| Bombyx mori is a beneficial and model insect in Lepidoptera.Now in China there are tweny million farmers feeding the silkworm for benefits.Regarded as ?successful parasites?,Microsporidia are a group of obligate intracellular and unicellular parasites related to Fungi that infect a broad range of hosts,including nematodes,fruit fly,silkworm,rabbit and human beings.To date,more than 1,400 species of Microsporidia belonging to 208 genera have been identified.Nosema bombycis,as the firstly identified microsporidian,can infect economic insect Bombyx mori through oral infection,and then germinate in midgut which causes a highly fatal silkworm disease,pébrine.Notably,transovarial transmission of N.bombycis leads to the embryo congenital infection.In history,the epidemic of pébrine caused the steep decline of the European silkworm industry in the 19 th Century.Today,this pathogen is responsible for substantial economic losses in many silkworm-producing countries,including China and India,etc.As a result,N.bombycis has been regarded as the only target of statutory quarantine inspection in silkworm egg-production in China due to its transoviral transmission.Therefore,the downstream events of N.bombycis transoviral transmission were focused on,including the proliferation characteristics of N.bombycis in silkworm embryos and the host innate immune responses induced by N.bombycis congenital infection,with the aim to illuminate the proliferative strategy utilized by N.bombycis and the host immune responses,and provide new clues on pébrine prevention,also provide the referential work for the studies of other congenital pathogens.The raw data generated in this study was submitted to the NCBI Sequence Read Archive(SRA,http://www.ncbi.nlm.nih.gov/sra)with the accession number PRJNA549766.Main results are as follows:1.Spatio-temporal proliferation characteristics of N.bombycis in congenitally infected silkworm embryoBy laser scanning confocal microscope,we observed that only a few N.bombycis of early developmental stages were localized around the yolk granules and germ band at1 day post ovipostion(dpo);At 2 dpo,clusters of immature spores and a few mature spores with a chitin layer emerged in yolk granules;meanwhile,a cluster of meronts/sporonts were observed in embryo tissues.More parasites continued to replicate around host yolk granules,and noticeable infection continued in embryo tissues at 3 dpo.With embryo intestinal lumen forming in the blastokinesis period(4dpo-6 dpo),some yolk granules with parasites were wrapped into embryo intestinal lumen.In addition to continuous accumulation in the yolk granules,N.bombycis was detected in the intestinal lumen at this stage.Additionally,a large number of mature spores were accumulated in the host intestinal lumen from 5dpo to 9 dpo.Finally,numerous mature spores were presented in the gut of newly hatched larva,and then were released into the environment via faeces for further horizontal transmission to other healthy silkworm larvae.TEM was also employed to characterize additional details of parasite replication.During the blastokinesis stage of embryonic development,parasites with thick spore wall were observed around yolk granules.At 8 dpo,parasites with polar tubes and smooth thick spore walls clustered among yolk granules.Finally,we observed that several meront/sporont-like and sporoblast-like parasites coexist in tissue cells of newly hatched larvae,indicative of intracellular parasitism caused by N.bombycis congenital infection.2.Transcriptome analyses of N.bombycis in congenitally infected silkworm embryoAfter preparation of N.bombycis congenital infected eggs,RNA-Seq were carried out to explore the expression pattern of pathogen and host in different time points during the infected embryo development.Totally,588.30 Gb data were obtained from seven time points in silkworm embryonic development,the number of reads maped to the unique location of silkworm reference genome is 2,345,559,311,and the number of reads maped to the unique location of N.bombycis reference genome is 5,759,867.Besides,a total of 95.73 Gb data were obtained from two time points in silkworm larval stages,the number of reads maped to the unique location of silkworm reference genome is 510,931,457,and the number of reads maped to the unique location of N.bombycis reference genome is 32,477,522.q RT-PCR was used to verify the differentially expressed genes,and the anastomotic rate with RNA-Seq was 70%,indicating that the transcriptome results were reliable.The evidence for the expression of 3,644(81.7%)of the total annotated genes in N.bombycis were obtained.Depending on principal component analysis,the transcription patterns of N.bombycis genes were grouped into three clusters.The first cluster included samples collected at 1 dpo,26 hpo and 2 dpo,corresponding to the early stages of embryo development.The second cluster was the sample collected at 4 dpo corresponding to the middle stages of embryo development.The third cluster consisted of samples collected at 6,8 and 9 dpo corresponding to the late stages of embryo development.Next we analyzed the top 100 highly expressed genes of N.bombycis from congenitally infected samples.Based on Venn diagrams,we identified 41 highly expressed core genes including ribosomal proteins,hypothetical proteins,histone proteins,transcription and translation factors,heat shock proteins and spore wall proteins.Meanwhile,we analyzed the expression pattern of N.bombycis genes with predicted signal peptides.In our gene expression data,we found a total of332 genes with predicted signal peptides had transcribed information,inclusive of 249 genes with hypothetical protein annotation,65 genes with known annotation continually expressed at all time-points.Among 65 genes,ADP/ATP carrier protein 1,serpins,Ricin B lectin,and some genes encoding N.bombycis structural proteins were found,such as SWPs and PTPs.Notably,serpin 2,serpin 3,serpin 6,serpin 10 with predicted signal peptides that clustered together phylogenetically exhibited a relatively high expression level compared to the other serpins expressed in the embryo stage.3.Transcriptome analyses of embryo and larva responses to N.bombycis congenital infectionIn this part,the transcription characteristics of host genes induced by congenital infection were analyzed.In general,the congenital infection of N.bombycis induces relatively weak embryo responses with a total of 527 DEGs including 365 up-regulated and 163 down-regulated in the seven timepoints of the whole embryo developmental stages.Volcano plot showed that number of the DEGs differed at each time point,the most number(314)of DEGs at embryo stages was present at 26 hpo,253 up-regulated genes such as caspase 8,dual oxidase,juvenile hormone esterase and Kruppel homolog1 were identified,meanwhile,61 down-regulated genes such as C-type lectin 16,serine protease inhibitor 10 were identified.Moreover,cuticular related genes were up-regulated at 1 and 6 dpo,but down-regulated at 4 dpo.Additionally,the expressions of lipase 3,facilitated trehalose transporter Tret1-like,pancreatic triacylglycerol lipase and alkaline phosphatase were down-regulated at 9 dpo.Pairwise correlation coefficients of transcripts across all eggs samples illustrate that both the uninfected groups and infected groups at same time-point were clustered together suggesting the congenital infection of N.bombycis has a little influence on embryonic development.On the contrary,strong larva responses with a total of 2631 DEGs including 1148up-regulated genes and 1483 down-regulated genes in larva stages(only two time-points).Gene Ontology(GO)enrichment analysis indicated that the infection of N.bombycis has a comprehensive influence on host especially in metabolic process.In the5 day post hatched(dph)and 6 dph DEGs library,enriched DEGs include those involved in metabolic process,cellular process,single-organism process,membrane,membrane part,cell,cell part,organelle,binding,catalytic activity,structural molecule activity and so on.4.Innate immunity analysis of embryo and larva responses to N.bombycis congenital infectionHere,we have screened most innate immunity related genes involved in pathogen recognition,modulation,signal transduction and effectors in embryo stages and larva stages,respectively.For the whole embryo stage,only a few DEGs involved in host immune responses including ?GRP1,SCRC,defencin,most of the immunity related genes were expressed in low level.MYD88,a universal adapter protein used by almost all Toll pathways was not detected in the whole embryo stages,most of AMPs were scarcely expressed at embryo stages.At the same time,a lysozyme and three lysozyme-like proteins were probably induced but not significantly between uninfected and infected embryo.Similarly,low level expression of prophenoloxidase 1 and prophenoloxidase 2,meanwhile,failing to get the data of pro PO-activating enzyme(PPAE)in RNA-seq implied that the innate immunity system was not well established in embryo stage.In newly hatched larvae(9 dpo),cec A1,glv3 were up-regulated but without significance.On the contrary,obvious immune responses of larvae were induced strongly by N.bombycis congenital infection,especially reflected by the significant expression changes of host's immunity related genes.For example,recognition related genes such as ?GRP 2,PGRP S1,PGRP S3,PGRP S5,PGRP S6,CTL 9 and CTL 19 were up-regulated,but CTL 1,CTL 2,CTL 4,CTL 7,CTL 12,CTL16 and CTL 18 were down-regulated.Moreover,modulation related genes such as CLIP4,CLIP 11,CLIP 12,SPN 5,SPN 7,SPN 19 and SPN 22 were up-regulated,however,CLIP 5,CLIP 14,SPN 10,SPN 12,SPN 15 and SPN 17 were down-regulated.Toll 9-1was up-regulated significantly together with TRAF 3,which suggested that N.bombycis had activated the Toll pathway.Interestingly,Cactus,a negative regulatory factor in Toll pathway was significantly up-regulated.Finally,the expressions of 13 antimicrobial peptides were up-regulated in high level such as cec,glv,mor LP,which suggested the innate immunity system have established and the serious N.bombycis infection induces an intense host innate immune responses.In summary,using histopathological approach and deep transcriptional profiling,we first illustrate the spatio-temporal proliferation characteristics of N.bombycis in silkworm embryos,including morphological and transcriptional characteristics,and then analyzed the embryo and larva immune responses to N.bombycis congenital infection.Our work sheds more lights on the downstream events of N.bombycis vertical transmission and provided clues for the interaction between pathogen and host.In addition,the transcriptome of embryo offers abundant referential data for the study on the development of innate immune system in insect embryo.In the sense of application,this thesis is specially beneficial for the proposing new ideas for prevention and control of pébrine in silkworm feeding industry. |
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