Mucosal organs exhibit distinct response signatures to hydrogen sulphide in Atlantic salmon (Salmo salar).

Hydrogen sulphide (H2S) is considered an immunotoxicant, and its presence in the water can influence the mucosal barrier functions of fish. However, there is a significant knowledge gap on how fish mucosa responds to low environmental H2S levels. The present study investigated the consequences of prolonged exposure to sub-lethal levels of H2S on the mucosal defences of Atlantic salmon (Salmo salar). Fish were continuously exposed to two levels of H2S (low: 0.05 µM; and high: 0.12 µM) for 12 days. Unexposed fish served as control. Molecular and histological profiling focused on the changes in the skin, gills and olfactory rosette. In addition, metabolomics and proteomics were performed on the skin and gill mucus. The gene expression profile indicated that the gills and olfactory rosette were more sensitive to H2S than the skin. The olfactory rosette showed a dose-dependent response, but not the gills. Genes related to stress responses were triggered at mucosal sites by H2S. Moreover, H2S elicited strong inflammatory responses, particularly in the gills. All mucosal organs demonstrated the key molecular repertoire for sulphide detoxification, but their temporal and spatial expression was not substantially affected by sub-lethal H2S levels. Mucosal barrier integrity was not considerably affected by H2S. Mucus metabolomes of the skin and gills were unaffected, but a matrix-dependent response was identified. Comparing the high-concentration group's skin and gills mucus metabolomes identified altered amino acid biosynthesis and metabolism pathways. The skin and gill mucus exhibited distinct proteomic profiles. Enrichment analysis revealed that proteins related to immunity and metabolism were affected in both mucus matrices. The present study expands our knowledge of the defence mechanisms against H2S at mucosal sites in Atlantic salmon. The findings offer insights into the health and welfare consequences of sub-lethal H2S, which can be incorporated into the risk assessment protocols in salmon land-based farms.

Mucosal and systemic physiological changes underscore the welfare risks of environmental hydrogen sulphide in post-smolt Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) might encounter toxic hydrogen sulphide (H2S) gas during aquaculture production. Exposure to this gas can be acute or chronic, with heightened levels often linked to significant mortality rates. Despite its recognised toxicity, our understanding of the physiological implications of H2S on salmon remains limited. This report details the mucosal and systemic physiological consequences in post-smolt salmon reared in brackish water at 12 ppt after prolonged exposure to elevated H2S levels over 4 weeks. The fish were subjected to two concentrations of H2S: 1 µg/L (low group) and 5 µg/L (high group). An unexposed group at 0 µg/L served as the control. Both groups exposed to H2S exhibited incremental mortality, with cumulative mortality rates of 4.7 % and 16 % for the low and high groups, respectively. Production performance, including weight and condition factors, were reduced in the H2S-exposed groups, particularly in the high group. Mucosal response of the olfactory organ revealed higher tissue damage scores in the H2S-exposed groups, albeit only at week 4. The high group displayed pronounced features such as increased mucus cell density and oedema-like vacuoles. Transcriptome analysis of the olfactory organ unveiled that the effects of H2S were more prominent at week 4, with the high group experiencing a greater magnitude of change than the low group. Genes associated with the extracellular matrix were predominantly downregulated, while the upregulated genes primarily pertained to immune response. H2S-induced alterations in the metabolome were more substantial in plasma than skin mucus. Furthermore, the number of differentially affected circulating metabolites was higher in the low group compared to the high group. Five core pathways were significantly impacted by H2S regardless of concentration, including the phenylalanine, tyrosine, and tryptophan biosynthesis. The plasma levels of phenylalanine and tyrosine were reduced following exposure to H2S. While there was a discernible distinction in the skin mucus metabolomes among the three treatment groups, only one metabolite - 4-hydroxyproline - was significantly impacted by H2S. Furthermore, this metabolite was significantly reduced in the plasma and skin mucus of H2S-exposed fish. This study underscores that prolonged exposure to H2S, even at concentrations previously deemed sub-lethal, has discernible physiological implications that manifest across various organisational levels. Given these findings, prolonged exposure to H2S poses a welfare risk, and thus, its presence must be maintained at low levels (<1 µg/L) in salmon land-based rearing systems.

