Single-Cell Atlas Reveals the Hemocyte Subpopulations and Stress Responses in Asian Giant Softshell Turtle during Hibernation

SIMPLE SUMMARY: Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In this study, we isolated and performed scRNA-seq analysis on P. cantorii hemocytes in the active state and during hibernation to classify the hemocyte types and obtain the relevant cell maps. We identified the marker genes of each cell population and the differentiation pathways associated with their maturation. We also discovered different immune-related genes that might play crucial roles in hemocyte differentiation before and after hibernation. Our study aims to provide a reference dataset for the unified classification of P. cantorii hemocytes and improve our understanding of the immune system of P. cantorii. ABSTRACT: Hibernation in turtle species is an adaptive survival strategy to colder winter conditions or food restrictions. However, the mechanisms underlying seasonal adaptions remain unclear. In the present study, we collected hemocytes from Pelochelys cantorii and compared the molecular signature of these cells between the active state and hibernation period based on single-cell RNA sequencing (scRNA-seq) analysis. We found six cell types and identified a list of new marker genes for each cell subpopulation. Moreover, several heat shock genes, including the Hsp40 family chaperone gene (DNAJ) and HSP temperature-responsive genes (HSPs), were upregulated during the hibernation period, which predicted these genes may play crucial roles in the stress response during hibernation. Additionally, compared to hemocytes in the active state, several upregulated differentially expressed immune-related genes, such as stat1, traf3, and socs6, were identified in hemocytes during the hibernation period, thus indicating the important immune function of hemocytes. Therefore, our findings provide a unified classification of P. cantorii hemocytes and identify the genes related to the stress response, thereby providing a better understanding of the adaptive mechanisms of hibernation.