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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Unlabelled Image Highlights • Mechanistic basis for rapid growth poorly understood in fish. • Comparative proteomic profiling done in fish strains showing highly enhanced growth. • Distinct basis for enhanced growth comparing transgenic and domesticated fish strains. • Highly distinct proteome profiles may explain outwardly similar growth phenotypes. • Study enhances understanding of how rapid growth is achieved in anthropogenic animal strains. [ABSTRACT FROM AUTHOR]

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 <0.85 excluded from the analysis, mean regional assignment accuracy of individuals via GSI was 98.4% over all 23 regions. A PBT-GSI or PBT system of identification will provide an alternate method of identification in the assessment and management of Canadian-origin coho salmon relative to the existing CWT program. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The first genetically modified ( GM) fish intended for human consumption has recently stimulated significant scientific discussion and regulatory scrutiny regarding food safety and environmental risks. Currently, no experiments with transgenic fish have been performed in nature, yet such data are needed to facilitate predictions of ecological consequences should engineered fish escape to the natural environment., To address this limitation, we conducted experiments under natural conditions but within a contained environment to assess the impact of invasion of growth-enhanced GM coho salmon Oncorhynchus kisutch ( Walbaum) on survival and growth of three naturally cohabitating fishes: Chinook salmon O. tshawytscha ( Walbaum), steelhead trout O. mykiss ( Walbaum) and conspecific wild-type coho salmon., We found that the impact of stream-reared GM coho salmon on invaded specimens was similar to the impact of non- GM coho salmon. However, GM fish significantly reduced survival and growth of the invaded populations if they were first allowed to grow larger under hatchery conditions before being released., Synthesis and applications. Our results show that the ecological impact of fish genetically modified ( GM) for rapid growth on closely related fish species may not be high in stream environments, unless these fish are first reared under culture conditions where they are able to realize their genetic growth potential. As such, first generation escapes of GM fish into the natural environment should be a main concern in the short term, whereas later generations, which are more similar to naturally occurring genotypes, are expected to have significantly weaker effects but which could persist for longer periods. [ABSTRACT FROM AUTHOR]

Abstract: Several different transgenic growth hormone (GH) gene constructs have been used to obtain accelerated growth in salmonids. However, there have been limited direct comparisons of these constructs in terms of the ability to achieve maximal growth in fish. We examined the effect of promoter type (sockeye salmon metallothionein-B or histone 3) fused to a growth hormone-1 coding region from the same species (OnMTGH1 and OnH3GH1 constructs respectively) on growth and plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I) in multiple strains of GH transgenic coho salmon (Oncorhynchus kisutch). Salmon transgenic for the OnMTGH1 construct had consistently greater overall weight than those containing the OnH3GH1 construct, although both groups possessed greatly accelerated growth over non-transgenic fish. However, there were strong strain effects, where some OnH3GH1 strains had similar weight to OnMTGH1 strains while others did not. Triploidy diminished growth acceleration and decreased condition factors in both a fast growing MT strain and slower growing H3 strain. Plasma GH levels did not correlate to weight in transgenic strains, and all but one transgenic strain had plasma GH levels similar to equal sized non-transgenic fish. In contrast, plasma IGF-I content correlated well to size in transgenic strains. The mechanism by which accelerated growth in transgenic fish is obtained appears to be due in part to an upregulation of GH action through increased circulating IGF-I levels, and promoter-type appears to influence potential for growth. [Copyright &y& Elsevier]

Abstract: The production of growth hormone (GH) transgenic animals has raised a host of social and scientific concerns regarding their potential impacts on ecosystems should they escape into nature. Indeed, theoretical models suggest that GH transgenic animals could, under specific conditions, decimate local populations. However, while there are numerous laboratory examinations of factors affecting survival of transgenic and wild animals, we know little about the competitive reproductive capacity of transgenic animals. Here, we examined the reproductive capabilities of cultured GH transgenic coho salmon (Oncorhynchus kisutch) when in competition with wild coho derived from nature using semi-natural mating arenas (within a contained facility). To account for the well-known reproductive impairments associated with culturing salmon in laboratory facilities we contrasted the competitive reproductive success of GH transgenic coho against that of cultured non-transgenic coho. We also performed in vitro sperm analyses to assess the postcopulatory competitive ability of GH transgenic coho. In competitive mating arenas, transgenic coho performed fewer courtship and aggressive behaviours than coho from nature and sired less than 6% of offspring. Non-transgenic cultured coho, despite their smaller body size, sired more than twice as many offspring than transgenic coho when competing against wild coho in mating arenas. Transgenic males also face a postcopulatory reproductive disadvantage as their ejaculates contained fewer sperm that swam slower and for shorter durations than sperm from wild males. Together, these findings suggest limited potential for the transmission of transgenes from cultured GH transgenic coho salmon through natural matings should they escape from a contained culture facility into nature and reproductively interact with a local wild coho salmon strain. However, as responses of wild-reared fish can differ greatly from those of cultured fish, we stress the importance of understanding genotype-by-environment interactions for reproductive phenotypes when developing risk assessment information. [Copyright &y& Elsevier]

