Unraveling the mechanism underlying the immune responses of Apis mellifera ligustica to Nosema ceranae stress based on small RNA omics analyses

Abstract:

[Objective] To reveal the mechanism underlying immune responses of Apis mellifera ligustica to Nosema ceranae stress at small RNA transcriptome level based on deep sequencing and omics analysis. [Methods] A. m. ligustica workers' midguts at 7 and 10 days post N. ceranae stress (Am7T, Am10T) and corresponding normal midguts (Am7CK, Am10CK) were sequenced using small RNA-seq. Related bioinformatic softwares were used to perform quality control of sequencing data, identification of known miRNAs and novel miRNAs and analysis of structural features of miRNAs. The expression of novel miRNAs was verified by Stem-loop RT-PCR. Differentially expressed miRNAs (DEmiRNAs) in Am7CK vs. Am7T and Am10CK vs. Am10T comparison groups were screened out following the standards of|log₂(Fold change)| ≥ 1 and P ≤ 0.05. Target mRNAs of DEmiRNAs were predicted followed by annotation in GO and KEGG databases using softwares, based on annotation information, summary and investigation of cellular and humoral immune-associated pathways and enriched target mRNAs were conducted. Regulatory networks of DEmiRNAs and differentially expressed mRNAs (DEmRNAs) related to immune pathways were constructed according to target binding relationship. RT-qPCR was performed to validate the sequencing data and differential expression of DEmiRNAs. [Results] Here, 165895574 raw reads and 132028990 clean tags were yielded, and average Pearson correlation coefficients among different biological replicas in every group were above 87.92%. 928 known miRNAs and 56 novel miRNAs were identified. The length of these miRNAs was among 18-28 nt, with the most abundant length 18 nt and 22 nt; the first base of most miRNAs had a U bias. The true expression of 12 novel miRNAs was verified. 48 up-regulated miRNAs and 36 down-regulated miRNAs were identified in Am7CK vs. Am7T, while 56 up-regulated miRNAs and 51 down-regulated miRNAs were identified in Am10CK vs. Am10T. These DEmiRNAs can respectively target 9827 and 10720 mRNAs, involving in 50 and 47 functional terms and 138 and 135 pathways. Analysis of regulatory networks of DEmiRNAs and target mRNAs related to immune pathways showed 26 DEmiRNAs in Am7CK vs. Am7T could target 10 DEmRNAs such as endocytosis, whereas 15 DEmiRNAs in Am10CK vs. Am10T can target 10 immune-associated pathways including MAPK signaling pathway. The reliability of sequencing data and differential expression trend of four DEmiRNA was validated. [Conclusion] Host DEmiRNAs may response to N. ceranae through regulating material and energy metabolisms, as well as cellular and humoral immune, but might not regulate the expression of antimicrobial peptide-encoded genes; miR-1-z was likely to participate in cell proliferation, apoptosis and immune processes; oxidative phosphorylation pathway may play a special role in host immune response and host-pathogen interaction.