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

Authors: ALDERSEY, JOHANNA - Orise Fellow; Abernathy, Jason; Beck, Benjamin; Lange, Miles

Submitted to: Frontiers in Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/14/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: 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 losses is due to different pathogenic diseases. Often time these disease processes occur systemically and involve the spleen an important site of adaptive immunity. To better understand the channel catfish systemic immune system, single-nuclei transcriptomes of sorted IgM+ cells were produced from adult channel catfish. Splenic cells were isolated from catfish and lymphocytes were isolated using a Ficoll gradient, IgM+ cells were then further recovered through flow cytometry cell sorting. The IgM+ cells from the individual fish were used to generate single-nuclei transcriptomes that were then sequenced. The transcriptomes were aligned to the channel catfish genome and then further analyzed and aggregated into a single population of data for cluster analysis. The cluster analysis identified 16 cell clusters which were classified as B cells, natural killer-like (NK-like) cells, T cells, hematopoietic stem cells, myeloid/epithelial cells and plasma cells. B cell clusters were then defined as immature, mature and cycling B cells and plasma cells. The plasma cells highly expressed secreted IgM. This atlas provides insight into the gene expression of IgM+ channel catfish lymphocytes.

Technical Abstract: 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 753.8 million reads were generated for 18,686 cells. After filtering, 10,632 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 (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. Further analysis of this rich data reveals important information regarding the gene expression of splenic immune cells.