Venom vesicles from the parasitoidGanaspis hookerifacilitate venom protein entry into host immune cells

The parasitoid waspGanaspis hookeriinfectsDrosophila melanogasterlarvae, 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 inG. hookeriis a venom- specific isoform of the SERCA (<underline>S</underline>arco/<underline>e</underline>ndoplasmic<underline>r</underline>eticulum<underline>C</underline>a²⁺-<underline>A</underline>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 ofG. hookerivenom. 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 suggestG. hookerivenom 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 whichG. hookerivenom proteins, including SERCA, are packaged into an array of venom vesicles, and transported into host cells for immune suppression.