepupa of its host Spodoptera littoralis (Noctuidae). Journal of Insect Physiology 45, 471–481. Stoltz, D.B., 1993. The polydnavirus life cycle. In: Beckage, N.E, Thompson, S.N., Federici, B.A., (Eds.), Parasites and Pathogens of Insects, Vol. 1. Academic Press, New York, pp. 167–187. Stoltz, D.B., Guzo, D., 1986. Apparent haemocytic transformations associated with parasitoid-induced inhibition of immunity in Malacosoma disstria larvae. Journal of Insect Physiology 32, 377–388. Stoltz, D.B., Guzo, D., Cook, D., 1986. Studies on polydnavirus transmission. Virology 155, 120–131. Strand, M.R., 1990. Characterization of larval development in Pseudoplusia includens (Lepidoptera: Noctuidae). Annals of the Entomological Society of America 83, 538–544. Strand, M.R., 1994. Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens hemocytes. Journal of General Virology 75, 3007–3020. Strand, M.R., Dover, B.A., 1991. Developmental disruption of Pseudoplusia includens and Heliothis virescens larvae by the calyx fluid and venom of Microplitis demolitor. Archives of Insect Biochemistry and Physiology 18, 131–145. Strand, M.R., Noda, T., 1991. Alterations in the haemocytes of Pseudoplusia includens after parasitism by Microplitis demolitor. Journal of Insect Physiology 37, 839–850. Strand, M.R., Wong, E.A., 1991. The growth and role of Microplitis demolitor teratocytes in parasitism of Pseudoplusia includens. Journal of Insect Physiology 37, 503–515. Strand, M.R., Pech, L.L., 1995a. Immunological basis for compatibility in parasitoid-host relationships. Annual Review of Entomology 40, 31–56.
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Strand, M.R., Pech, L.L., 1995b. Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens. Journal of General Virology 76, 283–291. Strand, M.R., Johnson, J.A., 1996. Characterization of monoclonal antibodies to hemocytes of Pseudoplusia includens. Journal of Insect Physiology 42, 21–31. Strand, M.R., Obrycki, J.J., 1996. Host specificity of insect parasitoids and predators. Bioscience 46, 422–429. Strand, M.R., Johnson, J.A., Culin, J.D., 1988. Developmental interactions between the parasitoid Microplitis demolitor (Hymenoptera: Braconidae) and its host Heliothis virescens (Lepidoptera: Noctuidae). Annals of the Entomological Society of America 81, 822–830. Strand, M.R., Witherell, A., Trudeau, D., 1997. Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain. Journal of Virology 71, 2146–2156.
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Strand, M.R., McKenzie, D.I., Grassl, V., Dover, B.A., Aiken, J.M., 1992. Persistence and expression of Microplitis demolitor polydnavirus in Pseudoplusia includens. Journal of General Virology 73, 1627–1635. Tautz, D., Pfeifle, C., 1989. A non-radioactive in situ hybridization method for the localization of specific RNAs by Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85. Vinson, S.B., Edson, K.M., Stoltz, D.B., 1979. Effect of a virus associated with the reproductive system of the parasitoid wasp, Campoletis sonorensis, on host weight gain. Journal of Invertebrate Pathology 34, 133–137. Whitfield, J.B., 1990. Parasitoids, polydnaviruses and endosymbiosis. Parasitology Today 6, 381–384.
A limited role in parasitism for Microplitis demolitor polydnavirus Dominique Trudeau, M.R. Strand
*
Department of Entomology, 237 Russell Labs, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA Received 1 October 1997; accepted 23 October 1997
Abstract Spodoptera frugiperda larvae stung by Microplitis demolitor undergo physiological alterations characteristic of parasitism. However, despite these physiological modifications, parasitized S. frugiperda larvae never yield adult wasps. Our original hypothesis that unsuccessful parasitism was due to a transcriptionally inactive polydnavirus proved untrue. Microplitis demolitor polydnavirus (MdPDV) successfully infected and expressed, albeit transiently, in S. frugiperda hemocytes. MdPDV expression was most abundant in the first three days of parasitism, then sharply declined on Day 4 post-parasitization and continued to decline for the remainder of the study. During the period of MdPDV expression, S. frugiperda hemocytes were non-adherent, incapable of spreading in vitro and did not encapsulate M. demolitor eggs in vivo. Concurrent with diminishing viral expresssion, S. frugiperda hemocytes regained their ability to adhere and spread in vitro and encapsulated M. demolitor eggs in vivo. Although MdPDV disrupted S. frugiperda’s encapsulation response for the first three days post-parasitization, M. demolitor was unable to develop in this noctuid species. Failure to develop was independant of viral activity, all M. demolitor eggs oviposited in S. frugiperda larvae failed to complete embryogenesis and died within 24 hour of oviposition. S. frugiperda larvae infected with MdPDV exhibited alterations in development very similar to other lepidopterans that are permissive hosts for M. demolitor. In addition, MdPDV DNA persisted in Spodoptera frugiperda hemocytes in the absence of viral expression. 1998 Elsevier Science Ltd. All rights reserved. Keywords: Parasitoid; Immunity; Hemocytes; Host suitability
