Comparative Transcriptomics in Atlantic Salmon Head Kidney and SHK-1 Cell Line Exposed to the Sea Louse Cr-Cathepsin

Abstract

The development of vaccines against sea lice in salmon farming is complex, expensive, and takes several years for commercial availability. Recently, transcriptome studies in sea louse have provided valuable information for identifying relevant molecules with potential use for fish vaccines. However, the bottleneck is the in vivo testing of recombinant protein candidates, the dosage, and the polyvalent formulation strategies. This study explored a cell-based approach to prospect antigens as candidate vaccines against sea lice by comparison with immunized fish. Herein, SHK-1 cells and Atlantic salmon head kidney tissue were exposed to the antigen cathepsin identified from the sea louse Caligus rogercresseyi. The cathepsin protein was cloned and recombinantly expressed in Escherichia coli, and then SHK-1 cell lines were stimulated with 100 ng/mL cathepsin recombinant for 24 h. In addition, Atlantic salmons were vaccinated with 30 ug/mL recombinant protein, and head kidney samples were then collected 30 days post-immunization. SHK-1 cells and salmon head kidney exposed to cathepsin were analyzed by Illumina RNA sequencing. The statistical comparisons showed differences in the transcriptomic profiles between SHK-1 cells and the salmon head kidney. However, 24.15% of the differentially expressed genes were shared. Moreover, putative gene regulation through lncRNAs revealed tissue-specific transcription patterns. The top 50 up and downregulated lncRNAs were highly correlated with genes involved in immune response, iron homeostasis, pro-inflammatory cytokines, and apoptosis. Also, highly enriched pathways related to the immune system and signal transduction were shared between both tissues. These findings highlight a novel approach to evaluating candidate antigens for sea lice vaccine development, improving the antigens screening in the SHK-1 cell line model.

Keywords: C. rogercresseyi; immune response; recombinant protein; transcriptome response.

Figure 1

Transcriptome profiling of mRNA from the SHK-1 cell line and salmon head kidney (HK) exposed to recombinant Cr-cathepsin. (A) Heatmaps for each treatment were constructed with the TPM (transcripts per million of reads) values of mRNAs and grouped by hierarchical clustering based on the Manhattan distance with average linkages. A red color indicates up-regulated mRNAs, and blue represents down-regulated transcripts. (B) Top-20 DEGs expressed in SHK-1 vs. control. (C) Venn diagrams of DEGs among tissues (SHK-1 cell line and HK exposed to Cr-cathepsin) vs. control group (|fold change| > 4, p-value < 0.05). Up/down-regulated transcripts were calculated from the comparisons between the SHK-1 cell line vs. control (17,825 transcripts), HK vs. control (16,563 transcripts), and shared (10,949 transcripts). (D) Top-20 DEGs in HK vs. control.

Figure 2

GO enrichment analysis for exclusive and common DEGs of the SHK-1 cell line and salmon HK exposed to recombinant Cr-cathepsin. (A) Top-20 of molecular function. (B) Top-20 of biological process.

Figure 3

KEGGs enrichment analysis for exclusive and common DEGs mRNA of SHK-1 cell line and salmon HK exposed to recombinant Cr-cathepsin. (A) Top pathways of exclusive DEGs mRNA of salmon HK. (B) Top pathways of exclusive DEGs mRNA of exclusive of SHK-1 cell line. (C) Top pathways of common DEGs mRNA salmon HK and SHK-1 cell line.

Figure 4

RNA-seq analysis of 30 immune-related genes selected from common DEGs in response to Cr-cathepsin. Heatmaps for each treatment were constructed with the TPM (transcripts per million of reads) values of mRNAs and grouped by hierarchical clustering based on Manhattan distance with average linkages. A red color indicates up-regulated mRNAs, and blue represents down-regulated transcripts.

Figure 5

Transcriptome profiling of lncRNA from the SHK-1 cell line and salmon HK exposed to recombinant Cr-cathepsin. (A) Heatmaps for each treatment were constructed with the TPM (transcripts per million of reads) values of lncRNAs and grouped by hierarchical clustering based on Manhattan distance with average linkages. A red color indicates up-regulated lncRNAs, and blue represents down-regulated transcripts. (B) A Venn diagram representing the significantly expressed lncRNA among tissues (SHK-1 cell line and salmon HK exposed to Cr-cathepsin) vs. control group (|fold change| > 4, p-value < 0.05). (C) LncRNA fold-change variation up/down-regulated in SHK-1 cell line and salmon HK exposed to Cr-cathepsin vs. the control group.

Figure 6

Correlation analyses among the top 55 upregulated shared lncRNA and selected coding transcripts related with immune response, iron homeostasis, and transcription factors. The correlation matrix was based on Pearson’s correlation calculation using transcript fold change values. Positive correlations of expression levels are represented in red, and negative correlations are represented in blue.