- Email: [email protected]
Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx
1. Asia-Pacific Entomol. 10(4): 357-361 (2007) www.entomology.or.kr
PHYSIOLOGY AND BIOCHEMISTRY
Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx Kim, Yonggyun* and Sanghoon Ryu' Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea lCentre for Connnon Research Facility, Andong National University, Andong 760-749, Korea
Abstract Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to an endoparasitoid, C. plutellae. Despite rich information on CpBV genome, there has been little known on its viral replication mode from proviral to episomal form. This study illustrates fine structures of the epithelial cells producing CpBV with a reference to non-producing ovarian epithelial cells. The ovarian epithelial cells of teneral females (within 12 h after emergence) were characterized by large nucleus and rich rough endoplasmic reticulum. CpBV particles were present only at the calyx region, in which follicle epithelial cells exhibited virogenic structures. Though a matured CpBV particle found in the calyx lumen was encapsidated in a single envelop containing multiple nucleocapsids, numerous free nucleocapsids were observed in the calyx epithelial cells and appeared to undergo assembly step to a fmal multiple capsid form. The multiple capsid forms appeared to be released into the oviduct lumen. The epithelial cells bordering the oviduct lumen showed phagocytosis presumably due to clearing cellular debris. At the calyx area close to the common oviduct, the epithelial cells appeared to maintain protein synthetic activity due to highly developed rough endoplasmic reticulum, but showed a marked decrease in the viral production.
Key wonts Cotesia plutellae, CpBV, Polydnavirus, Replication, TEM, Ultrastructure
Introduction Polydnavirus is a unique double stranded DNA virus symbiotic to some endoparasitoid wasps (Kim, 2006; Kim et al., 2007). It comprises of two genera, Ichnovirus (IV) and Bracovirus (BV), depending on host *Corresponding author. Email: [email protected] Tel: +82-54-820-5638; Fax: +82-54-823-1628 (Received October 2, 2007; Accepted December 7, 2007)
wasps, respectively, Ichneumonidae and Braconidae (Webb et al., 2000). These two viral groups are classified by viral morphology and serological or DNA hybridization analyses, suggesting their independent origins (Whitfield and Asgari, 2003). Polydnavirus is also unique in their viral transmission mode. It is located on host chromosome as a proviral form, which allows them to vertically transmit along with wasp generation (Blissard et al., 1987). Its replication into viral particles occurs only in ovarian calyx region during pupal stage in response to ecdysteroid signal (Webb and Summers, 1992; Wyder et al., 2002). IV virions are relatively uniform in size and contain biconvex nucleocapsids surrounded by two unit membranes, whereas BVs are highly variable in length and have cylindrical nucleocapsids surrounded by single envelope (Webb and Strand, 2005). The outer membrane of IV is acquired by budding off from calyx cells. In comparison, single envelop of BV suggests its release into the oviduct lumen by cell lysis (Wyler and Lanzrein, 2003). Cotesia plutellae (Braconidae: Hymenoptera) is a solitary endoparasitoid wasp that parasitizes the diamondback moth, Plutella xylostella (Bae and Kim, 2004). C. plutellae bracovirus (CpBV) has been identified and known to be replicated in the ovarian calyx during late pupal stage (Kim et al., 2004). CpBV has been regarded as a major factor to reduce host cellular innnune capacity in the parasitized P. xylostella (Basio and Kim, 2006; Ibrahim and Kim, 2006). For example, CpBVI5[3 gene effectively inhibits hemocyte-spreading behavior in P. xylostella (Madanagopal and Kim, 2007a). CpBV-lectin exhibits early expression and has been suspected to inhibit nonself recognition of P. xylostella (Madanagopal and Kim, 2006, 2007b). CpBV-PTPs impair cellular immune reactions in phagocytosis and encapsulation (Ibrahim et al., 2007; Ibrahim and Kim, 2007). CpBV-H4, CpBV-E94K and CpBV-ELPI are speculated to inhibit host immune capacity (Ibrahim et al., 2005; Lee et al., 2005). Despite significant roles of polydnavirus in the parasitism exhibited by the endoparasitoids, little has been known about molecular processes of polydnavirus repli-
358
J. Asia-Pacific Entomol. Vol. 10 (2007)
Materials and Methods
