Single-Cell Transcriptomic Analysis of Specific Responses of Different Cell Populations of Hemocytes to the Re-infection of Bacteria, a Case Study in Abalone

It is commonly believed that invertebrates lack immune memory due to the absence of immunoglobulins, related molecules, cells, and organs. However, our previous research demonstrated that Haliotis discus hannai , a prominent abalone species cultivated in China, often faces substantial economic losses due to diseases, particularly those caused by Vibrio sp . exhibited higher survival rates upon re-infection with Vibrio parahaemolyticus compared to the initial infection, implying the existence of immune memory. We hypothesized that hemocytes, which play a critical role in pathogen resistance in abalone, might be involved in the immune memory process. Therefore, we aimed to investigate the hemocyte response mechanism to V. parahaemolyticus re-infection to provide valuable insights for preventing and controlling abalone vibriosis and advancing sustainable abalone aquaculture. Additionally, our research aimed to contribute to understanding the origin and evolution of immune memory mechanisms. This study constructed a transcriptome map of abalone hemocytes using 10× Genomics single-cell RNA sequencing (scRNA-seq). Traditionally, abalone hemocytes were categorized into three cell types: hyalinocytes, semi-granulocytes, and granulocytes. The initial cell division resulted in the formation of 15 clusters further through subsequent analysis using scRNA-seq. Among these clusters, cluster_11 exhibited unique characteristics, indicating a more mature cluster of GRCs. This specific subpopulation displayed significant functionality as a core immune regulator, manifesting robust phagocytic and endocytic activities and substantial involvement in signal transduction and immune regulatory processes. Furthermore, we analyzed and detailed functional variances among different hemocyte types. Through the implementation of RNA interference technology, we validated the interplay between key signaling pathways. Interestingly, our findings suggested the potential existence of a classical TLR/NF-κB signaling pathway in abalone hemocytes, which may contribute to the immune regulation process in response to V. parahaemolyticus re-infection, as preliminarily confirmed in our study.