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Experiments examining potential impacts of growth hormone (GH) transgenesis in fish typically use a single source strain and do not address potential differential impacts in strains of different genetic backgrounds. Here, we examine the effects of differing genetic backgrounds when reared in culture on the growth of transgenic and nontransgenic coho salmon (Oncorhynchus kisutch) produced by mating sires from different rivers with transgenic dams from a single origin. We found a significant difference in size between offspring of sires originating from various river systems in British Columbia. This difference was independent of differences between transgenotypes (i.e., transgenic vs. nontransgenic offspring). However, the effects of strain or sire were relatively small compared to the effects of the transgene, which were consistent regardless of sire origin. Thus, results derived from studies of GH transgenic fish from a single source population can provide useful information for assessments of GH transgenic salmon from other systems. This has important implications for examining potential risks from introgression of a transgene into different populations.

Salmonids possess straying abilities that allows them to exploit open territory and establish new populations. Atlantic salmon (Salmo salar) are expanding their distribution primarily as first-generation escapes from aquaculture, whereas Pacific salmon (Oncorhynchus spp.) are expanding their range in polar and temperate regions due to both anthropogenic and natural influences. Here we utilize artificial intergeneric reciprocal crosses to assess the ability of seven species of Pacific salmon to hybridize with Atlantic salmon. Most cross types were found to produce low numbers of hatched embryos, but none survived to sexual maturation. Survivors consisted of diploids and triploids containing both Atlantic and Pacific salmon parental genomes. Thus, introgression of DNA between Pacific and Atlantic salmon may occur to form F1 hybrids, but transmission to subsequent generations is expected to be rare and occur only over evolutionary time scales. Further, the low viability observed for the most part at early stages of development and in both reciprocal crosses indicates that intergeneric crosses in nature between Atlantic and Pacific salmon are expected to have severe fitness consequences for both dams and sires.

DGS is a Serra Húnter Tenure-Track lecturer, and this work has been partially funded by the Ministerio de Ciencia y Tecnología grant number RTI2018-100757-B-100. RHD acknowledges support from the Canadian Regulatory System for Biotechnology (grant number 61740). 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 (TF) and non-transgenic (NT) fish of matching size were compared. Once morphometric parameters were normalized by animal length, we found that TF fish otoliths were smaller (-24%, -19%, -20% and -30%; P

Many natural and managed organisms will require substantial functional genetic variation to respond to selection in the face of rapid environmental change. Pacific salmon have experienced strong fluctuations in critical fitness traits over the past five decades. We examined genetic and phenotypic variability over three generations in a pedigreed hatchery population of coho salmon (Oncorhynchus kisutch) by monitoring seven fitness-related traits. Three-year-old adult return numbers varied more than fivefold, and jack (2-year-old males) numbers varied 13-fold. Body sizes of Inch Creek coho salmon decreased consistently such that fish were only 40.7% as heavy in 2015 as in 2006, and female reproductive traits also decreased. During the study period, the majority of families produced returning adult progeny, and effective population size was relatively constant. Heritability estimates for phenotypic traits were significantly greater than zero except for condition factor, and the estimated heritability for jacking was 0.42. The Inch Creek coho salmon population harbours substantial heritability for fitness and reproductive traits and thus likely retains substantial capacity for adaptation despite many years of hatchery propagation.

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 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 two chromosome-level genome assemblies and sequenced 24 RNA-seq libraries to better annotate the coho salmon genome assemblies. We also resequenced the genomes of 83 coho salmon across their North American range to identify nucleotide variants, characterize the broad effects of isolation-by-distance using a genome-wide association analysis approach, and understand the demographic histories of these salmon by modeling population size from genome-wide data. We observed that more than 13% of all SNPs were associated with latitude (before multiple test correction), likely an affect of isolation-by-distance. From demographic history modeling, we estimated that the SNP latitudinal gradient likely developed as recently as 8,000 years ago. In addition, we identified four genes each harboring multiple SNPs associated with latitude; all of these SNPs were also predicted to modify the function of the gene. Three of these genes have roles in cell junction maintenance and may be involved in osmoregulation. This signifies that ocean salinity may have been a factor influencing coho salmon recolonization after the last glaciation period – generating the current pattern of variation in these three genes.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s10126-020-10012-5.

