Recent studies suggest that the pea aphid (Acyrthosiphon pisum) has low immune defenses. However, its immune components are largely undescribed, and notably, extensive characterization of circulating cells has been missing. Here, we report characterization of five cell categories in hemolymph of adults of the LL01 pea aphid clone, devoid of secondary symbionts (SS): prohemocytes, plasmatocytes, granulocytes, spherulocytes and wax cells. Circulating lipid-filed wax cells are rare; they otherwise localize at the basis of the cornicles. Spherulocytes, that are likely sub-cuticular sessile cells, are involved in the coagulation process. Prohemocytes have features of precursor cells. Plasmatocytes and granulocytes, the only adherent cells, can form a layer in vivo around inserted foreign objects and phagocytize latex beads or Escherichia coli bacteria injected into aphid hemolymph. Using digital image analysis, we estimated that the hemolymph from one LL01 aphid contains about 600 adherent cells, 35% being granulocytes. Among aphid YR2 lines differing only in their SS content, similar results to LL01 were observed for YR2-Amp (without SS) and YR2-Ss (with Serratia symbiotica), while YR2-Hd (with Hamiltonella defensa) and YR2(Ri) (with Regiella insecticola) had strikingly lower adherent hemocyte numbers and granulocyte proportions. The effect of the presence of SS on A. pisum cellular immunity is thus symbiont-dependent. Interestingly, Buchnera aphidicola (the aphid primary symbiont) and all SS, whether naturally present, released during hemolymph collection, or artificially injected, were internalized by adherent hemocytes. Inside hemocytes, SS were observed in phagocytic vesicles, most often in phagolysosomes. Our results thus raise the question whether aphid symbionts in hemolymph are taken up and destroyed by hemocytes, or actively promote their own internalization, for instance as a way of being transmitted to the next generation. Altogether, we demonstrate here a strong interaction between aphid symbionts and immune cells, depending upon the symbiont, highlighting the link between immunity and symbiosis.

In several Hymenoptera, sexual fate is determined by the allelic composition at the complementary sex-determiner locus, a sex-determination mechanism that can strongly affect population dynamics. To date, the molecular identification of complementary sex determiner has only been achieved in the honeybee, where the complementary sex-determiner gene was reported to have arisen from duplication of the feminizer gene. Strikingly, the complementary sex-determiner gene was also proposed to be unique to the honeybee lineage. Here we identify feminizer and complementary sex-determiner orthologues in bumble bees and ants. We further demonstrate that the duplication of feminizer that produced complementary sex determiner occurred before the divergence of Aculeata species (~120 Myr ago). Finally, we provide evidence that the two genes evolved concertedly through gene conversion, complementary sex-determiner evolution being additionally shaped by mosaic patterns of selection. Thus, the complementary sex-determiner gene likely represents the molecular basis for single locus-complementary sex determination in the Aculeata infra-order, and possibly, in the entire Hymenoptera order.

Endoparasitoid wasps, which lay eggs inside the bodies of other insects, use various strategies to protect their offspring from the host immune response. The hymenopteran species of the genus Leptopilina, parasites of Drosophila, rely on the injection of a venom which contains proteins and peculiar vesicles (hereafter venosomes). We show here that the injection of purified L. boulardi venosomes is sufficient to impair the function of the Drosophila melanogaster lamellocytes, a hemocyte type specialized in the defense against wasp eggs, and thus the parasitic success of the wasp. These venosomes seem to have a unique extracellular biogenesis in the wasp venom apparatus where they acquire specific secreted proteins/virulence factors and act as a transport system to deliver these compounds into host lamellocytes. The level of venosomes entry into lamellocytes of different Drosophila species was correlated with the rate of parasitism success of the wasp, suggesting that this venosome-cell interaction may represent a new evolutionary level of host-parasitoid specificity.

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.

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.

Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs) to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.

Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts.

