Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera

Highlights

• A pilot transcriptome of the L. heterotoma venom gland complex yields 827 unigenes.

• More than 150 novel transcripts revealed, lacking significant known similarities.

• The remaining unigenes support conservation with venomous and stinging Hymenoptera.

• A subset of these reported unigenes likely contribute to venom and host control.

• A leading report of a figitid venom transcriptome targeting Drosophila hosts.

Abstract

Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma's predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation is among the first functional genomic studies for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts' immunity and shed light on the molecular basis of a natural arms race between these insects.

Introduction

The order Hymenoptera comprises approximately 130,000 insect species, with as many as 20% of these estimated to be parasitoid wasps in the Apocrita (Pennacchio and Strand, 2006). The reproductive strategies within this group target host development and viability, and contribute to community structure and ecology. Venom protein bioactivity has been studied since the early twentieth century, when the first snake (Noguchi, 1909) and scorpion venoms were investigated (Todd, 1909). The venom studies for pain-inflicting social insects such as bees, bumblebees, yellow jackets, and ants, have clarified the ontology of venom proteins and provided treatment applications (deGraaf et al., 2009, Hoffman, 1977, Peiren et al., 2005). In contrast to social insects, parasitoid wasps must apprehend and physiologically control their hosts to assure the success of their offspring. Early indications suggest that the venom pharmacopeia of these insects will prove to be richer (Danneels et al., 2010), paralleling the specific demands of host–parasite interactions.

Venom factors provide the armament for success in the host/parasitoid arms race. Venom proteins target host physiology and development to provide the developing parasitoid with a secure and nutrient-rich environment that will optimize its consumption of host resources (Rivers and Denlinger, 1994, Rivers and Denlinger, 1995). Hosts often are subdued through neuro-active venom components that may cause prolonged paralysis, particularly in ectoparasitic wasp attack (Rivers et al., 2002). Additionally, parasitic wasps protect their progeny either by passively evading the host immune system (e.g., Asobara tabida, (Prevost et al., 2009)) or by actively suppressing host immunity (e.g., Leptopilina spp. (Dubuffet et al., 2009, Lee et al., 2009)). Many studies in D. melanogaster have found that the cellular and humoral responses are predominantly under the control of Toll/NF-kappa B and JAK-STAT signaling pathways. Melanization of wasp egg also contributes to the host defense response (Govind, 2008, Lemaitre and Hoffmann, 2007, Schlenke et al., 2007). These molecular mechanisms appear to be active in other insects as well (Bitra et al., 2012), and are targets of inhibitors arising from venoms, polydnavirus gene expression, and calyx fluid (Nappi et al., 2009, Strand and Burke, 2012).

Leptopilina heterotoma (Lh), a member of a moderately sized genus (Allemand et al., 2002, Schilthuizen et al., 1998), successfully parasitizes most Drosophila species tested (Carton et al., 1986, Schlenke et al., 2007). It has been known for over fifty years that Lh strains must produce venom factors (Walker, 1959). The majority of the virulence activity is attributed to the action of virus-like particles (VLPs) that are produced and assembled in the long gland-reservoir complex (Chiu et al., 2006, Ferrarese et al., 2009, Morales et al., 2005, Rizki and Rizki, 1992). The long gland is a simple cylindrical organ lined peripherally with large, polyploid secretory cells. Internal and concentric to this cell layer is a single-celled layer of intimal cells, which lines the long gland lumen. A supracellular canal system of individual secretory units, one per secretory cell, feeds into the long gland lumen (Ferrarese et al., 2009). Antibody staining experiments have revealed that some VLP proteins are produced in the secretory cells; they enter the long gland lumen via secretory units and appear associated with small membranous structures. These structures undergo morphogenesis and assemble 3–6 spikes to assume unique stellate morphologies. Stellate VLPs and their constituent proteins block hemocyte-mediated wasp egg encapsulation by inducing cell lysis and apoptosis (Chiu and Govind, 2002, Chiu et al., 2006, Ferrarese et al., 2009, Morales et al., 2005, Rizki and Rizki, 1992).

L. heterotoma attack delays larval host development (Schlenke et al., 2007). The biological activities of venom components that contribute to the alteration of Drosophila development and immunity are largely unknown. We are interested in understanding not only the nature of bioactive molecules in the venom and those associated with VLPs, but also the process of VLP assembly and morphogenesis that occurs in the unique long gland-reservoir environment. We also want to know if the venom factors can contribute to immune suppression via an activating or adjuvant-type role, and whether VLPs have a viral origin.

To address these questions, we have initiated a cDNA-based transcriptome analysis of the venom gland. The enzymatic profile and KEGG terms of our Blast-based protein predictions suggest that in addition to conserved signaling, cell cycle, and housekeeping proteins, the Lh venom gland expresses hypothetical and unknown proteins that may help maintain the glandular environments for VLP and venom activities. Many enzymes with predicted biological activities that have been reported in studies of other parasitoid wasps, and in the stinging Aculeata, also appear to be utilized by Lh. Given the conservation among immune pathways in insects, of which Drosophila has been the classic model (Cherry and Silverman, 2006, Govind, 2008, Schmid-Hempel, 2005, Tanji and Ip, 2005), we predict that Lh venom factors with inhibitory functions in the D. melanogaster host will also modulate immune physiologies of other Drosophila species. A comprehensive understanding of the molecular strategies underlying the success of this natural Drosophilia parasitoid can potentially be used to target economically significant insect pests and pathogens.