Intergenerational effects of early life-stage temperature modulation on gene expression and DNA methylation in Atlantic cod (Gadus morhua).

After suffering several collapses, the cod farming industry is now in the process of trying to re-establish itself. We have used material from Norway's National Cod Breeding Program to study how different early life-stage temperature regimes affect DNA methylation and gene expression. Long-term effects were detected by sampling fish several weeks after the end of differential treatments, and offspring from the different exposure groups was also sampled. Many overlapping genes were found between the different exposure groups and generations, coupled with genes associated with differential CpG methylation levels. Genes involved in muscle fibre development, general metabolic processes and formation of deformities were significantly affected, and genes relevant for intergenerational transfer of epigenetic marks were also detected. We believe the use of environmental cues can be a useful strategy for improving the production of Atlantic cod.

Fast and slow releasing sulphide donors engender distinct transcriptomic alterations in Atlantic salmon hepatocytes.

Hydrogen sulphide (H2S) is a naturally occurring compound generated either endogenously or exogenously and serves both as a gaseous signalling molecule and an environmental toxicant. Though it has been extensively investigated in mammalian systems, the biological function of H2S in teleost fish is poorly identified. Here we demonstrate how exogenous H2S regulates cellular and molecular processes in Atlantic salmon (Salmo salar) using a primary hepatocyte culture as a model. We employed two forms of sulphide donors: the fast-releasing salt form, sodium hydrosulphide (NaHS) and the slow-releasing organic analogue, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Hepatocytes were exposed to either a low (LD, 20 µg/L) or high (HD, 100 µg/L) dose of the sulphide donors for 24 hrs, and the expression of key sulphide detoxification and antioxidant defence genes were quantified by qPCR. The key sulphide detoxification genes sulfite oxidase 1 (soux) and the sulfide: quinone oxidoreductase 1 and 2 (sqor) paralogs in salmon showed pronounced expression in the liver and likewise responsive to the sulphide donors in the hepatocyte culture. These genes were ubiquitously expressed in different organs of salmon as well. HD-GYY4137 upregulated the expression of antioxidant defence genes, particularly glutathione peroxidase, glutathione reductase and catalase, in the hepatocyte culture. To explore the influence of exposure duration, hepatocytes were exposed to the sulphide donors (i.e., LD versus HD) either transient (1h) or prolonged (24h). Prolonged but not transient exposure significantly reduced hepatocyte viability, and the effects were not dependent on concentration or form. The proliferative potential of the hepatocytes was only affected by prolonged NaHS exposure, and the impact was not concentration dependent. Microarray analysis revealed that GYY4137 caused more substantial transcriptomic changes than NaHS. Moreover, transcriptomic alterations were more marked following prolonged exposure. Genes involved in mitochondrial metabolism were downregulated by the sulphide donors, primarily in NaHS-exposed cells. Both sulphide donors influenced the immune functions of hepatocytes: genes involved in lymphocyte-mediated response were affected by NaHS, whereas inflammatory response was targeted by GYY4137. In summary, the two sulphide donors impacted the cellular and molecular processes of teleost hepatocytes, offering new insights into the mechanisms underlying H2S interactions in fish.

Non-synonymous variation and protein structure of candidate genes associated with selection in farm and wild populations of turbot (Scophthalmus maximus).

Non-synonymous variation (NSV) of protein coding genes represents raw material for selection to improve adaptation to the diverse environmental scenarios in wild and livestock populations. Many aquatic species face variations in temperature, salinity and biological factors throughout their distribution range that is reflected by the presence of allelic clines or local adaptation. The turbot (Scophthalmus maximus) is a flatfish of great commercial value with a flourishing aquaculture which has promoted the development of genomic resources. In this study, we developed the first atlas of NSVs in the turbot genome by resequencing 10 individuals from Northeast Atlantic Ocean. More than 50,000 NSVs where detected in the ~ 21,500 coding genes of the turbot genome, and we selected 18 NSVs to be genotyped using a single Mass ARRAY multiplex on 13 wild populations and three turbot farms. We detected signals of divergent selection on several genes related to growth, circadian rhythms, osmoregulation and oxygen binding in the different scenarios evaluated. Furthermore, we explored the impact of NSVs identified on the 3D structure and functional relationship of the correspondent proteins. In summary, our study provides a strategy to identify NSVs in species with consistently annotated and assembled genomes to ascertain their role in adaptation.