Bioinsecticides made from the bacterium Bacillus thuringiensis (Bt) are the bestselling bioinsecticide worldwide. Among Bt bioinsecticides, those based on the strain Bt subsp. kurstaki (Btk) are widely used in farming to specifically control pest lepidopteran larvae. Although there is much evidence of the lack of acute lethality of Btk products for non-target animals, only scarce data are available on their potential non-lethal developmental adverse effects. Using a concentration that could be reached in the field upon sprayings, we show that Btk products impair growth and developmental time of the non-target dipteran Drosophila melanogaster. We demonstrate that these effects are mediated by the synergy between Btk bacteria and Btk insecticidal toxins. We further show that Btk bioinsecticides trigger intestinal cell death and alter protein digestion without modifying the food intake and feeding behavior of the larvae. Interestingly, these harmful effects can be mitigated by a protein-rich diet or by adding the probiotic bacterium Lactobacillus plantarum into the food. Finally, we unravel two new cellular mechanisms allowing the larval midgut to maintain its integrity upon Btk aggression: First the flattening of surviving enterocytes and second, the generation of new immature cells arising from the adult midgut precursor cells. Together, these mechanisms participate to quickly fill in the holes left by the dying enterocytes.

Bioinsecticides based on Bacillus thuringiensis (Bt) spores and toxins are increasingly popular alternative solutions to control insect pests, with potential impact of their accumulation in the environment on non-target organisms. Here, we tested the effects of chronic exposure to commercial Bt formulations (Bt var. kurstaki and israelensis) on eight non-target Drosophila species present in Bt-treated areas, including D. melanogaster (four strains). Doses up to those recommended for field application (~ 106 Colony Forming Unit (CFU)/g fly medium) did not impact fly development, while no fly emerged at ≥ 1000-fold this dose. Doses between 10- to 100-fold the recommended one increased developmental time and decreased adult emergence rates in a dose-dependent manner, with species-and strain-specific effect amplitudes. Focusing on D. melanogaster, development alterations were due to instar-dependent larval mortality, and the longevity and offspring number of adult flies exposed to bioinsecticide throughout their development were moderately influenced. Our data also suggest a synergy between the formulation compounds (spores, cleaved toxins, additives) might induce the bioinsecticide effects on larval development. Although recommended doses had no impact on non-target Drosophila species, misuse or local environmental accumulation of Bt bioinsecticides could have side-effects on fly populations with potential implications for their associated communities.

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and β-subunits also form an (αβ)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and β for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps' success.

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.

Drosophila suzukii (the spotted-wing Drosophila) appears to be unsuitable for the development of most Drosophila larval endoparasitoids, be they sympatric or not. Here, we questioned the physiological bases of this widespread failure by characterizing the interactions between D. suzukii and various parasitoid species (Asobara japonica, Leptopilina boulardi, Leptopilina heterotoma and Leptopilina victoriae) and comparing them with those observed with D. melanogaster, a rather appropriate host. All parasitoids were able to oviposit in L1 and L2 larval stages of both hosts but their propensity to parasitize was higher on D. melanogaster. A. japonica and, to a much lesser extent, L. heterotoma, were the two species able to successfully develop in D. suzukii, the failure of the parasitism resulting either in the parasitoid encapsulation (notably with L. heterotoma) or the host and parasitoid deaths (especially with L. boulardi and L. victoriae). Compared to D. melanogaster, encapsulation in D. suzukii was strongly delayed and led, if successful, to the production of much larger capsules in surviving flies and, in the event of failure, to the death of both partners because of an uncontrolled melanization. The results thus revealed a different timing of the immune response to parasitoids in D. suzukii compared to D. melanogaster with a lose-lose outcome for parasitoids (generally unsuccessful development) and hosts (high mortality and possible reduction of the fitness of survivors). Finally, these results might suggest that some European endoparasitoids of Drosophila interact with this pest in the field in an unmeasurable way, since they kill their host without reproductive success.

Endoparasitoid wasps are essential regulators of insect pests in ecosystems as well as important biological control auxiliaries. Traits important for parasitism success, such as the injection of venom proteins at oviposition, have thus been mainly studied. However, identification of the key genes involved among the large number of genes identified was still prevented by the lack of functional approaches. Here, we report the development of RNA interference (RNAi) in Leptopilina boulardi, a figitid endoparasitoid that performs its entire development inside the Drosophila host. Having set up conditions for in vitro development of parasitoid late larval stages or pupae, we first targeted the cinnabar gene by microinjecting double-stranded RNA (dsRNA), leading to its silencing and production of red-eyed individuals. We then demonstrated that expression of the gene encoding LbGAP, a virulence factor found in a high amount in L. boulardi venom, could be specifically and almost completely silenced. Finally, a time-course analysis revealed that LbGAP silencing lasted during the entire lifetime of L. boulardi. This is the first report of the efficient silencing of venom protein-encoding genes in parasitoid wasps. Overall, RNAi opens the way for a large-scale functional analysis of parasitoid venom factors as well as other traits involved in parasitism success and more largely in the biology of these ecologically important organisms.

