Institute of Cell Biology, University of Berne, Baltzerstrasse 4, CH-3012 Bern, Switzerland
Correspondence
Beatrice Lanzrein
beatrice.lanzrein{at}izb.unibe.ch
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ABSTRACT |
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The EMBL accession numbers of the sequences in this paper are: Z58828, Z58831, Z58832, AJ278676, AJ278677, AJ319653 and AJ439880.
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INTRODUCTION |
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To address these questions we have made an extensive study, presented here and in the accompanying paper (Wyler & Lanzrein, 2003), encompassing histological, ultrastructural, endocrinological and molecular analyses of the process of ovary and calyx cell differentiation and polydnavirus formation. We used the braconid wasp C. inanitus and its polydnavirus (CiV) as a model system. The excision sites of two viral segments (CiV12 and CiV14) have been analysed and it has been shown that excision occurs through a site-specific recombinational event and that, after excision, flanking regions are rejoined (Gruber et al., 1996
; Wyder et al., 2002
). Proviral CiV segments appear to be clustered in the genome of the wasp, as flanking regions of CiV12 and CiV14 have been shown to hybridize to other viral segments (Wyder et al., 2002
). The morphogenesis of the ovary in the course of pupaladult development is strictly correlated with the pigmentation pattern of the pupae and has been subdivided into stages 16 (Albrecht et al., 1994
). Excision and circularization of viral DNA has been shown to commence at stage 3b (Gruber et al., 1996
; Wyder et al., 2002
).
In this paper, we have illustrated the morphological and histological changes of the ovary in the course of pupaladult development and the differentiation of the calyx cells. In the same period of time, we measured the copy number of viral DNA in the integrated and excised form and rejoined flanking DNA by real-time quantitative PCR; as a measure for replication of non-viral DNA, copy numbers of an actin gene were measured. In addition, the fluctuation of various ecdysteroids was measured in the course of pupaladult development and into adulthood. The data show that ecdysteroids were highest immediately after pupation when ovarian morphogenesis and calyx cell differentiation begin. Subsequently, the nuclei of calyx cells started to increase and the real-time PCR data indicated that calyx cells first become polyploid and that the proviral cluster is then selectively amplified followed by excision of viral segments. The excised circular segments appeared not to replicate. In the accompanying paper (Wyler & Lanzrein, 2003), we have described the DNA content and the ultrastructure of the various developmental stages of calyx cells on their way to becoming virus-producing cells and have related these changes to the molecular data presented here.
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METHODS |
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Laser confocal microscopy and DNA staining.
Ovaries were fixed in 2 % formaldehyde in PBS and rinsed twice in PBS containing 0·5 % Tween 80. RNA was digested by the addition of RNase A (3 mg ml-1). DNA was stained with propidium iodide (10 µg ml-1) in distilled water, rinsed in distilled water and mounted in Glycergel (Dako). Stained ovaries were analysed using a laser confocal microscope Zeiss 310; excitation lasers were set at 488 and 514 nm.
Histology.
Ovaries were fixed in sodium cacodylate buffer (pH 7·4) containing 2 % glutaraldehyde and 0·8 % formaldehyde. They were rinsed in sodium cacodylate buffer, dehydrated in an ethanol series and embedded in Technovit 7100 (Heraeus-Kulzer). Using glass knives, 2 µm sections were cut and stained using the Feulgen reaction and 1 % toluidine blue in distilled water.
Cloning of C. inanitus actin cDNA.
