Marylène Poirié

Summary: Legumes symbiosis with rhizobial nitrogen-fixing bacteria enable them to grow in nitrate-depleted soils. Rhizobial symbioses also induces systemic plant defence against bioagressors. We investigate how nitrogen-fixing symbiosis (NFS) in the legume Medicago truncatula can prime plant defence against the pea aphid Acyrthosiphon pisum . We analysed metabolite modification both by LC-MS and GC-MS and defence pathway gene expression by qPCR in leaves of both NFS and nitrate-fed (non-inoculated; NI) plants after aphid infestation (Amp). The accumulation of primary and secondary metabolites was modulated by both NFS and aphid infestation. 62 defense-related metabolites such as salicylate, pipecolate, gentisic acid and several soluble sugars were differentially regulated by aphid infestation in both NFS and NI conditions. 19 metabolites, including triterpenoid saponins, accumulated specifically in NFS_Amp conditions. Gene expression analysis showed that aphid-infested plants exhibited significantly higher expression of Chalcone isomerase , flavonol synthase , hydroxyisoflavone-O-methyl transferase and Pterocarpan synthase , while D-pinitol dehydrogenase was only significantly induced in NI infested leaves. Our data suggest that NFS, in addition to being a plant nitrogen provider, stimulates specific legume defenses upon pest attack and should also be considered as a potential tool in Integrated Pest Management strategy.

After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges.

Animal venoms and other protein-based secretions that perform a variety of functions, from predation to defense, are highly complex cocktails of bioactive compounds. Gene duplication, accompanied by modification of the expression and/or function of one of the duplicates under the action of positive selection, followed by further duplication to produce multigene families of toxins is a well-documented process in venomous animals. This evolutionary model has been less described in parasitoid wasps, which use maternal fluids, including venom, to protect their eggs from encapsulation by the host immune system. Here, we evidence the convergent recruitment and accelerated evolution of two multigene families of RhoGAPs presumably involved in virulence in two unrelated parasitoid wasp species, Leptopilina boulardi  (Figitidae) and Venturia canescens (Icheumonidae). In both species, these RhoGAPs are associated with vesicles that act as transport systems to deliver virulence factors, but are produced in different tissues: the venom gland in Leptopilina  sp. and the ovarian calyx in V. canescens . We show that the gene encoding the cellular RacGAP1 is at the origin of the virulent RhoGAP families found in Leptopilina sp. and V. canescens .   We also show that both RhoGAP families have undergone evolution under positive selection and that almost all of these RhoGAPs lost their GAP activity and GTPase binding ability due to substitutions in key amino acids. These results suggest an accelerated evolution and functional diversification of these vesicle-associated RhoGAPs in the two phylogenetically distant parasitoid species. The potential new function(s) and the exact mechanism of action of these proteins in host cells remain to be elucidated.

Behavioural avoidance has obvious benefits for animals facing environmental stressors such as pathogen-contaminated foods. Most current bioinsecticides are based on the environmental and opportunistic bacteriumBacillus thuringiensis(Bt) that kills targeted insect pests upon ingestion. While food and oviposition avoidance ofBtbioinsecticide by targeted insect species was reported, this remained to be addressed in non-target organisms, especially those affected by chronic exposure toBtbioinsecticide such asDrosophilaspecies. Here, using a two-choice oviposition test, we showed that female flies of threeDrosophilaspecies (four strains ofD. melanogaster,D. busckiiandD. suzukii) avoided laying eggs in the presence ofBtvar.kurstakibioinsecticide, with potential benefits for the offspring and female's fitness. Avoidance occurred rapidly, regardless of the fraction of the bioinsecticide suspension (spores and toxin crystals versus soluble toxins/compounds) and independently of the female motivation for egg laying. Our results suggest that, in addition to recent findings of developmental and physiological alterations upon chronic exposure to non-targetDrosophila, this bioinsecticide may modify the competitive interactions betweenDrosophilaspecies in treated areas and the interactions with their associated natural enemies.

Animal venoms and other protein-based secretions that perform a variety of functions, from predation to defense, are highly complex cocktails of bioactive compounds. Gene duplication, accompanied by modification of the expression and/or function of one of the duplicates under the action of positive selection, followed by further duplication to produce multigene families of toxins is a well-documented process in venomous animals. This evolutionary model has been less described in parasitoid wasps, which use maternal fluids, including venom, to protect their eggs from encapsulation by the host immune system. Here, we evidence the convergent recruitment and accelerated evolution of two multigene families of RhoGAPs presumably involved in virulence in two unrelated parasitoid wasp species, Leptopilina boulardi (Figitidae) and Venturia canescens (Icheumonidae). In both species, these RhoGAPs are associated with vesicles that act as transport systems to deliver virulence factors, but are produced in different tissues: the venom gland in Leptopilina sp. and the ovarian calyx in V. canescens . We show that the gene encoding the cellular RacGAP1 is at the origin of the virulent RhoGAP families found in Leptopilina sp. and V. canescens . We also show that both RhoGAP families have undergone evolution under positive selection and that almost all of these RhoGAPs lost their GAP activity and GTPase binding ability due to substitutions in key amino acids. These results suggest an accelerated evolution and functional diversification of these vesicle-associated RhoGAPs in the two phylogenetically distant parasitoid species. The potential new function(s) and the exact mechanism of action of these proteins in host cells remain to be elucidated.

