scRNA-seq of Penaeus japonicus hemocytes under environmentally-induced restriction of sand-diving behavior

Penaeus japonicus is an economically important marine shrimp species, but its sand-diving habit affects the further improvement of their culture yield. To investigate the molecular response to the restriction of sand-diving behavior, we designed three culture systems: the sandy group, the sandless group, and the sandless stress group (transfer the individuals to a sandless pond after a week in the sandy group). First, we analyzed the expression differences of stress-related (HSP60) and circadian rhythm genes (period, timeless) at different time points after stress induction, identifying 6 h post-transition as the timepoint with the most significant stress response (p < 0.05). A total of 25,371 highly expressed genes were detected across cell clusters, which were further classified into 13 distinct cell subpopulations. Manual annotation categorized these into granular cells (GCs), semi-granular cells (SGCs), hyaline cells (HCs), prohemocyte-like cells, and functional cells. qRT-PCR analysis confirmed the expression profiles of 13 highly expressed genes in GCs, SGCs, and HCs of P. japonicus. RNA in situ hybridization localized pxt, IGSF10, and IFI30 to GCs, HCs, and SGCs, respectively, validating the accuracy of cell clusters classification based on marker genes. Single-cell transcriptome differential expression analysis revealed significant gene expression differences among the three groups (p < 0.05), with most differentially expressed genes (DEGs) concentrated in cluster 2 (GCs) and cluster 8 (HCs), suggesting their close association with burrowing behavior regulation. qRT-PCR analysis of these DEGs in hemocytes from P. japonicus reared under different substrate conditions yielded results consistent with the scRNA-seq data, confirming the reliability of the transcriptomic findings. Furthermore, comparative analysis between groups identified key candidate DEGs, including trpa1, trpm, and the cut protein family, that may play pivotal roles in the response to environmental change and the restriction of natural behavior, though their specific functions require further validation. This study provides a theoretical foundation for understanding the molecular stress response to substrate deprivation in P. japonicus and identifies candidate genes for future functional studies on behavioral regulation.