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Parasitism by Cotesia plutellae inhibits imaginal wing disc development of diamondback moth, Plutella xylostella
Journal of Asia-Pacific Entomology 11 (2008) 83–87
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e
Parasitism by Cotesia plutellae inhibits imaginal wing disc development of diamondback moth, Plutella xylostella Sungwoo Bae, Yonggyun Kim ⁎ Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea
A R T I C L E
I N F O
Article history: Received 16 February 2008 Revised 5 April 2008 Accepted 22 April 2008 Keywords: Cotesia plutellae Hematopoietic organ Nutrition Parasitism Plutella xylostella Wing disc
A B S T R A C T Cotesia plutellae is an endoparasitoid that parasitizes the diamondback moth Plutella xylostella. Parasitized P. xylostella exhibits immunosuppression and developmental retardation, resulting in death before pupation after parasitoid emergence. Except digestive organ, most host internal organs of parasitized P. xylostella show poor development, suggesting nutrient deprivation by the parasitoid. Here we report another significant morphological aberration in the development of parasitized P. xylostella. Imaginal wing disc development was markedly inhibited, while nonparasitized larvae developed two pairs of wing discs (approximately 0.5 mm diameter) in the thorax at their final instar. Since P. xylostella wing disk development can begin at the late larval stage depending on their nutritional status, C. plutellae parasitism may prevent wing disc development indirectly by disrupting host nutrient usage. Also, the hypotrophied wing disc of parasitized P. xylostella may be associated with immunosuppression due to its structural association with the hematopoietic organ in most lepidopteran wing discs. This study showed a high correlation between wing disc development and total hemocyte population in P. xylostella. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2008. Published by Elsevier B.V. All rights reserved.
Introduction Various host physiological alterations are induced by endoparasitoid parasitism (Pennacchio and Strand, 2006). To defend against host immune responses, endoparasitoids should develop immune-evasive or suppressive factor(s). In Cotesia kariyai, an immune-evasive protein that is synthesized and coated on the parasitoid egg surface prevents the parasitoid eggs from being recognized by the host hemocyte surveillance (Tanaka et al., 2002). Several endoparasitoids use ovarian and venom proteins that act directly on hemocytes to induce significant host immunosuppression at an immediately early parasitization stage (Webb and Summers, 1990; Webb and Luckhart, 1994; Asgari, 2006). Polydnavirus-carrying endoparasitoids use the viral products in the parasitized host to inhibit immune responses (Kroemer and Webb, 2004). Moreover, koinobiotic endoparasitoids that allow their parasitized hosts to survive during wasp development alter host nutritional and developmental physiology (Dorn and Beckage, 2007), which result in a prolonged host larval period, inhibition of pupal metamorphosis, and diverting nutritional usage to the development of the parasitoid. An endoparasitoid, C. plutellae (Braconidae, Hymenoptera), is functionally classified into a koinobiotic solitary wasp against the diamondback moth, Plutella xylostella (Bae and Kim, 2004). C. plutellae parasitizes P. xylostella at early second instar, develops in the host ⁎ Corresponding author. Fax: +82 54 823 1628. E-mail address: [email protected] (Y. Kim).
hemocoel, and emerges from the host just before formation of pupal cocoon (Kim et al., 2004). With several parasitic factors including venom, ovarian proteins, teratocytes, and polydnavirus (Basio and Kim, 2005), C. plutellae induces host immunosuppression and extension of host larval period, and prevents metamorphosis from larva to pupal stage of the parasitized P. xylostella (Lee and Kim, 2004). Cellular immune responses of the parasitized P. xylostella are markedly malfunctioned and cannot form an effective hemocytic encapsulation against the parasitoid egg (Ibrahim and Kim, 2006). A polydnaviral product, CpBV-lectin, plays an important role in interrupting recognition of parasitoid eggs by hemocytes (Lee et al., 2008). Hemocytespreading behavior of the parasitized larvae can be inhibited by other polydnaviral factors such as CpBV-PTP (Ibrahim et al., 2007), CpBV15α/β (Nalini and Kim, 2007), and CpBV-H4 (Wael and Kim, in press). Moreover, total hemocyte population is markedly reduced in the parasitized P. xylostella due to the influence of another polydnaviral product, CpBV-ELP (Kwon and Kim, 2007). These parasitic factors including ovarian proteins, teratocytes, and polydnavirus act cooperatively to form a complete host immunosuppression (Basio and Kim, 2006). Another characteristic of C. plutellae parasitism is nutritional deprivation from host source (Kim and Son, 2006). More than 80% of major nutrients including carbohydrate, protein, and lipid of P. xylostella are used up to facilitate development of the endoparasitoid, resulting in poor development of host internal organs including fat body, malpighian tubules, and testes (Kim and Son, 2006). However, the parasitism does not change the digestive organ of the
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parasitized P. xylostella to maintain its feeding activity probably due to the koinobiotic survival strategy. Here we report another developmental change associated with C. plutellae parasitism, namely, tissue differentiation and growth of the imaginal wing discs during late larval stages of P. xylostella. Generally, imaginal discs are islands of embryonic tissue that remain undifferentiated until the onset of adult tissue development (Svácha, 1992). Nutritional status and endocrine signals are critical in determining imaginal disc development (Truman and Riddiford, 2007). The wing discs of some lepidopteran insects are closely attached to the hematopoietic organ (Teramoto and Tanaka, 2004). This paper reports the hypotrophied wing discs of P. xylostella parasitized by C. plutellae and discusses the physiological significance of the developmental aberration in terms of immunosuppression.
