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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INTRODUCTION |
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The characteristics of ichnoviruses and bracoviruses are described in Webb et al. (2000) and illustrate the differences in their morphology and the process of virion formation. Ichnovirus virions consist of nucleocapsids of uniform size having the form of a prolate ellipsoid, surrounded by two unit-membrane envelopes. The inner envelope appears to be assembled de novo within the nucleus of calyx cells, while the outer envelope is acquired by budding of virions through the plasma membrane into the oviduct lumen. Bracovirus virions consist of cylindrical nucleocapsids of uniform diameter but of variable length, surrounded by a single envelope, which appears to be assembled de novo within the nucleus. Depending on the species, a virion contains one or several nucleocapsids. The mode of release of bracoviruses is not clarified but probably involves lysis of virus-carrying cells. The genome of both ichnoviruses and bracoviruses is segmented and consists of various circles of double-stranded DNA. The viral genome is transmitted in proviral linear form integrated in the wasp's chromosomes (reviewed in Webb, 1998
; Belle et al., 2002
) and for two bracoviruses it was recently shown that proviral segments are clustered (Belle et al., 2002
; Wyder et al., 2002
).
Excision and circularization of viral DNA has been shown to begin at a particular stage in pupaladult development for the ichnovirus of Campoletis sonorensis (Webb & Summers, 1992) and the bracoviruses of Chelonus inanitus (Albrecht et al., 1994
; Gruber et al., 1996
; Wyder et al., 2002
) and Cotesia congregata (Savary et al., 1999
; Pasquier-Barre et al., 2002
). A comparison with ultrastructural observations (Norton & Vinson, 1983
; Pasquier-Barre et al., 2002
) suggests that excision of viral DNA is concomitant with the appearance of virions in calyx cells. Very little is known about the early development of calyx cells and the sequence of events leading to polydnavirus formation. In the accompanying paper (Marti et al., 2003
) on ovary development and polydnavirus morphogenesis in C. inanitus, we showed that calyx cells begin to differentiate and increase in size shortly after pupation, and that non-viral DNA (actin) and integrated proviral DNA are amplified to the same extent, which suggests polyploidization of calyx cells at this early stage of development. Calyx cells then massively increase in size, their nuclei become very large and proviral DNA is specifically amplified followed by excision of viral segments. Here we present a detailed ultrastructural investigation of the development of calyx cells and the processes associated with virion formation and release. We have also analysed the DNA content of calyx cell nuclei by measurement of fluorescence after Hoechst staining. The data indicate that calyx cells undergo dramatic developmental changes, which involve a massive increase in volume and DNA content of the nucleus, the appearance of viral envelopes and nucleocapsids in the nucleus and finally lysis of cells leading to release of virions into the lumen of the oviduct.
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METHODS |
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Preparation of ovaries.
Ovaries were fixed in 2 % glutaraldehyde and 0·8 % paraformaldehyde in 0·08 M sodium cacodylate buffer, pH 7·2, at 4 °C. After several washes in 0·1 M sodium cacodylate buffer, samples for LR-White embedding and for epoxy resin embedding were treated differently. LR-White was used as it has the advantage of allowing analysis of consecutive sections by fluorescence microscopy after Hoechst staining and electron microscopy. However, in combination with Hoechst staining, osmium tetroxide fixation cannot be used and thus membranes are not visualized. The LR-White samples (see Figs 2 and 3) were dehydrated through a graded series of ethanol and embedded in LR-White (Sigma). Polymerization was for 14 h at 60 °C. The epoxy resin samples (see Figs 1 and 4
7) were post-fixed in 1 % osmium tetroxide in sodium cacodylate buffer and stained in 0·5 % (w/v) uranyl acetate in 0·05 M maleate buffer, pH 5·0. They were washed with 0·05 M maleate buffer, dehydrated in acetone and embedded in low viscosity epoxy resin (Spurr, 1969
). Sections (1 µm for fluorescence microscopy and 60100 nm for electron microscopy) were cut on a Sorvall MT2-B ultramicrotome using diamond knifes (Diatome). Sections for electron microscopy were transferred on to carbon-coated Parlodion films on copper grids or on to single slot grids coated with Formvar films. LR-White-embedded sections were stained for 10 min in 2 % phosphotungstic acid, pH 6·8, and for 5 min in 1 % uranyl acetate in water. Epoxy resin-embedded sections were stained with lead citrate (Reynolds, 1963
). The sections were analysed on a Phillips EM300 electron microscope operated at 60 kV.
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For measurement of nuclear volumes, nuclei cut in the centre were selected. For the reference nuclei (non-virus-producing cells in the ovarial sheath or oviduct), the area was quantified using the AIDA software and the volume was calculated by assuming a spherical shape. For mature calyx cell nuclei, the length and width was measured and the volume was calculated assuming an ellipsoid shape.
