Viral Resistance and IFN Signaling in STAT2 Knockout Fish Cells.

IFN belong to a group of cytokines specialized in the immunity to viruses. Upon viral infection, type I IFN is produced and alters the transcriptome of responding cells through induction of a set of IFN stimulated genes (ISGs) with regulatory or antiviral function, resulting in a cellular antiviral state. Fish genomes have both type I IFN and type II IFN (IFN-γ), but no type III (λ) IFN has been identified. Their receptors are not simple counterparts of the mammalian type I/II IFN receptors, because alternative chains are used in type I IFN receptors. The mechanisms of the downstream signaling remain partly undefined. In mammals, members of the signal transducer and activator of family of transcription factors are responsible for the transmission of the signal from cytokine receptors, and STAT2 is required for type I but not type II IFN signaling. In fish, its role in IFN signaling in fish remains unclear. We isolated a Chinook salmon (Oncorhynchus tshawytscha) cell line, GS2, with a stat2 gene knocked out by CRISPR/Cas9 genome editing. In this cell line, the induction of ISGs by stimulation with a recombinant type I IFN is completely obliterated as evidenced by comparative RNA-seq analysis of the transcriptome of GS2 and its parental counterpart, EC. Despite a complete absence of ISGs induction, the GS2 cell line has a remarkable ability to resist to viral infections. Therefore, other STAT2-independent pathways may be induced by the viral infection, illustrating the robustness and redundancy of the innate antiviral defenses in fish.

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system.

BACKGROUND: Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. RESULTS: In the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. CONCLUSIONS: The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

Environmental and physiological factors shape the gut microbiota of Atlantic salmon parr (Salmo salar L.).

Gut microbes are key players in host immune system priming, protection and development, as well as providing nutrients to the host that would be otherwise unavailable. Due to this importance, studies investigating the link between host and microbe are being initiated in farmed fish. The establishment, maintenance and subsequent changes of the intestinal microbiota are central to define fish physiology and nutrition in the future. In fish, unlike mammals, acquiring intestinal microbes is believed to occur around the time of first feeding mainly from the water surrounding them and their microbial composition over time is shaped therefore by their habitat. Here we compare the distal intestine microbiota of Atlantic salmon parr reared in a recirculating laboratory aquarium with that of age matched parr maintained in cage culture in an open freshwater loch environment of a commercial fish farm to establish the microbial profiles in the gut at the freshwater stage and investigate if there is a stable subset of bacteria present regardless of habitat type. We used deep sequencing across two variable regions of the 16S rRNA gene, with a mean read depth of 180,144 ± 12,096 raw sequences per sample. All individual fish used in this study had a minimum of 30,000 quality controlled reads, corresponding to an average of 342 ± 19 Operational Taxonomic Units (OTUs) per sample, which predominantly mapped to the phyla Firmicutes, Proteobacteria, and Tenericutes. The results indicate that species richness is comparable between both treatment groups, however, significant differences were found in the compositions of the gut microbiota between the rearing groups. Furthermore, a core microbiota of 19 OTUs was identified, shared by all samples regardless of treatment group, mainly consisting of members of the phyla Proteobacteria, Bacteroidetes and Firmicutes. Core microbiotas of the individual rearing groups were determined (aquarium fish: 19 + 4 (total 23) OTUs, loch fish: 19 + 13 (total 32) OTUs), indicating that microbe acquisition or loss is occurring differently in the two habitats, but also that selective forces are acting within the host, offering niches to specific bacterial taxa. The new information gathered in this study by the Illumina MiSeq approach will be useful to understand and define the gut microbiota of healthy Atlantic salmon in freshwater and expand on previous studies using DGGE, TGGE and T-RFPL. Monitoring deviations from these profiles, especially the core microbes which are present regardless of habitat type, might be used in the future as early indicator for intestinal health issues caused by sub optimal feed or infectious diseases in the farm setting. STATEMENT OF RELEVANCE: The Microbiome is central to gut health, local immune function and nutrient up take. We have used deep sequencing approach to show differences in rearing conditions of Atlantic salmon. This work is of interest to aquaculture nutritionists.

Phylogeny and expression of tetraspanin CD9 paralogues in rainbow trout (Oncorhynchus mykiss).

CD9 is a member of the tetraspanin family, which is characterised by a unique domain structure and conserved motifs. In mammals, CD9 is found in tetraspanin-enriched microdomains (TEMs) on the surface of virtually every cell type. CD9 has a wide variety of roles, including functions within the immune system. Here we show the first in-depth analysis of the cd9 gene family in salmonids, showing that this gene has expanded to six paralogues in three groups (cd9a, cd9b, cd9c) through whole genome duplication events. We suggest that through genome duplications, cd9 has undergone subfunctionalisation in the paralogues and that cd9c1 and cd9c2 in particular are involved in antiviral responses in salmonid fish. We show that these paralogues are significantly upregulated in parallel to classic interferon-stimulated genes (ISGs) active in the antiviral response. Expression analysis of cd9 may therefore become an interesting target to assess teleost responses to viruses.

Seawater transfer alters the intestinal microbiota profiles of Atlantic salmon (Salmo salar L.).

Atlantic salmon undergo dramatic physiological changes as they migrate from freshwater to the marine environment. Osmoregulatory adaptation is the most crucial change, necessitating functional adaptations of the gills, kidney and intestine. Additionally, novel pathogens, microbes and dietary items are encountered in the saltwater environment, which suggests major changes in the intestinal microbiota following movement to saltwater. Here we compared the intestinal microbiota harboured in the distal digesta of Atlantic salmon freshwater fish (FW) kept in a commercial Scottish freshwater hatchery with that of their full-siblings after seawater acclimatisation (SW) by a 16S rRNA (V3-V4) high-throughput sequencing approach. Alpha- and beta-diversity were found significantly higher in FW compared to SW, both in terms of richness and diversity. Metastats analysis identified a higher number of Operational Taxonomic Units (OTUs) unique to FW compared to SW, with an additional 238 OTUs found at significantly different abundance. A core microbiota of 19 OTUs was identified in 100% of all fish, which indicates that certain microbes are maintained to fulfil minimal functions within the gut. Furthermore we show that the uniqueness of the respective microbial profiles can be correlated with significant differences in KEGG pathways including lipid and amino acid metabolism.

Transcriptomic responses in the fish intestine.

The intestine, being a multifunctional organ central to both nutrient uptake, pathogen recognition and regulating the intestinal microbiome, has been subjected to intense research. This review will focus on the recent studies carried out using high-throughput gene expression approaches, such as microarray and RNA sequencing (RNA-seq). These techniques have advanced greatly in recent years, mainly as a result of the massive changes in sequencing methodologies. At the time of writing, there is a transition between relatively well characterised microarray platforms and the developing RNA-seq, with the prediction that within a few years as costs decrease and computation power increase, RNA-seq related approaches will supersede the microarrays. Comparisons between the approaches are made and specific examples of how the techniques have been used to examine intestinal responses to pathogens, dietary manipulations and osmoregulatory challenges are given.

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.

CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.