Structural analysis of wing disc of P. xylostella
Materials and methods
Total hemocyte count
Insect rearing and parasitization
Different ages of larvae were surface-sterilized in 70% ethanol for a few seconds and rinsed with sterile water. An experimental unit of each age treatment represented individual larva and was replicated 10 times. After cutting off one of the abdominal prolegs with sterile scissors, the exuded hemolymph was collected with a glass capillary. To avoid hemocyte aggregation, hemolymph (1 μl) was mixed with 9 μl of anticoagulant buffer (98 mM NaOH, 186 mM NaCl, 17 mM Na2EDTA and 41 mM citric acid, pH 4.5) in a microfuge tube immediately after collection. Hemocytes were counted using a hemocytometer (Superior, Lauda, Germany) positioned in an Olympus BX41 phase contrast microscope (Olympus, Tokyo, Japan).
Larvae of P. xylostella were reared on cabbage leaves at 25 ± 1 °C under light:dark photoperiods of 16:8 h. For parasitization, early second instar larvae were exposed to their natural parasitoid wasp C. plutellae for 24 h. Propagation of both parasitoid and host was performed under the aforementioned rearing conditions. Wasp cocoons were collected and held in a plastic cage until their emergence. Emerged adult wasps were collected every day and allowed to mate for 24 h before being used for parasitization. Adult wasps were fed daily with 40% sucrose and the parasitized host was fed daily with cabbage leaves.
Wing discs isolated from fourth instar larvae were fixed in 2.5% glutaraldehyde 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, substituted with propylene oxide, and embedded in Epon 812 for 48 h at 60 °C. Sections (b90 nm thickness) were cut on a Leica Ultracut UCT ultramicrotome using a diamond knife (Diatome, Biel, Switzerland). Ultrathin sections were transferred onto 200 mesh copper grids and stained with uranyl acetate for 20 min and with lead citrate for 10 min. The sections were examined in a Hitachi H-7650 transmission electron microscope operating at 80 kV.
Fig. 1. Imaginal wing discs of P. xylostella. (A) A pair of wing discs was evident in each third (fore wing disc: ‘FWD’) and fourth (hind wing disc: ‘HWD’) thoracic segment of nonparasitized (‘NP’) larva, while no visible wing discs were evident in parasitized (‘P’) larva. (B) Magnified stereomicroscopic view (40×) of both wing discs with several branches of trachea. Scale bar indicates 0.3 mm.
S. Bae, Y. Kim / Journal of Asia-Pacific Entomology 11 (2008) 83–87
Statistical analyses Means and variances of control and experimental treatments were analyzed with a one-way ANOVA using PROC GLM (SAS Institute, 1989). Correlation analysis between wing disc development and total hemocyte population used PROC CORR. Results C. plutellae parasitism inhibits P. xylostella wing disc development A pair of wing discs was observed in each third and fourth thorax segment of nonparasitized P. xylostella larvae (Fig. 1). The fore wing disc of fourth instar larvae appeared to be larger than the hind wing disc, and was estimated to be 0.5 mm in diameter. Several tracheal branches developed in the wing discs. In contrast, no visible wing
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discs were evident in P. xylostella parasitized by C. plutellae, not even in the final instar (Fig. 1A). Ultrastructure of wing discs was analyzed by transmission electron microscopy. Examination of cross sections of wing discs revealed two surface layers separated by spaces (‘lacunae’) (Figs. 2A–C). The cell layers of the wing disc were enclosed by an epithelial sheath layer (Figs. 2B–D). When the epithelium was partly broken by artificial stress, the wing disc cells were released through the pores indicating the absence of a cell junction, probably due to rapid mitosis. The mitosis may have come from the medial part of the wing disc because the cells near the epithelial sheath appeared to be mature with rich cellular organelles, while other cells inside the disc were nascent with little organelle development (Fig. 2D). Wing disc development was associated with larval age (Fig. 3). In all tested stages, fore wing discs were significantly bigger than hind wing discs (F = 259.93; df = 1, 72; P b 0.0001). With larval development,
Fig. 2. Ultrastructure of P. xylostella imaginal wing discs. (A) Cross-sectional stereomicroscopic view of fore wing disc at 100× magnification. Lacunae (‘L’) are located between two surface layers. Transmission electron microscopy photos showing wing disc cells at magnifications of 300× (B), 1000× (C), and 2000× (D). A cell layer of epithelial sheath (denoted by arrows) encloses wing disc cells.
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both fore and hind wing discs displayed a significant increase (F = 574.12; df = 3, 72; P b 0.0001). However, fore wing disc development was faster than hind wing development (F = 17.12; df = 3, 72; P b 0.0001). P. xylostella wing disc development positively correlates with total hemocyte counts In some lepidopteran species including Pseudoplusia separata, the wing disc is attached to the hematopoietic organ and they develop concomitantly (Teramoto and Tanaka, 2004). However, we did not visually observe attachment of the hematopoietic organ to the wing disc in P. xylostella. To more rigorously assess any positive relationship between wing development and hematopoietic organ activity, we analyzed total hemocyte counts in both parasitized and nonparasitized P. xylostella (Fig. 4). Total hemocyte population was significantly higher in nonparasitized larvae (F = 2387.79; df = 1, 16; P b 0.0001) than in parasitized larvae that did not have any visible wing disc. With an increase of wing disc size, nonparasitized larvae displayed a significant increase in their total hemocyte counts (F = 338.62; df = 3, 16; P b 0.0001). Both developmental and immunological factors exhibited a high positive correlation (r = 0.9671; P = 0.0329).
Fig. 4. Developmental correlation between wing disc and total hemocyte population in P. xylostella. Total hemocyte counts were measured in a unit volume in both parasitized (‘P’) and nonparasitized (‘NP’) larvae of different ages. Larval age indicates instar and days in the instar; for example, ‘L4D2’ represents 2-day-old fourth instar. Each treatment used 10 discs. Disc size utilized the data of Fig. 3. Error bars indicate standard deviation.
