Specific Notch receptor and its ligand Delta interactions control antibacterial immunity by regulating hemocyte proliferation in Eriocheir sinensis

Infection or injury to aquatic invertebrates in aquaculture can lead to a significant loss of circulating hemocytes. Functional studies and single-cell RNA sequencing have revealed the key role of hemocyte proliferation in protecting invertebrates from pathogenic microbial attacks. However, molecular mechanisms underlying the regulation of hemocyte proliferation during the innate immune response remain unelucidated. In this study, we found that Notch receptor-specific ligand interactions in Chinese mitten crab (Eriocheir sinensis) mediate this process. We first confirmed the molecular underpinnings of EsNotch receptor and ligand EsDelta interactions. Coimmunoprecipitation and confocal analyses emphasized the important role of the epidermal growth factor (EGF)11–13 region of EsNotch and the key regions (DSL domain and first three EGF domains) of EsDelta in receptor-ligand specific binding. Gene expression analysis revealed that EsNotch and EsDelta are ubiquitously distributed in various tissues and both can regulate the expression of hemocyte proliferation-related genes. Subsequent studies reported that EsNotch and EsDelta were significantly induced after infection by Vibrio parahaemolyticus, whereas their silencing decreased crab survival and hemocyte proliferation while increasing bacterial concentrations in the hemolymph. Taken together, these results reveal the key role of Notch receptor-ligand interactions in the regulation of hemocyte proliferation after pathogen stimulation and provide new perspectives and theoretical foundations for a better understanding of disease control in crustacean breeding. •EsNotch and EsDelta interact through specific domains in Eriocheir sinensis.•EsNotch and EsDelta are involved in antibacterial immune responses.•EsNotch and EsDelta regulate hemocyte proliferation-related gene expression.•EsNotch and EsDelta promote the proliferation of hemocytes in E. sinensis.