Meteorus pulchricornis (Ichneumonoidea, Braconidae) is an endoparasitoid wasp of lepidopteran caterpillars. Its parasitic success relies on vesicles (named M. pulchricornis Virus-Like Particles or MpVLPs) that are synthesized in the venom gland and injected into the parasitoid host along with the venom during oviposition. In order to define the content and understand the biogenesis of these atypical vesicles, we performed a transcriptome analysis of the venom gland and a proteomic analysis of the venom and purified MpVLPs. About half of the MpVLPs and soluble venom proteins identified were unknown and no similarity with any known viral sequence was found. However, MpVLPs contained a large number of proteins labelled as metalloproteinases while the most abundant protein family in the soluble venom was that of proteins containing the Domain of Unknown Function DUF-4803. The high number of these proteins identified suggests that a large expansion of these two protein families occurred in M. pulchricornis. Therefore, although the exact mechanism of MpVLPs formation remains to be elucidated, these vesicles appear to be “metalloproteinase bombs” that may have several physiological roles in the host including modifying the functions of its immune cells. The role of DUF4803 proteins, also present in the venom of other braconids, remains to be clarified.

Temperature is particularly important for ectotherms, including endoparasitoid wasps that develop inside another ectotherm host. In this study, we tested the impact of three temperatures (20 °C, 25 °C and 30 °C) on the host–parasitoid immune interaction using two Drosophila host species (Drosophila melanogaster and D. yakuba) and two parasitoid lines of Leptopilina boulardi. Drosophila’s immune defense against parasitoids consists of the formation of a melanized capsule surrounding the parasitoid egg. To counteract this response, Leptopilina parasitoids rely on the injection of venom during oviposition. Here, we tested the effect of temperature on parasitic success and host encapsulation capacity in response to a parasitoid egg or other foreign body. Increased temperature either promoted or did not affect the parasitic success, depending on the parasitoid–host pairs considered. The mechanisms behind the higher success seemed to vary depending on whether the temperature primarily affected the host immune response or also affected the parasitoid counter-immune response. Next, we tested the effect of parasitoid rearing temperature on its success and venom composition. Venom composition varied strongly with temperature for both parasitoid lines, partially consistent with a change in their parasitic success. Overall, temperature may have a significant impact on the host–parasitoid immune interaction.

The pea aphid Acyrthosiphon pisum hosts different facultative symbionts (FS) which provide it with various benefits, such as tolerance to heat or protection against natural enemies (e.g., fungi, parasitoid wasps). Here, we investigated whether and how the presence of certain FS could affect phenoloxidase (PO) activity, a key component of insect innate immunity, under normal and stressed conditions. For this, we used clones of A. pisum of different genetic backgrounds (LL01, YR2 and T3‐8V1) lacking FS or harboring one or two (Regiella insecticola, Hamiltonella defensa, Serratia symbiotica + Rickettsiella viridis). Gene expression and proteomics analyses of the aphid hemolymph indicated that the two A. pisum POs, PPO1 and PPO2, are expressed and translated into proteins. The level of PPO genes expression as well as the amount of PPO proteins and phenoloxidase activity in the hemolymph depended on both the aphid genotype and FS species. In particular, H. defensa and R. insecticola, but not S. symbiotica + R. viridis, caused a sharp decrease in PO activity by interfering with both transcription and translation. The microinjection of different types of stressors (yeast, Escherichia coli, latex beads) in the YR2 lines hosting different symbionts affected the survival rate of aphids and, in most cases, also decreased the expression of PPO genes after 24 h. The amount and activity of PPO proteins varied according to the type of FS and stressor, without clear corresponding changes in gene expression. These data demonstrate that the presence of certain FS influences an important component of pea aphid immunity.

A bstract: Female endoparasitoid wasps usually inject venom into hosts to suppress their immune response and ensure offspring development. However, the parasitoid’s ability to evolve towards increased success on a given host simultaneously with the evolution of the composition of its venom has never been demonstrated. Here, we designed an experimental evolution to address this question. We crossed two parasitoid lines of Leptopilina boulardi differing both in parasitic success on different Drosophila hosts and venom composition. F ₂ descendants were reared on three different Drosophila species for nine generations. We tested for evolution of parasitic success over the generations and for the capacity of parasitoids selected on a given host to succeed on another host. We also tested whether the venom composition - based on a statistical analysis of the variation in intensity of the venom protein bands on SDS-PAGE 1D - evolved in response to different host species. Results showed a specialization of the parasitoids on their selection host and a rapid and differential evolution of the venom composition according to the host. Overall, data suggest a high potential for parasitoids to adapt to a new host, which may have important consequences in the field as well in the context of biological control.