Discussion Koinobiotic endoparasitoid wasps allow the host to continue to develop as their offspring mature (Askew and Shaw, 1986). Compared to ectoparasitic idiobionts, koinobiotic endoparasitoids have narrower host ranges due to their specific developmental interactions with the host (Shaw, 1994). Alteration of host nutritional physiology as well as immunosuppression by the endoparasitoid wasp are prerequisites for endoparasitoid development (Thompson, 1993; Thompson and Dahlman, 1998). Parasitism by C. plutellae significantly diverts use of host nutrients from the host to the parasitoid, which disrupts internal organ development (Kim and Son, 2006). The present study demonstrates that endoparasitoid parasitism prevents development of the imaginal wing disc of P. xylostella larvae. Imaginal discs are primordial organs that are destined to become adult tissues; they may be not necessary and indeed may even detract from optimal wasp development since they use some of the available nutrients. Additionally, the present study has revealed a high correlation between wing disc development and the total hemocyte population of P. xylostella. However, parasitized P. xylostella did not
Fig. 3. Development of P. xylostella imaginal wing discs. Larval age indicates instar and days in the instar; for example, ‘L4D2’ represents 2-day-old fourth instar. A maximum disc size diameter was evident using stereomicroscopy at 40× magnification. Each treatment used 10 discs. Error bars indicate standard deviations.
have any visible wing discs and displayed a markedly reduced total hemocyte population. The latter reduction can be explained by the breakdown of the circulating hemocytes and of the hematopoietic organ that generates the circulating hemocytes due to parasitic factors such as venom/ovarian proteins and polydnavirus (Teramoto and Tanaka, 2004). These factors would be enough for C. plutellae to inhibit the development of host wing discs. Parasitic factor(s) and the underlying mechanism(s) by which C. plutellae inhibits wing disc development of P. xylostella were not presently addressed and so remain unknown. However, based on related studies, we may explain the hypotrophied development of internal organs including wing disc in parasitized P. xylostella on the basis of poor nutritional status of parasitized P. xylostella due to biased nutrient usage for C. plutellae development. Accordingly, there may be not enough nutrient-related growth factors to stimulate wing disc development. However, it is also conceivable that the lack of growth factors does not explain wing disc development, given the observation that allatectomized larvae of Manduca sexta rescue disc development inhibited by starvation, but fail to rescue the development in the presence of juvenile hormone (JH) agonist (Truman et al., 2006). Nutritional changes due to parasitism are usually followed by changes in host endocrine physiology, in which the most common endocrine changes are increases in JH titers and a failure of 20-hydroxyecdysone (20E) to rise (Beckage and Gelman, 2004). P. xylostella parasitized by C. plutellae appears to maintain a high level of JH until the late larval stage, after which the level declines due to increased JH esterase activity (Lee and Kim, 2004). Just before the emergence of the parasitoid, parasitized larvae exhibit characteristic prepupal behaviors such as feeding cessation and migration to the site of cocoon-formation; these behaviors suggest an induction of larval to pupal metamorphic development due to the absence of JH. However, subsequent metamorphic development to pupation is halted, likely due to the lack of 20E. This is supported by absence of two characteristic gene expressions, namely Broad-Complex (BR-C) and hemolin, for the onset of pupal development in parasitized P. xylostella. BR-C is a 20E-induced early expressed gene that plays critical roles in larval to pupal metamorphosis (Kiss et al., 1988; Reza et al., 2004). A partially cloned BR-C gene in P. xylostella has been used to show that specific BR-C expression is observable during the late larval stage, during larval to pupal metamorphosis in nonparasitized larvae, while BR-C expression is undetectable in larvae parasitized by C. plutellae (data not shown). We do not yet know precisely how the 20E titer is decreased in the
S. Bae, Y. Kim / Journal of Asia-Pacific Entomology 11 (2008) 83–87
parasitized P. xylostella. It may be due to altered synthesis and release of prothoracicotropic hormone (PTTH) (Tanaka et al., 1987; Hayakawa, 1995), insensitivity of prothoracic glands (PTGs) to PTTH stimulation (Kelly et al., 1998), reduced biosynthetic activity of PTGs (Pennacchio et al., 1997, 1998), PTG cell death (Dover and Vinson, 1990), or altered ecdysteroid metabolism (Beckage and Gelman, 2004). In Toxoneuron nigriceps, a symbiotic polydnavirus encodes protein tyrosine phosphatase (PTP), which is considered to be a disrupter of host PTG function in ecdysteroid biosynthesis via the alteration of the phosphorylation status of key proteins regulating the PTTH signaling cascade (Falabella et al., 2006). In C. plutellae, the symbiotic polydnavirus (CpBV) contains more than 36 PTP genes, which may be responsible for the decreased 20E titer in parasitized P. xylostella. These data suggest that relatively prolonged high JH titers without 20E rise may prevent wing disc development of P. xylostella parasitized by C. plutellae. Acknowledgments This study was funded by Biogreen 21 of Rural Development Administration to Y. Kim. S. Bae was supported by the 2nd stage BK21 program of Ministry of Education and Human Resources Development, Korea. References Asgari, S., 2006. Venom proteins from polydnavirus-producing endoparasitoids: their role in host-parasite interactions. Arch. Insect Biochem. Physiol. 61, 146–156. Askew, R.R., Shaw, M.R., 1986. Parasitoid communities: their size, structure and development. J. Exp. Biol. 202, 225–263. Bae, S., Kim, Y., 2004. Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Comp. Biochem. Physiol. 138A, 39–44. Basio, N.A., Kim, Y., 2005. A short review of teratocytes and their characters in Cotesia plutellae (Braconidae: Hymenoptera). J. Asia-Pacific Entomol. 8, 211–217. Basio, N.A., Kim, Y., 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31, 341–347. Beckage, N.E., Gelman, D.B., 2004. Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control. Annu. Rev. Entomol. 49, 299–330. Dorn, S., Beckage, N.E., 2007. Superparasitism in gregarious hymenopteran parasitoids: ecological, behavioural and physiological perspectives. Physiol. Entomol. 32, 199–211. Dover, B.A., Vinson, S.B., 1990. Stage-specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands. Physiol. Entomol. 15, 405–414. Falabella, P., Caccialupi, P., Varricchio, P., Malva, C., Pennacchio, F., 2006. Protein tyrosine phosphatases of toxoneuron nigriceps bracovirus as potential disrupters of host prothoracic gland function. Arch. Insect Biochem. Physiol. 61, 157–169. Hayakawa, Y., 1995. Growth-blocking peptide: an insect biogenic peptide that prevents the onset of metamorphosis. J. Insect Physiol. 41, 1–6. Ibrahim, A.M.A., Kim, Y., 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella. J. Insect Physiol. 52, 943–950. Ibrahim, A.M.A., Choi, J.Y., Je, Y.H., Kim, Y., 2007. Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: sequence analysis, expression profile, and a possible biological role in host immunosuppression. Dev. Comp. Immunol. 31, 978–990. Kelly, T.J., Gelman, D.B., Reed, D.A., Beckage, N.E., 1998. Effects of parasitization by Cotesia congregata on the brain–prothoracic gland axis of its host, Manduca sexta. J. Insect Physiol. 44, 323–332.
