Characterization of two novel Microplitis demolitor polydnavirus mRNAs expressed in Pseudoplusia includens haemocytes

D. Trudeaub,1, R. A. Witherell1 and M. R. Strand1

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA1

Author for correspondence: Mike Strand. Fax +1 608 262 3322. e-mail mrstrand{at}facstaff.wisc.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The braconid wasp Microplitis demolitor carries M. demolitor polydnavirus (MdPDV) and parasitizes the larval stage of the moth Pseudoplusia includens. M. demolitor injects MdPDV into P. includens larvae when it lays an egg and the virus infects various cells including haemocytes. Two new MdPDV transcripts expressed in host haemocytes were characterized in this study. Screening of an MdPDV-infected haemocyte cDNA library identified a 0·4 kb cDNA encoding a predicted protein of 103 amino acids which was named Egf0·4. This protein contained a cysteine-rich epidermal growth factor (EGF)-like motif at its N terminus that was similar to the EGF-like domains in the previously identified MdPDV genes egf1·5 and egf1·0. Sequencing of the genomic clone pMd-10 indicated that it contained the egf0·4 gene, which consisted of two introns and three exons. This gene was located on MdPDV segment O and appeared to exist in multiple copies. A nucleic acid and expression screen identified a 1·8 kb cDNA encoding a predicted protein of 515 amino acids designated Glc1·8. This protein consisted of a heavily glycosylated central core of six tandemly arranged repeats flanked by hydrophobic N- and C-terminal domains. Northern blotting and in situ hybridization studies indicated that both egf0·4 and glc1·8 were expressed in MdPDV-infected host haemocytes. Immunocytochemical studies also indicated that Glc1·8 localized to the cell surface.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Certain parasitic wasps in the families Braconidae and Ichneumonidae carry polydnaviruses that are transmitted vertically to offspring as proviruses but which also replicate in female wasps in a region of the ovary called the calyx (Webb, 1998 ). Virions are stored in the lumen of the oviduct and the resulting suspension of virus and protein is called calyx fluid. The hosts of most polydnavirus-carrying parasitoids are larval stage Lepidoptera (moths and butterflies). When a female finds a host, she injects a quantity of calyx fluid, venom and one or more eggs into the haemocoel of the caterpillar. Virus then enters different host tissues with transcription, in the apparent absence of replication, occurring over the period of time required for the wasp’s progeny to complete development. Virus expression causes several physiological alterations in parasitized hosts including suppression of the immune system (Strand & Pech, 1995a ; Lavine & Beckage, 1995 ; Webb, 1998 ). In the absence of virus, the parasitoid’s progeny are encapsulated and killed by host immune cells (haemocytes), whereas progeny survive when virus is present (Davies et al., 1987 ; Edson et al., 1981 ; Guzo & Stoltz, 1985 , 1987 ; Stoltz & Guzo, 1986 ; Strand & Noda, 1991 ; Asgari et al., 1997 ).

The braconid Microplitis demolitor parasitizes several moths in the family Noctuidae including Pseudoplusia includens (Shepard et al., 1983 ). Like other polydnaviruses, the genome of M. demolitor polydnavirus (MdPDV) is segmented and consists of approximately 15 double-stranded circular DNAs that range in size from less than 2 kb (circle A) to greater than 30 kb (circle O) (Strand et al., 1992 ). Encapsulation in P. includens is mediated by two classes of haemocytes called granular cells and plasmatocytes (Pech & Strand, 1996 ). In P. includens parasitized by M. demolitor, granular cells undergo apoptosis while plasmatocytes lose the capacity to adhere to foreign surfaces (Strand & Pech, 1995b ). These haemocytes only exhibit these responses if they are directly infected by transcriptionally active MdPDV (Strand, 1994 ; Strand et al., 1999 ). Five size classes of viral mRNAs (ca. 0·4, 1·0, 1·5, 1·8 and 3·1 kb) are expressed in P. includens haemocytes and each mRNA is transcribed from genes located on viral DNA segment O (Strand et al., 1992 ; Strand, 1994 ). cDNAs corresponding to the 1·5 and 1·0 kb mRNAs were previously identified and found to encode predicted proteins of similar structure. The most distinguishing feature of the deduced proteins from these transcripts is an identical epidermal growth factor (EGF)-like motif at their N termini (Strand et al., 1997 ). As these proteins arise from different but closely related genes, we refer to this gene family as the MdPDV EGF-like genes and name the proteins Egf1·5 and Egf1·0, respectively. cDNAs corresponding to the 1·5 and 1·0 mRNAs were previously named MdPi59 and MdPi455, in reference to the plasmids from which they were identified (Strand et al., 1997 ), but these terms are no longer used.

