Immunosuppression Of Octodonta Nipae Encapsulation By Two Venom Proteins,neprilysin-like And 4-coumarate:CoA Ligase 4,from Tetrastichus Brontispae

The endoparasitoid Tetrastichus brontispae Ferriere(Hymenoptera:Eulophidae),a species devoid of polydnavirus,is a gregarious pupal endoparasitoid.Its poteintial effectiveness as a natural enemy of an invasive beetle,Octodonta nipae(Maulik),has been reported.During the antagonistic evolution between endoparasitoids and hosts,endoparasitoids have evolved original strategiest to alter the host immunity and/or manipulate its development and physiology.In endoparasitoids devoid of polydnavirus,venoms especially those with the most abundance play a key role in the virulence toward their hosts.Therefore,in the present study,we mainly focus on the immunosuppression of host encapsulation by two abundant venom protein,neprilysin-like(TbNEP)and 4-coumarate:CoA ligase 4(Tb4CL4)from T.brontispae.The main results were summarized as follows:1.TbNEP and Tb4CL4 encoded 722 and 576 amino acids,respectively.Sequence alignment showed that TbNEP is a soluble member of the neprilysin family,also containing a zinc binding motif(HEXXH)and two catalytical motifs(GENID and VNAFY)but with substitutions for the two E residues by Q and S,respectively.Tb4CL4,belonging to adenylate forming domain(AFD)Class Ⅰ superfamily,is an enzyme that catalyzes the activation of 4-coumarate and related substrates to the respective CoA esters,and also harbors three conserved catalytic residues Lys-Gln-Lys.The residues essential for substrate binding located between the putative AMP binding domain(Box Ⅰ)and the GEICIRG domain(Box Ⅱ).It is worth noting that most of the conserved residues for substrate binding in parasitoids were replaced compared with that of plants.2.Quantitative real time PCR results showed that TbNEP and Tb4CL4 indeed presented dramatically higher transcript levels in the venom apparatus compared with that in male parasitoid and other female tissues,such as head,ovary and abdomen.3.No peptidase and ligase activities were found for the recombinant TbNEP and Tb4CL4,respectively,by enzyme assay.This may be because of the substitution of key residues in the conserved motif.Encapsulation assay showed that TbNEP and Tb4CL4 could inhibit the host encapsulation,with encapsulation index and rate decreasing significantly 24 hours after injection of the recombinant TbNEP and Tb4CL4 to O.nipae pupae.The inhibition of host hemocyte-spreading by measuring F-actin contents was also observed 24 hours after injection of TbNEP and Tb4CL4,which indicated that the two venom protein could suppress the host encapsulation by the inhibition of hemocyte spreading ability.In conclusion,our results revealed that the two most abundant venom protein TbNEP and Tb4CL4 had an adverse effect on host encapsulation,which may be involved in the successful paratistim on O.nipae.The present study provides a knowledge of the interaction between T.brontispae and O.nipae,which enables us to utilize the venom for management of O.nipae in the future.