Molecular Mechanism On Early Maturation Of Silkworm Parasitized By Exorista Sorbillans And Characteristic Analysis Of Hatching Enzyme Gene

Exorista sorbillans Wiedemann parasitizes silkworm larvae and causes myiasis,which decreases sericulture production.At present,the control agent for myiasis is still the"Miecanying"popularized and used in the 1960s,and it is also the most effective agent for the control of myiasis so far.However,long-term use,E.sorbillans maggots have already developed some resistance to it.In order to find new methods or agents for the control of myiasis,the thesis mainly focus on the molecular mechanism of the early maturation of the parasitized silkworm and the characteristics of the hatching enzyme gene of the E.sorbillans.It provides a basis for further control of myiasis.The research on the hatching enzyme gene of E.sorbillans at the molecular level can provide a theoretical basis for the development of new insecticides targeting hatching,and at the same time provide new ideas and new approaches for biological control of agricultural pests.1.Molecular mechanism of E.sorbillans leading to early maturation of host silkworm.The silkworms will appear early maturation after being parasitized by E.sorbillans.The molting and metamorphosis development of silkworms are mainly composed of ecdysone and its active metabolite 20-hydroxyecdysone（20E）and the synergistic regulation of hormones such as Juvenile hormone（JH）.In order to clearly illustrate the molecular mechanism of early maturity in the silkworms parasitized by E.sorbitan,the titers of the juvenile hormone（JH）and 20-Hydroxyecdysone（20E）in the hemolymph were measured in the silkworm parasitized by E.sorbitan.The titers of JHⅢand 20E were both increased in the 5th instar silkworm larvae.The expression patterns of genes which were involved in the JH and 20E synthesis,metabolism,and signaling pathway were verified by RT-q PCR.The results showed that the Nvd,CYP302A1,and CYP307A1 genes involved in the synthesis of20E were significantly up-regulated after the silkworm was parasitized by E.sorbillans,while RNAi knockdown of the CYP302A1 and CYP307A1 genes respectively decreased the concentration of 20E,resulting in the prolongation of the fifth instar of silkworms.The related genes involved in JH synthesis and metabolism were significantly down-regulated in the late 5th instar larvae of the parasitized silkworm.Further RNAi knockdown of the JHE gene led to an increase in the concentration of JH III,and the 5th instar of the parasitic silkworm was prolonged.It is speculated that after the silkworm was parasitized,the up-regulated expression of key genes in the 20E synthesis pathway was induced,resulting in a significant increase in ecdysone of the silkworm hemolymph,and at the same time,the down-regulated expression of key genes in the JH metabolic pathway was induced,resulting in a significant increase in JHⅢin the late 5th instar of the parasitic silkworm,eventually leading to the early maturation of silkworms under the combined action of these two hormones.This provides a theoretical basis for further understanding the response mechanism of silkworm to parasitism.2.Cloning and Enzymatic Characterization of Hatching Enzyme Gene of E.sorbillans:（1）Cloning and Molecular Characterization of EsHE Gene:In order to study the hatching mechanism of E.sorbillans at the molecular level,total RNA was extracted from1.5-day-old fly eggs,and c DNA was synthesized by reverse transcription.Degenerate primers were designed according to the conserved sequence of the hatching enzyme gene of Diptera.The full-length c DNA of the hatching enzyme gene of E.sorbillans was obtained by RACE technology and named EsHE（Gen Bank accession number:ON042475）.The full-length c DNA of EsHE gene is 1518 bp,including 159 bp 5′UTR,423 bp 3′UTR,and936 bp complete ORF,encoding 312 amino acids.EsHE protein sequence contains the characteristic sequence of hatching enzyme HELMHVLGFMHE zinc-binding motif and YDYGSVMHY Met-turn motif.The phylogenetic tree analysis based on the amino acid sequence of EsHE and the homologous amino acid sequence of hatching enzymes of other21 animals showed that E.sorbillans and Lucilia sericata were most closely related.The ORF sequence of EsHE gene was cloned by using the genomic DNA of E.sorbillans as a template to obtain the intron of E.sorbillans.The 5′-terminal upstream