Primary Study On Molecular Mechanism Underlying Nosema Ceranae Infection Of Apis Mellifera Ligustica Worker

Nosema ceranae is a unicellular fungal pathogen that exclusively infects the epithelial cell of adult honeybee,leading to damage of gut tissue,disorder of digestive system,shortening of life-span,suppression of immune genes,inhibition of cell apoptosis,decrease of nectar-collecting quantity and so forth.At present,colonies in beekeeping countries all over the world have varying degrees of N.ceranae infection.However,the infection mechanism of N.ceranae has not yet been clarified until now,and the role N.ceranae plays during the infection process is largely unknown.In this project,to deeply reveal the mechanism regulating N.ceranae infection,study on the infection of N.ceranae to Apis mellifera ligustica worker was first conducted using approaches such as spore counting and RT-qPCR,and then,N.ceranae-infected and un-infected midguts of A.m.ligustica workers were sequenced using a combination of small RNA-seq and strand-specific library based lncRNA-seq technologies,followed by filtration of fungal pathogen's mRNA,miRNA and lncRNA omics datasets and comprehensive analysis combined with previously obtained whole transcriptome data of N.ceranae spore.1.A.m.ligustica workers were inoculated with N.ceranae under lab condition,PCR and electrophoresis result suggested N.ceranae can effectively infect A.m.ligustica worker;spore counting result showed the number of spores decreased during a range of 1 d-4 d,and gradually increased from 5 d post inoculation(dpi)to 14 dpi;N.ceranae mainly parasited in epithelial cells of workers' midguts,and the number of fungal spores continuously inceased with the infection time prolonged.2.A total of 5 816 465 and 28 501 225 high-quality clean reads of N.ceranae were filtered out from lncRNA-seq of infected workers' midguts;21 525 444 and 30 316 756 high-quality clean reads of N.ceranae were filtered out from sRNA-seq of infected workers' midguts3.Based on mRNA omics analysis,it's discovered 1 397,1 497 and 52 DEGs were respectively identified in NcCKM vs NcT1M,NcCKM vs NcT2M and NcT1M vs NcT2M comparison groups,involving in 23,23 and 11 GO terms as well as 80,79 and 27 KEGG pathways;five upregulated and two down-regulated genes in NcCKM vs NcT1M were enriched in MAPK signaling pathway,while NcCKM vs NcT2M two upregulated and five down-regulated genes were identified;toxin factors including spore wall protein,spore wall and anchoring disk complex protein gene,chitinase,polar tube protein and Ricin B lectin,and infectionrelated factors such as hexokinase,ATP/ADP transferase,ABC transporters and transcription factor ste12 may play a key role during pathogen proliferation.4.On basis of miRNA omics analysis,it's found that 164,122 and 60 DEmiRNAs were identified in NcCKS vs NcT1S,NcCKS vs NcT2S and NcT1S vs NcT2S comparison groups;a total of 1 885,1 733 and 1 524 mRNAs were predicted for the aforementioned DEmiRNAs,annotating to 27,25 and 26 GO terms,and among them the most abundant terms were metabolic processes,catalytic activity,cellular processes;the target mRNAs were also annotated to 84,84 and 84 KEGG pathways,and the largest pathways were metabolic pathway,ribosome,and secondary metabolite biosynthesis;there were 35,26 and 12 DEmiRNA target mRNAs associated with MAPK signaling pathway,49,40 and 17 DEmiRNA target mRNAs associated with glycoproteins/gluconeogenesis pathway;DEmiRNAs were likely to regulate the expression of genes encoding toxin factors such as ricin B lectin,apoptosis inhibitor,polar tube protein and spore wall protein,and infection-related factors including hexokinase and ATP/ADP transferase,ABC transporter and transcription factor ste12 to mediate the N.ceranae infection of A.m.ligustica worker.5.Based on lncRNA omics investigation,19,24 and four DElncRNAs were identified in NcCKL vs NcT1L?NcCKL vs NcT2L and NcT1L vs NcT2L comparison groups,regulating 26,27 and two upstream and downstream genes;partial DElncRNAs may mediate pathogen infection by regulating the expression of N.ceranae genes such as Argonaut through cis function;partial DElncRNAs can regulate 30,39 and 8 mRNAs through trans function,and most of these DElncRNAs can simultaneously one positively-related mRNA and one negatively-related mRNA;DElncRNAs may mediate N.ceranae infection via regulation of the expression of genes encoding ATP/ADP transferase and spore wall protein through trans function;15 DElncRNAs in NcCKL vs NcTL1 can bind to 195 miRNAs and 204 target mRNAs,while in NcCKL vs NcTL2 23 DElncRNAs can link to 211 miRNAs and 216 target mRNAs,four DElncRNAs can bind to 94 miRNAs and 73 target mRNAs;corresponding DElncRNAs may regulated the expression of genes encoding spore wall protein and ricin B lectin,thus affecting the N.ceranae infection of A.m.ligustica worker.