1. Introduction Parasitoids encounter many potential hosts when foraging. Some of these species will be completely suitable for offspring development, others will be marginally suitable, and most will be unsuitable (Godfray, 1994). Many parasitoids in the families Braconidae and Ichneumonidae carry polydnaviruses which replicate in the reproductive tract of female wasps and are injected into potential hosts along with one or more eggs at oviposition (Stoltz, 1993). In suitable hosts, polydnaviruses enter a variety of tissues with transcription in the apparent absence of replication occurring over the period required for the wasp’s progeny to complete development (Strand and Pech, 1995a). Transcriptionally active polydnaviruses induce several physiological alterations in hosts that are considered important for survival of the wasp’s progeny. Most notably, polydnaviruses have
* Corresponding author. Tel.: 1 608 262 6902; Fax: 1 608 2623322; E-mail: [email protected] 0022–1910 /98 /$19.00 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 1 2 - 2
immunosuppressive and juvenilizing effects on hosts (Whitfield, 1990; Fleming, 1992). In the presence of virus, host hemocytes are unable to encapsulate parasitoid progeny, whereas in the absence of virus hemocytes rapidly encapsulate and kill the parasitoid. Likewise, by delaying or inhibiting pupation, the presence of virus appears to maintain the host in a state that facilitates proper development and successful emergence of the parasitoid. Microplitis demolitor (Hymenoptera: Braconidae) is a solitary, polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens (Lepidoptera:Noctuidae) and several other species of noctuids (Shepard et al., 1983). Expression of Microplitis demolitor polydnavirus (MdPDV) in P. includens is detectable by 4 h post-parasitization or injection and continues over the 7 day period required for completion of development by the immature wasp (Strand et al., 1992; Strand, 1994; Strand et al., 1997). Although hemocytes are the primary target of infection, MdPDV-specific mRNAs are detectable in several tissues of P. includens (Strand et al., 1992; Strand and
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Pech, 1995a). Injection of a physiological dose of MdPDV into P. includens inhibits the ability of this host to encapsulate foreign targets and prevents normal pupation (Strand and Dover, 1991; Strand and Noda, 1991). Many polydnavirus-carrying wasps appear to successfully parasitize multiple species of hosts (Strand and Obrycki, 1996). Based on our studies of M. demolitor and those conducted on other species of polydnaviruscarrying wasps, we have long assumed that the role of polydnaviruses in parasitism is to secure a suitable host environment. That is, when MdPDV infects and expresses its normal complement of transcripts in a given host species, it induces physiological alterations that are required for successful parasitoid development. Thus, hosts successfully infected by MdPDV are suitable for development of M. demolitor, whereas species unsuitable for MdPDV are not. The fall armyworm, Spodoptera frugiperda, is a noctuid species that occurs in some of the same habitats as P. includens. In the laboratory, M. demolitor readily oviposits into S. frugiperda, and these larvae exhibit developmental alterations similar to those documented in P. includens. However, no M. demolitor ever emerges from S. frugiperda. The goal of this study was to determine whether the failure of M. demolitor to develop in S. frugiperda was due to a deficiency in MdPDV transcriptional activity. Here we report that MdPDV persists in S. frugiperda for at least 7 days post-oviposition, alters growth, and disrupts encapsulation. Despite these alterations, however, all M. demolitor die because they fail to successfully complete embryogenesis. The implications of these results in defining the role of polydnaviruses in parasitism are discussed.
