Distinct food-web transfers of 137Cs to fish in river and lake ecosystems: A case study focusing on masu salmon in the Fukushima evacuation zone.

This study was conducted to elucidate the spatial and size variations, and food-web transfer of 137Cs in freshwater fish in the upper reaches of the Ukedo River system, a highly contaminated river system flowing through the Fukushima evacuation zone. Fish collection and environmental surveys were conducted in the summer of 2020 at five forest rivers and at the Ogaki Dam reservoir (an artificial lake) with different air dose rates (mean 0.20-3.32 µSv/h). From the river sites, two salmonid species (masu salmon and white-spotted charr) were sampled, with masu salmon generally exhibiting higher 137Cs concentrations, ranging widely (10.6 Bq/kg-wet to 13.0 kBq/kg-wet) depending on the fish size (size effect) and site. The 137Cs concentrations in masu salmon were explained by the air dose rates, 137Cs concentrations in water, sediments (excluding the lake site), and primary producers, with site-specific variations. In the rivers, masu salmon (fluvial type with parr marks) mainly fed on terrestrial insects with higher 137Cs concentrations compared with those of aquatic insects, indicating that 137Cs was transferred mainly to fish through the allochthonous forest food-web during summer. In the lake, masu salmon (lake-run type with larger size and silvery body coloration) mainly preyed on smaller fish with lower 137Cs concentrations, demonstrating that 137Cs is transferred to fish through the autochthonous lake food-web with biomagnification. Differences in 137Cs concentrations among masu salmon (mean 441 Bq/kg-wet) and other fish species (mean 74.8 Bq/kg-wet to 2.35 kBq/kg-wet) were also found in the lake. The distinct 137Cs transfers to river and lake fish were supported by stable isotope analysis: δ15N and δ13C values enriched stepwisely through the food-webs were, respectively, higher and lower in the lake. Our results obtained using multiple approaches clearly revealed the distinct food-web transfer of 137Cs in river and lake ecosystems. These findings can contribute to prediction of radioactive contamination in freshwater fish in the Fukushima evacuation zone.

The cytokine genes of Oncorhynchus masou formosanus include a defective interleukin-4/13A gene.

Oncorhynchus masou formosanus (Formosa landlocked salmon) is a critically endangered salmonid fish endemic to Taiwan. To begin to understand how its drastic change in lifestyle from anadromous to exclusively river-dwelling is reflected in its immune genes, we characterized the genes encoding six cytokines (IL-2A, IL-2B, IL-4/13A, IL-4/13B1, IL-4/13B2, and IL-17A/F2a) important for T cell responses as no genomic data is available for this fish. Interestingly, all genes appeared homozygous indicative of a genetic bottleneck. The IL2 and IL17A/F2a genes and their products are highly similar to their characterized homologs in Oncorhynchus mykiss (rainbow trout) and other salmonid fish. Two notable differences were observed in IL4/13 family important for type 2 immune responses. First, O. m. formosanus carries not only one but two genes encoding IL-4/13B1 proteins and expansions of these genes are present in other salmonid fish. Second, the OmfoIL4/13A gene carries a 228 bp deletion that results in a premature stop codon and hence a non-functional IL-4/13A cytokine. This suggests a reduced ability for T cell responses against parasitic infections in this species.

Evolution of T cell receptor beta loci in salmonids.

T-cell mediated immunity relies on a vast array of antigen specific T cell receptors (TR). Characterizing the structure of TR loci is essential to study the diversity and composition of T cell responses in vertebrate species. The lack of good-quality genome assemblies, and the difficulty to perform a reliably mapping of multiple highly similar TR sequences, have hindered the study of these loci in non-model organisms. High-quality genome assemblies are now available for the two main genera of Salmonids, Salmo and Oncorhynchus. We present here a full description and annotation of the TRB loci located on chromosomes 19 and 25 of rainbow trout (Oncorhynchus mykiss). To get insight about variations of the structure and composition of TRB locus across salmonids, we compared rainbow trout TRB loci with other salmonid species and confirmed that the basic structure of salmonid TRB locus is a double set of two TRBV-D-J-C loci in opposite orientation on two different chromosomes. Our data shed light on the evolution of TRB loci in Salmonids after their whole genome duplication (WGD). We established a coherent nomenclature of salmonid TRB loci based on comprehensive annotation. Our work provides a fundamental basis for monitoring salmonid T cell responses by TRB repertoire sequencing.

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida.

