Rationed and satiated growth hormone transgenic Coho Salmon (Oncorhynchus kisutch) show tissue specific differences in energy stores.

Growth hormone transgenic coho salmon experience increased growth rates, driven primarily through elevated feed intake and feed conversion. However, neuropeptides that signal appetite stimulation have been shown to exhibit variable responses across fed states, suggesting a more complex system mediating growth in these fish. Studies have proposed that growth hormone may have a modulatory role on the energy reserves of fish, possibly through AMP-activated protein kinase (AMPK) activation. AMPK, an energy sensor in cells, has previously been shown to be upregulated in growth hormone transgenic salmon when compared to wild type, however, whether this effect is seen across fed states is unknown. Here, we tested the hypothesis that growth hormone induces an energetic deficit in metabolic tissues, leading to constitutive AMPK activation in growth hormone transgenic salmon. This study compared AMPK activity, ATP, and glycogen, of the liver, heart, and muscle of wild-type, and growth hormone transgenic salmon either fed to satiation or a wild-type ration. The results suggest that white muscle ATP levels in growth hormone salmon are elevated in satiation and rationed conditions. In the liver, growth hormone transgenic salmon fed a rationed wild-type diet experience reductions in ATP level and glycogen. In none of the tissues examined, did AMPK activity change. Taken together, these results indicate that growth hormone transgenic salmon experience metabolic duress when not fed to satiation.

Carotenoid pigmentation in salmon: variation in expression at BCO2-l locus controls a key fitness trait affecting red coloration.

Carotenoids are primarily responsible for the characteristic red flesh coloration of salmon. Flesh coloration is an economically and evolutionarily significant trait that varies inter- and intra-specifically, yet the underlying genetic mechanism is unknown. Chinook salmon (Oncorhynchus tshawytscha) represents an ideal system to study carotenoid variation as, unlike other salmonids, they exhibit extreme differences in carotenoid utilization due to genetic polymorphisms. Here, we crossed populations of Chinook salmon with fixed differences in flesh coloration (red versus white) for a genome-wide association study to identify loci associated with pigmentation. Here, the beta-carotene oxygenase 2-like (BCO2-l) gene was significantly associated with flesh colour, with the most significant single nucleotide polymorphism explaining 66% of the variation in colour. BCO2 gene disruption is linked to carotenoid accumulation in other taxa, therefore we hypothesize that an ancestral mutation partially disrupting BCO2-l activity (i.e. hypomorphic mutation) allowed the deposition and accumulation of carotenoids within Salmonidae. Indeed, we found elevated transcript levels of BCO2-l in white Chinook salmon relative to red. The long-standing mystery of why salmon are red, while no other fishes are, is thus probably explained by a hypomorphic mutation in the proto-salmonid at the time of divergence of red-fleshed salmonid genera (approx. 30 Ma).

Assessing the capacity for compensatory growth in growth-hormone transgenic coho salmon Oncorhynchus kisutch.

The effect of feed cycling (consisting of periods of starvation followed by periods of refeeding to satiation) on compensatory growth was evaluated in growth hormone transgenic and non-transgenic wild-type coho salmon Oncorhynchus kisutch. The specific growth rate (GSR ) of feed-restricted non-transgenic O. kisutch was not significantly different from the GSR of fully-fed non-transgenic O. kisutch during two refeeding periods, whereas the GSR of feed-restricted transgenic O. kisutch was significantly higher in relation to the GSR of fully-fed transgenic O. kisutch during the second refeeding period, but not during the first, indicating that growth compensation mechanisms are different between non-transgenic and growth-hormone (GH)-transgenic O. kisutch and may depend on life history (i.e. previous starvation). Despite the non-significant growth rate compensation in non-transgenic O. kisutch, these fish showed a level of body mass catch-up growth not displayed by transgenic O. kisutch.

Behaviour of growth hormone transgenic coho salmon Oncorhynchus kisutch in marine mesocosms assessed by acoustic tag telemetry.

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.

Gene--environment interactions influence feeding and anti-predator behavior in wild and transgenic coho salmon.