Impact of germ cell ablation on the activation of the brain-pituitary-gonadal axis in precocious Atlantic salmon (Salmo salar L.) males.

The germ cells are essential for sexual reproduction by giving rise to the gametes, but the importance of germ cells for gonadal somatic functions varies among vertebrates. The RNA-binding dead end (Dnd) protein is necessary for the specification and migration of primordial germ cells to the future reproductive organs. Here, we ablated the gametes in Atlantic salmon males and females by microinjecting dnd antisense gapmer oligonucleotides at the zygotic stage. Precocious maturation was induced in above 50% of both germ cell-depleted and intact fertile males, but not in females, by exposure to an off-season photoperiod regime. Sterile and fertile males showed similar body growth, but maturing fish tended to be heavier than their immature counterparts. Pituitary fshß messenger RNA levels strongly increased in maturing sterile and fertile males concomitant with the upregulated expression of Sertoli and Leydig cell markers. Plasma concentrations of 11-ketotestosterone and testosterone in maturing sterile males were significantly higher than the basal levels in immature fish, but lower than those in maturing fertile males. The study demonstrates that germ cells are not a prerequisite for the activation of the brain-pituitary-gonad axis and sex steroidogenesis in Atlantic salmon males, but may be important for the maintenance of gonadal somatic functions.

De novo transcriptome assemblies of red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) molting gland and eyestalk ganglia - Temperature effects on expression of molting and growth regulatory genes in adult red king crab.

Red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) are deep-sea crustaceans widely distributed in the North Pacific and Northwest Atlantic Oceans. These giant predators have invaded the Barents Sea over the past decades, and climate-driven temperature changes may influence their distribution and abundance in the sub-Arctic region. Molting and growth in crustaceans are strongly affected by temperature, but the underlying molecular mechanisms are little known, particularly in cold-water species. Here, we describe multiple regulatory factors in the two high-latitude crabs by developing de novo transcriptomes from the molting gland (Y-organ or YO) and eye stalk ganglia (ESG), in addition to the hepatopancreas and claw muscle of red king crab. The Halloween genes encoding the ecdysteroidogenic enzymes were expressed in YO, and the ESG contained multiple neuropeptides, including molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), and ion-transport peptide (ITP). Both crabs expressed a diversity of growth-related factors, such as mTOR, AKT, Rheb and AMPKα, and stress-responsive factors, including multiple heat shock proteins (HSPs). Temperature effects on the expression of key regulatory genes were quantified by qPCR in adult red king crab males kept at 4 °C or 10 °C for two weeks during intermolt. The Halloween genes tended to be upregulated in YO at high temperature, while the ecdysteroid receptor and several growth regulators showed tissue-specific responses to elevated temperature. Constitutive and heat-inducible HSPs were expressed in an inverse temperature-dependent manner, suggesting that adult red king crabs can acclimate to increased water temperatures.

The hemoglobin Gly16ß1Asp polymorphism in turbot (Scophthalmus maximus) is differentially distributed across European populations.

Turbot is an important flatfish widely distributed along the European coasts, whose fishery is centered in the North Sea. The commercial value of the species has boosted a successful aquaculture sector in Europe and China. Body growth is the main target of turbot breeding programs and is also a key trait related to local adaptation to temperature and salinity. Differences in growth rate and optimal growth temperature in turbot have been shown to be associated with a hemoglobin polymorphism reported more than 50 years ago. Here, we identified a Gly16Asp amino acid substitution in the ß1 globin subunit by searching for genetic variation in the five functional globin genes within the whole annotated turbot genome. We predicted increased stability of the turbot hemoglobin by the replacement of the conserved Gly with the negative charged Asp residue that is consistent with the higher rate of αß dimer assembly in the human J-Baltimore Gly16ß->Asp mutant than in normal HbA. The turbot Hbß1-Gly16 variant dominated in the northern populations examined, particularly in the Baltic Sea, while the Asp allele showed elevated frequencies in southern populations and was the prevalent variant in the Adriatic Sea. Body weight did not associate with the Hbß1 genotypes at farming conditions (i.e., high oxygen levels, feeding ad libitum) after analyzing 90 fish with high growth dispersal from nine turbot families. Nevertheless, all data at hand suggest that the turbot hemoglobin polymorphism has an adaptive significance in the variable wild conditions regarding temperature and oxygen availability.

