ABSTRACT: Purpose: To investigate any association between parasite infection and physiological status of the host we performed a comparative transcriptomic analysis of liver obtained from C. osculatum infected and non-infected cod. Methods: Samples for transcriptomic analyses were collected from cod caught by local fishermen. Infected cod: Live cod (ten specimens) obtained by trawling along the Coastline of Bornholm were brought ashore (Nexø, Bornholm island, Denmark) and stocked in fish tanks (volume 8 cubic meter) supplied with running saltwater from the Baltic (salinity 8 ppt, temperature 10 °C). Non-infected cod: Live cod (ten specimens) were caught on the coastline of Zealand and stocked in similar fish tanks at the fish keeping facility (Blue Planet, Kastrup, Denmark). Total RNA was extracted using RTN350 (Sigma-Aldrich), according to the manufacturer’s instruction and subsequently, DNase treated with DNase I (Thermo Scientific, USA). Quality and integrity of the total RNA were checked on an Agilent Bioanalyzer 2100 total RNA Nano series II chip (Agilent, Amstelveen, Netherlands). Illumina RNAseq libraries were prepared from 500 ng total RNA using the Illumina TruSeqTM Stranded mRNA LT Sample Prep Kit according to the manufacturer’s instructions (Illumina Inc. San Diego, CA, USA). All RNAseq libraries (150-750 bp inserts) were sequenced on an Illumina HiSeq2500 sequencer as 1 x 50 nucleotides single-end reads according to the manufacturer’s protocol. Image analysis and base calling were done using the Illumina pipeline. Reads were aligned to the Rainbow trout reference genome (http://www.genoscope.cns.fr/trout/data/) using TopHat (version 2.0.5) and on average 53.4% of the RNAseq reads could be mapped. The resulting files were filtered using SAMtools (version 0.1.18) to exclude secondary alignment of reads. For statistical comparison of gene expression levels between groups, aligned fragments per predicted gene were counted from SAM alignment files using the Python package HTSeq (version 0.5.3p9). To make comparisons across samples possible, these fragment counts were corrected for the total amount of sequencing performed for each sample. As a correction scalling factor, we employed library size estimates determined using the R/Bioconductor (release 2.11) package DESeq. Read counts were normalized by dividing the raw counts obtained from HTSeq by its scale factor. Correction for false positives is included in the statistical analysis of gene expression through DESeq. The cut-off for significance was set to adjusted p<0.05 and at least 2-fold change. Results: A total of 47,025 unigenes were identified from cod liver, of which 2,085 (4.43%) unigenes were differentially expressed in the infected liver when compared to non-infected. Of the differentially expressed unigenes (DEGs) 1,240 unigenes were up-regulated while 796 were down-regulated. The Gene Ontology (GO) enrichment analysis showed that 845 DEGs were highly represented in cellular process and single-organism process, cell and cell part, binding and catalytic activity. As determined by the Kyoto Encyclopedia of Gene and Genomes (KEGG) Pathways analysis, 241 DEGs were involved in 753 pathways. Eighty DEGs were related to metabolic pathways including carbohydrate, lipid, and amino acid metabolism. Twenty-four regulated genes were playing a role in immune response such as complement and coagulation cascades, B-cell receptor signalling, chemokine signalling and twenty-five genes (most of which were down-regulated) were associated with growth of Baltic cod Conclusion: a transcriptomic profile of rainbow trout gills exposed to the parasitic I. multifiliis was reported for the first time. A total of 3,355 differentially expressed unigenes were identified. Of these were 1,184 unigenes (mapped to 952 genes) annotated 282 KEGG pathways and 268 unigenes were associated with 16 immune pathways. Most unigenes were related to innate immune system pathways (Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration) although a number of unigenes was related to adaptive responses (antigen processing and presentation, T and B cell receptor signaling pathway). The present study gave a far better resolution of the immune response of rainbow trout exposed to a parasitic protozoan than has ever been presented previously. The identification of a series of immune genes involved in several but important was useful for understanding of immune mechanism of the rainbow trout responding to the parasite I. multifiliis. Our results provide tools to link innate and adaptive immune elements in the process and present basic information which will be useful in the future studies related to immunoprophylaxis.
