Molecular Characterization Of Venom From Pteromalus Puparum And Molecular Mechanism Of This Parasitoid Manipulation To Its Host

Parasitoids associated diversified range of virulence parasitic factors such as venom,polydnaviruse(PDV),virus-like particle(VLP),and teratocyte appear to interfere with host'simmune response or to disrupt host's development to insure the successful development of theiroffspring in the hemocoel or at the external surface of host insects.Given the specialphysiological functions,these parasitic factors would be potentially valuable resources ofnatural substance in developing new environmentally safe insect control agents or transgeneticcrops with modem biotechnologies.As a consequence,parasitoid-host interaction is a focalresearch point in the entomological physiology and biochemistry and biological control area,which aims to explore the nature of the parasitic factors and the mechanisms of parasitoidmanipulation of host that helpful to gain insights into the potential properties of the parasiticfactors for future practical use.Up to date,the biochemical and molecular basis of PDV andVLP have been well documented.The genome of PDV from near 10 parasitoids has beencompletely sequenced and lots of gene resources from them are available.Howeverover,onlylittle is known so far on the biochemical and molecular basis of venom.Regarding to themechanisms of parasitoid-host interactions,mostly investigations carried out at thephysiological and biochemical levels,but molecular dissection of parasitoid-host interactions isstill little known.In addition,in terms of parasitoid-host systems,the investigated parasitoids aremostly limited to ichneumonid and braconid,and are of egg-larval or larval stage specificparasitism.By consideration the historical perspective on previous issues on parasitoid-hostinteractions and on the basis of detailed biochemical characters and physiological functions ofvenom from Pteromalus puparum(Hymenoptera:Pteromalidae)(no other parasitoid associatedfactors other than venom are found in the female reproductive organ),a predominate pupalendoparasitoid of Pieris rapae(Lepidoptera:Pieridae),been well investigated in our laboratory,we here report on the outcomes of molecular characterization of venom from P.puparum andmolecular mechanism of this parasitoid manipulation to the host.1 Molecular characterization of venom from P.puparum(1)Morphology and ultrastructure of P.puparum venom apparatus.The venomapparatus of P.puparum was studied with light and electron microscope and was subjected tothe electrophoretic and immunohistochemical analyses.Typically its venom apparatus consistsof a venom gland and a venom reservoir,which is associated with a Dufour gland.The venomgland consists of four layers of basement membrane layer,secretory cell layer,duct cell layer and inner intima layer.Basement membrane and inner intima layers have evenly thickenedorganization.Secretory cells in the venom gland are characterized by extensive roughendoplasmic reticulum and numerous secretory vesicles,which is associated with an endapparatus to collect its secretions into the venom gland lumen.They also exhibit severalsecretory granules and vacuoles.Duct cells in the venom gland lack numerous cellularorganelles except secretory vesicles and their nucleus becomes smaller as compared to thenucleus in the secretory cell.The venom reservoir presents three distinct regions:an externallayer,composed by numerous fine muscle fibers;an internal layer,represented by epithelial cellwith large nucleus;and an intima portion,represented by thin and uniform organization.Theelectrophoretic and immunohistochemical results reveal that the rich proteinaceous componentsare present in the venom gland as that in venom reservoir.The venom proteins are firstly mainlyproduced in the venom gland,then drained to the lumen through the end apparatus,and arefinally collected and stored in the venom reservoir.(2)Proteomic analysis of P.puparum venom.Two-dimensional gel electrophoresisclearly defined 55 spots showing a major protein region(25-66.2 kDa,pI 4-7)were observed.All the venom protein spots were analyzed by mass spectrometry.Four venom components wereidentified:calreticulin,arginine kinase,serine protease and heat shock protein 70.