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

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

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

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

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

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

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

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

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

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

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

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

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

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

Significant differences in maternal carotenoid provisioning and effects on offspring fitness in Chinook salmon colour morphs.

In oviparous species, maternal carotenoid provisioning can deliver diverse fitness benefits to offspring via increased survival, growth and immune function. Despite demonstrated advantages of carotenoids, large intra- and interspecific variation in carotenoid utilization exists, suggesting trade-offs associated with carotenoids. In Chinook salmon (Oncorhynchus tshawytscha), extreme variation in carotenoid utilization delineates two colour morphs (red and white) that differ genetically in their ability to deposit carotenoids into tissues. Here, we take advantage of this natural variation to examine how large differences in maternal carotenoid provisioning influence offspring fitness. Using a full factorial breeding design crossing morphs and common-garden rearing, we measured differences in a suite of fitness-related traits, including survival, growth, viral susceptibility and host response, in offspring of red (carotenoid-rich eggs) and white (carotenoid-poor eggs) females. Eggs of red females had significantly higher carotenoid content than those of white females (6× more); however, this did not translate into measurable differences in offspring fitness. Given that white Chinook salmon may have evolved to counteract their maternal carotenoid deficiency, we also examined the relationship between egg carotenoid content and offspring fitness within each morph separately. Egg carotenoids only had a positive effect within the red morph on survival to eyed-egg (earliest measured trait), but not within the white morph. Although previous work shows that white females benefit from reduced egg predation, our study also supports a hypothesis that white Chinook salmon have evolved additional mechanisms to improve egg survival despite low carotenoids, providing novel insight into evolutionary mechanisms that maintain this stable polymorphism.

Growth hormone transgenesis in coho salmon disrupts muscle immune function impacting cross-talk with growth systems.

Suppression of growth during infection may aid resource allocation towards effective immune function. Past work supporting this hypothesis in salmonid fish revealed an immune-responsive regulation of the insulin-like growth factor (IGF) system - an endocrine pathway downstream of growth hormone (GH). Skeletal muscle is the main target for growth and energetic storage in fish, yet little is known about how its growth is regulated during an immune response. We addressed this knowledge gap by characterising muscle immune responses in size-matched coho salmon (Oncorhynchus kisutch) achieving different growth rates. We compared a wild-type strain with two GH transgenic groups from the same genetic background achieving either maximal or suppressed growth - a design separating GH's direct effects from its influence on growth rate and nutritional state. Fish were sampled 30 h post-injection with phosphate-buffered saline (control) or mimics of bacterial or viral infection. We quantified mRNA expression levels for genes from the GH, GH receptor, IGF hormone, IGF1 receptor and IGF-binding protein families, along with immune genes involved in inflammatory or antiviral responses and muscle growth status marker genes. We demonstrate dampened immune function in GH transgenics compared with wild-type. The muscle of GH transgenics achieving rapid growth showed no detectable antiviral response, coupled with evidence of a constitutive inflammatory state. GH and IGF system gene expression was strongly altered by GH transgenesis and fast growth, both for baseline expression and responses to immune stimulation. Thus, GH transgenesis strongly disrupts muscle immune status and normal GH and IGF system expression responses to immune stimulation.

Effect of growth hormone overexpression on gastric evacuation rate in coho salmon.

Growth hormone (GH) transgenic (T) coho salmon consistently show remarkably enhanced growth associated with increased appetite and food consumption compared to non-transgenic wild-type (NT) coho salmon. To improve understanding of the mechanism by which GH overexpression mediates food intake and digestion in T fish, feed intake and gastric evacuation rate (over 7 days) were measured in size-matched T and NT coho salmon. T fish displayed greatly enhanced feed intake levels (~ 2.5-fold), and more than 3-fold increase in gastric evacuation rates relative to NT coho salmon. Despite the differences in feed intake, no differences were noted in the time taken from first ingestion of food to stomach evacuation between genotypes. These results indicate that enhanced feed intake is coupled with an overall increased processing rate to enhance energy intake by T fish. To further investigate the molecular basis of these responses, we examined the messenger RNA (mRNA) levels of several genes in appetite- and gastric-regulation pathways (Agrp1, Bbs, Cart, Cck, Glp, Ghrelin, Grp, Leptin, Mc4r, Npy, and Pomc) by qPCR analyses in the brain (hypothalamus, preoptic area) and pituitary, and in peripheral tissues associated with digestion (liver, stomach, intestine, and adipose tissue). Significant increases in mRNA levels were found for Agrp1 in the preoptic area (POA) of the brain, and Grp and Pomc in pituitary for T coho salmon relative to NT. Mch and Npy showed significantly lower mRNA levels than NT fish in all brain tissues examined across all time-points after feeding. Mc4r and Cart for T showed significantly lower mRNA levels than NT in the POA and hypothalamus, respectively. In the case of peripheral tissues, T fish had lower mRNA levels of Glp and Leptin than NT fish in the intestine and adipose tissue, respectively. Grp, Cck, Bbs, Glp, and Leptin in stomach, adipose tissue, and/or intestine showed significant differences across the time-points after feeding, but Ghrelin showed no significant difference between T and NT fish in all tested tissues.

