Single-cell atlas of Exopalaemon carinicauda gills provides insights into pillar cell-mediated responses to low-salinity stress in crustaceans

Low-salinity stress exerts strong constraints on ion homeostasis in crustaceans cultivated in coastal environments. Gills function as central osmoregulatory organs in crustaceans, coordinating transepithelial ion flux and acid-base balance; however, the cellular mechanisms governing gill adaptation to reduced salinity remain poorly understood. Here, integrated ultrastructural characterization and single-cell RNA sequencing (scRNA-seq) were applied to define cellular responses to low-salinity exposure in Exopalaemon carinicauda gills. Low salinity induced pronounced morphological remodeling in pillar cells. Single-cell transcriptomic profiling resolved 11 transcriptionally distinct populations, including two ionocyte lineages represented by pillar cells and septal cells. Comparative analyses demonstrated a marked expansion of pillar cell subcluster 3 under low-salinity stress, accompanied by enrichment of differentially expressed genes (DEGs) in ion transport pathways, notably hydrogen ion transport. Weighted gene co-expression network analysis identified coordinated up-regulation of hub genes associated with pH regulation within pillar cells. Pseudotime reconstruction further indicated that gill adaptation to low-salinity stress proceeds through a defined differentiation trajectory (cell fate 1) originating from pillar cells. Core stress-responsive genes in pillar cells, including V(H⁺)-ATPases and cytoplasmic carbonic anhydrase, were predominantly associated with acid-base regulation. Collectively, these results delineate gill cellular heterogeneity and identify pillar cell-mediated regulatory mechanisms that underpin crustacean physiological adaptation to low-salinity environments.