Insights from a chum salmon (Oncorhynchus keta) genome assembly regarding whole-genome duplication and nucleotide variation influencing gene function.

Chum salmon are ecologically important to Pacific Ocean ecosystems and commercially important to fisheries. To improve the genetic resources available for this species, we sequenced and assembled the genome of a male chum salmon using Oxford Nanopore read technology and the Flye genome assembly software (contig N50: ∼2 Mbp, complete BUSCOs: ∼98.1%). We also resequenced the genomes of 59 chum salmon from hatchery sources to better characterize the genome assembly and the diversity of nucleotide variants impacting phenotype variation. With genomic sequences from a doubled haploid individual, we were able to identify regions of the genome assembly that have been collapsed due to high sequence similarity between homeologous (duplicated) chromosomes. The homeologous chromosomes are relics of an ancient salmonid-specific genome duplication. These regions were enriched with genes whose functions are related to the immune system and responses to toxins. From analyzing nucleotide variant annotations of the resequenced genomes, we were also able to identify genes that have increased levels of variants thought to moderately impact gene function. Genes related to the immune system and the detection of chemical stimuli (olfaction) had increased levels of these variants based on a gene ontology enrichment analysis. The tandem organization of many of the enriched genes raises the question of why they have this organization.

Population-size history inferences from the coho salmon (Oncorhynchus kisutch) genome.

Coho salmon (Oncorhynchus kisutch) are a culturally and economically important species that return from multiyear ocean migrations to spawn in rivers that flow to the Northern Pacific Ocean. Southern stocks of coho salmon in Canada and the United States have significantly declined over the past quarter century, and unfortunately, conservation efforts have not reversed this trend. To assist in stock management and conservation efforts, we generated a chromosome-level genome assembly. We also resequenced the genomes of 83 coho salmon across the North American range to identify nucleotide variants and understand the demographic histories of these salmon by modeling effective population size from genome-wide data. From demographic history modeling, we observed reductions in effective population sizes between 3,750 and 8,000 years ago for several northern sampling sites, which may correspond to bottleneck events during recolonization after glacial retreat.

Fast-growing growth hormone transgenic coho salmon (Oncorhynchus kisutch) show a lower incidence of vaterite deposition and malformations in sagittal otoliths.

In fish otoliths, CaCO3 normally precipitates as aragonite, and more rarely as vaterite or calcite. A higher incidence of vaterite deposition in otoliths from aquaculture-reared fish has been reported and it is thought that high growth rates under farming conditions might promote its deposition. To test this hypothesis, otoliths from growth hormone (GH) transgenic coho salmon and non-transgenic fish of matching size were compared. Once morphometric parameters were normalized by animal length, we found that transgenic fish otoliths were smaller (-24%, -19%, -20% and -30% for length, width, perimeter and area, respectively; P<0.001) and rounder (-12%, +13.5%, +15% and -15.5% in circularity, form factor, roundness and ellipticity; P<0.001) than otoliths from non-transgenic fish of matching size. Interestingly, transgenic fish had smaller eyes (-30% eye diameter) and showed a strong correlation between eye and otolith size. We also found that the percentage of otoliths showing vaterite deposition was significantly smaller in transgenic fish (21-28%) than in non-transgenic fish (69%; P<0.001). Likewise, the area affected by vaterite deposition within individual otoliths was reduced in transgenic fish (21-26%) compared with non-transgenic fish (42.5%; P<0.001). Our results suggest that high growth rates per se are not sufficient to cause vaterite deposition in all cases, and that GH overexpression might have a protective role against vaterite deposition, a hypothesis that needs further investigation.

Correction: The sockeye salmon genome, transcriptome, and analyses identifying population defining regions of the genome.

[This corrects the article DOI: 10.1371/journal.pone.0240935.].

The pink salmon genome: Uncovering the genomic consequences of a two-year life cycle.

