Experimental Handling Challenges Result in Minor Changes in the Phagocytic Capacity and Transcriptome of Head-Kidney Cells of the Salmonid Fish Coregonus maraena.

Aquaculture management involves regular handling procedures, but these can evoke stress responses in farmed fish. We compiled an extensive list of published parameters that indicate the most likely handling-induced physiological deviations from the norm. However, since these parameters are based almost exclusively on studies of rainbow trout and Atlantic salmon, we conducted a handling-challenge experiment with maraena whitefish (Coregonus maraena). This salmonid fish was sampled at either 3 or 24 h after a single 1-min handling or after 10 days of daily repeated 1-min handling. The cortisol levels were strongly elevated in some individuals at 3 h after the single handling challenge, but these elevations were not significantly different between the challenged and control cohorts. The phagocytic capacity of myeloid head-kidney cells stimulated with fluorophore-labeled, inactivated Aeromonas salmonicida was significantly decreased in maraena whitefish at 3 h after the handling challenge compared to control fish. Microarray analysis of head-kidney samples from the challenged and control fish revealed 12 differentially expressed genes at 3 h and 70 at 24 h after the single handling episode, but only 5 differentially expressed genes after 10 days of repeated daily handling. The identified genes were assigned to numerous stress- and immune-relevant functional pathways, including "glucocorticoid receptor signaling" (3 h post-challenge), "HIF1A signaling" (24 h post-challenge), or "complement system" (10 days of repeated challenge). Our data reveal the tight interconnection of immune and stress pathways in the head kidney of maraena whitefish and corroborate several parameters previously found regulated in other tissues of handling-stressed rainbow trout. These findings indicate that handling may compromise the health and welfare of maraena whitefish in aquaculture.

Evaluation of blood cell viability rate, gene expression, and O-GlcNAcylation profiles as indicative signatures for fungal stimulation of salmonid cell models.

Fungal diseases of fish are a significant economic problem in aquaculture. Using high-throughput expression analysis, we identified potential transcript markers in primary head kidney and secondary embryonic cells from salmonid fish after stimulation with the inactivated fungi Mucor hiemalis and Fusarium aveneacium and with purified fungal molecular patterns. The transcript levels of most of the 45 selected genes were altered in head-kidney cells after 24 h of stimulation with fungal antigens. Stimulation with the inactivated fungus M. hiemalis induced the most pronounced transcriptional changes, including the pathogen receptor-encoding genes CLEC18A and TLR22, the cytokine-encoding genes IL6 and TNF, and the gene encoding the antimicrobial peptide LEAP2. In parallel, we analyzed the total GlcNAcylation status of embryonic salmonid cells with or without stimulation with inactivated fungi. O-GlcNAcylation modulates gene expression, intracellular protein, and signal activity, but we detected no significant differences after a 3-h stimulation. A pathway analysis tool identified the "apoptosis of leukocytes" based on the expression profile 24 h after fungal stimulation. Fluorescence microscopy combined with flow cytometry revealed apoptosis in 50 % of head-kidney leukocytes after 3 h stimulation with M. hiemalis, but this level decreased by > 5% after 24 h of stimulation. The number of apoptotic cells significantly increased in all blood cells after a 3-h stimulation with fungal molecular patterns compared to unstimulated controls. This in vitro approach identified transcript-based parameters that were strongly modulated by fungal infections of salmonid fish.

Quality control in scRNA-Seq can discriminate pacemaker cells: the mtRNA bias.

