Components Of Pachycrepoideus Vindemiae Venoms And Its Regulations On Host Immune Responses

P.vindemiae(Hymenoptera:Pteromalidae)is a versatile and solitary pupal ectoparasitoid of many flies whose hosts range from Drosophilidae to Anthomyiidae,Calliphoridae,Muscidae,Sarcophagidae,Tachinidae,Tephritidae,and so on.As an important biological control organism for flies,it has been widely used on the control of D.suzukii.It injected venoms into the host while laying eggs that regulated the cellular and humoral immune responses of host Drosophila,mainly involving the changes of hemocyte numbers,disabilities in hemocyte spreading and adhesion in cellular aspects and the regulations in melanization of host hemolymph,Toll,Imd as well as Jak/Stat immune pathways in humoral aspects.Regarding that the immunology of D.melanogaster has been well explored,and to reveal the molecular mechanism of immune interactions between P.vindemiae and its host,it was used as a model to study the regulatory mechanism of P.vindemiae venoms on host immune responses.Besides,the identification and comparative analysis of P.vindemiae venom proteins were conducted.The functional characterization of three proteins,PvCRT,PvKazal and PvUn,as well as several unknown venoms were also revealed.It is hoped that our present study will provide a new insight into the biological control of pests using parasitic natural enemies.1 P.vindemiae regulates the cellular and humoral immunity of host D.melanogasterTo reveal the effects of P.vindemiae parasitism on host Drosophila cellular and humoral immune responses,the cell numbers,cell adherence,cell viability,hemolymph melanization and Toll,Imd,Jak/Stat immune pathways were thoroughly investigated in this chapter.The results strongly supported that parasitism significantly reduced the number of blood cells.In detail,venom had a significant inhibitory effect on lamellocyte adherence and induced plasmatocyte death.The determination of PO activity based on venom injection and in vitro incubation with host hemolymph showed that P.vindemiae venoms remarkably inhibited the melanization of host hemolymph in dose dependent manners.More in-depth investigation revealed that the Toll,Imd and Jak/Stat immune pathways were activated upon 1 h,6 h,12 h,24 h and 72 h parasitization.Additionally,by comparing the parasitic capabilities of P.vindemiae on different Drosophila lines,no significant differences were observed between the loss-of-function,gain-of-function of Toll,Imd,Jak/Stat immune pathways Drosophila and the wild type.2 Identification and comparative analysis of venom proteins in P.vindemiaeA combination of transcriptomic and proteomic methods was used to identify 64 putative venom proteins in P.vindemiae,including 37“knowns”and 27“unknowns”.The functional categories of the 37“knowns”fell into hydrolases,oxidoreductases,transferases,protease inhibitors,recognition and binding proteins,and others.Of the 37 categories,22 hydrolases occupied the majority.Expression analysis revealed that 20 tested venom proteins had 419-fold higher mean expression in the venom apparatus than in carcass,indicating their specialization to venom.Comparisons of venom proteins between P.vindemiae and other five species detected a core set of“ancient”orthologs in Pteromalidae.35 venom proteins of P.vindemiae were assigned to the orthologous groups by reciprocal best matches with venoms of other pteromalids,while the remaining 29 were not.Of the 35 categories,27 and 25 had orthologous relationships with N.vitripennis and P.puparum venom proteins,respectively.More distant relationships detected that 5 and 2 venom proteins of P.vindemiae were orthologous with venoms of two Figitidae parasitoids and a Braconidae representative,respectively.Moreover,22 venoms unique to P.vindemiae were also detected.The correlation analysis between the number of venom proteins and parasitic characteristics in 11 parasitoids indicated that solitary wasps had evolved more venom components compared to gregarious wasps during the co-evolution with their hosts.Further phylogenetic reconstruction based on a set of single-copy genes clustered P.vindemiae with P.puparum,N.vitripennis,and other members of the family Pteromalidae.3 A venom protein PvCRT regulates the host cellular immune responseHere,a protein calreticulin was identified in P.vindemiae venoms(PvCRT).PvCRT features a signal peptide and two conserved“Calreticulin”domains.Multiple sequence alignments showed that PvCRT shared 83.54%amino acid identities with CRTs from both P.puparum and N.vitripennis,further phylogenetic analysis revealed a close relationship among these three species.qPCR analysis detected a lower expression level of PvCRT(0.27-fold)in the venom apparatus compared to carcass,indicating its non-specific as a venom protein.Immunohistochemical localization revealed that PvCRT was ubiquitously expressed in venom glands.The transgenic Drosophila integrated with PvCRT was constructed and its heterologous expression in host immune tissues using UAS/Gal4 binary expression system reduced the self-encapsulation phenotype of tu(1)Sz~1 mutants.Additionally,both the quantification on antimicrobial peptides and survival experiments in hosts challenged by P.aeruginosa or S.aureus indicated that PvCRT did not act in the antimicrobial immune responses of host Drosophila.Taken together,PvCRT inhibited the host encapsulation.4 Venom proteins PvKazal and PvUn regulates the host humoral immune responseTwo proteins,PvKazal and PvUn,were identified from P.vindemiae venom components,acting in inhibiting the melanization of host hemolymph.qPCR analysis recorded a relatively higher transcription level of PvKazal in venom apparatus compared to carcass.A dramatic decrease in host PO activity was observed after its hemolymph was incubated with 0.01-1mg/ml recombinant PvKazal proteins.The transgenic Drosophila expressing PvKazal was constructed,and fewer crystal cells were detected in the larvae of PvKazal transgenic line compared to control.Similarly,the PO enzymatic activity in pupal transgenic Drosophila was relatively low.Besides,an unknown venom protein,PvUn,was also characterized,and it showed a 2600-fold higher expression in venom apparatus than carcass.PvUn played a role in suppressing the melanization of host hemolymph analogous to PvKazal.To clarify the venom proteins regulating the immune pathways of host Drosophila,a dual-luciferase reporter system was constructed and many candidates that activated the host Toll,Jak/Stat pathways,while suppressed the host Imd pathway were identified.All together,both the PvKazal and PvUn inhibited the melanization of host hemolymph,and several unknown venom components played vital roles in regulating the host Toll,Imd and Jak/Stat immune pathways.In conclusion,this study clarified that P.vindemiae venom led to decreases in hemocyte numbers,plasmatocyte death,disability in lamellocyte adherence,and inhibited the melanization of host hemolymph,while activated the host Toll,Imd and Jak/Stat immune pathways.A combinated transcriptomic and proteomic analyses identified 64 putative venom proteins in P.vindemiae,including 37“knowns”and 27“unknowns”.Comparisons of venom proteins between P.vindemiae and other five species detected a core set of orthologs in Pteromalidae.Functional studies on three venom proteins showed that PvCRT inhibited the host encapsulation,and PvKazal,PvUn suppressed the melanization of host hemolymph.Additionally,several venom components that activated the host Toll,Jak/Stat pathways,and inhibited the host Imd pathway were also revealed.