Forward (5'-gtggaagcttTCACCAACTGGGATGACATG-3') and reverse (5'-gtggaagcttACTCCTGCTTCGAGATCCAC-3') primers containing HindIII sites were designed in highly conserved coding regions of known insect actins with their 3' ends positioned in a non-degenerated codon. One µg of a C. inanitus first instar cDNA library in gt11 was used as a template in a 50 µl PCR reaction with 200 nM of each primer, 100 µM each dNTP and 2·5 units Taq polymerase (Qiagen). The reaction proceeded for 5 min at 95 °C, then for 35 cycles of 30 s at 95 °C, 30 s at 54 °C and 1 min at 72 °C, with 10 min termination at 72 °C. A PCR product of approximately 850 bp was gel-purified, digested with HindIII and ligated into a HindIII-cut pBluescript KS+ vector. Three positive clones were sequenced with the Thermo Sequenase Sequencing kit (Amersham) on a GeneReadIR 4200 apparatus (Licor). Sequence analysis was carried out using BLAST (Altschul et al., 1997
). Two clones were identical and were used to design primers for the real-time PCR. Their sequence showed significant similarity to known insect actins (>86 % at the mRNA level and 98 % at the protein level). The sequence is available under accession no. AJ439880. The third clone was similar to other actin sequences.
Isolation of ovarian DNA and real-time quantitative PCR.
Female pupae of stages P2, P3a, P3b, P4, P5, P6 and 4-day-old adults were anaesthetized on ice and their abdomina were cut away and placed in a drop of PBS. The ovaries were dissected out and the lower part containing only the calyx region and the oviducts was removed and placed in an Eppendorf tube with 200 µl of buffer (100 mM NaCl, 10 mM Tris/HCl, 25 mM EDTA, 0·5 % SDS, pH 8). Ovaries from three females per stage were combined. For each stage, three independent isolations were made. After addition of 10 µl proteinase K (10 mg ml-1), samples were incubated for 2 h at 37 °C. The DNA was purified by phenol and chloroform extractions and subsequent ethanol precipitation.
To determine the copy number of CiV12 and CiV14 in the integrated and excised form and the rejoined DNA, quantitative PCR was performed. For integrated CiV12, the primers used were 12RR/12RL, for excised CiV12, primers were 12LL/12RR and for the corresponding rejoined DNA, primers were 12LR/12RL, yielding amplicons of 232, 265 and 267 bp, respectively (see Gruber et al., 1996, and accession nos Z58828, Z58831 and Z58832). For integrated CiV14, primers were 14RL/14RR, for excised CiV14, primers were 14LL/14RR and for the corresponding rejoined DNA, primers were 14LR/14RL, yielding amplicons of 318, 264 and 386 bp, respectively (see Wyder et al., 2002
, and accession nos AJ278676, AJ278677 and AJ319653). To analyse amplification of non-viral wasp DNA, an actin gene of C. inanitus was used with the actin forward primer 5'-CCTTCAACAGCCCAGCCAT-3' and reverse primer 5'-CACGGACAATTTCTCGCTCA-3', yielding an amplicon of 255 bp. The PCR product obtained with these primers did not span an intron, as the size of the PCR product obtained with genomic DNA was the same as with cDNA.
PCR was performed with the Sybr Green I Reaction System (Eurogentec) in 96-well Optical Reaction Plates (Applied Biosystems). On each plate, each reaction was run in duplicate. To correct for variable primer efficiencies between different runs, a dilution series of P6 DNA was run on each plate as an internal standard. To allow determination of absolute copy numbers of integrated and excised CiV12 and CiV14 and their corresponding rejoined DNA, known amounts of clones were amplified on the same plate. In the case of CiV12, this was clone A21 for the integrated form, clone 1G10 for the excised form and clone pCR1 for the rejoined form (for description of clones, see Gruber et al., 1996
). In the case of CiV14, this was clone
1B231 for the integrated form, a 1300 bp HindIII subclone of 2A6 for the excised form and clone CiV14LR/RL for the rejoined form (for description of clones, see Wyder et al., 2002
). On each plate, non-template controls were carried out for each primer pair. For each reaction 2 µl of an appropriate dilution of template DNA was amplified in a volume of 30 µl containing 200 µM each dNTP, 3·5 mM MgCl2, 0·75 units HotGoldStar DNA polymerase, 200 nM of each primer and 0·9 µl of 1 : 2000-diluted Sybr Green I stock. Cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 1 min and 60 °C for 1 min. PCR analyses were performed with the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). The quality of primers was checked by analysis of melting curves and analysis of the PCR products on a 3·5 % MetaPhor (BioWhittaker) agarose gel. PCR data were analysed with the GeneAmp 5700 SDS Software, version 3.1. Each reaction was carried out in duplicate or triplicate with each of the three independent DNA isolations per stage.
