Comparative Analysis of the Transcriptome and Distribution of Putative SNPs in Two Rainbow Trout (Oncorhynchus mykiss) Breeding Strains by Using Next-Generation Sequencing.

Selective breeding can significantly improve the establishment of sustainable and profitable aquaculture fish farming. For rainbow trout (Oncorhynchus mykiss), one of the main aquaculture coldwater species in Europe, a variety of selected hatchery strains are commercially available. In this study, we investigated the genetic variation between the local Born strain, selected for survival, and the commercially available Silver Steelhead strain, selected for growth. We sequenced the transcriptome of six tissues (gills, head kidney, heart, liver, spleen, and white muscle) from eight healthy individuals per strain, using RNA-seq technology to identify strain-specific gene-expression patterns and single nucleotide polymorphisms (SNPs). In total, 1760 annotated genes were differentially expressed across all tissues. Pathway analysis assigned them to different gene networks. We also identified a set of SNPs, which are heterozygous for one of the two breeding strains: 1229 of which represent polymorphisms over all tissues and individuals. Our data indicate a strong genetic differentiation between Born and Silver Steelhead trout, despite the relatively short time of evolutionary separation of the two breeding strains. The results most likely reflect their specifically adapted genotypes and might contribute to the understanding of differences regarding their robustness toward high stress and pathogenic challenge described in former studies.

Evolutionary expression differences of creatine synthesis-related genes: Implications for skeletal muscle metabolism in fish.

The creatine/phosphocreatine system is the principal energy buffer in mammals, but is scarcely documented in fish. We measured the gene expression of major enzymes of this system, glycine amidinotransferase (GATM), guanidinoacetate N-methyltransferase (GAMT) and muscle-type creatine kinase (CKM) in kidney, liver, and muscle tissues of fish and mammals. CKM was expressed strongly in the muscles of all examined species. In contrast, GATM and GAMT were strongly expressed in the muscle tissue of fish, but not of mammals. This indicates that creatine synthesis and usage are spatially separated in mammals, but not in fish, which is supported by RNA-Seq data of 25 species. Differences in amino acid metabolism along with methionine adenosyltransferase gene expression in muscle from fishes but not mammals further support a central metabolic role of muscle in fish, and hence different organization of the creatine/phosphocreatine biosynthesis system in higher and lower vertebrates.

A molecular survey of programmed cell death in rainbow trout: Structural and functional specifications of apoptotic key molecules.

Apoptosis is an integral part of homeostasis and supports multiple physiological processes such as development and immune defense, thereby directly targeting damaged or unwanted cells without affecting neighbor cells. In the present study, we characterized the apoptotic key factors caspase-3, -7, and - 8 as well as regulator protein TPT1 (translationally-controlled tumor protein 1) from rainbow trout (Oncorhynchus mykiss). We identified multiple single-nucleotide changes in their coding sequences and showed that the CASP3 gene is present in at least three variants. Caspase genes were clustered to their orthologs in bony fish and human by using evolutionary analysis. Expression profiling in seven tissues of unchallenged adult fish revealed predominant transcript levels in the head kidney (CASP3, 7, and 8) or brain (TPT1). Further, we analyzed the expression of a more comprehensive panel of 16 trout genes encoding pro- and anti-apoptotic factors and associated proteins during development and upon stress exposure (in vitro temperature and staurosporine treatment). Previously published transcriptome data suggested that the induction of apoptotic processes is mirrored on the transcript level, but this could not be confirmed by the present gene-profiling study. Yet on the protein level, treatment of trout cell line RT-gill-W1 with 1 µM staurosporine for up to 120 min led to a significant increase of CASP3/7 activity. Moreover, a meta-analysis on published data showed that stress-related expression could only be detected sporadically for apoptotic key factors. In conclusion, there seems to be no reliable pattern or marker representing the stress-related induction of apoptosis in salmonids.

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.

Relative Abundance of Nitrotoga spp. in a Biofilter of a Cold-Freshwater Aquaculture Plant Appears To Be Stimulated by Slightly Acidic pH.