Pink salmon (Oncorhynchus gorbuscha) adults are the smallest of the five Pacific salmon native to the western Pacific Ocean. Pink salmon are also the most abundant of these species and account for a large proportion of the commercial value of the salmon fishery worldwide. A two-year life history of pink salmon generates temporally isolated populations that spawn either in even-years or odd-years. To uncover the influence of this genetic isolation, reference genome assemblies were generated for each year-class and whole genome re-sequencing data was collected from salmon of both year-classes. The salmon were sampled from six Canadian rivers and one Japanese river. At multiple centromeres we identified peaks of Fst between year-classes that were millions of base-pairs long. The largest Fst peak was also associated with a million base-pair chromosomal polymorphism found in the odd-year genome near a centromere. These Fst peaks may be the result of a centromere drive or a combination of reduced recombination and genetic drift, and they could influence speciation. Other regions of the genome influenced by odd-year and even-year temporal isolation and tentatively under selection were mostly associated with genes related to immune function, organ development/maintenance, and behaviour.

Incomplete reproductive isolation and strong transcriptomic response to hybridization between sympatric sister species of salmon.

Global change is altering ecosystems at an unprecedented rate. The resulting shifts in species ranges and reproductive timing are opening the potential for hybridization between closely related species which could dramatically alter the genetic diversity, adaptive capacity and evolutionary trajectory of interbreeding taxa. Here, we used behavioural breeding experiments, in vitro fertilization experiments, and whole-transcriptome gene expression data to assess the potential for and consequences of hybridization between Chinook and Coho salmon. We show that behavioural and gametic prezygotic barriers between socio-economically valuable Chinook and Coho salmon are incomplete. Postzygotically, we demonstrate a clear transcriptomic response to hybridization among F1 Chinook-Coho offspring. Genes transgressively expressed within hybrids were significantly enriched with genes encoded in the nucleus but localized to the mitochondrion, suggesting a potential role for mito-nuclear incompatibilities as a postzygotic mechanism of hybrid breakdown. Chinook and Coho salmon are expected to continue to respond to climate change with shifts in migration timing and habitat use, potentiating hybridization between these species. The downstream consequences of hybridization on the future of these threatened salmon, and the ecosystems they inhabit, is unknown.

Assessing the effects of genotype-by-environment interaction on epigenetic, transcriptomic, and phenotypic response in a Pacific salmon.

Genotype-by-environment (GxE) interactions are non-parallel reaction norms among individuals with different genotypes in response to different environmental conditions. GxE interactions are an extension of phenotypic plasticity and consequently studying such interactions improves our ability to predict effects of different environments on phenotype as well as the fitness of genetically distinct organisms and their capacity to interact with ecosystems. Growth hormone transgenic coho salmon grow much faster than non-transgenics when raised in tank environments, but show little difference in growth when reared in nature-like streams. We used this model system to evaluate potential mechanisms underlying this growth rate GxE interaction, performing RNA-seq to measure gene transcription and whole-genome bisulfite sequencing to measure gene methylation in liver tissue. Gene ontology (GO) term analysis revealed stress as an important biological process potentially influencing growth rate GxE interactions. While few genes with transcription differences also had methylation differences, in promoter or gene regions, many genes were differentially methylated between tank and stream environments. A GO term analysis of differentially methylated genes between tank and stream environments revealed increased methylation in the stream environment of more than 95% of the differentially methylated genes, many with biological processes unrelated to liver function. The lower nutritional condition of the stream environment may cause increased negative regulation of genes less vital for liver tissue function than when fish are reared in tanks with unlimited food availability. These data show a large effect of rearing environment both on gene expression and methylation, but it is less clear that the detected epigenetic marks are responsible for the observed altered growth and physiological responses.

Sexually Dimorphic Growth Stimulation in a Strain of Growth Hormone Transgenic Coho Salmon (Oncorhynchus kisutch).