Ecdysteroid analyses.
Solvents were of pro-analysis grade or high performance liquid chromatography (HPLC) grade and were purchased from Romil Chemicals or Merck. [23,24-3H(N)]-ecdysone (specific activity 58·8 Ci mmol-1) was purchased from New England Nuclear, and ecdysone (E) and 20-hydroxyecdysone (20E) were from Simes. Other ecdysteroids were kindly provided by R. Lafont (Paris). Standard abbreviations for ecdysteroids are used (Lafont et al., 1993).
The ecdysteroid analyses were in principle carried out as described in Grossniklaus-Bürgin & Lanzrein (1990a). For measurements of Sep-Pak-purified ecdysteroids (see Fig. 6
), three to five females were homogenized in 1 ml acetonitrile; for measurement of HPLC-purified ecdysteroids (see Fig. 7
), six to ten females were homogenized in 2 ml acetonitrile. After centrifugation the extract was washed once with 1 ml acetonitrile and twice with 1 ml 50 % acetonitrile in water. The supernatant was then partitioned against hexane to remove lipids and each phase was washed with an equal volume of the counterphase. The aqueous phase was evaporated in a Speed Vac concentrator and the residue taken up in 5 ml of water. The aqueous extract was then passed through a C18 Sep-Pak (Waters) cartridge and after washing with water, ecdysteroids were either eluted with 5 ml 100 % methanol and then separated by HPLC (see below) or else first eluted with 5 ml 25 % methanol and subsequently with 60 % methanol. Polar ecdysteroids, such as ecdysonoic acid (Eoic) and 20-hydroxyecdysonoic acid (20Eoic), elute in the 25 % methanol fraction, while mid-polar ecdysteroids, such as E, 20E and 20,26-dihydroxyecdysone (20,26E), elute in the 60 % methanol fraction (Grossniklaus-Bürgin & Lanzrein, 1990a
; Grossniklaus-Bürgin et al., 1998
). Aliquots of the 25 % and 60 % methanol fractions or the HPLC fractions were quantified by radioimmunoassay (RIA) as described previously (Grossniklaus-Bürgin & Lanzrein, 1990a
). Two different antibodies were used, one specific for the side chain (H-2) and the other specific for the ecdysone nucleus (H-22); both were generous gifts from L. I. Gilbert. Data were expressed in 20E equivalents or in their corresponding equivalents (Warren & Gilbert, 1986
).