In the present study, we sought to identify other MdPDV transcripts expressed in P. includens haemocytes. Here we report that the 0·4 kb mRNA is the third member of the EGF-like gene family that we name egf0·4. The 1·8 kb mRNA is a novel transcript characterized by two hydrophobic domains at its deduced N and C termini and multiple copies of a heavily glycosylated repeat element in its central domain. Based upon this glycosylated central domain, we name this protein Glc1·8 and suggest that it represents a second MdPDV gene family.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Insects.
P. includens and M. demolitor were reared as previously described on artificial diet at 27±1 °C with a 16 h light (L):8 h dark (D) photoperiod (Strand, 1990 ; Strand & Wong, 1991 ). For experiments, M. demolitor females were allowed to singly parasitize 8- to 12-h-old 4th instar P. includens larvae. Parasitized larvae were placed individually in plastic cups half filled with artificial diet.

{blacksquare} Calyx fluid.
Calyx fluid and venom were collected from wasps in Pringle’s saline (Pringle, 1938 ) by established methods (Strand et al., 1992 ). Calyx fluid and venom collected from a single wasp is referred to as one wasp equivalent. For selected experiments, 5th instar (24- to 30-h-old) P. includens larvae were injected with 0·05 wasp equivalents of calyx fluid plus venom, which is within the range of MdPDV and venom that wasps normally inject into hosts at oviposition (Strand et al., 1992 ; Strand, 1994 ). Although the venom itself does not affect host development or haemocyte behaviour, its presence synergizes the effects of MdPDV and other polydnaviruses associated with braconid wasps (Strand & Noda, 1991 ; Stoltz, 1993 ). MdPDV DNAs were isolated from calyx fluid as outlined by Strand et al. (1997) . Viral DNAs were extracted with 1 vol. of phenol:ether (1:1) and ethanol-precipitated in the presence of 0·25 M NaCl.

{blacksquare} Antibody production.
The three monoclonal antibodies, all IgGs, used in the study (MAbs 55F2G7, 49G3A3 and 49B8B10) were generated from the hybridoma screen of Strand & Johnson (1996) . Ascites fluid and spent media were produced and stored at -20 °C until use as previously described (Gardiner & Strand, 1999 ).

{blacksquare} DNA cloning and sequencing.
Two different approaches were used to identify MdPDV mRNAs expressed in P. includens haemocytes. Since all genes expressed in parasitized hosts reside on MdPDV segment O, our first approach was to screen, by Southern blotting, a previously constructed shotgun MdPDV genomic library (Strand et al., 1997 ) for clones that hybridized to this viral gene segment. Multiple clones were identified which were then used to probe Northern blots of haemocyte RNA from MdPDV-infected P. includens. A 2·6 kb genomic clone called pMd-10 was found to hybridize specifically to a 0·4 kb mRNA. Radiolabelled pMd-10 was then used to screen MdPDV-infected haemocyte cDNA libraries. These libraries had been constructed by synthesizing double-stranded cDNAs from polyadenylated mRNA isolated from P. includens haemocytes at 18 h post-infection with calyx fluid plus venom. Double-stranded cDNAs were then directionally cloned into Zap II Lambda arms (Stratagene) or the SmaI site of pGEM-3Z (Promega) as previously described (Strand et al., 1997 ). pMd-10 was labelled to high specific activity by random priming in the presence of [32P]dCTP (3000 Ci/nmol; Amersham) and recombinant plasmids or plaques containing cDNA inserts homologous to pMd-10 were identified by colony hybridization (Sambrook et al., 1989 ). pMd-10 and a cDNA clone named pMd201 were bidirectionally sequenced using the chain termination method and the ABI Prism cycle sequencing kit (Perkin Elmer). Sequence assembly and analysis was performed using the Genetics Computer Group package (Devereux et al., 1984 ).

Our second approach to identifying viral transcripts was to screen the aforementioned cDNA libraries with either 32P-labelled HindIII-digested MdPDV DNA or the MAb 55F2G7, which had been previously shown to specifically label MdPDV-infected haemocytes (Strand & Johnson, 1996 ). DNA probes were gel-purified and labelled to high specific activity by random priming, whereas MAb 55F2G7 was diluted in Blotto (1:500) plus 5% dry milk (Harlow & Lane, 1988 ). Colony hybridization filters were then prepared and screened. Studies focused on a cDNA clone named MdPi27 that was bidirectionally sequenced and analysed as described above.

