Differential Parasitic Capacity of Cotesia plutellae and C. glomerata on Diamondback Moth, Plutella xylostella and Dichotomous Taxonomic Characters

J. Asia-Pacific Entomol. 9(3):

293~300

(2006)

www.entomology.or.kr

Differential Parasitic Capacity of Cotesia plutellae and C. glomerata on Diamondback Moth, Plutella xylostella and Dichotomous Taxonomic Characters 2

Yonggyun Kim*, Ahmed M. A. Ibrahim, Sungchae Jung 1 and Min Kwoen

Department of Bioresource Sciences, College of Natural Sciences, Andong National University, Andong 760-749, Korea [Green AgroTech, Inc., Kyungsan 712-240, Korea 2Environment Management Division, National Institute of Highland Agriculture, RDA, Pyeongchang 232-955, Korea

Abstract Two closely-related endoparasitoids of Cotesia plutellae and C. glomerata parasitize the diamondback moth, Plutella xylostella. The parasitized hosts by either parasitoid species exhibited the extended larval period and died without further metamorphosis to pupal stage. However, two parasitoid species exhibited significantly different parasitic capacity and developmental rate, in which C. plutellae showed higher parasitism and faster development in the parasitized P. xylostella. To discriminate these two similar species, morphological and molecular differences were analyzed. Three dichotomous morphological characters including antennal flagellum, hind-leg femur, and terminal abdominal terga were determined. Based on the presumptive polydnaviral particles found in the ovarian calyx of C. glomerata, three genes similar to C. plutellae bracoviral genes were cloned in the C. glomerata genome and compared in their cDNA and the deduced amino acid sequences. Several polymorphic sites were detected to be applicable to design molecular markers to discriminate these two species. Key words Cotesia glomerata, C. plutellae, diamondback moth, parasitism, polydnavirus

Introduction Parasitoids spend their immature stage as parasites, but are present as free-living adults. Most (;:::; 80%) insect parasitoids belong to the order Hymenoptera, in which the monophyletic suborder Apocrita contains most species of parasitoids (Dowton and Austin, 1994; Whitfield, 1997). The Ichneumonoidea, a superfamily of Apocrita, are all parasitoids containing about *Corresponding author. E-mail: [email protected] Tel: +82-54-820-5638; Fax: +82-54-823-1628 (Received July 18, 2006; Accepted August I, 2006)

100,000 species and divided into the families Braconidae and Ichnewnonidae, some of which are known to possess their symbiotic polydnavirus (Webb, 1998). Polydnavirus is an insect DNA virus associated with the parasitoid wasp genome as a proviral form (Krell et al., 1982). It transmits vertically along with host parasitoid generations (Stoltz et al., 1993). When the host parasitoids lay eggs, the replicated polydnavirus particles move from the female reproductive tract to the parasitized insect hemocoel (Marti et al., 2003; Wyler and Lanzrein, 2003). The polydnavirus in the hemocoel infects target tissues mostly including hemocytes and fat body, and express its genes that are implicated with physiological alterations of the parasitized hosts (Kroemer and Webb, 2004). Polydnaviruses are divided into bracovirus ('BY') and ichnovirus ('IV') by their symbiotic host families (Webb, 1988). In Braconidae, four subfamilies (Microgastrinae, Cardiochilinae, Miracinae, and Cheloninae) are known to carry BYs and estimated to contain about 17,000 species that exhibit monophyletic lineage derived from non-BY-carrying ancestor at about 60 million years ago (Whitfield, 1997, 2000, 2002). Cotesia plutellae (Braconidae: Hymenoptera) is an endoparasitoid of the diamondback moth, Plutella xylostella, and possesses its symbiotic polydnavirus, CpBV (Bae and Kim, 2004; Choi et al., 2005). The parasitized P. xylostella shows an extended larval period and significant immunodepression (Lee and Kim, 2004; Ibrahim and Kim, 2006). In addition to the effect of C. plutellae teratocytes on the immunodepression (Basio and Kim, 2006), several CpBV genes have been regarded as putative inhibitory factors (Kim et al., 2006). CpBV-lectin and CpBV15a !G were cloned from the parasitized P. xylostella (Lee, 2004), which would be regarded as putative immunodepressive agents or host developmental regulators. CpBV-ELP1, CpBV-IkB, CpBV-PTPs, CpBV-E94a, and CpBV-H4 were identified from a full genome

