Early milk-feeding regimes in calves exert long-term effects on the development of ovarian granulosa cells.

BACKGROUND: Nutrition has not only an impact on the general wellbeing of an animal but can also affect reproductive processes. In cattle, feeding regimes can influence the age of puberty onset and alter gonadal development. We analyzed effects of different milk replacer (MR) feeding regimes during rearing on ovarian physiology with specific emphasis on the numbers as well as gene expression characteristics of granulosa cells (GCs) at the age of puberty onset. Two groups of calves received either 10% or 20% of bodyweight MR per day during their first 8 weeks. After weaning, both groups were fed the same mixed ration ad libitum until slaughter at 8 months. RESULTS: Animals of the 20% feeding group had a significantly higher body weight, but the proportion of animals having a corpus luteum at the time of slaughter was not different between groups, suggesting a similar onset of puberty. Calves of the 10% group showed a constant GC count regardless of the number of follicles (r = 0.23) whereas in the 20% group increasing numbers of GCs were detected with a higher follicle count (r = 0.71). As a first effort to find a possible molecular explanation for this unexpected limitation of GC numbers in the 10% group, we comparatively analyzed GC transcriptomes in both diet groups. The mRNA microarray analysis revealed a total of 557 differentially expressed genes comparing both groups (fold change > |1.5| and p < 0.05). OAS1X, MX2 and OAS1Z were among the top downregulated genes in the 20% vs. the 10% group, whereas top upregulated genes comprised BOLA and XCL1. All of these genes are known to be regulated by interferon. Subsequent signaling pathway analysis revealed the involvement of several immune response mechanisms in accordance with a number of interferons as upstream regulators. CONCLUSIONS: The results indicate that the plane of MR feeding in early life has an impact on the number and physiology of GCs later in life. This might influence the overall reproductive life initiated by the onset of puberty in cattle. In addition, the observed alterations in GCs of calves fed less MR might be a consequence of interferon regulated immunological pathways.

Are bold-shy personalities of European perch (Perca fluviatilis) linked to stress tolerance and immunity? A scope of harnessing fish behavior in aquaculture.

The sensitivity to stress and its impact on immunity are supposedly related to a fish's personality. In the present study, European perch (Perca fluviatilis) were exposed to an open-field and a novel-object test to identify distinctive shy and bold individuals. This series of cognitive tests revealed clear differences between proactive individuals with pronounced exploration behavior (bold personality) and reactive individuals that took a freeze-hide position (shy personality). A cohort of shy and bold perch was then exposed to elevated stocking density. Frozen activity and lower explorative behavior were related to higher basal and stocking-induced cortisol levels compared to proactive individuals. Since cortisol is a well-known modulator of immune-gene expression, we used multiplex real-time PCR to profile the differential immune responses to the intraperitoneal injection of Aeromonas hydrophila in the head kidney and peritoneal cells of bold and shy perch individuals. These expression differences between stimulated bold and shy perch were generally modest, except for the genes encoding the complement component c3 and the matrix metallopeptidase mmp9. The strong differential expression of these two bactericidal and inflammatory genes in the context of the modestly regulated features suggests that a fish's personality is linked to a particular immune-defense strategy. In conclusion, our approach, based on behavioral video observations, phagocytosis and enzyme assays, immunogene-expression profiling, and quantification of stress-relevant metabolites, revealed indications for divergent coping styles in cohorts of bold or shy European perch. This divergence could be exploited in future selective breeding programs.

The Discovery and Characterization of Conserved and Novel miRNAs in the Different Developmental Stages and Organs of Pikeperch (Sander lucioperca).