Growth hormone (GH) transgenic fish often exhibit remarkable transformations in growth rate and other phenotypes relative to wild-type. The 5750A transgenic coho salmon strain exhibits strong sexually dimorphic growth, with females possessing growth stimulation at a level typical of that seen for both sexes in other strains harbouring the same gene construct (e.g. M77), while males display a modest level of growth stimulation. GH mRNA levels were significantly higher in females than in males of the 5750A strain but equivalent in the M77 strain, indicating sex and transgene insertion locus altered transgene expression. We found that acute estradiol treatments did not influence GH expression in either strain (5750A and M77) or the transgene promoter (metallothionein-B), suggesting that estradiol level was not a significant factor influencing transgene activity. The feminization of XX and XY fish of the 5750A and M77 strains generated all-female groups and resulted in equalized growth of the two genetic sexes, suggesting that the presence of the Y chromosome was not directly capable of influencing the GH transgene-mediated growth in a physiological female conditions. These data suggest that the difference in growth rate seen between the sexes in the 5750A strain arises from non-estradiol-mediated sex influences on gene regulation at the transgene locus. This study shows how genetic factors and transgene insertion sites can influence transgene expression with significant consequent effects on phenotype.

The sockeye salmon genome, transcriptome, and analyses identifying population defining regions of the genome.

Sockeye salmon (Oncorhynchus nerka) is a commercially and culturally important species to the people that live along the northern Pacific Ocean coast. There are two main sockeye salmon ecotypes-the ocean-going (anadromous) ecotype and the fresh-water ecotype known as kokanee. The goal of this study was to better understand the population structure of sockeye salmon and identify possible genomic differences among populations and between the two ecotypes. In pursuit of this goal, we generated the first reference sockeye salmon genome assembly and an RNA-seq transcriptome data set to better annotate features of the assembly. Resequenced whole-genomes of 140 sockeye salmon and kokanee were analyzed to understand population structure and identify genomic differences between ecotypes. Three distinct geographic and genetic groups were identified from analyses of the resequencing data. Nucleotide variants in an immunoglobulin heavy chain variable gene cluster on chromosome 26 were found to differentiate the northwestern group from the southern and upper Columbia River groups. Several candidate genes were found to be associated with the kokanee ecotype. Many of these genes were related to ammonia tolerance or vision. Finally, the sex chromosomes of this species were better characterized, and an alternative sex-determination mechanism was identified in a subset of upper Columbia River kokanee.

Loci associated with variation in gene expression and growth in juvenile salmon are influenced by the presence of a growth hormone transgene.

BACKGROUND: Growth regulation is a complex process influenced by genetic and environmental factors. We examined differences between growth hormone (GH) transgenic (T) and non-transgenic (NT) coho salmon to elucidate whether the same loci were involved in controlling body size and gene expression phenotypes, and to assess whether physiological transformations occurring from GH transgenesis were under the influence of alternative pathways. The following genomic techniques were used to explore differences between size classes within and between transgenotypes (T vs. NT): RNA-Seq/Differentially Expressed Gene (DEG) analysis, quantitative PCR (qPCR) and OpenArray analysis, Genotyping-by-Sequencing, and Genome-Wide Association Study (GWAS). RESULTS: DEGs identified in comparisons between the large and small tails of the size distributions of T and NT salmon (NTLarge, NTSmall, TLarge and TSmall) spanned a broad range of biological processes, indicating wide-spread influence of the transgene on gene expression. Overexpression of growth hormone led to differences in regulatory loci between transgenotypes and size classes. Expression levels were significantly greater in T fish at 16 of 31 loci and in NT fish for 10 loci. Eleven genes exhibited different mRNA levels when the interaction of size and transgenotype was considered (IGF1, IGFBP1, GH, C3-4, FAS, FAD6, GLUT1, G6PASE1, GOGAT, MID1IP1). In the GWAS, 649 unique SNPs were significantly associated with at least one study trait, with most SNPs associated with one of the following traits: C3_4, ELA1, GLK, IGF1, IGFBP1, IGFII, or LEPTIN. Only 1 phenotype-associated SNP was found in common between T and NT fish, and there were no SNPs in common between transgenotypes when size was considered. CONCLUSIONS: Multiple regulatory loci affecting gene expression were shared between fast-growing and slow-growing fish within T or NT groups, but no such regulatory loci were found to be shared between NT and T groups. These data reveal how GH overexpression affects the regulatory responses of the genome resulting in differences in growth, physiological pathways, and gene expression in T fish compared with the wild type. Understanding the complexity of regulatory gene interactions to generate phenotypes has importance in multiple fields ranging from applications in selective breeding to quantifying influences on evolutionary processes.

