Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species

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

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2024
Publication Date: 9/26/2024
Citation: Aldersey, J., Lange, M.D., Beck, B.H., Abernathy, J.W. 2024. Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species. PLOS ONE. 19(9): e0309397. https://doi.org/10.1371/journal.pone.0309397.
DOI: https://doi.org/10.1371/journal.pone.0309397

Interpretive Summary: Given its role in food production, the channel catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the immune system, ARS Researchers in Auburn, Alabama performed single-cell RNA sequencing on nuclei isolated from whole channel catfish spleens, a major organ critical for immune responsiveness in teleost fish. Single nuclei sequencing libraries were prepared from multiple individual spleens, sequenced, aligned to the reference genome assembly, and gene features counted. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 20,121 cells, counts for 19,613 genes, with a median 665 genes/cell. This allowed for the identification and characterization of multiple cell types within this tissue, including erythroid cells, B-cells, T-cells, Natural Killer cells, myeloid cells, and hematopoietic stem cells. Each cell type was found to express specific genes for use in developing genetic markers. The dynamic processes that these cells undergo as part of their true biological nature was also modeled. These results will serve as the foundation for a cell atlas of this primary immune tissue in fish and allow for profiling of immune cell subsets to a higher-resolution than ever before.

Technical Abstract: The catfish industry is the largest sector of U.S. aquaculture production. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on nuclei isolated from whole spleens, a major lymphoid organ in teleost fish. Libraries were prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3’ Reagents and sequenced on an Illumina sequencer. Each demultiplexed sample was aligned to the CoCo_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually and as a normalized aggregate. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 20,121 cells, counts for 19,613 genes, with a median 665 genes/cell. Cluster analysis of all cells identified 17 clusters which were classified as erythroid, hematopoietic stem cells, B cells, T cells, myeloid cells, endothelial cells. Subcluster analysis was carried out on the immune cell clusters. Here distinct subclusters, such as immature B cells, mature B cells, plasma cells, Gamma-Delta T cells, dendritic cells and macrophages were identified. Differential gene expression analysis identified the top 10 genes for each cluster and subcluster. This dataset is a rich resource for investigating the channel catfish splenic immunome.