意大利蜜蜂响应东方蜜蜂微孢子虫胁迫的免疫应答

doi:10.3864/j.issn.0578-1752.2019.17.014

Abstract

Abstract:

【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 (Am7CK, Am7T, Am10CK and Am10T), DEGs in each comparison group were filtered out following the standard FDR≤1, P≤0.05 and |log₂fold 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-qPCR).【Result】 Differential expression analysis showed that there were 472 up-regulated and 385 down-regulated genes in Am7CK vs Am7T comparison group, and 611 up-regulated and 360 down-regulated genes in Am10CK vs Am10T 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 Am7CK vs Am7T 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 Am10CK vs Am10T 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 Am7CK vs Am7T 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, PI3K-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 PI3K-Akt signaling pathway were detected. Additionally, the up- and down-regulated genes in Am10CK vs Am10T 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 mRNA 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-qPCR 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.

Key words: Apis mellifera ligustica, Nosema ceranae, midgut, immune response, cellular immune, humoral immune

FU ZhongMin,CHEN HuaZhi,LIU SiYa,ZHU ZhiWei,FAN XiaoXue,FAN YuanChan,WAN JieQi,ZHANG Lu,XIONG CuiLing,XU GuoJun,CHEN DaFu,GUO Rui. Immune Responses of Apis mellifera ligustia to Nosema ceranae Stress[J].Scientia Agricultura Sinica, 2019, 52(17): 3069-3082.

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References 31

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)
Actin-F	CACTCCTGCTATGTATGTCGC
Actin-R	GGCAAAGCGTATCCTTCA
A-F	CATTGAGGATGAGCCCAT
A-R	CACGCTATCGTCAACTTCC
B-F	AGCGATTGAAGATGTGCC
B-R	ACGACAGGTGAAACCGAA
C-F	CATTGAGGATGAGCCCAT
C-R	GGTTTACCGCCATTCACA
D-F	TTCAGAACTCACGAGGCTG
D-R	AACCACAATGGCACCAGTA
E-F	CCTATGACAGGAGGGATGGT
E-R	GCACTGCCAGGTCTTCTACT
F-F	AACGAACCTTTGTCGGGT
F-R	TCCATCTGAATGCTGCCA

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
泛素介导的蛋白水解 Ubiquitin mediated proteolysis	ko04120	2	0
细胞凋亡 Apoptosis	ko04210	1	0
溶酶体 Lysosome	ko04142	0	1

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
PI3K-Akt信号通路 PI3K-Akt signaling pathway	ko04151	2	1
趋化因子信号通路 Chemokine signaling pathway	ko04062	1	0
NF-κB信号通路 NF-κB signaling pathway	ko04064	1	0
cAMP信号通路 cAMP signaling pathway	ko04024	0	1
Ras信号通路 Ras signaling pathway	ko04014	0	1
MAPK信号通路-酵母 MAPK signaling pathway - yeast	ko04011	0	1
FoxO信号通路 FoxO signaling pathway	ko04068	0	1

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
泛素介导的蛋白水解 Ubiquitin mediated proteolysis	ko04120	2	0
P450对外源物质代谢 Metabolism of xenobiotics by cytochrome P450	ko00980	2	0
药物代谢-细胞色素P450 Drug metabolism - cytochrome P450	ko00982	2	0
细胞凋亡 Apoptosis	ko04210	1	0
Fc-γ-R介导的吞噬作用 Fc-γ-R-mediated phagocytosis	ko04666	1	0
溶酶体 Lysosome	ko04142	1	0
内吞作用 Endocytosis	ko04144	1	1

通路名称 Pathway name	通路ID Pathway ID	上调基因 Up-regulated gene	下调基因 Down-regulated gene
NF-κB信号通路 NF-κB signaling pathway	ko04064	1	0
PI3K-Akt信号通路 PI3K-Akt signaling pathway	ko04151	0	1

Immune Responses of Apis mellifera ligustia to Nosema ceranae Stress

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