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.

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.

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The mammalian interleukin 1 receptor-like 1 receptor (IL1RL1), commonly known as ST2, is thought to downregulate TLR signalling by sequestering the signalling adapter MYD88 (myeloid differentiation primary response protein 88). ST2 sequences are known in several fish species, but none of them have functionally been examined. We characterised ST2 from rainbow trout (Oncorhynchus mykiss) and the structure of its encoding gene. The primary sequence of ST2 is only weakly conserved from fish to human. However, the amino acid sequences forming the interfaces for ST2 and MYD88 interaction are well conserved throughout evolution. High similarity of the gene segmentation unambiguously proves the common ancestry of fish and mammalian ST2. Trout ST2 and trout MYD88 genes were constitutively expressed in embryonic, larval and adult trout. In vivo infection with Aeromonas salmonicida did not modulate the mRNA levels of both factors. Overexpressing trout ST2 in the mammalian HEK-293 reconstitution system of TLR2 signalling quenched the Escherichia coli-induced activation of NF-κB and SAA promoters in a dose-dependent fashion. The expression of GFP-tagged trout ST2 in human HEK-293 or trout CHSE-214 cells surprisingly revealed that (i) ST2 localised abundantly at the nuclear membrane rather than at the cell membrane and (ii) the coexpression of both ST2 and MYD88 allowed the translocation of trout MYD88 from cytoplasm to nucleus, as assessed using confocal microscopy and Western blotting. Hence, we validated that trout ST2 is a dampener of TLR signalling and interacts with MYD88. The spatial distribution of these factors raises questions about how this repressive mechanism functions.

The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding irak3 from rainbow trout (Oncorhynchus mykiss), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related irak3 genes from rainbow trout and an irak3-sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking irak3 diversity. Infecting rainbow trout with a lethal dose of Aeromonas salmonicida enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1β and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms.