Special features of neuroendocrine interactions between stress and reproduction in teleosts.

Stress and reproduction are both essential functions for vertebrate survival, ensuring on one side adaptative responses to environmental changes and potential life threats, and on the other side production of progeny. With more than 25,000 species, teleosts constitute the largest group of extant vertebrates, and exhibit a large diversity of life cycles, environmental conditions and regulatory processes. Interactions between stress and reproduction are a growing concern both for conservation of fish biodiversity in the frame of global changes and for the development of sustainability of aquaculture including fish welfare. In teleosts, as in other vertebrates, adverse effects of stress on reproduction have been largely documented and will be shortly overviewed. Unexpectedly, stress notably via cortisol, may also facilitate reproductive function in some teleost species in relation to their peculiar life cyles and this review will provide some examples. Our review will then mainly address the neuroendocrine axes involved in the control of stress and reproduction, namely the corticotropic and gonadotropic axes, as well as their interactions. After reporting some anatomo-functional specificities of the neuroendocrine systems in teleosts, we will describe the major actors of the corticotropic and gonadotropic axes at the brain-pituitary-peripheral glands (interrenals and gonads) levels, with a special focus on the impact of teleost-specific whole genome duplication (3R) on the number of paralogs and their potential differential functions. We will finally review the current knowledge on the neuroendocrine mechanisms of the various interactions between stress and reproduction at different levels of the two axes in teleosts in a comparative and evolutionary perspective.

Acute Stress and an Electrolyte- Imbalanced Diet, but Not Chronic Hypoxia, Increase Oxidative Stress and Hamper Innate Immune Status in a Rainbow Trout (Oncorhynchus mykiss) Isogenic Line.

In aquaculture, fish may be exposed to sub-optimal rearing conditions, which generate a stress response if full adaptation is not displayed. However, our current knowledge of several coexisting factors that may give rise to a stress response is limited, in particular when both chronic and acute stressors are involved. This study investigated changes in metabolic parameters, oxidative stress and innate immune markers in a rainbow trout (Oncorhynchus mykiss) isogenic line exposed to a combination of dietary (electrolyte-imbalanced diet, DEB 700 mEq Kg-1) and environmental (hypoxia, 4.5 mg O2 L-1) challenges and their respective controls (electrolyte-balanced diet, DEB 200 mEq Kg-1 and normoxia, 7.9 or mg O2 L-1) for 49 days. At the end of this period, fish were sampled or subjected to an acute stressor (2 min of handling/confinement) and then sampled. Feeding trout an electrolyte-imbalanced diet produced a reduction in blood pH, as well as increases in cortisol levels, hepato-somatic index (HSI) and total energy content in the liver. The ratio between the lactate dehydrogenase (LDH) and isocitrate dehydrogenase (IDH) activities decreased in the liver of trout fed the DEB 700 diet, but increased in the heart, suggesting a different modulation of metabolic capacity by the dietary challenge. Several markers of oxidative stress in the liver of trout, mainly related to the glutathione antioxidant system, were altered when fed the electrolyte-imbalanced diet. The dietary challenge was also associated with a decrease in the alternative complement pathway activity (ACH50) in plasma, suggesting an impaired innate immune status in that group. Trout subjected to the acute stressor displayed reduced blood pH values, higher plasma cortisol levels as well as increased levels of metabolic markers associated with oxidative stress in the liver. An interaction between diet and acute stressor was detected for oxidative stress markers in the liver of trout, showing that the chronic electrolyte-imbalance impairs the response of rainbow trout to handling/confinement. However, trout reared under chronic hypoxia only displayed changes in parameters related to energy use in both liver and heart. Taken together, these results suggest that trout displays an adaptative response to chronic hypoxia. Conversely, the dietary challenge profoundly affected fish homeostasis, resulting in an impaired physiological response leading to stress, which then placed constraints on a subsequent acute challenge.

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.

Hypoxia, but not an electrolyte-imbalanced diet, reduces feed intake, growth and oxygen consumption in rainbow trout (Oncorhynchus mykiss).