aldehyde in 0.01 M phosphate buffer, pH 7.2, for 2 h at 4"C. After several washes in the phosphate buffer, samples were post-fixed by 1% osmium tetroxide for 30 min at 4"C and washed in the phosphate buffer. The samples were dehydrated through a graded series of ethanol and substituted with propylene oxide, and then embedded in Epon 812 for 48 h at 60"C. Sections «90 nm) were cut on a Leica Ultracut UCT ultramicrotome using a diamond knife (Diatome). Ultrathin sections were transferred onto 200 mesh copper grids and stained with uranyl acetate for 20 min, and with lead citrate for a further 10 min. The sections were examined in TEM (Hitachi, H-7650) operation at 80 kV
C. plutellae
Results and Discussion
cation. To understand the molecular events undergoing during polydnavirus replication, structural and genomic studies need to be thoroughly analyzed. To this end, this study performed the structural analysis of ovarian calyx cells of C. plutellae to understand replication and release of CpBV This study illustrates the CpBV replication only in ovarian calyx cells, not in other ovarian follicle cells. It also provides structural assembly of multiple nucleocapsids into an envelope, which is release into oviduct lumen.
Larvae of P. xylostella, as parasitized host of C. plutellae, were reared on cabbage leaves at 25±1 "C under a photoperiod of 16:8 (LD) h. For parasitization, early second instar larvae of P. xylostella were exposed to 1-4 days old C. plutellae adults for 24 h. Propagation of both parasitoid and host insects was performed at 25±1"C. The host was fed daily with cabbage leaves.
TEM (transmission electron microscope) analysis Newly emerged females (within 12 h) were used to isolate ovaries, which were fixed in 2.5% glutar-
CpBV begins to replicate during pupal stage of C. plutellae especially at adult tissue differentiation (Kim et al., 2004). When C. plutellae emerge, the oviduct lumen is filled with CpBV, which would be enough amounts for all parasitization during entire adult period. In this study, teneral females had fully grown ovaries with long germarium, thick vitellarium, and hypertrophied calyx region (Fig. 1). The calyx region was blue-eolored externally, presumably filled with CpBV In a sagittal section, mature oocytes were visible in vitellarium, some passing through the calyx region. Each ovariole in the germarium showed one layer epithelial structure and did not contain any oocyte
Fig. 1. Ultrastructure of epithelial cells ('Ep') at germarium and vitellarium of one day old Cotesia plutellae ovary. Oocyte ('Oc') is present in the vitellarium. Magnification is expressed in each TEM photo.
Ultrastructure of CpBV
and viral particles. The epithelial cells contained many patches of dense heterochromatin. Oocytes were frequently visible in the vitellarium and surrounded by follicle cells. Any viral production was not evident in the vitellarium and viral particles were not detected in the spaces between the oocytes and follicle cells. The calyx cells of C. plutellae were different from
Fig. 2. Ultrastructure of upper calyx cells bordering vitellarium of one day old Cotesia plutellae ovary, in which oocyte ('Dc') is visible. 'N' represents nucleus of the calyx cell, in which many virus particles are present in the cytosol. Magnification is expressed in a TEM photo.
359
ovarian epithelial cells in germarium or vitellarium in their large nuclei and richness of rough endoplasmic reticulum (Fig. 2). In Chelonus inanitus, the polydnavirus began to form during pupal stage, at which calyx cells become polyploidy and in the following phase integrated viral DNA is selectively and intensively amplified to be excised and circularized (Marti et al., 2003). Especially, when the calyx cells are differentiated from the remaining ovarian epithelial cells of C. inanitus, the 20-hydroxyexdysone (20E) titer is maximal during pupal stage (Marti et al., 2003). In comparison, Webb and Summers (1992) suggested that 20E is directly responsible for polydnaviral replication. Here we can interpret the effect of 20E on the polydnavirus production by its mediation of differentiation of the calyx epithelial cells into polydnavirus-producing state. Newly formed CpBV particles in the lumen were not attached to the surface of the mature eggs. Similar observations were made with C. marginiventris BV (Hamm et al., 1990), C. congregata BV (De Buron and Beckage, 1992) and the IVs of Diadegma terebrans (Krell, 1987) and Campoletis sonorensis (Norton et al., 1975). The calyx cells contained virions, some undergoing encapsidation, in which nucleus had virogenic stroma and many patches of dense heterochromatins (Fig. 3). Nucleocapsids were encapsidated singly or multiply.
