The Origin of Intraspecific Variation of Virulence in an Eukaryotic Immune Suppressive Parasite Dominique Colinet 1,2,3 *, Antonin Schmitz 1,2,3 , Dominique Cazes 1,2,3 , Jean-Luc Gatti 1,2,3 , Maryle `ne Poirie ´ 1,2,3 1 Institut National de la Recherche Agronomique, ESIM, INRA PACA, UMR 1301, Sophia Antipolis, France, 2 Centre National de la Recherche Scientifique, CNRS, UMR 6243, Sophia Antipolis, France, 3 Universite ´ Nice Sophia Antipolis, UFR Sciences, Sophia Antipolis, France Abstract 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. Citation: Colinet D, Schmitz A, Cazes D, Gatti J-L, Poirie ´ M (2010) The Origin of Intraspecific Variation of Virulence in an Eukaryotic Immune Suppressive Parasite. PLoS Pathog 6(11): e1001206. doi:10.1371/journal.ppat.1001206 Editor: David S. Schneider, Stanford University, United States of America Received April 20, 2010; Accepted October 22, 2010; Published November 24, 2010 Copyright: ß 2010 Colinet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work and D. Colinet’s post-doctoral position were supported by grants CLIMEVOL (ANR-08-BLAN-0231) and PARATOXOSE (ANR-09-BLAN-0243-01) from the French Agency for National Research, and the French National Institute for Agricultural Research (INRA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dominique.colinet@sophia.inra.fr Introduction Models of host-parasite coevolution all assume occurrence of genetic variation for host resistance and parasite virulence, such as well-described for plant-pathogen interactions [1] and in a few host-parasite models [2][3]. Advances in understanding coevolu- tionary interactions thus require unraveling the molecular bases of host resistance and parasite virulence, and then acquiring data on how polymorphism in genes controlling these traits affect parasitism success in the field [4]. This will enable direct observa- tion, rather than inference, of the host-parasite coevolutionary dynamics. One largely unaddressed question in eukaryotic parasites is the basis of intraspecific variation of virulence. Regarding immune aspects, the only described mechanism is, to our knowledge, the antigenic variation used by fungal and protozoan parasites to hide themselves from the mammalian immune system [5–10]. Whether changes associated with virulence variation in immune suppressive parasites are qualitative and/or quantitative and what is their nature, still remain to be assessed. The biological models of interacting species that satisfy all the requirements to study coevolutionary processes, from molecular tools to population polymorphisms, are scarce and Drosophila- parasitoid wasps interactions are undoubtedly among the best of them [11]. Variation in both Drosophila resistance and parasitoid virulence is observed in the field [12–14], and some major genes involved in these traits have been characterized [15–18]. Based on these data, we address here the question of the origin of intraspecific variation of virulence of Drosophila parasitoids. Endoparasitoid wasps develop inside the body of their arthropod host, which will die as a result of the interaction [19],[20]. To face the immune defense of the host, they have evolved original strategies ranging from displaying surface features that prevent their recognition to altering components of the host immune system using venom proteins, virus-like particles or wasp- specific viruses, polydnaviruses [21],[22]. However, occurrence of virulence variation together with molecular identification and characterization of the factors involved in immune suppres- sion has only been reported in the wasp Leptopilina boulardi [15],[16],[23],[24]. PLoS Pathogens | www.plospathogens.org 1 November 2010 | Volume 6 | Issue 11 | e1001206