Abstract:[Objective] Ascosphaera apis is a lethal fungal pathogen for Apis cerana cerana larvae. microRNA can participate in host-pathogen interaction processes by inhibition or degradation of mRNA via targeting at post-transcriptional level. The aim of this study was to analyze the differentially expressed miRNAs (DEmiRNAs) and their target genes in the 6-day-old larval gut of A. c. cerana under A. apis stress and reveal DEmiRNAs' roles in the stress response process. [Methods] Normal and A. apis-challenged 6-day-old larval guts of A. c. cerana (AcCK and AcT) were sequenced using Illumina MiSeq platform, followed by prediction and analysis of DEmiRNAs and their target genes using related softwares. Target genes of DEmiRNAs were annotated to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with Blast. Regulation networks between DEmiRNAs and target mRNAs were constructed using Cytoscape. Stem-loop RT-PCR and qPCR were conducted to verify the reliability of sequencing data. [Results] Deep sequencing of larval gut samples generated 537 miRNAs, the length of which was distributed between 16 nt and 35 nt. The first base bias of miRNAs with various length had apparent difference. The expression of 10 novel miRNAs were validated using Stem-loop RT-PCR. There were 54 DEmiRNAs in AcCK vs. AcT comparison group, including 31 up-regulated and 23 down-regulated miRNAs, which can respectively link 6170 and 8199 target genes. GO classification suggested that target genes of up-and down-regulated miRNAs were respectively involved in 47 and 47 terms, and the largest ones were binding, cellular process, and catalytic activity. KEGG pathway enrichment analysis demonstrated that target genes of up-and down-regulated miRNAs were respectively engaged in 134 and 126 pathways, and the mostly enriched ones were endocytosis and protein processing in endoplasmic reticulum. Analysis of regulation networks revealed that very complex regulation relationships existed between DEmiRNAs and corresponding target mRNAs; 31 miRNAs could bind 51 mRNAs associated with ubiquitin mediated proteolysis, and 18 miRNAs can bind 14 Jak-STAT signaling pathway-associated mRNAs; a total of 16 miRNAs, such as miR-1277-x, miR-26-x, miR-27-y, miR-30-x and miR-6052-x, can participate in regulating both of the above-mentioned immune pathways. Finally, three DEmiRNAs were randomly selected for qPCR, the result verified the reliability of our transcriptome sequencing data. [Conclusion] We first provided the expression profile and differential expression information of A. c. cerana miRNAs during the late stage of A. apis stress, revealed the complex interactions between A. apis and host at transcriptome level. As the core of regulation networks, miR-6052-x and miR-1277-x were likely to participate in host immune defense by affecting apoptosis, while miR-26-x and miR-30-x may join host responses to A. apis stress via regulation of Jak-STAT signaling pathway. Key DEmiRNAs screened in our study are expected to be used as potential molecular targets for chalkbrood control.
