Myomixer is expressed during embryonic and post-larval hyperplasia, muscle regeneration and differentiation of myoblats in rainbow trout (Oncorhynchus mykiss).

In contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as Myomaker or Myomixer. In this work, we identified a unique gene encoding a Myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish Myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts differentiation. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout.

Effect of increased embryonic temperature during developmental windows on survival, morphology and oxygen consumption of rainbow trout (Oncorhynchus mykiss).

Temperature is a crucial environmental factor that influences physiological functions in fishes, and increased temperature during development can shape an organism's phenotype. An active line of inquiry in comparative developmental physiology is whether short-term exposure to thermal changes have lasting phenotypic effects. This is the first study to apply a developmental 3-dimensional critical window experimental design for a vertebrate, using time, temperature, and phenotypic response (i.e., variables measured). Rainbow trout (Oncorhynchus mykiss) are an anadromous species for which resident populations occupy freshwater environments that are likely impacted by variable and rising temperatures, particularly during embryonic development. To assess thermal effects on fish development, we examined trout hatchling phenotypes following rearing in constant temperatures (5, 10, 15 and 17.5 °C) and following exposure to increased temperature above 5 °C during specific developmental windows. Time to 50% hatch, hatchling mass and body length showed general trends of decreasing with increasing constant temperature, and survival was highest in constant 10 °C incubation. Thermally shifting embryos into 17.5 °C during gastrulation and organogenesis reduced survival at hatch compared to 10 °C, and exposure to 15 and 17.5 °C only late in development produced lighter and shorter hatchlings. Oxygen consumption rate (V̇o2) at organogenesis differed between embryos incubated constantly in increased temperature or exposed only during organogenesis, but generally we found limited temperature effects on V̇o2 that may be due to high data variability. Collectively, these results suggest that survival of rainbow trout hatchlings is most sensitive to 17.5 °C exposure during gastrulation and organogenesis, while warm water exposure later in development has greater impacts on morphology. Thus, trait-specific critical windows of thermal sensitivity exist for rainbow trout embryos that alter the hatching phenotype.

Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout.

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction.

Breathing with fins: do the pectoral fins of larval fishes play a respiratory role?

Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O2) partial pressure (Po2) gradients that drive O2 uptake. Following hatch, larval fishes lack a developed gill and the skin is the dominant site of gas transfer, yet few studies have addressed the contribution of convective water flow to cutaneous O2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O2 transfer and thus aid in O2 uptake. In zebrafish (Danio rerio), the frequency of pectoral fin movements increased in response to hypoxia at 4 days postfertilization (dpf), but the response was blunted by 15 dpf, when the gills become the dominant site of O2 uptake, and was absent by 21 dpf. In rainbow trout (Oncorhynchus mykiss), Po2 measured at the skin surface of ventilating larvae was lower when the pectoral fins had been surgically removed, directly demonstrating that fins contribute to convective flow that dissipates cutaneous Po2 boundary layers. Lack of pectoral fins compromised whole animal O2 consumption in trout during hypoxia, but this effect was absent in zebrafish. Overall, our findings support a respiratory role of the pectoral fins in rainbow trout, but their involvement in zebrafish remains equivocal.

A glyphosate-based herbicide induces sub-lethal effects in early life stages and liver cell line of rainbow trout, Oncorhynchus mykiss.