Fig. 3. Virus production in the calyx cells of one day old Cotesia plutellae ovary. 'N' represents nucleus of the calyx cell, in which many virus particles are present in the cytosol. An arrow indicates a lysis of nuclear membrane. Singly or multiply encapsidated viral particles are shown lower panels. Magnification is expressed in each TEM photo.
360 1 Asia-Pacific Entomol. Vol. 10 (2007)
Single nucleocapsids varied in length and shape from round to square. Similar nucleocapsids were found in multiple packaged virions. In addition, all virions were multiply encapsidated in the calyx fluid, suggesting that the singly encapsidated particles would be further assembled. The assembled virions appeared to be transported into calyx fluid probably by cell rupture and the epithelial cells bordering the oviduct lumen exhibited phagocytosis to clear the cellular debris (Fig. 4). Some large vesicles containing several virions were also observed. Ultrastructural studies indicated that BVs were released by cell lysis with continued production of virus-producing cells from the calyx (Stoltz and Vinson, 1979). The lysed cellular debris was cleared by phagocytosis of the calyx epithelial cells bordering oviduct lumen (Wyler and Lanzrein, 2003). In C. plutellae, the epithelial cells bordering oviduct lumen also had
a various types of vacuoles (Fig. 5), suggesting phagocytic activity. Further clarification needs to determine the release mode of CpBV viral particles from the calyx cells to the ovarian lumen in C. plutellae. We have presented the first complete description of calyx cells and CpBV morphogenesis. Along with previous CpBV replication study (Kim et al., 2004), we may develop a complete picture of ovary and calyx cell development. During pupal stage, C. plutellae begins ovarian differentiation to form virus-producing calyx cells. This may be induced by rise of 20E. The virus production is confmed in the ovarian calyx cells. With oogenesis, matured oocytes are accumulated in the vitellarium, but free from CpBV particles. On adult emergence, the females are ready to parasitization with enough amounts of newly formed CpBV particles in the oviduct lumen. Acknowledgements This study was supported by Biogreen 21 project of RDA, Korea, and by the 2nd stage of BK21. We also appreciate Dr. Nalini Madanagopal for her preparing C. plutellae ovaries for TEM study.
Lumen
Literature Cited
Epithelium
Fig. 4. Phagocytosis of epithelial cells in of one day old Cotesia plutellae ovary. different phases of phagosome formation of membrane. Magnification is expressed in
the calyx region Arrows indicate the epithelial cell a TEM photo.
Sheath
Epithelium
Lumen
Fig. 5. The epithelial cells at the oviduct lumen filled with calyx fluid of day old Cotesia plutellae ovary. The cells are surrounded by the epithelial sheath. Magnification is expressed in a TEM photo.