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.

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.

Chum salmon (Oncorhynchus keta) is the species with the widest geographic range of the anadromous Pacific salmonids,. Chum salmon is the second largest of the Pacific salmon, behind Chinook salmon, and considered the most plentiful Pacific salmon by overall biomass. This species is of significant commercial and economic importance: on average the commercial chum salmon fishery has the second highest processed value of the Pacific salmon within British Columbia. The aim of this work was to establish genomic baseline resources for this species. Our first step to accomplish this goal was to generate a chum salmon reference genome assembly from a doubled-haploid chum salmon. Gene annotation of this genome was facilitated by an extensive RNA-seq database we were able to create from multiple tissues. Range-wide resequencing of chum salmon genomes allowed us to categorize genome-wide geographic variation, which in turn reinforced the idea that genetic differentiation was best described on a regional, rather than at a stock-specific, level. Within British Columbia, chum salmon regional groupings were described at the conservation unit (CU) level, and there may be substructure within particular CUs. Genome wide associations of phenotypic sex to SNP genetic markers identified two clear peaks, a very strong peak on Linkage Group 15, and another on Linkage Group 3. With these new resources, we were better able to characterize the sex-determining region and gain further insights into sex determination in chum salmon and the general biology of this species.

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 strict two-year life-history of most 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 centromere drive or a combination or 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.

The fine-scale behavioural activities of rainbow trout (Oncorhynchus mykiss) in nature are not well understood, but are of importance for identifying interactions with the ecosystem and of interest to conservationists and recreational anglers. We have undertaken a high-resolution acoustic telemetry study to identify the distinct movement patterns of 30 rainbow trout in a freshwater lake, specifically examining swim speed, area of movement, and site preference in both summer and winter. Activity levels were reduced in winter compared with summer across all fish, but ranking of individuals was consistent. In summer, 16/30 fish displayed diel movement, in which they travelled to a different area of the lake at dawn and returned at dusk, while other fish maintained their site preference regardless of the time of day or swam more randomly throughout the lake. These patterns were minimized in winter, where there was a reduction in cross-lake movement under ice and only 4/30 fish displayed diel movement. Winter conditions may limit the capability (physiological limitations) and (or) motivation (prey availability) for diel behaviours observed in summer.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5655-8) contains supplementary material, which is available to authorized users.

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 F 1 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.

Atlantic and Pacific salmon are frequently consumed species with very different economic values: farmed Atlantic salmon is cheaper than wild-caught Pacific salmons. Species replacements occur with the high valued Pacific species (Oncorhynchus keta, O. gorbuscha, O. kisutch, O. nerka and O. tshawytscha) substituted by cheaper farmed Atlantic salmon (Salmo salar) and Atlantic salmon by rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta). Here we use High-Resolution Melting Analysis (HRMA) to identify eight salmonid species. We designed primers to generate short amplicons of 72 and 116 bp from the fish barcode genes CO1 and CYTB. The time of analysis was under 70 min, after DNA extraction. Food processing of Atlantic salmon (fresh, “Bellevue”, “gravadlax”, frozen and smoked) did not impact the HRMA profiles allowing reliable identification. A blind test was conducted by three different institutes, showing correct species identifications irrespective of the laboratory conducting the analysis. Finally, a total of 82 retail samples from three European countries were analyzed and a low substitution rate of 1.2% was found. The developed tool provides a quick way to investigate salmon fraud and contributes to safeguard consumers.

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.

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.

Genetic alterations such as transgenesis can be useful, along with domestication and selective breeding, to improve aquaculture production efficiency and quality of end products. In particular, accelerated growth through growth hormone (GH) transgenesis has the potential to decrease production time and cost in a wide variety of aquaculture species. Increased GH production results in a cascade of endocrinological and physiological effects; indeed, few physiological processes are unaffected by GH transgenesis. Effects include alterations that directly benefit production such as fast growth, increased feed conversion efficiency, and improved utilization of some feed components. Other unintended physiological effects can be beneficial (e.g., improved performance in extreme environmental conditions) or detrimental to aquaculture production (e.g., impaired immune response in some species). The magnitude or direction of physiological alterations varies among species and lines, making overarching conclusions on the physiological impacts of GH transgenesis difficult. For aquaculture production, it is important to identify potential effects of GH transgenesis, both positive and negative, relative to available domesticated strains for a given species. Current commercial use of genetically engineered species in aquaculture is limited worldwide. However, continuing advancements in techniques (e.g., gene-editing), containment methods (e.g., land-based aquaculture and reproductive control), and refinements of husbandry practices to meet physiological needs of engineered lines are all anticipated to maximize production and enhance end products for aquaculture.

