Transcriptional dynamics and regulatory function of milRNAs in invading larvae.

In the present study, small RNA (sRNA) data from were filtered from sRNA-seq datasets from the gut tissues of -infected worker larvae, which were combined with the previously gained sRNA-seq data from spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem-loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between and milR-6882-x, as well as between and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of and a novel insight into the interaction between and honey bee larvae, but also provide candidate DEmilRNA-gene axis for further investigation.