Generation of a Litopenaeus vannamei hepatopancreas cell atlas from single nuclei transcriptomics using a new nuclei isolation method

Abstract

Background Crustacean aquaculture is one of the most important food sectors globally and projected to grow. It is a source of nutritious and economic animal protein in many countries. As the global demand for sea food increases, and with an increase in climatic and pathogenic threats to the industry, curating our current knowledge about crustaceans, as well as generating new tools and resources to help minimise the impact of various diseases on the sustainability of the industry is of the utmost importance to increase the resilience of the farmed animal stocks. The main aim of this pilot study was to create a new cell atlas for Pacific whiteleg shrimp (Litopenaeus vannamei) hepatopancreas which encapsulates both the different hepatocyte cell states as well as the various supporting cells found throughout the hepatopancreas, while developing a new method for nuclei isolation and data analysis in the species. Results We developed new protocols for TST-based nuclei isolation which could be used to successfully isolate and process crustacean-derived nuclei for single nuclei RNA-sequencing analysis with minimal nuclei degradation using frozen hepatopancreas tissue from healthy L. vannamei adults. The bioinformatic analysis that followed allowed us to create a new cell atlas for the hepatopancreas which details the different hepatocyte cell states. Additionally, we built up on the existing knowledge by also analysing the multiple supporting cell clusters such as IECs, fibroblasts and myocytes, which helps improve our understanding of the characteristics of this immune-related organ. Conclusion Overall, 4005 cells were assigned to nine different clusters. Distinct marker genes suggest unique functions of each hepatocyte subtypes and adjacent supportive cells. The new TST-based isolation method for frozen or archived nuclei is particularly useful for processing difficult samples, such as the hepatopancreas, minimise stress and dissociation bias while allowing for greater flexibility between tissue sampling and processing times. Combining the knowledge gained through this study with past and future work in other penaeid shrimp species will allow us to create a powerful resource that will help uncover new knowledge about these important species, especially in the field of stress and immunity.