Kaimin Zhou

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 shape ligand-biased extracellular isoform profiles that influence immune signaling efficiency. This research highlights extracellular Dscam alternative splicing as a mechanism linking bacterial ligand recognition to Dorsal activation and humoral antibacterial defense in the Chinese mitten crab.

Cyclins are core regulators of cell-cycle progression and also participate in transcriptional regulation and cellular stress responses. However, the cyclin family has not been systematically characterized in the Chinese mitten crab Eriocheir sinensis, and its potential role in hemocyte proliferation during antibacterial responses remains unclear. In this study, 18 cyclin family members were identified from the E. sinensis genome and designated as EsCyclins. Comparative analyses showed that these family members are distributed across 13 chromosomes and can be assigned to the major cyclin subfamilies on the basis of phylogenetic relationships, conserved motifs, gene structures, and domain organization. Representative cyclins, including EsCycA, EsCycB, EsCycB3, EsCycD, and EsCycE, displayed conserved sequence features and similar predicted core three-dimensional structures relative to invertebrate and vertebrate homologs. Tissue expression analysis further showed that these genes are broadly expressed but tissue biased, with relatively high transcript levels in hepatopancreas, testis, and, depending on the gene, stomach or muscle. After stimulation with heat-killed Vibrio parahaemolyticus, representative EsCyclin genes showed dynamic, gene-specific temporal expression patterns in synchronized primary hemocytes. Functional assays demonstrated that siRNA-mediated knockdown of EsCycA, EsCycD, or EsCycE significantly reduced hemocyte proliferation under bacterial stimulation, as assessed by flow cytometry and EdU incorporation. Together, this study provides the first systematic characterization of the cyclin family in E. sinensis and identifies EsCycA, EsCycD, and EsCycE as positive regulators of bacteria-induced hemocyte proliferation in vitro. These findings extend our understanding of cyclin-family evolution in crustaceans and provide a foundation for investigating how cell-cycle regulators participate in crustacean antibacterial cellular responses.

Antimicrobial peptides (AMPs) are crucial effectors in crustacean intestinal immunity, yet identifying novel crustacean AMPs is highly challenging due to rapid sequence divergence and poor homology with model organisms. In this study, an oral infection model of Vibrio parahaemolyticus (V. parahaemolyticus, Vp) was established to mimic bacterial enteritis in the Chinese mitten crab (Eriocheir sinensis). Through transcriptomic analysis, a significantly upregulated, previously uncharacterized transcript (LOC126997648) was discovered. Overcoming the limitations of traditional sequence-based annotation, we utilized AlphaFold 3 and Foldseek to reveal that this unannotated protein shares a highly conserved three-dimensional structural architecture with Drosophila Attacins, despite low amino acid sequence identity. Consequently, this novel structural homolog was designated as EsAttacin (EsAtt). Spatial and temporal expression profiles demonstrated that EsAtt was predominantly expressed in the intestine and acutely upregulated following Vp challenge. In vitro binding assays indicated that recombinant EsAtt (rEsAtt) exhibited dose-dependent binding activity to bacterial pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS) and peptidoglycan (PGN). In vivo functional analyses revealed that RNA interference (RNAi)-mediated knockdown of EsAtt significantly exacerbated bacterial loads and accelerated crab mortality. Conversely, the administration of rEsAtt facilitated bacterial clearance and significantly improved survival of infected crabs. Furthermore, the transcription of EsAtt was found to be strictly governed by the IMD signaling cascade, as the knockdown of EsRelish or EsImd significantly suppressed its expression. Collectively, our findings identify a structurally conserved but previously uncharacterized immune effector, EsAtt, and highlight the indispensable role of the IMD–Relish–Attacin axis in the crustacean intestinal defense against enteric bacterial infections.

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 exons, and three intracellular variable exons. However, how transmembrane (TM) and intracellular-domain (ICD) splice variants 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 exon (TM2) of the transmembrane domain, while bacterial challenge selectively remodeled splicing of the intracellular domain (ICD) toward truncated cytoplasmic tails, reshaping the TM–ICD landscape in the gut. Isoform-specific silencing demonstrated that Dscam isoforms containing TM2 dominate immune responses by promoting nuclear translocation of Dorsal, a nuclear factor κB (NF-κB) family transcription factor, and inducing stronger AMP expression than those with TM1. 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.

Integrins are evolutionarily conserved adhesion receptors that play pivotal roles in phagocytosis across metazoans, yet the specific αβ heterodimers involved in crustacean cellular immunity remain poorly defined. In this study, we identified and characterized an integrin α subunit, designated integrin αPS3 (EsITGAPS3), from the economically important Chinese mitten crab, Eriocheir sinensis. Bioinformatic analyses revealed that EsITGAPS3 shares structural similarity with arthropod integrin αPS3/αPS4/αPS5 subfamily members but exhibits low sequence conservation across distant taxa. EsITGAPS3 was ubiquitously expressed in crab tissues, with the highest levels in immune‑relevant organs such as hemocytes, hepatopancreas, and gills. Its transcription was significantly induced in hemocytes following in vitro challenge with Vibrio parahaemolyticus. Functional knockdown of EsITGAPS3 via RNA interference markedly impaired hemocyte phagocytosis of FITC‑labeled bacteria, demonstrating its essential role in antibacterial cellular immunity. Using AlphaFold‑based structural prediction, we identified integrin βPS‑like subunit integrin βPS1 (EsITGBPS1) as the strongest binding partner for EsITGAPS3, and co‑immunoprecipitation confirmed their physical interaction and heterodimer formation. Importantly, RNAi‑mediated silencing of EsITGBPS1 similarly compromised bacterial phagocytosis, indicating that the integrin αPS3βPS1 (EsITGAPS3-EsITGBPS1) heterodimer functions as a key receptor complex in crab hemocytes. Our findings define a specific integrin αPS3βPS1 pair required for efficient phagocytosis in E. sinensis, providing mechanistic insight into integrin‑mediated immune adaptation in crustaceans and highlighting a potential target for enhancing disease resistance in aquaculture.

Down syndrome cell adhesion molecule (Dscam) generates tens of thousands of isoforms by alternative splicing, thereby providing crucial functions during immune responses. In this study, a novel Dscam signaling pathway was investigated in crab, which remains poorly characterized in invertebrates. Bacterial infection induced the cytoplasmic cleavage of Dscam intracellular domains (ICDs) by γ-secretase, and then the released ICDs carrying specific alternatively spliced exons could directly interact with IPO5 to facilitate nuclear translocation. Nuclear imported ICDs thus promoted hemocyte proliferation and protect the host from bacterial infection. Protein-interaction studies revealed that the ectodomain of Dscam bound to a disintegrin and metalloprotease domain 10 (ADAM10) rather than ADAM17. Inhibition or overexpression of ADAM10 impaired or accelerated Dscam shedding activity post–bacterial stimulation, respectively. Moreover, the shedding signal then mediated Dscam with an intact cytoplasmic domain to promote the cleavage of ICDs by γ-secretase. Furthermore, the transcription of ADAM10 was regulated by Dscam-induced canonical signaling, but not nuclear imported ICDs, to serve as a feedback regulation between two different Dscam pathways. Thus, membrane-to-nuclear signaling of Dscam regulated hemocyte proliferation in response to bacterial infection.