(3)cDNA library construction and EST analysis of P.puparum venom apparatus.Theoriginal titration of library was 1.6×10~6 cfu with recombinant rate of 90.65%.The averagelength of insert cDNA fragment was between 0.5 kb and 2.5 kb.265 ESTs from the cDNAlibrary were obtained.After annotation,the results revealed that only 72 ESTs showedsignificant similarity to known genes from other organisms,and the other had no match to anydatabase sequence,indicating that mostly venom genes of P.puparum are new genes.Thevenom acid phospoatase and serine protease genes were obtained.A few contigs with homologyto virus structural protein were observed.Its open reading frame(ORF)was 2619 bp encoding873 amino acid residues with estimated molecular weight of 97.6 kDa.Its mature protein waswith three major capsid polyproteins(28,31 and 28 kDa)and a minor one(9.6 kDa).Phylogenetic evolution analysis exhibited that this virus belonged to the Cripavirus genus ofpathogenetic dicistroviruse and fell into same clade with Black queen cell virus.(4)Cloning and sequence analysis of cDNA encoding venom calreticulin gene of P.puparum.The 1722 bp full-length sequence had an open reading frame of 1212 bp encoding aprotein of 404 amino acids.The calculated molecular mass of the mature protein was 42.57 kDawith an estimated pI of 4.31.Sequence comparison showed that this venom protein has anoverall similarity of 69% to venom calreticulin of Cotesia rubecula. (5)Cloning and expression of venom arginine kinase gene of P.puparum.Thecomplete cDNA consisted of 1828 bp and contained an open reading frame of 1068 bp thatencoded 356 amino acid residues and had a predicted molecular weight of 39.9 kDa and pI 8.89.Sequence comparison showed that this venom protein has a highly similarity of 92% to venomarginine kinase of Cyphononyx dorsalis.This gene was expressed as a fusion protein usingpET-32a vector in Escherichia coli.(6)Molecular cloning and characterization of acid phosphatase in venom of P.puparum.The cDNA consisted of 1378 bp with 1215 bp open reading frame and encoded asequence of 405 amino acids.By multiple sequence alignment,the deduced amino acidsequence shared 88%,41%,31%,30% and 29% identity to its counterparts from Nasoniavitripennis,Apis mellifera,Tribolium castaneum,Aedes aegypti and Drosophila melanogaster.Using p-nitrophenyl phosphate(p-NPP)as substrate,the optimal pH and temperature for thisenzyme activity was measured to be 4.8 and 45℃,respectively.And NaF treatment waseffective way to inhibit the activity if this venom enzyme.Ultracytochemical analyses furtherrevealed that strong enzyme activity was located in the nuclei and secretory vesicles of thevenom gland secretory cells.Expression of the acid phosphatase gene was observed to beregulated at different developmental stages by RT-PCR analysis.Compared to the mRNAexpression,a time-course related enzyme activity in an individual venom apparatus was alsofound.(7)Molecular cloning and characterization of alkaline phosphatase in venom of P.puparum.Using chromogenic substrates 5-bromo-4-chloro-3'-indolyl phosphate(BCIP)andnitro blue tetrazolium(NBT),alkaline phosphatase was histochemically detected in the venomapparatus of P.puparum.Ultrastructural observations also demonstrated its presence in thesecretory vesicles and nuclei of the venom gland secretory cells.Using p-nitrophenyl phosphate(p-NPP)as substrate to measure the enzyme activity,this venom alkaline phosphatase wasfound to be temperature dependent and with bivalent cations effects.The full-length eDNAsequence of alkaline phosphatase was 2645 bp with 1623 bp open reading frame covered 541deduced amino acids with predicted molecular mass of 59.83 kDa and pI of 6.98.By multiplesequence alignment,the deduced amino acid sequence shared 91%,66%,48%,48% and 46%identity to its counterparts from N.vitripennis,A.mellifera,T.castaneum,A.aegypti and D.melanogaster.The transcript of alkaline phosphatase gene was detected by RT-PCR in venomapparatus with development related expression after adult wasp emergence.Compared to themRNA expression,a time-course related enzyme activity in an individual venom apparatus wasalso found. 