Single-cell RNA-sequencing (scRNA-seq) provides high-resolution insights into complex tissues. Cardiac tissue, however, poses a major challenge due to the delicate isolation process and the large size of mature cardiomyocytes. Regardless of the experimental technique, captured cells are often impaired and some capture sites may contain multiple or no cells at all. All this refers to "low quality" potentially leading to data misinterpretation. Common standard quality control parameters involve the number of detected genes, transcripts per cell, and the fraction of transcripts from mitochondrial genes. While cutoffs for transcripts and genes per cell are usually user-defined for each experiment or individually calculated, a fixed threshold of 5% mitochondrial transcripts is standard and often set as default in scRNA-seq software. However, this parameter is highly dependent on the tissue type. In the heart, mitochondrial transcripts comprise almost 30% of total mRNA due to high energy demands. Here, we demonstrate that a 5%-threshold not only causes an unacceptable exclusion of cardiomyocytes but also introduces a bias that particularly discriminates pacemaker cells. This effect is apparent for our in vitro generated induced-sinoatrial-bodies (iSABs; highly enriched physiologically functional pacemaker cells), and also evident in a public data set of cells isolated from embryonal murine sinoatrial node tissue (Goodyer William et al. in Circ Res 125:379-397, 2019). Taken together, we recommend omitting this filtering parameter for scRNA-seq in cardiovascular applications whenever possible.

Effects of Chronic Hypoxia on the Immune Status of Pikeperch (Sander lucioperca Linnaeus, 1758).

Inadequate oxygen saturation can induce stress responses in fish and further affect their immunity. Pikeperch, recently introduced in intensive aquaculture, is suggested to be reared at nearly 100% DO (dissolved oxygen), yet this recommendation can be compromised by several factors including the water temperature, stocking densities or low circulation. Herein, we aimed to investigate the effect of low oxygen saturation of 40% DO (±3.2 mg/L) over 28 days on pikeperch farmed in recirculating aquaculture systems. The obtained data suggest that-although the standard blood and health parameters did not reveal any significant differences at any timepoint-the flow cytometric analysis identified a slightly decreased proportion of lymphocytes in the HK (head kidney) of fish exposed to hypoxia. This has been complemented by marginally downregulated expression of investigated immune and stress genes in HK and liver (including FTH1, HIF1A and NR3C1). Additionally, in the model of acute peritoneal inflammation induced with inactivated Aeromonas hydrophila, we observed a striking dichotomy in the sensitivity to the low DO between innate and adaptive immunity. Thus, while the mobilization of myeloid cells from HK to blood, spleen and peritoneal cavity, underlined by changes in the expression of key proinflammatory cytokines (including MPO, IL1B and TNF) was not influenced by the low DO, hypoxia impaired the influx of lymphocytes to the peritoneal niche in the later phases of the immune reaction. Taken together, our data suggest high robustness of pikeperch towards the low oxygen saturation and further encourage its introduction to the intensive aquaculture systems.

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.

An ultra-high density SNP-based linkage map for enhancing the pikeperch (Sander lucioperca) genome assembly to chromosome-scale.

Pikeperch (Sander lucioperca) is a fish species with growing economic significance in the aquaculture industry. However, successful positioning of pikeperch in large-scale aquaculture requires advances in our understanding of its genome organization. In this study, an ultra-high density linkage map for pikeperch comprising 24 linkage groups and 1,023,625 single nucleotide polymorphisms markers was constructed after genotyping whole-genome sequencing data from 11 broodstock and 363 progeny, belonging to 6 full-sib families. The sex-specific linkage maps spanned a total of 2985.16 cM in females and 2540.47 cM in males with an average inter-marker distance of 0.0030 and 0.0026 cM, respectively. The sex-averaged map spanned a total of 2725.53 cM with an average inter-marker distance of 0.0028 cM. Furthermore, the sex-averaged map was used for improving the contiguity and accuracy of the current pikeperch genome assembly. Based on 723,360 markers, 706 contigs were anchored and oriented into 24 pseudomolecules, covering a total of 896.48 Mb and accounting for 99.47% of the assembled genome size. The overall contiguity of the assembly improved with a scaffold N50 length of 41.06 Mb. Finally, an updated annotation of protein-coding genes and repetitive elements of the enhanced genome assembly is provided at NCBI.

Time-Dependent Effects of Acute Handling on the Brain Monoamine System of the Salmonid Coregonus maraena.