87
Kim, Y., Son, Y., 2006. Parasitism of Cotesia plutellae alters morphological and biochemical characters of diamondback moth, Plutella xylostella. J. Asia-Pacific Entomol. 9, 37–42. Kim, Y., Bae, S., Lee, S., 2004. Polydnavirus replication and ovipositional habit of Cotesia plutellae. Kor. J. Appl. Entomol. 43, 225–231. Kiss, I., Beaton, A.H., Tardiff, J., Fristrom, D., Fristrom, J.W., 1988. Interactions and developmental effects of mutations in the broad-complex of Drosophila melanogaster. Genetics 118, 247–259. Kroemer, J.A., Webb, B.A., 2004. Polydnavirus and genome: emerging gene families and new insights into polydnavirus replication. Annu. Rev. Entomol. 49, 431–456. Kwon, S., Kim, Y., 2007. Immunosuppressive action of pyriproxyfen, a juvenile hormone analog, enhances pathogenicity of Bacillus thuringiensis subsp. kurstaki against diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Biol. Control 42, 72–76. Lee, S., Kim, Y., 2004. Juvenile hormone esterase of diamondback moth, Plutella xylostella, and parasitism of Cotesia plutellae. J. Asia-Pacific Entomol. 7, 283–287. Lee, S., Nalini, M., Kim, Y., 2008. A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocyte. Comp. Biochem. Physiol. A 149, 351–361. Nalini, M., Kim, Y., 2007. A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior. J. Insect Physiol. 53, 1283–1292. Pennacchio, F., Strand, M.R., 2006. Evolution of developmental strategies in parasitic hymenoptera. Annu. Rev. Entomol. 49, 233–258. Pennacchio, F., Sordetti, R., Falabella, P., Vinson, S.B., 1997. Biochemical and ultrastructural alterations in prothoracic glands of Heliothis virescens (F.) (Lepidoptera: Noctuidae) last instar larvae parasitized by Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae). Insect Biochem. Mol. Biol. 27, 439–450. Pennacchio, F., Falabella, P., Vinson, S.B., 1998. Regulation of Heliothis virescens prothoracic glands by Cardiochiles nigriceps polydnavirus (CnPDV). Arch. Insect Biochem. Physiol. 38, 1–10. Reza, A.M.S., Kanamori, Y., Shinoda, T., Shimura, S., Mita, K., Nakahara, Y., Kiuchi, M., Kamimura, M., 2004. Hormonal control of a metamorphosis-specific transcriptional factor broad-complex in silkworm. Comp. Biochem. Physiol. 139B, 753–761. SAS Institute, Inc., 1989. SAS/STAT user's guide. SAS Institute Inc., Cary, NC. Shaw, M., 1994. Parasitoid host ranges. In: Hawkins, B.A., Sheehan, W. (Eds.), Parasitoid community ecology. Oxford University Press, Oxford, UK, pp. 111–114. Svácha, P., 1992. What are and what are not imaginal disc: reevaluation of some basic concepts (Insect, Holometabola). Dev. Biol. 154, 101–117. Tanaka, T., Agui, N., Hiruma, K., 1987. The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release. Gen. Comp. Endocrinol. 67, 364–374. Tanaka, K., Matsumoto, H., Hayakawa, Y., 2002. Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp. Eur. J. Biochem. 269, 2557–2566. Teramoto, T., Tanaka, T., 2004. Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai. J. Insect Physiol. 50, 1103–1111. Thompson, S.N., 1993. Redirection of host metabolism and effects on parasite nutrition. In: Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds.), Parasites and pathogens of insects. Academic Press, New York, pp. 125–144. Thompson, S.N., Dahlman, D.L., 1998. Aberrant nutritional regulation of carbohydrate synthesis by parasitized Manduca sexta. J. Insect Physiol. 44, 745–754. Truman, J.W., Riddiford, L.M., 2007. The morphostatic actions of juvenile hormone. Insect Biochem. Mol. Biol. 37, 761–770. Truman, J.W., Hiruma, K., Allee, J.P., MacWhinnie, S.G., Champlin, D.T., Riddiford, L.M., 2006. Juvenile hormone is required to couple imagine disc formation with nutrition in insects. Science 312, 1385–1388. Wael, G., Kim, Y., in press. A viral histone H4 encoded by Cotesia plutellae bracovirus inhibits haemocyte-spreading behaviour of the diamondback moth, Plutella xylostella. J. Gen. Virol. Webb, B.A., Luckhart, S., 1994. Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens. Arch. Insect Biochem. Physiol. 26, 147–163. Webb, B.A., Summers, M.D., 1990. Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences. Proc. Natl. Acad. Sci. U. S. A. 87, 4961–4965.