2. Materials and methods 2.1. Insects S. frugiperda larvae were reared on the artificial diet of Greene et al. (1976). Larvae were maintained in 30 ml plastic cups with paper lids at 27 ± 1°C and a 16 h light (L):8 h dark (D) photoperiod as described by Strand (1990). S. frugiperda in our culture pupated after six instars. Moths were fed a 20% sucrose solution. M. demolitor was reared in P. includens at 27 ± 1°C and a 16L:8D photoperiod according to Strand and Wong (1991). The mixture of ovarian proteins and virus in oviducts of female wasps is referred to as calyx fluid while material from the wasp’s poison gland is called venom. Calyx fluid and venom were collected by established methods with quantities used to inject S. frugiperda larvae expressed in wasp equivalents (Strand and Wong, 1991; Strand et al., 1992). For experiments involving parasitization, M. demolitor females were allowed to
singly parasitize 8–12 h old fourth instar S. frugiperda larvae. Newly parasitized larvae were weighed and placed individually in plastic cups half filled with diet. Every day for seven days a cohort of 10 larvae was weighed, hemolymph collected and larvae dissected. At each time point, the number and developmental stage of the parasitoid progeny present per host was recorded. The number of encapsulated eggs was also recorded. Experiments were repeated three times. For convenience, S. frugiperda larvae stung by M. demolitor are referred to as parasitized. 2.2. Encapsulation assays To obtain M. demolitor eggs free of MdPDV, ovaries were carefully teased apart from female wasps into Pringle’s saline. Using sharp forceps, ovaries were opened and the eggs within collected. After rinsing to remove any calyx fluid, 5–7 wasp eggs were injected into individual fifth instar S. frugiperda larvae (12–24 h old) using a glass needle mounted on a micro manipulator. A total of 12 injected larvae were placed individually into plastic cups half filled with diet and incubated for 8 h at 27 ± 1°C prior to dissection. Following incubation, injected larvae were anaesthesized with CO2 and dissected in Pringle’s saline. The number of unencapsulated and encapsulated eggs recovered from each larva was then recorded. Eggs were scored as fully encapsulated if hemocytes covered their entire surface in multiple layers. Eggs with no or only a few adhering hemocytes were scored as unencapsulated. Encapsulation assays were also conducted using Dowex 1X-2 chromatography beads. Five S. frugiperda larvae, Day 7 post-parasitism, were injected with 7–10 beads then placed individually into plastic cups with diet and incubated at 27 ± 1°C for 22 h. Larvae were dissected as described above with the same criteria used to score encapsulation of beads. For each larva, the number of unencapsulated and encapsulated beads present was recorded. 2.3. Hemolymph collection and cell culture Parasitized larvae (Day 1 to Day 7 post-parasitism) were anaesthetized with CO2, surface sterilized with 70% ethanol, and bled from a cut proleg directly onto a piece of parafilm. From each parasitized larva, hemolymph aliquots of 0.6 l (104–105 cells) were transferred in quadruplicate to 96-well tissue culture plates containing 70 l of TC-100 medium (Sigma) plus 2 mM glutathione. The spreading behavior of hemocytes was observed by incubating one replicate plate at room temperature for 3 h. Wells containing aliquots of hemolymph from unparasitized larvae served as positive controls. Fifty cells from a randomly selected field in each well were examined by phase-contrast microscopy and the percentage of spread cells was scored as described
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by Pech et al. (1994). Briefly, granular cells were considered as spread when they became flattened, phasedark, and attained a diameter of 20–28 m. Plasmatocytes were scored as spread when they developed a flattened, phase-dark, fibroblastic morphology and were ⱖ 35 m along their longest axis (Pech et al., 1994). Cells were fixed in 10% formalin in phosphate buffer saline (PBS) for 8 min, rinsed 3 × in PBS, and resuspended in PBS containing 0.1% sodium azide. The replicate plates to be used for in situ hybridization and immunocytochemical analyses were fixed after a 15 min incubation period. Percentage and standard deviation (s.d.) are reported. 2.4. In situ hybridization Two replicate plates were used for in situ hybridization studies. Hemocytes in each plate were probed with either the viral subgenomic clone pMd-1010 or the cDNA clone MdPi506. pMd-1010 is a 3.0 Kb Hind III MdPDV genomic fragment that hybridizes to a MdPDV DNA of 10 Kb but does not recognize any expressed sequence in parasitized S. frugiperda larvae. MdPi506 was isolated from a P. includens hemocyte cDNA library (18 h post-parasitization) and encodes 572 bp region at the 3⬘ end of a 1.5 Kb viral mRNA expressed in P. includens hemocytes from 2 h to Day 7 post-parasitism (Strand, 1994; Strand et al., 1997). The clones pMd-1010 and MdPi506 were gel purified and labeled by random priming using digoxigenin-dUTP and a commercially available kit (Genius, Boehringer-Mainnheim). The resulting probe concentrations were determined by dot blot analysis against a known standard according to the manufacturer’s instructions. In situ hybridization conditions were performed as outlined by Tautz and Pfeifle (1989) with some modifications. Modifications included incubation of cells in 50 g/ml Proteinase K for only 90 sec and hybridization with 0.1 g/ml of digoxigeninlabeled probe for 36 h at 48°C. Washed cells were incubated for 1 h at room temperature with 1:2000 antidigoxigenin conjugate in PBT(PBS plus 0.1% Tween 20) and visualized with NBT (nitro blue tetrazolium chloride) and X-phosphate solution. Controls included hemocytes from unparasitized larvae, omission of probe or conjugated antibody. Fifty cells from a randomly selected field in each well were examined using a Nikon Diaphot epifluorescence microscope with Hoffman modulation contrast optics and the percentage of positive cells was recorded. Percentages and standard deviations (s.d.) are reported. 2.5. Immunocytochemistry One replicate plate was used for immunocytochemical studies using the monoclonal antibody Mab55F2. This antibody was isolated from a panel of monoclonal anti-