Abstract: Seasonal variation in daily food intake is a well-documented phenomenon in many organisms including wild-type coho salmon where the appetite is noticeably reduced during periods of decreased day length and low water temperature. This reduction may in part be explained by altered production of cholecystokinin (CCK) and growth hormone (GH). CCK is a hormone produced in the brain and gut that mediates a feeling of satiety and thus has an inhibitory effect on food intake and foraging behaviour. Growth hormone (GH) enhances feeding behaviour and consequently growth, but its production is reduced during winter. The objectives of this study were: first, to compare the seasonal feeding behaviour of wild and GH-transgenic coho salmon; second, to determine the behavioural effect of blocking the action of CCK (by using devazepide) on the seasonal food intake; and third, to measure CCK expression in brain and gut tissues between the two genotypes across seasons. We found that, in contrast to wild salmon, food intake in transgenic salmon was not reduced during winter indicating that seasonal control of appetite regulation has been disrupted by constitutive production of GH in transgenic animals. Blocking of CCK increased food intake in both genotypes in all seasons. The increase was stronger in wild genotypes than transgenic fish; however blocking CCK in wild-type fish in winter did not elevate appetites to levels observed in the summer. The response to devazepide was generally faster in transgenic than in wild salmon with more rapid effects observed during summer than during winter, possibly due to a higher temperature in summer. Overall, a seasonal effect on CCK mRNA levels was observed in telencephalon with levels during winter being higher compared to the summer in wild fish, but with no seasonal effect in transgenic fish. No differences in seasonal CCK expression were found in hypothalamus. Higher levels of CCK were detected in the gut of both genotypes in winter compared to summer. Thus, CCK appears to mediate food intake among seasons in both wild-type and GH-transgenic salmon, and an altered CCK regulation may be responsible at least in part for the seasonal regulation of food intake. [Copyright &y& Elsevier]

Aquaculture continues to be the fastest-growing animal food sector and accounts for over 50% of the world's fish consumption. This trend is projected to continue, highlighting the role that the aquaculture industry will play in ensuring future global food security. Enhancement of growth rates of fish can optimize aquaculture production and has been achieved through selective breeding programs producing fast-growing domesticated strains, or the development of growth hormone (GH) transgenic strains. Although the ability of selectively-bred domesticated and GH transgenic salmon to outgrow wild-type salmon is well documented, very little research has been reported that directly compares the growth rates of domesticated and GH transgenic strains. The aim of the present study was to compare the growth and survival of selectively-bred domesticated and GH transgenic coho salmon across a range of experimental conditions. We performed four experiments investigating the growth rates of domesticated and GH transgenic salmon by comparing these strains at different developmental stages and under different experimental rearing conditions, as well as under conditions when strains were competing and when they were reared separately. Results from all experiments showed that GH transgenic coho salmon grew faster and to a larger size than the selectively-bred domesticated coho salmon. This was true whether fish groups were reared post-smolt in freshwater or seawater, whether reared together or separately, and whether manipulated to be size-matched at early stages. Thus, GH transgenesis could become a viable complement to selective breeding to produce fast-growing salmon for aquaculture systems. While further research can continue to optimize the development and use of transgenic strains, the data presented here also are important for environmental risk assessments by providing data on the relative fitness and ecological consequences of selectively-bred domesticated and GH transgenic fish should they enter nature. • Compares selectively-bred domesticated salmon and GH transgenic salmon directly, over a range of life-stages and conditions. GH transgenic coho salmon grow to a larger size than selectively-bred domesticated coho salmon regardless of rearing conditions. • GH transgenesis could be a viable complement to selective breeding to produce fast-growing salmon for aquaculture systems. [ABSTRACT FROM AUTHOR]