Micro RNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional gene expression regulators. Genes regulated in vertebrates include those affecting growth and development or stress and immune response. Pikeperch (Sander lucioperca) is a species that is increasingly being considered for farming in recirculation aquaculture systems. We characterized the pikeperch miRNA repertoire to increase the knowledge of the genomic mechanisms affecting performance and health traits by applying small RNA sequencing to different developmental stages and organs. There were 234 conserved and 8 novel miRNA genes belonging to 104 families. A total of 375 unique mature miRNAs were processed from these genes. Many mature miRNAs showed high relative abundances or were significantly more expressed at early developmental stages, like the miR-10 and miR-430 family, let-7, the miRNA clusters 106-25-93, and 17-19-92. Several miRNAs associated with immune responses (e.g., slu-mir-731-5p, slu-mir-2188-5p, and slu-mir-8159-5p) were enriched in the spleen. The mature miRNAs slu-mir-203a-3p and slu-mir-205-5p were enriched in gills. These miRNAs are similarly abundant in many vertebrates, indicating that they have shared regulatory functions. There was also a significantly increased expression of the disease-associated miR-462/miR-731 cluster in response to hypoxia stress. This first pikeperch miRNAome reference resource paves the way for future functional studies to identify miRNA-associated variations that can be utilized in marker-assisted breeding programs.

Lost and Found: The Family of NF-κB Inhibitors Is Larger than Assumed in Salmonid Fish.

NF-κB signalling is largely controlled by the family of 'inhibitors of NF-κB' (IκB). The relevant databases indicate that the genome of rainbow trout contains multiple gene copies coding for iκbα (nfkbia), iκbε (nfkbie), iκbδ (nkfbid), iκbζ (nfkbiz), and bcl3, but it lacks iκbß (nfkbib) and iκbη (ankrd42). Strikingly, three nfkbia paralogs are apparently present in salmonid fish, two of which share a high sequence identity, while the third putative nfkbia gene is significantly less like its two paralogs. This particular nfkbia gene product, iκbα, clusters with the human IκBß in a phylogenetic analysis, while the other two iκbα proteins from trout associate with their human IκBα counterpart. The transcript concentrations were significantly higher for the structurally more closely related nfkbia paralogs than for the structurally less similar paralog, suggesting that iκbß probably has not been lost from the salmonid genomes but has been incorrectly designated as iκbα. In the present study, two gene variants coding for iκbα (nfkbia) and iκbε (nfkbie) were prominently expressed in the immune tissues and, particularly, in a cell fraction enriched with granulocytes, monocytes/macrophages, and dendritic cells from the head kidney of rainbow trout. Stimulation of salmonid CHSE-214 cells with zymosan significantly upregulated the iκbα-encoding gene while elevating the copy numbers of the inflammatory markers interleukin-1-beta and interleukin-8. Overexpression of iκbα and iκbε in CHSE-214 cells dose-dependently quenched both the basal and stimulated activity of an NF-κB promoter suggesting their involvement in immune-regulatory processes. This study provides the first functional data on iκbε-versus the well-researched iκbα factor-in a non-mammalian model species.

Immunological insights into the resistance of Nile tilapia strains to an infection with tilapia lake virus.

The emergence of viral diseases affecting fish and causing very high mortality can lead to the disruption of aquaculture production. Recently, this occurred in Nile tilapia aquaculture where a disease caused by a systemic infection with a novel virus named tilapia lake virus (TiLV) caused havoc in cultured populations. With mortality surpassing 90% in young tilapia, the disease caused by TiLV has become a serious challenge for global tilapia aquaculture. In order to partly mitigate the losses, we explored the natural resistance to TiLV-induced disease in three genetic strains of tilapia which were kept at the University of Göttingen, Germany. We used two strains originating from Nilotic regions (Lake Mansala (MAN) and Lake Turkana (ELM)) and one from an unknown location (DRE). We were able to show that the virus is capable of overcoming the natural resistance of tilapia when injected, providing inaccurate mortality results that might complicate finding the resistant strains. Using the cohabitation infection model, we found an ELM strain that did not develop any clinical signs of the infection, which resulted in nearly 100% survival rate. The other two strains (DRE and MAN) showed severe clinical signs and much lower survival rates of 29.3% in the DRE strain and 6.7% in the MAN strain. The disease resistance of tilapia from the ELM strain was correlated with lower viral loads both at the mucosa and internal tissues. Our results suggest that the lower viral load could be caused by a higher magnitude of a mx1-based antiviral response in the initial phase of infection. The lower pro-inflammatory responses also found in the resistant strain might additionally contribute to its protection from developing pathological changes related to the disease. In conclusion, our results suggest the possibility of using TiLV-resistant strains as an ad hoc, cost-effective solution to the TiLV challenge. However, as the fish from the disease-resistant strain still retained significant virus loads in liver and brain and thus could become persistent virus carriers, they should be used within an integrative approach also combining biosecurity, diagnostics and vaccination measures.\.

Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus.

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.

PIAS Factors from Rainbow Trout Control NF-κB- and STAT-Dependent Gene Expression.

Four 'protein inhibitors of activated STAT' (PIAS) control STAT-dependent and NF-κB-dependent immune signalling in humans. The genome of rainbow trout (Oncorhynchus mykiss) contains eight pias genes, which encode at least 14 different pias transcripts that are differentially expressed in a tissue- and cell-specific manner. Pias1a2 was the most strongly expressed variant among the analysed pias genes in most tissues, while pias4a2 was commonly low or absent. Since the knock-out of Pias factors in salmonid CHSE cells using CRISPR/Cas9 technology failed, three structurally different Pias protein variants were selected for overexpression studies in CHSE-214 cells. All three factors quenched the basal activity of an NF-κB promoter in a dose-dependent fashion, while the activity of an Mx promoter remained unaffected. Nevertheless, all three overexpressed Pias variants from trout strongly reduced the transcript level of the antiviral Stat-dependent mx gene in ifnγ-expressing CHSE-214 cells. Unlike mx, the overexpressed Pias factors modulated the transcript levels of NF-κB-dependent immune genes (mainly il6, il10, ifna3, and stat4) in ifnγ-expressing CHSE-214 cells in different ways. This dissimilar modulation of expression may result from the physical cooperation of the Pias proteins from trout with differential sets of interacting factors bound to distinct nuclear structures, as reflected by the differential nuclear localisation of trout Pias factors. In conclusion, this study provides evidence for the multiplication of pias genes and their sub-functionalisation during salmonid evolution.

Insights into early ontogenesis: characterization of stress and development key genes of pikeperch (Sander lucioperca) in vivo and in vitro.

There are still numerous difficulties in the successful farming of pikeperch in the anthropogenic environment of various aquaculture systems, especially during early developmental steps in the hatchery. To investigate the physiological processes involved on the molecular level, we determined the basal expression patterns of 21 genes involved in stress and immune responses and early ontogenesis of pikeperch between 0 and 175 days post hatch (dph). Their transcription patterns most likely reflect the challenges of growth and feed conversion. The gene coding for apolipoprotein A (APOE) was strongly expressed at 0 dph, indicating its importance for yolk sac utilization. Genes encoding bone morphogenetic proteins 4 and 7 (BMP4, BMP7), creatine kinase M (CKM), and SRY-box transcription factor 9 (SOX9) were highly abundant during the peak phases of morphological changes and acclimatization processes at 4-18 dph. The high expression of genes coding for peroxisome proliferator-activated receptors alpha and delta (PPARA, PPARD) at 121 and 175 dph, respectively, suggests their importance during this strong growth phase of juvenile stages. As an alternative experimental model to replace further in vivo investigations of ontogenetically important processes, we initiated the first approach towards a long-lasting primary cell culture from whole pikeperch embryos. The present study provides a set of possible biomarkers to support the monitoring of pikeperch farming and provides a first basis for the establishment of a suitable cell model of this emerging aquaculture species.

Characterisation of the teleostean κB-Ras family: The two members NKIRAS1 and NKIRAS2 from rainbow trout influence the activity of NF-κB in opposite ways.