The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly defined in fishes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control fish with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identified the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including γδ T cells, and candidate populations of monocytes/macrophages and dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell specific manner, reflecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inflammation, B cell differentiation and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver immune and non-immune cells in host defense and metabolic remodeling following infection and provides many novel cell-specific marker genes to empower future studies of this organ in fishes.

Profiling the T Cell Receptor Alpha/Delta Locus in Salmonids.

In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/ß or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family Salmonidae) include two key teleost species for aquaculture, rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in Salmo, the majority with a close counterpart in Oncorhynchus. Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.

Effect of autochthonous lactobacilli on immunologically important molecules of rainbow trout after bacterial infection studied on intestinal primoculture.

Nowadays, the aquaculture industry is one of the fastest growing industries. Intensive aquaculture has a negative impact on fish health. Probiotic bacteria are often used due to beneficial effect to health of host, e.i. decrease of diseases outbreaks, immunomodulatory effect or better utilization of feed. The aim of this work was to study the influence of probiotic bacteria on the immune response of trout intestinal cells in primoculture infected with pathogenic bacteria. In the experiment, we tested the effect of pre-treatment of intestinal cells with an autochthonous strain of Lactobacillus plantarum R2 Biocenol™ (CCM 8674) following infection with the most serious salmonid pathogens - Aeromonas salmonicida subsp. salmonicida (CCM 1307) and Yersinia ruckeri (CCM 6093). Tested probiotic strain reduced inflammation after A. salmonicida infection through decreased expression of pro-inflammatory cytokines and increased expression of anti-inflammatory cytokines. In contrast, after infection with Y. ruckeri, which causes immunosuppression, the probiotic strain stimulated immunity by up-regulation of expression of proinflammatory cytokines and suppressed the expression of anti-inflammatory cytokines. These results are a prerequisite for the immunomodulatory potential of the strain, but its action must be confirmed in subsequent in vivo experiments.

Development of a Multiplex PCR Assay for Genotyping the Fish Pathogen Piscirickettsia salmonis Through Comparative Genomics.

Piscirickettsia salmonis is a bacterial pathogen that severely impact the aquaculture in several countries as Canada, Scotland, Ireland, Norway, and Chile. It provokes Piscirickettsiosis outbreaks in the marine phase of salmonid farming, resulting in economic losses. The monophyletic genogroup LF-89 and a divergent genogroup EM-90 are responsible for the most severe Piscirickettsiosis outbreaks in Chile. Therefore, the development of methods for quick genotyping of P. salmonis genogroups in field samples is vital for veterinary diagnoses and understanding the population structure of this pathogen. The present study reports the development of a multiplex PCR for genotyping LF-89 and EM-90 genogroups based on comparative genomics of 73 fully sequenced P. salmonis genomes. The results revealed 2,322 sequences shared between 35 LF-89 genomes, 2,280 sequences in the core-genome of 38 EM-90 genomes, and 331 and 534 accessory coding sequences each genogroup, respectively. A total of 1,801 clusters of coding sequences were shared among all tested genomes of P. salmonis (LF-89 and EM-90), with 253 and 291 unique sequences for LF-89 and EM-90 genogroups, respectively. The Multiplex-1 prototype was chosen for reliable genotyping because of differences in annealing temperatures and respective reaction efficiencies. This method also identified the pathogen in field samples infected with LF-89 or EM-90 strains, which is not possible with other methods currently available. Finally, the genome-based multiplex PCR protocol presented in this study is a rapid and affordable alternative to classical sequencing of PCR products and analyzing the length of restriction fragment polymorphisms.

Evidence for Introgressive Hybridization between Native Dolly Varden (Salvelinus curilus (syn. Salvelinus malma)) and Introduced Brook Trout (Salvelinus fontinalis) in the Nishibetsu River of Hokkaido, Japan.

Hybridization is one of the negative outcomes for the introduction of non-native species, which can lead to rapid displacement and genetic extinction of native species. Salmonid fishes have been widely introduced outside of their native ranges for food supply and recreational fishing. Here, we investigate the occurrence of introgressive hybridization among native Dolly Varden (Salvelinus curilus (syn. Salvelinus malma)), white-spotted charr (Salvelinus leucomaenis), and introduced brook trout (Salvelinus fontinalis), in streams of the Nishibetsu River, Hokkaido, Japan. Microsatellite DNA analysis detected five hybrids between native Dolly Varden and introduced brook trout. This is the first evidence for hybridization between native Dolly Varden and introduced brook trout, while the latter has been known to hybridize with many other salmonids. Furthermore, incongruence between mitochondrial DNA and microsatellite DNA analyses suggested introgression among the three Salvelinus species. Further studies to estimate the hybrid fitness are necessary to understand how hybridization among the three species affects the native species.

Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

Assessing the impacts of skin mucus from Salmo salar and Oncorhynchus mykiss on the growth and in vitro infectivity of the fish pathogen Piscirickettsia salmonis.

Piscirickettsiosis is a fish disease caused by the facultative intracellular bacterium, Piscirickettsia salmonis. Even though entry routes of P. salmonis in fish are not fully clear yet, the skin seems to be the main portal in some salmonid species. Despite the importance of fish mucous skin barrier in fighting waterborne pathogens, the interaction between salmonid skin mucus and the bacterium is unknown. This study seeks to determine the in vitro changes in the growth of two Chilean P. salmonis strains (LF-89-like and EM-90-like genotypes) and the type strain LF-89T under exposures to skin mucus from Salmo salar and Oncorhynchus mykiss, as well as changes in the cytotoxic effect of P. salmonis on the SHK-1 cells following exposures. The results suggest that the growth of three P. salmonis strains was not significantly negatively affected under exposures to skin mucus (adjusted at 100 µg total protein ml-1 ) of O. mykiss (69 ± 18 U lysozyme ml-1 ) and S. salar (48 ± 33 U lysozyme ml-1 ) over time. However, the cytotoxic effect of P. salmonis, pre-exposed to salmonid skin mucus, on the SHK-1 cell line was reliably identified only towards the end of the incubation period, suggesting that the mucus had a delaying effect on the cytotoxic response of the cell line to the bacterium. These results represent a baseline knowledge to open new avenues of research intended to understand how P. salmonis faces the fish mucous skin barrier.

Genetic and Morphological Characteristics in the Local Population of the Landlocked Salmon Oncorhynchus masou Originally Distributed in Kanagawa Prefecture, Japan.

Oncorhynchus masou, including subspecies of Oncorhynchus masou masou (yamame) and Oncorhynchus masou ishikawae (amago), is one of the salmonid groups impacted by human activity such as dam construction and release of non-native salmonids. In this study, we investigated the genetic structure of O. masou populations in the Sakawa and Sagami Rivers, Japan, by sequencing the mitochondrial control region. We hoped to identify genetically the O. masou populations specific to and originally native to Kanagawa Prefecture, where the two subspecies are thought to be present. The populations found in the upstream tributaries, where there has been no human impact and no upstream migration of fishes, were assumed to be descendants of the local O. masou populations in both river systems, and the morphological features seen here were similar to amago and yamame. However, both populations were genetically related to amago. In addition, only six haplotypes were detected in 315 individuals collected from 20 localities in the two river systems. Furthermore, haplotype diversity and nucleotide diversity of these populations were low, and high FST values were observed. These results suggest that the population size is restricted and genetic diversity is decreasing in the O. masou populations of the Sakawa and Sagami Rivers.

Standardized IMGT® Nomenclature of Salmonidae IGH Genes, the Paradigm of Atlantic Salmon and Rainbow Trout: From Genomics to Repertoires.

In teleost fish as in mammals, humoral adaptive immunity is based on B lymphocytes expressing highly diverse immunoglobulins (IG). During B cell differentiation, IG loci are subjected to genomic rearrangements of V, D, and J genes, producing a unique antigen receptor expressed on the surface of each lymphocyte. During the course of an immune response to infections or immunizations, B cell clones specific of epitopes from the immunogen are expanded and activated, leading to production of specific antibodies. Among teleost fish, salmonids comprise key species for aquaculture. Rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) are especially important from a commercial point of view and have emerged as critical models for fish immunology. The growing interest to capture accurate and comprehensive antibody responses against common pathogens and vaccines has resulted in recent efforts to sequence the IG repertoire in these species. In this context, a unified and standardized nomenclature of salmonid IG heavy chain (IGH) genes is urgently required, to improve accuracy of annotation of adaptive immune receptor repertoire dataset generated by high-throughput sequencing (AIRRseq) and facilitate comparisons between studies and species. Interestingly, the assembly of salmonids IGH genomic sequences is challenging due to the presence of two large size duplicated IGH loci and high numbers of IG genes and pseudogenes. We used data available for Atlantic salmon to establish an IMGT standardized nomenclature of IGH genes in this species and then applied the IMGT rules to the rainbow trout IGH loci to set up a nomenclature, which takes into account the specificities of Salmonid loci. This unique, consistent nomenclature for Salmonid IGH genes was then used to construct IMGT sequence reference directories allowing accurate annotation of AIRRseq data. The complex issues raised by the genetic diversity of salmon and trout strains are discussed in the context of IG repertoire annotation.