The release or escape of genetically modified fish from confined aquaculture facilities presents a potential risk to wild fish stocks and surrounding ecosystems. Thus, reliable biocontainment methods for aquaculture-relevant species are needed. Since physical barriers are not 100% effective, it is critical to develop reliable genetic-based methods for biocontainment. RNA interference (RNAi) could regulate gene expression and target genes that are essential for fish viability and/or reproduction. One such target gene, deadend (dnd), is required for primordial germ cell (PGC) development, migration and survival. While the use of RNAi technology has been successfully reported in C. elegans, M. musculus, and D. melanogaster , its use in zebrafish (Danio rerio) has been met with variable success. We targeted dnd using two different short-hairpin RNA (shRNA) approaches in zebrafish embryos, namely, 1) CMV-driven or a Gal4/UAS constructs with one double short-hairpin RNA stem-loop design consisting of two different target sites, and 2) CMV-driven to test two separate target sites independently flanked by the 5′ and 3′ sequences of the pri-miR-30e miRNA. The first shRNA strategy did not completely nor consistently abolish PGCs, based on vasa -expression, but low vasa expression was observed in some fish at 6 dpf. In contrast, the second shRNA strategy resulted in reduced and/or ectopic positioning of PGCs based on vasa expression at 2 and 6 dpf. However, neither strategy affected fertility in adult fish. • shRNA constructs targeting the deadend (dnd) gene were produced. • shRNA knockdown of dnd in zebrafish impaired primordial germ cell development and/or migration. • shRNA knockdown of dnd did not markedly impair fertility of resulting adult zebrafish. [ABSTRACT FROM AUTHOR]

The Fraser River once supported massive salmon returns. However, over the last century, the largest returns have consistently been less than half of the recorded historical maximum. There is substantial interest from surrounding communities and governments to increase salmon returns for both human use and functional ecosystems. To generate resources for this endeavor, we resequenced genomes of Chinook (Oncorhynchus tshawytscha), coho (O. kisutch), and sockeye salmon (O. nerka) from the Fraser River at moderate coverage (∼16x). A total of 954 resequenced genomes were analyzed, with 681 collected specifically for this study from tissues sampled between 1997 and 2021. An additional 273 were collected from previous studies. At the species level, Chinook salmon appeared to have 1.6-2.1x more SNPs than coho or sockeye salmon, respectively. This difference may be attributable to large historical declines of coho and sockeye salmon. At the population level, three Fraser River genetic groups were identified for each species using principal component and admixture analyses, which is consistent with previous research and supports the continued use of these groups in conservation and management efforts. Environmental factors and a migration barrier were identified as major factors influencing the boundaries of these genetic groups. Additionally, 20 potentially adaptive loci were identified among the genetic groups. This information may be valuable in new management and conservation efforts. Furthermore, the resequenced genomes are an important resource for contemporary genomics research on Fraser River salmon and have been made publicly available., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., Competing Interests: Conflicts of interest statement The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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.

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.

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.

Polyploidy is an important driver of evolutionary change (generally via tetraploidy) and also serves a practical role in aquaculture and fisheries management (via triploidy). Fundamental changes in cell size and number that accompany polyploidy are predicted to affect cellular and whole-animal physiology due to constraints placed on surface-mediated processes at the cellular level, potentially altering environmental tolerances and optima. The aim of this study was to determine whether the documented reduction in thermal tolerance of aquatic polyploids is a result of their being less hypoxia tolerant. This was assessed by holding diploid and triploid rainbow trout for 1 h above their thermal optima in separate trials at eight temperatures between 20° and 27°C and then rapidly reducing the oxygen tension (Po 2 ) of the water and determining the nonlethal Po 2 at which fish lost equilibrium. As expected, there was a highly significant ([Formula: see text]) effect of temperature on Po 2 at loss of equilibrium. Although there was also a significant ([Formula: see text]) effect of ploidy on Po 2 at loss of equilibrium, with triploid values higher than diploid, post hoc analyses showed no significant effect of ploidy at any specific temperature. Oxygen availability alone therefore does not appear to play a major role in determining the thermal tolerance of polyploids.