Low Oxygen Stress During Early Development Influences Regulation of Hypoxia-Response Genes in Farmed Atlantic Salmon (Salmo salar).

Survival and growth of developing salmonids are negatively affected by low oxygen levels within gravel nests in natural streams, and hypoxic stress is often experienced by farmed Atlantic salmon (Salmo salar) within hatcheries. Exposure to hypoxia during early development may have long-lasting effects by altering epigenetic marks and gene expression in oxygen regulatory pathways. Here, we examine the transcriptomic response to low dissolved oxygen (DO) in post-hatch salmon reared continuously in 30%, 60% or 100% DO from fertilization until start of feeding. RNA sequencing revealed multiple differentially expressed genes, including oxygen transporting hemoglobin embryonic α subunit (hbae) and EGLN3 family hypoxia-inducible factor 3 (egln3) which regulates the stability of hypoxia inducible factor 1α (HIF-1α). Both hbae and egln3 displayed expression levels inversely correlated to oxygen concentration, and DNA methylation patterns within the egln3 promoter were negatively associated with the transcript levels. These results suggest that epigenetic processes are influenced by low oxygen levels during early development in Atlantic salmon to upregulate hypoxia-response genes.

A Nanopore Based Chromosome-Level Assembly Representing Atlantic Cod from the Celtic Sea.

Currently available genome assemblies for Atlantic cod (Gadus morhua) have been constructed from fish belonging to the Northeast Arctic Cod (NEAC) population; a migratory population feeding in the Barents Sea. These assemblies have been crucial for the development of genetic markers which have been used to study population differentiation and adaptive evolution in Atlantic cod, pinpointing four discrete islands of genomic divergence located on linkage groups 1, 2, 7 and 12. In this paper, we present a high-quality reference genome from a male Atlantic cod representing a southern population inhabiting the Celtic sea. The genome assembly (gadMor_Celtic) was produced from long-read nanopore data and has a combined contig length of 686 Mb with an N50 of 10 Mb. Integrating contigs with genetic linkage mapping information enabled us to construct 23 chromosome sequences which mapped with high confidence to the latest NEAC population assembly (gadMor3) and allowed us to characterize, to an extent not previously reported large chromosomal inversions on linkage groups 1, 2, 7 and 12. In most cases, inversion breakpoints could be located within single nanopore contigs. Our results suggest the presence of inversions in Celtic cod on linkage groups 6, 11 and 21, although these remain to be confirmed. Further, we identified a specific repetitive element that is relatively enriched at predicted centromeric regions. Our gadMor_Celtic assembly provides a resource representing a 'southern' cod population which is complementary to the existing 'northern' population based genome assemblies and represents the first step toward developing pan-genomic resources for Atlantic cod.

Multiplicity and Polymorphism of Fish Hemoglobins.

The diversity of fish hemoglobins and the association with oxygen availability and physiological requirements during the life cycle has attracted scientists since the first report on multiple hemoglobin in fishes (Buhler and Shanks 1959). The functional heterogeneity of the fish hemoglobins enables many species to tolerate hypoxic conditions and exhausting swimming, but also to maintain the gas pressure in the swim bladder at large depths. The hemoglobin repertoire has further increased in various species displaying polymorphic hemoglobin variants differing in oxygen binding properties. The multiplicity of fish hemoglobins as particularly found in the tetraploid salmonids strongly contrasts with the complete loss of hemoglobins in Antarctic icefishes and illustrates the adaptive radiation in the oxygen transport of this successful vertebrate group.

Early Developmental Stress Affects Subsequent Gene Expression Response to an Acute Stress in Atlantic Salmon: An Approach for Creating Robust Fish for Aquaculture?

