Bacterial outer membrane proteins modulate extracellular Dscam isoforms and Dorsal-mediated immunity in Eriocheir sinensis.

The Down syndrome cell adhesion molecule (Dscam) is a highly diverse immune receptor produced through extensive alternative splicing, but whether specific bacterial surface ligands can guide stimulus-specific Dscam splicing and subsequent immune signaling in crustaceans remains unclear. In this study, we examined the role of the Chinese mitten crab (Eriocheir sinensis) Dscam in hemocyte responses to two outer membrane proteins of Vibrio parahaemolyticus, OmpA0764 and OmpA1186. Bacterial challenge increased Dscam expression in hemocytes. Both recombinant proteins significantly elevated Dscam and multiple antimicrobial peptide (AMP) gene levels and also promoted Dorsal nuclear translocation. Conversely, RNA interference-mediated knockdown of Dscam markedly reduced these responses. Exon-targeted amplicon sequencing further revealed that OmpA0764 and OmpA1186 triggered distinct exon usage patterns within the extracellular exon 4 and exon 6 variable clusters of Dscam. OmpA0764 favored isoforms containing Exon4.25 and Exon6.15, while OmpA1186 favored Exon4.17 and Exon6.41. Functional studies showed that silencing these ligand-enriched exons significantly impaired Dorsal nuclear accumulation and downstream AMP expression. Collectively, these results suggest that bacterial outer membrane proteins not only induce Dscam expression but also modulate ligand-biased extracellular isoform profiles that are associated with immune signaling efficiency. This research highlights extracellular Dscam alternative splicing as a potential mechanism linking bacterial ligand-induced isoform remodeling to Dorsal activation and humoral antibacterial defense in the Chinese mitten crab.