Anadromous Arctic Char Microbiomes: Bioprospecting in the High Arctic.

Northern populations of Arctic char (Salvelinus alpinus) can be anadromous, migrating annually from the ocean to freshwater lakes and rivers in order to escape sub-zero temperatures. Such seasonal behavior demands that these fish and their associated microbiomes adapt to changes in salinity, temperature, and other environmental challenges. We characterized the microbial community composition of anadromous S. alpinus, netted by Inuit fishermen at freshwater and seawater fishing sites in the high Arctic, both under ice and in open water. Bacterial profiles were generated by DNA extraction and high-throughput sequencing of PCR-amplified 16S ribosomal RNA genes. Results showed that microbial communities on the skin and intestine of Arctic char were statistically different when sampled from freshwater or saline water sites. This association was tested using hierarchical Ward's linkage clustering, showing eight distinct clusters in each of the skin and intestinal microbiomes, with the clusters reflecting sampling location between fresh and saline environments, confirming a salinity-linked turnover. This analysis also provided evidence for a core composition of skin and intestinal bacteria, with the phyla Proteobacteria, Firmicutes, and Cyanobacteria presenting as major phyla within the skin-associated microbiomes. The intestine-associated microbiome was characterized by unidentified genera from families Fusobacteriaceae, Comamonadaceae, Pseudomonadaceae, and Vibrionaceae. The salinity-linked turnover was further tested through ordinations that showed samples grouping based on environment for both skin- and intestine-associated microbiomes. This finding implies that core microbiomes between fresh and saline conditions could be used to assist in regulating optimal fish health in aquaculture practices. Furthermore, identified taxa from known psychrophiles and with nitrogen cycling properties suggest that there is additional potential for biotechnological applications for fish farm and waste management practices.

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.

Transmission routes maintaining a viral pathogen of steelhead trout within a complex multi-host assemblage.

This is the first comprehensive region wide, spatially explicit epidemiologic analysis of surveillance data of the aquatic viral pathogen infectious hematopoietic necrosis virus (IHNV) infecting native salmonid fish. The pathogen has been documented in the freshwater ecosystem of the Pacific Northwest of North America since the 1950s, and the current report describes the disease ecology of IHNV during 2000-2012. Prevalence of IHNV infection in monitored salmonid host cohorts ranged from 8% to 30%, with the highest levels observed in juvenile steelhead trout. The spatial distribution of all IHNV-infected cohorts was concentrated in two sub-regions of the study area, where historic burden of the viral disease has been high. During the study period, prevalence levels fluctuated with a temporal peak in 2002. Virologic and genetic surveillance data were analyzed for evidence of three separate but not mutually exclusive transmission routes hypothesized to be maintaining IHNV in the freshwater ecosystem. Transmission between year classes of juvenile fish at individual sites (route 1) was supported at varying levels of certainty in 10%-55% of candidate cases, transmission between neighboring juvenile cohorts (route 2) was supported in 31%-78% of candidate cases, and transmission from adult fish returning to the same site as an infected juvenile cohort was supported in 26%-74% of candidate cases. The results of this study indicate that multiple specific transmission routes are acting to maintain IHNV in juvenile fish, providing concrete evidence that can be used to improve resource management. Furthermore, these results demonstrate that more sophisticated analysis of available spatio-temporal and genetic data is likely to yield greater insight in future studies.

Functional Annotation of All Salmonid Genomes (FAASG): an international initiative supporting future salmonid research, conservation and aquaculture.

We describe an emerging initiative - the 'Functional Annotation of All Salmonid Genomes' (FAASG), which will leverage the extensive trait diversity that has evolved since a whole genome duplication event in the salmonid ancestor, to develop an integrative understanding of the functional genomic basis of phenotypic variation. The outcomes of FAASG will have diverse applications, ranging from improved understanding of genome evolution, to improving the efficiency and sustainability of aquaculture production, supporting the future of fundamental and applied research in an iconic fish lineage of major societal importance.

Lineage-specific rediploidization is a mechanism to explain time-lags between genome duplication and evolutionary diversification.