Sex-biased predation, a predator’s bias for one prey sex over the other, can have important demographic impacts on prey species of conservation concern. Yet, it is difficult to measure in the wild. Molecular scatology has been used to indirectly determine the proportion of prey items consumed in the diet, and it may be possible to apply similar approaches to determine the sex-biased consumption of prey items. We developed a molecular method to indirectly examine sex-specific predation employing scat, focusing on predator–prey interactions between Chinook salmon (Oncorhynchus tshawytscha) and harbor seals (Phoca vitulina richardii). We established that the proportions of male and female Chinook DNA can be determined in a controlled sample by measuring a Y-linked marker, growth hormone pseudogene, using qPCR. We then applied the assay to harbor seal scat samples. Although the assay amplified in 83% of scat samples, 27% of samples quantified had an estimated male proportion > 1, which may have been due to a lack of robustness of the PCR assay in samples. Lastly, we constructed a biomass calibration curve to determine whether DNA measurements could estimate the proportions of male and female biomass consumed. The calibration curve was skewed by high male DNA density precluding our ability to quantify the relative amounts. We demonstrated that nuclear prey markers can be amplified in predator scat, however, contamination and extreme DNA density differences between the prey sexes may pose practical difficulties to estimate the relative amounts of male to female biomass consumed.

Application of fluorescent protein transgenes in tropical freshwater fish is used in research (e.g., in zebrafish) and in the commercial ornamental aquarium trade. To assess the overwinter potential of such fish in Canada, we examined the minimum temperature tolerance of three wild-type species (zebrafish, Danio rerio (Hamilton, 1822); black tetra, Gymnocorymbus ternetzi (Boulenger, 1895); tiger barb, Puntius tetrazona (Bleeker, 1855)) used as fluorescent models in the aquarium trade, as well as four lines of green fluorescent protein (GFP) transgenic and wild-type zebrafish used in research. Both tetra and barb had lower temperature tolerance limits that were higher than surveyed winter temperatures in Canada, and the lower tolerance limits of zebrafish lines was only within the range of a few surveyed lakes in some years. One line of GFP zebrafish had a lower temperature tolerance limit that was significantly higher than those of other lines, suggesting that some fluorescent transgene constructs may alter tolerance to extreme temperatures. When lines of zebrafish were reared at 8, 10, or 28 °C for 4 months, all lines were capable of producing viable embryos after cold rearing. These results indicate that tropical transgenic fish models used in research and in the aquarium trade are not expected to persist over winter in Canadian water systems.

As the world increasingly relies on aquaculture operations to meet rising seafood demands, reliable biocontainment measures for farmed fish stocks are desired to minimize ecological impacts arising from interactions of cultured fish with wild populations. One possible biocontainment strategy is to induce a dietary dependence on a vitamin, such as thiamine (vitamin B1), required for survival. Fish expressing thiaminase (an enzyme that degrades thiamine) within a confined aquaculture facility could receive supplemental thiamine to allow survival and normal growth, whereas escapees lacking this dietary rescue would die from thiamine deficiency. To test the concept and efficacy of such a dietary dependency system (for potential future use in larger aquaculture species), we expressed thiaminase in zebrafish as a test model. We drove the expression of thiaminase under the strong ubiquitous and constitutive control of the CMV promoter which resulted in non-viable fish, indicating that the thiaminase sequence kills fish. However, the CMV promoter is too strong to allow conditional survival since the lethality could not be rescued by exogenous thiamine provided as a supplement to typical food. In addition, microinjection of 0.5 pg of thiaminase mRNA in zebrafish embryos at the one-cell stage resulted in 50% larval mortality at 5 days post-fertilization (dpf), which was partially rescued by thiamine supplementation. Evaluating the efficacy of biocontainment strategies helps assess which methods can reliably prevent ecological impacts arising from breaches in physical containment systems that release engineered organisms to nature, and consequently provides critical information for use in regulatory risk assessment processes.