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RESULTS |
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In the accompanying paper (Marti et al., 2003), the Feulgen-stained ovaries showed that nuclei of mature calyx cells have a very high DNA content. We thus investigated the DNA content of calyx cell nuclei at various stages of their differentiation into virus-producing cells by treating ovaries of stages 3a6 with the Hoechst DNA stain. Fig. 2
shows that at stage 3a, the nuclei of the peripheral elongated and roundish central cells are much larger and brighter than the nuclei of the septum, oviduct or ovarial sheath, indicating an increase in DNA content. At stage 3b, several nuclei in the lower part of the calyx have become excessively large and bright, indicating a further increase in DNA content. At stage 4, the proportion of enlarged and very bright nuclei has increased and at stage 6 these large and bright nuclei predominate. At this stage, oocytes appear in the calyx and the oviduct is bright, indicating the presence of calyx fluid. To characterize the various stages of calyx cells seen with the Hoechst stain, we analysed consecutive sections by either Hoechst staining or electron microscopy. For this purpose, LR-White-embedded ovaries had to be used (see Methods). A direct comparison of Hoechst-stained cells with the ultrastructural analysis is shown in Fig. 3
, illustrating a region of the stage 6 ovary presented in Fig. 2
, which contains several developmental stages of calyx cells (from left to right). The partially enlarged nuclei, which appear roundish and dotted in the Hoechst stain, are also roundish in the electron microscope and the dots represent patches of dense heterochromatin. The irregularly shaped nuclei of intermediate size and brightness in the Hoechst stain are either nuclei that are highly lobulated and rich in heterochromatin (early intermediate stage, I 1) or swollen and lobulated and with a similar electron-density in the nucleus and cytoplasm (late intermediate stage, I 2). The very large and bright nuclei in the Hoechst stain appear as highly swollen and lobulated in the electron microscope and contain virogenic stroma and virions (VC). Details are shown in Fig. 4
. To get an idea of the increase of DNA in calyx cells in the course of their development, densities of Hoechst-stained sections of ovaries at stages 3b, 4 and 6 were quantified (Table 1
). The densities per area are presented relative to that of non-virus cells, which were given the value 1. The average density in nuclei of intermediate-stage calyx cells (I 1 and I 2) was about 1·5 times higher than that in nuclei of non-virus cells. In virus-containing nuclei, the density was between 2·15 (stage 3b) and 2·65 (stage 6) times higher than in non-virus cell nuclei. In the calyx fluid, the density was lower than in virus-containing nuclei and was similar to that of intermediate-stage calyx cells. We also measured the increase in volume of calyx cell nuclei. For non-virus cells, the mean volume of 40 measured nuclei was 109·5±32·5 µm3, while for virus-containing calyx cells the mean volume of 20 measured nuclei was 4807·5±2305·3 µm3. Thus, the nuclear volume increased approximately 45-fold. Along with the increase in density of approximately 2·65 (Table 1
), this means that the DNA content in calyx cell nuclei increased approximately 120-fold.
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The position of the various stages of calyx cells together with the epithelial layer and the calyx fluid in a 1-day-old adult female are shown in Fig. 5. The overview of the lower outer part of the calyx (Fig. 5a
) shows that the oviduct lumen is filled with virions (in the calyx fluid) and that various stages of calyx cells are present, the latest stage being situated nearest to the oviduct lumen and the younger stages in the upper part. The epithelial cells forming the border of the oviduct lumen have differentiated into cells with apparently phagocytic activity. The higher magnifications (Fig. 5b, c
) show that these cells have protrusions into the oviduct lumen and contain lysosome-like vesicles and various types of vacuoles, some also containing virions. The release of the virions from the calyx cell nuclei into the oviduct is illustrated in Fig. 6
. A calyx cell in which the nuclear membrane has disintegrated is shown in Fig. 6(a)
and (b). At this stage virions and cytoplasmic organelles are mixed. The next step is illustrated in Fig. 6(c)
and (d) showing a calyx cell at the border of the oviduct lumen with a disintegrated nuclear mebrane and a partially disintegrated plasma membrane. Toward the oviduct lumen, this disintegrating cell is lined by one of the phagocytic epithelial cells. We assume that these cells remove cellular debris from lysed calyx cells, which results in the very homogeneous calyx fluid consisting only of virions. This is the first illustration of the process of release for a bracovirus. Another section of the calyx region of a 1-day-old adult female, where eggs passing through the calyx region are visible, is shown in Fig. 7
(a). This reveals that mature eggs with a chorion pass between the calyx cells and the layer of phagocytic cells on the border of the oviduct lumen. The higher magnification of two adjacent eggs (Fig. 7b
) clearly shows that virions are not attached to the surface of the egg chorion. A comparison of the density of virions in a mature calyx cell, where virogenesis is complete, and in calyx fluid shows that the density is higher in calyx fluid (Fig. 7c
). Fig. 7(d)
shows a higher magnification of C. inanitus bracovirus particles in calyx fluid. The virions have a roundish to pear-like shape and contain a single cylindrical nucleocapsid of varying length.
