Vergleichende Charakterisierung von Genen für eine differente Stresstoleranz bei der Regenbogenforelle (Oncorhynchus mykiss)

Die an Brackwasser adaptierte lokale Regenbogenforellenlinie BORN hat sich im direkten Vergleich mit kommerziell in der Aquakultur eingesetzten Importforellen als besonders robust in ihrer Reaktion auf Stressoren gezeigt. Ziel dieser Arbeit war es, Gene und Stoffwechselwege zu identifizieren (u.a. vergleichende Transkriptomanalysen) und molekulargenetisch zu charakterisieren (u.a. Strukturanalyse, vergleichende Expressionsanalysen), die zusätzlich zu klassischen Stressgenen an der differenten Stressantwort der BORN-Forelle beteiligt sind.The local rainbow trout selection strain BORN has been shown to be less susceptible towards different stressors than imported breeding-lines commercially used in aquaculture farms. The aim of this thesis was to identify and characterize candidate genes that are, additionally to already known genes responding to stress, involved in the different stress response of BORN trout. The candidate genes were molecularly characterized; comparative expression analyses in healthy trout as well as after stress caused by infection and challenging temperatures reflect strain specific expression profiles

The synergistic interaction of thermal stress coupled with overstocking strongly modulates the transcriptomic activity and immune capacity of rainbow trout (Oncorhynchus mykiss)

The objective of the present study is to identify and evaluate informative indicators for the welfare of rainbow trout exposed to (A) a water temperature of 27 °C and (B) a stocking density of 100 kg/m3 combined with a temperature of 27 °C. The spleen-somatic and condition index, haematocrit and the concentrations of haemoglobin, plasma cortisol and glucose revealed non-significant differences between the two stress groups and the reference group 8 days after the onset of the experiments. The transcript abundance of almost 1,500 genes was modulated at least twofold in in the spleen of rainbow trout exposed to a critical temperature alone or a critical temperature combined with crowding as compared to the reference fish. The number of differentially expressed genes was four times higher in trout that were simultaneously challenged with high temperature and crowding, compared to trout challenged with high temperature alone. Based on these sets of differentially expressed genes, we identified unique and common tissue- and stress type-specific pathways. Furthermore, our subsequent immunologic analyses revealed reduced bactericidal and inflammatory activity and a significantly altered blood-cell composition in challenged versus non-challenged rainbow trout. Altogether, our data demonstrate that heat and overstocking exert synergistic effects on the rainbow trout’s physiology, especially on the immune system.publishedVersio

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

Abstract 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

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

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

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.publishedVersio

Gradual and Acute Temperature Rise Induces Crossing Endocrine, Metabolic, and Immunological Pathways in Maraena Whitefish (Coregonus maraena)

The complex and still poorly understood nature of thermoregulation in various fish species complicates the determination of the physiological status on the basis of diagnostic marker genes and indicative molecular pathways. The present study aimed to compare the physiological impacts of both gradual and acute temperature rise from 18 to 24°C on maraena whitefish in aquaculture. Microarray-based transcriptome profiles in the liver, spleen and kidney of heat-stressed maraena whitefish revealed the modulation of a significantly higher number of genes in those groups exposed to gradually rising temperatures compared with the acutely stressed groups, which might reflect early adaptation mechanisms. Moreover, we suggest a common set of 11 differentially expressed genes that indicate thermal stress induced by gradual or acute temperature rise in the three selected tissues. Besides the two pathways regulated in both data sets unfolded protein response and aldosterone signaling in epithelial cells, we identified unique tissue- and stress type-specific pathways reflecting the crossroads between signal transduction, metabolic and immunologic pathways to cope with thermal stress. In addition, comparing lists of differentially regulated genes with meta-analyzed published data sets revealed that “acute temperature rise”-responding genes that encode members of the HSP70, HSP90, and HSP40 families; their functional homologs; co-chaperones and stress-signal transducers are well-conserved across different species, tissues and/or cell types and experimental approaches

Microarray-predicted marker genes and molecular pathways indicating crowding stress in rainbow trout (Oncorhynchus mykiss)

