| Parasitoid wasps,a kind of important biological control resources,include many members.To resist the host immune response,parasitoid wasps have evolved a variety of venom genes encoding immune regulatory factors,which target host cellular and humoral immune pathways and have diverse functions,such as interrupting host signal transduction of immune cells,inducing immune cell death and so on.Pachycrepoideus vindemiae,a pupal ectoparasitoid,has the capacity to parasitize a variety of flies including Drosophila melanogaster.How the venom,sole parasitic factor of this wasp,regulates host immunity has not been fully understood.Thus,this thesis uses the P.vindemiae and D.melanogaster as model to explore the mechanism of venom on host immune regulation and clarify the spatio-temporal expression dynamics of these components,and ultimately explore the function of serpin.1 The effects of P.vindemiae venom on host innate immunityThis chapter explored the effect of P.vindemiae venom on cellular and humoral immunity of D.melanogaster.In vitro experiments showed that the venom significantly enhanced the phagocytosis,but inhibited the spreading of host blood cells.The coincubation of gene expression products of C-type lectin,an protein facilitating host encapsulation,beads and crude venoms,showed that low doses of venom reduced the encapsulation rate of host hemocytes.In addition,venom induced the high expression of host apoptosis-related genes.It was also indicated that the downstream effector genes of Toll,IMD and JAK/STAT pathways significantly up-regulated after parasitism,demonstrating that the three immune pathways were activated even in the early stage of parasitism.2 Analysis of the expression pattern of P.vindemiae venom genesBased on the expression data of P.vindemiae venom components in both venom glands and carcasses,a clustered heat map was drawn.The transcription levels of 64 venom genes in venom glands was significantly higher than those in carcasses.Twentyone genes were selected and their tissue specificity(head,thorax,ovary,carcass,venom apparatus,female)expression was verified by RT-qPCR,showing that most genes were specifically and highly expressed in venom apparatus.The transcriptional levels of these genes after 1-7 day eclosion were also detected,and results showed that their transcription levels were higher at 3rd and 7th days.3 Functional analysis of the venom protein Serpin in P.vindemiaeA serine protease inhibitor PvSPN was identified.Sequence alignment and phylogenetic analysis indicated it is embedded with a serpin domain highly conserved in hymenopteran insects.RT-qPCR results indicated that the expression levels of PvSPN are not different between venom apparatus and other tissues,and there was a higher transcription level after 4-6 days eclosion.Prokaryotic expression of PvSPN manifested that 0.01 mg/mL recombinant proteins significantly inhibited the host melanization.The subsequent determination of phenoloxidase activity verified the above results,indicating that PvSPN plays an important role in disrupting the host melanization cascade of host hemolymph.In conclusion,this study clarified that the venom of P.vindemiae enhanced the host’s blood cell phagocytosis,inhibited the spreading and encapsulation of blood cells.The venom activated the host Toll,IMD and JAK/STAT immune pathways.RT-qPCR analyzed the temporal and spatial expression patterns of 21 venom genes.It was identified that a venom protein PvSPN can inhibit the melanization of host hemolymph. |
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