Stress during early life has potential to program and alter the response to stressful events and metabolism in later life. Repeated short exposure of Atlantic salmon to cold water and air during embryonic (E), post-hatch (PH) or both phases of development (EPH) has been shown to alter the methylome and transcriptome and to affect growth performance during later life compared to untreated controls (CO). The aim of this study was to investigate how the transcriptome of these fish responds to subsequent acute stress at the start feeding stage, and to describe methylation differences that might steer these changes. EPH treated fish showed the strongest down-regulation of corticotropin releasing factor 1, up-regulation of glucocorticoid receptor and 3-oxo-5-alpha-steroid 4-dehydrogenase 2 gene expression and a suppressed cortisol response 3 hr after the acute stress, differences that could influence hormesis and be affecting how EPH fish cope and recover from the stress event. Growth hormone 2 and insulin-like growth factor 1 were more strongly down-regulated following acute stress in EPH treated fish relative to E, PH and CO fish. This indicates switching away from growth toward coping with stress following stressful events in EPH fish. Genes implicated in immune function such as major histocompatibility class 1A, T-cell receptor and toll-like receptor also responded to acute stress differently in EPH treated fish, indicating that repeated stresses during early life may affect robustness. Differential DNA methylation was detected in regions mapping <500 bases from genes differentially responding to acute stress suggesting the involvement of epigenetic mechanisms. Stress treatments applied during early development therefore have potential as a husbandry tool for boosting the productivity of aquaculture by affecting how fish respond to stresses at critical stages of production.

Influence of feed ration size on somatic and muscle growth in landlocked dwarf and farmed Atlantic salmon Salmo salar.

We examined the possible adaptation of the dwarf Bleke population of Atlantic salmon Salmo salar from Lake Byglandsfjord in southern Norway to limited food resources. The growth performance and muscle development in juvenile Bleke and farmed S. salar under satiated or restricted (50%) feeding were examined for 10 months, starting 3 weeks after first-feeding stage. Four-thousand fish were divided into four replicated groups and random samples of 16-40 fish per group were measured six times during the experiment. The two strains showed no significant difference in mean body mass when fed restricted ration, but the individual variation was considerably higher in the farmed fish. Both Bleke and farmed S. salar grew significantly faster when fed to satiation, but the farmed S. salar showed much higher gain in mass and were three times heavier (201.5 g vs 66.7 g) and possessed twice as many fast muscle fibres (179,682 vs 84,779) compared with landlocked S. salar after 10 months. Farmed fish fed full ration displayed both hypertrophic and hyperplasic muscle growth, while the increased growth in Bleke S. salar was entirely associated with a larger fibre diameter. The landlocked Bleke strain has apparently adapted to low food availability by minimising the metabolic costs of maintenance and growth through reduced dominance hierarchies and by an increase in average muscle fibre diameter relative to the ancestral condition.

Characterization of a male specific region containing a candidate sex determining gene in Atlantic cod.

The genetic mechanisms determining sex in teleost fishes are highly variable and the master sex determining gene has only been identified in few species. Here we characterize a male-specific region of 9 kb on linkage group 11 in Atlantic cod (Gadus morhua) harboring a single gene named zkY for zinc knuckle on the Y chromosome. Diagnostic PCR test of phenotypically sexed males and females confirm the sex-specific nature of the Y-sequence. We identified twelve highly similar autosomal gene copies of zkY, of which eight code for proteins containing the zinc knuckle motif. 3D modeling suggests that the amino acid changes observed in six copies might influence the putative RNA-binding specificity. Cod zkY and the autosomal proteins zk1 and zk2 possess an identical zinc knuckle structure, but only the Y-specific gene zkY was expressed at high levels in the developing larvae before the onset of sex differentiation. Collectively these data suggest zkY as a candidate master masculinization gene in Atlantic cod. PCR amplification of Y-sequences in Arctic cod (Arctogadus glacialis) and Greenland cod (Gadus macrocephalus ogac) suggests that the male-specific region emerged in codfishes more than 7.5 million years ago.

Training the salmon's genes: influence of aerobic exercise, swimming performance and selection on gene expression in Atlantic salmon.