Bae, S. and Y Kim. 2004. Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Compo Biochem. Physiol. 138B: 39-44. Basio, NA and Y Kim. 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31: 341-347. Blissard, G.W., O.P., Smith and MD. Summers. 1987. Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome. Virology 160: 120-134. De Buron, 1. and N.E. Beckage. 1992. Characterization of a polydnavirus (PDV) and virus-like filamentous particle (VLFP) in the braconid wasp Cotesia congregata (Hymenoptera: Braconidae). 1 Invertebr. Pathol. 59: 315-327. Hamm, 11, E.L. Styer and W.I Lewis. 1990. Comparative virogenesis of filamentous virus and polydnavirus in the female reproductive tract of Cotesia marginiventris (Hymenoptera, Braconidae). 1 Invertebr. Pathol. 55: 357-374. Ibrahim, AMA and Y Kim. 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth Plutella xylostella. 1 Insect Physiol. 52: 943-950. Ibrahim, AMA and Y Kim. 2007. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses. Naturwissenschaften (In press) Ibrahim, AMA, IY Choi, YH. Je and Y Kim. 2005. Structure and expression profile of two putative Cotesia plutellae bracovirus genes (CpBV-H4 and CpBV-E94a) in parasitized Plutella xylostella. 1 Asia-Pac. Entomol. 8: 359-366. Ibrahim, AMA, IY Choi, YH. Je and Y Kim. 2007. Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: sequence analysis, expression profile, and a possible
Ultrastructure of CpBV
biological role in host immunosuppression. Dev. Compo Irnrnunol. 31: 978-990. Kim, Y 2006. Polydnavirus and its novel application to insect pest control. Kor. 1 Appl. Entomol. 45: 241-259. Kim, Y, S. Bae and S. Lee. 2004. Polydnavirus replication and oviposition habit of Cotesia plutellae. Kor. 1 Appl. Entomol. 43: 225-231. Kim, Y, r.v. Choi and YH. Je. 2007. Cotesia plutellae bracovirus genome and its function in altering insect physiology. 1 Asia-Pac. Entomol. 10: 181-191. Krell, P.l 1987. Replication of long virus-like particles in the reproductive tract of the ichneumonid wasp Diadegma terebrans. 1 Gen. Virol. 68: 1477-1484. Lee, K., S. Cho, H. Lee, r.v. Choi, YH. Je and Y Kim. 2005. Gene expression pattern of Cotesia plutellae bracovirus EPI-like protein (CpBV-ELPl) in parasitized diamondback moth Plutellae xylostella. 1 Asia- Pac. Entomol. 8: 249-255. Madanagopal, N. and Y Kim. 2006. Parasitism by Cotesia glomerata induces immunosuppression of Pieris rapae: effects of ovarian protein and polydnavirus. 1 Asia-Pac. Entomol. 9: 339-346. Madanagopal, N. and Y Kim. 2007a. A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior. 1 Insect Physiol. 53: 1283-1292. Madanagopal, N. and Y Kim. 2007b. Biochemical characterization of C-type lectin in the diamondback moth, Plutella xylostella. 1 Asia-Pac. Entomol. 10: 229-237. Marti, D., C. Grossniklaus-Burgin, S. Wyder, T. Wyler and B. Lanzrein. 2003. Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. I. Ovary morphogenesis, amplification of viral DNA and
361
ecdysteroid titres. 1 Gen. Virol. 84: 1141-150. Norton, W.N., S.B. Vinson and D.B. Stoltz. 1975. Nuclear secretory particles associated with calyx cells of the ichneumonid parasitoid Campoletis sonorensis (Cameron). Cell Tissue Res. 162: 195-208. Webb, BA, N.E. Beckage, Y. Hayakawa, P.l Krell, B. Lanzrein, D.B. Stoltz, MR Strand and MD. Summers. 2000. Polydnaviridae. pp. 253-260, in Virus taxonomy, Eds. MHV van Regenmortel, C.M Fauquet, DHL. Bishop, E.B. Carstens, MK. Estes, S.M Lemon, 1 Maniloff, MA Mayo, D.l McGeoch, C.R. Pringle and RB. Wickner. Academic Press, New York. Webb, BA and MR Strand. 2005. The biology and genomics of polydnaviruses. pp. 323-360, in Comprehensive molecular insect science, Eds. L.I. Gilbert, K. Iatrou and S.S. Gill. Elsevier, New York. Webb, BA and MD. Summers. 1992. Stimulation of polydnavirus replication by 20-hydroxyecdysone. Experientia 48: 1018-1022. Whitfield, lB. and S. Asgari. 2003. Virus or nor? Phylogenetics of polydnaviruses and their wasp carriers. 1 Insect Physiol. 49: 397-405. Wyder, S., A. Tschannen, A. Hochuli, A. Gruber, V. Saladin, S. Zumbach and B. Lanzrein. 2002. Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering. 1 Gen. Virol. 83: 247-256. Wyler, T. and B. Lanzrein. 2003. Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. 2. Ultrastructure analysis of calyx cell development, virion formation and release. 1 Gen. Virol. 84: 1151-1163.
