Dscam alternative splicing tunes intestinal immunity and microbiota homeostasis in the Chinese mitten crab

Intestinal immunity in crustaceans must control invading pathogens while maintaining a stable microbial community, yet the molecular mechanisms that connect immune recognition to tissue-specific signaling outputs remain poorly understood. In the Chinese mitten crab (Eriocheir sinensis), Dscam generates vast receptor diversity via alternative splicing across three extracellular exon clusters, two transmembrane (TM) exons, and three intracellular variable exons. However, how transmembrane and intracellular domain splice variants (ICD) are paired in the intestine and how these combinations influence antibacterial defense remain unknown. Here, we developed an oral intestinal infection model in the Chinese mitten crab using bacteria Vibrio parahaemolyticus and Staphylococcus aureus. Oral challenge led to broad induction of antimicrobial peptides (AMPs) and increased intestinal transmembrane Dscam expression. Systemic double-stranded RNA (dsRNA) knockdown of transmembrane Dscam resulted in higher culturable bacterial loads in the gut and significantly decreased survival after infection. Splicing analysis showed a persistent bias toward the second transmembrane exon (TM2), whereas bacterial challenge selectively remodeled ICD splicing to produce ICD-truncated isoforms, thereby altering the ICD composition of the predominantly TM2-containing receptor pool in the gut. Isoform-specific silencing demonstrated that TM2-containing Dscam isoforms dominate immune responses by promoting nuclear translocation of Dorsal, a nuclear factor κB (NF-κB) family transcription factor, and by inducing stronger AMP expression than TM1-containing isoforms. Lastly, TM-specific knockdown disrupted gut microbial structure and reduced diversity, with the greatest dysbiosis observed following TM2 depletion. Overall, these results reveal that TM-ICD combinatorial splicing acts as a dynamic regulatory layer linking intestinal Dscam isoform composition to NF-κB-dependent antibacterial immunity and microbiota homeostasis in a decapod crustacean.