STATEMENT OF RELEVANCE: The comprehensive recording of animal-based biomarkers is becoming increasingly important for the further development of the aquaculture industry with importance for fish well-being and consumer opinion. Previous studies on crowding stress in rainbow trout have used single or multi-gene approaches to assess the relevance of putative biomarkers, but transcriptomic approaches to define crowding stress-induced pathways in salmonids are overdue. The present study compares the transcriptomic profiles of rainbow trout exposed to different stocking densities and correlates these data sets with classical plasma parameters and behavioural observations.Stocking density is considered as a key factor determining the productivity of fish aquaculture systems. The transcriptomic response to crowding stress is, however, still poorly investigated. We aimed to identify potential biomarker genes via microarray analyses to gain insight into molecular pathways modulated by density-induced stress in farmed rainbow trout Oncorhynchus mykiss. Transcriptome profiling in the liver, kidney, and gills was complemented by analysis of classical plasma parameters and behavioural observations. Individuals of two trout strains were exposed for nine days to definite stocking densities, ~ 10 kg/m3 (low density); ~ 30 kg/m3 (moderate); ~ 60 kg/m3 (elevated); and ~ 120 kg/m3 (high). Only the PYGMA gene, encoding the key enzyme of glycogenolysis, was upregulated in gills and kidney, while about 90 genes were differentially regulated in the liver of trout exposed to high density compared with the moderate-density group. Pathway enrichment analyses predicted the upregulation of HIF1A signalling in liver, contributing to glucose homeostasis during stress conditions. This is consistent with the increased plasma glucose levels detected in crowding-stressed trout. The mTOR and PI3K/AKT signalling pathways were impaired in liver at higher stocking densities indicating stress at the cellular level. Contrary to our expectations, protein ubiquitination and cholesterol biosynthesis pathways were found to be underexpressed. Cholesterol is a precursor of the stress hormone cortisol, whose plasma concentrations were indeed within the normal physiological range in all treatment groups. In addition, we did not observe any aggressive interactions, which are known to be triggered through cortisol release. Taken together, we identified a relatively modest number of rainbow trout-specific crowding markers suggesting that farmed rainbow trout have gained enhanced stress-tolerant phenotypes through decades of selective breeding

Proteomic Comparison of Two Invasive Polychaete Species and Their Naturally Occurring F₁-hybrids

The mud worm genus <i>Marenzelleria</i> is highly invasive and is therefore studied intensively. In recently invaded habitats, sympatric populations of the sibling species <i>Marenzelleria viridis</i> and <i>Marenzelleria neglecta</i> are found. In these secondary contact zones, hybridization occurs frequently, revealing incomplete reproductive isolation between these recently diverged species. Two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometric methods were applied for a comparative analysis of these species and their F₁-hybrids. Nineteen proteins were identified by cross-species identification strategies. A low degree of interindividual variability within either species allowed characterizing qualitative species-specific differences in 2-DE spot patterns as well as in peptide maps. Species-specific peptides were found in tryptic digests of various proteins, such as glyceraldehyde-3-phosphate dehydrogenase, troponin C, gelsolin-like protein, and peroxiredoxin-1. F₁-hybrids of <i>M. viridis</i> and <i>M. neglecta</i> showed additivity of protein spot patterns, and the presence of both parental traits was confirmed by mass spectrometric data. This study is one of few dealing with global protein expression in polychaetes and is the first proteomic description of natural F₁-hybrids in polychaetes. It furthermore indicates the feasibility of proteomic methods for analyses of speciation in <i>Marenzelleria</i> siblings as well as of hybridization events in secondary contact zones in general

The synergistic interaction of thermal stress coupled with overstocking strongly modulates the transcriptomic activity and immune capacity of rainbow trout (Oncorhynchus mykiss)

The objective of the present study is to identify and evaluate informative indicators for the welfare of rainbow trout exposed to (A) a water temperature of 27 °C and (B) a stocking density of 100 kg/m3 combined with a temperature of 27 °C. The spleen-somatic and condition index, haematocrit and the concentrations of haemoglobin, plasma cortisol and glucose revealed non-significant differences between the two stress groups and the reference group 8 days after the onset of the experiments. The transcript abundance of almost 1,500 genes was modulated at least twofold in in the spleen of rainbow trout exposed to a critical temperature alone or a critical temperature combined with crowding as compared to the reference fish. The number of differentially expressed genes was four times higher in trout that were simultaneously challenged with high temperature and crowding, compared to trout challenged with high temperature alone. Based on these sets of differentially expressed genes, we identified unique and common tissue- and stress type-specific pathways. Furthermore, our subsequent immunologic analyses revealed reduced bactericidal and inflammatory activity and a significantly altered blood-cell composition in challenged versus non-challenged rainbow trout. Altogether, our data demonstrate that heat and overstocking exert synergistic effects on the rainbow trout’s physiology, especially on the immune system