Impact of Temperature on the Immune Interaction between a Parasitoid Wasp and Drosophila Host Species.

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.

Functional insights from the GC-poor genomes of two aphid parasitoids, Aphidius ervi and Lysiphlebus fabarum.

BACKGROUND: 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. RESULTS: We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes. CONCLUSIONS: These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org.

Rapid and Differential Evolution of the Venom Composition of a Parasitoid Wasp Depending on the Host Strain.

Parasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with the crosses of two lines of Leptopilinaboulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance/susceptibility to the parental parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results made evident a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened.

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma.

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 ß-subunits also form an (αß)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and ß 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.

Comparative venomics of Psyttalia lounsburyi and P. concolor, two olive fruit fly parasitoids: a hypothetical role for a GH1 ß-glucosidase.

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 ß-glucosidase and a family of leucine-rich repeat (LRR) proteins. Olive is extremely rich in glycoside compounds that are hydrolyzed by ß-glucosidases into defensive toxic products in response to phytophagous insect attacks. Assuming that Psyttalia host larvae sequester ingested glycosides, the injected venom GH1 ß-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.

Recurrent DNA virus domestication leading to different parasite virulence strategies.

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.

Identification of the main venom protein components of Aphidius ervi, a parasitoid wasp of the aphid model Acyrthosiphon pisum.

BACKGROUND: 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. RESULTS: A combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified. CONCLUSIONS: Our data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms.

Development of RNAi in a Drosophila endoparasitoid wasp and demonstration of its efficiency in impairing venom protein production.

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.

Insights into function and evolution of parasitoid wasp venoms.

Most species in the order Hymenoptera are parasitoids that lay eggs and develop in or on the body of arthropod hosts. Several factors contribute to successful parasitism including venoms that wasps inject into hosts when ovipositing. Here, we review the composition, function and diversity of parasitoid venoms with emphasis on studies of wasps that parasitize hosts in the genus Drosophila. The comparative literature indicates that some closely related species parasitizing the same host do not share any abundant venom protein while unrelated species sometimes have the same major venom component. Within species, studies also identify intraspecific variation that suggests parasitoid venoms may rapidly evolve. Overall, however, our picture of venom function remains largely unclear and will require additional comparative data on the composition of venoms from a greater diversity of species than exists currently. Further advances will come mainly from experimental data using functional tools, such as RNA interference.

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.

Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins.

The venom gland is a conserved organ in Hymenoptera that shows adaptations associated with life-style diversification. Few studies have investigated venom components and function in the highly diverse parasitic wasps and all suggest that the venom regulates host physiology. We explored the venom of the endoparasitoid Hyposoter didymator (Campopleginae), a species with an associated polydnavirus produced in the ovarian tissue. We investigated the effects of the H. didymator venom on two physiological traits of the host Spodoptera frugiperda (Noctuidae): encapsulation response and growth rate. We found that H. didymator venom had no significant effect on host cellular immunity or development, suggesting that it does not contribute to parasitism success. The host physiology seemed to be modified essentially by the ovarian fluid containing the symbiotic polydnaviruses. Proteomic analyses indicated that the H. didymator venom gland produces a large variety of proteins, consistent with the classical hymenopteran venom protein signature, including: reprolysin-like, dipeptidyl peptidase IV, hyaluronidase, arginine kinase or allergen proteins. The venom extracts also contained novel proteins, encoded by venom genes conserved in Campopleginae ichneumonids, and proteins with similarities to active molecules identified in other parasitoid species, such as calreticulin, reprolysin, superoxide dismutase and serpin. However, some of these proteins appear to be produced only in small amounts or to not be secreted. Possibly, in Campopleginae carrying polydnaviruses, the host-modifying activities of venom became redundant following the acquisition of polydnaviruses by the lineage.

Variability of venom components in immune suppressive parasitoid wasps: from a phylogenetic to a population approach.

Endoparasitoid wasps develop at the expense of other insects, leading to their death. Eggs deposited inside the host body induce an immune response, which results in the formation of a melanized cellular capsule around the egg. To evade or counteract this response, endoparasitoids have evolved different strategies, the most often reported being injection into the host of immunosuppressive factors, notably venom proteins, along with the egg. The analysis of venom components has been performed independently in species of different taxa, but the present picture is far from complete. Intriguingly, the question of the level of venom variability inside species has been neglected, although it may partly determine the potential for parasitoid adaptation. Here, we present a short review of our present knowledge of venom components in endoparasitoids, as well as of the only well-known example of intraspecific variability in a venom immune suppressive protein being responsible for variation in parasitoid virulence. We then present data evidencing inter-individual variation of venom protein profiles, using a gel electrophoresis approach, both in laboratory strains and field populations of a figitid and a braconid species. Whether occurrence of such variability may permit a selection of parasitoid venom components driven by the host remains to be tested, notably in the context of the production and use of biological control auxiliaries.

Tracing back the nascence of a new sex-determination pathway to the ancestor of bees and ants.

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.

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.

Involvement of the cytokine MIF in the snail host immune response to the parasite Schistosoma mansoni.

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions.

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.

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens.

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.