The immediate stress response involves the activation of the monoaminergic neurotransmitter systems including serotonin, dopamine and noradrenaline in particular areas of the fish brain. We chose maraena whitefish as a stress-sensitive salmonid species to investigate the influence of acute and chronic handling on the neurochemistry of monoamines in the brain. Plasma cortisol was quantified to assess the activation of the stress axis. In addition, we analyzed the expression of 37 genes related to the monoamine system to identify genes that could be used as markers of neurophysiological stress effects. Brain neurochemistry responded to a single handling (1 min netting and chasing) with increased serotonergic activity 3 h post-challenge. This was accompanied by a modulated expression of monoaminergic receptor genes in the hindbrain and a significant increase of plasma cortisol. The initial response was compensated by an increased monoamine synthesis at 24 h post-challenge, combined with the modulated expression of serotonin-receptor genes and plasma cortisol concentrations returning to control levels. After 10 days of repeated handling (1 min per day), we detected a slightly increased noradrenaline synthesis and a down-regulated expression of dopamine-receptor genes without effect on plasma cortisol levels. In conclusion, the changes in serotonergic neurochemistry and selected gene-expression profiles, together with the initial plasma cortisol variation, indicate an acute response and a subsequent recovery phase with signs of habituation after 10 days of daily exposure to handling. Based on the basal expression patterns of particular genes and their significant regulation upon handling conditions, we suggest a group of genes as potential biomarkers that indicate handling stress on the brain monoamine systems.

Indication of Premelanosome Protein (PMEL) Expression Outside of Pigmented Bovine Skin Suggests Functions Beyond Eumelanogenesis.

The premelanosome protein (PMEL) is important for fibril formation within melanosomes during vertebrate melanogenesis. Fibrils form a matrix for pigment deposition within pigmented tissues such as skin and hair. PMEL mutations are known to modulate eumelanic pigmentation in vertebrates. However, in bovines, PMEL mutations were also found to alter pheomelanic pigmentation resulting in coat color dilution. Furthermore, epistatic effects of a mutated PMEL allele were detected in the phenotypic expression of the bovine hair defect "rat-tail syndrome" (RTS) characterized by charcoal coat color and hair deformation. Reports about PMEL gene expression in non-pigmented tissues raised the hypothesis that there may be unknown functions of the PMEL protein beyond eumelanin deposition to PMEL fibrils. In our study, we analysed the PMEL protein expression in pigmented skin and non-pigmented bovine tissues (non-pigmented skin, thyroid gland, rumen, liver, kidney, and adrenal gland cortex). We found that a processed form of the bovine PMEL protein is expressed in pigmented as well as in non-pigmented tissues, which is in line with gene expression data from targeted RT-PCR and whole transcriptome RNAseq analysis. The PMEL protein is located in membranes and within the cytosol of epithelial cells. Based on our data from bovine tissues, we concluded that at least in cattle PMEL potentially has additional, yet unexplored functions, which might contribute to effects of PMEL mutations on pheomelanin coat color dilution and charcoal coat color in RTS animals. However, indication of PMEL protein in unpigmented cells and tissues will require further confirmation in the future, because there have been no confirmed reports before, which had detected bovine PMEL protein with specific antibodies either in pigmented or unpigmented tissue.

Integrative Cluster Analysis of Whole Hearts Reveals Proliferative Cardiomyocytes in Adult Mice.

The recent development and broad application of sequencing techniques at the single-cell level is generating an unprecedented amount of data. The different techniques have their individual limits, but the datasets also offer unexpected possibilities when utilized collectively. Here, we applied snRNA-seq in whole adult murine hearts from an inbred (C57BL/6NRj) and an outbred (Fzt:DU) mouse strain to directly compare the data with the publicly available scRNA-seq data of the tabula muris project. Explicitly choosing a single-nucleus approach allowed us to pin down the typical heart tissue-specific technical bias, coming up with novel insights on the mammalian heart cell composition. For our integrated dataset, cardiomyocytes, fibroblasts, and endothelial cells constituted the three main cell populations accounting for about 75% of all cells. However, their numbers severely differed between the individual datasets, with cardiomyocyte proportions ranging from about 9% in the tabula muris data to around 23% for our BL6 data, representing the prime example for cell capture technique related bias when using a conventional single-cell approach for these large cells. Most strikingly in our comparison was the discovery of a minor population of cardiomyocytes characterized by proliferation markers that could not be identified by analyzing the datasets individually. It is now widely accepted that the heart has an, albeit very restricted, regenerative potential. However there is still an ongoing debate where new cardiomyocytes arise from. Our findings support the idea that the renewal of the cardiomyocyte pool is driven by cytokinesis of resident cardiomyocytes rather than differentiation of progenitor cells. We thus provide data that can contribute to an understanding of heart cell regeneration, which is a prerequisite for future applications to enhance the process of heart repair.