Predation, metabolic priming and early life-history rearing environment affect the swimming capabilities of growth hormone transgenic rainbow trout.

The period of first feeding, when young salmonid fishes emerge from natal stream beds, is one fraught with predation risk. Experiments conducted in semi-natural stream mesocosms have shown that growth hormone transgenic salmonids are at greater risk of predation than their non-transgenic siblings, due partly to the higher metabolic demands associated with transgenesis, which force risky foraging behaviours. This raises questions as to whether there are differences in the swim-performance of transgenic and non-transgenic fishes surviving predation experiments. We tested this hypothesis in wild-origin rainbow trout (Oncorhynchus mykiss) that were reared from first feeding in semi-natural stream mesocosms characterized by complex hydrodynamics, the presence of predators and oligotrophic conditions. Using an open-flume raceway, we swam fish and measured their capacity for burst-swimming against a sustained flow. We found a significant genotype effect on burst-performance, with transgenic fish sustaining performance longer than their wild-type siblings, both in predator and predator-free stream segments. Importantly, this effect occurred before differences in growth were discernable. We also found that mesocosm-reared fish had greater burst-performance than fish reared in the controlled hatchery environment, despite the latter being unexposed to predators and having abundant food. Our results suggest a potential interaction between predation and metabolic priming, which leads to greater burst capacity in transgenic trout.

Red and white Chinook salmon: genetic divergence and mate choice.

Chinook salmon (Oncorhynchus tshawytscha) exhibit extreme differences in coloration of skin, eggs and flesh due to genetic polymorphisms affecting carotenoid deposition, where colour can range from white to bright red. A sympatric population of red and white Chinook salmon occurs in the Quesnel River, British Columbia, where frequencies of each phenotype are relatively equal. In our study, we examined evolutionary mechanisms responsible for the maintenance of the morphs, where we first tested whether morphs were reproductively isolated using microsatellite genotyping, and second, using breeding trials in seminatural spawning channels, we tested whether colour assortative mate choice could be operating to maintain the polymorphism in nature. Next, given extreme difference in carotenoid assimilation and the importance of carotenoids to immune function, we examined mate choice and selection between colour morphs at immune genes (major histocompatibility complex genes: MHC I-A1 and MHC II-B1). In our study, red and white individuals were found to interbreed, and under seminatural conditions, some degree of colour assortative mate choice (71% of matings) was observed. We found significant genetic differences at both MHC genes between morphs, but no evidence of MHC II-B1-based mate choice. White individuals were more heterozygous at MHC II-B1 compared with red individuals, and morphs showed significant allele frequency differences at MHC I-A1. Although colour assortative mate choice is likely not a primary mechanism maintaining the polymorphisms in the population, our results suggest that selection is operating differentially at immune genes in red and white Chinook salmon, possibly due to differences in carotenoid utilization.

Regulation of feeding behavior and food intake by appetite-regulating peptides in wild-type and growth hormone-transgenic coho salmon.

Survival, competition, growth and reproductive success in fishes are highly dependent on food intake, food availability and feeding behavior and are all influenced by a complex set of metabolic and neuroendocrine mechanisms. Overexpression of growth hormone (GH) in transgenic fish can result in greatly enhanced growth rates, feed conversion, feeding motivation and food intake. The objectives of this study were to compare seasonal feeding behavior of non-transgenic wild-type (NT) and GH-transgenic (T) coho salmon (Oncorhynchus kisutch), and to examine the effects of intraperitoneal injections of the appetite-regulating peptides cholecystokinin (CCK-8), bombesin (BBS), glucagon-like peptide-1 (GLP-1), and alpha-melanocyte-stimulating hormone (α-MSH) on feeding behavior. T salmon fed consistently across all seasons, whereas NT dramatically reduced their food intake in winter, indicating the seasonal regulation of appetite can be altered by overexpression of GH in T fish. Intraperitoneal injections of CCK-8 and BBS caused a significant and rapid decrease in food intake for both genotypes. Treatment with either GLP-1 or α-MSH resulted in a significant suppression of food intake for NT but had no effect in T coho salmon. The differential response of T and NT fish to α-MSH is consistent with the melanocortin-4 receptor system being a significant pathway by which GH acts to stimulate appetite. Taken together, these results suggest that chronically increased levels of GH alter feeding regulatory pathways to different extents for individual peptides, and that altered feeding behavior in transgenic coho salmon may arise, in part, from changes in sensitivity to peripheral appetite-regulating signals.