Most pesticides used in agriculture end up in the aquatic environment through runoff and leaching of treated crops. One of the most commonly used herbicides is glyphosate. This compound or its metabolites are frequently detected in surface water in Europe. In the present study, in vivo and in vitro studies were carried out using the early life stages of rainbow trout (Oncorhynchus mykiss) and the cell line RTL-W1 (a liver cell line from rainbow trout) to characterize the toxic effects of glyphosate at environmentally-realistic concentrations. Both studies were performed using the commercial formulation Roundup® GT Max, and technical-grade glyphosate for the in vitro study. Eyed-stage embryos were exposed for 3 weeks to sub-lethal concentrations (0.1 and 1 mg/L) of glyphosate using Roundup. Numerous toxicity endpoints were recorded such as survival, hatching success, larval biometry, developmental abnormalities, swimming activity, genotoxicity (formamidopyrimidine DNA-glycosylase Fpg-modified comet assay), lipid peroxidation (TBARS), protein carbonyls and target gene transcription. Concentrations neither affected embryonic or larval survival nor increased developmental abnormalities. However, a significant decrease was observed in the head size of larvae exposed to 1 mg/L of glyphosate. In addition, a significant increase in mobility was observed for larvae exposed to glyphosate at 0.1 mg/L. TBARS levels were significantly decreased on larvae exposed to 1 mg/L (a.i.), and cat and cox1 genes were differently transcribed from controls. DNA damage was detected by the Fpg-modified comet assay in RTL-W1 cell line exposed to the technical-grade glyphosate and Roundup formulation. The results suggest that chronic exposure to glyphosate, at environmental concentrations, could represent a potential risk for early life stages of fish.

Evaluating the role of fish as surrogates for amphibians in pesticide ecological risk assessment.

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (rs) = 0.88], body weight (rs = 0.86), and length (rs = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages.

Are magnetic and electromagnetic fields of anthropogenic origin potential threats to early life stages of fish?

The number of underwater cables transferring electric current in sea and freshwater environments is constantly increasing. As a result, the risk of negative effects of magnetic fields generated in the vicinity of those cables on fish eggs and larvae is also growing. This is especially the case for species that settle on the bottom for certain periods of time during early development. To study those effects, eggs and larvae of rainbow trout, Oncorhynchus mykiss, were subjected under experimental conditions to a static magnetic field (MF) of 10 m T and a 50 Hz electromagnetic field (EMF) of 1 m T for a period of 36 days (i.e., from eyed egg stage to approximately 26 days post hatching, dph). Neither MF nor EMF had significant effect on embryonic or larval mortality, hatching time, larval growth, or the time of larvae swim-up from the bottom. However, both MF and EMF enhanced the yolk-sac absorption rate. Although it was not related directly to magnetic field effect, it was also shown that larvae with absorbed yolk-sacs by the time of swim-up were less efficient in taking advantage of available food at first feeding (i.e., obtained smaller weight at age). That indicates the importance of processes affecting yolk-sac absorption rate.

Formation of intramuscular connective tissue network in fish: first insight from the rainbow trout (Oncorhynchus mykiss).

The formation of the intramuscular connective tissue was investigated in rainbow trout Oncorhynchus mykiss by combining histological and in situ gene-expression analysis. Laminin, a primary component of basement membranes, surrounded superficial slow and deep fast muscle fibres in O. mykiss as soon as the hatching stage (c. 30 days post fertilization (dpf)). In contrast, type I collagen, the primary fibrillar collagen in muscle of vertebrates, appeared at the surface of individual slow and fast muscle fibres only at c. 90 and 110 dpf, respectively. The deposition of type I collagen in laminin-rich endomysium ensheathing individual muscle fibres correlated with the late appearance of collagen type 1 α 1 chain (col1α1) expressing fibroblasts inside slow and then fast-muscle masses. Double in situ hybridization indicated that coll1α1 expressing muscle resident fibroblasts also expressed collagen type 5 α 2 chain (col5α2) transcripts, showing that these cells are a major cellular source of fibrillar collagens within O. mykiss muscle. At c. 140 dpf, the formation of perimysium-like structure was manifested by the increase of type I collagen deposition around bundles of myofibres concomitantly with the alignment and elongation of some collagen-expressing fibroblasts. Overall, this study shows that the formation of O. mykiss intramuscular connective tissue network is completed only in aged fry when fibroblast-like cells expressing type I and V collagens arise inside of the growing myotome.

Endogenous regulation of 11-deoxycorticosterone (DOC) and corticosteroid receptors (CRs) during rainbow trout early development and the effects of corticosteroids on hatching.