{blacksquare} Southern analysis.
To map genomic or cDNA clones to MdPDV DNAs, inserts were gel-purified, labelled with [32P]dCTP and hybridized to Southern blots of undigested or restriction enzyme-digested (HindIII and EcoRI) viral DNA. Undigested or restriction enzyme-digested viral DNAs were size-fractionated on 0·7% agarose gels and transferred to nylon in 10x SSC (1xSSC equals 0·15 M NaCl plus 0·015 M sodium citrate) by standard methods (Strand et al., 1997 ). Blots were prehybridized for 4  h at 65 °C in hybridization buffer (0·5 M Na2HPO4 pH 7·2, 7% SDS and 1 mM EDTA). 32P-labelled DNA probes were added to the hybridization buffer at a concentration of 0·5x106 c.p.m./ml and hybridized for 12 to 16 h at 65 °C. Blots were washed under conditions of high stringency in 40 mM Na2HPO4 pH 7·2, 1% SDS and 1 mM EDTA for 1 h at 65 °C and autoradiographed at -80 °C with an intensifying screen.

{blacksquare} RNA extraction and Northern analysis.
After parasitization, haemocytes were collected from P. includens larvae every 24 h. For each time-point, ten larvae were bled from a cut proleg into anticoagulant buffer (98 mM NaOH, 0·19 M NaCl, 17 mM EDTA and 41 mM citric acid, pH adjusted to 4·5). The pooled haemolymph was transferred to a 1·5 ml centrifuge tube and spun at 250 g for 1 min. The cell pellet contained all four classes of haemocytes normally present in circulation (granular cells, plasmatocytes, spherule cells and oenocytoids) and was referred to as unseparated haemocytes. This pellet of unseparated haemocytes was washed once in anticoagulant and total RNA was isolated by homogenizing the pellet in an 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 vol. of 2 M sodium acetate) (Sambrook et al., 1989 ).

For Northern blots, total RNAs (2 µg per lane) from unseparated P. includens haemocytes were fractionated on 6% formaldehyde–1·0% agarose gels and transferred to nylon membranes in 10xSSC (Sambrook et al., 1989 ). Samples were isolated from larvae injected 1 to 7 days previously with calyx fluid plus venom as well as from non-parasitized larvae (controls). Filters were then hybridized using MdPDV DNA probes prepared by random priming using [32P]dCTP. Hybridization conditions followed the method outlined by Lee et al. (1992) . Filters were prehybridized for 4 h at 65 °C in 1 M sodium phosphate pH 7·2, 7·5% SDS, 1% BSA and 50 mM EDTA. Probes were added to filters at 0·5x106 c.p.m./ml and hybridized for 16 h at 65 °C. Filters were washed under conditions of high stringency in 0·1xSSC and 1% SDS at 65 °C for 45 min.

{blacksquare} In situ hybridization and immunocytochemistry.
In situ hybridization studies were conducted with digoxigenin-labelled cDNAs. cDNAs were labelled by random priming using digoxigenin–dUTP and a commercially available kit (Genius, Boehringer Mannheim). Probe concentration was estimated by slot-blot analysis against a known standard according to the manufacturer’s instructions. Hybridization was carried out by established methods (Tautz & Pfeifle, 1989 ; Strand, 1994 ). Haemocytes from normal, parasitized or calyx fluid plus venom-injected larvae were placed into 96-well culture plates containing 70 µl of Ex-Cell 400 medium at a density of 2·0x104 cells per well. Haemocytes were allowed to settle for 15 to 30 min and then fixed for 20 min with an equal volume of 10% formalin and 50 mM EDTA in PBS. Haemocytes were then permeabilized with PBT (PBS plus 0·1% Tween 20) with subsequent incubation of cells in 50 µg/ml of proteinase K for 90 s. Cells in each well were prehybridized in hybridization buffer for 1 h at 48 °C followed by the addition of digoxigenin-labelled probe at a concentration of 100 ng/ml for 24 h. Hybridized cells were washed for 3 h (30 min per wash) in PBT. Washed cells were incubated for 1 h at room temperature with 1:2000 anti-digoxigenin conjugate in PBT and positive cells visualized with a NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) solution. Controls included probing of haemocytes from non-parasitized larvae and omission of probe or conjugated antibody from each type of sample. One hundred 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.

