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.

Spätzle 是 Toll 受体的细胞外配体，参与甲壳类动物的先天免疫。在这项研究中，从日本沼虾中克隆了四个Spätzle基因，并设计为MnSpz1、MnSpz2、MnSpz2-isoform和MnSpz3。四个Spätzle的编码区基因均包含一个内含子和两个外显子，根据序列相似性和系统发育树预测它们是通过基因复制产生的。预测的 MnSpz1、MnSpz2 和 MnSpz3 蛋白都包含一个信号肽和一个 Spätzle 结构域。与MnSpz2相比，由于 50 bp 核苷酸缺失引起的移码突变，在 MnSpz2 异构体中不存在信号肽，但存在 Spätzle 结构域。定量实时聚合酶链反应 (RT-qPCR) 分析表明MnSpz1、MnSpz2和MnSpz3在所有检测到的M. nipponense和MnSpz2组织中均有表达。被发现是心脏、鳃、胃和肠中的主要异构体。副溶血性弧菌、金黄色葡萄球菌或白斑综合征病毒(WSSV)刺激后， MnSpz1、MnSpz2和MnSpz3的表达水平发生变化。鉴于 MnSpz1-3 之间的高度相似性，使用MnSpz1的dsRNA 的 RNA 干扰 (RNAi)抑制了三个Spätzle基因（MnSpz1、MnSpz2和MnSpz3）的表达。MnSpz1-3的沉默下调了九种抗菌肽的表达水平（AMP ) 基因在M. nipponense中。敲除MnSpz后，体内M. nipponense中副溶血性弧菌、金黄色葡萄球菌和WSSV拷贝数显着增加。我们的结果表明Spätzle s 参与了日本分枝杆菌的先天免疫。通过基因复制扩展MnSpz基因有利于增强日本分枝杆菌的先天免疫防御能力。