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DISCUSSION |
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At stage 3b, the first calyx cells with virions appear; the DNA content in the nuclei of these cells is very high (Figs 2 and 3, Table 1
), ploidy has reached the final level of approximately 30 and viral DNA is now selectively and intensely amplified and excised (Marti et al., 2003
). The volume of these nuclei is approximately 45 times greater than that of cells in the ovarial sheath or oviduct and together with the DNA density measurements (Table 1
), a total increase in DNA content of approximately 120 can be calculated. Up to stage 6, amplification of proviral DNA and excision of viral DNA continue (Marti et al., 2003
) and more and more calyx cells reach the final stage of development, which is characterized by the presence of mature virions in the nucleus. The increase in average relative DNA content per area of virus-containing cells from 2·15 in stage 3b to 2·65 in stage 6 (Table 1
), along with the fact that excised viral DNA does not replicate (Marti et al., 2003
), suggests that amplification of proviral DNA and excision of viral DNA and encapsidation can occur in parallel. This is in accordance with ultrastructural data, which show that young virus-containing calyx cells contain mainly envelopes and few virions, while the oldest virus-containing cells (often located nearest to the oviduct) are filled with virions.
At the end of stage 5, some mature calyx cells in the proximity of the oviduct begin to disintegrate whereby first the nuclear membrane and then the plasma membrane fall apart leading to the release of virions into the oviduct (Figs 5 and 6), which has been demonstrated here for the first time. The calyx fluid in the oviduct consists of very densely packed virions, the density being even higher than in the nucleus (Fig. 7c
). It appears that the layer of epithelial cells that form the border of the oviduct lumen play an important role and remove debris (Figs 5 and 6
).
A comparison with other bracoviruses revealed the following. The situation in C. inanitus is similar to that in Chelonus texanus (Stoltz et al., 1976, Stoltz & Vinson, 1977
), as only one type of virion is formed and as each virion contains only one nucleocapsid. In C. inanitus we could show that each nucleocapsid contained only one segment of viral DNA (Albrecht et al., 1994
). In C. congregata, Cotesia marginiventris and Cotesia kariyai virions contain several nucleocapsids (Stoltz & Vinson, 1977
; Hamm et al., 1990
; De Buron & Beckage, 1992
; Tanaka et al., 2002
) and it is not known whether single nucleocapsids consist of single segments of viral DNA. In C. congregata, middle and late pupal stages and adults have been analysed and ovary development was correlated with a characteristic increase in pigmentation (Pasquier-Barre et al., 2002
) as is the case with C. inanitus (Albrecht et al., 1994
). Similarly, a stage of calyx cell development with a highly lobulated nucleus and patches of heterochromatin was also seen, followed by a stage with the appearance of virogenic stroma (Pasquier-Barre et al., 2002
). In C. marginiventris only adult females were analysed (Hamm et al., 1990
), but the different stages of calyx cells described and the changes in shape and size of the nucleus were similar to those seen in C. inanitus. These authors also proposed that mature calyx cells disintegrate and mentioned epithelial phagocytic cells; phagocytic cells were also seen in C. congregata (De Buron & Beckage, 1992
) but their function remained unclear. In C. kariyai, a 13-fold increase in the rate of DNA synthesis in the calyx region was observed in the course of pupaladult development (Tanaka et al., 2002
).
A comparison with other ichnoviruses revealed the following. In two ichneumonids, ovary development and ichnovirus morphogenesis have been analysed, namely Campoletis sonorensis (Norton et al., 1975, Norton & Vinson, 1983
) and Hyposoter didymator (Volkoff et al., 1995
). As in C. inanitus, the development of the ovary and the appearance of polydnaviruses and excision of viral DNA are correlated with the pigmentation pattern of the pupae (Norton & Vinson, 1983
; Webb & Summers, 1992
; Volkoff et al., 1995
) and the calyx cell differentiation begins shortly after pupation (Volkoff et al., 1995
). Calyx cell nuclei of H. didymator first increase in size, then pass through a lobulated stage with patches of heterochromatin followed by a stage with virogenic stroma and less heterochromatin (Volkoff et al., 1995
), similar to the situation in C. inanitus. However, the final stages of virogenesis are different, as release of ichnoviruses involves budding through the nuclear and plasma membranes (Norton et al., 1975
; Volkoff et al., 1995
). A major difference in the situation with C. inanitus is the finding that some calyx cells also appear to be secretory in addition to producing polydnaviruses and that the calyx fluid contains flocculent material in addition to polydnaviruses (Volkoff et al., 1995
).
In C. inanitus, polydnaviruses are not attached to the surface of the mature eggs (Fig. 7a, b). Similar observations were made with the C. marginiventris bracovirus (Hamm et al., 1990
), the C. congregata bracovirus (De Buron & Beckage, 1992
) and the ichnoviruses of Diadegma terebrans (Krell, 1987
) and C. sonorensis (Norton et al., 1975
). In contrast, the ichnovirus of Tranosema rostrale was found to form a particulate coat around the egg (Cusson et al., 1998
). In conclusion, it appears that ovarian morphogenesis and the early steps in calyx cell development are similar in polydnavirus-producing braconids and ichneumonids. Thus, despite the fact that the association of braconids and ichneumonids with viruses seems to have occurred independently (Whitfield, 1997
), the way of propagating these viruses is similar and involves the development of a particular cell type, the calyx cells, which are directed to produce these unique viruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 September 2002;
accepted 20 January 2003.