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J. Asia-Pacific Entomol. Vol. 9 (2006)

sequencing project (Choi et al., 2005) and regarded as another sources of host physiology regulators (Ibrahim et al., 2005; Lee et al., 2005; Kim et al., 2006). Another closely related microgastrinid species, C. glomerata, has been known to parasitize P. xylostella (Jung et al., 2006) in addition to its main natural hosts, Pieris brassicae and P. rapae (Laing and Levin, 1982). C. glomerata is a gregarious endoparasitoid that has from three to 158 brood sizes, and the brood size increases with superparasitism in Pieris hosts (Gu et al., 2003). However, only one C. glomerata cocoon was formed in the parasitized P. xylostella as did C. plutellae (Jung et al., 2006). Though these two Cotesia species are so much similar in their external shape, they seem to be different in some biological characters such as parasitism and developmental rate. For practical purpose to use these biological agents against P. xylostella, their parasitic functional comparison needs to be analyzed. Then the better biological agent should be selected for mass-rearing to release because the contamination of the inferior species in the natural field may cause some wasteful species-competition. This study focused on comparing the two Cotesia species in terms of parasitic capacity. Then we determined the morphological and molecular characters to discriminate these closely related species.

Materials and Methods Insect rearing and parasitization P. xylostella larvae were reared under 25±I"C and

16:8 h (L : D) photoperiod with cabbage leaves. Adults were fed 10% sucrose solution. Late second instal' larvae were parasitized by C. plutellae or C. glomerata at 1:2 (wasp: DBM) density for 24 h under the rearing condition. Then, the parasitized larvae were fed the cabbage leaves and incubated at the rearing environment. After emergence, adult wasps were allowed to mate for 24 h and then used for parasitization.

Determination of parasitic capacity To compare the parasitism capacity between two wasp species, ten larvae of the second instal' (4 days old after oviposition) P. xylostella were exposed to a single mated female (2 days old after emergence) of C. plutellae or C. glomerata in a plastic box (10x12x 15 em) for 4 h under the rearing condition. Following parasitization host larvae were dissected and presence of parasitoid embryo was observed and counted as parasitized larva under a stereo microscope (Olympus

SZ30, Olympus, Japan). Each measurement was replicated three times independently. The developmental periods of wasps were measured in both egg-larval stages and pupal stage at the rearing environment. The egg-larval period was measured from the parasitization to the wasp larval egression from the host. Three hundred parasitized larvae were used for an experimental unit and replicated three times. The pupal period was measured from the cocoon formation to adult emergence at the rearing environment. Ten pupae were used for an experimental unit and replicated three times.

Morphological analysis Three morphological characters of adults were observed under a stereomicroscope and their structures were taken using Fluoview image system (Olympus, Tokyo, Japan). Antennal structure was focused on the distal flagellar area. The integument darkening was observed on the hind leg femur. Terminal abdominal terga of females were compared in size and form. The tergal structure was photographed at 2.5 kV with a scanning electron microscope (SEM6400, JEOL Ltd., Tokyo, Japan).

RT-peR (reverse transcriptasepolymerase chain reaction) and eDNA sequence analysis Total RNA was extracted from the C. glomerata parasitized larvae (5 days after parasitization) using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and followed by its precipitation with isopropanol. Then the resulting RNA pellet was washed in 70 % ethanol and resuspended in DEPC treated water. To synthesize the first strand of cDNA, 1 ug of the total RNA reacted with RT-premix (Bioneer, Daejon, Korea) using an oligo (dT) primer (5' -CCAGT GAGCA GAGTG ACGAG GACTC GAGCT CAAGC TTTTT TTTTT TTTTT T-3') and followed by the treatment of RNase H. This cDNA was used as template to amplify C. glomerata bracovirus (CgBV) genes using specific primers (Table 1). The PCR product then was cloned into PCR2.1 cloning vector (Invitrogen). The insert sequences were analyzed by a DNA sequencing company (Macrogen, Daejon, Korea). The CgBV genes were aligned with those of CpBV using Clustal V of DNAstar program (Version 5.01, DNAstar Inc, Madison, WI, USA). The characteristic restriction polymorphic locus was confirmed by restriction digestion with the PCR products using the method of Kim et al. (1998).

Cotesia plutellae versus C. glomerata

295

Results

df = 4; P = 0.4216): 2.23±0.21 days for C. plutellae and 2.37±0.15 days for C. glomerata.