The functioning of recirculation aquaculture systems (RAS) is essential to maintain water quality for fish health, and one crucial process here is nitrification. The investigated RAS was connected to a rainbow trout production system and operated at an average temperature of 13°C and pH 6.8. Community analyses of the nitrifying biofilm revealed a coexistence of Nitrospira and Nitrotoga, and it is hypothesized that a slightly acidic pH in combination with lower temperatures favors the growth of the latter. Modification of the standard cultivation approach toward lower pH values of 5.7 to 6.0 resulted in the successful enrichment (99% purity) of Nitrotoga sp. strain HW29, which had a 16S rRNA sequence similarity of 99.0% to Nitrotoga arctica. Reference cultures of Nitrospira defluvii and the novel Nitrotoga sp. HW29 were used to confirm differentiation of these nitrite oxidizers in distinct ecological niches. Nitrotoga sp. HW29 revealed pH and temperature optima of 6.8 and 22°C, respectively, whereas Nitrospira defluvii displayed the highest nitrite oxidation rate at pH 7.3 and 32°C. We report here the occurrence of Nitrotoga as one of the main nitrite-oxidizing bacteria in freshwater aquaculture systems and indicate that a slightly acidic pH, in addition to temperatures below 20°C, can be applied as a selective isolation criterion for this microorganism.

Impact of Thermal Stress on Kidney-Specific Gene Expression in Farmed Regional and Imported Rainbow Trout.

Seasonal water temperatures can be stressful for fish in aquaculture and can therefore negatively influence their welfare. Although the kidney is the crucial organ associated with the primary stress response, knowledge about the stress-modulated kidney transcriptome in salmonids is limited. In the present study, we used a comparative microarray approach to characterize the general gene expression profiles of rainbow trout trunk kidney after a 2-week acclimation to mild heat (23 °C) and cold stress (8 °C). Hypothesizing that local adaptation influences stress performance, we aimed to identify differences in the temperature-induced gene expression in the regional trout strain BORN, in addition to a common imported strain. Moderate temperature challenge provoked typical stress response clusters, including heat-shock proteins or cold-inducible factors, in addition to altered energy metabolism in trout kidney. Mild cold, in particular, enhanced renal protein degradation processes, as well as mRNA and protein synthesis, while it also triggered fatty acid biosynthesis. Mild heat led to cytoskeleton-stabilizing processes and might have facilitated cell damage and infection. Furthermore, both breeding lines used different strategies for energy provision, cellular defense, and cell death/survival pathways. As a main finding, the genes involved in energy provision showed generally higher transcript levels at both temperatures in BORN trout compared to imported trout, indicating adjusted metabolic rates under local environmental conditions. Altogether, this study provides a general overview of stress-induced transcriptional patterns in rainbow trout trunk kidney, in addition to identifying genes and networks that contribute to the robustness of the BORN strain. Our analyses suggest SERPINH1 and CIRBP as general marker genes for heat stress and cold stress in trout, respectively.

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.

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.

Creatine metabolism differs between mammals and rainbow trout (Oncorhynchus mykiss).

Creatine plays an important role in the cell as an energy buffer. As the energy system is a basic element of the organism it may possibly contribute to differences between rainbow trout strains selected for the traits growth and robustness, respectively. The cDNA sequences of creatine-related genes encoding glycine amidinotransferase (GATM), guanidinoacetate N-methyltransferase (GAMT), creatine kinase muscle-type (CKM) and creatine transporter 1 (CT1, encoded by gene solute carrier family 6, member 8 (SLC6A8)) were characterized in rainbow trout. Transcripts of the respective genes were quantified in kidney, liver, brain and skeletal muscle in both trout strains that had been acclimated to different temperatures. Several differences between the compared trout strains were found as well as between temperatures indicating that the energy system may contribute to differences between both strains. In addition to that, the expression data showed clear differences between the creatine system in rainbow trout and mammals, as the spatial distribution of the enzyme-encoding gene expression was clearly different from the patterns described for mammals. In rainbow trout, creatine synthesis seems to take place to a big extent in the skeletal muscle.

Transcriptome profiling reveals insight into distinct immune responses to Aeromonas salmonicida in gill of two rainbow trout strains.

The fish gills represent a crucial organ for the communication with the aquatic environment. Transcriptional changes in gills of two hatchery rainbow trout strains in response to injection with the potent pathogen Aeromonas salmonicida were detected by global gene expression profiling using a 4×44K oligonucleotide microarray. Emphasis was placed on "day 3 postinfection" representing a decisive time point for the resolution of inflammation. The comparison of features and pathways differentially regulated in branchial tissues revealed that the local breeding strain BORN and imported American rainbow trout apply common and specific immune strategies. In gills of infected BORN trout, we observed a dynamic regulation of genes controlling NF-κB pathways and the induction of factors promoting the development of myeloid cells, whereas an increased expression of lysozyme and immunoglobulin genes was obvious in gills of infected import trout. In order to prove the relevance of the array-predicted candidates as well as well-known immune genes for gill immunity, a subsequent in vitro experiment was conducted. Altogether, we uncovered dynamic but moderate changes in the expression of a broad range of immune-relevant features implying the gill's involvement in pathogen defense strategies.

