Differential Proteomic Analysisin Haemolymph Of Bombyx Mori Induced By The Larva Of Exorista Sorbillans Wiedemann Parasite Infection

Exorista sorbillans Wiedemann parasitized in silkworm larvae between the bodywall and the muscles,which leads to the multivoltine maggoty disease induced greatharms to the sericultural production. In recent decades, The existing research aremainly concentrated on the Exorista sorbillans biology and prophylaxis andtreatment.But the research on the physiological and biochemical changes in the bodyof the silkworm parasitized by Exorista sorbillans was less. In molecular level, theexploration about growing and developmenting process and molecular mechanism ofthe immune response of silkworm parasitized by Exorista sorbillans wasvacant.Therefore we mainly study the effect and mechanism of silkworm growth andimmune after it parasitized by Exorista sorbillans from nucleic and protein level.Andelaborate the biological problems such as the silkworm molecular mechanism ofimmune response and the impact of silkworm parasitized by Exorista sorbillans.Looking for the relevant points between multivoltine maggoty disease prevention and thesilkworm physiology.The research could provid important reference on prophylaxisand treatmentand improving the economic benefits of sericulture production andbreeding for disease resistance. The results are as follows:1. Survey of the harm on silkworm parasitized by Exorista sorbillans and parasitic siteobservationSeen from the data on mortality of silkworm after parasitized，most of parasitiedsilkworms in the early days of fourth and fifth instar were died.But the mortality ofsilkworm on the day3th of fourth instar and the day3th or4th of fifth instar wassignificantly reduced. Moreover, we observed and recorded the differentcharacteristics about parasitic sites which along with the change of developmentingtime. From dissections of silkworm, which would hatched maggots,we observedparasitic maggots.From dissections of the pupal parasitized by Exoristasorbillans.we found that the silkworm generated a lot of wrappages to encircle themaggots and led it to died.2. Proteomic analysis on the haemolymph of silkworm parasitized by ExoristasorbillansSilkworm parasitized by Exorista sorbillans on the day1stof fifth instar, usingSDS-PAGE to analysis the blood protein change between parasitic and normal of theday4thin fifth instar, the day6thin fifth instar, the day2ndin pupal,the day4ndin pupal and the day6ndin pupal. the results showed: the blood proteins constituent content ofthe day2ndin pupal stage changed significantly. Moreover, the silkworm stop eatingmulberry at this time,all the required energy of silkworm and Exorista sorbillansWiedemann came from the stored energy material of silkworm itself.Therefore weselected the parasitic blood group and the control group of on the day2ndin pupalstage in the same situation to establised establish the two-dimensional electrophoresispatterns, Identified the significantly different protein spots and subsequently analyzedby mass spectrometry. Clarified the blood proteins which are relevant to the immuneresponses and the process of physiological and pathological changes while silkworminfected by Exorista sorbillans Wiedemann.49differentially expressed proteins thatwere successfully identified by mass spectrometry,35proteins were up-regulated,including peptidoglycan recognition protein S1, C-type lectin10and phenoloxidase2which play important roles in the innate immune system.14proteins weredown-regulated, including some proteins relevant to the growth and metabolism. Theresults show that: the innate immune system was activated while the silkworminfected by the Exorista sorbillans Wiedemann, the growth and metabolism were alsoaffected.Gene Ontology tools were used to analyze the identified proteins to present anoverall view on the functional categories of proteins. The results indicate that37ofthe49identified proteins show at least one matched GO annotation. The proteinswere classified into cellular component, molecular function, and biological processaccording to the GO hierarchy using WEGO. Those proteins were mapped to theKEGG ortholog level for the KEGG analysis. These pathways were classified intometabolism, genetic information processing, environmental information processing,cellular processes, organismal systems, and human diseases，which mainly relevant toabsorption and digestion of protein,carbohydrate metabolism, detoxificationmetabolism and the immune response process.3. The verification of Real-time PCRTo the differentially expressed proteins identified by2-DE, We chosing theseveral key proteins-peptidoglycan recognition protein-S1,C-type lectin-10,phenoloxidase-2and ecdysteroid-regulated16kDa protein precursor and theircorresponding genes,using bioinformatics method combined with the genetic data andgene arrays data of silkworm to conduct the analysis, And making use of the methodof Real-time PCR to further confirm the conclusion. Experimental results show that: only Phenoloxidase-2have not any signal peptide sequence; the expression of abovefour genes were high in fat body.The expression of peptidoglycan recognitionprotein-S1gene, C-type lectin-10gene and Phenoloxidase-2gene were increased,Onthe contrary,the expression of ecdysteroid-regulated16kDa protein precursor genewere declined. This experimental results confirm that the protein changes in theregulation of transcription and translation level change was almost the same.Namely,the result of Real-time PCR and2-DE were consistent.