Two structurally similar NF-κB-inhibitor-interacting Ras-like proteins (NKIRAS) regulate the activity of the transcription factor NF-κB and thereby control several early immune mechanisms in mammals. We identified the orthologous sequences of NKIRAS1 and NKIRAS2 from the rainbow trout Oncorhynchus mykiss. The level of sequence identity was similarly high (≥68%) between the two and in comparison to their mammalian counterparts. Strikingly, NKIRAS2 was present as four transcript variants. These variants differed only in length and in the nucleotide composition of their 5' termini and were most likely generated by splicing along unconventional splice sites. The shortest NKIRAS2 variant was most strongly expressed in a lymphocyte-enriched population, while NKIRAS1 was most strongly expressed in cells of myeloid origin. Fluorescent-labelled NKIRAS1 and NKIRAS2 proteins from rainbow trout were detected in close association with the p65 subunit of NF-κB in the nucleus and cytoplasm of CHSE-214 cells. Subsequent reporter-gene experiments revealed that NKIRAS1 and a longer NKIRAS2 variant in rainbow trout decreased the level of activated NF-κB, while the two shortest NKIRAS2 variants increased the NF-κB activity. In addition, the overexpression of the shortest NKIRAS2 variant in CHSE-214 cells induced a stronger transcription of the genes encoding the pro-inflammatory cytokines TNF, CXCL8, and IL1B compared to non-transfected control cells. This is the first characterisation of NKIRAS orthologues in bony fish and provides additional information to the as yet underexplored inhibition pathways of NF-κB in lower vertebrates.

Early response of salmonid head-kidney cells to stress hormones and toll-like receptor ligands.

The functional spectrum of the teleostean head kidney covers haematopoietic, immune and endocrine signalling pathways with physiological effects that are likely to conflict if activated at the same time. An in vivo experiment on the salmonid fish maraena whitefish (Coregonus maraena) revealed that the head kidney shows a remarkably strong response after injection of Aeromonas salmonicida within 48 h. In order to investigate the potential influence of endocrine signalling on the initiation of immune responses, we established a primary culture of head-kidney cells of maraena whitefish. For the characterisation of this model system, we used flow cytometry complemented with an extensive panel of immunological/haematological and stress-physiological/neuroendocrinological qPCR assays. More than one third of the cells expressed the characteristic signature of myeloid cells, while more than half of the cells expressed those genes typical for lymphocytes and monocytes. In parallel, we quantified the expression of genes encoding endocrine receptors and identified ADRA2D as by far the most highly expressed adrenergic-receptor gene in head-kidney cells. The stimulation of the head-kidney cells with toll-like receptor ligands induced the expression of typical immune genes (IL1B, CXCL8, TNF, SAA) after only 1 h. The incubation with the stress hormones cortisol, adrenaline and noradrenaline also had an immune-activating effect, though less pronounced. However, cortisol had the strongest suppressive effect on the stimulation-induced immune response, while adrenaline exerted a comparably weaker effect and noradrenaline was almost ineffective. Moreover, we found that cortisol reduced the expression of genes coding for adrenergic and some glucocorticoid receptors, while noradrenaline increased it. In conclusion, the primary head-kidney cells of maraena whitefish reflect the immunological and neuroendocrinological diversity of the entire organ. This in vitro system allowed thus identifying the correlative changes between the activities of hormones and immune factors in salmonid fish in order to contribute to a better understanding of the regulation circuit between stress and immune defence.

Vertebrate Alpha2,8-Sialyltransferases (ST8Sia): A Teleost Perspective.