Novel insights into the peritoneal inflammation of rainbow trout (Oncorhynchus mykiss).

The peritoneal cavity has been extensively used as a laboratory model of inflammation in many species, including the teleost fish. Although, the peritoneal cavity of rainbow trout (Oncorhynchus mykiss) was previously shown to contain a resident population of leukocytes, closer information about their exact composition and their functional response to pathogens is still missing. In the presented work, flow cytometric analysis using monoclonal antibodies was performed to characterize this cell population and evaluate its traffic during the first 72 h after antigenic stimulation and infection with Aeromonas salmonicida. Obtained results indicate that the unstimulated peritoneal cavity represents rather a lymphoid niche, dominated by the IgM(+) B cells. Expectedly, the composition changed rapidly after stimulation, which resulted in two complete changes of dominant cell type within first 72 h post injection. While the first stage of inflammation was dominated by myeloid cells, lymphocytes predominated at the later time points, with IgM(+) B cells representing more than two thirds of all cells. Later, the infection experiment elucidated the peritoneal infection and identified the key differences to the antigenic stimulation. Additionally, the data indicate that the resolution of the inflammation depends more on the bacterial clearance by myeloid cells than on regulation by lymphocytes. Taken together, obtained results represent the first complete description of the immune reaction protecting the peritoneal cavity of the fish and shed some light on the conservation of these processes during the evolution.

Transcriptome profiling of gill tissue in regionally bred and globally farmed rainbow trout strains reveals different strategies for coping with thermal stress.

Thermal stress can pose a major challenge to salmonid fish. A 4x44K oligonucleotide microarray approach was used to screen for genetically determined variations of a temperature stress response during acclimation in fish gills, a highly specialized and complex organ responsible for gas and electrolyte exchange as well as excretion. The comparison addressed transcriptional changes in the local breeding strain BORN and imported (TCO) rainbow trout after graded 2-week acclimation to 8 and 23 °C. Besides well-characterized mediators of thermoregulation such as genes encoding cold-inducible RNA-binding protein and heat shock proteins, the present microarray study suggests several new candidate genes commonly regulated in gills of the two trout lines. Having identified the differential expression of thermoregulated genes as duplicated paralogues, they were subsequently validated in a gill cell model. Moreover, the comparison of transcriptome profiles provides evidence for distinctively employed expression patterns. The induction of genes encoding factors of the early innate immunity in BORN trout upon warming contrasts with the increased expression of adaptive immune genes in import trout. Cold acclimation induced genes assigned to the functional categories "cell death" and "ion channel activity" in import trout, but repressed "lipid metabolism." This manuscript provides an overview of the genes of the multifunctional gills in rainbow trout that are mandated after temperature change, suggesting links between the different temperature-dependent pathways and gene networks.

Iron-sulfur cluster scaffold (ISCU) gene is duplicated in salmonid fish and tissue and temperature dependent expressed in rainbow trout.

The iron-sulfur cluster protein ISCU is a scaffold protein tasked with the building and mediation of iron-sulfur [Fe-S]-clusters. These are crucial for [Fe-S]-enzymes, which are involved in essential biological cell processes like metabolism or ion transport. Analysis of ISCU in rainbow trout (Oncorhynchus mykiss) and maraena whitefish (Coregonus maraena) revealed the existence of two gene variants in each of the two salmonids. This study presents the characterization of the duplicated ISCU cDNA sequences in both species as well as the comparative functional analysis of the genes in healthy and affected fish of two rainbow trout strains differing in trait robustness under regional aquaculture conditions. Coding sequences of trout ISCUA and ISCUB genes are spanning over five exons. Open reading frames (ORF) of trout (ISCUA: 495bp, ISCUB: 498bp) and whitefish (ISCUA and ISCUB: 495bp) genes encode for evolutionary highly conserved proteins and share 72% sequence similarity with human ISCU. Transcriptome analyses comparing healthy fish of the local rainbow trout strain BORN and the import strain TCO revealed strain-specific expression patterns for ISCU. Expression analyses by quantitative RT-PCR indicated remarkable differences between the transcript level of the gene variants ISCUA and ISCUB. Moderate temperature challenge (8°C and 23°C) suggests a generally higher transcript level of the two gene variants at 8°C in the liver, spleen, and gill of both strains. However, no remarkable differences between the strains occurred in the temperature-dependent ISCU gene expression profiles. The experimental infection with Aeromonas salmonicida resulted in a different ISCU gene expression in the gill and trunk kidney of both strains after two weeks, suggesting a specific role of the scaffold gene in rainbow trout strain BORN, regarding the recovery after infection. Although results partially reflect the expected strain- and tissue-specific ISCUA and ISCUB regulation in rainbow trout, the data do not support the assumed association of ISCU with the trait robustness.

