Regulation Of Heliothis Virescens Ascovirus 3h On The Melanization Of Spodoptera Exigua

Melanization is an important immune defense mechanism in insect hemolymph,which can kill and eliminate the invading pathogenic microorganisms and parasites.Melanization is mainly mediated by serine protease(SP)cascades and is strictly regulated by serine protease inhibitors(serpins).Ascoviruses are a kind of insect-specific viruses with biological control potential.They firstly invade into the hemolymph of host larvae through the oviposition behavior of parasitoids,and can significantly inhibit the growth and development of host.The specific interactions between virus and immune response mechanism of host may influence the survival and reproduction of virus.Therefore,in this study,Heliothis virescens ascovirus 3h(Hv AV-3h)and its host Spodoptera exigua larvae were as the main research objects.Through the means of molecular biology,aiming at the melanization of hemolymph and the effect of negative regulatory factor serpin of S.exigua larvae,the influence of the infection of ascoviruses on the melanization which was an important immune response in host S.exigua larvae was explored.The main research results are as follows:1.Infection of Hv AV-3h promoted the melanization of S.exigua larval hemolymph.After infected with Hv AV-3h,the speed of melanization in infected S.exigua larval hemolymph was accelerated,phenoloxidase(PO)activity of hemolymph in larvae infected with Hv AV-3h increased significantly.And,the transcription level of gene encoding Spodoptera exigua prophenoloxidase-1(Se PPO-1)was upregulated dramatically in fat body during the middle stage of infection.2.Melanization is beneficial to the replication of Hv AV-3h.Melanization in hemolymph of S.exigua larvae or Sf9 cells was inhibited or promoted,then Hv AV-3h was inoculated,and the replication of the virus was detected by quantitative PCR.The results showed that within the first 48 hours post infection,whether in the hemolymph of larvae(in vivo)or in Sf9 cells(in vitro),the melanization was inhibited or promoted,correspondingly,the replication of virus was also inhibited or promoted.3.Screening and bioinformatics analysis of S.exigua serpin involved in immune response induced by Hv AV-3h infection.Based on the transcriptome data and immune stimulation of different pathogens,screening and obtaining a serine protease inhibitor of S.exigua(Seserpin-2.1 like)with significantly increased transcription level under Hv AV-3h infection.The open reading frame(ORF)of Seserpin-2.1 like contains 1218 pb and encodes405 amino acid residues protein with a predicted molecular weight of 45.37 k Da.Phylogenetic analysis and sequence alignment displayed the Seserpin-2.1 like sequence was similar to serpin 2.1 like from Spodoptera litura.There were a typical SERPIN domain and the RCL which are conserved signatures of the serpin superfamily in sequence of Se Serpin-2.1 like.Therefore,we speculate that it is a serpin protein with inhibitory activity.4.Prokaryotic expression and polyclonal antibody preparation of Seserpin-2.1 like.The ORF of Seserpin-2.1 like gene was cloned into expression vector p ET-32a(+).The recombinant Seserpin-2.1 like protein was expressed in the prokaryotic expression system(Escherichia coli),and the rabbit anti-Seserpin-2.1 like polyclonal antibody was prepared with the purified protein.5.Tissue distribution and expression patterns of Seserpin-2.1 like in different developmental stages of S.exigua.The results indicated that Seserpin-2.1 like was expressed in all tested tissues(including hemolymph,fat body,midgut,cuticle and head),and expressed at a higher level in fat body,hemolymph and head.In terms of developmental stages,the expression of it was found in all developmental stages.The higher expression level of it like was detected in 5th instar larvae and eggs with the lowest expression in 1st instar larvae.6.Influence of Hv AV-3h infection on the expression of Seserpin-2.1 like protein in S.exigua larval hemolymph.Western blot analysis showed that Seserpin-2.1 like protein can express normally in hemolymph of mock-infected larvae.And,the expression of it in Hv AV-3h-infected larval hemolymph could be detected at 0,24 and48 hpi,but could not be detected at 72,96 and 120 hpi.In this study,we first confirmed that the infection of Hv AV-3h would stimulate the occurrence of the host hemolymph melanization,and the melanization is beneficial to the replication of Hv AV-3h.In addition,we also obtained a S.exigua serpin(Seserpin-2.1 like)which transcription level in hemolymph was significantly upregulated,but its expression decreased or even disappeared after infection.The above study provided basic information for the study of the interaction between ascoviruses and host insect melanization.