Cloning and function of prophenoloxidase activating factors gene from the epidermis of Macrobrachium nipponense

Prophenoloxidase activation is a crucial innate immune response in crustaceans against pathogenic microorganisms infections, with prophenoloxidase-activating factors (PPAFs) being pivotal in this activation. Despite their importance, crustacean epidermal PPAFs have been seldom studied. To elucidate the role of epidermal PPAFs in crustaceans immunity, a novel PPAF gene, MnPPAF1 (GenBank accession: OP784577), was cloned and characterized from the epidermis of Macrobrachium nipponense using PCR and RACE techniques, based on prior transcriptome data. Bioinformatics, qPCR and RNAi methods were employed to analyze the gene sequence, spatial and temporal expression patterns, transcription levels of epidermal MnPPAF1, phenoloxidase (PO) activity, and shrimps mortality following Aeromonas hydrophila challenge. The complete MnPPAF1 cDNA sequence is 1 718 bp, encoding a 460-amino acid protein with clip-domain serine proteinase (Clip-SP) and trypsin-like serine protease (Tryp-SP) domains. MnPPAF1 expression was detechted in various tissues, including ventral and dorsal epidermis, hemocytes, gills, stomach, heart, hepatopancreas, with epidermal expression correlating to the molting cycle. Expression levels in the later premolt stage (D₄ stage) were 857% higher than in the intermolt stage (C stage). After A. hydrophila challenge, epidermal MnPPAF1 expression peaked at 6 h, increasing by 227% over the control group, while epidermal PO activity peaked at 12 h, increasing by 24%. 3 μg of dsRNA solution was injected into the second segment of the abdomen and back every 12 h, 3 injections in total. RNAi has the highest interference efficiency 12 h after the last injection, where the expression of epidermal MnPPAF1 decreased by 71.79%, compared to the control group, and after 24 h, the activity of epidermal PO decreased by 72.31%. Challenge at the time point with the highest interference efficiency and within 120 h, the cumulative mortality of shrimp in the challenged + RNAi group increased by 26% compared with that in the challenged + non-RNAi group. These findings indicate that MnPPAF1, an important immune factor in the epidermis, was involved in regulating the activation of the prophenoloxidase system. The down-regulation of this gene expression can significantly increase the mortality of shrimp infected by A. hydrophila. This study contributes to a better understanding of the epidermis's role in the crustaceans immune system.