Nathan T. Mortimer

The parasitoid wasp Ganaspis hookeri infects Drosophila melanogaster larvae, laying an egg and injecting venom directly into the host body cavity . While infected hosts mount an immune response in an attempt to eliminate the parasitoid egg, parasitoid venom proteins act to inhibit these host responses and manipulate host physiology to ensure infection success. A key immune suppressive venom protein in G. hookeri is a venom-specific isoform of the SERCA (Sarco/endoplasmic reticulum calcium-ATPase) calcium pump. However, SERCA is a large hydrophobic protein, and the mechanism by which it and other venom proteins are transported into host cells is not well understood. Electron microscopy and nanoparticle tracking analysis of G. hookeri venom revealed the presence of venom extracellular vesicles (vEVs) as a putative transport mechanism. We characterized the biophysical properties of vEVs, and found that G. hookeri venom contains multiple vesicle types that are distinguishable by size, zeta potential, protein content, and density. A subset of these vEVs contain SERCA, and we found that fluorescently labeled vEVs are able to enter into host immune cells and alter host intracellular calcium levels, and that disruption of vesicle integrity results in the loss of this SERCA activity in host cells. Together, our data support a model in which G. hookeri venom proteins, including SERCA, are packaged into an array of venom vesicles, and are transported into host cells for immune suppression.

This protocol will allow you to fluorescently label parasitoid wasp venom extracellular vesicles (vEVs), and conduct entry and subcellular localization assays in Drosophila immune cells. Labeling is performed using ExoSparkler (Dojindo) with a modified version of the manufacturer's protocol. After incubation with labeled vEVs, Drosophila immune cells are fixed and stained as appropriate for the intended cellular assay.

This protocol was developed for the isolation of insect membrane proteins from purified vesicles and tissues. We utilize Triton X-114 for inducing cloud-point separation, partitioning membrane proteins into the detergent phase and retaining soluble protein in the aqueous phase of the extraction. This allows for downstream analysis of membrane (detergent phase) and soluble (aqueous phase) proteins via immunoblot, mass spectrometry, or biochemical assay.

Parasitoid wasps are a large and diverse group of species that infect a wide variety of insect hosts. In response, hosts have evolved numerous defensive strategies to protect against infection. Here, we review the immune and behavioral defense responses of the fruit fly Drosophila melanogaster against parasitoid wasps, the best-characterized dipteran system for host–parasitoid interactions. The melanotic encapsulation of parasitoid eggs is a highly conserved immune response that defends hosts against both coevolving and novel parasitoid species while simultaneously protecting against self-inflicted immune damage. Behavioral defense mechanisms include parental behaviors to protect offspring from infection and adaptive alterations in infected juveniles. We discuss the genetic basis and conserved mechanisms of these responses and identify exciting questions for future research.

Many insect species use self-medication, the consumption of an environmental compound with antipathogen activity, as a defense against pathogen infection. One well-studied example is the interaction between Drosophila melanogaster and parasitoid wasps, in which D. melanogaster larvae consume ethanol-laden food to kill the developing parasitoid. Despite research into self-medication as a behavioral immune response to parasitoid infection, the parasitoid-killing mechanism remains elusive. To test the impact of ethanol consumption and infection on host metabolism, we used untargeted Nuclear Magnetic Resonance (NMR) metabolomics of hemolymph samples isolated from naïve and parasitoid infected larvae fed ethanol-containing or control food. Surprisingly, we found that the consumption of dietary ethanol did not result in an elevated hemolymph ethanol abundance. Instead, we found evidence that host carbohydrate-derived energy production and amino acid metabolism were altered by ethanol consumption. Our results suggest that these ethanol-mediated changes in host metabolism, rather than a direct effect of dietary ethanol, confers parasitoid resistance.

SUMMARY: Mounting an immune response requires energy, but how that energy is reallocated at the organismal level remains poorly understood. In Drosophila melanogaster , infection by a parasitoid wasp triggers a systemic metabolic switch known as immunometabolism which is characterized by a shift in metabolic activity and the redistribution of resources away from organismal development and toward the production of a cellular immune response. We identify the PDGF/VEGF (PVF) signaling pathway as a key initiator of this immunometabolic switch. Genetic manipulation of PVF signaling alters infection outcomes, modulates systemic metabolite profiles, and reveals a direct trade-off between immune function and development. Our findings establish the Drosophila- parasitoid wasp system as a genetically tractable model for understanding the molecular basis of immunometabolism.

The parasitoid wasp Ganaspis hookeri infects Drosophila melanogaster larvae, laying an egg and injecting venom directly into the host body cavity. While infected hosts mount an immune response in an attempt to eliminate the parasitoid egg, parasitoid venom proteins act to inhibit these host immune responses and manipulate host physiology to ensure infection success. A key immune suppressive venom protein in G. hookeri is a venom- specific isoform of the SERCA ( S arco/ e ndoplasmic r eticulum C a ²⁺ - A TPase) calcium pump. However, SERCA is a large hydrophobic protein, and the mechanism by which it and other venom proteins are transported into the host is not well understood. We used a variety of biophysical, biochemical, and cell biological approaches to assess the properties of G. hookeri venom. Electronic microscopy and nanoparticle tracking analysis revealed the presence of venom vesicles as a putative transport mechanism. We used tunable resistive pulse sensing (TRPS) to biophysically characterize these vesicles, and our TRPS data suggest G. hookeri venom is composed of multiple vesicle types that are distinguishable by size, zeta potential, and density. Finally, we fluorescently labeled venom vesicles to test for entry into host immune cells. We observed that these vesicles interact with immune cells membranes and are internalized into the cell. Our data support a model in which G. hookeri venom proteins, including SERCA, are packaged into an array of venom vesicles, and transported into host cells for immune suppression.

Innate immune responses that allow hosts to survive infection depend on the action of multiple conserved signaling pathways. Pathogens and parasites in turn have evolved virulence factors to target these immune signaling pathways in an attempt to overcome host immunity. Consequently, the interactions between host immune molecules and pathogen virulence factors play an important role in determining the outcome of an infection. The immune responses of Drosophila melanogaster provide a valuable model to understand immune signaling and host-pathogen interactions. Flies are commonly infected by parasitoid wasps and mount a coordinated cellular immune response following infection. This response is characterized by the production of specialized blood cells called lamellocytes that form a tight capsule around wasp eggs in the host hemocoel. The conserved JAK-STAT signaling pathway has been implicated in lamellocyte proliferation and is required for successful encapsulation of wasp eggs. Here we show that activity of Stat92E, the D. melanogaster STAT ortholog, is induced in immune tissues following parasitoid infection. Virulent wasp species are able to suppress Stat92E activity during infection, suggesting they target JAK-STAT pathway activation as a virulence strategy. Furthermore, two wasp species (Leptopilina guineaensis and Ganaspis xanthopoda) suppress phenotypes associated with a gain-of-function mutation in hopscotch, the D. melanogaster JAK ortholog, indicating that they inhibit the activity of the core signaling components of the JAK-STAT pathway. Our data suggest that parasitoid wasp virulence factors block JAK-STAT signaling to overcome fly immune defenses.