Oxygen limitation and dietary imbalances are key aspects influencing feed intake (FI) and growth performance in cultured fish. This study investigated the combined effects of hypoxia and dietary electrolyte balance on the growth performance, body composition and nutrient utilization in a rainbow trout (Oncorhynchus mykiss) isogenic line. Fish were fed ad libitum two experimental diets: electrolyte-balanced or -imbalanced diets (DEB 200 or 700 mEq kg-1, respectively) and exposed to normoxia or hypoxia (7.9 or 4.5 mg O2 l-1, respectively) for 42 days. DEB did not affect FI, growth performance or body composition. Nevertheless, hypoxia had a negative impact, reducing FI (6%), growth rate (8%), oxygen consumption (19%), energy (5%) and lipid (42%) contents. Digestible energy intake and heat production were higher in normoxic fish (40% and 23%, respectively), retaining 64% more energy in lipid or protein. Hypoxia reduced the apparent digestibility of dry matter, ash, protein, lipid, carbohydrates and energy. Trout fed DEB 700 diet were energetically less efficient, reflected in higher heat production and energy requirements for maintenance. FI was inhibited by low dissolved oxygen levels, but not by electrolyte-imbalanced diet, in spite of the higher energy requirements for maintenance. This study highlights the importance that dietary-electrolyte content and DO levels have on energy balance and growth performance when fish are fed to satiation.

Regulation of the corticosteroid signalling system in rainbow trout HPI axis during confinement stress.

This study aims to shed light on corticosteroid regulation of stress in teleost fish with focus on the corticosteroid signalling system. The role of the mineralocorticoid-like hormone 11-deoxycorticosterone (DOC) in fish is still enigmatic, as is the function of the mineralocorticoid receptor, MR. Low plasma DOC levels and ubiquitous tissue distribution of MR question the physiological relevance of the mineralocorticoid-axis. Furthermore, the particular purpose of each of the three corticosteroid receptors in fish, the glucocorticoid receptors, GR1 and GR2, and the MR, is still largely unknown. Therefore we investigate the regulation of cortisol and DOC in plasma and mRNA levels of MR, GR1 and GR2 in the HPI-axis tissues (hypothalamus, pituitary and interrenal gland) during a detailed confinement stress time-course. Here we show a sustained up-regulation of plasma DOC levels during a confinement stress time-course. However, the low DOC levels compared to cortisol measured in the plasma do not favour an activity of DOC through MR receptors. Furthermore, we show differential contribution of the CRs in regulation and control of HPI axis activity following confinement stress. Judged by the variation of mRNA levels negative feedback regulation of cortisol release occurs on the level of the pituitary via MR and on the level of the interrenal gland via GR2. Finally, asa significant effect of confinement stress on CR expressions was observed in the pituitary gland, we completed this experiment by demonstrating that corticosteroid receptors (GR1, GR2 and MR) are co-expressed in the ACTH cells located in the adenohypophysis. Overall, these data suggest the involvement of these receptors in the regulation of the HPI axis activity by cortisol.

Genetic variability of environmental sensitivity revealed by phenotypic variation in body weight and (its) correlations to physiological and behavioral traits.

Adaptive phenotypic plasticity is a key component of the ability of organisms to cope with changing environmental conditions. Fish have been shown to exhibit a substantial level of phenotypic plasticity in response to abiotic and biotic factors. In the present study, we investigate the link between environmental sensitivity assessed globally (revealed by phenotypic variation in body weight) and more targeted physiological and behavioral indicators that are generally used to assess the sensitivity of a fish to environmental stressors. We took advantage of original biological material, the rainbow trout isogenic lines, which allowed the disentangling of the genetic and environmental parts of the phenotypic variance. Ten lines were characterized for the changes of body weight variability (weight measurements taken every month during 18 months), the plasma cortisol response to confinement stress (3 challenges) and a set of selected behavioral indicators. This study unambiguously demonstrated the existence of genetic determinism of environmental sensitivity, with some lines being particularly sensitive to environmental fluctuations and others rather insensitive. Correlations between coefficient of variation (CV) for body weight and behavioral and physiological traits were observed. This confirmed that CV for body weight could be used as an indicator of environmental sensitivity. As the relationship between indicators (CV weight, risk-taking, exploration and cortisol) was shown to be likely depending on the nature and intensity of the stressor, the joint use of several indicators should help to investigate the biological complexity of environmental sensitivity.

On the Use of a Simple Physical System Analogy to Study Robustness Features in Animal Sciences.