Comparison of C. plutellae and C. glomerata in parasitic capacity

Comparison of C. plutellae and C. glomerata in adult female morphology

Two endoparasitoids were compared in their capacity of parasitism on P. xylostella (Fig. I). Both species parasitized P. xylostella but showed significant difference in the parasitism rates, in which C. plutellae were superior to C. glomerata. From the parasitized P. xylostella by either C. plutellae or C. glomerata, the fully developed late instar wasp larvae began to egress from the moribund host larvae and showed a peak cocoon formation at 8 days after the parasitism in C. plutellae, but 9 days in C. glomerata (Fig. 2). The average larval periods (from the time of parasitization to the time of the egression) were 8.05±0.15 days for C. plutellae and 8.77±0.13 days for C. glomerata, both of which were significantly different (t = 6.36; df = 4; P = 0.0031). However, pupal periods in 25°C were not significantly different (t = 0.89;

The fact that the two Cotesia species exhibit different parasitic efficiency suggests a need to discriminate these two closely related species with dichotomous characters. Three morphological characters were selected because of their significant differences (Fig. 3). The antennal flagellum was tapering in width in C. glomerata whereas it did not show significant change in C. plutellae. Both species showed a tanned area on the hind-leg femur, in which the tanned area was broader in C. glomerata than in C. plutellae. The last two abdominal terga were significantly different. C. plutellae had a large triangular penultimate tergum and a small ring of the last tergum. In contrast, C. glomerata had a large ring-shaped penultimate tergum and a triangular last tergum.

Table 1. Primer sequences used to clone three polydnaviral genes of Cotesia glomerata bracovirus Genes

Direction

ELP]

Forward

5'-ATG TTC AAC AAA GTA GTC TTC-3'

Reverse

5'-TTG CAG CCG ATA ACT ATC CAT TTG-3'

Forward

5'-ATG GCT GAT CAT CCT AAA GG-3'

Reverse

5'-ACC TCC ATA ACC ATA GAT CAT AC-3'

Forward

5'-ATG GGC GCG AAA TTC ACT AA-3'

Reverse

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200

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e

0 0 u 0

70 60

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150

'0

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40 30

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10

50

.... C. pluteflae

C. glomerata

I~ ~ J

n 10

Days after parasitization

Fig. 1. Comparison of parasitism on Plutella xylostella between Cotesia plutellae and C. glomerata. See Materials and Methods for the details of parasitism assay. Different letters above the standard error bars indicate significant difference between the means at Type I error = 0.05 (LSD test).

Fig. 2. Comparison of developmental rates in the parasitized host, Plutella xylostella, between Cotesia plutellae and C. glomerata. See Materials and Methods for the details of the assay. The error bars represent standard deviation of three replications.

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J. Asia-Pacific Entomol. Vol. 9 (2006)

Comparison of C. plutellae and C. glomerata in three polydnaviral gene sequences The taxonomic relatedness and the fact that all microgastrinid species investigated possess polydnavirus (Whitfield, 2002) suggest that C. glomerata genome contains polydnavirus ('CgBV'). When the reproductive organ of C. glomerata was observed, the characteristic hypertrophied ovarian calyx was detected (Fig.

4). Even though we did not investigate the viral particles using electron microscope, the calyx lumen may contain the CgBV particles. This was supported by the cloning of polydnaviral genes. The parasitized P. xylostella by C. glomerata was used to construct cDNA, which was used as a template to clone the genes (ELPl, H4, and PTPl) corresponding to CpBV. The RT-PCR products were sequenced and compared with those of CpBV (Fig. 5). Among these three genes, the highest homology

Antennal flagellum

Hind-leg femur

Abdominal tip

Fig. 3. Comparison of three morphological characters of female Cotesia plutellae and C. glomerata. See Materials and Methods for the details. Antennal and hind leg characters were observed at 500 x under a stereomicroscope. The abdominal tip was observed under SEM at 1,500 x

Fig. 4. Female reproductive organ of Cotesia glomerata. 'C' represents ovarian calyx, presumably filled with polydnavirus. 'OVP' represents ovipositor.

Cotesia plutellae versus C. glomerata

297

To develop a molecular marker to discriminate two species, a restriction polymorphic site was chosen in Pst I and tested after peR of ELP1 locus. As expected, ELP1 of C. glomerata was cut into two segments (71 and 658 bp), but that of C. plutellae was not (data not shown).

between two wasp species was found in PTP 1 gene (Table 2), in which only 3 nucleotides out of 900 base pairs. ELP1 gene showed the highest variation between two species especially in 5' open-reading frame region. The nucleotide changes included several numbers of transition and transversion point mutations (Table 2).