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bodies generated against infected and non-infected P. includens hemocytes and recognizes virally infected hemocytes (Strand and Johnson, 1996). Briefly, fixed hemocytes were rinsed in PBS and permeabilized for 15 min in PBS plus 0.1% Triton X-100. Cells were blocked for 1 h in 3% bovine serum albumin(BSA) in PBT followed by a 1 h incubation with the monoclonal antibody Mab55F2 diluted 1:20 in PBT plus 3% BSA. After rinsing 4 × in PBT, hemocytes were incubated with FITC-conjugated goat anti-mouse secondary antibody (IgG; Kirkegaard and Perry) diluted 1:20 in PBT plus 3% BSA. Hemocytes were rinsed 4 × in PBS. Fifty cells from a randomly selected field in each well were examined as described previously and the percentage of positive cells was recorded. Percentages are reported with their standard deviation (s.d.). 2.6. RNA extraction and Northern analysis Every 24 h for seven days, cohorts of 10 parasitized larvae were anaesthetized with CO2 and bled through a proleg into 500 l of anticoagulant buffer (0.098 M NaOH, 0.186 M NaCl, 0.017 M EDTA and 0.041 M citric acid pH 4.5). The pooled hemolymph was then transferred to a 1.5 ml centrifuge tube and spun at 250 g for 1 min. The cell pellet was washed once in anticoagulant and total RNA isolated by homogenizing the pellet in equal volume of phenol-chloroform (1:1) and extraction buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl, 0.1% 2-mercaptoethanol and 0.1 volume of 2 M sodium acetate) as outlined by Sambrook et al. (1989). Northern blots were probed with either total MdPDV DNA, the viral genomic clone pMd1010 or the cDNA clone MdPi506. For Northern blots, total RNA (2 g/lane) from S. frugiperda hemocytes was fractionated on 6% formaldehyde, 1.0% agarose gels and transferred to nylon in 10 × SSC (Sambrook et al., 1989). Hybridization conditions followed the method outlined by Lee et al. (1992). Filters were prehybridized for 4 hours at 65°C in 1 M sodium phosphate, pH 7.2, 7.5% SDS, 1% bovine serum albumin(BSA) and 50 mM EDTA. Probes were added to filters at 0.5 × 106 cpm/ml and hybridized for 16 h at 65°C. Filters were washed under conditions of high stringency in 0.1 × SSC, 1% SDS at 65°C for 45 min.
3. Results 3.1. Development and survival of M. demolitor M. demolitor females readily parasitized 0–12 h 4th instar S. frugiperda larvae. Dissection of 30 S. frugiperda larvae immediately after oviposition revealed that female wasps laid 1–3 eggs per parasitization event with an average of 1.8 ± 0.7 eggs per larva. However, no M.
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demolitor eggs hatched. Embryogenesis proceeded up to germband extension but thereafter development ceased between 12 and 24 h (Fig. 1). Out of 210 parasitized S. frugiperda larvae dissected in the course of this study, not a single first instar parasitoid larva was observed. Unhatched eggs remained in circulation in the host’s hemocoel for several days. From Day 1 to Day 3 postparasitism, eggs were free of adhering hemocytes. By Day 4 post-oviposition, however, 50 ± 10% of parasitized S. frugiperda larvae contained encapsulated eggs. The proportion of larvae that contained encapsulated eggs increased to 80 ± 10% on Day 5 and reached 93.3 ± 5.8% by Day 7 post-parasitism (Fig. 2). To determine whether M. demolitor eggs were encapsulated in S. frugiperda in the absence of MdPDV, twelve 5th instar larvae were injected with 5–7 parasitoid eggs in the absence of virus. Eight hour post-injection, an average of 3.8 ± 1.5 encapsulated M. demolitor eggs were recovered per larva. An unencapsulated parasitoid egg was only detected in one S. frugiperda larva. No melanization was found associated with capsules of M. demolitor eggs. Whether parasitized S. frugiperda larvae could encapsulate targets other then M. demolitor eggs was investigated using Dowex 1X-2 beads. Seven to ten Dowex
Fig. 2. Mean percentage ( ± s.d.) of parasitised S. frugiperda larvae containing encapsulated parasitoid eggs. A total of ten larvae were dissected at each time point and the number of hosts that contained an encapsulated M. demolitor egg was recorded.
Fig. 1. Fate of M. demolitor eggs oviposited in S. frugiperda. (a) 12 h post-oviposition, the parasitoid egg has completed germ band extension. (b) Day 1 post-oviposition the embryo is dead and tissue histolysis has begun. (c) Day 2 post-oviposition, the egg is necrotic. (d) Day 4 postoviposition, the egg (E) is encapsulated by multiple layers of hemocytes (arrow). Bar = 145 m.
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1X-2 beads were injected into each of five 5th instar S. frugiperda larvae Day 7 post-parasitism. Twenty-two hours post-injection, an average of 5.3 ± 1.6 encapsulated beads and 1.6 ± 1.1 unencapsulated beads were recovered per larva. Out of a total of 33 beads recovered from injected S. frugiperda larvae, 75.8% were fully encapsulated whereas 24.2% were free of adhering hemocytes. Unlike M. demolitor eggs, all recovered beads were melanized whether they were encapsulated or unencapsulated.
