摘 要：
【Objective】The objective of this study is to reveal the cellular and humoral immune responses of Apis mellifera ligusitca worker's midgut to Nosema ceranae stress, and to provide a basis for screening and functional study of key immune response genes by deep investigation of host differentially expressed genes(DEGs) and immune pathways.【Method】Base on the previously obtained transcriptome datasets of normal and N. ceranae-stressed midguts of A. m. ligustica 7-and 10-day-old workers' midgut samples(Am7 CK, Am7 T, Am10 CK and Am10 T), DEGs in each comparison group were filtered out following the standard FDR≤1, P≤0.05 and |log2 fold change|≥1. Additionally, Pearson correlations, Venn analysis, GO classification and KEGG pathway enrichment analysis were conducted by using related bioinformatic softwares. Further, DEGs enriched in immune pathways were summarized and analyzed. Finally, the reliability of transcriptome data was verified via real-time quantitative PCR(RT-q PCR).【Result】Differential expression analysis showed that there were 472 up-regulated and 385 down-regulated genes in Am7 CK vs Am7 T comparison group, and 611 up-regulated and 360 down-regulated genes in Am10 CK vs Am10 T comparison group. Venn analysis suggested that the number of specific DEGs of the aforementioned two comparison groups was 739 and 853, respectively, and 118 DEGs were shared. GO classification indicated that the up-and down-regulated genes in Am7 CK vs Am7 T were respectively annotated to 23 and 29 functional terms; among them, the top five terms of up-regulated genes were binding, catalytic activity, metabolic process, cellular process and single tissue process; while the top five terms of down-regulated genes were metabolic process, single tissue process, catalytic activity, cellular process and binding. In addition, the up-and down-regulated genes in Am10 CK vs Am10 T were respectively involved in 36 and 26 functional terms; among them, the top five terms of up-regulated genes were single process, binding, cellular process, catalytic activity and metabolic activity; while the top five terms of down-regulated genes were binding, cellular process, catalytic activity, metabolic process and single process. KEGG metabolic pathway enrichment analysis showed that the up-and down-regulated genes in Am7 CK vs Am7 T were respectively enriched in 38 and 33 pathways, and among them the top five enriched pathways of up-regulated genes were bile secretion, endoplasmic reticulum protein processing, ubiquitin-mediated proteolysis, PI3 K-Akt signaling pathway and neurotrophic factor signaling pathway; while the top five enriched pathways of down-regulated genes were cytoplasmic DNA-sensing pathway, purine metabolism, pyrimidine metabolism, RNA polymerase and ribosome; three cellular immune pathways including ubiquitin-mediated proteolysis, and seven humoral pathways including PI3 K-Akt signaling pathway were detected. Additionally, the up-and down-regulated genes in Am10 CK vs Am10 T were respectively associated with 54 and 43 pathways, and among them the top five enriched pathways of up-regulated genes were Hippo signaling pathway, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, ubiquitin-mediated proteolysis, and sphingolipid metabolism; while the top five enriched pathways of down-regulated genes were m RNA surveillance pathway, sphingolipid signaling pathway, fructose and mannose metabolism, galactose metabolism, and sphingolipid metabolism; seven cellular immune pathways including ubiquitin-mediated proteolysis, and two humoral pathways including NF-κB signaling pathway were found. The result of RT-q PCR verification demonstrated the expression trend of six randomly selected DEGs was consistent with that of the sequencing data, which confirmed the reliability of the transcriptome data. Further analysis noted that NF-κB signaling pathway was activated by N. ceranae in both 7-and 10-day-old workers' midguts of A. m. ligustica, followed by immediate initiation of three antimicrobial peptides such as apidaecin, defensin-1 and hymenoptaecin, indicating their key importance in host defense against N. ceranae invasion.【Conclusion】The immune responses of A. m. ligusitca worker's midgut to N. ceranae were uncovered at transcriptomic level, and it was revealed that the host made both cellular and humoral immune responses at the early stage of N. ceranae stress. The host cellular immune responses may play a major role in resisting pathogen invasion. The host cellular immune continued to increase at the later stage of fungal stress, while the humoral immune drastically weakened. The ubiquitin mediated proteolysis and enriched DEGs, NF-κB signaling pathway and enriched DEGs, and antibacterial peptide-encoded genes such as apidaecin, defensin-1 and hymenoptaecin were likely to play pivotal roles in host immune response to N. ceranae stress.
