Identification of four Spätzle genes (MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3) and their roles in the innate immunity of Macrobrachium nipponense

Abstract

Spätzle, an extracellular ligand of the Toll receptor, is involved in the innate immunity of crustaceans. In this study, four Spätzle genes were cloned from Macrobrachium nipponense and designed as MnSpz1, MnSpz2, MnSpz2-isoform, and MnSpz3. The coding region of the four Spätzle genes all contained one intron and two exons, and they were predicted to be produced by gene duplication based on sequence similarities and phylogenetic tree. The predicted MnSpz1, MnSpz2, and MnSpz3 proteins all contained a signal peptide and a Spätzle domain. No signal peptide but a Spätzle domain existed in MnSpz2-isoform because of frameshift mutation caused by 50 bp nucleotide deletion compared with MnSpz2. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that MnSpz1, MnSpz2, and MnSpz3 were expressed in all the detected tissues of M. nipponense, and MnSpz2 was found to be the major isoform in the heart, gills, stomach, and intestine. After stimulation by Vibrio parahaemolyticus, Staphylococcus aureus, or White spot syndrome virus (WSSV), the expression levels of MnSpz1, MnSpz2, and MnSpz3 changed. Given the high similarities among MnSpz1-3, RNA interference (RNAi) using dsRNA of MnSpz1 inhibited the expression of the three Spätzle genes (MnSpz1, MnSpz2 and MnSpz3). Silencing of MnSpz1-3 down-regulated the expression levels of nine antimicrobial peptide (AMP) genes in M. nipponense. After Knockdown of MnSpzs, the number of V. parahaemolyticus, S. aureus and WSSV copies in M. nipponense increased significantly in vivo. Our results suggest that Spätzles are involved in the innate immunity of M. nipponense. The expansion of MnSpz genes through gene duplication is beneficial to enhance the innate immune defense ability of M. nipponense.