2 Molecular mechanism of P.puparum manipulation of P.rapae(1)Proteome changes in the plasma ofP.rapae induced by P.puparum parasitization.By examining the differential expression of plasma proteins in the parasitized and unparasitizedhosts pupae after 24 h by two-dimensional electrophoresis,7 proteins were found to vary inrelation to parasitization compared to unparasitized control samples.All of them were submittedto identification by mass spectrometry coupled with a database search.Six were identified asmasquerade-like serine proteinase homolog(MasSPH),enolase(ENO),imaginal disc growthfactor(IDGF),bilin-binding protein(BBP),ornithine decarboxylase(ODC)and cellular retinoicacid binding protein(CRABP).Next to known function proteins,one was identified asunannotated proteins.The modulated proteins were found to fall into the following functionalgroups:humoral or cellular immunity,detoxification,energy metabolism,and others.Degenerate primers designed based on peptides obtained from mass spectral analysis were usedfor PCR amplification of the fragment of the differential expressed proteins.And their fulllength eDNA were derived from RACE amplification.The gene of MasSPH,ENO and BBPwere cloned and sequenced.RT-PCR analysis indicated that parasitization by P.puparuminduced increased transcripts of MasSPH and BBP,and had no apparent impact on the level ofIDGF and ENO transcripts in the fat body of P.rapae pupae.(2)cDNA of an arylphorin-type storage protein from P.rapae with parasitisminducible expression by P.puparum.The cDNA of a storage protein,PraAry,from P.rapaewas cloned and its expression regulated by parasitization of P.puparum was investigated.Thefull-length cDNA of PraAry was 2270 nucleotides and contains a 2121 bp open reading frameencoding 707 amino acids with calculated molecular weights of approximately 83 kDa.Analysisof the primary protein sequence revealed that it possesses a signal peptide of 16 amino acids atthe N-terminus and contains two highly conserved storage protein signature motifs.Accordingto both phylogenetic analysis and the criteria for amino acid composition,PraAry belongs to thesubfamily of arylphorin-type storage protein(1.42% methionine and 18.82% aromatic aminoacids).RT-PCR analysis indicated that the transcriptional level of PraAry mRNA in P.rapaepupae fat body is inducible upregulation in response to parasitization by P.puparum.(3)Transcriptional changes of calreticulin from P.rapae after parasitization by P.puparum.The cDNA of calreticulin from P.rapae was cloned and its expression regulated byparasitization of P.puparum was investigated.Sequence analysis showed that P.rapaecalreticulin was of 1816 bp in full length contained an 1194 bp open reading frame whichpredicted a 398 amino acid protein.The calculated molecular mass and pI were 45.75 kDa and4.4,respectively.The deduced amino acid sequence contained N,P and C domains with special characters similar to that of other species,sharing 74%,70% and 69% homology with thecounterparts of A.mellifera,Anopheles gambiae and D.melanogaster.Expression analyses ofthe calreticulin gene in the haemocyte of P.rapae pupae parasitized and unparasitized by P.puparum were performed by RT-PCR,which indicated down regulated transcription underparasitization.(4)Transcriptional changes of cytoskeleton related proteins of P.rapae afterparasitization by the endoparasitoid wasp P.puparum.The cDNA of actin,actindepolymerisation factor and tubulin from P.rapae was cloned and their expression regulated byparasitization of P.puparum was investigated.P.rapae actin contained an open reading frameof 1131 bp encoding for 377 amino acid residues with predicted molecular mass and pI of 41.78kDa and 5.29,respectively.The homology of P.rapae actin with other insect actin genes wasgreater than 90% in amino acids.P.rapae actin was classified as a cytoplasmic type by absenceof muscle-specific amino acids and phylogenetic tree analysis.The 1243 bp cDNA sequence ofP.rapae actin depolymerisation factor contained an open reading frame of 447 bp encoding anneutral(pI 7.11)protein of 149 amino acids predicted molecular mass of 16.97 kDa,sharing97%,87%,89% and 72% homology with the counterparts