BACKGROUND: The functional divergence of duplicate genes (ohnologues) retained from whole genome duplication (WGD) is thought to promote evolutionary diversification. However, species radiation and phenotypic diversification are often temporally separated from WGD. Salmonid fish, whose ancestor underwent WGD by autotetraploidization ~95 million years ago, fit such a 'time-lag' model of post-WGD radiation, which occurred alongside a major delay in the rediploidization process. Here we propose a model, 'lineage-specific ohnologue resolution' (LORe), to address the consequences of delayed rediploidization. Under LORe, speciation precedes rediploidization, allowing independent ohnologue divergence in sister lineages sharing an ancestral WGD event. RESULTS: Using cross-species sequence capture, phylogenomics and genome-wide analyses of ohnologue expression divergence, we demonstrate the major impact of LORe on salmonid evolution. One-quarter of each salmonid genome, harbouring at least 4550 ohnologues, has evolved under LORe, with rediploidization and functional divergence occurring on multiple independent occasions >50 million years post-WGD. We demonstrate the existence and regulatory divergence of many LORe ohnologues with functions in lineage-specific physiological adaptations that potentially facilitated salmonid species radiation. We show that LORe ohnologues are enriched for different functions than 'older' ohnologues that began diverging in the salmonid ancestor. CONCLUSIONS: LORe has unappreciated significance as a nested component of post-WGD divergence that impacts the functional properties of genes, whilst providing ohnologues available solely for lineage-specific adaptation. Under LORe, which is predicted following many WGD events, the functional outcomes of WGD need not appear 'explosively', but can arise gradually over tens of millions of years, promoting lineage-specific diversification regimes under prevailing ecological pressures.

Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish: Proof of concept for key components of the insulin-like growth factor axis.

High-throughput sequencing has revolutionised comparative and evolutionary genome biology. It has now become relatively commonplace to generate multiple genomes and/or transcriptomes to characterize the evolution of large taxonomic groups of interest. Nevertheless, such efforts may be unsuited to some research questions or remain beyond the scope of some research groups. Here we show that targeted high-throughput sequencing offers a viable alternative to study genome evolution across a vertebrate family of great scientific interest. Specifically, we exploited sequence capture and Illumina sequencing to characterize the evolution of key components from the insulin-like growth (IGF) signalling axis of salmonid fish at unprecedented phylogenetic resolution. The IGF axis represents a central governor of vertebrate growth and its core components were expanded by whole genome duplication in the salmonid ancestor ~95Ma. Using RNA baits synthesised to genes encoding the complete family of IGF binding proteins (IGFBP) and an IGF hormone (IGF2), we captured, sequenced and assembled orthologous and paralogous exons from species representing all ten salmonid genera. This approach generated 299 novel sequences, most as complete or near-complete protein-coding sequences. Phylogenetic analyses confirmed congruent evolutionary histories for all nineteen recognized salmonid IGFBP family members and identified novel salmonid-specific IGF2 paralogues. Moreover, we reconstructed the evolution of duplicated IGF axis paralogues across a replete salmonid phylogeny, revealing complex historic selection regimes - both ancestral to salmonids and lineage-restricted - that frequently involved asymmetric paralogue divergence under positive and/or relaxed purifying selection. Our findings add to an emerging literature highlighting diverse applications for targeted sequencing in comparative-evolutionary genomics. We also set out a viable approach to obtain large sets of nuclear genes for any member of the salmonid family, which should enable insights into the evolutionary role of whole genome duplication before additional nuclear genome sequences become available.

Energy stores, lipid mobilization and leptin endocrinology of rainbow trout.

The physiological role of leptin in fish is not fully elucidated. In the present study, the involvement of the leptin system in lipid deposition and mobilization in rainbow trout during feeding and 1, 2 and 4 weeks of fasting was investigated in two lines of rainbow trout with different muscle and visceral adiposity: a fat line (FL) with high total energy reserves, high muscle adiposity, but low visceral adiposity and a lean line (LL) with lower total energy reserves and lower muscle adiposity, but higher visceral adiposity. During 4 weeks of fasting, muscle lipids decreased by 63 % in the FL fish, while no such energy mobilization from muscle occurred in the LL fish. On the other hand, lipid stores in liver and visceral adipose tissue was utilized to a similar extent by the two fish lines during fasting. Under normal feeding conditions, plasma leptin levels were higher in the LL than the FL fish, suggesting a possible contribution of visceral adipocytes to plasma leptin levels. Plasma leptin-binding protein levels did not differ between the lines and were not affected by fasting. After 4 weeks of fasting, the long leptin receptor and the leptin-binding protein isoforms 1 and 3 muscle expression increased in the LL fish, as well as hepatic expression of leptin A1 and the two binding protein isoforms. These responses were not seen in the FL fish. The data suggest that the Lep system in rainbow trout is involved in regulation of energy stores and their mobilization.

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.