Introduction
Sea lice parasitism is one of the greatest economic, environmental, a nd animal welfare issues facing the Atlantic salmon ( Salmo salar) aquaculture industry (Torrissen et al. 2013) . A potential solution may exist in coho salmon (Oncorhynchus kisutch), a species that mounts a massive inflammatory response to sea lice, resulting in their swift detachment from the skin (Fast et al. 2002) . However, it is not known which of the many cell types pre sent in fish skin underlie this remarkable resistance. We therefore employed single-nuclei RNA sequencing, a technique facilitating transcriptomic profiling of thousands of individual cells, to investigate the cell types and gene expression patterns ch aracterizing the response of coho and Atlantic salmon to sea lice.
Materials and Methods
A tlantic and coho salmon were reared in a RAS at the Centre for Aquaculture Technologies (PEI, Canada) . Juvenile f ish were exposed to copepodid stage sea lice (Lepeophtheirus salmonis) and pelvic fin and skin where lice had attached were sampled at 12h, 24h, 36h, 48h, and 60h after exposure . F in and skin were also taken from unexposed control fish reared in identical conditions. Nuclei were isolated from one skin and one fin sample from the control and each of the five treatment time points for each species (N = 24 samples total) using a custom protocol (Ruiz Daniels et al. 2023). Samples were processed with Chromium (10X Genomics) and sequenced with Illumina technologies. L ibrary mapping was performed with STAR (Kaminow et al. 2021). Resulting outputs were analysed with Seurat (Stuart et al. 2019). Samples were analysed by species and also integrated into a single dataset using 1:1 orthologs identified with OrthoFinder (Emms & Kelly 2019).
Results
The variety of cell types we detected (Fig.1 ) were largely consistent in identity and marker genes across species. Integration of both species data using 649 4 1:1 orthologous genes detected most of the cell types observed in each of the species-specific analyses . These results suggest the same cell types are present in the skin and fin of both species.
We identified 4567 and 1799 unique genes in Atlantic and coho salmon , respectively, which were differentially expressed between control and any of the treatment time points (Fig.2) . Both species upregulated immune genes in response to lice, intriguingly in both immune and non-immune cell types. However, coho salmon uniquely demonstrated a down-regulation of iron-sequestration related genes in red blood cells, potentially to starve lice. Coho salmon keratinocytes also dramatically upregulated genes associated with inflammation and immunity. Our results confirm the importance of previously identified candidate genes associated with lice-resistance but also identify new candidates that may have been missed by previous bulk RNAseq studies due to their expression in multiple cell types.
Discussion
Clearly, multiple cell types, common to both species, are involved in sea lice response in Atlantic and coho s almon. Our results also suggest that coho salmon use multiple strategies (with different cell types and genes) to repel lice. This may explain why a single locus for lice resistance has proven elusive. Yet , the candidate genes we found to underlie coho salmon’s multiple resistance strategies to lice could be targeted in isolation or in combination via gene editing to confer this innate immunity to Atlantic salmon.
References
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Kaminow et al. (2021). STARsolo: accurate, fast and versatile mapping/quantification of single-cell and single-nucleus RNA-seq data. Biorxiv, 2021-05.
Ruiz Daniels et al. (2022). A versatile nuclei extraction protocol for single nucleus sequencing in fish species–optimization in various Atlantic salmon tissues. Protocols.io. dx.doi.org/10.17504/protocols.io.261genwm7g47/v1
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