of Bombyx mori,T.castaneum,A.mellifera and Maconellicoccus hirsutus.Nucleotide sequence analysis of P.rapae tubulinshowed full length cDNA of 1757 bp with open reading frames of 1344 bp encoding proteins of448 amino acids with a predicted molecular weight and pI of 50.38 kDa and 4.86,respectively.The cDNA-deduced amino acid sequence of P.rapae tubulin showed 97%,97%,87% and 93%homology with that of B.mori beta-tubulin 1,2,3 and 4.P.rapae tubulin was classified as betatype by phylogenetic evolution analysis.RT-PCR analysis showed that P.rapae actin,actindepolymerisation factor and tubulin gene were affected by P.puparum parasitization indicatingdecreased expression.(5)Endocrine changes in the hemolymph ofP.rapae indiced by venom of P.puparum.The changes of hemolymph juvenile hormone(JH)(only JHⅢdetected),ecdysteroid andjuvenile hormone esterase activity(JHE)over 72 h in parasitized and venom microinjected P.rapae pupae were monitored.Non-parasisized and PBS microinjected P.rapae served ascontrols.Results showed that JH titers were significant higher in parasitized and venommicroinjected pupae than that in control pupae during 24 to 72 h.After 12 h,JH titers weresignificantly promoted by parasitization and venom microinjection.JHE activities ofnon-parasitized and PBS microinjected pupae were significant higher than that of parasitizedand venom microinjected which was with a peak at 12 h(parasitized pupae)or 24 h(venommicroinjected pupae)during 6 to 48 and 12 to 36 h respectively.The hemolymph titers of ecdysteroid in non-parasitized and PBS microinjected pupae increased rapidly during 12 to 36 hwith a peak at 36 h,and were higher than treatments before 48 h,while presenting significantdifference at 24 to 48 h between the treatments and controls.The results demonstrate that venomalone of this parasitoid wasp can disrupt its host's endocrine system.(6)Proteome changes in the plasma of Papilio xuthus induced by P.puparumparasitization.By examining the differential expression of plasma proteins in the parasitizedand non-parasitized hosts pupae after 24 h by two-dimensional electrophoresis,16 proteins werefound to vary in relation to parasitization compared to unparasitized control samples.Among ofthem,8 proteins were elevated,5 showed decreased expression,and 1 disappeared,and 2 newlyoccurred after parasitization.All of them were submitted to identification by mass spectrometrycoupled with a database search.The modulated proteins were found to fall into the followingfunctional groups:humoral or cellular immunity,detoxification,energy metabolism,and others.In this paper,the microstructure and ultrastructure of venom apparatus of P.puparum wasclearly defined.The venom proteins of calreticulin,arginine kinase,serine protease,heat shockprotein 70,acid phosphatase and alkaline phosphatase were firtly identified from the venom ofpupal stage specific parasitoid or parasitoid belonging to Pteromalidae.The cDNA encodingvenom calreticulin,arginine kinase,acid phosphatase and alkaline phosphatase genes werecloned and sequenced,and the tissue location,biochemical characters and time-course relatedgene expression of acid and alkaline phosphatase were defined,which provided the firstevidence for the detail investigation of parasitoid venom acid and alkaline phosphatase.A novelpathogenic small RNA virus belonged to the Cripavirus genus of Dicistroviridae was reportedfrom the parasitoid.The results first revealed that the parasitization by the parasitoid onlyassociated with venom induced the proteins in the host plasma related to humoral or cellularimmunity,detoxification,energy metabolism differentially expressed,the the transcription ofnutrition metabolism related arylphorin-type storage protein gene up regulated,and thetranscription of cellular immune related calreticulin,actin,actin depolymerisation factor andtubulin genes decreased.In addition,the results firstly demonstrated that venom alone ofparasitoid can disrupted its host's endocrine system.These results not only revealed themolecular mechanisms of the interaction between parasitoid and host to a large extent,but alsomade a great contribution to gain insights into exploiting parasitoid venom for insect control.