To enhance understanding of triploid gene expression, the transcriptome information from bighead catfish (Clarias macrocephalus Günther, 1864) was studied using the paired-end Illumina HiSeq™ 2000 sequencing platform. In total, 68,227,832 raw reads were generated from liver tissues and 53,149 unigenes were assembled, with an average length of 765 bp and N50 length of 1283 bp. Of these unigenes, 33,428 (62.89%) could be annotated according to their homology with matches in the NCBI non-redundant (Nr), NCBI nucleotide (Nt), Swiss-Prot, Clusters of Orthologous Groups (COG), gene ontology (GO), or Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Relative expression of liver genes between diploid and triploid bighead catfish revealed more than 90% of the annotated unigenes similarly expressed, regardless of ploidy, whereas 362 upregulated and 83 downregulated with at least a twofold change in triploid relative to diploid. Quantitative real-time PCR of 15 differentially expressed growth-related genes showed consistency between the expression profiles of those genes with the results from RNA-seq analysis. Our results showed that genes in C. macrocephalus liver responded independently to triploidy with the majority showing similar expression levels between diploid and triploid (a dosage compensation phenomenon). The underlying mechanism of the varying gene expression patterns was discussed. Notably, 5 of the top 20 upregulated genes associated with stress response and thus may reflect stress caused by triploidy. The present study adds a substantial contribution to the sequence data available for C. macrocephalus and hence provides valuable resources for further studies. Furthermore, it gives information that may enhance understanding of triploid physiology.

Growth hormone (GH) transgenic salmon possesses markedly increased metabolic rate, appetite, and feed conversion efficiency, as well as an increased ability to compete for food resources. Thus, the ability of GH-transgenic fish to withstand periods of food deprivation as occurs in nature is potentially different than that of nontransgenic fish. However, the physiological and genetic effects of transgenic GH production over long periods of food deprivation remain largely unknown. Here, GH-transgenic coho salmon (Oncorhynchus kisutch) and nontransgenic, wild-type coho salmon were subjected to a 3-month food deprivation trial, during which time performance characteristics related to growth were measured along with proximate compositions. To examine potential genetic effects of GH-transgenesis on long-term food deprivation, a group of genes related to muscle development and liver metabolism was selected for quantitative PCR analysis. Results showed that GH-transgenic fish lose weight at an increased rate compared to wild-type even though proximate compositions remained relatively similar between the groups. A total of nine genes related to muscle physiology (cathepsin, cee, insulin-like growth factor, myostatin, murf-1, myosin, myogenin, proteasome delta, tumor necrosis factor) and five genes related to liver metabolism (carnitine palmitoyltransferase, fatty acid synthase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glucokinase) were shown to be differentially regulated between GH-transgenic and wild-type coho salmon over time. These genetic and physiological responses assist in identifying differences between GH-transgenic and wild-type salmon in relation to fitness effects arising from elevated growth hormone during periods of long-term food shortage.

Whole genome duplication has been implicated in evolutionary innovation and rapid diversification. In salmonid fishes, however, whole genome duplication significantly pre-dates major transitions across the family, and re-diploidization has been a gradual process between genomes that have remained essentially collinear. Nevertheless, pairs of duplicated chromosome arms have diverged at different rates from each other, suggesting that the retention of duplicated regions through occasional pairing between homeologous chromosomes may have played an evolutionary role across species pairs. Extensive chromosomal arm rearrangements have been a key mechanism involved in re-dipliodization of the salmonid genome; therefore, we investigated their influence on degree of differentiation between homeologs across salmon species. We derived a linkage map for coho salmon and performed comparative mapping across syntenic arms within the genus Oncorhynchus, and with the genus Salmo, to determine the phylogenetic relationship between chromosome arrangements and the retention of undifferentiated duplicated regions. A 6596.7 cM female coho salmon map, comprising 30 linkage groups with 7415 and 1266 nonduplicated and duplicated loci, respectively, revealed uneven distribution of duplicated loci along and between chromosome arms. These duplicated regions were conserved across syntenic arms across Oncorhynchus species and were identified in metacentric chromosomes likely formed ancestrally to the divergence of Oncorhynchus from Salmo. These findings support previous studies in which observed pairings involved at least one metacentric chromosome. Re-diploidization in salmon may have been prevented or retarded by the formation of metacentric chromosomes after the whole genome duplication event and may explain lineage-specific innovations in salmon species if functional genes are found in these regions., (Copyright © 2014 Kodama et al.)

There are genetically based differences in growth and behavior between domestic and wild (W) trout and salmon, although the molecular underpinnings of the physiological alterations have not been identified. To test for genetically based alterations in the GH/IGF-I axis, which is thought to mediate some of the differences in growth and behavior, we measured circulating concentrations of GH, IGF-I, and thyroid hormone (T(3)), as well as mRNA levels for GH, IGF-I, and GH receptor, from multiple tissues and from fish reared under different environments. Both age-matched and size-matched individuals were examined to overcome difficulties examining strains with inherently different growth rates (and, thus, body size at age). A principal components analysis detected four factors that explained over 70% of the variation in the data; of these, a factor composed of mRNA expression of GH receptor in the liver, IGF-I in the liver, and circulating IGF-I was most strongly correlated with genotype. W coho salmon families responded to environmental alteration with a lower level of plasma IGF-I detected in the seminatural (reduced food) environment relative to the culture environment, whereas no environmental response was detected in the domestic families. The results suggest that genetically based differences in hormone expression and regulation, particularly for IGF-I, are present in response to anthropogenic selection pressures in salmon and trout. In addition, although rearing environment alone can alter relative hormone expression, domestication appears to have reduced the physiological response to environment relative to W fish.