Spiroplasma citri is a cell wall-less bacterium that infects plants. It is transmitted by the leafhopper Circulifer haematoceps, which hosts this bacterium in the haemocel and insect tissues. Bacterial factors involved in spiroplasma colonization of the insect host have been identified, but the immune response of the leafhopper to S. citri infection remains unknown. In this study, we showed that C. haematoceps activates both humoral and cellular immune responses when challenged with bacteria. When infected by S. citri, C. haematoceps displayed a specific immune response, evidenced by activation of phagocytosis and upregulation of a gene encoding the protein hexamerin. S. citri infection also resulted in decreased phenoloxidase-like activity. Inhibition of hexamerin by RNA interference resulted in a significant reduction in phenoloxidase-like activity and increased mortality of infected leafhoppers. Therefore, the gene hexamerin is involved in S. citri control by interfering with insect phenoloxidase activity.

Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences.

Relics of ancient infections are abundant in eukaryote genomes, but little is known about how they evolve when they confer a functional benefit on their host. We show here, for the first time, that the virus-like particles shown to protect Venturia canescens eggs against host immunity are derived from a nudivirus genome incorporated by the parasitic wasp into its own genetic material. Nudivirus hijacking was also at the origin of protective particles from braconid wasps. However, we show here that the viral genes produce "liposomes" that wrap and deliver V. canescens virulence proteins, whereas the particles are used as gene transfer agents in braconid wasps. Our findings indicate that virus domestication has occurred repeatedly during parasitic wasp evolution but with different evolutionary trajectories after endogenization, resulting in different virulence molecule delivery strategies.

Venosomes are extracellular vesicles found in the venom of Leptopilina endoparasitoids wasps, which transport and target virulence factors to impair the parasitoid egg encapsulation by the lamellocytes of their Drosophila melanogaster host larva. Using the co-immunolocalization of fluorescent L. boulardi venosomes and one of the putative-transported virulence factors, LbGAP, with known markers of cellular endocytosis, we show that venosomes endocytosis by lamellocytes is not a process dependent on clathrin or macropinocytosis and internalization seems to bypass the early endosomal compartment Rab5. After internalization, LbGAP colocalizes strongly with flotillin-1 and the GPI-anchored protein Atilla/L1 (a lamellocyte surface marker) suggesting that entry occurs via a flotillin/lipid raft-dependent pathway. Once internalized, venosomes reach all intracellular compartments, including late and recycling endosomes, lysosomes, and the endoplasmic reticulum network. Venosomes therefore enter their target cells by a specific mechanism and the virulence factors are widely distributed in the lamellocytes' compartments to impair their functions.

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 bacterium Bacillus thuringiensis (Bt) that kills targeted insect pests upon ingestion. While food and oviposition avoidance of Bt bioinsecticide by targeted insect species was reported, this remained to be addressed in non-target organisms, especially those affected by chronic exposure to Bt bioinsecticide such as Drosophila species. Here, using a two-choice oviposition test, we showed that female flies of three Drosophila species (four strains of D. melanogaster, D. busckii and D. suzukii) avoided laying eggs in the presence of Bt var. kurstaki bioinsecticide, 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-target Drosophila, this bioinsecticide may modify the competitive interactions between Drosophila species in treated areas and the interactions with their associated natural enemies.

Venom composition of parasitoid wasps attracts increasing interest - notably molecules ensuring parasitism success on arthropod pests - but its variation within and among taxa is not yet understood. We have identified here the main venom proteins of two braconid wasps, Psyttalia lounsburyi (two strains from South Africa and Kenya) and P. concolor, olive fruit fly parasitoids that differ in host range. Among the shared abundant proteins, we found a GH1 β-glucosidase and a family of leucine-rich repeat (LRR) proteins. Olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. Assuming that Psyttalia host larvae sequester ingested glycosides, the injected venom GH1 β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. Venom LRR proteins are similar to truncated Toll-like receptors and may possibly scavenge the host immunity. The abundance of one of these LRR proteins in the venom of only one of the two P. lounsburyi strains evidences intraspecific variation in venom composition. Altogether, venom intra- and inter-specific variation in Psyttalia spp. were much lower than previously reported in the Leptopilina genus (Figitidae), suggesting it might depend upon the parasitoid taxa.

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.