Clear evidence for a physiological role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) and the mineralocorticoid receptor (MR) in fish is still lacking. Efforts to demonstrate an osmoregulatory role for this hormone has so far not been conclusive, while a few scattered studies have indicated a role for DOC in development and reproduction. In this study, we investigate the onset of de novo DOC synthesis in parallel with endogenous corticosteroid receptor mRNA production from fertilization to the swim-up stage in rainbow trout. Whole egg DOC content decreased from fertilization until hatching followed by an increase to pre-fertilization levels just after hatching. Onset of de novo transcription of corticosteroid receptor mRNA's was observed shortly after the midblastula transition; initially glucocorticoid receptor 2 (GR2) followed by MR and then GR1. Non-invasive introduction of DOC or cortisol at fertilization resulted in altered corticosteroid receptor regulation and accelerated hatching date, suggesting a regulatory role in trout ontogenesis of both hormones through MR signaling pathway. The results presented in this study suggest a possible physiological role of the DOC-MR signaling pathway during fish ontogenesis, at fertilization and just after hatching.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

Exposure to an acute hypoxic stimulus during early life affects the expression of glucose metabolism-related genes at first-feeding in trout.

Rainbow trout (Oncorhynchus mykiss) is considered a "glucose-intolerant" species. With the aim of programming trout to improve their metabolic use of dietary carbohydrates, we hypothesised that a hypoxic stimulus applied during embryogenesis could later affect glucose metabolism at the first-feeding stage. An acute hypoxic stimulus (2.5 or 5.0 mg·L-1 O2) was applied for 24 h to non-hatched embryos or early hatched alevins followed by a challenge test with a high carbohydrate diet at first-feeding. The effectiveness of the early hypoxic stimulus was confirmed by the induction of oxygen-sensitive markers such as egln3. At first-feeding, trout previously subjected to the 2.5 mg·L-1 O2 hypoxia displayed a strong induction of glycolytic and glucose transport genes, whereas these glucose metabolism-related genes were affected much less in trout subjected to the less severe (5.0 mg·L-1 O2) hypoxia. Our results demonstrate that an acute hypoxic stimulus during early development can affect glucose metabolism in trout at first-feeding.

Lhx8 interacts with a novel germ cell-specific nuclear factor containing an Nbl1 domain in rainbow trout (Oncorhynchus mykiss).

Lhx8 is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. In this study, we report the characterization of rainbow trout lhx8 genes and identification of a novel germ cell-specific nuclear factor that interacts with Lhx8. Two lhx8 genes, lhx8a and lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Collectively, results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout.

Modifications in the proteome of rainbow trout (Oncorhynchus mykiss) embryo and fry as an effect of triploidy induction.

Two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry, and database searching were used to analyze the effects of triploidization heat shock treatment on protein expression in rainbow trout eyed embryo and fry. After fertilization, the eggs were incubated at 10 °C for 10 min. Half of the eggs were then subjected to heat shock for 10 min submerged in a 28 °C water bath to induce triploidy. The remainder was incubated normally and used as diploid controls. Specimens of eyed embryos and fry were taken on 18 and 76 days post-fertilization, respectively. In the eyed embryo extracts, seven protein spots were significantly changed in abundance between the control and heat-shocked groups and one of these was decreased while the others were increased in the heat shock-treated group. Of the spots that were shown to change in abundance in the eyed embryos with heat shock treatment, two were identified as vitellogenin, while the others were creatine kinase and angiotensin I. In the 2-DE from the fry muscle extraction, 23 spots were significantly changed in abundance between the diploid and triploid groups. Nineteen of these showed a decreased abundance in diploids, while the remaining four spots had an increased abundance. Triploidization caused differential expression of muscle metabolic proteins including triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and beta-enolase. Myosin heavy chain as a structural protein was also found to change in abundance in triploids. The altered expression of both structural and metabolic proteins in triploids was consistent with their increased cell size and lower growth performance.

Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

Cryopreservation of fish sperm offers the practical applications in the selective breeding and biodiversity conservation. However, because of the lack of cryopreservation methods for fish eggs and embryos, maternally inherited cytoplasmic compartments cannot be successfully preserved. We previously developed an alternative method to derive functional eggs and sperm from cryopreserved whole testis by transplanting testicular cells into female and male recipients. However, if target fish had ovaries, the previous method employing male-derived germ cells would be ineffective. Here, we aimed to generate functional gametes from cryopreserved whole ovaries by transplanting ovarian germ cells into peritoneal cavity of sterile hatchlings. Cryopreservation conditions for rainbow trout ovaries (1.0 M DMSO, 0.1 M trehalose, and 10% egg yolk) were optimized by testing several different cryoprotective agents. Ovarian germ cells from thawed ovaries were intraperitoneally transplanted into allogeneic triploid hatchlings. Transplanted germ cells migrated toward and were incorporated into recipient gonads, where they underwent gametogenesis. Transplantation efficiency of ovarian germ cells remained stable after cryopreservation period up to 1185 days. Although all triploid recipients that did not undergo transplantation were functionally sterile, 5 of 25 female recipients and 7 of 25 male recipients reached sexual maturity at 2.5 years post-transplantation. Inseminating the resultant eggs and sperm generated viable offspring displaying the donor characteristics of orange body color, green fluorescence, and chromosome numbers. This method is thus a breakthrough tool for the conservation of endangered fish species that are crucial to cryopreserve the genetic resources of female fish.

Androgenetic development of X- and Y-chromosome bearing haploid rainbow trout embryos.

Haploid fish embryos are important in studies regarding role of the recessive traits during early ontogeny. In fish species with the male heterogamety, androgenetic haploid embryos might be also useful tool in studies concerning role of the sex chromosomes during an embryonic development. Morphologically differentiated X and Y chromosomes have been found in a limited number of fish species including rainbow trout (Oncorhynchus mykiss Walbaum 1792). To evaluate role of the sex chromosomes during rainbow trout embryonic development, survival of the androgenetic haploids in the presence of X or Y sex chromosomes has been examined. Androgenetic haploid rainbow trout were produced by fertilization of X-irradiated eggs with spermatozoa derived from the normal males (XY) and neomales, that is, sex-reversed females (XX) to produce X- and Y-bearing haploids, and all X-bearing haploids, respectively. Survival rates of the androgenetic progenies of normal males and neomales examined during embryogenesis and at hatching did not differ significantly. However, all haploids died within next few days after hatching. Cytogenetic analysis of the androgenetic embryos confirmed their haploid status. Moreover, apart from the intact paternal chromosomes, residues of the irradiated maternal chromosomes observed as chromosome fragments were identified in some of the haploids. Provided results suggested that rainbow trout X and Y chromosomes despite morphological and genetic differences are at the early stage of differentiation and still share genetic information responsible for the proper embryonic development.

Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions.

Effects of triploidy induction on physiological and immunological characteristics of rainbow trout (Oncorhynchus mykiss) at early developmental stages (fertilized eggs, eyed eggs and fry).

The aim of this study was to compare effects of triploidy induction on basal physiological and immunological characteristics in rainbow trout at three developmental stages including fertilized eggs, eyed eggs and fry. Eggs and milt were taken from eight females and six males. The gametes were pooled to minimize the individual differences. After insemination, the eggs were incubated at 10°C for 10min. Half of the fertilized eggs were then subjected to heat shock for 10min submerged in a 28°C water bath to induce triploidy. The remainder were incubated normally and used as diploid controls. Three batches of eggs were randomly selected from each group and were incubated at 10-11°C under the same environmental conditions in hatchery troughs until the fry stage. The first-feeding offspring were also reared under the same environmental and nutritional conditions for 38 days. Triplicate samples of 30 eggs (10 eggs per trough) from each group were selected 1.5h post-fertilization and at the eyed stage. Based on red blood cell analysis, nine diploid and nine triploid fish were also selected for study. The triploidy induction success rate was 87.1%. While diploid fish had greater body weights than those in the heat-shock treatment group, weight gain (WG%) was not different between the fry of the diploid and heat-shock treatment groups. Of thyroid hormones measured, 3,5,3'-triiodo-l-thyronine (T3) was less (P<0.05) in eyed eggs of the heat-shock treatment group, but thyroxine (T4) was greater in fry of the heat-shock treatment group as compared to those that were diploid. Cortisol concentration was greater in fry of the heat-shock treatment group as compared to those that were diploid suggesting that fry in the triploid state may be more susceptible to stressors. Concentrations of immune variables (lysozyme, ACH50, albumin, IgM, total protein, globulin and complement) were either comparable or greater in fry of the heat-shock treatment group suggesting that the immune system is not impaired in fish as a result of triploidy induction.

