The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca (hymenoptera: ichneumonidae) involves Ca+2-dependent cell death pathways

The endoparasitoid Pimpla hypochondriaca injects venom during oviposition to condition its lepidopteran hosts. Venom is a complex mixture of proteins and polypeptides, many of which have been identified as enzymes, including phenoloxidase, endopeptidase, aminopeptidase, hydrolase, and angiotensin-converting enzyme. Constituents of the venom have been shown to possess cytolytic and paralytic activity, but the modes of action of factor(s) responsible for exerting such effects have not been deciphered. In this study, we examined the mode of action of isolated venom using cultured cells (BTI-TN-5B1-4). A series of blockage and inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti-calreticulin antibodies. Monolayers exposed to venom alone were highly susceptible with more than 84.6+/-2.3% dead within 15 min. Susceptible cells displayed a retraction of cytoplasmic extensions, rounding, and swelling prior to lysis in more than half (55.7+/-1.7%) of the dying cells. Within 15 min of exposure to venom, cells displayed qualitative increases in [Ca(+2)](i) as evidenced by staining with the calcium-sensitive probe fluo-4 AM, and mitochondrial membrane potential (DeltaPsi(m)) was undetectable by 5 min post-treatment with venom. These venom-mediated changes occurred regardless of whether an external source of calcium was present, or whether venom was pre-treated with PTU. In contrast, venom toxicity was attenuated by treatment with anti-calreticulin antibodies. Not only did fewer cells die when exposed to antibody-treated venom but also cell swelling diminished and no increases in intracellular calcium were detected. A possible mode of action for the venom is discussed.