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
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Abstract |
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Introduction |
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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.
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Methods |
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Calyx fluid.
Calyx fluid and venom were collected from wasps in Pringles 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.
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
).
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.
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.
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% formaldehyde1·0% 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.
In situ hybridization and immunocytochemistry.
In situ hybridization studies were conducted with digoxigenin-labelled cDNAs. cDNAs were labelled by random priming using digoxigenindUTP and a commercially available kit (Genius, Boehringer Mannheim). Probe concentration was estimated by slot-blot analysis against a known standard according to the manufacturers 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.
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.
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Results |
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Discussion |
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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 ligandreceptor 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.
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Acknowledgments |
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Footnotes |
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b Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 947023102, USA.
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References |
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Received 15 May 2000;
accepted 14 August 2000.