Abstract

As a cytokine-like ligand, Spätzle activates the Toll signaling pathway through binding to Toll-like receptors (TLRs), thereby participating in the innate immune defense of crustaceans against diverse pathogens. A novel Spätzle (MrSpz5) was cloned from Macrobrachium rosenbergii in this study. The MrSpz5 cDNA is 1,900 bp, comprising a 972-bp open reading frame (ORF) encoding 323 amino acids, a 662-bp 5' UTR, and a 266-bp 3' UTR. MrSpz5 was constitutively expressed in various tissues, with the highest expression in the gills. Knockdown of MrSpz5 via RNA interference in M. rosenbergii significantly down-regulated the expression of MrTLR1, MrTLR5, MrALF4, MrALF5, and MrCrustin1-4 after Vibrio parahaemolyticus infection. Concurrently, phenoloxidase (PO) concentration, alkaline phosphatase (AKP) and acid phosphatase (ACP) enzymatic activities, as well as the expression of MrSpz, MrTLR3, and MrALF1 increased obviously in the same treatment group. Similarly, in MrSpz5-knockdown prawns challenged with Vibrio alginolyticus, PO concentration, AKP activity, and the expression of MrTLR3, MrALF1, MrALF2, and MrSpz were significantly enhanced. In contrast, the expression of MrTLR1, MrTLR4, MrTLR5, MrALF3, MrALF4, MrALF5, and MrCrustin1-4 was notably suppressed. Furthermore, MrSpz5-knockdown M. rosenbergii exhibited significantly higher cumulative mortality compared to all other treatments after challenge with either V. parahaemolyticus or V. alginolyticus. Moreover, the dual-luciferase reporter assay demonstrated that MrSpz5 was the target gene of miR-56. MiR56 knockdown induced an obvious up-regulation of MrSpz5 and other immune gene expression in M. rosenbergii infected with either V. parahaemolyticus or V. alginolyticus. Collectively, these findings indicated that MrSpz5 mediated immune defense to V. alginolyticus and V. parahaemolyticus in the prawns.