Identification of the main venom protein components of Aphidius ervi, a parasitoid wasp of the aphid model Acyrthosiphon pisum

Abstract

Background: Endoparasitoid wasps are important natural enemies of the widely distributed aphid pests and are mainly used as biological control agents. However, despite the increased interest on aphid interaction networks, only sparse information is available on the factors used by parasitoids to modulate the aphid physiology. Our aim was here to identify the major protein components of the venom injected at oviposition by Aphidius ervi to ensure successful development in its aphid host, Acyrthosiphon pisum.

Results: A combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified.

Conclusions: Our data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms.

Figure 1

Comparison of venom gland and reservoir protein profiles, and proteomic analysis. Proteins from A. ervi venom glands and reservoirs were separated on a 6-16% SDS-PAGE under reducing conditions and visualized by silver staining. All stained protein bands numbered on the gel were excised and submitted for protein identification by LC-MS-MS. Molecular mass is in kDa.

Figure 2

Venn diagram showing the repartition of unisequences found in proteomics between venom glands (VG) and reservoirs (R). The green and red rectangles highlight the number of unisequences for which sequence was complete and that were predicted to be secreted (S) or predicted not to be secreted (NS), respectively. The blue ellipse corresponds to considered “putative venom proteins”.

Figure 3

Multiple alignment of γ-GT sequences. The three A. ervi γ-GT sequences identified were aligned with the published A. ervi γ-GT sequence [GenBank: CAL69624] and the human γ-GT1 sequence [Swiss-Prot:P19440]. Residues identical or similar are highlighted in black and grey, respectively. Stars indicate mutations in the Aerv_CL1Contig6 that were described to affect the enzymatic activity of human γ-GT1. Aerv, A.ervi; Hsap, H. sapiens.

Figure 4

Maximum-likelihood phylogenetic tree of hymenopteran γ-GT sequences. The blue, orange, and green vertical lines correspond to the three major clades (A, B and C) obtained for hymenopteran γ-GT sequences. A. ervi and N. vitripennis venomous γ-GT sequences are marked with blue and orange rectangles respectively. Numbers at corresponding nodes are bootstrap support values (1000 bootstrap replicates). The outgroup is the human γ-GT6 sequence [Swiss-Prot: Q6P531]. Aech, Acromyrmex echinatior; Aerv, Aphidius ervi; Aflo, Apis florea; Amel, Apis mellifera; Bimp, Bombus impatiens; Bter, Bombus terrestris; Cflo, Camponotus floridanus; Hsal, Harpegnathos saltator; Hsap, Homo sapiens; Mrot, Megachile rotundata; Nvit, Nasonia vitripennis.

Figure 5

Multiple alignment of toxin-like sequences. The three A. ervi toxin-like sequences were aligned with the mature peptide sequence corresponding to each BLAST best hit (SwissProt database). Residues identical or similar are highlighted in black and grey, respectively. The predicted signal peptide is underlined in red, the six conserved cysteine residues are identified by red stars. Theraphotoxin: U8-theraphotoxin-Cj1a from Chilobrachys jingzhao [Swiss-Prot: B1P1C0]; Conotoxin_Vi: Conotoxin Vi11.3 from Conus vitelinus [Swiss-Prot: C7DQX8]; Conotoxin_Ab: Conotoxin AbVIN from Conus abbreviatus [Swiss-Prot: Q9TVQ6].