Isolation of an imaginal disc growth factor homologue from Pieris rapae and its expression following parasitization by Cotesia rubecula

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Abstract

Endoparasitoid insects introduce maternal factors into the body of their host at oviposition to suppress cellular defences for the protection of the developing parasitoid. We have shown that transient expression of polydnavirus genes from a hymenopteran parasitoid Cotesia rubecula (CrPDV) is responsible for the inactivation of hemocytes from the lepidopteran host Pieris rapae. Since the observed downregulation of CrPDV genes in infected host tissues is not due to cis-regulatory elements at the CrV1 gene locus, we speculated that the termination of CrPDV gene expression may be due to cellular inactivation caused by the CrV1-mediated immune suppression of infected tissues. To test this assumption, we isolated an imaginal disc growth factor (IDGF) that is expressed in fat body and hemocytes, the target of viral infection and expression of CrPDV genes. Time-course experiments showed that the level of P. rapae IDGF is not affected by parasitization and polydnavirus infection. However, the amount of highly expressed genes, such as storage proteins, arylphorin and lipophorin, are significantly reduced following parasitization.

Introduction

Insect parasitoids manipulate their host physiology by introducing into the body of their hosts, mostly immature stages of other insect species, factors that interfere with host defence functions to secure the successful development of the developing endoparasitoid. Inactivation of the host immune system is vital for the survival of the egg and emerging larva, protecting it from encapsulation reactions by host hemocytes. In various host–parasitoid systems maternal factors such as virus-like particles, venom or accessory gland secretions are the outcome of evolutionary adaptations that target key elements of the natural host defence system. Polydnaviruses (PDVs) produced in the calyx region of braconid and ichneumonid wasps have been shown to actively suppress host immune components and interfere with host physiology (Turnbull and Webb, 2002). PDVs do not replicate in the host but their genes are expressed following the infection of various host tissues. The PDV genes are mainly involved in immune suppression by causing breakdown of cell cytoskeleton (Asgari et al., 1996) or apoptosis in host hemocytes (Strand and Pech, 1995).

In addition to inactivation of the host immune system, PDVs have been shown to cause developmental arrest. Koinobiont parasitoids, which allow the host to continue its development until the growing wasp larva has reached its maximum size (Naumann, 1991), have evolved mechanisms to retard host development until the parasitoid has completed larval development. Several lines of evidence indicate that PDVs reduce ecdysteroid titres in parasitised larvae (Dahlman et al., 1990, Lawrence and Lanzrein, 1993). This is mediated by viral infection resulting in the deterioration of the prothoracic gland (Dover et al., 1988) or regulating fat body cells, which produce protein factors that affect the responsiveness of the prothoracic gland (Dahlman et al., 1990, Dover et al., 1987).

Cotesia rubecula PDVs (CrPDVs) are unique among parasitoid systems in that only a limited number of genes are expressed by the large virus genome. These genes are transiently expressed between 2 and 12 h after parasitism due to strong induction and subsequent repression of CrPDV gene transcription (Asgari et al., 1996, Asgari et al., 1997). Using cloned CrV1 gene fragments, the strong induction of CrV1 gene expression was confirmed by promoter studies (Asgari and Schmidt, 2001). However, these experiments did not reveal any cis-regulatory elements within the examined DNA region that could be responsible for the downregulation. We therefore speculated that the abrupt termination of CrPDV gene expression may be caused by an inactivation of infected host tissues and cells, similar to the observed immune suppression in hemocytes. To test this assumption, we isolated a host gene that is expressed in the same tissues as the viral genes, an imaginal disc growth factor homologue (PrIDGF), and monitored its expression after parasitization in conjunction with storage proteins, arylphorin and lipophorin. We hypothesize that if termination of viral transcription were due to general inactivation of host cell functions, the host gene would be expected to discontinue expression together with viral genes. Data presented here indicate that PrIDGF is expressed in fat body cells and hemocytes and secreted into the hemolymph. Importantly, the level of IDGF expression was not affected following parasitization by C. rubecula and infection by CrPDVs. In contrast, expression of arylphorin and lipophorin was significantly down-regulated following parasitism. This suggests that CrPDV infections differentially regulate host protein expression based on virus-specific mechanisms.

Section snippets

Insect cultures

The parasitoid C. rubecula and its host were maintained at 25 °C, on a 14 h:10 h (light/dark) photoperiod. P. rapae larvae were reared on cabbage plants. Adult C. rubecula wasps were fed with honey–water solution.

Isolation of hemocytes, fat body and hemolymph

Fourth instar P. rapae larvae were surface sterilized in 70% ethanol and bled onto multi-well microscope slides. The wells contained PBS saturated with phenyl-thiuorea to prevent melanization.

Hemocytes were collected from P. rapae by bleeding larvae as above, followed by centrifugation

Isolation and characterization of PrIDGF

A 52 kDa protein from P. rapae was identified as a protein present in both fat body and hemocytes, tissues where CrPDV genes are expressed. In order to identify the protein, we isolated the protein from the gel and digested it with trypsin. Digested peptides were separated on rpHPLC, two of which were sequenced at N-terminal by Edman degradation method (Fig. 1, peptides underlined). Preliminary sequence similarity searches using the peptide sequences indicated homology to imaginal disc growth

Discussion

Several studies have shown the effects of PDVs on host protein expression, which point towards virus-related interference at the translational level. In comprehensive studies using Campoletis sonorensis PDVs, it was demonstrated that CsIVs specifically inhibit translation of certain growth-associated proteins from Heliothis virescens such as arylphorin (Shelby and Webb, 1994), riboflavin binding hexamer and juvenile hormone esterase (Shelby and Webb, 1997). However, the plasma titres of

Acknowledgements

This study was supported by an Australian Research Fellowship, a University of Queensland grant to S. Asgari and an ARC grant to OS.

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Genbank accession number: The NCBI/Genbank accession number for the sequence reported here is AY616136.

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