Single-cell RNA sequencing reveals the heterogeneity of hepatopancreas cells and their association with gonadal development in the red swamp crayfish Procambarus clarkii

The hepatopancreas plays a crucial role in synthesizing yolk substances during ovarian development in crustaceans, but the diverse types of hepatopancreas cells and their specific roles in ovarian development in Procambarus clarkii remain unclear. During ovarian development, the hepatopancreatic index increased from 6.5 % to 8.14 % between stages I and IV, then decreased to 4.35 % by stage VI. To reveal the heterogeneity of hepatopancreas cells and their association with ovarian development in P. clarkii , single-cell transcriptome sequencing was performed on the hepatopancreas from three distinct phases of ovarian development: the large growth phase (stage III), the mature phase (stage V), and the recovery phase (stage VI), analyzing a total of 16,626 cells. The cells were classified into five types: R-cells, E -cells, B-cells, F-cells, and M-cells. R-cells had the highest proportion (52.86 %), followed by E-cells (24.68 %). The proportion of R-cells varied from 65.10 % at stage III to 26.42 % at stage V, and 50.62 % at stage VI. Pseudo-time analysis indicated that E -cells were the first to differentiate, gradually giving rise to other cell types. Key pathways related to ovarian development were enriched in R- and E -cells, such as “oxidative phosphorythlation”, “protein processing in the endoplasmic reticulum” and “antigen processing and presentation”. By screening for differentially expressed genes within these pathways, potential functional marker genes linked to ovarian development in the hepatopancreas were identified and validated through qPCR , including Lysophosphatidylcholine Acyltransferase and Vitelline Membrane Outer Layer Protein I . These findings may provide insights into the molecular regulatory mechanisms governing ovarian development in crustaceans. • ScRNA-seq was performed on P. clarkii hepatopancreas during ovarian developmental stages. • Hepatopancreatic cell types and marker genes were identified. • Gene expression & cell composition changed notably during ovarian development.