BACKGROUND: Farmed and wild Atlantic salmon are exposed to many infectious and non-infectious challenges that can cause mortality when they enter the sea. Exercise before transfer promotes growth, health and survival in the sea. Swimming performance in juveniles at the freshwater parr stage is positively associated with resistance to some diseases. Genetic variation is likely to affect response to exercise. In this study we map genetic differences associated with aerobic exercise, swimming performance and genetic origin. Eggs from the selectively bred Bolaks salmon and wild Lærdal River salmon strains were reared until parr in a common environment. Swimming performance was assessed by subjecting the fish to either continuous hard exercise or control conditions for 18 days. Heart was sampled for examination of gene expression using RNA-seq (~60 fish/treatment). RESULTS: Lower expression of genes affecting immune function was found in domesticated than wild parr. Among wild parr under control exercise the expression of a large number of genes involved in general metabolism, stress and immune response was lower in superior swimmers suggesting that minimisation of energy expenditure during periods of low activity makes parr better able to sustain bursts of swimming for predator avoidance. A similar set of genes were down-regulated with training among wild parr with inferior swimming performance. These parr react to training in a way that their cardiac expression patterns become like the superior performing wild parr under control exercise conditions. Diversifying selection caused by breeding of domesticated stock, and adaptive pressures in wild stock, has affected the expression and frequency of single nucleotide polymorphisms (SNPs) for multiple functional groups of genes affecting diverse processes. SNPs associated with swimming performance in wild parr map to genes involved in energetic processes, coding for contractile filaments in the muscle and controlling cell proliferation. CONCLUSIONS: Domesticated parr have less phenotypic plasticity in response to training and lower expression of genes with functions affecting immune response. The genetic response to training is complex and depends on the background of parr and their swimming ability. Exercise should be tailored to the genetics and swimming performance of fish.

Resolving the complexity of vitellogenins and their receptors in the tetraploid Atlantic salmon (Salmo salar): Ancient origin of the phosvitin-less VtgC in chondrichthyean fishes.

Egg yolk proteins are mainly derived from vitellogenin (Vtg), and serve as essential nutrients during early development in oviparous organisms. Vertebrate Vtgs are predominantly synthesized in the liver of maturing females, and are internalized by the oocyte after binding to specific surface receptors (VtgR). Here, we clarify the evolutionary history of vertebrate Vtgs, including the teleost VtgC, which lacks phosvitin, and investigate the repertoire of Vtgs and VtgRs in the tetraploid Atlantic salmon (Salmo salar). Conserved synteny of the vtg genes in elephant fish (Callorhinchus milii) strongly indicates that the vtg gene cluster was present in the ancestor of tetrapods and ray-finned fish. The shortened phosvitin in the VtgC ortholog of this chondrichthyean fish may have resulted from early truncation events that eventually allowed the total disappearance of phosvitin in teleost VtgC. In contrast, the tandem-duplicated VtgCs identified in the spotted gar (Lepisosteus oculatus) both contain the phosvitin domain. The Atlantic salmon genome harbors four vtg genes encoding the complete VtgAsa1, phosvitin-less VtgC, and truncated VtgAsb proteins; vtgAsa2 is a pseudogene. The three vtg genes were mainly expressed in the liver of maturing females, and the vtgAsa1 transcript predominated prior to spawning. The splice variant lacking the O-linked sugar domain dominated ovarian expression of vtgr1 and vtgr2. Strongly increased vtgAsa1 expression during vitellogenesis contrasted with the peaks of vtgr1 and vtgr2 in the previtellogenic oocytes, which gradually decreased over the same period. Recycling of the oocyte VtgRs is probably not sufficient to maintain receptor number during vitellogenesis.

Impacts of Early Life Stress on the Methylome and Transcriptome of Atlantic Salmon.

Exposure to environmental stressors during early-life stages can change the rate and timing of various developmental processes. Epigenetic marks affecting transcriptional regulation can be altered by such environmental stimuli. To assess how stress might affect the methylome and transcriptome in salmon, fish were treated using cold-shock and air-exposure from the eye-stage until start-feeding. The fish were either stressed prior to hatching (E), post-hatching (PH), pre- and post-hatching (EPH) or not stressed (CO). Assessing transcriptional abundances just prior to start feeding, E and PH individuals were found to have modified the expression of thousands of genes, many with important functions in developmental processes. The EPH individuals however, showed expression similar to those of CO, suggesting an adaptive response to extended periods of stress. The methylome of stressed individuals differed from that of the CO, suggesting the importance of environment in shaping methylation signatures. Through integration of methylation with transcription, we identified bases with potential regulatory functions, some 10s of kb away from the targeted genes. We then followed fish growth for an additional year. Individuals in EPH showed superior growth compared to other treatment groups, highlighting how stress can potentially have long-lasting effects on an organism's ability to adapt to environmental perturbations.

