Evolutionary, comparative, and functional analyses of STATs and regulation of the JAK-STAT pathway in lumpfish upon bacterial and poly(I:C) exposure.

Background: The Janus kinase/signal transducers and activators of transcription (JAK-STAT) system regulates several biological processes by affecting transcription of genes as a response to cytokines and growth factors. In the present study, we have characterized the STAT genes in lumpfish (Cyclopterus lumpus L.), belonging to the order Perciformes, and investigated regulation of the JAK-STAT signaling pathway upon exposure to bacteria (Vibrio anguillarum) and poly(I:C), the latter mimicking antiviral responses. Methods: Characterization and evolutionary analyses of the STATs were performed by phylogeny, protein domain, homology similarity and synteny analyses. Antibacterial and antiviral responses were investigated by performing KEGG pathway analysis. Results: We observed that lumpfish have stat1a, 2, 3, 4, 5a, 5b, and 6. Transcriptome-wide analyses showed that most components of the JAK-STAT pathway were present in lumpfish. il-6, il-10, il-21, iκBα and stat3 were upregulated 6 hours post exposure (hpe) against bacteria while type I interferons (IFNs), irf1, irf3, irf10, stat1 and 2 were upregulated 24 hpe against poly(I:C). Conclusions: Our findings shed light on the diversity and evolution of the STATs and the data show that the STAT genes are highly conserved among fish, including lumpfish. The transcriptome-wide analyses lay the groundwork for future research into the functional significance of these genes in regulating critical biological processes and make an important basis for development of prophylactic measure such as vaccination, which is highly needed for lumpfish since it is vulnerable for both bacterial and viral diseases.

Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C).

Background: Both bacterial and viral diseases are a major threat to farmed fish. As the antiviral immune mechanisms in lumpfish (Cyclopterus lumpus L.) are poorly understood, lumpfish leukocytes were stimulated with poly(I:C), a synthetic analog of double stranded RNA, which mimic viral infections, and RNA sequencing was performed. Methods: To address this gap, we stimulated lumpfish leukocytes with poly(I:C) for 6 and 24 hours and did RNA sequencing with three parallels per timepoint. Genome guided mapping was performed to define differentially expressed genes (DEGs). Results: Immune genes were identified, and transcriptome-wide analyses of early immune responses showed that 376 and 2372 transcripts were significantly differentially expressed 6 and 24 hours post exposure (hpe) to poly(I:C), respectively. The most enriched GO terms when time had been accounted for, were immune system processes (GO:0002376) and immune response (GO:0006955). Analysis of DEGs showed that among the most highly upregulated genes were TLRs and genes belonging to the RIG-I signaling pathway, including LGP2, STING and MX, as well as IRF3 and IL12A. RIG-I was not identified, but in silico analyses showed that genes encoding proteins involved in pathogen recognition, cell signaling, and cytokines of the TLR and RIG-I signaling pathway are mostly conserved in lumpfish when compared to mammals and other teleost species. Conclusions: Our analyses unravel the innate immune pathways playing a major role in antiviral defense in lumpfish. The information gathered can be used in comparative studies and lay the groundwork for future functional analyses of immune and pathogenicity mechanisms. Such knowledge is also necessary for the development of immunoprophylactic measures for lumpfish, which is extensively cultivated for use as cleaner fish in the aquaculture for removal of sea lice from Atlantic salmon (Salmo salar L.).

Interleukin-1 Ligands and Receptors in Lumpfish (Cyclopterus lumpus L.): Molecular Characterization, Phylogeny, Gene Expression, and Transcriptome Analyses.

