Immunologic Profiling of the Atlantic Salmon Gill by Single Nuclei Transcriptomics

Abstract

Anadromous salmonids begin life adapted to the freshwater environments of their natal streams before a developmental transition, known as smoltification, transforms them into marine-adapted fish. In the wild, smoltification is a photoperiod-regulated process, involving radical remodeling of gill function to cope with the profound osmotic and immunological challenges of seawater (SW) migration. While prior work has highlighted the role of specialized "mitochondrion-rich" cells (MRCs) and accessory cells (ACs) in delivering this phenotype, recent RNA profiling experiments suggest that remodeling is far more extensive than previously appreciated. Here, we use single-nuclei RNAseq to characterize the extent of cytological changes in the gill of Atlantic salmon during smoltification and SW transfer. We identify 20 distinct cell clusters, including known, but also novel gill cell types. These data allow us to isolate cluster-specific, smoltification-associated changes in gene expression and to describe how the cellular make-up of the gill changes through smoltification. As expected, we noted an increase in the proportion of seawater mitochondrion-rich cells, however, we also identify previously unknown reduction of several immune-related cell types. Overall, our results provide fresh detail of the cellular complexity in the gill and suggest that smoltification triggers unexpected immune reprogramming.

Keywords: Atlantic salmon (Salmo salar); gill; immune cells; photoperiod; single nuclei RNA sequencing; smoltification.

Figure 1

Single nuclei RNAseq analysis of Atlantic salmon gill tissue. (A) Gill tissue processing. Pooled duplicates from all T2, T3 and T4 collection points are integrated against T1 as a reference set. (B) UMAP plot of pooled cell data from 18844 cells representing eight samples from four collection states. The plot indicates 20 separate cell clusters. (C) Expression of marker genes in 20 cell clusters. From left to right: hierarchical relatedness of difference cell clusters; total cells in each cluster; UMI number in each cell cluster; gene features in each cell cluster; violin plots showing expression pattern of marker genes for each cluster. ACs, accessory cells; DCs, dendritic cells; ECs, epithelial cells; fib, fibrocytes; GCs, goblet cells; LCs, lymphatic cells; Ms, monocytes; MRC, mitochondrion-rich cells; NDCs, non-differentiated cells; PVCs, pavement cells; RBCs, red blood cells (erythrocytes); TCs, T cells; VCs, vascular cells.

Figure 2

Comparative abundance of cell clusters at different sampling points. (A) Experimental design. Fish were kept in constant light (LL) from hatching then transferred to short photoperiod (SP; 8L:16D) for 8 weeks before being returned to constant light (LL) for 8 weeks. Finally the fish were transferred to sea water for 24h. Sample points are indicated T1-T4. (B) Subset of cell clusters from T2, T3 or T4 (orange and blue dots) overlaid on T1 cells (grey dots). (C) Increasing abundance of sea-water mitochondrion-rich cells (MRCs SW) and vascular cells (VC 3) during smoltification (D) Decreasing abundance of leukocytes and immune-associated cells during smoltification.

Figure 3

Photoperiodic changes in gill gene expression and localized cell cluster expression. (A) Heat map representing 9746 genes differentially regulated (FDR <0.01) from T1-T3. Regulatory patterns for 5 major cluster are shown as amplitude index and 95% confidence limits. Major gene ontology terms for each cluster are shown. (B) RNAseq data for immune-associated genes differentially expressed by smoltification (FDR <0.01). (C) RNAseq data for “classical” smoltification-related genes and violin plots showing their cluster specific expression.

Figure 4

Sea-water transfer-associated changes in gill gene expression and localized cell cluster expression. (A) Genes differentially regulated (FDR <0.01) by 24h seawater transfer. Major gene ontology terms for each cluster are shown. (B) RNAseq data for immune-associated genes suppressed by seawater transfer (FDR <0.01) (C) RNAseq data for sea-water transfer-related genes and violin plots from the snRNAseq dataset showing their cluster specific expression.