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burrowed into the artificial diet in preparation for pupation and pupated by Day 10. In contrast, the majority of parasitized larvae molted to the 5th instar on Day 4 and reached the 6th instar on Day 7. From Day 7 on, the course of development of parasitized S. frugiperda larvae varied greatly. Out of a total of sixty parasitized S. frugiperda larvae, fifty eight percent died as either larvae or larval-pupal intermediates by Day 17 post-parasitization. The remaining larvae successfully pupated between Day 11 and 13 and gave rise to adult moths.
3.2. Development of parasitized S. frugiperda larvae 3.3. Northern analysis From Day 1 to Day 7 post-parasitism, S. frugiperda larvae fed minimally and gained little weight (Fig. 3). Parasitized larvae did not exhibit weight gain in the first two days of parasitization but showed a modest increase in weight between Day 3 and Day 7. The mean larval weight at the time of parasitization was 13.73 ± 2.59 mg and by Day 7 post-parasitization was 105.49 ± 67.65 mg. Control larvae more than tripled in weight in the first two days of the experiment and reached a maximum mean weight on Day 6 of 432.88 ± 72.71 mg. Control larvae molted to the 5th instar on Day 2 and reached the 6th instar on Day 4. By Day 7, unparasitized larvae
To determine whether expression of MdPDV transcripts could be detected in S. frugiperda hemocytes, total hemocyte RNA was isolated from parasitized fourth instar larvae, at 24 h intervals, for 7 days. Northern blots of this time course were prepared and then hybridized with selected probes. Northern analysis using 32P-labeled MdPDV DNA revealed the presence of several viral transcripts in infected hemocytes. Viral messages ranged in size from 400 bp to 3.3 Kb with a 1.5 and 3.3 Kb transcripts being the most abundant (Fig. 4a). Viral expression was strongest Day 1 to 3 post-parasitism, then sharply declined on Day 4 and continued to decline for the remainder of the sampling period. The expression profile of MdPDV was unique on Day 2 post-parasitism in that two abundant transcripts, a 2.0 Kb and a 3.3 Kb transcripts, were not present. To determine whether the cDNA clone MdPi506 was a reliable and specific marker for monitoring the expression of the 1.5 Kb transcript in infected hemocytes, a time course Northern analysis was performed with this clone. 32P-labeled MdPi506 gave a single hybridization signal that corresponded with the 1.5 Kb mRNA. The temporal expression of the 1.5 Kb mRNA followed that of the other viral transcripts detected using 32 P labeled-MdPDV DNA (Fig. 4b). Briefly, the 1.5 Kb message was most abundant Day 1 to Day 3 post-parasitization then declined on Day 4 and was barely detectable by Day 7 post-parasitization. Total RNA isolated from unparasitized larvae did not give a hybridization signal. 3.4. Hemocyte spreading behavior post-parasitism
Fig. 3. Mean weight ( ± s.d.) of parasitized (open circles) and nonparasitized (closed circles) S. frugiperda larvae Day 0–7 post-parasitism. M. demolitor females were allowed to singly parasitize seventy 4th instar (0–12 h) S. frugiperda larvae. Larvae were weighed immediately following parasitization, then placed in plastic cups with diet (one individual per cup) and maintained at 27°C ± 1°C. Every 24 h for seven days, larval weight and instar were recorded. Controls consisted of a cohort of seventy unparasitized 0–12 h 4th instar larvae placed individually in plastic cups and maintained at 27°C ± 1°C. Unparasitized larvae were weighed and staged every 24 h for seven days. Each datum point represents the mean weight of 10 larvae.
Whether parasitism affected the function of the major hemocyte morphotypes was investigated by observing the in vitro behavior of granular cells and plasmatocytes. Typically, granular cells and plasmatocytes from noctuids like S. frugiperda first adhere to the bottom of the tissue culture vessel and then spread in a morphotypespecific fashion as previously described (Strand and Pech, 1995a; Gillespie et al., 1997). To assess whether MdPDV affected the spreading behavior of S. frugiperda hemocytes, mixed hemocyte populations were collected
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Fig. 5. (a) Mean percentage ( ± s.d.) of S. frugiperda hemocytes spread Day 0–7 post-parasitism by M. demolitor. (b) Spread hemocytes from an unparasitized S. frugiperda larva. (c) Hemocytes from a parasitized larva 24 h post-parasitism. (d) Hemocytes from a parasitized larva seven days post-parasitism. Hemocytes from individual larvae were placed into individual wells of a 96 well culture plate. After 3 h, a total of 50 hemocytes was counted per well and the proportion of hemocytes that had spread was recorded. Each datum point represents the mean proportion of spread cells from 10 larvae. Bar = 40 m.