Environmental perturbations can affect the health, welfare, and fitness of animals. Being able to characterize and phenotype adaptive capacity is therefore of growing scientific concern in animal ecology and in animal production sciences. Terms borrowed from physics are commonly used to describe adaptive responses of animals facing an environmental perturbation, but no quantitative characterization of these responses has been made. Modeling the dynamic responses to an acute challenge was used in this study to facilitate the characterization of adaptive capacity and therefore robustness. A simple model based on a spring and damper was developed to simulate the dynamic responses of animals facing an acute challenge. The parameters characterizing the spring and the damper can be interpreted in terms of stiffness and resistance to the change of the system. The model was tested on physiological and behavioral responses of rainbow trout facing an acute confinement challenge. The model has proven to properly fit the different responses measured in this study and to quantitatively describe the different temporal patterns for each statistical individual in the study. It provides therefore a new way to explicitly describe, analyze and compare responses of individuals facing an acute perturbation. This study suggests that such physical models may be usefully applied to characterize robustness in many other biological systems.

Interaction between the trout mineralocorticoid and glucocorticoid receptors in vitro.

The salmonid corticosteroid receptors (CRs), glucocorticoid receptors 1 and 2 (GR1 and GR2) and the mineralocorticoid receptor (MR) share a high degree of homology with regard to structure, ligand- and DNA response element-binding, and cellular co-localization. Typically, these nuclear hormone receptors homodimerize to confer transcriptional activation of target genes, but a few studies using mammalian receptors suggest some degree of heterodimerization. We observed that the trout MR confers a several fold lower transcriptional activity compared to the trout GRs. This made us question the functional relevance of the MR when this receptor is located in the same cells as the GRs and activated by cortisol. A series of co-transfection experiments using different glucocorticoid response elements (GREs) containing promoter-reporter constructs were carried out to investigate any possible interaction between the piscine CRs. Co-transfection of the GRs with the MR significantly reduced the total transcriptional activity even at low MR levels, suggesting interaction between these receptors. Co-transfection of GR1 or GR2 with the MR did not affect the subcellular localization of the GRs, and the MR-mediated inhibition seemed to be independent of specific activation or inhibition of the MR. Site-directed mutagenesis of the DNA-binding domain and dimerization interface of the MR showed that the inhibition was dependent on DNA binding but not necessarily on dimerization ability. Thus, we suggest that the interaction between MR and the GRs may regulate the cortisol response in cell types where the receptors co-localize and propose a dominant-negative role for the MR in cortisol-mediated transcriptional activity.

A multivariate analysis using physiology and behavior to characterize robustness in two isogenic lines of rainbow trout exposed to a confinement stress.

Robustness is a complex trait difficult to characterize and phenotype. In the present study, two features of robustness in rainbow trout were investigated: sensitivity and resilience to an acute stressor. For that purpose, oxygen consumption, cortisol release, group dispersion and group activity of two isogenic lines of juvenile rainbow trout were followed before and after an environmental challenge. The effect of a 4h confinement protocol (~140kg/m(3)), which is generally considered as a highly stressful challenge, was investigated. Temporal patterns produced by this experiment were analyzed using multivariate statistics on curve characteristics to describe physiological and behavioral adaptive systems for each isogenic line. The two isogenic lines were found to be highly divergent in their corticosteroid reactivity. However, no correlation between physiological and behavioral sensitivity or resilience was observed. Furthermore, the multivariate analysis results indicated two separate and independent fish group coping strategies, i.e. by favoring either behavioral or physiological responses. In addition, considerable intra-line variabilities were observed, suggesting the importance of micro-environment effects on perturbation sensitivities. In this context, cortisol release rate variability was found to be related to the pre-stress social environment, with a strong correlation between pre-stress aggressiveness and cortisol release rate amplitude. Overall, this approach allowed us to extract important characteristics from dynamic data in physiology and behavior to describe components of robustness in two isogenic lines of rainbow trout.

Transcriptional assessment by microarray analysis and large-scale meta-analysis of the metabolic capacity of cardiac and skeletal muscle tissues to cope with reduced nutrient availability in Gilthead Sea Bream (Sparus aurata L.).