promoter sequence and the 3′-terminal downstream sequence of EsHE genomic DNA were cloned by Genome Walking technology,and the complete genome DNA structure of the hatching enzyme of E.sorbillans was obtained by sequence splicing.The full-length EsHE genomic DNA is 8974bp,including the upstream sequence of 2429 bp,the first exon of 1005 bp,the intron of2638 bp,and the second exon of 501 bp,and the downstream sequence of 2401 bp.RT-q PCR detection of EsHE gene showed the expression level began to increase sharply at36 h after egg laying and reached the highest level at 48 h before hatching.It indicated that EsHE was closely related to the embryonic development and hatching of E.sorbillans.The EsHE gene was highly expressed in the third instar larvae,and the expression level reached the highest value in the whole generation development stage.It is suggested that the EsHE gene may also play the role of cocoon lysing enzyme to help soften the cocoon shell,which is beneficial for fly maggots to burrow out of the cocoon shell.Bioinformatics predicted that EsHEpro contains transcription factors GATAd and HSF associated with embryonic development.The above research results lay a foundation for further elucidating the molecular mechanism of the hatchling of E.sorbillans,and provide a theoretical basis for the research and development of insecticides targeting hatching-related genes.（2）Enzymatic properties of EsHE:In vitro expression of EsHE protein found that EsHE protein mainly exists in E.coli in the form of inclusion bodies.After purification and renaturation of three kinds of EsHE,the degradation activity of EsHEb on casein and Leu-Leu-Glu-MCA amino acid peptide chain was investigated.The results show that the degradation activity of the mature enzyme EsHEb was the highest.The optimum temperature for degrading eggshells of EsHEb is about 40℃,and the optimum p H value for degrading eggshells is about 7.0.The Km value of EsHEb for casein was 8.913 mg/m L.In view of the hydrolysis of purified EsHEb protein,the effect of different protease inhibitors on its activity was investigated.The results showed that 5,5′-dimethyl-2,2′-bipyridine significantly inhibited the activity of EsHEb,and the inhibition rate reached 82.9%.Finally,using the eggshell of E.sorbillans and the silkworm sericin cocoon as substrates,the degradation activity of EsHEb on natural substrates was detected.The results showed that the degradation activities of EsHE on the eggshell of E.sorbillans and the silkworm sericin cocoon were 343.44±78.56 and 223.48±13.11ΔA280 10^-3min^-1mg^-1,respectively.Both the eggshell of E.sorbillans and the silkworm sericin cocoon have high degradation activities,indicating that EsHE may participate in the hatching process of E.sorbillans,meanwhile it may be involved in the function of dissolving the cocoon of B.mori.（3）Application of hatching enzyme inhibitor,bipyridine in the control of silkworm myiasis:Based on the combination of the characteristics of bipyridine itself（that is,the chelation of metal ions）and the characteristics of the E.sorbillans parasitic silkworm（that is,the transformation from eggs to larvae needs to go through the process of"incubation"）,bipyridine significantly inhibited the activity of EsHEb,the application technology of bipyridine in silkworm myiasis was preliminarily studied.The experimental results showed that the therapeutic effect of 0.5-3m M bipyridine on the myiasis of the silkworm could reach 90%-100%,which was higher than the therapeutic effect of the“Miecanying”on the myiasis（83.4%）.For different concentrations of bipyridine,the four treatment methods of dipping and body spraying,dipping,body spraying,and feeding have certain therapeutic effects on myiasis,among which the treatment method of dipping and body spraying has the best effect.The effective period of the drug treatment of bipyridine to control myiasis is after spawning and before the larvae hatch and enter the silkworm body,that is,the best drug treatment period is 0-36 hours after the E.sorbillans lay eggs.0.5～3m M bipyridine is safe for the vitality of silkworms,and can correspondingly prolong the fifth instar period of silkworms,thereby increasing the total cocoon volume and cocoon layer volume of silkworms,and increasing the economic efficiency of sericulture.Bipyridine has great potential application prospects in the control of silkworm myiasis.