PHYSIOLOGY AND BIOCHEMISTRY
Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx Kim, Yonggyun* and Sanghoon Ryu' Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea lCentre for Connnon Research Facility, Andong National University, Andong 760-749, Korea
Abstract Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to an endoparasitoid, C. plutellae. Despite rich information on CpBV genome, there has been little known on its viral replication mode from proviral to episomal form. This study illustrates fine structures of the epithelial cells producing CpBV with a reference to non-producing ovarian epithelial cells. The ovarian epithelial cells of teneral females (within 12 h after emergence) were characterized by large nucleus and rich rough endoplasmic reticulum. CpBV particles were present only at the calyx region, in which follicle epithelial cells exhibited virogenic structures. Though a matured CpBV particle found in the calyx lumen was encapsidated in a single envelop containing multiple nucleocapsids, numerous free nucleocapsids were observed in the calyx epithelial cells and appeared to undergo assembly step to a fmal multiple capsid form. The multiple capsid forms appeared to be released into the oviduct lumen. The epithelial cells bordering the oviduct lumen showed phagocytosis presumably due to clearing cellular debris. At the calyx area close to the common oviduct, the epithelial cells appeared to maintain protein synthetic activity due to highly developed rough endoplasmic reticulum, but showed a marked decrease in the viral production.
Key wonts Cotesia plutellae, CpBV, Polydnavirus, Replication, TEM, Ultrastructure
Introduction Polydnavirus is a unique double stranded DNA virus symbiotic to some endoparasitoid wasps (Kim, 2006; Kim et al., 2007). It comprises of two genera, Ichnovirus (IV) and Bracovirus (BV), depending on host *Corresponding author. Email: [email protected] Tel: +82-54-820-5638; Fax: +82-54-823-1628 (Received October 2, 2007; Accepted December 7, 2007)
wasps, respectively, Ichneumonidae and Braconidae (Webb et al., 2000). These two viral groups are classified by viral morphology and serological or DNA hybridization analyses, suggesting their independent origins (Whitfield and Asgari, 2003). Polydnavirus is also unique in their viral transmission mode. It is located on host chromosome as a proviral form, which allows them to vertically transmit along with wasp generation (Blissard et al., 1987). Its replication into viral particles occurs only in ovarian calyx region during pupal stage in response to ecdysteroid signal (Webb and Summers, 1992; Wyder et al., 2002). IV virions are relatively uniform in size and contain biconvex nucleocapsids surrounded by two unit membranes, whereas BVs are highly variable in length and have cylindrical nucleocapsids surrounded by single envelope (Webb and Strand, 2005). The outer membrane of IV is acquired by budding off from calyx cells. In comparison, single envelop of BV suggests its release into the oviduct lumen by cell lysis (Wyler and Lanzrein, 2003). Cotesia plutellae (Braconidae: Hymenoptera) is a solitary endoparasitoid wasp that parasitizes the diamondback moth, Plutella xylostella (Bae and Kim, 2004). C. plutellae bracovirus (CpBV) has been identified and known to be replicated in the ovarian calyx during late pupal stage (Kim et al., 2004). CpBV has been regarded as a major factor to reduce host cellular innnune capacity in the parasitized P. xylostella (Basio and Kim, 2006; Ibrahim and Kim, 2006). For example, CpBVI5[3 gene effectively inhibits hemocyte-spreading behavior in P. xylostella (Madanagopal and Kim, 2007a). CpBV-lectin exhibits early expression and has been suspected to inhibit nonself recognition of P. xylostella (Madanagopal and Kim, 2006, 2007b). CpBV-PTPs impair cellular immune reactions in phagocytosis and encapsulation (Ibrahim et al., 2007; Ibrahim and Kim, 2007). CpBV-H4, CpBV-E94K and CpBV-ELPI are speculated to inhibit host immune capacity (Ibrahim et al., 2005; Lee et al., 2005). Despite significant roles of polydnavirus in the parasitism exhibited by the endoparasitoids, little has been known about molecular processes of polydnavirus repli-
358
J. Asia-Pacific Entomol. Vol. 10 (2007)
Materials and Methods
aldehyde in 0.01 M phosphate buffer, pH 7.2, for 2 h at 4"C. After several washes in the phosphate buffer, samples were post-fixed by 1% osmium tetroxide for 30 min at 4"C and washed in the phosphate buffer. The samples were dehydrated through a graded series of ethanol and substituted with propylene oxide, and then embedded in Epon 812 for 48 h at 60"C. Sections «90 nm) were cut on a Leica Ultracut UCT ultramicrotome using a diamond knife (Diatome). Ultrathin sections were transferred onto 200 mesh copper grids and stained with uranyl acetate for 20 min, and with lead citrate for a further 10 min. The sections were examined in TEM (Hitachi, H-7650) operation at 80 kV
C. plutellae
Results and Discussion
cation. To understand the molecular events undergoing during polydnavirus replication, structural and genomic studies need to be thoroughly analyzed. To this end, this study performed the structural analysis of ovarian calyx cells of C. plutellae to understand replication and release of CpBV This study illustrates the CpBV replication only in ovarian calyx cells, not in other ovarian follicle cells. It also provides structural assembly of multiple nucleocapsids into an envelope, which is release into oviduct lumen.
