Abstract: 【Aim】 This study aims to reveal the molecular mechanism underlying Nosema ceranae infection of Apis mellifera ligustica at the transcriptome level by investigation of differentially expressed genes (DEGs), virulence factors and infection-associated factors. 【Methods】 Following the criteria of P≤0.05 and |log₂(Fold change)|≥1, DEGs within NcCK vs NcT1, NcCK vs NcT2 and NcT1 vs NcT2 comparison groups were screened out via the comparative analysis based on our previously obtained high-quality transcriptome datasets from purified spores of N. ceranae (NcCK) and N. ceranae in the midgut of A. m. ligustica workers at 7 d and 10 d post infection (NcT1 and NcT2, respectively). Venn analysis, GO classification and KEGG pathway enrichment analysis of DEGs were conducted using the related bioinformatic software, virulence factors and infection-associated factors of N. ceranae were summarized and analyzed based on Nr database annotations, KEGG database annotations and related literature documentations. The transcriptomic data and expression trends of DEGs were verified by RT-qPCR. 【Results】 In total, 1 397, 1 497 and 52 DEGs were identified in NcCK vs NcT1, NcCK vs NcT2 and NcT1 vs NcT2, respectively. Venn analysis showed that 10 up-regulated genes and one down-regulated gene were shared by various comparison groups. GO functional classification results showed the largest functional terms of DEGs in NcCK vs NcT1 and NcCK vs NcT2 were metabolic process, cellular process, single-organism process, cell, cell part, organelle, catalytic activity and binding, while DEGs in NcT1 vs NcT2 were mostly enriched in metabolic process, cellular process, single-organism process, catalytic activity and binding. In addition, KEGG pathway enrichment analysis indicated that DEGs within NcCK vs NcT1 and NcCK vs NcT2 were enriched in 80 and 79 pathways, respectively. The number of up-regulated genes enriched in glycolysis/gluconeogenesis and MAPK signaling pathway was more than that of down-regulated genes. Investigation of virulence factors displayed that spore wall protein 9 encoding gene and spore wall protein 12 encoding gene were down-regulated in NcCK vs NcT1 and NcCK vs NcT2, while spore wall protein 8 encoding gene was down-regulated only in NcCK vs NcT1. In additional, the expression levels of spore wall protein precursor encoding gene, spore wall and anchoring disk complex protein encoding gene, chitinase encoding gene, polar tube protein encoding gene, and ricin B lectin encoding gene were all up-regulated in NcCK vs NcT1 and NcCK vs NcT2. Moreover, the infection-associated factor analysis demonstrated that in NcCK vs NcT1 and NcCK vs NcT2 three key enzyme genes engaged in glycolytic pathway were up-regulated, while in NcCK vs NcT1 and NcCK vs NcT2 three ATP/ADP transferase-associated genes were up-regulated but one down-regulated. Two ABC transporterassociated genes were up-regulated in NcCK vs NcT1 and NcCK vs NcT2, while four down-regulated. Finally, the RT-qPCR results verified the authenticity of the transcriptomic data and expression trends of DEGs. 【Conclusion】 In this study the transcriptomic dynamics of N. ceranae infecting the A. m. ligustica worker was deciphered through the comparative analyses. Our findings revealed that genes encoding virulence factors including spore wall protein, spore wall and anchoring disk complex protein, chitinase, polar tube protein and ricin B lectin, and genes encoding infection-associated factors such as hexokinase, pyruvate kinase, 6-phosphofructokinase, ATP/ADP transferase and ABC transporters, are likely to play key roles in pathogen proliferation, providing a basis for clarifying the infection mechanism of N. ceranae.

Key words: Apis mellifera ligustica, Nosema ceranae, transcriptome, differentially expressed gene, infection mechanism, virulence factor, infectionassociated factor