The interleukin (IL)-1 family play a fundamental role as immune system modulators. Our previous transcriptome-analyses of leukocytes from lumpfish (Cyclopterus lumpus L.) showed that IL-1ß was among the most highly upregulated genes following bacterial exposure. In the present study, we characterized IL-1 signaling pathways, identified and characterized four ligands of the IL-1 family in lumpfish; IL-1ß type I and type II, IL-18, and the novel IL-1 family members (nIL-1F), both at mRNA and gene levels. The two IL-1ß in lumpfish is termed IL-1ß1 (type II) and IL-1ß2 (type I). Furthermore, a comprehensive phylogenetic analysis of 277 IL-1 ligands showed that nIL-1F, in common with IL-1ß, likely represents an ancestral gene, as representatives for nIL-1F were found in cartilaginous and lobe-finned fish, in addition to teleosts. This shows that nIL-1F is not exclusively present in teleosts as previously suggested. Our analyses of exon-intron structures, intron phases, phylogeny and synteny clearly show the separation of IL-1ß into groups; type I and type II, which likely is a result of the third whole genome duplication (3R WGD). The phylogenetic analysis shows that most teleosts have both type I and type II. Furthermore, we have determined transcription levels of the IL-1 ligands in leukocytes and 16 different tissues, and their responses upon in vitro stimulation with seven different ligands. In addition, we have identified the IL-1 receptors IL-1R1, IL-1R2, IL-1R4 (ST2/IL-33 receptor/IL-1RL), IL-1R5 (IL-18R1), and partial sequences of DIGIRR and IL-1R3 (IL-RAcP). Identification of immune molecules and description of innate responses in lumpfish is interesting for comparative and evolutionary studies and our study constitutes a solid basis for further functional analyses of IL-1 ligands and receptors in lumpfish. Furthermore, since lumpfish are now farmed in large numbers to be used as cleaner fish for removal of sea lice on farmed salmon, in-depth knowledge of key immune molecules, signaling pathways and innate immune responses is needed, as the basis for design of efficient immune prophylactic measures such as vaccination.

The proinflammatory cytokines TNF-α and IL-6 in lumpfish (Cyclopterus lumpus L.) -identification, molecular characterization, phylogeny and gene expression analyses.

The proinflammatory cytokines TNF-α and IL-6 are important mediators of inflammatory reactions and orchestrators of the immune system in vertebrate. In this study, we have identified TNF-α and IL-6 in lumpfish, molecular characterized them at mRNA and gene level, performed homology modelling and measured their gene expression in different tissues and upon in vitro stimulation. A comprehensive phylogenetic analysis of TNF-α teleost sequences give novel insight into the TNF -α biology. Interestingly, we identified two isoforms of luIL-6. In normal tissue and leukocyte, the level of luTNF-α transcripts was higher than luIL-6. The expression pattern were parallel, except for brain, eye and gonad, and they displayed a similar induction pattern upon exposure to PAMPs, being most highly upregulated by flagellin. This is the first in-depth characterization of TNF and IL-6 in lumpfish. In recent years, lumpfish has become an important species for the aquaculture industry and establishment of qPCR-assays of luTNF-α and luIL-6 provide a valuable tool to measure effect of immune modulation, such as vaccination, microbiological disease and physiological trials. Lumpfish is also interesting for comparative studies as it represent a phylogenetic group that is poorly described immunologically.

Transcriptome-wide mapping of signaling pathways and early immune responses in lumpfish leukocytes upon in vitro bacterial exposure.

We performed RNA sequencing, identified components of the immune system and mapped early immune responses of lumpfish (Cyclopterus lumpus) leukocytes following in vitro exposure to the pathogenic bacterium Vibrio anguillarum O1. This is the first characterization of immune molecules in lumpfish at the gene level. In silico analyses revealed that genes encoding proteins involved in pathogen recognition, cell signaling and cytokines in mammals and teleosts are conserved in lumpfish. Unique molecules were also identified. Pathogen recognition components include 13 TLRs, several NLRs and complement factors. Transcriptome-wide analyses of immune responses 6 and 24 hours post bacterial exposure revealed differential expression of 9033 and 15225 genes, respectively. These included TLR5S, IL-1ß, IL-8, IL-6, TNFα, IL-17A/F3, IL-17C and several components of the complement system. The data generated will be valuable for comparative studies and make an important basis for further functional analyses of immune and pathogenicity mechanisms. Such knowledge is also important for design of immunoprophylactic measures in lumpfish, a species of fish now farmed intensively for use as cleaner-fish in Atlantic salmon (Salmo salar) aquaculture.