Contents lists available at ScienceDirect
Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j a p e
Parasitism by Cotesia plutellae inhibits imaginal wing disc development of diamondback moth, Plutella xylostella Sungwoo Bae, Yonggyun Kim ⁎ Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea
A R T I C L E
I N F O
Article history: Received 16 February 2008 Revised 5 April 2008 Accepted 22 April 2008 Keywords: Cotesia plutellae Hematopoietic organ Nutrition Parasitism Plutella xylostella Wing disc
A B S T R A C T Cotesia plutellae is an endoparasitoid that parasitizes the diamondback moth Plutella xylostella. Parasitized P. xylostella exhibits immunosuppression and developmental retardation, resulting in death before pupation after parasitoid emergence. Except digestive organ, most host internal organs of parasitized P. xylostella show poor development, suggesting nutrient deprivation by the parasitoid. Here we report another significant morphological aberration in the development of parasitized P. xylostella. Imaginal wing disc development was markedly inhibited, while nonparasitized larvae developed two pairs of wing discs (approximately 0.5 mm diameter) in the thorax at their final instar. Since P. xylostella wing disk development can begin at the late larval stage depending on their nutritional status, C. plutellae parasitism may prevent wing disc development indirectly by disrupting host nutrient usage. Also, the hypotrophied wing disc of parasitized P. xylostella may be associated with immunosuppression due to its structural association with the hematopoietic organ in most lepidopteran wing discs. This study showed a high correlation between wing disc development and total hemocyte population in P. xylostella. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2008. Published by Elsevier B.V. All rights reserved.
Introduction Various host physiological alterations are induced by endoparasitoid parasitism (Pennacchio and Strand, 2006). To defend against host immune responses, endoparasitoids should develop immune-evasive or suppressive factor(s). In Cotesia kariyai, an immune-evasive protein that is synthesized and coated on the parasitoid egg surface prevents the parasitoid eggs from being recognized by the host hemocyte surveillance (Tanaka et al., 2002). Several endoparasitoids use ovarian and venom proteins that act directly on hemocytes to induce significant host immunosuppression at an immediately early parasitization stage (Webb and Summers, 1990; Webb and Luckhart, 1994; Asgari, 2006). Polydnavirus-carrying endoparasitoids use the viral products in the parasitized host to inhibit immune responses (Kroemer and Webb, 2004). Moreover, koinobiotic endoparasitoids that allow their parasitized hosts to survive during wasp development alter host nutritional and developmental physiology (Dorn and Beckage, 2007), which result in a prolonged host larval period, inhibition of pupal metamorphosis, and diverting nutritional usage to the development of the parasitoid. An endoparasitoid, C. plutellae (Braconidae, Hymenoptera), is functionally classified into a koinobiotic solitary wasp against the diamondback moth, Plutella xylostella (Bae and Kim, 2004). C. plutellae parasitizes P. xylostella at early second instar, develops in the host ⁎ Corresponding author. Fax: +82 54 823 1628. E-mail address: [email protected] (Y. Kim).
hemocoel, and emerges from the host just before formation of pupal cocoon (Kim et al., 2004). With several parasitic factors including venom, ovarian proteins, teratocytes, and polydnavirus (Basio and Kim, 2005), C. plutellae induces host immunosuppression and extension of host larval period, and prevents metamorphosis from larva to pupal stage of the parasitized P. xylostella (Lee and Kim, 2004). Cellular immune responses of the parasitized P. xylostella are markedly malfunctioned and cannot form an effective hemocytic encapsulation against the parasitoid egg (Ibrahim and Kim, 2006). A polydnaviral product, CpBV-lectin, plays an important role in interrupting recognition of parasitoid eggs by hemocytes (Lee et al., 2008). Hemocytespreading behavior of the parasitized larvae can be inhibited by other polydnaviral factors such as CpBV-PTP (Ibrahim et al., 2007), CpBV15α/β (Nalini and Kim, 2007), and CpBV-H4 (Wael and Kim, in press). Moreover, total hemocyte population is markedly reduced in the parasitized P. xylostella due to the influence of another polydnaviral product, CpBV-ELP (Kwon and Kim, 2007). These parasitic factors including ovarian proteins, teratocytes, and polydnavirus act cooperatively to form a complete host immunosuppression (Basio and Kim, 2006). Another characteristic of C. plutellae parasitism is nutritional deprivation from host source (Kim and Son, 2006). More than 80% of major nutrients including carbohydrate, protein, and lipid of P. xylostella are used up to facilitate development of the endoparasitoid, resulting in poor development of host internal organs including fat body, malpighian tubules, and testes (Kim and Son, 2006). However, the parasitism does not change the digestive organ of the
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2008. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2008.04.008
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parasitized P. xylostella to maintain its feeding activity probably due to the koinobiotic survival strategy. Here we report another developmental change associated with C. plutellae parasitism, namely, tissue differentiation and growth of the imaginal wing discs during late larval stages of P. xylostella. Generally, imaginal discs are islands of embryonic tissue that remain undifferentiated until the onset of adult tissue development (Svácha, 1992). Nutritional status and endocrine signals are critical in determining imaginal disc development (Truman and Riddiford, 2007). The wing discs of some lepidopteran insects are closely attached to the hematopoietic organ (Teramoto and Tanaka, 2004). This paper reports the hypotrophied wing discs of P. xylostella parasitized by C. plutellae and discusses the physiological significance of the developmental aberration in terms of immunosuppression.