We identified and analyzed α2,8-sialyltransferases sequences among 71 ray-finned fish species to provide the first comprehensive view of the Teleost ST8Sia repertoire. This repertoire expanded over the course of Vertebrate evolution and was primarily shaped by the whole genome events R1 and R2, but not by the Teleost-specific R3. We showed that duplicated st8sia genes like st8sia7, st8sia8, and st8sia9 have disappeared from Tetrapods, whereas their orthologues were maintained in Teleosts. Furthermore, several fish species specific genome duplications account for the presence of multiple poly-α2,8-sialyltransferases in the Salmonidae (ST8Sia II-r1 and ST8Sia II-r2) and in Cyprinus carpio (ST8Sia IV-r1 and ST8Sia IV-r2). Paralogy and synteny analyses provided more relevant and solid information that enabled us to reconstruct the evolutionary history of st8sia genes in fish genomes. Our data also indicated that, while the mammalian ST8Sia family is comprised of six subfamilies forming di-, oligo-, or polymers of α2,8-linked sialic acids, the fish ST8Sia family, amounting to a total of 10 genes in fish, appears to be much more diverse and shows a patchy distribution among fish species. A focus on Salmonidae showed that (i) the two copies of st8sia2 genes have overall contrasted tissue-specific expressions, with noticeable changes when compared with human co-orthologue, and that (ii) st8sia4 is weakly expressed. Multiple sequence alignments enabled us to detect changes in the conserved polysialyltransferase domain (PSTD) of the fish sequences that could account for variable enzymatic activities. These data provide the bases for further functional studies using recombinant enzymes.

Under control: The innate immunity of fish from the inhibitors' perspective.

The innate immune response involves a concerted network of induced gene products, preformed immune effectors, biochemical signalling cascades and specialised cells. However, the multifaceted activation of these defensive measures can derail or overshoot and, if left unchecked, overwhelm the host. A plenty of regulatory devices therefore mediate the fragile equilibrium between pathogen defence and pathophysiological manifestations. Over the past decade in particular, an almost complete set of teleostean sequences orthologous to mammalian immunoregulatory factors has been identified in various fish species, which prove the remarkable conservation of innate immune-control concepts among vertebrates. This review will present the current knowledge on more than 50 teleostean regulatory factors (plus additional fish-specific paralogs) that are of paramount importance for controlling the clotting cascade, the complement system, pattern-recognition pathways and cytokine-signalling networks. A special focus lies on those immunoregulatory features that have emerged as potential biomarker genes in transcriptome-wide research studies. Moreover, we report on the latest progress in elucidating control elements that act directly with immune-gene-encoding nucleic acids, such as transcription factors, hormone receptors and micro- and long noncoding RNAs. Investigations into the function of teleostean inhibitory factors are still mainly based on gene-expression profiling or overexpression studies. However, in support of structural and in-vitro analyses, evidence from in-vivo trials is also available and revealed many biochemical details on piscine immune regulation. The presence of multiple gene copies in fish adds a degree of complexity, as it is so far hardly understood if they might play distinct roles during inflammation. The present review addresses this and other open questions that should be tackled by fish immunologists in future.

Polysialic Acid in Human Plasma Can Compensate the Cytotoxicity of Histones.

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.

Multiple gene and transcript variants encoding trout C-polysaccharide binding proteins are differentially but strongly induced after infection with Aeromonas salmonicida.

Two 'trout C-polysaccharide-binding proteins,' TCBP1 and -2, with relevance to early inflammatory events have been discovered in the last century. The present study characterises the respective cDNA sequences from rainbow trout (Oncorhynchus mykiss), including multiple TCBP1 transcript variants. These variants are generated either by the use of alternative splice sites or the exclusion of exons. The longest mRNA isoform, TCBP1-1, encodes a 245-aa protein with a large signal peptide and a complement component C1q domain. The shortest mRNA isoform, TCBP1-5, contains a premature termination codon and hence fails to encode a functional factor. The 224-aa-long TCBP2 protein consists of a comparably shorter signal peptide and a pentraxin domain. Evolutionary analyses clearly separated TCBP1 and -2 because of distinctive protein motifs. Expression profiling in the liver, spleen, and head kidney tissues of healthy trout revealed that TCBP2 mRNA concentrations were higher than the concentrations of all five TCBP1 mRNA isoforms together. The hepatic levels of these TCBP1 variants increased significantly upon infection with Aeromonas salmonicida, whereas TCBP2 transcript levels rose moderately. As the biological function of TCBP1 is barely understood, we tagged this factor with the green fluorescent protein and visualised its expression in HEK-293 cells. Overexpression of TCBP1 increased the level of active NF-κB factors and induced cell death, indicating its involvement in proapoptotic NF-κB-dependent signalling routes.