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RESULTS |
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Replication of non-viral DNA and integrated and excised viral DNA in pupaladult development
We measured replication of non-viral and viral DNA in the course of pupaladult development. To analyse whether replication of viral DNA occurred in the integrated proviral or the excised form, or both, real-time quantitative PCR was carried out with primers specific for integrated and excised viral segment CiV12 and for the rejoined flanking DNA of CiV12. Replication of non-viral DNA was measured with primers specific for an actin gene of C. inanitus. DNA from calyx regions of ovaries from pupae at stages 26 and from 4-day-old adult females was used as templates and the data are shown in Table 1 and Fig. 3
. As the calyx region is not yet separated from the reproductive part of the ovary at stage 1 (see Figs 1a and 2
), analyses obtained with this stage were not included. The amplicon copy number of actin and integrated CiV12 increased from stage 2 to 3a to a similar extent, i.e. tenfold, to approximately 4·6x105 and 1·5x105, respectively, while excised CiV12 and corresponding flanking DNA were absent. Since in this phase many calyx cell nuclei have greatly increased in size and DNA content (Fig. 2
; see also Wyler & Lanzrein, 2003
), this increase in both actin and integrated CiV12 suggested that initially calyx cells become polyploid. In stage 3b, actin had increased in copy number to 106 per ovary, while integrated CiV12 had increased even more to 1·3x107; excised CiV12 and corresponding rejoined DNA appeared with very similar copy numbers of approximately 1·1x105 and 2·5x105, respectively. From stage 3b up to stage 6, actin remained at a similar level while integrated CiV12 increased massively up to 5x108, suggesting selective amplification of the proviral cluster; excised CiV12 and rejoined flanking DNA both increased by three orders of magnitude to 3x108 and 2x108, respectively. The fact that excised CiV12 and rejoined flanking DNA are present in similar quantities up to stage 6 suggests that CiV12 is only amplified before excision. In 4-day-old adult females, excised CiV12 was highest, integrated CiV12 somewhat lower and rejoined DNA dropped to 2·6x105 and actin to 2·9x103. To investigate whether the situation is similar for CiV14, quantitative PCR measurements were carried out with CiV14 primers. Fig. 4
shows that at stage 6, copy numbers of integrated and excised CiV14 and corresponding flanking DNA were in a similar range to those of CiV12 although slightly lower. From all these data the following hypothetical model for CiV DNA replication is proposed (Fig. 5
): (i) calyx cells first become polyploid; (ii) the proviral cluster is then selectively amplified; this could be due to local amplification of chromosomal DNA or a rolling circle mechanism; (iii) after amplification single segments are excised and circularized by a recombination process. Excised circular viral segments are not further amplified.
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DISCUSSION |
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From stage 3a onwards, integrated proviral CiV12 was approximately 100 times more amplified than actin, as it reached a copy number of around 5x108 compared with 4x106 for actin. This indicates selective amplification of proviral CiV12. As CiV segments appeared to be clustered in the wasp's genome (Wyder et al., 2002) and since proviral CiV14 reached a similar copy number (Fig. 4
), it seems likely that after stage 3a, the entire proviral cluster is specifically amplified. Similarly, Drosophila follicle cells first become polyploid and then specifically amplify regions containing the chorion genes (Osheim et al., 1988
; Calvi et al., 1998
). In baculoviruses, palindromic structures have been reported to act as origins of replication (Pearson et al., 1992
; Ahrens et al., 1995
; Kool et al., 1995
). In CiV, several inverted repeats have been found on all sequenced segments (Wyder et al., 2002
) and it is conceivable that some of the palindromic structures might serve as origins of replication. A large palindromic structure was also found in the EP1 circle of the C. congregata bracovirus (Savary et al., 1997
). Another way of replication of the viral cluster could be a rolling circle mechanism in which DNA synthesis proceeds unidirectionally (Fig. 5
). In this case, a large circular molecule containing the viral cluster and an origin of replication should exist. For the bracovirus of C. congregata, it was recently shown that a large molecule containing at least two viral segments and downstream sequences is amplified prior to excision of individual segments (Pasquier-Barre et al., 2002
).
Excised circular CiV12 DNA and corresponding rejoined flanking DNA was first detected at stage 3b and both then increased in a similar manner (Table 1, Fig. 3
). At stage 6, copy numbers of excised circular and flanking rejoined DNA were similar for both CiV12 and CiV14 (Fig. 4
). This suggests that excised circular segments do not replicate. Also recent observations with the C. congregata bracovirus support this assumption, as the quantity of excised EP1 never exceeded that of the large precursor molecule containing EP1 (Pasquier-Barre et al., 2002
). Coincident with the first appearance of excised circular viral DNA in stage 3b, calyx cells with a huge and swollen nucleus containing viral envelopes and virogenic stroma appear (Wyler & Lanzrein, 2003
); these cells appear to be the ones in which amplification of the proviral cluster and excision of viral DNA take place.