Genetic background and embryonic temperature affect DNA methylation and expression of myogenin and muscle development in Atlantic salmon (Salmo salar).

The development of ectothermic embryos is strongly affected by incubation temperature, and thermal imprinting of body growth and muscle phenotype has been reported in various teleost fishes. The complex epigenetic regulation of muscle development in vertebrates involves DNA methylation of the myogenin promoter. Body growth is a heritable and highly variable trait among fish populations that allows for local adaptations, but also for selective breeding. Here we studied the epigenetic effects of embryonic temperature and genetic background on body growth, muscle cellularity and myogenin expression in farmed Atlantic salmon (Salmo salar). Eggs from salmon families with either high or low estimated breeding values for body growth, referred to as Fast and Slow genotypes, were incubated at 8°C or 4°C until the embryonic 'eyed-stage' followed by rearing at the production temperature of 8°C. Rearing temperature strongly affected the growth rates, and the 8°C fish were about twice as heavy as the 4°C fish in the order Fast8>Slow8>Fast4>Slow4 prior to seawater transfer. Fast8 was the largest fish also at harvest despite strong growth compensation in the low temperature groups. Larval myogenin expression was approximately 4-6 fold higher in the Fast8 group than in the other groups and was associated with relative low DNA methylation levels, but was positively correlated with the expression levels of the DNA methyltransferase genes dnmt1, dnmt3a and dnmt3b. Juvenile Fast8 fish displayed thicker white muscle fibres than Fast4 fish, while Slow 8 and Slow 4 showed no difference in muscle cellularity. The impact of genetic background on the thermal imprinting of body growth and muscle development in Atlantic salmon suggests that epigenetic variation might play a significant role in the local adaptation to fluctuating temperatures over short evolutionary time.

Multiplicity of Buc copies in Atlantic salmon contrasts with loss of the germ cell determinant in primates, rodents and axolotl.

BACKGROUND: The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is induced by zygotic signals, maternally delivered germ cell determinants specify the PGCs in birds, frogs and teleost fish. Assembly of the germ plasm in the oocyte is organized by the single Buc in zebrafish, named Velo1 in Xenopus, and by Oskar in Drosophila. Secondary loss of oskar in several insect lineages coincides with changes in germline determination strategies, while the presence of buc in mammals suggests functions not associated with germline formation. RESULTS: To clarify the evolutionary history of buc we searched for the gene in genomes available from various chordates. No buc sequence was found in lamprey and chordate invertebrates, while the gene was identified in a conserved syntenic region in elephant shark, spotted gar, teleosts, Comoran coelacanth and most tetrapods. Rodents have probably lost the buc gene, while a premature translation stop was found in primates and in Mexican axolotl lacking germ plasm. In contrast, several buc and buc-like (bucL) paralogs were identified in the teleosts examined, including zebrafish, and the tetraploid genome of Atlantic salmon harbors seven buc and bucL genes. Maternal salmon buc1a, buc2a and buc2b mRNAs were abundant in unfertilized eggs together with dnd and vasa mRNAs. Immunostained salmon Buc1a was restricted to cleavage furrows in 4-cell stage embryos similar to a fluorescent zebrafish Buc construct injected in salmon embryos. Salmon Buc1a and Buc2a localized together with DnD, Vasa and Dazl within the Balbiani body of early oocytes. CONCLUSIONS: Buc probably originated more than 400 million years ago and might have played an ancestral role in assembling germ plasm. Functional redundancy or subfunctionalization of salmon Buc paralogs in germline formation is suggested by the maternally inherited mRNAs of three salmon buc genes, the localized Buc1a in the cleavage furrows and the distribution of Buc1a and Buc2a in the Balbiani body during oogenesis. The extra-ovarian expression of salmon buc genes and the presence of a second zebrafish bucL gene suggest additional functions not related to germ cell specification.