Single-nuclei transcriptomics provides insights into cellular heterogeneity and communications in the glacial relict Hucho bleekeri under heat exposure

Rising temperatures driven by global warming and industrial thermal pollution are threatening the survival of both wild and farmed fish, which has emerged as one of the central concerns in international aquaculture. Understanding the physiological and behavioral responses of fish to heat exposure is critical for enhancing their survival. However, most studies rely on bulk-omic analyses at the tissue level which fail to reveal cellular heterogeneity. In the present study, ultrastructural observation and single-nuclei RNA sequencing (snRNA-seq) were applied to systematically study the physiological changes and the mechanisms of the Hucho bleekeri under acute heat stress. Histological analysis showed focal hemorrhage and lamellae broken in gill after heat stress. Ultrastructural observation revealed that after heat exposure, gill epithelial cells shed and the morphology of cell organelle damaged in pavement and mitochondria-rich cells. SnRNA-seq of gill generated 28 clusters, with the number of cells in each cluster ranging from 129 to 5590. After heat stress, epithelial (GobC) and endothelial cell proportion decreased significantly while neutrophil increased. Specifically, the change of cell proportion and pseudotime analysis indicated the occurrence of endothelial-to-mesenchymal transition during heat stress, and the expression of inflammation-related genes increased along the pseudotime axis. Differentially expressed genes (DEGs) analysis revealed that genes in heat shock protein and haemoglobin families were up-regulated in most cell type, whereas each cell also displayed specific DEGs profiles. Transcription factors analysis revealed increased activity of CEBPD, JUNB and CEBPA in most of the cell type after heat stress. Cell communication analysis showed interactions of CXCL12A-CXCR4B as well as CLDN11A-CLDN11A after heat stress. In addition, the results of snRNA-seq were validated through real-time PCR and fluorescence in situ hybridization analysis. Our study provides insights into cellular heterogeneity and physiological changes of H. bleekeri in response to heat stress, and lay a foundation for future studies on the mechanism of environmental stress on salmonid fish.