The copy number of proviral and excised viral DNA increases massively from stage 3b up to stage 6 (Table 1, Fig. 3
). A comparison with histological (Fig. 2
) and ultrastructural observations (Wyler & Lanzrein, 2003
) indicates that in this phase calyx cells of all developmental stages occur, the most advanced being located near the oviduct. The proportion of virion-containing cells increases steadily, and at stage 6 release of virions has commenced and calyx fluid, which contains virions, can be seen in the oviduct. In 4-day-old adult females, which were not allowed to oviposit, the copy number of integrated proviral CiV12 had dropped to 3·6x107 suggesting that amplification of integrated proviral DNA decreases in the adult. The copy number of excised circular CiV12 was still at 1·5x108, while that of rejoined flanking DNA had dropped to 2·6x105 and that of actin to 2·9x103. This shows that in the adult stage the excised circular form of viral DNA is predominant. This is the form that is encapsidated in viral particles and released into the oviduct as calyx fluid (Fig. 2
). All these data show that virus production is most intense in the second half of pupaladult development and diminishes in the adult, which agrees with ultrastructural observations (Wyler & Lanzrein, 2003
). A comparison with the egg-laying behaviour of the wasp revealed the following. Under our rearing conditions, adult females live for 2530 days and may lay eggs until they die. On average a female lays 1757±945 eggs (n=12) in its lifetime but some lay up to 2800 eggs (F. von Niederhäusern & B. Lanzrein, unpublished). With DNA dot blot analyses it was found that the calyx fluid collected from one female contained 150250 ng viral DNA at adult eclosion (F. von Niederhäusern & S. Wyder, unpublished) and extrapolation from copy numbers of excised CiV12 gave values in the range of 600 ng per ovary. As we have shown that approximately 0·1 ng viral DNA is injected along with each egg (Lanzrein et al., 2001
), the amount of virus present at adult eclosion would already be sufficient for all ovipositions in the life-time of a female. In C. congregata, the intensity of polydnavirus replication remained high until 3 days after adult eclosion (Pasquier-Barre et al., 2002
).
Ecdysteroids increased immediately after the pupal moult and 20E was predominant; thereafter ecdysteroids decreased (Figs 6 and 7). Here we have used data obtained with whole body homogenates, as collection of haemolymph, especially from young pupae, is difficult. However, for stages 2, 3a, 3b and 5, some analyses with haemolymph have been made, which indicated that the majority of ecdysteroids in the 60 % methanol fraction circulates in haemolymph (data not shown). These are the first data on ecdysteroid identification and quantification in pupaladult development of a polydnavirus-carrying wasp. In the ichneumonid C. sonorensis, it was proposed, on the basis of in vitro application of 20E, that this hormone regulates polydnavirus replication (Webb & Summers, 1992
). However, the titre data shown here do not support this hypothesis, since at stage 3b, when excised viral DNA first appears, 20E has already decreased and continues to drop, whereas polydnavirus replication is most intense up to stage 6. The level of 20E is highest when the reproductive tract is in a phase of intensive cell proliferation and differentiation, which suggests a role in early ovarian morphogenesis, as has been reported for Drosophila (Audit-Lamour & Busson, 1981
; Hodin & Riddiford, 1998
). Furthermore, ecdysteroid titres were also highest shortly after pupation in pupae of an ichneumonid ectoparasitoid, which does not harbour polydnaviruses (Gelman et al., 2000
). Thus, a specific role of 20E in regulating polydnavirus replication appears rather unlikely; it is conceivable that the in vitro 20E application made by Webb & Summers (1992)
accelerated calyx cell differentiation and thus indirectly caused an increase in viral DNA.
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ACKNOWLEDGEMENTS |
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Received 16 September 2002;
accepted 20 January 2003.