Introduction
The Atlantic salmon (S. salar) industry covers approximately 90% of the total salmonid culture, with an annual worldwide production of ~1,000,000 tonnes . Chile is the second largest Atlantic salmon worldwide producer, representing its second most important economic activity. Among their challenges, the industry has to deal with sanitary issues, such as s ea lice (C. rogercresseyi ) infestations causing significant economic losses and social consequences for the aquaculture industry worldwide. Sea lice is an ectoparasite that infests the skin mucosa of Atlantic salmon in Chile. Despite its relevance , few studies have focused on evaluating the health status of the skin mucosa of Atlantic salmon reared in sea cage farms. In addition, e nvironmental fluctuations such as temperature, a parameter of particular relevance in fish due to their poikilothermic characteristics, have not been considered either. For this reason, in this study, we evaluated the epithelial skin mucosa transcriptome in summer (at the maximum seawater surface temperature) and autumn ( at the descending ramp temperature between the highest temperature and the lowest temperature) of Atlantic salmon infested with C. rogercresseyi.
Materials and methods
We performed a transcriptomic profile of the epithelial skin mucosa in Atlantic salmon infested and non-infested with sea lice sampled from the same seawater cage of a farm located in the fjords of the Aysén Region (Chile). We conducted the sampling in two different seasons: summer (during the peak of maximum water temperature; 10 weeks after seawater transfer) and autumn (at the half-descending ramp temperature;23 weeks after seawater transfer) . For transcriptomic analysis (RNA-Seq), t otal RNA was obtained from the epithelial skin tissue. We used a pooling strategy for the non-infested and infested fish group (n= 3 pools per condition; n= 5 fish per pool).
We verified the quality of each sequencing library with FastqQC (Andrews, 2010), a software package that estimates the number of uncallable and low-quality bases. We mapped the skin mucosa transcriptome to the Salmo salar reference genome (Ssal_v3.1) using STAR (Dobin, 2013), a high-performance community standard aligner. We used transcripts per million (TPM) values as gene expression levels for all the analyse s. The differential gene expression analysis was based on the negative binomial distribution using the DESeq2 package (Love, 2014). Pathways enrichment analysis was performed using the Gene Ontology Consortium database (Gene Ontology Consortium, 2019) and STRING database (Szklarczyk, 2019).
Results
We mapped our data to a total of 14,831 genes. We observed a different expression profile between phenotypes (infested; non-infested) and seasons (summer; autumn). We identified only 19 differential expressed genes (DEGs) in the summer. By contrast, in autumn, the DEGs augmented to 102 DEGs. The representation of common DEGs between summer and autumn was minimal, with o nly 3 DEGs. For the exclusive DEGs, the summer showed 11 upregulated and 6 downregulated DEGs. On the other hand, the autumn showed 66 upregulated and 33 downregulated DEGs.
We compared the functionality of the upregulated and downregulated DEGs between the infested and non-infested Atlantic salmon. The enrichment analysis showed the preference of the extracellular matrix process for the upregulated DEGs during summer. Conversely, the downregulated DEGs during summer showed no enrichment. In autumn, we observed upregulated DEGs associated with cellular response to corticotropin-releasing hormone stimulus, metabolic processes , cell proliferation, and nitric oxide biosynthetic process regulation , among others. On the other hand, the downregulated DEGs were associated with processes like riboflavin transport and mitochondrion organization.
Conclusions
Our analysis revealed that sea lice infestation does not appear to dominate the differential expression profile in summer, suggesting that both infested and non-infested samples are more concerned with seawater environmental adaptation . On the contrary , during autumn, the infested and non-infested Atlantic salmon show a differential expression profile and biological processes in response to sea lice. In this way , it stands out t he physiological response to stress orchestrated by the cellular response to glucocorticoid stimulus.
Acknowledgments
Fondecyt (1211841; 11221308) and DICYT-USACH (082344RL_Postdoc; 082344RL_Ayudante) grants.
References
Andrews, S. (2010). FastQC: a quality control tool for high throughput sequence data.
Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., ... & Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29(1), 15-21.
Gene Ontology Consortium. (2019). The gene ontology resource: 20 years and still GOing strong. Nucleic acids research, 47(D1), D330-D338.
Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology, 15(12), 1-21.
Szklarczyk, D., Gable, A. L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., ... & Mering, C. V. (2019). STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic acids research, 47(D1), D607-D613.