Haemocytes were processed for antibody staining as outlined by Gardiner & Strand (1999) . Briefly, haemocytes were collected from parasitized or calyx fluid plus venom-injected larvae and placed into 96-well culture plates. Cells were fixed in 5% formalin in PBS, permeabilized with 0·1% Triton X-100 and then blocked in 3% BSA in PBS. Cells were then incubated for 1 h with the MAb 55F2G7, rinsed with PBS and incubated with a 1:100 dilution of either fluorescein isothiocyanate (FITC)- or Texas red-conjugated goat anti-mouse IgG secondary antibody (Jackson Laboratory) in blocking buffer. In some experiments, haemocytes were double-labelled with MAb 55F2G7 and either MAb 49G3A3 or 49B8B10, which specifically label plasmatocytes and granular cells, respectively (Gardiner & Strand, 1999 ). To double-label cells, haemocytes were incubated with MAb 55F2G7 and a Texas red-conjugated secondary antibody, rinsed and then incubated with MAb 49G3A3 or 49B8B10 and a FITC-conjugated secondary antibody. We also labelled living cells by collecting haemocytes from parasitized larvae and placing them into culture wells containing TC-100 medium (Sigma). After 30 min in culture, MAb 55F2G7 was added for a further 30 min. Cells were then rinsed with fresh TC-100 medium and incubated for 1 h with Texas red-conjugated secondary antibody. Controls for these experiments included using haemocytes from non-parasitized larvae and omitting the primary antibody from the reaction.

{blacksquare} Image processing.
Microscope images were captured using Metamorph software (Metamorph 1.0) interfaced with a Photometrics high resolution camera. Autoradiograms and gels were scanned at a resolution of 300 d.p.i. using an Eagle Eye documentation station (Stratagene). Files were printed from Adobe photoshop (5.0) using a Tektronix Phaser IISDX dye sublimation printer.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
egf0·4 is the third member of the MdPDV EGF-like gene family
Screening of our MdPDV-infected haemocyte cDNA libraries with the MdPDV genomic clone pMd-10 identified multiple recombinants that contained an identically sized insert of 0·4 kb. Sequencing of the clone pMd201 revealed a 402 bp cDNA with a 103 amino acid open reading frame (ORF) coding for a protein with a predicted molecular mass of 11·7 kDa (Fig. 1a). The predicted protein began with a strongly hydrophobic region of 17 amino acids typical of a eukaryotic signal sequence. This was followed by a cysteine-rich domain of 52 amino acids from nucleotides 122 to 288, another hydrophobic domain of 16 amino acids from nucleotides 289 to 329 and a short 3' untranslated region (UTR) that included a single polyadenylation signal. The spacing of the eight cysteine residues immediately suggested that this cDNA contained an EGF-like motif similar to those present in egf1·5 and egf1·0 (Strand et al., 1997 ). Alignment confirmed that the position of each deduced cysteine residue was identical and that overall these domains were 71% similar to one another (Fig. 1b). Based upon this similarity, we concluded that the 0·4 kb mRNA represented the third member of the MdPDV EGF-like gene family and we named the deduced protein Egf0.4. Comparison with sequences in the SWISS-PROT and GenBank/EMBL databases indicated that the EGF-like domain of Egf0·4 was also 44% similar to the cysteine-rich region of the anticoagulant protein AcAPc2 from the hookworm, Ancylostoma caninum (Stanssens et al., 1996 ) (Fig. 1b). Sequencing of pMd-10 revealed that it aligned identically with the egf0·4 cDNA and confirmed that this genomic clone contained the corresponding egf0·4 gene (Fig. 1c). This gene consisted of two introns (118 and 193 bp) and three exons with a single ORF of 103 amino acids that agreed precisely with the predicated amino acid sequence for the corresponding cDNA (Fig. 1c). The egf0·4 gene also had a TATA and a CAAT box located 52 and 68 nucleotides upstream of the 5' end of the identified cDNA (Fig. 1c).



