Integrative Approach Reveals Composition of Endoparasitoid Wasp Venoms

<div><p>The fruit fly <i>Drosophila melanogaster</i> and its endoparasitoid wasps are a developing model system for interactions between host immune responses and parasite virulence mechanisms. In this system, wasps use diverse venom cocktails to suppress the conserved fly cellular encapsulation response. Although numerous genetic tools allow detailed characterization of fly immune genes, lack of wasp genomic information has hindered characterization of the parasite side of the interaction. Here, we use high-throughput nucleic acid and amino acid sequencing methods to describe the venoms of two related Drosophila endoparasitoids with distinct infection strategies, <i>Leptopilina boulardi</i> and <i>L. heterotoma</i>. Using RNA-seq, we assembled and quantified libraries of transcript sequences from female wasp abdomens. Next, we used mass spectrometry to sequence peptides derived from dissected venom gland lumens. We then mapped the peptide spectral data against the abdomen transcriptomes to identify a set of putative venom genes for each wasp species. Our approach captured the three venom genes previously characterized in <i>L. boulardi</i> by traditional cDNA cloning methods as well as numerous new venom genes that were subsequently validated by a combination of RT-PCR, blast comparisons, and secretion signal sequence search. Overall, 129 proteins were found to comprise <i>L. boulardi</i> venom and 176 proteins were found to comprise <i>L. heterotoma</i> venom. We found significant overlap in <i>L. boulardi</i> and <i>L. heterotoma</i> venom composition but also distinct differences that may underlie their unique infection strategies. Our joint transcriptomic-proteomic approach for endoparasitoid wasp venoms is generally applicable to identification of functional protein subsets from any non-genome sequenced organism.</p></div

Proteomics results.

<p>Proteomics results.</p

Analyzing venom and body open reading frames for secretion signal sequences, PFAM and gene ontology (GO) annotations, and molecular function enrichment analyses based on GO annotations.

<p>Analyzing venom and body open reading frames for secretion signal sequences, PFAM and gene ontology (GO) annotations, and molecular function enrichment analyses based on GO annotations.</p

Wasp venom proteins with annotated ‘glycolysis’ activity (GO:0006096).

<p>Wasp venom proteins with annotated ‘glycolysis’ activity (GO:0006096).</p

E-value distributions of BLASTx results of indicated transcript subset blasted against the known venom database: (A) <i>L.</i> <i>boulardi</i> venom, (B) <i>L. boulardi</i> body, (C) <i>L. heterotoma</i> venom, and (D) <i>L. heterotoma</i> body sequences.

<p>Venom and body E-value distributions were compared using Chi-squared tests. (E) Venn diagram illustrating overlap of unique conserved domains identified from <i>L. boulardi</i> and <i>L. heterotoma</i> venoms and the known venom database.</p

Venom and control genes for RT-PCR from <i>L. heterotoma</i>.

<p>Venom and control genes for RT-PCR from <i>L. heterotoma</i>.</p

<i>L. heterotoma</i> venom proteins showing homology to known venom proteins.

<p><i>L. heterotoma</i> venom proteins showing homology to known venom proteins.</p

<i>L. boulardi</i> venom proteins showing homology to known venom proteins.

<p><i>L. boulardi</i> venom proteins showing homology to known venom proteins.</p

Assembly statistics for the abdomen transcriptomes of each species.

<p>Assembly statistics for the abdomen transcriptomes of each species.</p

RT-PCR of putative venom genes from <i>L.</i> <i>boulardi</i> (A) and <i>L. heterotoma</i> (B). Sex- (top panel) and tissue- (bottom panel) specific RT-PCRs of venom (1–10) and control (c1–c4) genes.

<p>Gene ID numbers correspond to those listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064125#pone-0064125-t003" target="_blank">Tables 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064125#pone-0064125-t004" target="_blank">4</a>.</p