Spätzle maintains homeostasis of hemolymph microbiota in Scylla paramamosain through Toll2

The Toll pathway is crucial for innate immune responses in organisms (including Drosophila and mammals). The Spätzle protein outside of cells acts as a ligand for Toll receptors, enabling the transfer of signals from outside the cell to the inside. However, the function of Spätzle in the immune system of mud crab (Scylla paramamosain) remains unclear. This research discovered a novel Spätzle gene (Sp-Spz) in mud crab, which showed extensive expression in all the tissues that were examined. The RNA interference exhibited the correlation between Sp-Spz and the anti-lipopolysaccharide factors (ALFs). Knockdown of Sp-Spz decreased the expression of Sp-Toll2 but not Sp-Toll1. In Drosophila Schneider 2 cells, Sp-Spz was found interacted with Sp-Toll2. Moreover, the depletion of Sp-Spz caused the separation of hepatic lobules from the basement membrane, resulting in the disruption of the structural coherence of hepatopancreatic cells. Additionally, the knockdown of Sp-Spz resulted in changes to the composition of the hemolymph microbiota, specifically affecting the proportions of different phylum and family levels. The findings indicated that Sp-Spz may promote the synthesis of ALFs via Sp-Toll2, thereby influencing the homeostasis of microbiota in the hemolymph. In this study, novel insights into mud crab immunity are provided.