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Fig. 1. Nucleotide and predicted amino acid sequences of Egf0·4. (a) The putative signal peptide is shown in italics, the start codon and polyadenylation signal are single underlined and the EGF-like motif is double underlined. (b) Alignment of the deduced EGF-like motif in Egf0·4 with the deduced EGF-like motifs in Egf1·0 (accession no. U76033), Egf1·5 (accession no. U76034) and the anticoagulant protein AcAPc2 (accession no. U30793) from A. caninum. Based on this alignment, common cysteine residues are underlined while identical and similar amino acids are shaded. (c) Schematic of the genomic clone pMd-10, which contains the egf0·4 gene. This gene consists of three exons (boxes) and two introns (I and II). The location of the EGF-like domain in Egf0·4 is indicated by the shaded region of the second and third exons. TATA and CAAT boxes are indicated at the 5' end. The putative start and termination codons for egf0·4 are also indicated. The numbers above the schematic indicate the total length of pMd-10 (2614 bp) and nucleotide positions of each exon and intron. Numbers below the schematic indicate the nucleotide positions for the egf0·4 transcript.

 
The egf0·4 cDNA hybridized specifically to MdPDV DNA segment O on Southern blots of undigested viral DNA and to five (12·9, 9·2, 4·8, 4·5 and 2·6 kb) and six (11·3, 7·9, 6·3, 5·5, 4·8 and 4·5 kb) fragments, respectively, when MdPDV DNAs were digested with HindIII or EcoRI (Fig. 2a). This cDNA also hybridized specifically to the 0·4 kb mRNA on Northern blots of haemocyte RNA from MdPDV-infected P. includens (Fig. 2b).



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Fig. 2. Identification of MdPDV DNAs homologous to egf0·4. (a) Hybridization of the egf0·4 cDNA to uncut (U), HindIII-digested (H) and EcoRI-digested (E) DNAs from MdPDV. Undigested and restriction enzyme-digested MdPDV DNAs were separated on 0·7% agarose gels (left panel), transferred to a nylon membrane and probed with radiolabelled insert (right panel). Filters were washed under conditions of high stringency and exposed to autoradiographic film at -80 °C for 4 h. The position of DNA segment O is indicated. (b) Hybridization of the egf0·4 cDNA to mRNAs from M. demolitor-parasitized haemocytes. The cDNA was radiolabelled and hybridized to Northern blots of total haemocyte RNA (2 µg per lane) from P. includens larvae parasitized 24 h previously by M. demolitor. Filters were washed under conditions of high stringency and exposed to autoradiographic film at -80 °C for 16 h. A size marker is shown on the left.

 
glc1·8 represents a new MdPDV gene family
Library screening with 32P-labelled MdPDV DNA or MAb 55F2G7 identified multiple cDNA clones. Most of the clones identified with radiolabelled MdPDV DNA were not detected by MAb 55F2G7. Partial sequencing and Southern blotting studies indicated that these clones contained cDNAs for the already identified EGF-like genes and were not considered further. However, both MAb 55F2G7 and radiolabelled MdPDV DNA identified another set of clones that contained an insert of 1·8  kb. Sequencing of the clone MdPi27 identified a 1838 bp cDNA that contained a single potential initiation codon and encoded a predicted protein of 515 amino acids with a molecular mass of 56·1 kDa (Fig. 3). The deduced protein has two hydrophobic domains, one at the N terminus and the other at the C terminus, flanking a central core of five near perfect tandem repeats of 78 amino acids. By the rules of von Heijne (1987) , the N-terminal hydrophobic domain is likely to be a signal peptide with the cleavage site at the carboxyl side of Gly-14 (Fig. 3). The C-terminal hydrophobic domain may serve to anchor the protein to the cell membrane. This sequence has the features of glycosyl-phosphatidylinositol (GPI) anchor signals: a region of strongly hydrophobic residues preceded by a few hydrophilic residues (Englund, 1993 ). Although sequence similarity to known proteins was not detected by BLAST searches, the O-glycbase database (Gupta et al., 1999 ) and Motifs program of the Genetics Computer Group package identified 11 putative O-glycosylation and 29 N-glycosylation sites in the central domain of the deduced protein. Based upon the heavily glycosylated central domain, we named this protein Glc1·8. Previous study indicated that MAb 55F2G7 recognizes a ca. 78 kDa protein on Western blots of MdPDV-infected haemocyte proteins separated under reducing conditions on SDS–PAGE (Strand & Johnson, 1996 ). This value is higher than the predicted molecular mass (56·1 kDa) of Glc1·8 but we suggest this discrepancy is probably due to glycosylation.




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Fig. 3. Nucleotide and predicted amino acid sequences of Glc1·8. The N- and C-terminal hydrophobic protein domains are double underlined. The putative cleavage site of the signal peptide is indicated by a vertical arrow. Each direct repeat in the central domain is shown by horizontal arrows. Potential N-glycosylation sites are singly underlined while O-glycosylation sites are indicated with residues in bold and underlined. The putative start codon, termination codon and polyadenylation signal are also in bold.