Environmental conditions are known to affect phenotypic development in many organisms, making the characteristics of an animal reared under one set of conditions not always representative of animals reared under a different set of conditions. Previous results show that such plasticity can also affect the phenotypes and ecological interactions of different genotypes, including animals anthropogenically generated by genetic modification. To understand how plastic development can affect behavior in animals of different genotypes, we examined the feeding and risk-taking behavior in growth-enhanced transgenic coho salmon (with two- to threefold enhanced daily growth rates compared to wild type) under a range of conditions. When compared to wild-type siblings, we found clear effects of the rearing environment on feeding and risk-taking in transgenic animals and noted that in some cases, this environmental effect was stronger than the effects of the genetic modification. Generally, transgenic fish, regardless of rearing conditions, behaved similar to wild-type fish reared under natural-like conditions. Instead, the more unusual phenotype was associated with wild-type fish reared under hatchery conditions, which possessed an extreme risk averse phenotype compared to the same strain reared in naturalized conditions. Thus, the relative performance of genotypes from one environment (e.g., laboratory) may not always accurately reflect ecological interactions as would occur in a different environment (e.g., nature). Further, when assessing risks of genetically modified organisms, it is important to understand how the environment affects phenotypic development, which in turn may variably influence consequences to ecosystem components across different conditions found in the complexity of nature.

Accuracy of nonmolecular identification of growth-hormone- transgenic coho salmon after simulated escape.

Concerns with transgenic animals include the potential ecological risks associated with release or escape to the natural environment, and a critical requirement for assessment of ecological effects is the ability to distinguish transgenic animals from wild type. Here, we explore geometric morphometrics (GeoM) and human expertise to distinguish growth-hormone-transgenic coho salmon (Oncorhynchus kisutch) specimens from wild type. First, we simulated an escape of 3-month-old hatchery-reared wild-type and transgenic fish to an artificial stream, and recaptured them at the time of seaward migration at an age of 13 months. Second, we reared fish in the stream from first-feeding fry until an age of 13 months, thereby simulating fish arising from a successful spawn in the wild of an escaped hatchery-reared transgenic fish. All fish were then assessed from 'photographs by visual identification (VID) by local staff and by GeoM based on 13 morphological landmarks. A leave-one-out discriminant analysis of GeoM data had on average 86% (72-100% for individual groups) accuracy in assigning the correct genotypes, whereas the human experts were correct, on average, in only 49% of cases (range of 18-100% for individual fish groups). However, serious errors (i.e., classifying transgenic specimens as wild type) occurred for 7% (GeoM) and 67% (VID) of transgenic fish, and all of these incorrect assignments arose with fish reared in the stream from the first-feeding stage. The results show that we presently lack the skills of visually distinguishing transgenic coho salmon from wild type with a high level of accuracy, but that further development-of GeoM methods could be useful in identifying second-generation,fish from nature as a nonmolecular approach.

Upper thermal tolerance of wild-type, domesticated and growth hormone-transgenic coho salmon Oncorhynchus kisutch.

In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26·9° C, CTmax ) were observed among size-matched wild-type, domesticated, growth hormone (GH)-transgenic fish fed to satiation, and GH-transgenic fish on a ration-restricted diet. Instead, GH-transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17·3 ± 0·1° C, TAB ). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH-transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits.

Effects of domestication and growth hormone transgenesis on mRNA profiles in rainbow trout (Oncorhynchus mykiss).

Growth rate can be genetically modified in many vertebrates by domestication and selection and more recently by transgenesis overexpressing growth factor genes [e.g., growth hormone (GH)]. Although the phenotypic end consequence is similar, it is currently not clear whether the same modifications to physiological pathways are occurring in both genetic processes or to what extent they may interact when combined. To investigate these questions, microarray analysis has been used to assess levels of mRNA in liver of wild-type and growth-modified strains of rainbow trout (Oncorhynchus mykiss). This species has been used as a model because nondomesticated wild strains are available as comparators to assess genetic and physiological changes that have arisen both from domestication and from GH transgenesis. The analysis examined pure wild-type and pure domesticated strains as well as 2 different GH transgenes (with markedly different growth effects) both in pure wild and in wild × domesticated hybrid backgrounds. Liver mRNA showed highly concordant changes (Pearson correlations; r>0.828; P<0.001) in levels in domesticated and GH transgenic fish, relative to wild-type, for both up- and downregulated genes. Furthermore, among domesticated, transgenic, and their hybrid genotypes, a strong correlation (P<0.001) was found between growth rate and the number of genes affected (r=0.761 for downregulated mRNA and r=0.942 for upregulated mRNA) or between growth rate and mRNA levels relative to wild-type (r=0.931 for downregulated mRNA and r=0.928 for upregulated mRNA). One GH transgenic strain was found to affect growth and mRNA levels similar to domestication whereas effects of the other GH transgenic strain were much stronger. For both GH transgenes, a hybrid domesticated×wild background influenced growth rate and mRNA levels to only a small extent relative to the transgenes in a pure wild-type genetic background. Functional analysis found that genes involved in immune function, carbohydrate metabolism, detoxification, transcription regulation, growth regulation, and lipid metabolism were affected in common by domestication and GH transgenesis. The common responses of mRNAs in domesticated and GH transgenic strains is consistent with the GH pathway or its downstream effects being upregulated in domesticated animals during their modification from wild-type growth rates.