Fig. 4. (a) Hybridization of MdPDV DNA to RNA from hemocytes of parasitized S. frugiperda larvae. 32P-labelled MdPDV DNA was hybridized to Northern blots of total RNA (2 g per lane) from hemocytes from unparasitized larvae (C) and larvae Day 1 to 7 post-paratism. Size markers are shown at the left in kilobases. Blots were washed under conditions of high stringency and exposed to autoradiograph film using an intensifying screen at −80°C for 24 h. (b) Hybridization of MdPi506 to RNA from hemocytes of S. frugiperda larvae Day 1 to Day 7 post-parasitism. Insert from MdPi506 was gel purified, radiolabeled, and hybridized to Northern blots of total hemocyte RNA (2 m per lane). RNA samples and hybridization conditions were as stated above.
from nonparasitized larvae and at selected intervals Day 1 to Day 7 post-parasitism. Eighty-three percent of hemocytes from nonparasitized larvae were spread 3 h after placement in culture, whereas only 21.5 ± 8.9% of hemocytes from Day 1 parasitized larvae were spread (Fig. 5a,Fig. 5b). Neither granular cells nor plasmatocytes from Day 1 parasitized larvae were able to adhere to the bottom of the tissue culture plate and many membrane bound vesicles, suggestive of apoptosis, were present (Fig. 5c). The number of spread hemocytes in parasitized individuals steadily increased over the seven day obser-
vation period. By Day 4 post-parasitism, 53.8 ± 17.4% of hemocytes were able to adhere and spread on the foreign surface and this proportion reached near control levels seven days post-parasitism (Fig. 5d). 3.5. Viral expression in hemocytes To determine whether hemocyte dysfunction correlated with viral expression, in situ hybridization experiments were conducted using the cDNA clone MdPi506. The percentage of hemocytes positive for the 1.5 Kb mRNA was highest from Day 1 to Day 3 post-parasitism and then declined by Day 4 post-parasitization. This decline persisted over time so that by Day 7 post-parasitism only 27.5 ± 18.0% of hemocytes were labeled when probed with MdPi506 (Fig. 6a). A nuclear and cytoplasmic signal was detected in hemocytes positive for MdPi506 (Fig. 6b). This distribution is consistent with MdPi506 recognizing the 1.5 Kb transcript. No hybridization signal was detected when hemocytes from nonparasitized individuals were used or when probe or antibody were omitted (data not shown). Viral expression in hemocytes was also characterized using the monoclonal antibody Mab55F2 (Fig. 6c). The spatial and temporal pattern of viral expression obtained using Mab55F2 closely
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3.6. Persistence of viral DNA To determine whether viral DNA persisted in infected hemocytes in the absence of viral expression, in situ hybridization experiments were performed using the MdPDV genomic clone pMd-1010. In situ experiments with pMd-1010 revealed that greater than 80% of hemocytes were positive for MdPDV DNA throughout the course of parasitization (Fig. 7a). Thus unlike viral expression which declined 4 days post-parasitization, viral DNA persisted in hemocytes. Consistent with pMd1010 being a non-expressed viral sequence, only a nuclear signal was detected in hemocytes (Fig. 7b). No hybridization signal was detected in hemocytes from nonparasitized larvae or when either probe or antibody were omitted (data not shown).
4. Discussion
Fig. 5. Continued.
followed that of the in situ experiments with 70–75% of hemocytes positive in the first three days following parasitization by M. demolitor but then declining Day 4 to Day 7 post-parasitization (Fig. 6a). No labeling was observed among cells from nonparasitized individuals or when either the primary or secondary antibodies were omitted (data not shown).
We have long assumed that the developmental and immunological alterations observed in P. includens larvae, as a result of MdPDV infection, to be necessary and sufficient for parasitoid survival and development. Previous studies have shown that M. demolitor eggs, by themselves, are readily encapsulated when injected into a permissive host, like P. includens, in the absence of any wasp derived factors (Strand and Noda, 1991). However, in parasitized P. includens or P. includens injected with MdPDV, M. demolitor eggs are never encapsulated (Strand and Noda, 1991). The inability to encapsulate the parasitoid is due to the inability of plasmatocytes to spread and the death of granular cells via apoptosis. Both of these alterations in hemocyte function are due to direct infection of hemocytes by MdPDV and subsequent transcription of MdPDV specific genes (Strand et al., 1992; Strand and Pech, 1995b; Strand et al., 1997). Concurrent with immunosuppression, infected P. includens larvae exhibit a severe reduction in weight gain, temporal delays in larval to larval molts and inhibition of normal pupation (Strand and Dover, 1991; Dover et al., 1995; Balgopal et al., 1996). Host juvenilization is believed to contribute to successful parasitism by providing an adequate time frame for completion of parasitoid development whereas immunosuppression ensures wasp survival by inhibiting parasitoid encapsulation (Strand and Obrycki, 1996). As documented in P. includens, MdPDV also induced significant changes in the cellular immune system of S. frugiperda larvae. Viral transcripts detected in hemocytes Day 1 to Day 4 post-parasitism were identical in size and, with the exception of Day 2 post-parasitism, in abundance to those reported for P. includens. Consistent with viral expression, S. frugiperda hemocytes became nonadherent, incapable of spreading and did not encapsulate M. demolitor eggs in vivo. Since viral tran-
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Fig. 6. (a) Mean percentage ( ± s.d.) of S. frugiperda hemocytes positive for the monoclonal antibody Mab55F2 and hybridizing with the digoxigenin-labelled MdPi506 Day 0–7 post-parasitism. Hemocytes from individual larvae were placed into individual wells of a 96 well tissue culture plate. Following in situ hybridization or immunocytochemistry (performed as described in Materials and Methods), a total of 50 hemocytes were examined per well and the proportion of hemocytes that had a positive signal was recorded. Each datum point is the mean percentage of cells exhibiting a positive signal from 10 different larvae. (b) In situ hybridization of S. frugiperda hemocytes infected in vivo with MdPDV and hybridized with the dioxigenin-labeled MdPi506. Note that the hybridization signal is both nuclear and cytoplasmic (arrow). Uninfected cells are not labeled (asterisk) (c) S. frugiperda hemocytes infected in vivo with MdPDV and labeled with the monoclonal antibody, Mab55F2. Bar = 40 m.
