RhoA, a small GTPase, inhibits bacterial infection through regulated phagocytosis in the Chinese mitten crab,...

Small GTPase RhoA is a pivotal regulator of cytoskeletal dynamics and phagocytosis in mammalian phagocytes, yet its functional role in crustacean immunity remains poorly characterized. In this study, we identified and characterized RhoA from Eriocheir sinensis (designated EsRhoA), demonstrating its essential role in hemocyte phagocytosis and antibacterial defense. The EsRhoA gene encodes a 257-amino-acid protein containing a conserved RHO domain and displays over 90 % sequence similarity to orthologs in both vertebrates and invertebrates. Transcriptional analysis revealed widespread expression of EsRhoA across various tissues, with a significant upregulation observed in hemocytes following infection with Vibrio parahaemolyticus. RNA interference (RNAi)-mediated knockdown of EsRhoA in hemocytes led to decreased expression of phagocytosis-related effectors ROCK2 and Arp2/3, resulting in a 62 % reduction in bacterial phagocytosis as measured by flow cytometry. Immunocytochemistry analysis confirmed that EsRhoA protein translocates to the hemocyte membrane during pathogen-induced phagocytosis, suggesting a mechanistic similarity to integrin-mediated internalization in mammalian cells. Importantly, in vivo knockdown of EsRhoA significantly increased bacterial loads in hemolymph and reduced crab survival after infection. Collectively, these findings establish EsRhoA as an evolutionarily conserved regulator of hemocyte phagocytosis that provides critical protection against bacterial pathogens in crustaceans, offering novel insights into the evolution of immune cell phagocytosis mechanisms and potential applications in crustacean aquaculture disease management.