Inside the heads of David and Goliath: environmental effects on brain morphology among wild and growth-enhanced coho salmon Oncorhynchus kisutch.

Transgenic and wild-type individual coho salmon Oncorhynchus kisutch were reared in hatchery and near-natural stream conditions and their brain and structure sizes were determined. Animals reared in the hatchery grew larger and developed larger brains, both absolutely and when controlling for body size. In both environments, transgenics developed relatively smaller brains than wild types. Further, the volume of the optic tectum of both genotypes was larger in the hatchery animals and the cerebellum of transgenics was smaller when reared in near-natural streams. Finally, wild types developed a markedly smaller telencephalon under hatchery conditions. It is concluded that, apart from the environment, genetic factors that modulate somatic growth rate also have a strong influence on brain size and structure.

Growth and endocrine effects of recombinant bovine growth hormone treatment in non-transgenic and growth hormone transgenic coho salmon.

To examine the relative growth, endocrine, and gene expression effects of growth hormone (GH) transgenesis vs. GH protein treatment, wild-type non-transgenic and GH transgenic coho salmon were treated with a sustained-release formulation of recombinant bovine GH (bGH; Posilac). Fish size, specific growth rate (SGR), and condition factor (CF) were monitored for 14 weeks, after which endocrine parameters were measured. Transgenic fish had much higher growth, SGR and CF than non-transgenic fish, and bGH injection significantly increased weight and SGR in non-transgenic but not transgenic fish. Plasma salmon GH concentrations decreased with bGH treatment in non-transgenic but not in transgenic fish where levels were similar to controls. Higher GH mRNA levels were detected in transgenic muscle and liver but no differences were observed in GH receptor (GHR) mRNA levels. In non-transgenic pituitary, GH and GHR mRNA levels per mg pituitary decreased with bGH dose to levels seen in transgenic salmon. Plasma IGF-I was elevated with bGH dose only in non-transgenic fish, while transgenic fish maintained an elevated level of IGF-I with or without bGH treatment. A similar trend was seen for liver IGF-I mRNA levels. Thus, bGH treatment increased fish growth and influenced feedback on endocrine parameters in non-transgenic but not in transgenic fish. A lack of further growth stimulation of GH transgenic fish suggests that these fish are experiencing maximal growth stimulation via GH pathways.

Effects of temperature and growth hormone on individual growth trajectories of wild-type and transgenic coho salmon Oncorhynchus kisutch.

In this study, individual growth patterns of wild-type and growth-enhanced coho salmon Oncorhynchus kisutch at 8, 12 and 16 degrees C water temperature were followed. Despite large differences among individuals in growth rates, there was generally little variation in the shape of the growth curves among O. kisutch individuals of both genotypes and at all temperatures. Typically, individuals that were relatively large initially were also relatively large at the end of the growth period. The limitation in variation was more pronounced in the growth-enhanced O. kisutch than in the wild type, where the relative size of some individuals reared at 12 and 8 degrees C changed by the end of the trial. As a warmer temperature seems to decrease the plasticity of growth trajectories in wild-type fish, it is possible that global warming will influence the ability of wild fish to adapt their growth to changing conditions.

Growth hormone transgenesis influences carbohydrate, lipid and protein metabolism capacity for energy production in coho salmon (Oncorhynchus kisutch).

Growth hormone (GH) transgenesis results in increased growth, feed intake and consequent metabolic rates in fish, and alters the utilization of dietary and stored carbohydrates, lipid and protein. However, the manner in which GH transgenesis differentially alters these energy sources in fish has not been well explored. We examined the effects of GH transgenesis and dietary carbohydrate, lipid and protein levels on metabolic enzyme activity in coho salmon (Oncorhynchus kisutch). In white muscle, increased activities of glycolytic enzymes and decreased activities of lipolytic enzymes in transgenic fish indicate a sparing of lipids through the preferential use of carbohydrates for energy production. In liver, transgenic fish showed increased activity of lipid synthesis enzymes and a shift in amino acid metabolism from catabolic to synthetic roles, suggesting a larger emphasis on anabolic pathways in transgenic fish to support accelerated growth. Unlike nontransgenic fish, transgenic fish fed a diet high in carbohydrates maintained growth rates, had increased capacity for lipid synthesis, and increased potential for biosynthetic roles of amino acids. GH transgenesis influences metabolic reactions in coho salmon by emphasizing carbohydrate degradation for energy production and lipid synthesis, and increasing utilization of lipids and proteins for synthetic roles necessary to maintain accelerated growth.

Endocrine effects of growth hormone overexpression in transgenic coho salmon.

