Noxious Components of Venom from the Pupa-Specific ParasitoidPimpla hypochondriaca

Abstract

Venom from the endoparasitoid waspPimpla hypochondriacainduced a lethal paralysis when injected into the hemocoel of larval, pupal, and adult stages of the tomato mothLacanobia oleraceaand was also toxic to the adult stages of the houseflyMusca domesticaand the cockroachBlatella germanica.Heating the venom to 58°C did not reduce its potency againstM. domestica.Phenoloxidase activity was detected in diluted venom using dihydroxyphenylalanine as the substrate and occurred independently of activators. Enzyme activity was strongly inhibited by 2.0 μM phenylthiocarbamide (PTC), a known inhibitor of phenoloxidase, and was not detectable in venom that had been heated to 58°C for 10 min. Venom treated to remove phenoloxidase was shown to inhibit phenoloxidase activity in untreated venom, with a potency likely to be sufficient to prevent enzyme activity in the parasitoid's venom sac. The venom was found to be cytotoxic to an insect cell line (SF21) fromSpodoptera frugiperda,and this activity was not reduced when the cells were cultured in the presence of 20 μM PTC. Venom constituents were separated according to size using high-resolution gel filtration. Three venom components: phenoloxidase, a factor which caused paralysis ofM. domestica,and a factor which was cytotoxic to SF 21 cells were detected and found to have maximum activity in fractions 19, 40–42, and 50–51, respectively. Phenoloxidase and the factor causing paralysis ofM. domesticahad estimated apparent molecular masses of 390 and 27 kDa, respectively, while the cytotoxic factor had an apparent molecular mass of less than 13 kDa. Assay for inhibitors of venom phenoloxidase identified two major inhibitory components having highest activity in fractions 48 and 60, respectively. The larger of these two factors had an estimated apparent molecular mass of 12 kDa, but the other factor was considerably smaller than the lowest molecular mass standard (13.7 kDa) used to calibrate the column.