Effect Of The Endoparasitoid Wasp Pteromalus Puparum On The Lipid Metabolism In The Host Pieris Rapae And The Functions Of Relevant Venom Proteins

To investigate how endoparasitoids wasps affect host lipid metabolism and the functions of its venom proteins,the pupal endoparasitoids Pteromalus puparum(with only one single parasitism associated factor)and its natural host the cabbage butterfly Pieris rapae were used as a system model to perform the following studies in this work.Understanding the pathways and mechanism of the interaction between the parasitic wasp and the host lipid metabolism contributes to the further development of parasitic wasp conservation and artificial rearing.Furthermore,studying the potential functions of its relevant venom proteins benefits of the understanding of the functional diversity of parasitoid venom proteins.It contributes to the exploitation of new insecticidal gene resources that may be used in biological control of pests.1.Lipidomics analysis of the host fat body post-parasitizationUPLC-MS-based lipidomics approach was used to analyze the identities and concentrations of lipids in the fat body of host P.rapae infected by the pupal endoparasitoid P.puparum.During the infection,host fat body levels of highly unsaturated,soluble triacylglycerides(TAGs)increased while less unsaturated,less soluble forms decreased.These data suggest that wasp venom induces host fat body TAGs to be transformed into lower melting point(more liquid)forms and released into the host hemolymph following infection,allowing simple absorption and nutritional acquisition by wasp larvae.In order to further investigate what causes the desaturation of TAGs in host fat bodies,the annotation of predicted desaturase genes was performed using P.puparum genome.Combining the analysis of P.puparum transcriptome data of its different developmental stages and tissues,a putative desaturase encoding gene PPU08555 was found with especially high expression in P.puparum venom gland.It is believed to be involved in inducing the conversion of the highly unsaturated TAGs to other forms.In addition,levels of a large number of phospholipids increase the host fat body post-infection by P.puparum suggesting that fat body cells are destroyed.2.Lipidomics analysis of the host hemolymph post-parasitizationUPLC-MS-based lipidomics approach was used to analyze the identities and concentrations of lipids in the hemolymph of host P.rapae infected by the pupal endoparasitoid P.puparum.In the infected host hemolymph,overall levels of TAG and phospholipids increased,suggesting that fat body cells are destroyed and their contents are dispersed.Combining with the transcriptome data analysis of host P.rapae,the expression of a diacylglycerol acyltransferase gene(DGAT2)involved in the glyceride metabolism pathway was found significantly up-regulated in an hour post-infection by P.puparum.Therefore,it is believed that the increase of TAG levels in host hemolymph are converted from DAGs.In addition,cholesteryl esters(CEs)increased in host hemolymph following infection with no concomitant decrease in host cholesterol,implying that the wasp may provide this necessary food resource to its offspring via its venom.3.Genome-wide annotation and functional analysis of the lipase genes from P.puparumLipases play essential roles in digestion,transport,and processing of dietary lipids in insects.For those parasitoid wasps with a unique life cycle,lipase functions could be multitudinous in particular.Here,P.puparum genome and transcriptome data were interrogated to annotate a total of 64 predicted lipase genes belonging to five lipase families and suggest that 8 venom and 4 salivary lipases could mainly regulate the host nutrition environment post-parasitization.According to the results of multiple sequence alignments,many putative venom lipases were found with incomplete catalytic triads,relatively long ?9 loops,and short lid domains,suggesting the loss of catalytic activity and weak triacylglycerol(TAG)hydrolytic activity.Phylogenetic trees indicate various predicted functions of lipases in P.puparum.4.Expression of P.puparum venom lipases and the activity detectionSix lipase genes identified in P.puparum venom were cloned and expressed in Escherichia coli.The optimal expression conditions of these lipase genes in the vector p Cold-tf were screened out and their protein features were shown.Using the improved cholesterol esterase activity detection method,the cholesterol esterase activity in P.puparum venom and the purified PPU16612 recombinant protein was observed.PPU16612 is the venom lipase gene with the highest relative expression in P.puparum venom and capable of hydrolyzing cholesterol esterase as predicted based on the KEGG orthology databases.While the expression of PPU16612 was suppressed via RNA interference,the cholesterol esterase activity of P.puparum venom significantly reduced.According to the transcriptome data of the parasitized and unparasitized host P.rapae,the esterification of cholesterol was suppressed following infection.