Single-nuclei transcriptome analysis of IgM + cells isolated from channel catfish (Ictalurus punctatus) spleen

Johanna E Aldersey ^1,2 Jason W Abernathy ² Benjamin H Beck ² Miles D Lange ^2*

• ¹ Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, Tennessee, United States
• ² Aquatic Animal Health Research, Agricultural Research Service (USDA), Auburn, United States

Catfish production is the primary aquaculture sector in the United States, and the key cultured species is channel catfish (Ictalurus punctatus). The major cause of production losses are pathogenic diseases, and the spleen, an important site of adaptive immunity, is implicated in these diseases. To examine the channel catfish immune system, single-nuclei transcriptomes of sorted and captured IgM + cells were produced from adult channel catfish. Three channel catfish (~1 kg) were euthanized, the spleen dissected, and tissue dissociated. The lymphocytes were isolated using a Ficoll gradient, then IgM + cells were sorted with flow cytometry. The IgM + cells were lysed and single-nuclei libraries generated using a Chromium Next GEM Single Cell 3' GEM Kit and the Chromium X Instrument (10x Genomics) and sequenced with the Illumina NovaSeq X Plus sequencer. The reads were aligned to the I. punctatus reference assembly (Coco_2.0) using Cell Ranger, and normalization, cluster analysis and differential gene expression analysis were carried out with Seurat. Across the three samples approximately 753.5 million reads were generated for 18,686 cells. After filtering, 10,637 cells remained for the cluster analysis. The cluster analysis identified 16 clusters which were classified as B cells (10,276), natural killer-like (NK-like) cells (178), T cells or natural killer cells (45), hematopoietic stem and progenitor cells (HSPC) (66), myeloid/epithelial cells (40) and plasma cells (32). The B cell clusters were further defined as immature B cells, mature B cells, cycling B cells and plasma cells. The plasma cells highly expressed ighm and we demonstrate that the secreted form of the transcript was largely being expressed by these cells. This atlas provides an insight into the gene expression of IgM + immune cells in the channel catfish. The atlas is publicly available and could be used garner more important information regarding the gene expression of splenic immune cells.