Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera
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.
Section snippets
Insect stocks
L. heterotoma strain New York USA (Chiu et al., 2006, Schlenke et al., 2007) were raised in house at 25 °C on the y w strain of D. melanogaster on standard corn meal and yeast diet.
Transcriptome library preparation and sequencing
500 Lh females were anesthetized by CO2 and washed with 70% alcohol. Their long gland-reservoir-ovipositor complexes (called venom glands here), were removed simply by pulling the ovipositor, and frozen at − 70 °C. Eight micrograms of total RNA were extracted and used to prepare a standard cDNA library (Evrogen) in the
Overview
More than 950 original clone sequences from Lh venom gland expression were cleaned and assembled using pred/phrap methodology (Ewing and Green, 1998, Ewing et al., 1998) to yield 827 preliminary unigenes. 153 (145 singlets and 8 contigs) of the 827 are novel, lacking reliable domain identifications and/or significant similarity to published sequences. An additional 42 sequences (37 singlets and 5 contigs) are similar to hypothetical proteins that lack annotation. Here, we present 281 unique
Concluding remarks
Parasitism requires bioactive venom proteins and peptides for immune evasion or immune suppression, to facilitate nutrient acquisition, and to cause some level of host subdual (Rivers et al., 2002). The most critical determinants of venom protein profiles in relation to host strategy and host range have remained intractable until recently. Powerful transcriptomic and venom proteomic approaches (deGraaf et al., 2009) are now providing thorough characterizations to understand the roles of
Conflict of interest
The authors declare that no conflicts of interest exist.
Acknowledgments
We thank undergraduate students of spring 2010 Bioinformatics class (Bio31312) and C. Chand for initial analyses of the sequence data. Thanks to W. Qiu (Hunter College) and R. Walsh (CUNY High Performance Computing Center) for help with bioinformatics and computing; to I. Paddibhatla and Z. Papadopol for help with experiments; and to A. Berkov and V. Gokhman for feedback on the manuscript. This publication was made possible by grants from NSF (1121817), USDA (NRI/USDA CSREES 2006-03817 and
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2022, Current Opinion in Insect ScienceCitation Excerpt :Parasitoids can also actively counter or obstruct different components of the host immune responses. The venoms that parasitoids inject during oviposition have multifaceted immunomodulatory properties, and the diverse cocktails of venoms have been characterized with transcriptomic and proteomic approaches [12,84•,85–87]. The mechanisms by which individual venom components can suppress host immune responses include factors that antagonize the hosts’ intracellular calcium signalling to stop haemocyte differentiation, serpins that inhibit serine protease cascades and melanisation, toxins that inactivate hosts’ RhoGAPs that are required for proper lamellocyte functioning, and proteins that cause lysis of the hosts’ lymph gland or destroy the fully differentiated haemocytes [60,84•,88–92].
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Present address: Medgar Evers College, City University of New York, 1650 Bedford Avenue, Brooklyn, NY 11225, USA.