Identification and Annotation of Potential Function of Regulatory Antisense Long Non-Coding RNAs Related to Feed Efficiency in Bos taurus Bulls.

Long non-coding RNAs (lncRNAs) can influence transcriptional and translational processes in mammalian cells and are associated with various developmental, physiological and phenotypic conditions. However, they remain poorly understood and annotated in livestock species. We combined phenotypic, metabolomics and liver transcriptomic data of bulls divergent for residual feed intake (RFI) and fat accretion. Based on a project-specific transcriptome annotation for the bovine reference genome ARS-UCD.1.2 and multiple-tissue total RNA sequencing data, we predicted 3590 loci to be lncRNAs. To identify lncRNAs with potential regulatory influence on phenotype and gene expression, we applied the regulatory impact factor algorithm on a functionally prioritized set of loci (n = 4666). Applying the algorithm of partial correlation and information theory, significant and independent pairwise correlations were calculated and co-expression networks were established, including plasma metabolites correlated with lncRNAs. The network hub lncRNAs were assessed for potential cis-actions and subjected to biological pathway enrichment analyses. Our results reveal a prevalence of antisense lncRNAs positively correlated with adjacent protein-coding genes and suggest their participation in mitochondrial function, acute phase response signalling, TCA-cycle, fatty acid ß-oxidation and presumably gluconeogenesis. These antisense lncRNAs indicate a stabilizing function for their cis-correlated genes and a putative regulatory role in gene expression.

Single-Nucleus Sequencing of an Entire Mammalian Heart: Cell Type Composition and Velocity.

: Analyses on the cellular level are indispensable to expand our understanding of complex tissues like the mammalian heart. Single-nucleus sequencing (snRNA-seq) allows for the exploration of cellular composition and cell features without major hurdles of single-cell sequencing. We used snRNA-seq to investigate for the first time an entire adult mammalian heart. Single-nucleus quantification and clustering led to an accurate representation of cell types, revealing 24 distinct clusters with endothelial cells (28.8%), fibroblasts (25.3%), and cardiomyocytes (22.8%) constituting the major cell populations. An additional RNA velocity analysis allowed us to study transcription kinetics and was utilized to visualize the transitions between mature and nascent cellular states of the cell types. We identified subgroups of cardiomyocytes with distinct marker profiles. For example, the expression of Hand2os1 distinguished immature cardiomyocytes from differentiated cardiomyocyte populations. Moreover, we found a cell population that comprises endothelial markers as well as markers clearly related to cardiomyocyte function. Our velocity data support the idea that this population is in a trans-differentiation process from an endothelial cell-like phenotype towards a cardiomyocyte-like phenotype. In summary, we present the first report of sequencing an entire adult mammalian heart, providing realistic cell-type distributions combined with RNA velocity kinetics hinting at interrelations.

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.

Gene Profiling in the Adipose Fin of Salmonid Fishes Supports its Function as a Flow Sensor.

