Abstract

ADP-ribosylation factor 6 (Arf6) is a small GTPase that coordinates membrane trafficking and cytoskeletal dynamics, processes essential for innate immune responses. However, its specific role in crustacean immunity remains largely unexplored. In this study, an Arf6 homolog from the Chinese mitten crab ( Eriocheir sinensis ), designated Es Arf6, was identified and functionally characterized. Es Arf6 encodes a 175-amino-acid protein that is highly conserved across vertebrate and invertebrate species. Quantitative real-time PCR analysis revealed that Es Arf6 is ubiquitously expressed in crab tissues, with highest transcript levels detected in hemocytes and hepatopancreas. Following challenge with either Staphylococcus aureus or Vibrio parahaemolyticus , Es Arf6 expression was significantly upregulated in hemocytes in a time-dependent manner. Functional analyses demonstrated that RNA interference (RNAi)-mediated silencing of Es Arf6 markedly reduced the phagocytic capacity of hemocytes toward both Gram-positive and Gram-negative bacteria in vitro . Consistently, in vivo knockdown of Es Arf6 resulted in elevated bacterial loads in the hemolymph and significantly decreased host survival following bacterial infection. These findings indicate that Es Arf6 is required for efficient antibacterial defense in E. sinensis , primarily through the regulation of hemocyte-mediated phagocytosis. Overall, this study provides new insight into the conserved role of Arf6 in invertebrate innate immunity and expands current understanding of immune regulatory mechanisms in crustaceans.