Rapid growth accelerates telomere attrition in a transgenic fish.

BACKGROUND: Individuals rarely grow as fast as their physiologies permit despite the fitness advantages of being large. One reason may be that rapid growth is costly, resulting for example in somatic damage. The chromosomal ends, the telomeres, are particularly vulnerable to such damage, and telomere attrition thus influences the rate of ageing. Here, we used a transgenic salmon model with an artificially increased growth rate to test the hypothesis that rapid growth is traded off against the ability to maintain somatic health, assessed as telomere attrition. RESULTS: We found substantial telomere attrition in transgenic fish, while maternal half-sibs growing at a lower, wild-type rate seemed better able to maintain the length of their telomeres during the same time period. CONCLUSIONS: Our results are consistent with a trade-off between rapid growth and somatic (telomere) maintenance in growth-manipulated fish. Since telomere erosion reflects cellular ageing, our findings also support theories of ageing postulating that unrepaired somatic damage is associated with senescence.

RNAseq analysis of fast skeletal muscle in restriction-fed transgenic coho salmon (Oncorhynchus kisutch): an experimental model uncoupling the growth hormone and nutritional signals regulating growth.

BACKGROUND: Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon. RESULTS: Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish. CONCLUSIONS: TR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling.

Selection for upper thermal tolerance in rainbow trout (Oncorhynchus mykiss Walbaum).

Rainbow trout (Oncorhynchus mykiss Walbaum) in southern Western Australia have undergone passive selection for over 19 generations to survive high water temperatures. Based on the conceptual model of 'oxygen- and capacity-limited thermal tolerance', we measured critical thermal maximum (CTmax), maximum heart rate (fH,max) and aerobic scope to test the hypothesis that these rainbow trout can maintain aerobic scope at high temperatures through a robust cardiac performance supporting oxygen delivery. Across five family groups CTmax averaged 29.0±0.02°C. Aerobic scope was maximized at 15.8±0.3°C (Topt), while the upper pejus temperature (Tpej, set at 90% of maximum aerobic scope) was 19.9±0.3°C. Although aerobic scope decreased at temperatures above Topt, the value at 25°C remained well over 40% of the maximum. Furthermore, pharmacologically stimulated fH,max increased with temperature, reaching a peak value between 23.5±0.4 and 24.0±0.4°C (Tmax) for three family groups. The Arrhenius breakpoint temperature (TAB) for fH,max was 20.3±0.3 to 20.7±0.4°C, while the average Q10 breakpoint temperature (TQB, when the incremental Q10<1.6) for fH,max was 21.6±0.2 to 22.0±0.4°C. Collectively, fH,max progressively became less temperature dependent beyond 20°C (TAB and TQB), which coincides with the upper Tpej for aerobic scope. Although upper thermal performance indices for both aerobic scope and fH,max were compared among family groups in this population, appreciable differences were not evident. Compared with other populations of rainbow trout, the present assessment is consistent with the prediction that this strain has undergone selection and shows the ability to tolerate higher water temperatures.

Muscle fibre size optimisation provides flexibility for energy budgeting in calorie-restricted coho salmon transgenic for growth hormone.

Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (GH) show substantially faster growth than wild-type (WT) fish. We fed GH-transgenic salmon either to satiation (1 year; TF) or the same smaller ration of wild-type fish (2 years; TR), resulting in groups matched for body size to WT salmon. The myotomes of TF and WT fish had the same number and size distribution of muscle fibres, indicating a twofold higher rate of fibre recruitment in the GH transgenics. Unexpectedly, calorie restriction was found to decrease the rate of fibre production in transgenics, resulting in a 20% increase in average fibre size and reduced costs of ionic homeostasis. Genes for myotube formation were downregulated in TR relative to TF and WT fish. We suggest that muscle fibre size optimisation allows the reallocation of energy from maintenance to locomotion, explaining the observation that calorie-restricted transgenics grow at the same rate as WT fish whilst exhibiting markedly higher foraging activity.

Growth and endocrine effect of growth hormone transgene dosage in diploid and triploid coho salmon.

