Components And Functions Of Cotesia Chilonis Venom And Ovarian Proteins

The striped stem borer,Chilo suppressalis(Walker)(Lepidoptera:Crambidae)is one of the most economically important rice pests.Cotesia chilonis(Matsumura)(Hymenoptera:Braconidae),an obligate larval endoparasitoid that effectively regulates C.suppressalis populations,is a potential biological control agent.To facilitate the survive and development of progenies in the insect hosts successfully,C.chilonis injects parasitism factors into C.suppressalis with eggs to regulate host physiology.The parasitism factors includes polydnavirus(PDV),venom,calyx proteins and teratocytes.To study the components and functions of parasitism factors could lead us to better understandings in host-parasitoid molecular relationships and discoveries of new chemistries,new proteins and new genes,some of which may become products useful in agriculture,pharmacology and many other areas.In this thesis,the venom and ovary composition of C,chilonis are investigated,and the functions of some related proteins from venom and ovary are explored.1 Effects of C.chilonis parasitism on host C.suppressalis immune responsesWe studied the effects of C.chilonis parasitism,venom,and calyx fluid on cellular and humoral immunity of its host C.suppressalis larvae.Total hemocyte counts were higher in parasitized larvae than in unparasitized larvae in the late stages following parasitization.While both plasmatocyte and granulocyte fractions and hemocyte mortality did not differ between parasitized and unparasitized hosts,in vitro spreading behavior of hemocytes was inhibited significantly by parasitism throughout the course of parasitoid development.C.chilonis parasitism suppressed the encapsulation response and melanization in the early stages.Venom alone did not alter cellular immune responses,including effects on total hemocyte counts,mortality,hemocyte compositioncell spreading and encapsulation,but venom did inhibit humoral immunity by reducing melanization within 6 h after injection.In contrast to venom,calyx fluid had a significant effect on cell spreading,encapsulation and melanization from 6 h after injection.Dose—response injection studies indicated the effects of venom and calyx fluid synergized,showing a stronger and more persistent reduction in immune system responses than the effect of either injected alone.2 Combined transcriptomic and proteomic analyses of C.chilonis venomWe used the combined transcriptomic and proteomic approaches to identify 37 possible venom proteins from the PDV-carrying endoparasitoid C.chilonis.The most abundant proteins were hydrolases,such as proteases,peptidases,esterases,glycosyl hydrolase,and endonucleases.Some components are classical parasitoid venom proteins with known functions,including extracellular superoxide dismutase 3,serine protease inhibitor(serpin)and calreticulin(CRT).The venom contains novel proteins,not recorded from any other parasitoid species,including tolloid-like proteins,chitooligosaccharidolytic β-N-acetylglucosaminidase(NAG),FK506-binding protein 14,corticotropin-releasing factor-binding protein and vascular endothelial growth factor receptor 2.3 Analysis of two venom proteins from C.chilonisTo ensure successful parasitism,endoparasitoids inject venom into host to suppress the host immune responses.Serpin and Calreticulin have been reported to inhibit the host melanization.We cloned Serpin(CcSPN)and Calreticulin(CcCRT)venom proteins from C.chilonis.The complete opening reading frame(ORF)of CcSPN is 1221 nucleotides long encoding a protein of 406 amino acids,which includes a predicted signal peptide and a Serpin domain.Western blot showed that CcSPN would be expressed almost exclusively in the venom apparatus.GFP-CcSPN inhibited host melanization in vitro in a dose-dependent manner.The ORF of CcCRT is 1212 nucleotides long encoding a protein of 403 amino acids,which includes a predicted signal peptide and conserved CRT family signatures.Western blot indicated that CcCRT was detected in all the tissues tested.Recombinant CcCRT could not inhibit host hemolymph melanization and hemocyte spreading.However,it could enter host granulocytes.The function of CcCRT need to be further studied.4 Combined transcriptomic and proteomic analyses of ovarian fluids from C.chilonisThe ovarian fluids were injected into the host with endoparasitoid eggs.We analyzed the ovarian fluids from C.chilonis by combining transcriptomic and proteomic approaches.1,565 genes were upregulated and 1682 genes were downregulated compared to the FPOvary.Up regulated genes in FPOvary were enriched in the pathways related to ovarian development,oogenesis and immune regulation.The most abundant proteins were enzymes.Some components are similar to venom,such as serine protease,Tolloid-like protein 2,aminopeptidase/carboxypeptidase,esterase,NAG,disulfide-isomerase and serpin.Some ovarian components were identified in other parasitoid venoms instead of C.chilonis,including Neprilysin,venom acid phosphatase,aspartylglucosaminidase,chitinase,conotoxin and odorant-binding protein.Other proteins were not recorded from any other parasitoid venoms,including kynurenine-oxoglutarate transaminase,diphosphomevalonate decarboxylase,and protein yellow.Here,the only one ovarian protein reproted to be involved in successful parasitism is immunoevasive protein(IEP).5 Analysis of Crp32-like ovarian proteins from C.chilonisTo develop successfully in the host hemocoel,endoparasitoids have evolved diverse strategies in different parasitoid-host systems,which include active and passive strategies.We identified three homologs of IEP:IEP-1,IEP-2A and IEP-2B.We also found three homologs of Crp32:Cip32A,Crp32B and Crp32C.qPCR results showed that IEP-1,IEP-2A and Crp32A-C transcripts were mainly expressed in the ovary,while IEP-2B transcript was mainly expressed in the venom gland.A time-course analysis showed that IEP-2A,Crp32A and Crp32B mRNA levels reached a peak at day 4 post-pupation.IEP-1 mRNA level reached a peak at day 1 post-eclosion and IEP-2B,Crp32C mRNA levels reached a peak at day 3 post-pupation.Western blot indicated that Crp32B protein was only detected in the ovary of female adult and was increased in ovary staring at day 3 post-pupation,reached a peak at day 2 post-eclosion.RNA-FISH indicated that Crp32B was transcripted in all parts of the ovary,especially in the calyx.Immunohistochemistry analysis revealed Crp32B was localized in all parts of the ovary and on egg surface.Moreover,encapsulation of rCrp32B-covered resin beads decreased significantly in a dose-dependent manner.