Parentage-based tagging (PBT) and genetic stock identification (GSI) were used to identify individual coho salmon (Oncorhynchus kisutch) to specific populations and brood years. In total, 20 242 individuals from 117 populations were genotyped at 304 single nucleotide polymorphisms (SNPs) via direct sequencing of amplicons. Coho salmon from 15 populations were assigned via parentage analysis that required the genotypes of both parents. The overall accuracy of assignment for 1939 coho salmon to the correct population was 100%, and to correct brood year within population was also 100%. Inclusion of individuals requiring only a single parental genotype for identification resulted in assignments of 2101 individuals, with an accuracy of 99.95% (2000–2001) to population and 100.0% to age. With 23 regions defined by the coded-wire tag (CWT) program, and individuals displaying an assignment probability

Growth hormone (GH) is involved in the parr–smolt transformation of salmonid fishes and is known to improve salinity tolerance. This study compared the capacity for seawater acclimation of GH transgenic coho salmon (Oncorhynchus kisutch (Walbaum, 1792)) to that of wild-type fish, allowing examination of responses to sustained (chronic) exposure to elevated GH. GH transgenic fish (GH TG) smolted 1 year in advance of wild-type salmon and showed a greater capacity to hypo-osmoregulate in seawater. As GH TG fish were much larger than the wild-type fish, a second experiment was conducted with three size-matched groups of coho salmon (a 1+-year-old wild-type group, a 1+-year-old ration-restricted GH TG group, and a 0+-year-old fully fed GH TG group). When size-matched, the effect of GH transgenesis was not as dramatic, but the feed-rationed TG1+ group exhibited smaller deviations in plasma ion and osmolality levels following seawater exposure than did the other groups, suggesting a somewhat improved hypo-osmoregulatory ability. These results support a role for GH in the development of seawater tolerance by salmonid fishes independent of fish size.

Growth hormone (GH) transgenic fish have been proposed for use in aquaculture to enhance production efficiency. As part of a risk analysis for use of such fish, the influence of GH transgenesis on the potential to persist and succeed in natural ecosystems is being examined in confined laboratory conditions. GH transgenesis can greatly accelerate growth and, in culture conditions, is associated with secondary effects such as poor swimming capacity and spawning success. However, standard culture has also been shown to negatively affect fitness components of wild-type fish, raising the question of whether culture conditions influence fitness components of transgenic fish in a similar way. To examine factors influencing the phenotype of marine-stage GH transgenic salmon (T), and to determine if genotype-by-environment interactions exist at this life stage, we grew T and wild-type (NT) coho salmon ( Oncorhynchus kisutch ) over six cohort years in 350,000 L seawater tanks (termed mesocosms) designed to minimize effects of standard culture conditions. Mesocosm rearing partially facilitated development of normal size and morphology of NT fish relative to nature-reared counterparts, but altered overall body shape, indicating mesocosm conditions do not fully mimic natural environmental effects on coho salmon phenotype. T fish reared in mesocosms had larger mass at maturity than mesocosm- or nature-reared NT fish, indicating GH transgenesis can alter maximum obtainable mass in salmon. Unlike NT, T fish obtained maximum size at maturity across environments, suggesting marine environmental conditions may affect T growth less than NT growth. Screening parents for a common disease agent ( Renibacterium salmoninarum ) improved seawater survival, and T fish had lower survival than NT fish when from unscreened parents and inconsistent relative survival when from screened parents, indicating GH transgenesis may constitute an advantage or disadvantage in terms of survival. Transgenic salmon had lower swimming capacity and aerobic scope, but similar routine metabolic rate and thermal tolerance, demonstrating transgenesis can have different influences depending on what phenotype is examined. Using an alternate strain of T fish in phenotypic comparisons did not greatly influence most fitness components, although had a strong effect on female fecundity. The inconsistent influence of GH transgenesis on different fitness components, and existence of genotype-by-environment interactions during the marine life stage, complicates extrapolation of laboratory data for transgenic fish to natural environments. However, current and previous data do not provide evidence that overall increased performance of GH transgenic salmon over wild-type fish would arise in the marine environment. Statement of relevance Rearing in seawater mesocosms demonstrate that growth hormone transgenesis has inconsistent effects on marine fitness components in coho salmon.

