Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

Time-course study of the protection induced by an interferon-inducible DNA vaccine against viral haemorrhagic septicaemia in rainbow trout.

The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively. Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.

Effects of temperature on Paramoeba perurans growth in culture and the associated microbial community.

Population growth, in vitro, of three Paramoeba perurans cultures, one polyclonal (G) and two clonal (B8, CE6, derived from G), previously shown to differ in virulence (B8 > G > CE6), was compared at 10 and 15 °C. B8 showed a significantly higher increase in attached and in suspended amoebae over time at 15 and 10 °C, respectively. CE6 and G also had significantly higher numbers of suspended amoebae at 10 °C compared with 15 °C at experiment termination. However, in contrast to B8, numbers of attached amoebae were significantly higher at 10 °C in CE6 but showed a similar trend in G at the end of the experiment. Numbers of both suspended and attached amoebae were lower in B8 compared with CE6 and G. Significant differences in bacterial community composition and/or relative abundances were found, between cultures, between temperatures and between the same culture with and without amoebae, based on 16S rRNA Illumina MiSeq sequencing. Bacterial diversity was lower in B8 and CE6 compared with G, possibly reflecting selection during clonal isolation. The results indicate that polyclonal P. perurans populations may contain amoebae displaying different growth dynamics. Further studies are required to determine if these differences are linked to differences seen in the bacterial communities.

Gene expression analysis of isolated salmonid GALT leucocytes in response to PAMPs and recombinant cytokines.

Increased knowledge of the immune response of the intestine, a physiologically critical organ involved in absorption, secretion and homeostasis in a non-sterile environment, is needed to better understand the mechanisms involved in the induction of long-lasting immunity and, subsequently, the development of efficacious gastrointestinal immunization approaches. To this end, analysis of isolated gut cells will give an insight into the cell types present and their immune capability. Hence, in this study we first optimised a method for salmonid gut leucocyte isolation and characterised the cells on the basis of their expression of a range of selected cell markers associated with T & B cells and dendritic cells. The GALT leucocytes were then stimulated with a variety of PAMPs, recombinant cytokines and PHA, as a means to help characterise the diversity of the immune repertoire present in such cells. The stimulants tested were designed to examine the nature of the antibacterial, antiviral and T cell type responses in the cells (at the transcript level) using a panel of genes relevant to innate and adaptive immunity. The results showed distinct responses to the stimulants, with a clear delineation seen between the stimulant used (eg viral or bacterial PAMP) and the pathway elicited. The changes in the expression patterns of the immune genes in these cells indicates that the salmonid intestine contains a good repertoire of competent immune cells able to respond to different pathogen types. Such information may aid the development of efficient priming by oral vaccination in salmonids.

Atlantic salmon post-smolts adapted for a longer time to seawater develop an effective humoral and cellular immune response against Salmonid alphavirus.

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection.

Atlantic salmon adapted to seawater for 9 weeks develop a robust immune response to salmonid alphavirus upon bath challenge.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra-muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM-B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI-B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM-B and BI-B groups. Transcription of the analysed genes peaked at 7 dpi in the IM-B group and at 14 dpi in the BI-B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI-B group showed higher transcription of IL-1ß, IFNγ and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM-B group. Histopathological changes in the heart were restricted to the IM-B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non-infected control fish in Phase B (CT-B) was also significantly different when compared to Phase A control fish (CT-A).

Effect of repeated exposure to AQUI-S® on the viability and growth of Neoparamoeba perurans.

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28-day period to AQUI-S® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post-smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non-attached amoeba in the culture was assessed with a vital stain. AQUI-S® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.

Immune gene profiles in Atlantic salmon (salmo salar L.) post-smolts infected with SAV3 by bath-challenge show a delayed response and lower levels of gene transcription compared to injected fish.