Glucose metabolism ontogenesis in rainbow trout (Oncorhynchus mykiss) in the light of the recently sequenced genome: new tools for intermediary metabolism programming.

The rainbow trout (Oncorhynchus mykiss), a carnivorous fish species, displays a 'glucose-intolerant' phenotype when fed a high-carbohydrate diet. The importance of carbohydrate metabolism during embryogenesis and the timing of establishing this later phenotype are currently unclear. In addition, the mechanisms underlying the poor ability of carnivorous fish to use dietary carbohydrates as a major energy substrate are not well understood. It has recently been shown in trout that duplicated genes involved in glucose metabolism may participate in establishing the glucose-intolerant phenotype. The aim of this study was therefore to provide new understanding of glucose metabolism during ontogenesis and nutritional transition, taking into consideration the complexity of the trout genome. Trout were sampled at several stages of development from fertilization to hatching, and alevins were then fed a non-carbohydrate or a high-carbohydrate diet during first feeding. mRNA levels of all glucose metabolism-related genes increased in embryos during the setting up of the primitive liver. After the first meal, genes rapidly displayed expression patterns equivalent to those observed in the livers of juveniles. g6pcb2.a (a glucose 6-phosphatase-encoding gene) was up-regulated in alevins fed a high-carbohydrate diet, mimicking the expression pattern of gck genes. The g6pcb2.a gene may contribute to the non-inhibition of the last step of gluconeogenesis and thus to establishing the glucose-intolerant phenotype in trout fed a high-carbohydrate diet as early as first feeding. This information is crucial for nutritional programming investigations as it suggests that first feeding would be too late to programme glucose metabolism in the long term.

Retene causes multifunctional transcriptomic changes in the heart of rainbow trout (Oncorhynchus mykiss) embryos.

Fish are particularly sensitive to aryl hydrocarbon receptor (AhR)-mediated developmental toxicity. The molecular mechanisms behind these adverse effects have remained largely unresolved in salmonids, and for AhR-agonistic polycyclic aromatic hydrocarbons (PAHs). This study explored the cardiac transcriptome of rainbow trout (Oncorhynchus mykiss) eleuteroembryos exposed to retene, an AhR-agonistic PAH. The embryos were exposed to retene (nominal concentration 32 µg/L) and control, their hearts were collected before, at and after the onset of the visible signs of developmental toxicity, and transcriptomic changes were studied by microarray analysis. Retene up- or down-regulated 122 genes. The largest Gene Ontology groups were signal transduction, transcription, apoptosis, cell growth, cytoskeleton, cell adhesion/mobility, cardiovascular development, xenobiotic metabolism, protein metabolism, lipid metabolism and transport, and amino acid metabolism. Together these findings suggest that retene affects multiple signaling cascades in the heart of rainbow trout embryos, and potentially disturbs processes related to cardiovascular development and function.

Stressor timing, not cortisol, is an important embryo viability determinant in female rainbow trout Oncorhynchus mykiss.

The objective of this study was to determine whether the timing of elevated cortisol levels in adult female rainbow trout Oncorhynchus mykiss, achieved by silastic implants within the body cavity, had an effect on embryo viability. Cortisol-implanted fish experienced a significant increase in circulating levels of plasma cortisol compared with sham-implanted fish, and plasma cortisol remained elevated relative to sham-implanted fish for 4 months in each of the three treatment groups (0-4, 4-8 and 8-12 months). There were no significant differences in embryo viability, egg diameters or plasma 17ß-oestradiol levels between the cortisol and sham-implanted treatments in any of the groups. There was a significant difference in the number of subfertile females (<80% embryo viability) when the three treatments were compared. The majority of the females (75%) implanted immediately postspawn (0-4 months) produced subfertile eggs, which was significantly higher than those treated 4-8 (33%) or 8-12 (17%) months postspawn. These results imply that silastic implantation can affect oocyte development, independent of plasma cortisol levels, leading to a reduction in embryo viability.