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.

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.

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.

Characterization of igf1 and igf2 genes during maraena whitefish (Coregonus maraena) ontogeny and the effect of temperature on embryogenesis and igf expression.

The insulin-like growth factors IGF-1 and IGF-2 play important roles in the growth, development, and metabolism of teleost fish. We isolated cDNA sequences of igf1, and igf2 genes from maraena whitefish. We quantified the mRNA and protein expressions of IGFs in different tissues of marketable juvenile maraena whitefish. Moreover, we analyzed the gene expression profiles during maraena whitefish development from unfertilized egg to fingerling and examined the effect of incubation temperature on igf1, and igf2 gene expression during embryonic and early larval development. Transcripts encoding IGF-1 or IGF-2 were detected in all tested tissues, with the greatest abundance in the liver. We measured higher igf2 than igf1 copy numbers in all tissues and at all developmental stages examined, even at advanced juvenile stages. Using the Western blot technique, we demonstrated that several isoforms of IGF-1 are expressed in the liver and gills but not in muscle tissue, indicating tissue-specific protein expression of IGF-1. We observed an accelerated embryonic development with increasing temperature, resulting in shortened hatching periods. Out of the three tested temperatures, we observed the highest hatching rate, larval hatching size, and larval growth at 6 °C. At 9 °C, hatching rate, larval hatching size and larval growth were reduced compared to the values we observed at 4 °C and 6 °C, since incubation temperature might have exceeded the optimum. To our knowledge, our data show for the first time that both igf1 and igf2 expression were upregulated due to elevated incubation temperature within embryonic development of fish. Further, we found significantly higher igf expression for the best-developing larvae (6 °C group) at specific life stages of maraena whitefish.

Adverse Husbandry of Maraena Whitefish Directs the Immune System to Increase Mobilization of Myeloid Cells and Proinflammatory Responses.

Adverse life circumstances evoke a common "conserved transcriptional response to adversity" (CTRA) in mammalian leukocytes. To investigate whether this pattern is preserved in lower vertebrates, maraena whitefish (Coregonus maraena) were exposed for 9 days to different stocking densities: ~10 kg/m3 (low density), ~33 kg/m3 (moderate), ~60 kg/m3 (elevated), and ~100 kg/m3 (high). Transcriptome profiling in the liver and kidney of individuals from each group suggested that crowding conditions activate stress-related signaling and effector pathways. Remarkably, about one-quarter of the genes differentially expressed under crowding conditions were involved in the activation of immune pathways such as acute-phase response and interleukin/TNF signaling attended by the simultaneous reduction of antiviral potency. Network analysis confirmed the complex interdigitation of immune- and stress-relevant pathways with interleukin-1 playing a central role. Antibody-based techniques revealed remarkable changes in the blood composition of whitefish and demonstrated the correlation between increasing stocking densities and elevated number of myeloid cells together with the increased phagocytic activity of peripheral blood leukocytes. In line with current studies in mammals, we conclude that crowding stress triggers in whitefish hallmarks of a CTRA, indicating that the stress-induced molecular mechanisms regulating the immune responses not only are conserved within mammals but were established earlier in evolution.

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.