Profiling and functional validation of LPS-responsive miRNAs in the giant freshwater prawn, Macrobrachium rosenbergii

MicroRNAs (miRNAs) are key post-transcriptional regulators involved in crustacean immune responses, yet their roles during pattern recognition receptor activation remain poorly understood. In this study, we investigated the miRNA response of Macrobrachium rosenbergii to lipopolysaccharide (LPS), a conserved bacterial pathogen-associated molecular pattern (PAMP). High-throughput small RNA sequencing identified 155 differentially expressed miRNAs (DEMs) in the hepatopancreas following LPS injection. Pathway enrichment analysis revealed that target genes of these DEMs were significantly associated with immune pathways, including the ubiquitin–proteasome pathway (UPP) and endocytosis. Network analysis highlighted mro-miR-365-1-5p as a central regulator targeting key genes such as Rpn10, Rab11, Vps2, and ArfGAP3. Temporal expression profiling revealed rapid downregulation of mro-miR-365-1-5p after LPS exposure. The regulatory role of mro-miR-365-1-5p was validated by dual-luciferase reporter assays and an in vivo challenge experiment. In vivo overexpression of mro-miR-365-1-5p significantly increased mortality in prawns challenged with Vibrio harveyi, indicating impaired immune defense. These findings provide novel insights into the regulatory roles of LPS-responsive miRNAs in crustaceans and suggest that modulation of specific miRNAs may be crucial for effective immune activation during bacterial infection.