Salmonid alphavirus (SAV) causes pancreatic disease (PD) in salmonids in Northern Europe which results in large economic losses within the aquaculture industry. In order to better understand the underlying immune mechanisms during a SAV3 infection Atlantic salmon post-smolts were infected by either i.m.-injection or bath immersion and their immune responses compared. Analysis of viral loads showed that by 14 dpi i.m.-injected and bath immersion groups had 95.6% and 100% prevalence respectively and that both groups had developed the severe pathology typical of PD. The immune response was evaluated by using RT-qPCR to measure the transcription of innate immune genes involved in the interferon (IFN) response as well as genes associated with inflammation. Our results showed that IFNa transcription was only weakly upregulated, especially in the bath immersion group. Despite this, high levels of the IFN-stimulated genes (ISGs) such as Mx and viperin were observed. The immune response in the i.m.-injected group as measured by immune gene transcription was generally faster, and more pronounced than the response in the bath immersion group, especially at earlier time-points. The response in the bath immersion group started later as expected and appeared to last longer often exceeding the response in the i.m-injected fish at later time-points. High levels of transcription of many genes indicative of an active innate immune response were present in both groups.

Impact of selenium supplementation on fish antiviral responses: a whole transcriptomic analysis in rainbow trout (Oncorhynchus mykiss) fed supranutritional levels of Sel-Plex®.

BACKGROUND: Selenium (Se) is required for the synthesis of proteins (selenoproteins) with essential biological functions. Selenoproteins have a crucial role in the maintenance of cellular redox homeostasis in nearly all tissues, and are also involved in thyroid hormone metabolism, inflammation and immunity. Several immune processes rely on Se status and can be compromised if this element is present below the required level. Previous work has supported the notion that when Se is delivered at levels above those deemed to be the minimal required but below toxic concentrations it can have a boosting effect on the organism's immune response. Based on this concept Se-enriched supplements may represent a valuable resource for functional feeds in animal farming, including aquaculture. RESULTS: In this study we tested the effects of Se supplemented as Sel-Plex during an immune challenge induced by polyinosinic:polycytidylic acid (poly(I:C)), a pathogen-associated molecular pattern (PAMP) that mimics viral infection. Trout were fed two diets enriched with 1 or 4 mg Se Kg(-1) of feed (dry weight) by Sel-Plex addition and a commercial formulation as control. The whole trout transcriptomic response was investigated by microarray and gene ontology analysis, the latter carried out to highlight the biological processes that were influenced by Sel-Plex supplementation in the head kidney (HK) and liver, the main immune and metabolic organs in fish. Overall, Sel-Plex enrichment up to 4 mg Se Kg(-1) induced an important response in the trout HK, eliciting an up-regulation of several genes involved in pathways connected with hematopoiesis and immunity. In contrast, a more constrained response was seen in the liver, with lipid metabolism being the main pathway altered by Se supplementation. Upon stimulation with poly(I:C), supplementation of 4 mg Se Kg(-1) increased the expression of principal mediators of the antiviral defences, especially IFN-γ, and down-stream molecules involved in the cell-mediated immune response. CONCLUSIONS: Supplementation of diets with 4 mg Se Kg(-1) using Sel-Plex remarkably improved the fish response to viral PAMP stimulation. Sel-Plex, being a highly bioavailable supplement of organic Se, might represent a suitable option for supplementation of fish feeds, to achieve the final aim of improving fish fitness and resistance against immune challenges.

Identification and characterisation of TLR18-21 genes in Atlantic salmon (Salmo salar).

Teleost fish possess many types of toll-like receptor (TLR) some of which exist in other vertebrate groups and some that do not (ie so-called "fish-specific" TLRs). In this study, we identified in Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs seven TLRs that are not found in mammals, including six types of fish-specific TLRs (one TLR18, one TLR19, and four TLR20 members (two of which are putative soluble forms (s)) and one TLR21. Phylogenetic analysis revealed that teleost TLR19-21 are closely related with murine TLR11-TLR13, whilst teleost TLR18 groups with mammalian TLR1, 2, 6 and 10. A typical TLR protein domain structure was found in all these TLRs with the exception of TLR20b(s) and TLR20c(s). TLR-GFP expression plasmids transfected into SHK-1 cells showed that salmon TLR19, TLR20a and TLR20d were preferentially localised to the intracellular compartment. Real time PCR analysis suggested that salmon TLR19-TLR21 are mainly expressed in immune related organs, such as spleen, head kidney and gills, while TLR18 transcripts are more abundant in muscle. In vitro stimulation of primary head kidney cells with type I IFN, IFNγ and IL-1ß had no impact on TLR expression. Infectious salmon anaemia virus (ISAV) infection, in vivo, down-regulated TLR20a, TLR20b(s), TLR20d and TLR21 in infected salmon kidney tissue. In contrast, up-regulation of TLR19 and TLR20a expression was found in posterior kidney in rainbow trout with clinical proliferative kidney disease (PKD).

