Immunoevasive property of a polydnaviral product, CpBV-lectin, protects the parasitoid egg from hemocytic encapsulation of Plutella xylostella (Lepidoptera: Yponomeutidae)

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Abstract

Immunosuppression is the main pathological symptom of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae), parasitized by an endoparasitoid wasp, Cotesia plutellae (vestalis, Hymenoptera: Braconidae). C. plutellae bracovirus (CpBV), which is a symbiotic virus of C. plutellae, has been known to be the main parasitic factor in the host–parasitoid interaction. CpBV-lectin, encoded in the viral genome and expressed in P. xylostella during early parasitization stage, was suspected to play a role in immunoevasion of defense response. Here we expressed CpBV-lectin in Sf9 cells using a recombinant baculovirus for subsequent functional assays. The recombinant CpBV-lectin exhibited hemagglutination against vertebrate erythrocytes. Its hemagglutinating activity increased with calcium, but inhibited by adding EDTA, indicating its C-type lectin property. CpBV-lectin showed specific carbohydrate-binding affinity against N-acetyl glucosamine and N-acetyl neuraminic acid. The role of this CpBV-lectin in immunosuppression was analyzed by exposing hemocytes of nonparasitized P. xylostella to rat erythrocytes or FITC-labeled bacteria pretreated with recombinant CpBV-lectin, which resulted in significant reduction in adhesion or phagocytosis, respectively. The immunosuppressive activity of CpBV-lectin was further analyzed under in vitro encapsulation response of hemocytes against parasitoid eggs collected at 1- or 24-h post-parasitization. Hemocytic encapsulation was observed against 1-h eggs but not against 24-h eggs. When the 1-h eggs were pretreated with the recombinant CpBV-lectin, encapsulation response was completely inhibited, where CpBV-lectin bound to the parasitoid eggs, but not to hemocytes. These results suggest that CpBV-lectin interferes with hemocyte recognition by masking hemocyte-binding sites on the parasitoid eggs.

Introduction

An endoparasitoid wasp, Cotesia plutellae (vestalis, Hymenoptera: Braconidae), induces significant immunosuppression of parasitized host, Plutella xylostella (Lepidoptera: Yponomeutidae) (Bae and Kim, 2004, Ibrahim and Kim, 2006). Several parasitic factors of C. plutellae have been identified and include venom proteins, ovarian proteins, teratocytes, and polydnavirus (Basio and Kim, 2006, Kim and Ryu, 2007). Though these parasitic factors coordinate together for successful parasitization, the symbiotic polydnavirus (C. plutellae bracovirus: CpBV) has been considered to play a major role in inducing the host immunosuppression due to at least 125 putative encoded genes and their expression through parasitization period (Kim et al., 2007).

Bracovirus is alternative type among the two groups of polydnavirus, which are classified by their host insect family and viral morphology (Webb et al., 2000). Polydnavirus is present in a provirus state on host chromosome and forms viral particles by replication in the ovarian calyx (Fleming and Summers, 1986, Wyler and Lanzrein, 2003). These viral particles are released into the hemocoel of parasitized insect by the parasitoid during oviposition (Stoltz, 1993). In the parasitized host, polydnavirus expresses its own genes, which play vital roles in immunosuppression and other alterations in the host developmental status (Webb and Strand, 2005).

Several putative genes encoded in CpBV genome have been identified and classified into characteristic polydnaviral gene families (Choi et al., 2005, Kim et al., 2007). At least 36 CpBV-PTPs (protein tyrosine phosphatases) represent the largest gene family and their expression in hemocytes causes inhibition of cell spreading behavior in response to pathogen challenge (Ibrahim et al., 2007, Ibrahim and Kim, 2007). At least eight CpBV-IkBs (inhibitors of NFkB) are encoded and may play an inhibitory role in host antiviral activity (Kim et al., 2006). At least seven EP1-like genes are encoded in CpBV genome and induce significant decrease in total hemocyte population of P. xylostella (Kwon and Kim, 2008). CpBV-H4 (histone H4) is expressed in hemocytes of parasitized P. xylostella and inhibits hemocyte-spreading behavior (Gad and Kim, 2008). CpBV15β, a late expressed gene, suppresses hemocyte-spreading behavior by inhibiting protein synthesis activity (Nalini and Kim, 2007).

In addition to the immunosuppressive agents encoded in CpBV genome, a viral lectin gene, designated as CpBV-lectin, has been identified in the parasitized P. xylostella and expresses during early parasitization period (Lee et al., 2008). Insect immune responses are triggered by innate immune recognition, which is mediated by pattern recognition molecules or receptors (Janeway, 1992, Janeway and Medzhitov, 2002). Among the various pattern recognition receptors, lectins are known to possess the ability to act as recognition molecules inside cells, on cell surfaces, and in physiological fluids (Sharon and Lis, 2004). Multimeric lectins are adhesion molecules that promote attachment and spreading on surface glycodeterminants (Vasta et al., 1999, Sharon, 2007). The assumption is that CpBV-lectin may compete with host lectin-like molecule(s) to bind pathogenic target so as to interfere with pathogen recognition of host immune system. This study was performed to determine functional binding of CpBV-lectin against nonself including parasitoid egg, leading to failure of hemocytic encapsulation. To address this hypothesis, we expressed CpBV-lectin using Sf9 cells through a recombinant baculovirus. The recombinant CpBV-lectin was analyzed for its biochemical properties by hemagglutination and inhibition assays. Subsequently, the functional form of CpBV-lectin was used to analyze cellular immune responses such as nonself adherence of hemocytes, phagocytosis, and encapsulation.

Section snippets

Insects and parasitization

Larvae of diamondback moth, P. xylostella, were reared on cabbage leaves at 25 ± 1 °C under a photoperiod of 16:8 (L:D) h. For parasitization early second instar larvae were exposed to their natural parasitoid wasp, C. plutellae, for 24 h. Propagation of both parasitoid and the hosts were performed under the above rearing conditions. Wasp cocoons were collected and held in plastic cage until their emergence. The emerged adult wasps were collected every day and allowed to mate for 24 h before using

Expression of a recombinant CpBV-lectin in Sf9 cells

A CpBV-lectin was expressed in Sf9 cells using a recombinant baculovirus (Fig. 1). The cell extract of the recombinant showed a specific band by an immunoblotting using CpBV-lectin antibody at the expected size (20,240 Da: Lee et al., 2008), while the extract from Sf9 cells infected with EGFP-recombinant baculovirus did not show any immunoreactivity. However, the recombinant CpBV-lectin was not detectable in the cultured medium, even though it possessed a signal peptide.

Biochemical properties of CpBV-lectin expressed in Sf9 cells

CpBV-lectin expressed in

Discussion

A previous study suggests that CpBV-lectin may interfere with hemocyte binding to pathogens including C. plutellae eggs to inhibit encapsulation (Lee et al., 2008). CpBV-lectin is present in the plasma of the parasitized P. xylostella during initial period for at least 48 h (Lee et al., 2008). C. plutellae eggs hatch at 36-h post-parasitization at 25 °C (Basio and Kim, 2005). This indicates that C. plutellae eggs can be exposed to CpBV-lectin except a few hours just before CpBV-lectin expression

Acknowledgements

This study was funded by Korea Research Foundation Grant (MOEHRD) (KRF-2006-311-F00042) to Y. Kim. M. Nalini was supported by the 2nd Stage BK21 program of Ministry of Education and Human Resources Development, Korea.

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