Larvae of P. xylostella, as parasitized host of C. plutellae, were reared on cabbage leaves at 25±1 "C under a photoperiod of 16:8 (LD) h. For parasitization, early second instar larvae of P. xylostella were exposed to 1-4 days old C. plutellae adults for 24 h. Propagation of both parasitoid and host insects was performed at 25±1"C. The host was fed daily with cabbage leaves.
TEM (transmission electron microscope) analysis Newly emerged females (within 12 h) were used to isolate ovaries, which were fixed in 2.5% glutar-
CpBV begins to replicate during pupal stage of C. plutellae especially at adult tissue differentiation (Kim et al., 2004). When C. plutellae emerge, the oviduct lumen is filled with CpBV, which would be enough amounts for all parasitization during entire adult period. In this study, teneral females had fully grown ovaries with long germarium, thick vitellarium, and hypertrophied calyx region (Fig. 1). The calyx region was blue-eolored externally, presumably filled with CpBV In a sagittal section, mature oocytes were visible in vitellarium, some passing through the calyx region. Each ovariole in the germarium showed one layer epithelial structure and did not contain any oocyte
Fig. 1. Ultrastructure of epithelial cells ('Ep') at germarium and vitellarium of one day old Cotesia plutellae ovary. Oocyte ('Oc') is present in the vitellarium. Magnification is expressed in each TEM photo.
Ultrastructure of CpBV
and viral particles. The epithelial cells contained many patches of dense heterochromatin. Oocytes were frequently visible in the vitellarium and surrounded by follicle cells. Any viral production was not evident in the vitellarium and viral particles were not detected in the spaces between the oocytes and follicle cells. The calyx cells of C. plutellae were different from
Fig. 2. Ultrastructure of upper calyx cells bordering vitellarium of one day old Cotesia plutellae ovary, in which oocyte ('Dc') is visible. 'N' represents nucleus of the calyx cell, in which many virus particles are present in the cytosol. Magnification is expressed in a TEM photo.
359
ovarian epithelial cells in germarium or vitellarium in their large nuclei and richness of rough endoplasmic reticulum (Fig. 2). In Chelonus inanitus, the polydnavirus began to form during pupal stage, at which calyx cells become polyploidy and in the following phase integrated viral DNA is selectively and intensively amplified to be excised and circularized (Marti et al., 2003). Especially, when the calyx cells are differentiated from the remaining ovarian epithelial cells of C. inanitus, the 20-hydroxyexdysone (20E) titer is maximal during pupal stage (Marti et al., 2003). In comparison, Webb and Summers (1992) suggested that 20E is directly responsible for polydnaviral replication. Here we can interpret the effect of 20E on the polydnavirus production by its mediation of differentiation of the calyx epithelial cells into polydnavirus-producing state. Newly formed CpBV particles in the lumen were not attached to the surface of the mature eggs. Similar observations were made with C. marginiventris BV (Hamm et al., 1990), C. congregata BV (De Buron and Beckage, 1992) and the IVs of Diadegma terebrans (Krell, 1987) and Campoletis sonorensis (Norton et al., 1975). The calyx cells contained virions, some undergoing encapsidation, in which nucleus had virogenic stroma and many patches of dense heterochromatins (Fig. 3). Nucleocapsids were encapsidated singly or multiply.