Toll-like receptors in maraena whitefish: Evolutionary relationship among salmonid fishes and patterns of response to Aeromonas salmonicida.

Toll-like receptors (TLRs) interact directly with particular pathogenic structures and are thus highly important to innate immunity. The present manuscript characterises a suite of 14 TLRs in maraena whitefish (Coregonus maraena), a salmonid species with increasing importance for aquaculture. Whitefish TLRs were structurally and evolutionary analysed. The results revealed a close relationship with TLRs from salmonid fish species rainbow trout and Atlantic salmon. Profiling the baseline expression of TLR genes in whitefish indicated that mainly members of the TLR11 family were highly expressed across all investigated tissues. A stimulation model with inactivated Aeromonas salmonicida was used to induce inflammation in the peritoneal cavity of whitefish. This bacterial challenge induced the expression of pro-inflammatory cytokine genes and evoked a strong influx of granulated cells of myeloid origin into the peritoneal cavity. As a likely consequence, the abundance of TLR-encoding transcripts increased moderately in peritoneal cells, with the highest levels of transcripts encoding non-mammalian TLR22a and a soluble TLR5 variant. In the course of inflammation, the proportion of granulated cells increased in peripheral blood accompanied by elevated TLR copy numbers in spleen and simultaneously reduced TLR copy numbers in head kidney at day 3 post-stimulation. Altogether, the present study provides in-vivo evidence for relatively modest TLR response patterns, but marked trafficking of myeloid cells as an immunophysiological consequence of A. salmonicida inflammation in whitefish. The present results contribute to improved understanding of the host-pathogen interaction in salmonid fish.

Comprehensive and comparative transcription analyses of the complement pathway in rainbow trout.

The complement system is one of the most ancient and most essential innate immune cascades throughout the animal kingdom. Survival of aquatic animals, such as rainbow trout, depends on this early inducible, efficient immune cascade. Despite increasing research on genes coding for complement components in bony fish, some complement-related genes are still unknown in salmonid fish. In the present study, we characterize the genes encoding complement factor D (CFD), CD93 molecule (CD93), and C-type lectin domain family 4, member M (CLEC4M) from rainbow trout (Oncorhynchus mykiss). Subsequently, we performed comprehensive and comparative expression analyses of 36 complement genes including CFD, CD93, and CLEC4M and further putative complement-associated genes to obtain general information about the functional gene interaction within the complement pathway in fish. These quantification analyses were conducted in liver, spleen and gills of healthy fish of two rainbow trout strains, selected for survival (strain BORN) and growth (Import strain), respectively. The present expression study clearly confirms for rainbow trout that liver represents the primary site of complement expression. Spleen and gills also express most complement genes, although the mean transcript levels were generally lower than in liver. The transcription data suggest a contribution of spleen and gills to complement activity. The comparison of the two rainbow trout strains revealed a generally similar complement gene expression. However, a significantly lower expression of numerous genes especially in spleen seems characteristic for the BORN strain. This suggests a strain-specific complement pathway regulation under the selected rearing conditions.

Cloning and characterization of the proximal promoter region of rainbow trout (Oncorhynchus mykiss) interleukin-6 gene.