Domestication has been extensively used in agricultural animals to modify phenotypes such as growth rate. More recently, transgenesis of growth factor genes [primarily growth hormone (GH)] has also been explored as a rapid approach to accelerating performance of agricultural species. Growth rates of many fishes respond dramatically to GH gene transgenesis, whereas genetic engineering of domestic mammalian livestock has resulted in relatively modest gains. The most dramatic effects of GH transgenesis in fish have been seen in relatively wild strains that have undergone little or no selection for enhanced growth, whereas genetic modification of livestock necessarily has been performed in highly domesticated strains that already possess very rapid growth. Such fast-growing domesticates may be refractory to further stimulation if the same regulatory pathways are being exploited by both genetic approaches. By directly comparing gene expression in wild-type, domestic, and GH transgenic strains of coho salmon, we have found that domestication and GH transgenesis are modifying similar genetic pathways. Genes in many different physiological pathways show modified expression in domestic and GH transgenic strains relative to wild-type, but effects are strongly correlated. Genes specifically involved in growth regulation (IGF1, GHR, IGF-II, THR) are also concordantly regulated in domestic and transgenic fish, and both strains show elevated levels of circulating IGF1. Muscle expression of GH in nontransgenic strains was found to be elevated in domesticated fish relative to wild type, providing a possible mechanism for growth enhancement. These data have implications for genetic improvement of existing domesticated species and risk assessment and regulation of emerging transgenic strains.

When the biochemical characteristics of coho salmon SHBG (csSHBG) plasma were examined, two different steroid-binding profiles were obtained corresponding to recombinant csSHBG-alpha and csSHBG-beta. These SHBG paralogs share only 24% sequence identity, and this explains their unique steroid-binding properties. Both proteins bind testosterone, but csSHBG-alpha also binds androstenedione (Kd = 2.8 nm) and ethinylestradiol with high affinity, whereas csSHBG-beta binds estradiol (Kd = 0.8 nm) preferentially. When analyzed by gel filtration, csSHBG-alpha displays the properties of a 153-kDa homodimer, whereas csSHBG-beta elutes as a 68-kDa monomer. The unique steroid-binding properties of csSHBG-alpha and csSHBG-beta allowed us to develop an assay for their measurements in immature (pre-smolt) and mature coho salmon blood. Plasma csSHBG-alpha levels were 3- to 4-fold higher than those of csSHBG-beta irrespective of developmental stage or sex and correlate with each other. The major site of csSHBG-alpha expression in pre-smolts and mature fish is the liver, but low levels of csSHBG-alpha mRNA are present in stomach/intestine of mature fish. In pre-smolts, high levels of csSHBG-beta mRNA are present in gills and ovary, whereas csSHBG-beta mRNA is most abundant in muscle and stomach/intestine of mature fish. Based on the differences in csSHBG-alpha and csSHBG-beta plasma levels and their tissue expression profiles, we conclude that gills and/or muscle contribute mainly to plasma SHBG-beta in coho salmon. The assays we have developed will enable studies of how SHBG-alpha/SHBG-beta biosynthesis is regulated throughout the salmonid life cycle and how they influence steroid hormone action in these fish.

Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.

Recent advances in gene technology have been applied to create fast-growing transgenic fish, which are of great commercial interest owing to their potential to shorten production cycles and increase food production. However, there is growing concern and speculation over the impact that escaped growth hormone (GH)-transgenic fish may have on the natural environment. To predict these risks it is crucial to obtain empirical data on the relative fitness of transgenic and non-transgenic fish under nature-like conditions. Using landscaped stream aquaria with live food and predators, we show that the predation mortality of newly hatched GH-transgenic coho salmon fry (Oncorhynchus kisutch) is much higher than in non-transgenic conspecifics, and that this difference is amplified when food abundance decreases. The growth rate of transgenic and non-transgenic fish is similar at high food levels, whereas transgenic fish grow more slowly than non-transgenic fish when food abundance is reduced. Our results suggest that the fitness of young GH-transgenic coho salmon in the wild will be determined by both predation pressure and food availability.