Structural analysis of wing disc of P. xylostella
Materials and methods
Total hemocyte count
Insect rearing and parasitization
Different ages of larvae were surface-sterilized in 70% ethanol for a few seconds and rinsed with sterile water. An experimental unit of each age treatment represented individual larva and was replicated 10 times. After cutting off one of the abdominal prolegs with sterile scissors, the exuded hemolymph was collected with a glass capillary. To avoid hemocyte aggregation, hemolymph (1 μl) was mixed with 9 μl of anticoagulant buffer (98 mM NaOH, 186 mM NaCl, 17 mM Na2EDTA and 41 mM citric acid, pH 4.5) in a microfuge tube immediately after collection. Hemocytes were counted using a hemocytometer (Superior, Lauda, Germany) positioned in an Olympus BX41 phase contrast microscope (Olympus, Tokyo, Japan).
Larvae of P. xylostella were reared on cabbage leaves at 25 ± 1 °C under light:dark photoperiods of 16:8 h. For parasitization, early second instar larvae were exposed to their natural parasitoid wasp C. plutellae for 24 h. Propagation of both parasitoid and host was performed under the aforementioned rearing conditions. Wasp cocoons were collected and held in a plastic cage until their emergence. Emerged adult wasps were collected every day and allowed to mate for 24 h before being used for parasitization. Adult wasps were fed daily with 40% sucrose and the parasitized host was fed daily with cabbage leaves.
Wing discs isolated from fourth instar larvae were fixed in 2.5% glutaraldehyde 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, substituted with propylene oxide, and embedded in Epon 812 for 48 h at 60 °C. Sections (b90 nm thickness) were cut on a Leica Ultracut UCT ultramicrotome using a diamond knife (Diatome, Biel, Switzerland). Ultrathin sections were transferred onto 200 mesh copper grids and stained with uranyl acetate for 20 min and with lead citrate for 10 min. The sections were examined in a Hitachi H-7650 transmission electron microscope operating at 80 kV.
Fig. 1. Imaginal wing discs of P. xylostella. (A) A pair of wing discs was evident in each third (fore wing disc: ‘FWD’) and fourth (hind wing disc: ‘HWD’) thoracic segment of nonparasitized (‘NP’) larva, while no visible wing discs were evident in parasitized (‘P’) larva. (B) Magnified stereomicroscopic view (40×) of both wing discs with several branches of trachea. Scale bar indicates 0.3 mm.
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Statistical analyses Means and variances of control and experimental treatments were analyzed with a one-way ANOVA using PROC GLM (SAS Institute, 1989). Correlation analysis between wing disc development and total hemocyte population used PROC CORR. Results C. plutellae parasitism inhibits P. xylostella wing disc development A pair of wing discs was observed in each third and fourth thorax segment of nonparasitized P. xylostella larvae (Fig. 1). The fore wing disc of fourth instar larvae appeared to be larger than the hind wing disc, and was estimated to be 0.5 mm in diameter. Several tracheal branches developed in the wing discs. In contrast, no visible wing
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discs were evident in P. xylostella parasitized by C. plutellae, not even in the final instar (Fig. 1A). Ultrastructure of wing discs was analyzed by transmission electron microscopy. Examination of cross sections of wing discs revealed two surface layers separated by spaces (‘lacunae’) (Figs. 2A–C). The cell layers of the wing disc were enclosed by an epithelial sheath layer (Figs. 2B–D). When the epithelium was partly broken by artificial stress, the wing disc cells were released through the pores indicating the absence of a cell junction, probably due to rapid mitosis. The mitosis may have come from the medial part of the wing disc because the cells near the epithelial sheath appeared to be mature with rich cellular organelles, while other cells inside the disc were nascent with little organelle development (Fig. 2D). Wing disc development was associated with larval age (Fig. 3). In all tested stages, fore wing discs were significantly bigger than hind wing discs (F = 259.93; df = 1, 72; P b 0.0001). With larval development,
Fig. 2. Ultrastructure of P. xylostella imaginal wing discs. (A) Cross-sectional stereomicroscopic view of fore wing disc at 100× magnification. Lacunae (‘L’) are located between two surface layers. Transmission electron microscopy photos showing wing disc cells at magnifications of 300× (B), 1000× (C), and 2000× (D). A cell layer of epithelial sheath (denoted by arrows) encloses wing disc cells.
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both fore and hind wing discs displayed a significant increase (F = 574.12; df = 3, 72; P b 0.0001). However, fore wing disc development was faster than hind wing development (F = 17.12; df = 3, 72; P b 0.0001). P. xylostella wing disc development positively correlates with total hemocyte counts In some lepidopteran species including Pseudoplusia separata, the wing disc is attached to the hematopoietic organ and they develop concomitantly (Teramoto and Tanaka, 2004). However, we did not visually observe attachment of the hematopoietic organ to the wing disc in P. xylostella. To more rigorously assess any positive relationship between wing development and hematopoietic organ activity, we analyzed total hemocyte counts in both parasitized and nonparasitized P. xylostella (Fig. 4). Total hemocyte population was significantly higher in nonparasitized larvae (F = 2387.79; df = 1, 16; P b 0.0001) than in parasitized larvae that did not have any visible wing disc. With an increase of wing disc size, nonparasitized larvae displayed a significant increase in their total hemocyte counts (F = 338.62; df = 3, 16; P b 0.0001). Both developmental and immunological factors exhibited a high positive correlation (r = 0.9671; P = 0.0329).
Fig. 4. Developmental correlation between wing disc and total hemocyte population in P. xylostella. Total hemocyte counts were measured in a unit volume in both parasitized (‘P’) and nonparasitized (‘NP’) larvae of different ages. Larval age indicates instar and days in the instar; for example, ‘L4D2’ represents 2-day-old fourth instar. Each treatment used 10 discs. Disc size utilized the data of Fig. 3. Error bars indicate standard deviation.