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.

Identification of the sex chromosome pair in chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha).

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in the karyotypes of chum salmon (Oncorhynchus keta) and pink salmon (Oncorhynchus gorbuscha). The sex chromosome pair is a small acrocentric chromosome pair in chum salmon and the smallest metacentric chromosome pair in pink salmon. Both of these chromosome pairs are morphologically different from the sex chromosome pairs in chinook salmon (Oncorhynchus tshawytscha) and coho salmon (Oncorhynchus kisutch). The 5S rRNA genes are on multiple chromosome pairs including the sex chromosome pair in chum salmon, but at the centromeres of two autosomal metacentric pairs in pink salmon. The sex chromosome pairs and the chromosomal locations of the 5S rDNA appear to be different in all five of the North American Pacific salmon species and rainbow trout. The implications of these results for evolution of sex chromosomes in salmonids are discussed.

Genetically enhanced growth causes increased mortality in hypoxic environments.

Rapid growth and development are associated with several fitness-related benefits. Yet, organisms usually grow more slowly than their physiological maximum, suggesting that rapid growth may carry costs. Here we use coho salmon (Oncorhynchus kisutch) eggs of wild and transgenic genotypes to test whether rapid growth causes reduced tolerance to low levels of oxygen (hypoxia). Eggs were exposed to four different durations of hypoxia, and survival and growth were recorded until the end of the larval stage. Survival rates decreased with increasing duration of hypoxia, but this decrease was most pronounced for the transgenic group. Larval mass was also negatively affected by hypoxia; however, transgenic genotypes were significantly larger than wild genotypes at the end of the larval stage. Oxygen can be a limiting factor for survival and development in a wide range of organisms, particularly during the egg stage. Thus, the reduced ability of fast-growing genotypes to cope with low oxygen levels identified in the present study may represent a general constraint on evolution of rapid growth across taxa.

Identification of the sex chromosome pair in coho salmon (Oncorhynchus kisutch): lack of conservation of the sex linkage group with chinook salmon (Oncorhynchus tshawytscha).

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in coho salmon (Oncorhynchus kisutch). The sex chromosome pair is morphologically similar to chinook salmon (Oncorhynchus tshawytscha) with the GH-Y localized to the small short arm of the largest subtelocentric chromosome pair. FISH experiments with probes containing sex-linked genes in rainbow trout (Oncorhynchus mykiss) (SCAR163) and chinook salmon (Omy7INRA) showed that the coho sex linkage group is different from chinook and rainbow trout and this was confirmed by segregation analysis for the Omy7INRA locus. The telomeric location of the SEX locus, the presence of shared male-specific markers in coho and chinook salmon, and the lack of conservation of sex-linkage groups suggest that transposition of a small male-specific region may have occurred repeatedly in salmonid fishes of the genus Oncorhynchus.

Evolution of duplicated growth hormone genes in autotetraploid salmonid fishes.

A defining character of the piscine family Salmonidae is autotetraploidy resulting from a genome-doubling event some 25-100 million years ago. Initially, duplicated genes may have undergone concerted evolution and tetrasomic inheritance. Homeologous chromosomes eventually diverged and the resulting reduction in recombination and gene conversion between paralogous genes allowed the re-establishment of disomic inheritance. Among extant salmonine fishes (e.g. salmon, trout, char) the growth hormone (GH) gene is generally represented by two functional paralogs, GH1 and GH2. Sequence analyses of salmonid GH genes from species of subfamilies Coregoninae (whitefish, ciscos) and Salmoninae were used to examine the evolutionary history of the duplicated GH genes. Two divergent GH gene paralogs were also identified in Coregoninae, but they were not assignable to the GH1 and GH2 categories. The average sequence divergence between the coregonine GH genes was more than twofold lower than the corresponding divergence between the salmonine GH1 and GH2. Phylogenetic analysis of the coregonine GH paralogs did not resolve their relationship to the salmonine paralogs. These findings suggest that disomic inheritance of two GH genes was established by different mechanisms in these two subfamilies.

Recent advances in our knowledge of the Myxozoa.

In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis. and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete. Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that: 1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

Identification of the Y chromosome in chinook salmon (Oncorhynchus tshawytscha).

The Y chromosome in chinook salmon, Oncorhynchus tshawytscha, was identified using fluorescence in situ hybridization (FISH) with a probe to a male-specific repetitive sequence isolated from this species. The probe highlights the distal end of the short arm of an acrocentric chromosome with a DAPI-bright interstitial band of variable size. The proximal portion of the short arm of the Y chromosome contains 5S rDNA sequences, which are also found on the short arms of six other acrocentric chromosomes in this species.