Heat-responsive ame-miR-1-3p modulates thermotolerance in honeybees (Apis mellifera L.)

Thermal stress significantly harms the survival, physiology and development of honeybees (Apis mellifera L.). miRNAs are emerging as key regulators of environmental stress responses. This study investigated honeybee's miRNA expression under heat stress and clarified the role of ame-miR-1-3p in thermotolerance. Global miRNA expression profiles were analysed in forager bees exposed to 45°C for 2 and 4 h using small RNA sequencing (sRNA-seq). To explore the effects of ame-miR-1-3p, young bees were fed miRNA mimics or antagonists to either overexpress or knock down its expression. The impact on target gene expression (Hsp90 and LOC413120) and the bees' heat susceptibility was then assessed. A dual luciferase reporter assay was used to confirm the direct targeting relationship between ame-miR-1-3p and LOC413120. The results showed that exposure to 45°C led to seven and five differentially expressed miRNAs (DEMs) at 2 and 4 h, respectively, all of which were suppressed. Bioinformatic analyses indicated that DEM target genes were enriched in stress-related signalling pathways including MAPK, phosphatidylinositol and Notch. ame-miR-1-3p, the most abundant DEM, was significantly down-regulated under heat exposure, as validated by qRT-PCR. Conversely, the expression of LOC413120 (encoding DnaJ homologue subfamily B member 12), a potential target of ame-miR-1-3p, was upregulated. Increasing ame-miR-1-3p expression significantly suppressed LOC413120 expression and reduced heat resistance, whereas decreasing ame-miR-1-3p expression significantly enhanced LOC413120 expression and increased heat resistance. Furthermore, dual luciferase analysis confirmed that LOC413120 is a direct target of ame-miR-1-3p. These findings reveal a novel function of miRNAs in honeybee heat resistance.