 
The glc1·8 cDNA hybridized to MdPDV DNA segment O on Southern blots of undigested viral DNA, to fragments of 12·9 and 9·2 kb when MdPDV DNAs were digested with HindIII and to fragments of 11·3 and 7·9 kb when MdPDV DNAs were digested with EcoRI (Fig. 4a glc1·8 also hybridized to both a 1·8 kb and a 3·1 kb mRNA on Northern blots of MdPDV-infected haemocyte RNA (Fig. 4b).



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Fig. 4. Identification of MdPDV DNAs homologous to the cDNA clone glc1·8. (a) Hybridization of glc1·8 to uncut (U), HindIII-digested (H) and EcoRI-digested (E) DNAs from MdPDV. Samples were processed and analysed as described in Fig. 2(a). (b) Hybridization of glc1·8 to mRNAs from M. demolitor-parasitized haemocytes. Samples were processed and analysed as described in Fig. 2(b).

 
egf0·4 and glc1·8 are abundantly expressed in host haemocytes infected by MdPDV
To determine temporal patterns of expression in host haemocytes, the egf0·4 and glc1·8 cDNAs were hybridized to haemocyte RNA from P. includens larvae parasitized 1 to 7 days earlier by M. demolitor. Haemocytes from uninfected P. includens served as the control. The egf0·4 cDNA hybridized with greatest strength to the 0·4 kb mRNA at 24 h post-parasitization (p.p.). The hybridization signal then declined from days 2 to 4 and no hybridization signal was detected 5 to 7 days p.p. (Fig. 5a) unless autoradiograms were exposed to film for an extended period (7 to 9 days) (data not presented). The glc1·8 cDNA detected both a 1·8 kb and a 3·1 kb mRNA from infected haemocytes but did not hybridize to any mRNAs from non-infected haemocytes (Fig. 5b). Hybridization signals were again strongest at 24 h p.p. and declined to undetectable levels thereafter.



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Fig. 5. Temporal expression of egf0.4 (a) and glc1·8 (b) in P. includens haemocytes. cDNAs were gel-purified, radiolabelled and hybridized to Northern blots of total haemocyte RNA (2 µg per lane) from non-parasitized P. includens (C) and larvae injected 1 to 7 days previously with 0·05 equivalents of calyx fluid plus venom. Transcript sizes are shown on the right. Size markers (kb) are shown on the left in (b). Blots were washed under conditions of high stringency and exposed to autoradiographic film by using an intensifying screen at -80 °C for 2 days.

 
In situ hybridization was used to determine the percentage of haemocytes expressing egf0·4 and glc1·8. At 2 h p.p. approximately 13% of haemocytes exhibited both a cytoplasmic and a nuclear hybridization signal when probed with the egf0·4 cDNA (Fig. 6). More than 80% of haemocytes were labelled at 24 h p.p. but the percentage of labelled cells then declined at 72 and 144 h (Fig. 6a). Visual inspection of haemocytes indicated that egf0·4 was expressed in all of the haemocyte types present in circulation (data not presented). A similar percentage of haemocytes from parasitized hosts were labelled when probed with digoxigenin-labelled glc1·8 or MAb 55F2G7 (Fig. 6b). Again, visual inspection and double-labelling of haemocytes with anti-haemocyte MAbs indicated that granular cells, plasmatocytes and other haemocyte types expressed this virus gene product (data not presented). Virtually identical results were obtained when egf0·4, glc1·8 or MAb 55F2G7 was used to probe haemocytes from larvae injected with calyx fluid plus venom. However, no hybridization or antibody labelling signal was detected in haemocytes from non-parasitized larvae or when either the primary or secondary antibodies were omitted from the reactions (data not presented).



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Fig. 6. Mean percentages±SD of P. includens haemocytes stained by digoxigenin-labelled egf0·4(a), digoxigenin-labelled glc1·8 (b) and the MAb 55F2G7 (b). Probes and antibodies were incubated with haemocytes collected at selected intervals from parasitized larvae. Each datum point is the mean percentage of haemocytes labelled from three different larvae.