Underwater acoustic tag telemetry was used to assess behavioural differences between juvenile wild-type (i.e. non-transgenic, NT) and growth hormone (GH) transgenic (T) coho salmon Oncorhynchus kisutch in a contained simulated ocean environment. T O. kisutch were found across days to maintain higher baseline swimming speeds than NT O. kisutch and differences in response to feeding were detected between T and NT genotypes. This is the first study to assess behaviour of GH transgenic salmonids in a marine environment and has relevance for assessing whether behavioural effects of GH overexpression seen in freshwater environments can be extrapolated to oceanic phases of the life cycle.

The linkage between the Y-chromosomal DNA marker GH-Y (growth hormone pseudogene) and the Y-associated sex-determining locus was examined in 31 populations of Coho Salmon Oncorhynchus kisutch, 29 populations of Chum Salmon O. keta, and 14 populations of Pink Salmon O. gorbuscha from North America (British Columbia [BC] and Yukon, Canada; Oregon and Washington, USA) and Japan to examine the stability of Y-chromosome sequences in salmonids. This marker is used for genetic sexing of multiple salmon species both for population studies and for aquaculture strain development. We found a strong association between GH-Y and sex in Coho Salmon (with rare exceptions seen in Washington and Oregon only), whereas the association of GH-Y and sex was weak in Chum Salmon and Pink Salmon. Up to 100% of males lacking GH-Y were found in some BC Chum Salmon populations, whereas there were no exceptional (GH-Y-negative) males in Japan, the Yukon, or Washington, indicating strong geographic clustering of GH-Y marker de...

5′adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. This study identified expansions in the AMPK-α, -β 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.

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.

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.

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.

Growth in fish is regulated in part by the growth hormone (GH)-insulin-like growth factor (IGF) axis, and salmon transgenic for GH are known to show dramatic increased growth. However, little is known concerning the in vivo global levels of metabolites and the mechanism of enhancement of growth in GH transgenic vertebrates. The present study examined the charged metabolites levels in GH transgenic coho salmon (Oncorhynchus kisutch) overexpressing GH by metabolomic analysis. Triplicate groups of size-matched (0 year-old, approx. 60 g) and age-matched (1.5 years-old) GH transgenic (T) and non-transgenic (NT) wild salmon were quantitatively assessed for levels of approximately 200 metabolites in both muscle and liver. The most notable difference found between T and NT fish was that glycolysis metabolite levels were increased in the muscle of transgenic fish. In addition, an increase in some metabolite levels in the transgenic fish muscle was found to be enhanced by ration-restriction. However, these effects observed in muscle were different from that seen in liver. The results suggest that GH transgenesis can improve the use of carbohydrates as a source of energy associated with rapid growth. These effects are likely to depend on the level of total digestible energy intake and type of tissue in transgenic fish.

Next generation sequencing data can be used for rapid identification of genetic markers linked to phenotypes of interest for use in conservation, resource management or breeding. Here we report on the use of genotyping by sequencing (GBS) to develop SNP markers linked with phenotypic sex and with a transgenic construct in coho salmon (Oncorhynchus kisutch). Associations between phenotypes and genotypes were confirmed using high-resolution melting analysis (HRMA). A total of 56 SNPs were associated with phenotypic sex after analysis with the software TASSEL. Concordance between phenotypes and genotypes were tested for 28 SNPS, and of these, seven had moderate to high concordance across test populations. However, only one SNP showed 100 % concordance across all test populations. Fifteen SNPs were associated with the transgenotype using a TASSEL analysis of the GBS data; of these, four showed moderate to high concordance with the transgenotype using HRMA. This type of approach could be used to identify SNPs associated with phenotypes of interest in species with few previously developed genetic resources.