Fluorescent protein transgenesis has varied effects on behaviour and cold tolerance in a tropical fish (Gymnocorymbus ternetzi): implications for risk assessment.

Fluorescent protein (FP) transgenesis is used in the ornamental aquarium trade to produce new colour morphs in tropical fish. Understanding whether such genetic modification could alter ability to survive temperate waters, or interactions with native fish, should such fish be released to natural systems is critical in developing policy on their commercial use. We examined the competitive foraging ability and cold tolerance of unrelated pet-trade sourced adult green FP transgenic tetra and non-transgenic white tetra (Gymnocorymbus ternetzi), as well as white non-transgenic and green FP transgenic juvenile progeny of these groups. FP transgenesis did not affect the foraging success or aggressive behaviour in either adult or juvenile fish, indicating FP transgenesis may not influence potential hazards through this pathway. During a cold temperature tolerance trial, adult green tetras had greatly diminished cold tolerance relative to unrelated adult white fish, while sibling juvenile offspring of these groups had intermediate cold tolerance between adult fish groups that were not affected by FP transgenesis. This data suggests background genetics, rearing history and/or life stage may play larger roles in cold tolerance than FP transgenesis in this species. Unexpectedly, both adult and juvenile white tetras were 3.8 times more likely to take refuge in shelters when temperature declined than green tetras. These data indicate FP transgenic fish may pose equal or lesser risk than non-transgenic fish, should they be released to natural environments. Results also demonstrate that unrelated pet-trade sourced fish may not always be appropriate models for examining effects of FP transgenesis.

Effect of growth rate on transcriptomic responses to immune stimulation in wild-type, domesticated, and GH-transgenic coho salmon.

BACKGROUND: Transcriptomic responses to immune stimulation were investigated in coho salmon (Oncorhynchus kisutch) with distinct growth phenotypes. Wild-type fish were contrasted to strains with accelerated growth arising either from selective breeding (i.e. domestication) or genetic modification. Such distinct routes to accelerated growth may have unique implications for relationships and/or trade-offs between growth and immune function. RESULTS: RNA-Seq was performed on liver and head kidney in four 'growth response groups' injected with polyinosinic-polycytidylic acid (Poly I:C; viral mimic), peptidoglycan (PGN; bacterial mimic) or PBS (control). These groups were: 1) 'W': wild-type, 2) 'TF': growth hormone (GH) transgenic salmon with ~ 3-fold higher growth-rate than W, 3) 'TR': GH transgenic fish ration restricted to possess a growth-rate equal to W, and 4) 'D': domesticated non-transgenic fish showing growth-rate intermediate to W and TF. D and TF showed a higher similarity in transcriptomic response compared to W and TR. Several immune genes showed constitutive expression differences among growth response groups, including perforin 1 and C-C motif chemokine 19-like. Among the affected immune pathways, most were up-regulated by Poly I:C and PGN. In response to PGN, the c-type lectin receptor signalling pathway responded uniquely in TF and TR. In response to stimulation with both immune mimics, TR responded more strongly than other groups. Further, group-specific pathway responses to PGN stimulation included NOD-like receptor signalling in W and platelet activation in TR. TF consistently showed the most attenuated immune response relative to W, and more DEGs were apparent in TR than TF and D relative to W, suggesting that a non-satiating ration coupled with elevated circulating GH levels may cause TR to possess enhanced immune capabilities. Alternatively, TF and D salmon are prevented from acquiring the same level of immune response as TR due to direction of energy to high overall somatic growth. Further study of the effects of ration restriction in growth-modified fishes is warranted. CONCLUSIONS: These findings improve our understanding of the pleiotropic effects of growth modification on the immunological responses of fish, revealing unique immune pathway responses depending on the mechanism of growth acceleration and nutritional availability.