Fig. 3. Virus production in the calyx cells of one day old Cotesia plutellae ovary. 'N' represents nucleus of the calyx cell, in which many virus particles are present in the cytosol. An arrow indicates a lysis of nuclear membrane. Singly or multiply encapsidated viral particles are shown lower panels. Magnification is expressed in each TEM photo.
360 1 Asia-Pacific Entomol. Vol. 10 (2007)
Single nucleocapsids varied in length and shape from round to square. Similar nucleocapsids were found in multiple packaged virions. In addition, all virions were multiply encapsidated in the calyx fluid, suggesting that the singly encapsidated particles would be further assembled. The assembled virions appeared to be transported into calyx fluid probably by cell rupture and the epithelial cells bordering the oviduct lumen exhibited phagocytosis to clear the cellular debris (Fig. 4). Some large vesicles containing several virions were also observed. Ultrastructural studies indicated that BVs were released by cell lysis with continued production of virus-producing cells from the calyx (Stoltz and Vinson, 1979). The lysed cellular debris was cleared by phagocytosis of the calyx epithelial cells bordering oviduct lumen (Wyler and Lanzrein, 2003). In C. plutellae, the epithelial cells bordering oviduct lumen also had
a various types of vacuoles (Fig. 5), suggesting phagocytic activity. Further clarification needs to determine the release mode of CpBV viral particles from the calyx cells to the ovarian lumen in C. plutellae. We have presented the first complete description of calyx cells and CpBV morphogenesis. Along with previous CpBV replication study (Kim et al., 2004), we may develop a complete picture of ovary and calyx cell development. During pupal stage, C. plutellae begins ovarian differentiation to form virus-producing calyx cells. This may be induced by rise of 20E. The virus production is confmed in the ovarian calyx cells. With oogenesis, matured oocytes are accumulated in the vitellarium, but free from CpBV particles. On adult emergence, the females are ready to parasitization with enough amounts of newly formed CpBV particles in the oviduct lumen. Acknowledgements This study was supported by Biogreen 21 project of RDA, Korea, and by the 2nd stage of BK21. We also appreciate Dr. Nalini Madanagopal for her preparing C. plutellae ovaries for TEM study.
Lumen
Literature Cited
Epithelium
Fig. 4. Phagocytosis of epithelial cells in of one day old Cotesia plutellae ovary. different phases of phagosome formation of membrane. Magnification is expressed in
the calyx region Arrows indicate the epithelial cell a TEM photo.
Sheath
Epithelium
Lumen
Fig. 5. The epithelial cells at the oviduct lumen filled with calyx fluid of day old Cotesia plutellae ovary. The cells are surrounded by the epithelial sheath. Magnification is expressed in a TEM photo.
