Eriocheir sinensis 14-3-3ζ regulates hemocyte phagocytosis, apoptosis and antimicrobial peptide expression during Spiroplasma eriocheiris infection.

Tremor disease, caused by Spiroplasma eriocheiris in the Chinese mitten crab (Eriocheir sinensis), is highly destructive, leading to mass mortality and substantial economic losses in crab aquaculture. Our previous studies have confirmed that E. sinensis 14-3-3ζ (Es14-3-3ζ) contributes to host defense against S. eriocheiris infection in crabs. However, the detailed mechanism by which Es14-3-3ζ regulates host resistance to S. eriocheiris requires further investigation. In this study, we demonstrated that miR-2309 inhibited hemocyte phagocytic activity toward S. eriocheiris via targeting the 3' UTR of Es14-3-3ζ. Similarly, knockdown of Es14-3-3ζ by dsRNA also resulted in compromised hemocyte phagocytosis. In contrast, overexpression of Es14-3-3ζ in Drosophila S2 cells significantly promoted S. eriocheiris internalizationand subsequent intracellular transport to lysosomes. Confocal analysis revealed that Es14-3-3ζ colocalized with F-actin and lysosomes in the cytosol. Furthermore, transfection of miR-2309 mimics significantly promoted hemocyte apoptosis, whereas treatment with miR-2309 inhibitors decreased the apoptotic rate. In Drosophila S2 cells, Es14-3-3ζ overexpression evidently inhibited S. eriocheiris infection-induced apoptosis. Additionally, in hemocytes, both miR-2309-mediated repression of Es14-3-3ζ and RNA interference significantly downregulated immune-related genes including Rab7, ERK, Relish and anti-lipopolysaccharide factors. In conclusion, our findings revealed a critical role of Es14-3-3ζ in controlling innate immunity by promoting phagocytosis and antimicrobial peptide production while inhibiting apoptosis, offering a potential therapeutic target against pathogenic infection in aquatic crustaceans.