Growth hormone regulates opsin expression in the retina of a salmonid fish.

Colour vision relies on retinal photoreceptors that express a different predominant visual pigment protein (opsin). In several vertebrates, the primary opsin expressed by a photoreceptor can change throughout ontogeny, although the molecular factors that influence such regulation are poorly understood. One of these factors is thyroid hormone which, together with its receptors, modulates opsin expression in the retinas of multiple vertebrates including rodents and salmonid fishes. In the latter, thyroid hormone induces a switch in opsin expression from SWS1 (ultraviolet light sensitive) to SWS2 (short wavelength or blue light sensitive) in the single cone photoreceptors of the retina. The actions of other hormones on opsin expression have not been investigated. In the present study, we used a transgenic strain of coho salmon (Oncorhynchus kitsutch) with enhanced levels of circulating growth hormone compared to that of wild siblings to assess the effects of this hormone on the SWS1 to SWS2 opsin switch. Transgenic fish showed a developmentally accelerated opsin switch compared to size-matched controls as assessed by immunohistological and in situ hybridisation labelling of photoreceptors and by quantification of transcripts using quantitative polymerase chain reaction. This accelerated switch led to a different spectral sensitivity maximum, under a middle to long wavelength adapting background, from ultraviolet (λmax  ~ 380 nm) in controls to short wavelengths (λmax  ~ 430 nm) in transgenics, demonstrating altered colour vision. The effects of growth hormone over-expression were independent of circulating levels of thyroid hormone (triiodothyronine), the hormone typically associated with opsin switches in vertebrates.

The AMPK system of salmonid fishes was expanded through genome duplication and is regulated by growth and immune status in muscle.

5'adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. This study identified expansions in the AMPK-α, -ß and -γ families of salmonid fishes due to a history of genome duplication events, including five novel salmonid-specific AMPK subunit gene paralogue pairs. We tested the hypothesis that the expanded AMPK gene system of salmonids is transcriptionally regulated by growth and immunological status. As a model, we studied immune-stimulated coho salmon (Oncorhynchus kisutch) from three experiment groups sharing the same genetic background, but showing highly-divergent growth rates and nutritional status. Specifically, we compared wild-type and GH-transgenic fish, the latter achieving either enhanced or wild-type growth rate via ration manipulation. Transcript levels for the fifteen unique salmonid AMPK subunit genes were quantified in skeletal muscle after stimulation with bacterial or viral mimics to alter immune status. These analyses revealed a constitutive up-regulation of several AMPK-α and -γ subunit-encoding genes in GH-transgenic fish achieving accelerated growth. Further, immune stimulation caused a decrease in the expression of several AMPK subunit-encoding genes in GH-transgenic fish specifically. The dynamic expression responses observed suggest a role for the AMPK system in balancing energetic investment into muscle growth according to immunological status in salmonid fishes.

Effect of triploidy on liver gene expression in coho salmon (Oncorhynchus kisutch) under different metabolic states.

BACKGROUND: Triploid coho salmon are excellent models for studying gene dosage and the effects of increased cell volume on gene expression. Triploids have an additional haploid genome in each cell and have fewer but larger cells than diploid coho salmon to accommodate the increased genome size. Studying gene expression in triploid coho salmon provides insight into how gene expression may have been affected after the salmonid-specific genome duplication which occurred some 90 MYA. Triploid coho salmon are sterile and consequently can live longer and grow larger than diploid congeners in many semelparous species (spawning only once) because they never reach maturity and post-spawning mortality is averted. Triploid fishes are also of interest to the commercial sector (larger fish are more valuable) and to fisheries management since sterile fish can potentially minimize negative impacts of escaped fish in the wild. RESULTS: The vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon, indicating that the same amount of mRNA transcripts were being produced per gene copy (positive gene dosage effects) within a larger volume cell. Several genes related to nutrition and compensatory growth were differentially expressed between diploid and triploid salmon, indicating that some loci are sensitive to cell size and/or DNA content per cell. To examine how robust expression between ploidies is under different conditions, a genetic/metabolic modifier in the form of different doses of a growth hormone transgene was used to assess gene expression under conditions that the genome has not naturally experienced or adapted to. While many (up to 1400) genes were differentially expressed between non-transgenic and transgenic fish, relatively few genes were differentially expressed between diploids and triploids with similar doses of the transgene. These observations indicate that the small effect of ploidy on gene expression is robust to large changes in physiological state. CONCLUSIONS: These findings are of interest from a gene regulatory perspective, but also valuable for understanding phenotypic effects in triploids, transgenics, and triploid transgenics that could affect their utility in culture conditions and their fitness and potential consequences of release into nature.

