Hemolymph exosomes inhibit Spiroplasma eriocheiris infection by promoting Tetraspanin‐mediated hemocyte phagocytosis in crab

Abstract Exosomes released from infected cells are thought to play an important role in the dissemination of pathogens, as well as in host‐derived immune molecules during infection. As an intracellular pathogen, Spiroplasma eriocheiris is harmful to multiple crustaceans. However, the immune mechanism of exosomes during Spiroplasma infection has not been investigated. Here, we found exosomes derived from S. eriocheiris ‐infected crabs could facilitate phagocytosis and apoptosis of hemocytes, resulting in increased crab survival and suppression of Spiroplasma intracellular replication. Proteomic analysis revealed the altered abundance of Es Tetraspanin may confer resistance to S. eriocheiris , possibly by mediating hemocyte phagocytosis in Eriocheir sinensis . Specifically, knockdown of Es Tetraspanin in E. sinensis increased susceptibility to S. eriocheiris infection and displayed compromised phagocytic ability, whereas overexpression of Es Tetraspanin in Drosophila S2 cells inhibited S. eriocheiris infection. Further, it was confirmed that intramuscular injection of recombinant LEL domain of Es Tetraspanin reduced the mortality of S. eriocheiris ‐infected crabs. Blockade with anti‐ Es Tetraspanin serum could exacerbate S. eriocheiris invasion of hemocytes and impair hemocyte phagocytic activity. Taken together, our findings prove for the first time that exosomes modulate phagocytosis to resist pathogenic infection in invertebrates, which is proposed to be mediated by exosomal Tetraspanin, supporting the development of preventative strategies against Spiroplasma infection.