| Exorista sorbillans(Wiedemann)is a parasitic dipteran insect that parasitizes the silkworm and causes the silkworm myiasis,causing great economic losses to the sericulture industry in China.The successful completion of the entire generation of E.sorbillans is regulated by a variety of genes,and obtaining information on key genes during reproduction and development is extremely important for the development of control methods for E.sorbillans.However,the activities of wild E.sorbillans have significant seasonality,that limits the study of genes related to their reproductive development.Therefore,This article began with investigated the in vitro culture method of E.sorbillans,and on this basis,explored the key genes of reproduction and development of the E.sorbillans to provide basic information for potential targets of silkworm myiasis control.The main research and results of this article are as follows:1.Establishment and optimization of in vitro culture methods for E.sorbillans:On the basis of the artificial base medium for Exorista larvarum,investigated the effect of adding the haemolymph of the silkworm at different times on the in vitro culture of the E.sorbillans,obtained a medium with short development time and high fly formation rate,the components were: L5D6 silkworm haemolymph 10.0%,skimmed milk 55.0%,sucrose1.7%,yeast extract 5.0%,egg yolk 10.0%,sterile water 18.3% and support material sterile skimmed cotton.As there is no natural host for E.sorbillans in winter and spring,obtaining a large number of eggs is a challenge.In this study,four insects were selected as alternative oviposition carriers: Tenebrio molitor,Antheraes pernyi pupae,Hermetia illucens larvae and third instar larvae of E.sorbillans to attract E.sorbillans to oviposit concentratedly.The high number of bacteria on Hermetia illucens larvae made it difficult for the eggs to hatch,while the surface eggs of the other three insects hatched successfully and the hatching rate was not significantly different from that of the surface eggs of the fifth instar silkworms.The Antheraes pernyi pupae are the most suitable alternative egg-laying vehicle because they are large and easy to retrieve during the winter and spring that are duration of dispause for Antheraes pernyi pupae.Further investigation into the appropriate time for egg collection revealed that egg collection within 6-24 h after ovipositing had no effect on the hatching rate of the eggs.The parasitism rate of E.sorbillans obtained based on this culture system was not significantly different,although the pupal weight was slightly lower than that of the wild type.The above results indicate that this method can successfully complete the rapid culture of E.sorbillans in winter and spring and maintain the parasitic performance of the next generation,which provides the experimental conditions for the subsequent molecular biological research of E.sorbillans.2.Cloning and identification of genes involved in reproductive development in E.sorbillans :Under the basic conditions established by the laboratory method of artificially generate E.sorbillans,further cloned and identified two types of protease genes involved in the reproductive development of E.sorbillans,and to analyse their mechanisms of action in the reproductive development process,so as to provide new ideas for the prevention and control methods of silkworm myiasis.(1)Cloning and analysis of the seminal metalloproteinase gene(EsSemp)of E.sorbillans: Two EsSemp full length cDNA sequences,named as EsSemp 1 and EsSemp 2,were obtained using the RACE technique,with full cDNA lengths of 996 bp and 1055 bp,ORFs of 771 bp and 765 bp,respectively,encoding 256 and 255 amino acid residues,respectively.The complete genome structure was obtained using Genome Walking method,and EsSemp 1 and EsSemp 2 both contain one intron and two exons.Protein sequence analysis revealed that EsSemp 1 and EsSemp 2 share typical conserved motifs for astacin.Phylogenetic tree analysis showed that EsSemp 1 is highly homologous to EsSemp 2 and has high homology to seminal proteases of various insects.Investigation of EsSemp 1 and EsSemp 2 expression by RT-q PCR at different periods and different tissues of E.sorbillansin.The results showed that EsSemp 1 and EsSemp 2 were expressed weakly in the sex gonad of male flies and were mainly highly expressed in the midgut and malpighian tubule of maggots and adult flies,it is hypothesized that EsSemp 1 and EsSemp 2 may act on gonads after expression in other tissues.Meanwhile,EsSemp 1 was highly expressed at 0 h of spawning and late egg stage,and EsSemp 2 was weaker than EsSemp 1 in egg stage overall,but the expression trends were the same.It is assumed that EsSemp 1 and EsSemp 2play a role in egg fertilization and hatching of E.sorbillansin.The EsSemp 1 protein was expressed in vitro,and EsSemp 1 was hydrolytically active against the Leu-Leu-Glu-MCA amino acid peptide chain,a specific substrate for metalloproteinases,and screening for EsSemp 1 protease inhibitors may be a new avenue for the development of myiasis control agents.(2)Cloning and functional analysis of the serine protease gene(EsSP)of E.sorbillans:The degenerated primers were designed based on the evolutionarily conserved serine protease 2 gene in insects and the conserved region of EsSP was cloned.The 1179 bp full length cDNA sequence of EsSP was obtained by RACE technique,with an ORF of 981 bp,encoding 326 amino acid residues.The complete genome structure of EsSP was obtained by Genome Walking technique,sequence alignment and splicing,and EsSP was identified to contain 3 introns and 4 exons.Alignment of amino acid sequences showed that EsSP is highly homologous to the 26 k Da protein of Sarcophaga peregrina.Investigation of the expression pattern of EsSP of E.sorbillans by RT-q RCR,the results showed that EsSP was mainly expressed in 1-day-old pupae,and the expression increased in female flies after mating.In vitro expression of EsSP full length protein and mature enzyme protein reacted with silkworm epidermal protein(Bm CPAP)and showed that the EsSP mature protein had a significant hydrolytic effect on Bm CPAP.After RNAi knockdown of EsSP,black lesions appeared in the midgut of the pupae on the fourth day post treatment.The above results show that EsSP is influenced by mating behaviour and is female specific,suggesting that EsSP has a regulatory function on the physiological metabolism and oviposition of female flies after mating.In addition,EsSP may be involved in the metamorphosis of E.sorbillans larvae to pupae,playing an important role in the degradation of larval epidermis and midgut remodelling during the pupal stage.In summary,this study established an in vitro culture method for rapid reproduction of E.sorbillans without natural hosts in winter and spring,and successfully obtained three genes related to the reproduction and development of E.sorbillansin from this experimental materials,and initially elucidated the physiological functions of the three genes in the reproductive development of E.sorbillansin,which provided basic information for the targets of E.sorbillansin control. |
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