Effects of recombinant salmon type II growth hormone and bovine growth hormone on growth of coho salmon (Oncorhynchus kisutch).

A comparison of the efficacy of salmon and bovine growth hormone to stimulate growth of coho salmon juveniles was performed. Oncorhynchus nerka (sockeye salmon) type II growth hormone (nGH2) was produced using a bacterial expression system, yielding approximately 25 mg of refolded recombinant protein per litre of cells. The purified nGH2 and bovine growth hormone (bGH) were tested in juvenile O. kisutch (coho salmon) over 24 weeks. Weekly intraperitoneal injections of 0.1 and 0.5 µg/g nGH2 resulted in a dose-dependent increase in weight and fork length compared to control fish injected with bovine serum albumin (BSA). Application of 0.5 µg/g bGH resulted in the same stimulation of growth as did 0.5 µg/g nGH2, indicating these proteins were equipotent. Following 6 weeks of treatment and a subsequent rest period of 7 weeks, coho salmon were further treated with bGH at 0.5 µg/g. A prior treatment with bGH did not reduce growth-promoting activity of bGH in subsequent treatments. Throughout the experiment, condition factor decreased at similar rates for all treatment groups. These data show that bGH, which is widely available, can be used to elevate growth rate in juvenile salmon comparably to homologous GH, and validate the use of bGH in physiological or ecological experiments where rapid growth is desired compared to that seen in wild type.

Importance of Experimental Environmental Conditions in Estimating Risks and Associated Uncertainty of Transgenic Fish Prior to Entry into Nature.

Salmonids show a high degree of phenotypic plasticity that can differ among genotypes, and this variation is one of the major factors contributing to uncertainty in extrapolating laboratory-based risk assessment data to nature. Many studies have examined the relative growth and survival of transgenic and non-transgenic salmonids, and the results have been highly variable due to genotype × environment interactions. The relative survival of fast- and slow-growing strains can reverse depending on the environment, but it is not clear which specific environmental characteristics are driving these responses. To address this question, two experiments were designed where environmental conditions were varied to investigate the contribution of rearing density, food amount, food type, habitat complexity, and risk of predation on relative growth and survival of fast-growing transgenic and slow-growing wild-type coho salmon. The first experiment altered density (high vs. low) and food amount (high vs. low). Density impacted the relative growth of the genotypes, where transgenic fish grew more than non-transgenic fish in low density streams, regardless of food level. Density also affected survival, with high density causing increased mortality for both genotypes, but the mortality of transgenic relative to non-transgenic fish was lower within the high-density streams, regardless of food level. The second experiment altered habitat complexity (simple vs. complex), food type (artificial vs. natural), amount of food (normal vs. satiation), and risk of predation (present vs. absent). Results from this experiment showed that genotype affected growth and survival, but genotype effects were modulated by one or more environmental factors. The effect of genotype on survival was influenced by all examined environmental factors, such that no predictable trend in relative survival of transgenic versus non-transgenic fry emerged. This was primarily due to variations in survival of non-transgenic fish under different environmental conditions (non-transgenic fry had highest survival in hatchery conditions, and lowest survival in complex conditions with natural food fed at a normal level with or without predators). Transgenic fry survival was only significantly influenced by predator presence. The effects of genotype on mass and length were significantly modulated by food type only. Transgenic fry were able to gain a large size advantage over non-transgenic fish when fed artificial food under all habitat types. These experiments support the observations of dynamic responses in growth and survival depending on the environment, and demonstrate the challenge of applying laboratory-based experiments to risk assessment in nature.