Red mark syndrome in rainbow trout Oncorhynchus mykiss: investigation of immune responses in lesions using histology, immunohistochemistry and analysis of immune gene expression.

Red mark syndrome (RMS) is an economically significant disease which affects farmed rainbow trout in the United Kingdom, in the US and in mainland Europe. From the pattern of incidence, it appears to be transmissable, although no causative agent has yet been identified. RMS presents as a severe lymphocytic infiltration centred on the dermis and an alternative, host-focused approach was taken to understand the disease through investigating immune responses occurring in the lesion. Lesion and non-lesion skin at different stages of lesion development were examined using histochemistry and immunohistochemistry on paraffin sections. Expression of immune-related genes was compared between lesion and non-lesion skin. Investigation of early stage lesions suggested that the initial immune response is targeted at the region of the scale pocket, with lymphocyte infiltration and anti-tumour necrosis factor (TNF)-α staining of the stratum spongiosum, and increased numbers of major histocompatibility complex (MHC) II-positive cells immediately adjacent to the scale pocket. Gene expression analysis suggested a counterbalancing T helper (Th)1 and T regulatory (Treg) - type response is occurring in the lesion, with repression of Th2 and Th17-type responses.

Isolation of RAG-1 and IgM transcripts from the striped trumpeter (Latris lineata), and their expression as markers for development of the adaptive immune response.

A partial sequence of the recombination activating gene-1 (RAG-1) and several full length sequences of the immunoglobulin M (IgM) heavy chain mRNA were obtained from the striped trumpeter (Latris lineata). The RAG-1 fragment consisted of 205 aa and fell within the core region of the open reading frame. The complete IgM heavy chain sequences translated into peptides ranging between 581 and 591 aa. Both genes showed good homology to other vertebrate sequences. The expression of the two genes was assessed throughout the early developmental stages of striped trumpeter larvae (5-100 dph) and used as markers to follow the ontogeny of the adaptive immune response. Using RT-PCR, RAG-1 mRNA expression was detectable at 5 dph and remained so until 80 dph, before becoming undetectable at 100 dph. IgM expression was also detectable at 5 dph, and remained so throughout. These patterns of expression may suggest that the striped trumpeter possess mature B cells with surface IgM at 100 dph. However, complete immunological competence is likely not reached until some time later. The early detection of IgM mRNA at 5 dph led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

Cloning and expression analysis of three striped trumpeter (Latris lineata) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, in response to infection by the ectoparasitic, Chondracanthus goldsmidi.

This study reports the cloning and sequencing of three striped trumpeter (Latris lineata Forster) pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-8, as well as their differential expression in response to an infection by the ectoparasite Chondracanthus goldsmidi. The striped trumpeter TNF-alpha transcript consisted of 1093 bp, including a 759 bp ORF which translated into a 253 aa transmembrane peptide. The sequence contained a TACE cut site, that would produce a 167 aa soluble peptide containing the TNF ligand family signature. The IL-1beta sequence consisted of 963 bp, including a 774 bp ORF which translated into a 258 aa protein. The protein lacked both a signal peptide and an ICE cleavage site, but did contain the IL-1 family signature. The sequence for the chemokine IL-8 contained 906 bp, with an ORF of 297 bp, which translated into a 99 aa protein. The protein lacked an ELR motif as is common with many teleost IL-8 sequences. The differential expression of the three cytokine genes in parasitized fish was investigated via quantitative real-time PCR. A significant up-regulation of all three pro-inflammatory cytokines was found in the gills, which were the site of parasite attachment. Examination of head kidney cells revealed a significant up-regulation of TNF-alpha, but not IL-1beta or IL-8. Conversely, the spleen cells showed significant up-regulation of both IL-1beta and IL-8, but not TNF-alpha. These findings allow for more detailed investigations of the striped trumpeter immune response.

