The Physiological Functions And The Purification Of Active Components Of Venom From Pteromalus Puparum (Hymenoptera: Ptermalidae)

The physiological changes of host hemocytes induced by the venom of Pteromalus puparum were be studied further on the basis of the physiological functions and biochemistry characters of venom from Pteromalus puparum. We design the methods of partially and completely isolation of the factors of inhibiting the spreading and encapsulation capability of cells. The results were summarized as follows:1 Effects of venom on the spreading and encapsulation capability of hemocytes from the different instars Pieris rapaeThe immune response of hosts included cellular and humoral immunity. The venom influenced the spreading of cells of hosts differently with treatment of different instars and no humoral or not. The difference was significant at 0.5 h. It suggested that the interaction effects of venom, cellular and humoral immunity were significant. They had accumulation effect. In contrast, they were no significant at 4 h. The phenomena indicated that there was no accumulation effect. It could be associated with the characteristics of cell spreading. Three factors, venom, different instars of hosts and humoral of hosts, had no combined effect on cell encapsulation. It might be associated with encapsulation, complicated affected by multi-factors.2 Effects of venom on the apoptosis, mortality and viability of hemocytes from Pieris rapaeCells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface after initiating early apoptosis. PS was tested whether the apoptosis, mortality and viability of hemocytes from different instars of Pieris rapae were affected by venom. The results showed the variations of the apoptosis, mortality and viability of hemocytes after the treatment of venom were unstable in non-parasitized higher instar larvae and prepupa. The late instar larvae apoptosis rate reduced sometimes, but the viability of cells could not increase. The mortality of cells increased or unchanged. Compared with controls, there were no difference between control group and treatment. The apoptosis of cells of prepupa was invariant or decreased sometimes. The viability of cells from prepupa increased or unchanged sometime. There was no effect on the mortality of cells. The apoptosis rate of cells of pupa of 12 h and 24 h decreased after the treatment of venom. There were significantly difference between control and treatment. The viability of cells increased. There were significantly difference between control and treatment. It indicated that venom significantly inhibited the cell apoptosis. It was favor of survival of parasitoid wasps.3 Effects of venom on the granularity and size of hemocytes from Pieris rapaeThe granularity and size of the cells was analyzed by flow cytometry. We investigated the volume and granularity of hemocytes from the different instars cabbage butterfly after venom treatment. The results showed that the cell granularity of late instars larvae and prepupa after treatment of venom for 1 h increased. There were significantly difference between control and treatment. Meanwhile, the cell size was unchanged. There were no difference between control and treatment. The cell granularity of pupation of 12 h after venom treatment for 0.5 h decreased. There were significantly difference between control and treatment. The cell size of pupation of 12 h after venom treatment for 1 h and the cell volume of pupation of 24 h after venom treatment for 4 h increased. There were significantly difference between control and treatment. The changes of cell size and granularity were different from the apoptotic cells. It suggested that the cells from different instars P. rape may be no apoptosis or decreased apoptosis rate indirectly.4 Effects of partial purification of venom on the spreading and encapsulation capability of hemocytes from Pieris rapae The parasite wasps injected the venoms before oviposition. The venom inhibited the host immune reaction. The wasps parasitize the hosts successfully. The eggs develop maturation. Venom is the important factor of suppressing the host immune defense. The venom from Pteromalus puparum inhibited the cell spreading and encapsulation. The venom is abundant in species and quantity. However, it was known little that which components were active. We designed a few methods to partially purify the venom proteins so as to find the active components. The results showed that the activity of partial purification of venom increased significantly. We found that the protein with molecular weight of 98 kDa by Native-PAGE. Meanwhile, we found the protein with molecular weight of 24 kDa with SDS-PAGE. The protein may be a potential active factor of venom.5 Isolation of venom and effect of purification on the spreading and encapsulation capability of hemocytes from Pieris rapaeIn hymenopteran parasioits devoid of symbiotic viruses, venom proteins appear to play a major role in host immune suppression and host regulation. Not much is known about the active components of venom proteins in these parasitoids, especially those that have the functions involved in the suppression of host cellular immunity. Here, it was reported that the isolation and characterization of a venom protein Vn. 11 with 24.1 kDa in size from Pteromalus puparum, a pupa-specific endoparasitoid of Pieris rapae. The Vn.11 venom protein is isolated with the combination of ammonium sulfate precipitation and anion exchange chromatography, and its purity is verified using SDS-PAGE analysis. Like crude venom, the Vn.11 venom protein significantly inhibits the spreading behavior and encapsulation ability of host hemocytes in vitro. It is suggested that this protein is an actual component of P.puparum crude venom as host cellular-immune suppressive factor.