Discussion Koinobiotic endoparasitoid wasps allow the host to continue to develop as their offspring mature (Askew and Shaw, 1986). Compared to ectoparasitic idiobionts, koinobiotic endoparasitoids have narrower host ranges due to their specific developmental interactions with the host (Shaw, 1994). Alteration of host nutritional physiology as well as immunosuppression by the endoparasitoid wasp are prerequisites for endoparasitoid development (Thompson, 1993; Thompson and Dahlman, 1998). Parasitism by C. plutellae significantly diverts use of host nutrients from the host to the parasitoid, which disrupts internal organ development (Kim and Son, 2006). The present study demonstrates that endoparasitoid parasitism prevents development of the imaginal wing disc of P. xylostella larvae. Imaginal discs are primordial organs that are destined to become adult tissues; they may be not necessary and indeed may even detract from optimal wasp development since they use some of the available nutrients. Additionally, the present study has revealed a high correlation between wing disc development and the total hemocyte population of P. xylostella. However, parasitized P. xylostella did not
Fig. 3. Development of P. xylostella imaginal wing discs. Larval age indicates instar and days in the instar; for example, ‘L4D2’ represents 2-day-old fourth instar. A maximum disc size diameter was evident using stereomicroscopy at 40× magnification. Each treatment used 10 discs. Error bars indicate standard deviations.
have any visible wing discs and displayed a markedly reduced total hemocyte population. The latter reduction can be explained by the breakdown of the circulating hemocytes and of the hematopoietic organ that generates the circulating hemocytes due to parasitic factors such as venom/ovarian proteins and polydnavirus (Teramoto and Tanaka, 2004). These factors would be enough for C. plutellae to inhibit the development of host wing discs. Parasitic factor(s) and the underlying mechanism(s) by which C. plutellae inhibits wing disc development of P. xylostella were not presently addressed and so remain unknown. However, based on related studies, we may explain the hypotrophied development of internal organs including wing disc in parasitized P. xylostella on the basis of poor nutritional status of parasitized P. xylostella due to biased nutrient usage for C. plutellae development. Accordingly, there may be not enough nutrient-related growth factors to stimulate wing disc development. However, it is also conceivable that the lack of growth factors does not explain wing disc development, given the observation that allatectomized larvae of Manduca sexta rescue disc development inhibited by starvation, but fail to rescue the development in the presence of juvenile hormone (JH) agonist (Truman et al., 2006). Nutritional changes due to parasitism are usually followed by changes in host endocrine physiology, in which the most common endocrine changes are increases in JH titers and a failure of 20-hydroxyecdysone (20E) to rise (Beckage and Gelman, 2004). P. xylostella parasitized by C. plutellae appears to maintain a high level of JH until the late larval stage, after which the level declines due to increased JH esterase activity (Lee and Kim, 2004). Just before the emergence of the parasitoid, parasitized larvae exhibit characteristic prepupal behaviors such as feeding cessation and migration to the site of cocoon-formation; these behaviors suggest an induction of larval to pupal metamorphic development due to the absence of JH. However, subsequent metamorphic development to pupation is halted, likely due to the lack of 20E. This is supported by absence of two characteristic gene expressions, namely Broad-Complex (BR-C) and hemolin, for the onset of pupal development in parasitized P. xylostella. BR-C is a 20E-induced early expressed gene that plays critical roles in larval to pupal metamorphosis (Kiss et al., 1988; Reza et al., 2004). A partially cloned BR-C gene in P. xylostella has been used to show that specific BR-C expression is observable during the late larval stage, during larval to pupal metamorphosis in nonparasitized larvae, while BR-C expression is undetectable in larvae parasitized by C. plutellae (data not shown). We do not yet know precisely how the 20E titer is decreased in the
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parasitized P. xylostella. It may be due to altered synthesis and release of prothoracicotropic hormone (PTTH) (Tanaka et al., 1987; Hayakawa, 1995), insensitivity of prothoracic glands (PTGs) to PTTH stimulation (Kelly et al., 1998), reduced biosynthetic activity of PTGs (Pennacchio et al., 1997, 1998), PTG cell death (Dover and Vinson, 1990), or altered ecdysteroid metabolism (Beckage and Gelman, 2004). In Toxoneuron nigriceps, a symbiotic polydnavirus encodes protein tyrosine phosphatase (PTP), which is considered to be a disrupter of host PTG function in ecdysteroid biosynthesis via the alteration of the phosphorylation status of key proteins regulating the PTTH signaling cascade (Falabella et al., 2006). In C. plutellae, the symbiotic polydnavirus (CpBV) contains more than 36 PTP genes, which may be responsible for the decreased 20E titer in parasitized P. xylostella. These data suggest that relatively prolonged high JH titers without 20E rise may prevent wing disc development of P. xylostella parasitized by C. plutellae. Acknowledgments This study was funded by Biogreen 21 of Rural Development Administration to Y. Kim. S. Bae was supported by the 2nd stage BK21 program of Ministry of Education and Human Resources Development, Korea. References Asgari, S., 2006. Venom proteins from polydnavirus-producing endoparasitoids: their role in host-parasite interactions. Arch. Insect Biochem. Physiol. 61, 146–156. Askew, R.R., Shaw, M.R., 1986. Parasitoid communities: their size, structure and development. J. Exp. Biol. 202, 225–263. Bae, S., Kim, Y., 2004. Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella. Comp. Biochem. Physiol. 138A, 39–44. Basio, N.A., Kim, Y., 2005. A short review of teratocytes and their characters in Cotesia plutellae (Braconidae: Hymenoptera). J. Asia-Pacific Entomol. 8, 211–217. Basio, N.A., Kim, Y., 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31, 341–347. Beckage, N.E., Gelman, D.B., 2004. Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control. Annu. Rev. Entomol. 49, 299–330. Dorn, S., Beckage, N.E., 2007. Superparasitism in gregarious hymenopteran parasitoids: ecological, behavioural and physiological perspectives. Physiol. Entomol. 32, 199–211. Dover, B.A., Vinson, S.B., 1990. Stage-specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands. Physiol. Entomol. 15, 405–414. Falabella, P., Caccialupi, P., Varricchio, P., Malva, C., Pennacchio, F., 2006. Protein tyrosine phosphatases of toxoneuron nigriceps bracovirus as potential disrupters of host prothoracic gland function. Arch. Insect Biochem. Physiol. 61, 157–169. Hayakawa, Y., 1995. Growth-blocking peptide: an insect biogenic peptide that prevents the onset of metamorphosis. J. Insect Physiol. 41, 1–6. Ibrahim, A.M.A., Kim, Y., 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella. J. Insect Physiol. 52, 943–950. Ibrahim, A.M.A., Choi, J.Y., Je, Y.H., Kim, Y., 2007. Protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus: sequence analysis, expression profile, and a possible biological role in host immunosuppression. Dev. Comp. Immunol. 31, 978–990. Kelly, T.J., Gelman, D.B., Reed, D.A., Beckage, N.E., 1998. Effects of parasitization by Cotesia congregata on the brain–prothoracic gland axis of its host, Manduca sexta. J. Insect Physiol. 44, 323–332.