Using problem formulation for fit-for-purpose pre-market environmental risk assessments of regulated stressors.

Pre-market/prospective environmental risk assessments (ERAs) contribute to risk analyses performed to facilitate decisions about the market introduction of regulated stressors. Robust ERAs begin with an explicit problem formulation, which involves among other steps: (1) formally devising plausible pathways to harm that describe how the deployment of a regulated stressor could be harmful; (2) formulating risk hypotheses about the likelihood and severity of such events; (3) identifying the information that will be useful to test the risk hypotheses; and (4) developing a plan to acquire new data for hypothesis testing should tests with existing information be insufficient for decision-making. Here, we apply problem formulation to the assessment of possible adverse effects of RNA interference-based insecticidal genetically modified (GM) plants, GM growth hormone coho salmon, gene drive-modified mosquitoes and classical biological weed control agents on non-target organisms in a prospective manner, and of neonicotinoid insecticides on bees in a retrospective manner. In addition, specific considerations for the problem formulation for the ERA of nanomaterials and for landscape-scale population-level ERAs are given. We argue that applying problem formulation to ERA maximises the usefulness of ERA studies for decision-making, through an iterative process, because: (1) harm is defined explicitly from the start; (2) the construction of risk hypotheses is guided by policy rather than an exhaustive attempt to address any possible differences; (3) existing information is used effectively; (4) new data are collected with a clear purpose; (5) risk is characterised against well-defined criteria of hypothesis corroboration or falsification; and (6) risk assessment conclusions can be communicated clearly. However, problem formulation is still often hindered by the absence of clear policy goals and decision-making criteria (e.g. definition of protection goals and what constitutes harm) that are needed to guide the interpretation of scientific information. We therefore advocate further dialogue between risk assessors and risk managers to clarify how ERAs can address policy goals and decision-making criteria. Ideally, this dialogue should take place for all classes of regulated stressors, as this can promote alignment and consistency on the desired level of protection and maximum tolerable impacts across regulated stressors.

Proteomic comparison of selective breeding and growth hormone transgenesis in fish: Unique pathways to enhanced growth.

In fish used for food production and scientific research, fast growth can be achieved via selective breeding or induced instantaneously via growth hormone (GH) transgenesis (GHT). The proteomic basis for these distinct routes towards a similar higher phenotype remains uncharacterized, as are associated implications for health parameters. We addressed this knowledge gap using skeletal muscle proteomics in coho salmon (Oncorhynchus kisutch), hypothesising that i) selective breeding and GHT are underpinned by both parallel and unique changes in growth systems, and ii) rapidly-growing fish strains have lowered scope to allocate resources towards immune function. Quantitative profiling of GHT and growth-selected strains was done in comparison to wild-type after injection with PBS (control) or Poly I:C (to mimic infection). We identified remodelling of the muscle proteome in each growth-enhanced strain that was strikingly non-overlapping. GHT was characterized by focal upregulation of systems driving protein synthesis, while the growth-selected fish presented a larger and more diverse set of changes, consistent with complex alterations to many metabolic and cellular pathways. Poly I:C had little detectable effect on the muscle proteome. This study demonstrates that distinct proteome profiles can explain outwardly similar enhanced growth phenotypes, improving our understanding of growth mechanisms in anthropogenic animal strains. SIGNIFICANCE: This work provides the first proteomic insights into mechanisms underpinning different anthropogenic routes to rapid growth in salmon. High-throughput proteomic profiling was used to reveal changes supporting enhanced growth, comparing skeletal muscle of growth hormone transgenic (GHT) and selectively-bred salmon strains with their wild-type counterparts. Contrasting past mRNA-level comparisons of the same fish strains, our data reveals a surprisingly substantial proteomic divergence between the GHT and selectively bred strains. The findings demonstrate that many unique molecular mechanisms underlie growth-enhanced phenotypes in different types of fish strain used for food production and scientific research.