 
Inspection of haemocytes labelled by MAb 55F2G7 indicated that Glc1.8 accumulates primarily at the cell surface (Fig. 7a, b). To further corroborate the association of this protein with the haemocyte surface, we stained living haemocytes from MdPDV-infected hosts with MAb 55F2G7 and Texas red-conjugated goat anti-mouse IgG. Fig. 7(c) shows a prominent ring around most cells, confirming that the epitope recognized by this antibody is present on the surface of haemocytes.



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Fig. 7. Immunocytochemical localization of Glc1·8 in P. includens haemocytes. (a) Haemocytes were collected, fixed and permeabilized from a larva parasitized 24 h previously by M. demolitor. Cells were then incubated with MAb 55F2G7 and FITC-conjugated goat anti-mouse secondary antibody. Representative haemocytes stained positively (green) by the antibody are indicated by arrows. Bar, 200 µM. (b) Higher magnification image of a fixed and permeabilized plasmatocyte double-labelled by the MAbs 55F2G7 (orange-red) and 49G3A3 (yellow-green). The haemocyte sample was collected from a larva injected 24 h earlier with 0·05 equivalents of M. demolitor calyx fluid plus venom. Labelling by MAb 55F2G7 is distinctly around the periphery of the plasmatocyte (arrow) while labelling by MAb 49G3A3 is localized to the cytoplasm. Bar, 75 µm. (c) Immunological localization of Glc1·8 on living haemocytes collected from a larva parasitized 24 h previously by M. demolitor. Haemocytes were placed into primary culture and incubated with MAb 55F2G7 followed by incubation with a Texas red-conjugated goat anti-mouse IgG secondary antibody. The surface of most haemocytes is strongly labelled red (arrows). Bar, 200 µm.

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
We have isolated and molecularly characterized two new MdPDV transcripts expressed in P. includens haemocytes. Structural features and the high abundance of these transcripts in haemocytes suggest that they may be involved in causing the alterations that occur in P. includens haemocytes following infection by MdPDV. In particular, the membrane association of Glc1·8 raises the possibility that this factor plays a role in the inability of MdPDV-infected haemocytes to adhere to foreign surfaces.

The egf1·5 and egf1·0 genes were previously mapped to MdPDV DNA segment O and were found to be expressed in infected host haemocytes (Strand et al., 1997 ). The deduced proteins encoded by these genes feature an identical EGF-like motif at their N termini and multiple copies of a 35 amino acid repeat element in their central domain. However, these proteins differ in the number of repeats they contain, they have unrelated C-terminal ends and they are encoded by different genes (Strand et al., 1997 ). In the present study, we found that egf0·4 is also expressed in P. includens haemocytes. Egf0·4 contains a similar EGF-like motif to Egf1·5 and Egf1·0 but is encoded by a different gene that is also located on MdPDV DNA segment O. We currently have only partial sequence information on MdPDV DNA segment O, which we estimate to be 32·1 kb. As found previously for egf1·5 and egf1·0 (Strand et al., 1997 ), egf0·4 hybridizes to multiple restriction fragments when viral DNA is digested with HindIII or EcoRI. Collectively, these results suggest that multiple copies of the EGF-like genes exist on DNA segment O.

We also note that the EGF-like motif of Egf0·4 is 43% similar to the cysteine-rich domain in the small anticoagulant protein AcAPc2 from the hookworm, A. caninum. AcAPc2 and related proteins from A. caninum are 62 to 85 amino acids in length and represent a unique subset of serine protease inhibitors (serpins) known from only nematodes (Stanssens et al., 1996 ). AcAPc2 appears to interfere with coagulation of mammalian blood by inhibiting the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The region of similarity between Egf0·4 and AcAPc2 spans the EGF-like motif in which the putative reactive site of AcAPc2 resides. Other classes of serpins have been found in the haemolymph of insects and arthropods (Jiang et al., 1998 ). Although the function of these inhibitors is not completely understood, these proteins are known to play critical roles in regulating proteinases involved in the phenoloxidase cascade and haemolymph coagulation (Jiang et al., 1998 ).

We identified the glc1·8 cDNA using both nucleic acid probes and the MAb 55F2G7. Since this clone is the same size (1838 bp) as the 1·8 kb mRNA detected on Northern blots, we conclude that this cDNA corresponds to the 1·8 kb viral mRNA. However, we also note that this cDNA hybridizes to the 3·1 kb mRNA that is also expressed in MdPDV-infected haemocytes. We do not know at this time the structural relationship between glc1·8 and the 3·1 kb mRNA or whether these transcripts are encoded by the same or different genes on MdPDV DNA segment O. In light of the similarities between the EGF-like genes, however, we hypothesize that the 3·1 kb and 1·8 kb mRNAs will be similar in structure and are likely to represent a second MdPDV gene family. This possibility is currently under investigation.