Comparison of splenic transcriptome activity of two rainbow trout strains differing in robustness under regional aquaculture conditions.

The spleen plays a crucial role in innate and adaptive immunity in bony fish. Consequently, this organ is well suited to assess the immune competence of an organism and hence provides useful information for comparison and classification of production traits of food fish, such as robustness and susceptibility. To gain information about differences in the basal splenic transcriptome activity, healthy rainbow trout of the commercially important strain TCO and the local selection strain BORN have been compared in a holistic expression analysis using the GRASP 16K cDNA microarray. Nearly all differentially expressed genes (n = 807) in the spleen were on a lower expression level (n = 802) in BORN trout compared to the TCO strain. Global gene ontology analysis revealed that most genes are involved in fundamental biological processes like cellular growth, vesicular trafficking and energy metabolism. Surprisingly, only 7 % of splenic differentially expressed genes are associated with functions of the immune system like TLR signaling, acute phase response and complement system. MARCH3 is one lower expressed gene of interest in BORN trout. This gene, coding for an E3 ubiquitin ligase, is involved in three metabolic functions: immune system, vesicular trafficking and ubiquitination. Since MARCH genes are furthermore differently regulated in the two strains after viral infection and assumed to be potentially active in regulation of immune receptors in fish, MARCH3 was chosen for a closer structural analysis. In concert with the data interpretation of the achieved comparative transcriptome analysis for the involved rainbow trout strains, we provide the full mRNA sequence of trout MARCH3 and its hypothetical protein structure.

Identification of differentially expressed protective genes in liver of two rainbow trout strains.

Since 1975, the rainbow trout strain BORN (Germany) has been bred in brackish water from a coastal form imported from Denmark. Accompanying phenotypic monitoring of the adapted BORN trout until now revealed that this selection strain manifested a generally elevated resistance towards high stress and pathogenic challenge including lower susceptibility towards Aeromonas salmonicida infections in comparison to other trout strains in local aqua farms. We focus on the elucidation of both, genetic background and immunological basis for the increased survivorship to infections. A first comparison of gene expression profiles in liver tissue of healthy rainbow trout from the local selection strain BORN and imported trout using a GRASP 16K cDNA microarray revealed six differentially expressed genes evoking pathogen and wounding responses, LEAP2A (encoding for liver-expressed antimicrobial peptide), SERPINA1 (alpha-1 antitrypsin), FTH1 (middle subunit of ferritin), FGL2 (fibroleukin), CLEC4E (macrophage-inducible C-type lectin), and SERPINF2 (alpha-2 antiplasmin). Since the latter gene is not described in salmonid species so far, our first aim was to characterize the respective sequence in rainbow trout. Two trout SERPINF2 genes were identified, which share only 48% identical amino acid residues and a characteristic SERPIN domain. Second, we aimed to analyse the expression of those genes after temperature challenge (8 °C and 23 °C). Only FTH1 was upregulated in BORN and import trout after increase of temperature, while SERPINA1 and FGL2 were only elevated in import trout. Third, the expression of all named genes was analyzed after pathogen challenge with A. salmonicida subsp. salmonicida. As a main finding, we detected a comparably faster regeneration of LEAP2A mRNA abundance in BORN trout following bacterial infection. Ingenuity Pathways Analysis suggested a functional interplay among the mentioned factors and the pro-inflammatory cytokine TNF, whose stronger expression was validated in liver of BORN trout. This data indicate that the examined genes contribute to an improved first barrier against invading pathogens in BORN trout.