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298

J. Asia-Pacific Entomol. Vol. 9 (2006)

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Fig. 5. Sequence alignment of cDNA and its deduced amino acid in three polydnaviral genes: CpBV (Cotesia plutellae bracovirus) and CgBV (c. glomerata bracovirus). 'ELPI', 'H4' and 'PTPI' represent EPl-like I, histone subunit 4, and protein tyrosine phosphatase 1, respectively. The three CgBV genes were uploaded on the NCB I Genbank with accession numbers of DQ844603 ('ELPI '), DQ839630 ('PTPI '), and DQ839631 ('H4'). Only point mutations are shown in open boxes.

Table 2. Sequence similarity and difference in three polydnaviral genes of Cotesia plutellae and C. glomerata Viral genes

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Discussion The vast majority of polydnavirus-carrying parasitoids have restricted host ranges, usually being able to parasitize only a few species of hosts (Cui et al., 2000; Whitfield and Asgari, 2003). In this study, we showed that two Cotesia species of C. plutellae and C.

glomerata parasitized P. xylostella in addition to their alternative natural hosts. C. plutellae also parasitizes Hyphantria cunea and successfully develops into a cocoon (Kim et al., 2004). C. glomerata parasitizes Pieris rapae or P. brassicae and develop multiple cocoons from a parasitized host (Gu et al., 2003), though it produced a cocoon from the parasitized P. xylostella. The superparasitism occurred in laboratory

Cotesia plutellae versus C. glomerata

and field populations of C. glomerata on the Pieris hosts allows a favorable survival strategy for the species by giving rise to increase the brood size of the wasp progeny (Tagawa, 2000; Gu et al., 2003). The switch of C. glomerata from a general gregarious in Pieris species to a solitary parasitism in P. xylostella has been unclear, but seems to depend on the host size and nutritional value, considering the high variation in the brood sizes usually exhibited in the gregarious parasitism. P. xylostella parasitized by C. glomerata may not provide enough food resource for more than one progeny. The resource limitation causes intra-specific competition among individual wasps (Harvey et al., 2000), which may result in a solitary parasitism in P. xylostella. C. plutellae can be regarded as the better biological control agent against P. xylostella than C. glomerata because C. plutellae showed higher parasitism and shorter developmental period. Observational data indicate that C. glomerata parasitizes P. xylostella infesting cabbage cultivated in high altitude whereas C. plutellae does in normal low land-cultivating areas (MK, unpublished data). The survival strategy of C. glomerata in extreme environment can be supported by its diapause capacity to overcome low temperatures (Tagawa et al., 1984). Thus, this differential property of two biological agents allows an optimal choice between these two parasitoids depending on field condition. However, the mixture of these two species may decrease the expected biological control efficacy. Though the super-parasitism of C. glomerata on P. xylostella parasitized by C. plutellae was not analyzed in this study, it may decrease the parasitic capacity in the subsequent generation. Three morphological characters were chosen to discriminate the two species because of their clear differences that can be used even by non-taxonomists. For molecular markers, we analyzed three polydnaviral genes. Polydnaviral gene markers are more versatile than other wasp genome markers because it can be used in both wasp and the parasitized host because the polydnaviral genome, in general, moves into the parasitized host as viral particles along with the wasp egg during oviposition (Wyler and Lanzrein, 2003). This study suggests for the first time that C. glomerata contains its symbiotic polydnavirus ('CgBV') by morphological evidence showing a characteristic hypertrophied calyx in the female reproductive organ (Marti et al., 2003) and the presence of the three related polydnaviral genes by RT-PCR in the parasitized host. ELPI has been found in CpBV genome (Lee et al., 2005) and showed high similarity with EPI gene of other Cotesia-associated polydnavirus, in which EPI is expressed at early parasitization period and increases its hemolymph titer (15% of the total he-