The central domain of Glc1·8 is highly glycosylated and we hypothesize that the hydrophobic domain at the C terminus functions to anchor this virus product to the surface of infected haemocytes. Our immunocytochemical results with MAb 55F2G7 are consistent with this latter conclusion since they show that Glc1·8 localizes primarily to the surface of infected cells. Sequence analysis of Glc1·8 also indicates that this protein shares some features with mucins, which are a large heterogeneous group of glycoproteins that can be either secreted or membrane associated. van Klinken et al. (1995) note that mucins typically contain a high percentage of pro, thr and ser residues (20 to 55%) concentrated in one or several regions of the polypeptide. The ser and thr residues of mucins tend to be heavily O-glycosylated, resulting in carbohydrates accounting for 40 to 80 % of the weight of the mature protein. Mucins also commonly have strongly hydrophobic domains at their N and/or C termini that serve as signal peptides or anchor sequences. Glc1·8 has a pro-thr-ser (PTS) content of 28·5%, with a large number of ser and thr residues predicted to be O-glycosylated. The estimated molecular mass of this protein as determined by Western blotting (78 kDa) (Strand & Johnson, 1996 ) is also correspondingly higher than the molecular mass (56 kDa) predicted from the primary structure of Glc1·8. Unlike most mucins, Glc1·8 does not exhibit any long PTS-rich repetitive units. However, Shen et al. (1999) recently isolated a mucin expressed in the midgut of the mosquito Anopheles gambiae with shorter and less numerous PTS repeats than is typically observed in mammals.

Mucins anchored to the cell surface form an extended rigid structure that may protrude several nanometers above the plasma membrane (Hilkens et al., 1992 ). Membrane-associated mucins have also been shown to possess anti-adhesive and anti-aggregation activities (Ardman et al., 1992 ; Hayes et al., 1990 ; Ligtenberg et al., 1992 ; van de Wiel-Kemanade et al., 1993 ; Wesseling et al., 1995 ). These mucins are believed to block adhesion by physically hindering ligand–receptor interactions (Hilkens et al., 1992 ; Ligtenberg et al., 1992 ; Wesseling et al., 1995 ). Currently we do not know whether Glc1·8 is involved in altering the adhesive properties of host haemocytes. Nonetheless, the structural properties and spatial distribution of this protein on the surface of haemocytes suggest such a possibility.

Several polydnavirus isolates have been reported to induce alterations in haemocytes that in turn compromise the ability of hosts to encapsulate the developing parasitoid (reviewed by Strand & Pech, 1995a ; Lavine & Beckage, 1995 ; Webb, 1998 ). However, the molecular mechanisms underlying changes in haemocyte function remain unclear. Studies with the ichneumonid Campoletis sonorensis and braconid Cotesia rubecula suggest that haemocyte function is disrupted by virus gene products secreted into the plasma of parasitized hosts (Li & Webb, 1994 ; Asgari et al., 1997 ). In contrast, in vitro and in vivo studies indicate that plasma from M. demolitor-parasitized P. includens does not affect the ability of haemocytes to spread on foreign surfaces or disrupt encapsulation (Strand, 1994 ; Strand & Johnson, 1996 ; Strand & Noda, 1991 ; Strand et al., 1999 ). Granular cells undergo apoptosis and plasmatocytes lose the capacity to attach to foreign surfaces only if directly infected with transcriptionally active MdPDV (Strand, 1994 ; Strand & Pech, 1995b ). This suggests that one or more MdPDV gene products endogenously expressed in haemocytes are responsible for immunosuppression of P. includens. We have now isolated and sequenced four of the five viral transcripts expressed in P. includens haemocytes. Functional studies now in progress will undoubtedly clarify how these factors disrupt granular cell and plasmatocyte function.


   Acknowledgments
 
We thank K. Kadash for assistance in sequencing selected clones. Grants from the NIH (AI32617) and the USDA Hatch Program to M.R.S. supported this research. D.T. was a recipient of fellowships from the Natural Sciences and Engineering Research Council (Canada) and from FCAR (Quebec, Canada).


   Footnotes
 
The GenBank accession numbers of the sequences reported in this paper are AF267174 and AF267175.

b Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94702–3102, USA.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 15 May 2000; accepted 14 August 2000.