Toxic Switch and Key Effectors of Cu in Zebrafish Gills: Coupling Single-Cell RNA Sequencing and Redox Imaging

Copper (Cu) pollution poses a great threat to aquatic organisms, with gills serving as the primary interface for toxicant uptake and detoxification. However, the toxic switch leading to massive fish mortality under a low Cu burden in the fish gills remains unresolved. Here, we employed single-cell RNA sequencing (scRNA-seq) to systematically identify the Cu-sensitive cellular and molecular response mechanisms in zebrafish gills by focusing on the toxicity switch and key toxic effectors. Six cell populations significantly changed in the zebrafish gills after Cu exposure. Our results revealed that the Cu redox state switch controlled the activation process of Cu toxicity. This toxic switch involves macrophage, pillar, and HR cells, serving as central regulators through ESAM and COLLAGEN LT, and GALECTIN pathways. Cu redox state transformation gene (Steap4) and Cu transportation genes (Cox17 and slc31a) serve as the potential biomarkers of Cu pollution. The novel in situ fluorescence probes enabled simultaneous visualization of Cu(I)/Cu(II) spatial distribution, validating cluster-specific Cu redox states dynamics. The most prominent toxic effects observed were the respiratory inhibition and epithelial hyperplasia. Intriguingly, Cu overload reprogrammed intercellular communication networks with severe pathological injury. Overall, these findings illustrate an essential role of redox state switch in ecological risk of Cu in aquatic organisms.