scription is required for the induction of immunosuppression in many polydnavirus-host systems (Davies et al., 1987; Edson et al., 1981; Guzo and Stoltz, 1985, 1987; Stoltz and Guzo, 1986; Strand and Noda, 1991; Strand and Pech, 1995a; Asgari et al., 1996), we suggest that the alterations observed in S. frugiperda hemocytes are also likely due to MdPDV transcription. Supporting this conclusion, we found that disruption of hemocyte spreading is correlated with high levels of viral expression whereas the subsequent ability of hemocytes to spread at Day 7 post-parasitism is correlated with cessation of viral expression. In Lepidoptera, granular cells and plasmatocytes are the principal hemocyte morphotypes involved in capsule formation (reviewed by Gillespie et al., 1997). Normally, these cells circulate freely in the hemocoel in a nonadhesive state. However, upon recognition of foreigness, granular cells and plasmatocytes become activated whereby they attach to and spread on foreign surfaces such as tissue culture plates (Davies et al., 1988). In P. includens, granular cells are responsible for both the initiation and termination of encapsulation whereas plasmatocytes make up the bulk of capsules by forming concentric layers of flattened cells around the target (Pech and Strand, 1996). Although the role of individual hemocyte morphotypes in encapsulation may vary between noctuid species, all encapsulation reactions require hemocytes to change from non-adhesive cells to adhesive, flattened cells that strongly adhere to the foreign target and one another (Pech and Strand, 1995). In MdPDVinfected hemocytes, the loss of adhesive capability directly interferes with the ability of granular cells and plasmatocytes to participate in encapsulation of foreign targets, assuring survival of the M. demolitor egg or larva. Permissive hosts have traditionally been defined as those that fulfill all the developmental requirements of parasitoids. The present study suggests that, contrary to our original assumption, the ability of MdPDV to infect and express in S. frugiperda did not make this host suitable for M. demolitor development. That is, host suitability is not defined solely by the transcriptional activity
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Fig. 7. (a) Mean percentage ( ± s.d.) of S. frugiperda hemocytes hybridizing with digoxigenin-labelled pMd-1010 Day 0–7 post-parasitism. Hemocytes were collected from individual larvae at selected intervals post-parasitism (Day 1 to Day 7) and placed into individual wells of a 96 well tissue culture plate. Following in situ hybridization (performed as outlined in Materials and Methods), a total of 50 hemocytes per well were examined and the proportion of cells exhibiting a positive signal was recorded. Each datum point is the mean percentage of positive cells from 10 different larvae. (b) In situ hybridization of digoxigenin-labelled pMd-1010 to hemocytes from parasitized S. frugiperda larvae. Note that the hybridization signal is restricted to the nucleus (arrow). Uninfected cells are not labeled (asterisk). Bar = 40 m.
