Mechanisms That Polyembryonic Parasitoids Microcentrus Cingulum Resist The Immune Reaction Of Host Ostrinia Furnacalis

Polyembryonic wasp, Microcentrus cigululm may parasitize the larvae of Ostrinia furnacalis successfully. As one of the important factors that parasitoids avoid the immune system of host, materials in wasps egg surface play an important role in protecting parasitoids from host's immune reaction. This paper includes researches on physiology, biochemistry and molecular biology in the fields of hemocyte immune reactions of Ostrinia furnacalis larvae, effects of parasitization by Microcentrus cigulum on the immune reaction of host's hemocytes and strategies that wasps avoid host's immune system. The results are as follows: 1. Using phase contrast microscopy and electronic transmission microscopy, five types of hemocytes were determined in the hemolymph of Ostrinia furnacalis larvae, namely Granular hemocytes, Plasmatocytes, Oenocytoids, Prohemocytes and Spherule cells. Total hemocyte counts (THC) and differential hemocyte counts (DHC) were compared in the hemolymph of the larvae. From the late stage of third instar till fifth day of fifth instar, it is interesting to note that THC increased shortly before and after ecdysis and thereafter decreased to a basal level. During the first day to the fifth day of the fifth instar, the amount of plasmatocytes increased before the third day and decreased after that but granular hemocytes showed a reversed profile. In vitro test both plastomatocytes and granularcytes showed spreading and adhesive behaviour but their spreading abilities were different. When Sephadex A-25 beads were injected into the fifth-instarlarvae they were soon encapsulated and some of them were melanized. Hemocytes were also able to encapsulate the latex beads in vitro test. No difference of the capsule structure could be seen.2. Comparing hemocyte counts ( THC and DHC), morphologies, spreading behavior, encapsulation on Sephadex A-25 and melanization of capsules of hemocytes in parasitized host larvae and normal larvae, no distinct differences were observed. Therefore, parasitization of Macrocentrus cingulum has no significant effect on the immune reaction of the hemcoytes of host, Ostrinia furnacalis larvae.3. Immature egg excised from ovariole of the female wasp has a sheath of follicular epithelium but mature egg from lateral oviducts does not have it. Injected M. cingulum mature eggs were not encapsulated, while immature eggs and Driselase treated eggs provoked an encapsulation response. Inspection of eggs by transmission electron microscopy revealed that there is a fibrous layer on the surface of mature eggs and Driselase collapsed the surface fibrous layer of the eggs, indicating that surface fibrous layer may play a role in protecting eggs from host's immune attack. Egg, embryonic and larvae of M. cingulum can be labelled by FITC-lectin, but labelling on larvae was weak. A 97kDa protein in eggs and embryonics can be conjugated with FITC-lectin, speculating this 97kDa protein play an important role in wasp resisting host's immune reaction.4. A cDNA library of Macrocentrus cingulum embryos, excised from their host Ostrinia furnacalis 6 days after parasitism has been constructed. After synthesizing the first-strand cDNA using limited amount of mRNA isolated from total RNA, the double-strand cDNA was amplified with long-distance PCR method. Then the ligation of the Sfi I digested cDNA to the Sfi I digested vector λTripIEX2 was perpormed and the phage was packaged to construct the full longth cDNA library. It was determined that the titer of unamplified library was 0.75(106 pfu?ml-1 and thepercentage of recombinant clones was 96.77% and the titer of amplified library was 0.5(1010 pfu?ml-1 by plating E.coli XL1- Blue. 5. 97 kDa protein combined with other proteins extracted from embryo was run SDS-polyacrylamide electrophoresis and 97kDa protein was cut from gel to immunize BALB/c mice directly. ELISA and Dot-blotting were used to determine the titer of the above prepared antisera. Using polyclonal antibody as immune probe to screen cDNA libaray, a positive plaque was pi...