In stock enhancement and sea-ranching procedures, the adipose fin of hundreds of millions of salmonids is removed for marking purposes annually. However, recent studies proved the significance of the adipose fin as a flow sensor and attraction feature. In the present study, we profiled the specific expression of 20 neuron- and glial cell-marker genes in the adipose fin and seven other tissues (including dorsal and pectoral fin, brain, skin, muscle, head kidney, and liver) of the salmonid species rainbow trout Oncorhynchus mykiss and maraena whitefish Coregonusmaraena. Moreover, we measured the transcript abundance of genes coding for 15 mechanoreceptive channel proteins from a variety of mechanoreceptors known in vertebrates. The overall expression patterns indicate the presence of the entire repertoire of neurons, glial cells and receptor proteins on the RNA level. This quantification suggests that the adipose fin contains considerable amounts of small nerve fibers with unmyelinated or slightly myelinated axons and most likely mechanoreceptive potential. The findings are consistent for both rainbow trout and maraena whitefish and support a previous hypothesis about the innervation and potential flow sensory function of the adipose fin. Moreover, our data suggest that the resection of the adipose fin has a stronger impact on the welfare of salmonid fish than previously assumed.

Biological Network Approach for the Identification of Regulatory Long Non-Coding RNAs Associated With Metabolic Efficiency in Cattle.

Background: Genomic regions associated with divergent livestock feed efficiency have been found predominantly outside protein coding sequences. Long non-coding RNAs (lncRNA) can modulate chromatin accessibility, gene expression and act as important metabolic regulators in mammals. By integrating phenotypic, transcriptomic, and metabolomic data with quantitative trait locus data in prioritizing co-expression network analyses, we aimed to identify and functionally characterize lncRNAs with a potential key regulatory role in metabolic efficiency in cattle. Materials and Methods: Crossbred animals (n = 48) of a Charolais x Holstein F2-population were allocated to groups of high or low metabolic efficiency based on residual feed intake in bulls, energy corrected milk in cows and intramuscular fat content in both genders. Tissue samples from jejunum, liver, skeletal muscle and rumen were subjected to global transcriptomic analysis via stranded total RNA sequencing (RNAseq) and blood plasma samples were used for profiling of 640 metabolites. To identify lncRNAs within the indicated tissues, a project-specific transcriptome annotation was established. Subsequently, novel transcripts were categorized for potential lncRNA status, yielding a total of 7,646 predicted lncRNA transcripts belonging to 3,287 loci. A regulatory impact factor approach highlighted 92, 55, 35, and 73 lncRNAs in jejunum, liver, muscle, and rumen, respectively. Their ensuing high regulatory impact factor scores indicated a potential regulatory key function in a gene set comprising loci displaying differential expression, tissue specificity and loci overlapping with quantitative trait locus regions for residual feed intake or milk production. These were subjected to a partial correlation and information theory analysis with the prioritized gene set. Results and Conclusions: Independent, significant and group-specific correlations (|r| > 0.8) were used to build a network for the high and the low metabolic efficiency group resulting in 1,522 and 1,732 nodes, respectively. Eight lncRNAs displayed a particularly high connectivity (>100 nodes). Metabolites and genes from the partial correlation and information theory networks, which each correlated significantly with the respective lncRNA, were included in an enrichment analysis indicating distinct affected pathways for the eight lncRNAs. LncRNAs associated with metabolic efficiency were classified to be functionally involved in hepatic amino acid metabolism and protein synthesis and in calcium signaling and neuronal nitric oxide synthase signaling in skeletal muscle cells.

Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection.

The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1-3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/ß-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line.

Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

Transcriptome sequencing of maraena whitefish (Coregonus maraena).

Maraena whitefish (Coregonus maraena, Bloch, 1779) is a high-quality food fish belonging to the family Salmonidae with considerable economic relevance in the Baltic area. Aquaculture of this species is fundamental for its successful conservation and thus sustainable fisheries. Robust fishes obtained from breeding lines build the basis for effective aquaculture. Doubtless, the utilization of transcriptome sequencing and identification of genetic markers contribute to this aim. 454 FLX Titanium Sequencing provided 1.31 million sequence reads representing a first insight into the C. maraena transcriptome. The 454 Newbler Assembly arranged 29,094 contigs with an average length of 798bp. We found a whole series of transcripts highly probably resulting from ancient genome duplication and annotated 2887 different transcripts with an average length of 812bp. Functional annotation obtained a transcript composition predominantly comprising enzyme-coding genes.

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.