Rainbow trout interleukin-2: cloning, expression and bioactivity analysis.

In this study the rainbow trout (Oncorhynchus mykiss) interleukin-2 (IL-2) cDNA has been cloned, and its expression and bioactivity analysed in head kidney leucocytes. The IL-2 precursor encoded an open reading frame of 429 bp, that translates into a predicted protein of 142 aa, with a 20 aa signal peptide. The trout IL-2 had moderate protein homology (30.9% identity/48.3% similarity) with Fugu IL-2, the only IL-2 homologue identified in fish to date, with lower homology to avian (17.8% identity/23.2% similarity) and mammalian (34.2 identity/46.5% similarity) IL-2s. IL-2 expression was induced by the T cell mitogen PHA and by the mixed leucocyte reaction, where leucocytes from pairs of fish were cultured together for four days. Expression was also induced in vivo during bacterial (Yersinia ruckeri) infection. The Escherichia coli produced recombinant IL-2 was shown to increase the expression of two transcription factors, STAT5 and Blimp-1, known to be involved in IL-2 signalling in mammals, as well as IFN-gamma, gIP and IL-2 itself. The potential signalling pathways involved and possible use as an adjuvant for fish vaccines are discussed.

Identifying potential virulence determinants in viral haemorrhagic septicaemia virus (VHSV) for rainbow trout.

We identified viral haemorrhagic septicaemia virus (VHSV) isolates classified within Genotype Ib which are genetically similar (>99.4% glycoprotein amino acid identity) yet, based on their isolation history, were suspected to differ in virulence in juvenile rainbow trout. The virulence of an isolate recovered in 2000 from a viral haemorrhagic septicaemia disease episode in a marine rainbow trout farm in Sweden (SE-SVA-1033) was evaluated in juvenile rainbow trout via intraperitoneal injection and immersion challenge alongside 3 isolates recovered from wild-caught marine fish (DK-4p37, DK-5e59 and UKMLA98/6HE1) suspected of being of low pathogenicity to trout. Mortality data revealed that isolate SE-SVA-1033 caused VHSV-specific mortality in both intraperitoneal and immersion challenges (75.0 and 15.4%, respectively). The remaining Genotype Ib isolates caused significantly lower mortalities using the same experimental infection routes (<35.0 and <2.0%, respectively). Having identified VHSV isolates with clear differences in their pathogenicity, coding and inter-genic non-coding regions of 2 isolates (SE-SVA-1033 and DK-4p37) were determined and compared in order to identify potential markers responsible for the observed differences in virulence. Only 4 predicted amino acid substitutions were identified across the genome sequenced; these occurred in the N (R46G), G (S113G), NV (L12F) and L (S56A) proteins. These findings form the basis for further studies aimed at determining the biological significance of these mutations and suggest that small changes at the molecular level can cause significant changes in the virulence properties of VHSV isolates.

Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: functional analysis and transcriptional modulation.

Interferon regulatory factors (IRF) 3 and 7 in mammals are known to be crucial in regulating the type I interferon (IFN) response to viral infection as part of transcriptional complexes binding to IRF-binding elements (IRF-Es) and interferon stimulatory response elements (ISREs) within IFN and interferon-stimulated genes (ISGs). Here we report the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt)IRF7 and, for the first time in fish, IRF3. RtIRF3 consists of 2127 bp with a 159 bp 5'-UTR-containing two upstream AUGs and a 573 bp 3'-UTR. RtIRF7 was found to be 2055 bp, with a 102 bp 5'-UTR and a 705 bp 3'-UTR. The open reading frames (ORFs) translate into 464 amino acid and 415 amino acid proteins, respectively, each possessing a putative DNA-binding domain (DBD) containing a tryptophan cluster, which is characteristic of all IRF family members. The presence of putative IRF association domain (IAD)s, serine-rich C terminal domains (poorly conserved in trout IRF3), and phylogenetic analysis places the two genes in the IRF3 subfamily. Both genes were found to be upregulated by poly I:C, type I recombinant rainbow trout (r) IFN (second isoform, type I rIFN), type II rIFN (rIFNgamma), LPS, and rIL-1beta in the trout macrophage cell line, RTS-11. Poly I:C and type I rIFN also induced IRF3 and IRF7 expression in a trout fibroblast cell line (RTG-2). Transient transfection of RTG-2 cells with each IRF fused to GFP revealed a predominant cytoplasmic distribution found most intensely around the nucleus and, to a lesser extent, within cell nuclei. Transient transfection of rtIRF3 in the Mx-1-luciferase reporter cell line, RTG-P1, revealed a modest increase in luciferase activity relative to the vehicle control, which was lost in cells over-expressing a DBD-truncated form of rtIRF3. Both full-length and DBD-truncated forms of rtIRF7 increased reporter activity relative to the control, although to a non-significant extent. Electromobility shift assays (EMSAs) did not reveal a specific interaction between each IRF and the ISRE element found in the Mx-1 promoter, although the Mx-1 ISRE bound specifically to endogenous transcriptional complexes. These data support the premise that rtIRF3 and rtIRF7 are important molecules in the regulation of antiviral responses in fish, with the impact of rIFNgamma on rtIRF3/7 expression implying a role for these IRFs in immune processes other than type I IFN-driven antiviral responses.