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and persistence of MdPDV. Studies with H. fugitivus, H. rivalis and selected polydnaviruses in L. dispar also support this finding (Guzo and Stoltz, 1985; Stoltz et al., 1986). Although MdPDV alone does not make a host suitable for M. demolitor, MdPDV gene products are still critical to successful parasitism. Several studies, indicate that polydnaviruses play an essential role in protecting their associated parasitoid from encapsulation. Successful development of Cotesia kariyai in parasitized Pseudaletia separata but not in parasitized Spodoptera litura correlated with differential infectivity of Cotesia kariyai virus for each host (Hayakawa et al., 1994). With Campoletis sonorensis, host suitability correlated with the presence of a polydnavirus gene product in the host’s hemolymph. Host species in which viral gene products could not be detected almost always encapsulated wasp progeny (Cook et al., 1984). Furthermore, Lymantria dispar which normally encapsulates Hyposoter rivalis eggs was rendered immunologically permissive with prior parasitization by an habitual parasite of this insect, the polydnavirus-carrying wasp, Cotesia melanoscela (Guzo and Stoltz, 1985). Even transient polydnavirus expression has been shown to contribute to successful parasitism. The braconid wasp, Cotesia rubecola, evades its host encapsulation response by combining passive protection and polydnavirus activity (Asgari et al., 1996). As for MdPDV, since neither the egg nor larval stages of M. demolitor appear capable of passive immune evasion, survival of wasp progeny in the absence of viral expression is unlikely (Strand and Noda, 1991, Trudeau and Strand, unpublished). Parasitized S. frugiperda larvae also exhibited symptoms characteristic of developmental arrest. A similar response to parasitism has previously been reported for P. includens and Heliothis virescens, both suitable hosts for M. demolitor (Strand and Dover, 1991; Strand et al., 1988). Developmental arrest, like immunosuppression, has been found to correlate with the transcriptional activity of several polydnaviruses including MdPDV (Beckage et al., 1987, 1994; Doucet and Cusson, 1996; Dover et al., 1988a, b; Hayakawa et al., 1994; Soller and Lanzrein, 1996; Strand and Dover, 1991; Strand and Wong, 1991; Vinson et al., 1979). Parasitized S. frugiperda larvae remained developmentally arrested even after viral expression had ceased in hemocytes. Perhaps continuous viral expression takes place in tissues or cells other than hemocytes (Strand et al., 1992; Asgari et al., 1996). Most importantly though, the effect of parasitism on S. frugiperda’s development persisted for the seven days required for wasp development. In summary, the present study suggests that successful infection of a host by a polydnavirus does not assure that the host will also be suitable for successful development of the associated parasitoid. Studies are currently underway to determine whether host species permissive
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for M. demolitor development are reciprocally always permissive to infection by MdPDV. Acknowledgements We thank J. Johnson for assistance with figures and A. Witherell for screening of an expression library with Mab55F2. This research was supported in part by the National Institute of Health grant AI32617. D. T. was a recipient of postgraduate fellowships from the Natural Sciences and Engineering Research Council of Canada (NSERC) and from Fonds pour la formation de Chercheurs et l’Aide a la Recherche (FCAR) (Canada). References Asgari, S., Hellers, M., Schmidt, O., 1996. Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2352–2662. Balgopal, M.M., Dover, B.A., Goodman, W.G., Strand, M.R., 1996. Parasitism by Microplitis demolitor induces alterations in the juvenile hormone titers and juvenile hormone esterase activity of its host Pseudoplusia includens. Journal of Insect Physiology 42, 337–345. Beckage, N.E., Templeton, T.J., Nielsen, B.D., Cook, D.I., Stoltz, D.B., 1987. Parasitism-induced hemolymph polypeptides in Manduca sexta (L.) larvae parasitized by the braconid wasp Cotesia congregata (Say). Insect Biochemistry 17, 439–455. Beckage, N.E., Tan, F.F., Schleifer, K.W., Lane, R.D., Cherubin, L.L., 1994. Characterization and biological effects of Cotesia congregata polydnavirus on host larvae of the tobacco hornworm, Manduca sexta. Archives of Insect Biochemistry and Physiology 26, 165– 195. Cook, D.I., Stoltz, D.B., Vinson, S.B., 1984. Induction of a new haemolymph glycoprotein in larvae of permissive hosts parasitized by Campoletis sonorensis. Insect Biochemistry 14, 45–50. Davies, D.H., Strand, M.R., Vinson, S.B., 1987. Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. Journal of Insect Physiology 33, 143–153. Davies, D.H., Hayes, T.K., Vinson, S.B., 1988. Preliminary characterization of in vitro encapsulation promoting factor: a peptide that mediates insect haemocyte adhesion. Developmental and Comparative Immunology 12, 241–253. Doucet, D., Cusson, M., 1996. Alteration of developmental rate and growth of Choristoneura fumiferana parasitized by Tranosema rostrale: role of calyx fluid. Entomologia experimentalis et applicata 81, 12–30. Dover, B.A., Davies, D.H., Vinson, S.B., 1988a. Degeneration of last instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus. Journal of Invertebrate Pathology 51, 80–91. Dover, B.A., Davies, D.H., Vinson, S.B., 1988b. Dose-dependent influence of Campoletis sonorensis polydnavirus on the development and ecdysteroid titers of last-instar Heliothis virescens larvae. Archives of Insect Biochemistry and Physiology 8, 113–126. Dover, B.A., Menon, A., Brown, R.C., Strand, M.R., 1995. Suppression of juvenile hormone esterase in Heliothis virescens by Microplitis demolitor calyx fluid. Journal of Insect Physiology 41, 809–817. Edson, K.M., Vinson, S.B., Stoltz, D.B., Summers, M.D., 1981. Virus in a parasitoid wasp: suppression of the cellular immune response in the parasitoid’s host. Science 211, 582–583.
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