Interleukin-6 (IL6) is a pleiotropic cytokine with important immunoregulatory functions. Its expression is inducible in immune cells and tissues of several fish species. We also found that IL6 mRNA abundance was significantly increased in spleen, liver, and gill of rainbow trout after experimental infection with Aeromonas salmonicida. Genomic DNA sequences of IL6 orthologs from three salmonid species revealed a conserved exon/intron structure and a high overall nucleotide identity of >88%. To uncover key mechanisms regulating IL6 expression in salmonid fish, we amplified a fragment of the proximal IL6 promoter from rainbow trout and identified in-silico conserved binding sites for NF-κB and CEBP. The activity of this IL6 promoter fragment was analyzed in the established human embryonic kidney line HEK-293. Luciferase- and GFP-based reporter systems revealed that the proximal IL6 promoter is activated by Escherichia coli. Essentially, both reporter systems proved that NF-κB p50, but not NF-κB p65 or CEBP, activates the IL6 promoter fragment. Truncation of this fragment caused a significant decrease in IL6 promoter activation. This characterization of the proximal promoter of the IL6-encoding gene provides basic knowledge about the IL6 gene expression in rainbow trout.

Identification and de novo sequencing of housekeeping genes appropriate for gene expression analyses in farmed maraena whitefish (Coregonus maraena) during crowding stress.

Maraena whitefish (Coregonus maraena; synonym Coregonus lavaretus f. balticus) is a high-quality food fish in the Southern Baltic Sea belonging to the group of salmonid fishes. Coregonus sp. is successfully kept in aquaculture throughout northern Europe (e.g. in Finland, Germany, Russia) and North America. In this regard, the molecular and immunological characterisation of stress response in maraena whitefish contributes to the development of robust and fast-growing maraena whitefish breeding strains for aquaculture. Thus, in the present study, the potential housekeeping genes beta actin (ACTB), elongation factor 1 alpha (EEF1A1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), ribosomal protein 9 (RPL9), ribosomal protein 32 (RPL32) and ribosomal protein S20 (RPS20) were de novo sequenced and tested concerning their applicability as reference genes in quantitative real-time PCR (qPCR) in maraena whitefish under different stocking densities. For this purpose, tissue samples of liver, kidney, gills, head kidney, skin, adipose tissue, heart and dorsal fin were investigated. qPCR data were analysed with Normfinder tool to determine gene expression stability. DNA sequencing exposed transcribed paralogous EEF1A1A and EEF1A1B genes differing in their putative protein structure. Normfinder analysis revealed RPL9 and RPL32 as most stable, GAPDH and ACTB as least stable genes for qPCR analyses, respectively. This is the first study that provides a subset of seven de novo sequenced housekeeping genes usable as reference genes in studies of stress response in maraena whitefish.

Characterization of the interleukin 1 receptor-associated kinase 4 (IRAK4)-encoding gene in salmonid fish: the functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells.

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells.

GRP94 is encoded by two differentially expressed genes during development of rainbow trout (Oncorhynchus mykiss).

The glucose-regulated protein, 94 kDa (GRP94), is an endoplasmic reticulum (ER)-localized heat shock protein that plays among other functions a crucial role in folding and exports of Toll-like receptors (TLRs) and some other immune-relevant factors. We identified two copies of the GRP94-encoding gene in rainbow trout sharing 91% DNA sequence identity. The conceptually translated ORFs encode a 795-aa GRP94a and a 510-aa GRP94b protein variant, respectively, with characteristic domains and amino acid residues. However, the shorter variant lacks motifs required for its localization in the ER and might thus represent an isoform of the putative mammalian ortholog GRP94a. Heat stress only slightly affects the expression of the two GRP94-encoding trout genes, as reported for mammals. We recorded the abundances of transcripts coding for both GRP94 variants as well as for a broad panel of TLRs representing their potential targets. In embryonic and larval trout, only the mRNAs encoding TLR1, -2, -9, and -20 were found in significant concentrations, while the expression of nine other TLRs was hardly detectable. The GRP94a-encoding gene showed constantly high expression levels indicating that this isoform is vitally required throughout the life cycle of rainbow trout. The concentration of the GRP94b-encoding mRNA was only ~0.1% compared to the GRP94a mRNA level. These structural and gene expression data together suggest that the two GRP94 gene products fulfill different physiological roles.