Precursor and Mature miRNA Sequences from the Hepatopancreas of Macrobrachium rosenbergii under Low-Temperature Stress

All Macrobrachium rosenbergii individuals used in this study were sourced from Jiangsu Shufeng Aquatic Seed Industry Co., Ltd. A total of 675 healthy prawns (average body length 4.5 ± 0.5 cm, body weight 2.6 ± 0.5 g), aged three months and randomly selected from 27 families of the selectively bred G04 generation, were subjected to acute cold stress treatment. The prawns were housed in a 4 m² indoor tank, where water temperature was gradually decreased from 26 °C to 0 °C at a rate of approximately 1 °C every 4 hours by incremental addition of ice. This cooling process continued until all experimental individuals succumbed to the cold stress.A matched control group of 675 prawns, from the same families and maintained at the same rearing density, was held at a constant temperature of 26 ± 0.2 °C. Throughout the 48-hour experimental period, all tanks were continuously aerated, feed was withheld, and water quality parameters—including pH, dissolved oxygen, ammonia nitrogen, and nitrite—were kept stable, with temperature as the sole variable. Prior heritability analyses indicated no significant difference in cold tolerance among the 27 families used.Based on mortality timing during the cold stress trial, individuals were categorized into three groups: the control hepatopancreas default group (CHD) maintained at 26 °C; the early death group (EH), representing the earliest 10% mortality under cold stress; and the late death group (LH), representing the latest 10% mortality. From each group, three prawns were randomly selected for hepatopancreas tissue collection. Tissues were immediately flash-frozen in liquid nitrogen and stored at –80 °C.Total RNA was extracted from these hepatopancreas samples for small RNA sequencing. Both precursor and mature miRNA sequences were identified and analyzed to detect differential expression patterns in response to acute low-temperature stress. Subsequent target gene prediction and functional enrichment analyses were performed to elucidate the molecular regulatory roles of miRNAs in the cold stress adaptation mechanisms of Macrobrachium rosenbergii.