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray.

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.

Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus).

This paper reports the cloning and sequencing of two pro-inflammatory cytokines, interleukin (IL)-1beta and IL-8, in haddock (Melanogrammus aeglefinus) by homology cloning. The complete transcript of the haddock IL-1beta was sequenced and contained 1043 bp, including a 762 bp open reading frame. The 3' end of the gene includes a polyadenylation signal 13 bp upstream of the poly(A) tail, along with 10 instability motifs. The predicted protein of 253aa revealed the presence of the IL-1 family signature and the absence of an ICE cut site. The cDNA of the chemokine IL-8 was sequenced in haddock and contained 903 bp of which 306 bp are the open reading frame. Interestingly, the predicted protein sequence of 101aa, contains an ELR motif preceding the CXC signature, common in all vertebrate IL-8 molecules but absent in all teleost genes sequenced to date. The expression of both haddock cytokines was studied in four different tissues: head kidney, spleen, liver and gill. Tissues were obtained from both healthy fish and fish stimulated in vivo with four commercial serotypes of LPS, namely Escherichia coli 026:B6, 055:B5, 0111:B4 and 0127:B8 and PMA. Haddock IL-1beta was not constitutively expressed and expression was only observed following stimulation. However, this expression was stimulant dependent and only PMA and LPS 026:B6 induced high levels of expression in the head kidney. The haddock IL-8 gene on the other hand, showed a constitutive expression, that could be up or down-regulated depending on the immunostimulant used, although to a lesser extent than IL-1beta.

The discovery and comparative expression analysis of three distinct type I interferons in the perciform fish, meagre (Argyrosomus regius).

Type I interferons (IFN) play an important role in anti-viral responses. In teleost fish multiple genes exist, that are classified by group/subgroup. That multiple subgroups are present in Acanthopterygian fish has only become apparent recently, and 3 subgroups are now known to be expressed, including a new subgroup termed IFNh. However, the potential to express multiple IFN subgroups and their interplay is not well defined. Hence this study aims to clarify the situation and undertook the first in-depth analysis into the nature and expression of IFNc, IFNd and IFNh in the perciform fish, meagre. Constitutive expression was analysed initially during larval development and in adult tissues (gills, mid-gut, head kidney, spleen). During early ontogeny IFNc was the highest expressed IFN, and this was also the case in adult tissues with the exception of gills where IFNd was highest. However, comparison between tissues for individual isoforms showed that spleen had high transcript levels of all three IFNs, IFNd/IFNh were also highly expressed in gills. The expression of each sub-group was increased significantly in the four tissues following injection of poly I:C, however, this increase was only seen in the mid-gut for IFNh. Following in vitro stimulation with poly I:C again all three isoforms were upregulated, although with differences in kinetics and the cell source used. For example, early induction was seen for IFNc/IFNh in gill cells, IFNd/IFNh in splenocytes and all three isoforms in head kidney cells. Induction was sustained in splenocytes and head kidney cells, but in gut cells only a late induction was seen. These results demonstrate a complex pattern of regulation between the different IFN isoforms present in meagre and highlights potential sub-functionalisation of these IFN subgroups during perciform anti-viral responses.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.