| Microsporidia are obligate intracellular parssitic eukaryote,it has a wide range of host domains in nature with numerous and widely distributed.Nosema bombycis is the typical species of the Microspore genus,which can trigger Pebrine.The disease can be transmitted by sub-infection and embryo infection.It is a devastating disease in the production of sericulture.Therefore,it is listed as the legal quarantine object of the sericulture producing country.There are many factors influencing the infection of Pebrine.In addition to the infection caused by contamination of the feeding environment and sericulture tools and cross infection of microsporidia in the wild,it may also be caused by the diseased female moth.Prevention and control of silkworm micro-particle disease is the focus and difficulty of sericulture production.At present,in addition to strengthening inspection,quarantine and elimination for poisonous silkworm,the methods for controlling the silkworm disease are broadly divided into two categories: chemical control and physical control.Chemical control methods are relatively mature,including chemical disinfection and chemical drug treatment.However,the disinfectant is generally highly corrosive and has no control effect on the pathogen in the poisonous silkworm egg.The long-term using of drug treatment is toxic to the silkworm and affecting the quality of the silkworm eggs.The drug treatment.The physical control methods that have been reported mainly include the warm bath method,the high-temperature instant acid method,and the hot-air treatment method.Early high-temperature instant acid methods for multifilament silkworm varieties' eggs in the world have been found to have a control effect on the disease.The high temperature instant acid method has obvious effects and is more practical.However,the above high temperature instant acid test is carried out with the research on multifilament silkworm varieties.So the technology of disease in silkworm eggs controlled by high-temperature instant acid of dimorphic silkworm varieties has not yet been established and remains to be studied.In this study,the embryo-infected silkworm eggs(Hereinafter referred to as the poisonous silkworm eggs)were prepared by artificial inoculation.We observed the development of microsporidia in silkworm eggs by Giemsa method,detected the inactivation effect of Nb in silkworm eggs from 24 to 240 hours after oviposition by real-time PCR,and analyzed the DEGs after high temperature instant acid treatment by high-throughput sequencing.We want to elucidate the mechanism of high temperature instant acid in the prevention and control of the embryonic infection of Pebrine,and provide scientific basis for the application of high-temperature instant acid to prevent and control the disease.The following research results were obtained:1.Treated by high-temperature instant acid for the bivoltine quaternary hybrid variety Liangguang No.2,After hatching,the eggs were raised to 3rd and 5th silkworm larvae for microscopic examination of Nb,microsporidia infection rate of poisonous silkworm egg with high-temperature instant acid treatment are 3.55±1.55% and 13.10 ±5.90% respectively compared with conventional instant acid treatment,the relative control effects are 92.30±2.95% and 81.80±0.06%,respectively.This shows that high-temperature instant acid has significant control effect for Pebrine.The results showed that high temperature instant acid treatment has obvious effects on controlling the vertical transmission of pebrine,and it has significant therapeutic effect for pebrine2.Observation on the developmental of microsporidia in silkworm eggs with hightemperature instant acid period(18 to 22 h of egg age)by Giemsa staining,it found tha the microsporidia was observed as dual core or quad core in the early silkworm eggs.After instant acid treatment of 48 h,schizonts were still observed in the poisonous silkworm eggs of the positive control group and the conventional instant acid group,but in the same period of high-temperature instant acid treatment group,no microsporidia were found.It is speculated that the target of high-temperature instant acid for the control of Pebrine is the schizont stage of Nb.3.A standard curve for the expression of Tubulin gene of Nb was established by realtime PCR.The number of copies of microsporidia by absolute quantitative method were calculated in poisonous silkworm eggs from 24 to 240 hours after oviposition which treated with positive control,conventional instant acidicking and high-temperature instant acid.It was found that the amount of pathogen increased sharply within 24 to 48 hours,and it is speculated that this period is an active period of microsporidia.The quantitative results showed that both instant acid treatment had the effect of killing microsporidia,and the hightemperature treatment effect was more remarkable.It was confirmed that high-temperature instant acid had significant control effect for Pebrine.After soaking in two different temperatures,the pathogens in the eggs of the venom of the larvae showed a similar change from 24 h to 240 h,which was consistent with the proliferation and development of the microsporidia.It is speculated that high-temperature instant acid treatment can inactivate most of the schizonts in silkworm eggs,and the inactivated mature spores and a few schizonts can continue to grow and develop.4.Through RNA-seq,there were 400 of differentially expressed genes in the GO functional group which compared within positive control and conventional instant acidicking,differentially expressed genes in Pathway significant enrichment analysis were significantly enriched in Fatty acid elongation,Endocytosis,Cysteine and methionine metabolism,and JAK-STAT signaling pathway;And there were 1999 of differentially expressed genes in the GO functional group which compared within positive control and high-temperature treatment,and the gene expression of each small class was abundant,the differentially expressed genes in Pathway significant enrichment analysis significantly enriched Ribosomes and Oxidative phosphorylation.Analysis of the transcriptome of microsporidia showed that 38 and 20 differentially expressed genes were obtained by conventional instant soaking and high-temperature instant acidicking,respectively.In the Pathway significant enrichment analysis,the differentially expressed genes in the former were Protein export,Protein processing in endoplasmic reticulum,Ether lipid metabolism and Phagosomes were significantly enriched,and the differentially expressed genes of the latter pathogen were significantly enriched in Glycosylphosphatidylinositol(GPI)-anchor biosynthesis and Protein export. |
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