299

molymph protein) during the initial period (Harwood and Beckage, 1994). The function of EPI has not been known, but can be regarded as an immunosuppressor by protecting the wasp egg from hemocyte encapsulation (Harwood et al., 1998). H4 is a viral histone subunit 4 that contains N-terminal extension and has been found only in CpBV (Ibrahim et al., 2005). It has been speculated that the CpBV-H4 may interrupt a normal chromosomal rearrangement in response to gene expression signal. PTPI is a protein tyrosine phosphatase found in CpBV (YK, unpublished data). PTPs regulate tyrosyl phosphorylation in numerous signaling pathways and have been targeted by selected prokaryotic pathogens to disrupt phagocytosis by mammalian immune cells (Neel and Tonks, 1997; Comelis, 2002). PTPs have also been suggested to play a role in disrupting ecdysteroid biosynthesis in parasitized hosts (Falabella et al., 2006). The occurrence of these three genes in the CgBV genome and expression in the parasitized P. xylostella partially explains its host physiology alteration, as parasitized P. rapae and P. brassicae larvae consume more food and grow faster than nonparasitized larvae (Rahman, 1970; Slansky, 1978; Coleman et al., 1999). All these three genes were found in both Cotesia species and showed significant numbers of point mutations between two species. The most polymorphic sites were found in ELPl between C. plutellae and C. glomerata. Pst I can be an RFLP marker in this locus because it cut only CgBV-ELPl. This PCR-RFLP provides a molecular tool to discriminate these two species in field and laboratory populations. Acknowledgments This study was supported by Biogreen 21 project of RDA, Korea. AMAI and SJ were financially nd supported by the 2 stage of BK21. We also appreciate Youngim Song for her valuable help and encouragement.

Literature Cited 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. l38A: 39-44. Basio, N.A.M. and Y. Kim. 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31: 1-7. Choi, 1.Y., J.Y. Rho, J.N. Kang, H.J. Shim, S.D. Woo, B.R. Jin, M.S. Li and Y.H. Je. 2005. Genomic segments cloning analysis of Cotesia plutellae polydnavirus using plasmid capture system. Biochem. Biophys. Res. Cornm. 332: 487-493. Coleman, R.A., A.M. Barker and M. Fenner. 1999. Parasitism of the herbivore Pieris brassicae L. (Lep., Pieridae) by

300

J. Asia-Pacific Entomol. Vol. 9 (2006)

Cotesia glomerata L. (Hym. Braconidae) does not benefit the host plant by reduction of herbivory. I. Appl. Entomol. 123: 171-177. Cornelis, G.R. 2002. Yersinia type III secretion: send in the effectors. J. Cell BioI. 158: 401-408. Cui, L., AI. Soldevila and B.A Webb. 2000. Relationships between PDV gene expression and host range of the parasitoid wasp Campoletis sonorensis. J. Insect Physiol. 46: 1397-1407. Dowton, M. and AD. Austin. 1994. Molecular phylogeny of the insect order Hymenoptera: apocritain relationships. Proc. Natl. Acad. Sci. USA 91: 9911-9915. Falabella, P., P. Caccialupi, P. Varricchio, C. Malva and F. Pennacchio. 2006. Protein tyrosine phosphatases of Toxoneuron nigriceps bracovirus as potential disrupters of host prothoracic gland function. Arch. Insect Biochem. Physiol. 61: 157-169. Gu, H., W. Qun and S. Dom. 2003. Superparasitism in Cotesia glomerata: response of hosts and consequences for parasitoids. Ecol. Entomol. 28: 422-431. Harvey, J.A, K. Kadash and M.R. Strand. 2000. Differences in larval feeding behavior correlate with altered developmental strategies in two parasitic wasps: implications for the size-fitness hypothesis. Oikos 88: 621-629. Harwood, S.H. and N.E. Beckage. 1994. Purification and characterization of an early-expressed polydnavirus-induced protein from the hemolymph of Manduca sexta larvae parasitized by Cotesia congregata. Insect Biochem. Mol. BioI. 24: 685-698. Harwood, S.H., I.S. McElfresh, A. Nguyen, CA Conlan and N.E. Beckage. 1998. Production of early expressed parasitism-specific protein in alternate sphingid hosts of the braconid wasp Cotesia congregata. J. Invertebr. Pathol. 71: 271-279. Ibrahim, A.MA, I.Y. Choi, Y.H. Je and Y. Kim. 2005. Structure and expression profiles of two putative Cotesia plutellae bracovirus genes (CpBV-H4 and CpBV-E94a) in parasitized Plutella xylostella. J. Asia-Pacific Entomol. 8: 359-366. Ibrahim, AM.A and Y. Kim. 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella. J. Insect Physiol. (In Press). Jung, S., M. Kwoen, J.Y. Choi, Y.H. Je and Y. Kim. 2006. Parasitism of Cotesia spp. enhances susceptibility of Plutella xylostella to other pathogens. J. Asia-Pacific Entomol. 9: (in press). Kim, Y., S. Bae and S. Lee. 2004. Polydnavirus replication and ovipositional habit of Cotesia plutellae. Korean J. Appl. Entomol. 43: 225-231. Kim, Y., M. Lee and C. Chung. 1998. Study on the genetic variation of the mitochondrial DNA in the beet armywonn, Spodoptera exigua (Hubner), using PCR-RFLP. Korean J. Appl. Entomol. 37: 23-30. Kim, Y., NA Basio, A.MA Ibrahim and S. Bae. 2006. Gene structure of Cotesia plutellae bracovirus (CpBV)-IkB and its expression pattern in the parasitized diamondback moth, Plutella xylostella. Korean J. Appl. Entomol. 45: 15-24. Krell, P.I., M.D. Summers and S.B. Vinson. 1982. Virus with a multipartite superhelical DNA genome from the ichneumonid parasitoid, Campoletis sonorensis. I. Virol. 43: 859870. Kroemer, I.A. and BA Webb. 2004. Polydnavirus and genome:

emerging gene families and new insights into polydnavirus replication. Annu. Rev. Entomol. 49: 431-456. Laing, J.E. and D.B. Levin. 1982. A review of the biology and a bibliography of Apanteles glomeratus (L.) (Hymenoptera: Braconidae). Biocon. News Inform. 3: 7-23. Lee, K., S. Cho, H. Lee, I.Y. Choi, Y.H. Je and Y. Kim. 2005. Gene expression of Cotesia plutellae bracovirus EPI-like protein (CpBV-ELPl) in parasitized diamondback moth, Plutella xylostella. J. Asia-Pacific Entomol. 8: 249-255. Lee, S. 2004. Study on polydnavirus-derived genes and their functions related with parasitism by Cotesia plutellae against diamondback moth, Plutella xylostella. MS Thesis, l57pp. Andong National University, Andong, Korea. Lee, S. and Y. Kim. 2004. Juvenile hormone esterase of diamondback moth, Plutella xylostella, and. parasitism of Cotesia plutellae. J. Asia-Pacific Entomo1. 7: 283-287. Marti, D., C. Grossniklaus-Brugin, 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 ecdysteroid titres. J. Gen. Virol. 84: 1141-1150. Neel, B.G. and N.K. Tonks. 1997. Protein tyrosine phosphatase in signal transduction. Curro Biol, 9: 193-204. Rahman, M. 1970. Effect of parasitism on food consumption of Pieris rapae larvae. J. Econ. Entomol. 63: 820-821. Slansky, F. 1978. Utilization of energy and nitrogen by larvae of the imported cabbageworm. Pieris rapae, as affected by parasitism by Apanteles glomeratus. Environ. Entomol. 17: 179-185. Stoltz, D.B. 1993. The PDV life cycle. pp. 167-187, in Parasites and pathogens of insects, Vol. 1, Parasites, Eds. S.N. Thompson, B.A Federici and N.E. Beckage. Academic Press, San Diego, CA. Tagawa, J. 2000. Sex allocation and clutch size in the gregarious larval endoparasitoid wasp, Cotesia glomerata. Entomol. Exp. Appl. 97: 193-202. Tagawa, J., M. Ishii and Y. Sato. 1984. Diapause in the braconid wasp, Apanteles glomeratus L. I. Evidence of diapause in overwintering prepupae. Appl. Entomol. Zool. 19: 396-399. Webb, BA 1988. Polydnavirus biology, genome structure and evolution. pp. 105-139, in The insect virus, Eds. L.K. Miller and A Ball. Plenum Press, New York. Whitfield, J.B. 1997. Molecular and morphological data suggests a single origin of the PDVs among braconid wasps. Naturwissenschaften 84: 502-507. Whitfield, J.B. 2000. Phylogeny of Microgastroid Braconid wasps, and it tells us about polydnavirus evolution. pp. 97-105, in Hymenoptera: evolution, biodiversity and biological control. CSIRO publishing, Collingwood, Australia. Whitfield, J.B. 2002. Estimating the age of the polydnavirus/ braconid wasp symbiosis. Proc Natl. Acad. Sci. USA 99: 7508-7513. Whitfield, J.B. and S. Asgari. 2003. Virus or not? Phylogenetics of polydnaviruses and their wasp carriers. I. Insect Physiol. 49: 397-405. Wyler, T. and B. Lanzrein. 2003. Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. II. Ultrastructural analysis of calyx cell development, virion formation and release. I. Gen. Virol. 84: 11511163.