Extracellular superoxide dismutase in insects: characterization, function, and interspecific variation in parasitoid wasp venom.

Endoparasitoid wasps inject venom proteins with their eggs to protect them from the host immune response and ensure successful parasitism. Here we report identification of Cu,Zn superoxide dismutase (SOD) transcripts for both intracellular SOD1 and extracellular SOD3 in the venom apparatus of two Leptopilina species, parasitoids of Drosophila. Leptopilina SODs show sequence and structure similarity to human SODs, but phylogenetic analyses indicate that the extracellular SODs are more related to cytoplasmic vertebrate SODs than to extracellular SODs, a feature shared by predicted insect extracellular SODs. We demonstrate that L. boulardi SOD3 is indeed secreted and active as monomeric glycosylated forms in venom. Our results also evidence quantitative variation in SOD3 venom contents between closely related parasitoid species, as sod3 is 100-fold less expressed in Leptopilina heterotoma venom apparatus and no protein and SOD activity are detected in its venom. Leptopilina recombinant SOD3s as well as a mammalian SOD in vitro inhibit the Drosophila phenoloxidase activity in a dose-dependent manner, demonstrating that SODs may interfere with the Drosophila melanization process and, therefore, with production of cytotoxic compounds. Although the recombinant L. boulardi SOD3 quantity needed to observe this effect precludes a systemic effect of the wasp venom SOD3, it is still consistent with a local action at oviposition. This work provides the first demonstration that insect extracellular SODs are indeed secreted and active in an insect fluid and can be used as virulence factors to counteract the host immune response, a strategy largely used by bacterial and fungal pathogens but also protozoan parasites during infection.

The origin of intraspecific variation of virulence in an eukaryotic immune suppressive parasite.

Occurrence of intraspecific variation in parasite virulence, a prerequisite for coevolution of hosts and parasites, has largely been reported. However, surprisingly little is known of the molecular bases of this variation in eukaryotic parasites, with the exception of the antigenic variation used by immune-evading parasites of mammals. The present work aims to address this question in immune suppressive eukaryotic parasites. In Leptopilina boulardi, a parasitic wasp of Drosophila melanogaster, well-defined virulent and avirulent strains have been characterized. The success of virulent females is due to a major immune suppressive factor, LbGAP, a RacGAP protein present in the venom and injected into the host at oviposition. Here, we show that an homologous protein, named LbGAPy, is present in the venom of the avirulent strain. We then question whether the difference in virulence between strains originates from qualitative or quantitative differences in LbGAP and LbGAPy proteins. Results show that the recombinant LbGAPy protein has an in vitro GAP activity equivalent to that of recombinant LbGAP and similarly targets Drosophila Rac1 and Rac2 GTPases. In contrast, a much higher level of both mRNA and protein is found in venom-producing tissues of virulent parasitoids. The F1 offspring between virulent and avirulent strains show an intermediate level of LbGAP in their venom but a full success of parasitism. Interestingly, they express almost exclusively the virulent LbGAP allele in venom-producing tissues. Altogether, our results demonstrate that the major virulence factor in the wasp L. boulardi differs only quantitatively between virulent and avirulent strains, and suggest the existence of a threshold effect of this molecule on parasitoid virulence. We propose that regulation of gene expression might be a major mechanism at the origin of intraspecific variation of virulence in immune suppressive eukaryotic parasites. Understanding this variation would improve our knowledge of the mechanisms of transcriptional evolution currently under active investigation.

Amount of venom that Leptopilina species inject into Drosophila melanogaster larvae in relation to parasitic success.

The Drosophila endoparasitoid wasps Leptopilina boulardi and L. heterotoma (Hymenoptera: Cynipidae) are pro-ovigenic species, i.e., females contain their lifetime number of mature eggs at emergence. They are therefore able to immediately parasitize many hosts when present. In response to parasitoid oviposition, the larval host D. melanogaster can mount an immune response, encapsulation, that can destroy the parasitoid eggs. This response is counteracted by the venom the wasp injects during oviposition. Here, we estimated the amount of venom injected into a D. melanogaster host larva using immunodetection of venom proteins and we attempted to correlate this amount with the number of eggs a female can lay on successive days. The venom reservoir of L. boulardi contains enough venom for at least 100 ovipositions while that of L. heterotoma contains venom for about 16 ovipositions. While a female L. boulardi may have enough venom for three days of parasitism when 20 or 40 larval hosts were presented each day, L. heterotoma certainly needs to synthesize new venom to parasitize the number of hosts offered. Interestingly, parasitism stopped (L. boulardi), egg protection (L. heterotoma) and egg hatching decreased (both species) after three days of parasitism. Thus, although venom does not appear to be a limiting factor for parasitism, our data suggest that it may have less effectiveness on the egg protection and on egg/host development after high repetitive egg laying.

Extensive inter- and intraspecific venom variation in closely related parasites targeting the same host: the case of Leptopilina parasitoids of Drosophila.

The arms race between immune suppressive parasites that produce virulence factors and hosts that evolve resistance to these factors is suggested to be a key driver for the diversification of both partners. However, little is known regarding the diversity of virulence factors in closely related parasites or the mechanisms underlying the variation of virulence. One of the best-described model to address this issue is the interaction between Leptopilina parasitic wasps and their Drosophila hosts, in which variation of virulence is well documented. Thanks to a combined transcriptomic and proteomic approach, we have identified the main secreted proteins in the venom of Leptopilina heterotoma (Gotheron strain, 66 proteins) and of two well-characterized strains of Leptopilina boulardi, ISm and ISy (65 and 49 proteins, respectively). Results revealed significant quantitative differences in venom components between the L. boulardi strains, in agreement with their different virulence properties. Strikingly, the two related Leptopilina species did not share any abundant venom protein. The main identified proteins in L. boulardi were RhoGAPs and serpins while an aspartylglucosaminidase (AGA) was found abundant in L. heterotoma. The extensive quantitative variation observed between these species may be related with their use of different virulence strategies and/or to differences in their host range (specialist versus generalist). Altogether, our data suggests that parasitoid venom can quickly evolve, mainly through rapid changes in regulation of gene expression. It also evidences venom evolutionary processes largely described in other venomous animals i.e. the convergent recruitment of venom proteins between phylogenetically unrelated organisms, and the role of duplications in the emergence of multigenic families of virulence factors.

A serpin from the parasitoid wasp Leptopilina boulardi targets the Drosophila phenoloxidase cascade.

The insect phenoloxidase (PO) cascade is known to be tightly regulated by serine proteases and serine protease inhibitors of the serpin family. As a key component of the insect immune system, it is also suspected to be inhibited by several endoparasitoid wasps, insects that develop inside other arthropods as hosts. However, the underlying mechanisms of this inhibition are largely undescribed. Here, we report the characterization of a gene encoding a serpin, LbSPNy, highly expressed in the venom of the wasp Leptopilina boulardi (IS(y) type), and we show that either the venom or the recombinant LbSPNy inhibit the PO cascade in the hemolymph of Drosophila yakuba host larva. Altogether, our results identify the first serpin used as a virulence factor by a parasitoid wasp and show that it disrupts the activation pathway of the PO in the Drosophila host.