Growth-hormone transgene dosage, polyploidy, and parental effects on growth and endocrine responses have been assessed in coho salmon. Diploid fry with one or two transgene doses grew equally, whereas later-stage juvenile homozygotes grew faster than hemizygotes. In contrast, homozygotes and hemizygotes grew equally after smoltification, both in sea water and fresh water. Triploid transgenic salmon showed impaired growth which could not be fully overcome with additional transgene copies. Levels of muscle GH mRNA were elevated in two vs. one transgene dose diploids, but in triploids, a dosage effect was observed in muscle but not for animals carrying three transgene doses. IGF-I mRNA levels were elevated in transgenic vs. non-transgenic animals, but a dosage effect was not observed. Diploids and triploids with two transgenes had higher plasma GH levels than one-dose animals, but three-dose triploids showed no further elevation. Circulating IGF-I levels also showed a dosage effect in diploids, but not among any transgene doses in triploids. The present study reveals complex interactions among transgene dosage, maternal effects, developmental stage, and ploidy on growth and endocrine parameters in GH transgenic coho salmon. Specifically, GH transgenes do not always express nor have effects on growth that are directly correlated with the number of transgenes. Further, the reduced growth rate seen in triploid transgenic animals could not be fully overcome by increasing transgene dosage. The findings have relevance for understanding growth physiology, transgene function, and for environmental risk assessments that require understanding phenotypes of hemizygous vs. homozygous transgenic animals in populations.

Glucose metabolic gene expression in growth hormone transgenic coho salmon.

Salmonids are generally known to be glucose intolerant. However, previous studies have shown that growth hormone (GH) transgenic coho salmon display modified nutritional regulation of glycolysis and lipogenesis compared to non-transgenic fish, suggesting the potential for better use of glucose in GH transgenic fish. To examine this in detail, GH transgenic and non-transgenic coho salmon were subjected to glucose tolerance test and subsequent metabolic assessments. After intra-peritoneal injection of 250mg/kg glucose, we analysed post-injection kinetics of glycaemia and expression of several key target genes highly involved in glucose homeostasis in muscle and liver tissues. Our data show no significant differences in plasma glucose levels during peak hyperglycaemia (3-6h after injection), demonstrating a similar glucose tolerance between transgenic and non transgenic. However, and unrelated to the hyperglycaemic episode, GH transgenic fish return to a slightly lower basal glycaemia values 24h after injection. Correspondingly, GH transgenic fish exhibited higher mRNA levels of glucokinase (GK) and glucose-6-phosphate dehydrogenase (G6PDH) in liver, and glucose transporter (GLUT4) in muscle. These data suggest that these metabolic actors may be involved in different glucose use in GH transgenic fish, which would be expected to influence the glucose challenge response. Overall, our data demonstrate that GH transgenic coho salmon may be a pertinent animal model for further study of glucose metabolism in carnivorous fish.

Influence of developmental stage and genotype on liver mRNA levels among wild, domesticated, and hybrid rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Release of domesticated strains of fish into nature may pose a threat to wild populations with respect to their evolved genetic structure and fitness. Understanding alterations that have occurred in both physiology and genetics as a consequence of domestication can assist in evaluating the risks posed by introgression of domesticated genomes into wild genetic backgrounds, however the molecular causes of these consequences are currently poorly defined. The present study has examined levels of mRNA in fast-growing pure domesticated (D), slow-growing age-matched pure wild (Wa), slow-growing size-matched pure wild (Ws), and first generation hybrid cross (W/D) rainbow trout (Oncorhynchus mykiss) to investigate the influence of genotype (domesticated vs. wild, and their interactions in hybrids) and developmental stage (age- or size-matched animals) on genetic responses (i.e. dominant vs. recessive) and specific physiological pathways. RESULTS: Highly significant differences in mRNA levels were found between domesticated and wild-type rainbow trout genotypes (321 mRNAs), with many mRNAs in the wild-domesticated hybrid progeny showing intermediate levels. Differences were also found between age-matched and size-matched wild-type trout groups (64 mRNAs), with unique mRNA differences for each of the wild-type groups when compared to domesticated trout (Wa: 114 mRNAs, Ws: 88 mRNAs), illustrating an influence of fish developmental stage affecting findings when used as comparator groups to other genotypes. Analysis of differentially expressed mRNAs (found for both wild-type trout to domesticated comparisons) among the genotypes indicates that 34.8% are regulated consistent with an additive genetic model, whereas 39.1% and 26.1% show a recessive or dominant mode of regulation, respectively. These molecular data are largely consistent with phenotypic data (growth and behavioural assessments) assessed in domesticated and wild trout strains. CONCLUSIONS: The present molecular data are concordant with domestication having clearly altered rainbow trout genomes and consequent phenotype from that of native wild populations. Although mainly additive responses were noted in hybrid progeny, the prevalence of dominant and non-additive responses reveals that introgression of domesticated and wild genotypes alters the type of genetic control of mRNA levels from that of wild-type, which may lead to disruption of gene regulation systems important for developing phenotypes for optimal fitness in nature. A clear influence of both fish age and size (developmental stage) on mRNA levels was also noted in this study, which highlights the importance of examining multiple control samples to provide a comprehensive understanding of changes observed between strains possessing differences in growth rate.