87
Kim, Y., Son, Y., 2006. Parasitism of Cotesia plutellae alters morphological and biochemical characters of diamondback moth, Plutella xylostella. J. Asia-Pacific Entomol. 9, 37–42. Kim, Y., Bae, S., Lee, S., 2004. Polydnavirus replication and ovipositional habit of Cotesia plutellae. Kor. J. Appl. Entomol. 43, 225–231. Kiss, I., Beaton, A.H., Tardiff, J., Fristrom, D., Fristrom, J.W., 1988. Interactions and developmental effects of mutations in the broad-complex of Drosophila melanogaster. Genetics 118, 247–259. Kroemer, J.A., Webb, B.A., 2004. Polydnavirus and genome: emerging gene families and new insights into polydnavirus replication. Annu. Rev. Entomol. 49, 431–456. Kwon, S., Kim, Y., 2007. Immunosuppressive action of pyriproxyfen, a juvenile hormone analog, enhances pathogenicity of Bacillus thuringiensis subsp. kurstaki against diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Biol. Control 42, 72–76. Lee, S., Kim, Y., 2004. Juvenile hormone esterase of diamondback moth, Plutella xylostella, and parasitism of Cotesia plutellae. J. Asia-Pacific Entomol. 7, 283–287. Lee, S., Nalini, M., Kim, Y., 2008. A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocyte. Comp. Biochem. Physiol. A 149, 351–361. Nalini, M., Kim, Y., 2007. A putative protein translation inhibitory factor encoded by Cotesia plutellae bracovirus suppresses host hemocyte-spreading behavior. J. Insect Physiol. 53, 1283–1292. Pennacchio, F., Strand, M.R., 2006. Evolution of developmental strategies in parasitic hymenoptera. Annu. Rev. Entomol. 49, 233–258. Pennacchio, F., Sordetti, R., Falabella, P., Vinson, S.B., 1997. Biochemical and ultrastructural alterations in prothoracic glands of Heliothis virescens (F.) (Lepidoptera: Noctuidae) last instar larvae parasitized by Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae). Insect Biochem. Mol. Biol. 27, 439–450. Pennacchio, F., Falabella, P., Vinson, S.B., 1998. Regulation of Heliothis virescens prothoracic glands by Cardiochiles nigriceps polydnavirus (CnPDV). Arch. Insect Biochem. Physiol. 38, 1–10. Reza, A.M.S., Kanamori, Y., Shinoda, T., Shimura, S., Mita, K., Nakahara, Y., Kiuchi, M., Kamimura, M., 2004. Hormonal control of a metamorphosis-specific transcriptional factor broad-complex in silkworm. Comp. Biochem. Physiol. 139B, 753–761. SAS Institute, Inc., 1989. SAS/STAT user's guide. SAS Institute Inc., Cary, NC. Shaw, M., 1994. Parasitoid host ranges. In: Hawkins, B.A., Sheehan, W. (Eds.), Parasitoid community ecology. Oxford University Press, Oxford, UK, pp. 111–114. Svácha, P., 1992. What are and what are not imaginal disc: reevaluation of some basic concepts (Insect, Holometabola). Dev. Biol. 154, 101–117. Tanaka, T., Agui, N., Hiruma, K., 1987. The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release. Gen. Comp. Endocrinol. 67, 364–374. Tanaka, K., Matsumoto, H., Hayakawa, Y., 2002. Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp. Eur. J. Biochem. 269, 2557–2566. Teramoto, T., Tanaka, T., 2004. Mechanism of reduction in the number of the circulating hemocytes in the Pseudaletia separata host parasitized by Cotesia kariyai. J. Insect Physiol. 50, 1103–1111. Thompson, S.N., 1993. Redirection of host metabolism and effects on parasite nutrition. In: Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds.), Parasites and pathogens of insects. Academic Press, New York, pp. 125–144. Thompson, S.N., Dahlman, D.L., 1998. Aberrant nutritional regulation of carbohydrate synthesis by parasitized Manduca sexta. J. Insect Physiol. 44, 745–754. Truman, J.W., Riddiford, L.M., 2007. The morphostatic actions of juvenile hormone. Insect Biochem. Mol. Biol. 37, 761–770. Truman, J.W., Hiruma, K., Allee, J.P., MacWhinnie, S.G., Champlin, D.T., Riddiford, L.M., 2006. Juvenile hormone is required to couple imagine disc formation with nutrition in insects. Science 312, 1385–1388. Wael, G., Kim, Y., in press. A viral histone H4 encoded by Cotesia plutellae bracovirus inhibits haemocyte-spreading behaviour of the diamondback moth, Plutella xylostella. J. Gen. Virol. Webb, B.A., Luckhart, S., 1994. Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens. Arch. Insect Biochem. Physiol. 26, 147–163. Webb, B.A., Summers, M.D., 1990. Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences. Proc. Natl. Acad. Sci. U. S. A. 87, 4961–4965.