Bae, S. and Y Kim. 2004. Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Compo Biochem. Physiol. 138B: 39-44. Basio, NA and Y Kim. 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31: 341-347. Blissard, G.W., O.P., Smith and MD. Summers. 1987. Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome. Virology 160: 120-134. De Buron, 1. and N.E. Beckage. 1992. Characterization of a polydnavirus (PDV) and virus-like filamentous particle (VLFP) in the braconid wasp Cotesia congregata (Hymenoptera: Braconidae). 1 Invertebr. Pathol. 59: 315-327. Hamm, 11, E.L. Styer and W.I Lewis. 1990. Comparative virogenesis of filamentous virus and polydnavirus in the female reproductive tract of Cotesia marginiventris (Hymenoptera, Braconidae). 1 Invertebr. Pathol. 55: 357-374. Ibrahim, AMA and Y Kim. 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth Plutella xylostella. 1 Insect Physiol. 52: 943-950. Ibrahim, AMA and Y Kim. 2007. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses. Naturwissenschaften (In press) Ibrahim, AMA, IY Choi, YH. Je and Y Kim. 2005. Structure and expression profile of two putative Cotesia plutellae bracovirus genes (CpBV-H4 and CpBV-E94a) in parasitized Plutella xylostella. 1 Asia-Pac. Entomol. 8: 359-366. Ibrahim, AMA, IY Choi, YH. Je and Y Kim. 2007. Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: sequence analysis, expression profile, and a possible
Ultrastructure of CpBV
biological role in host immunosuppression. Dev. Compo Irnrnunol. 31: 978-990. Kim, Y 2006. Polydnavirus and its novel application to insect pest control. Kor. 1 Appl. Entomol. 45: 241-259. Kim, Y, S. Bae and S. Lee. 2004. Polydnavirus replication and oviposition habit of Cotesia plutellae. Kor. 1 Appl. Entomol. 43: 225-231. Kim, Y, r.v. Choi and YH. Je. 2007. Cotesia plutellae bracovirus genome and its function in altering insect physiology. 1 Asia-Pac. Entomol. 10: 181-191. Krell, P.l 1987. Replication of long virus-like particles in the reproductive tract of the ichneumonid wasp Diadegma terebrans. 1 Gen. Virol. 68: 1477-1484. Lee, K., S. Cho, H. Lee, r.v. Choi, YH. Je and Y Kim. 2005. Gene expression pattern of Cotesia plutellae bracovirus EPI-like protein (CpBV-ELPl) in parasitized diamondback moth Plutellae xylostella. 1 Asia- Pac. Entomol. 8: 249-255. Madanagopal, N. and Y Kim. 2006. Parasitism by Cotesia glomerata induces immunosuppression of Pieris rapae: effects of ovarian protein and polydnavirus. 1 Asia-Pac. Entomol. 9: 339-346. Madanagopal, N. and Y Kim. 2007a. A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior. 1 Insect Physiol. 53: 1283-1292. Madanagopal, N. and Y Kim. 2007b. Biochemical characterization of C-type lectin in the diamondback moth, Plutella xylostella. 1 Asia-Pac. Entomol. 10: 229-237. Marti, D., C. Grossniklaus-Burgin, S. Wyder, T. Wyler and B. Lanzrein. 2003. Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. I. Ovary morphogenesis, amplification of viral DNA and
361
ecdysteroid titres. 1 Gen. Virol. 84: 1141-150. Norton, W.N., S.B. Vinson and D.B. Stoltz. 1975. Nuclear secretory particles associated with calyx cells of the ichneumonid parasitoid Campoletis sonorensis (Cameron). Cell Tissue Res. 162: 195-208. Webb, BA, N.E. Beckage, Y. Hayakawa, P.l Krell, B. Lanzrein, D.B. Stoltz, MR Strand and MD. Summers. 2000. Polydnaviridae. pp. 253-260, in Virus taxonomy, Eds. MHV van Regenmortel, C.M Fauquet, DHL. Bishop, E.B. Carstens, MK. Estes, S.M Lemon, 1 Maniloff, MA Mayo, D.l McGeoch, C.R. Pringle and RB. Wickner. Academic Press, New York. Webb, BA and MR Strand. 2005. The biology and genomics of polydnaviruses. pp. 323-360, in Comprehensive molecular insect science, Eds. L.I. Gilbert, K. Iatrou and S.S. Gill. Elsevier, New York. Webb, BA and MD. Summers. 1992. Stimulation of polydnavirus replication by 20-hydroxyecdysone. Experientia 48: 1018-1022. Whitfield, lB. and S. Asgari. 2003. Virus or nor? Phylogenetics of polydnaviruses and their wasp carriers. 1 Insect Physiol. 49: 397-405. Wyder, S., A. Tschannen, A. Hochuli, A. Gruber, V. Saladin, S. Zumbach and B. Lanzrein. 2002. Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering. 1 Gen. Virol. 83: 247-256. Wyler, T. and B. Lanzrein. 2003. Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. 2. Ultrastructure analysis of calyx cell development, virion formation and release. 1 Gen. Virol. 84: 1151-1163.