The effects of nutrient availability on the transcriptome of cardiac and skeletal muscle tissues were assessed in juvenile gilthead sea bream fed with a standard diet at two feeding levels: (1) full ration size and (2) 70 % satiation followed by a finishing phase at the maintenance ration. Microarray analysis evidenced a characteristic transcriptomic profile for each muscle tissue following changes in oxidative capacity (heart > red skeletal muscle > white skeletal muscle). The transcriptome of heart and secondly that of red skeletal muscle were highly responsive to nutritional changes, whereas that of glycolytic white skeletal muscle showed less ability to respond. The highly expressed and nutritionally regulated genes of heart were mainly related to signal transduction and transcriptional regulation. In contrast, those of white muscle were enriched in gene ontology (GO) terms related to proteolysis and protein ubiquitination. Microarray meta-analysis using the bioinformatic tool Fish and Chips ( http://fishandchips.genouest.org/index.php ) showed the close association of a representative cluster of white skeletal muscle with some of cardiac and red skeletal muscle, and many GO terms related to mitochondrial function appeared to be common links between them. A second round of cluster comparisons revealed that mitochondria-related GOs also linked differentially expressed genes of heart with those of liver from cortisol-treated gilthead sea bream. These results show that mitochondria are among the first responders to environmental and nutritional stress stimuli in gilthead sea bream, and functional phenotyping of this cellular organelle is highly promising to obtain reliable markers of growth performance and well-being in this fish species.

Assessment of the role of cortisol and corticosteroid receptors in epidermal ionocyte development in the medaka (Oryzias latipes) embryos.

Cortisol is a pleiotropic glucocorticoid hormone that acts through the intracellular glucocorticoid receptors (GR). Cortisol affects many important biological functions in mammals, including immune function, behavior, stress, metabolism, growth and organogenesis. In fishes, cortisol has an additional function in the osmoregulatory activity of ionocytes (ICs). Although much progress has been made toward understanding cortisol action at the levels of adult osmoregulatory tissues, the developmental functions of cortisol and its receptors in ICs remain to be clarified. We first analyzed the total contents of both cortisol and corticosteroid receptor mRNAs (GR1, GR2 and MR) during medaka development. Although low levels of cortisol were detected during development of the medaka embryo, maternal GR1, GR2 and MR transcripts were detected at higher levels than zygotic transcripts. We investigated the effect of exogenous cortisol on IC number during medaka embryogenesis. We observed that cortisol treatment induced an earlier expansion of the IC population but did not modify the final IC number. Using functional genomic approaches, we also tested the involvement of GR1, GR2 and mineralocorticoid receptor (MR) in IC development by systematic knock-down with translation-blocking morpholinos. Only GR2 knock-down led to a reduction of the total number of ICs in the epidermis. In addition, a GR2 splice-blocking morpholino did not have any effect on the biogenesis of ICs, underscoring the importance of maternally inherited GR2 mRNAs. We propose that maternal GR2, but not GR1 or MR, is a major pathway in the IC biogenesis in medaka most likely through cortisol activation, and that cortisol exposition fine-tunes their developmental timing. These findings provide a framework for future research on the regulatory functions of corticosteroids in euryhaline fishes and provide medaka as an advantageous model to further elucidate the underlying molecular regulatory mechanisms of IC development.

Characterization of rainbow trout gonad, brain and gill deep cDNA repertoires using a Roche 454-Titanium sequencing approach.

Rainbow trout, Oncorhynchus mykiss, is an important aquaculture species worldwide and, in addition to being of commercial interest, it is also a research model organism of considerable scientific importance. Because of the lack of a whole genome sequence in that species, transcriptomic analyses of this species have often been hindered. Using next-generation sequencing (NGS) technologies, we sought to fill these informational gaps. Here, using Roche 454-Titanium technology, we provide new tissue-specific cDNA repertoires from several rainbow trout tissues. Non-normalized cDNA libraries were constructed from testis, ovary, brain and gill rainbow trout tissue samples, and these different libraries were sequenced in 10 separate half-runs of 454-Titanium. Overall, we produced a total of 3million quality sequences with an average size of 328bp, representing more than 1Gb of expressed sequence information. These sequences have been combined with all publicly available rainbow trout sequences, resulting in a total of 242,187 clusters of putative transcript groups and 22,373 singletons. To identify the predominantly expressed genes in different tissues of interest, we developed a Digital Differential Display (DDD) approach. This approach allowed us to characterize the genes that are predominantly expressed within each tissue of interest. Of these genes, some were already known to be tissue-specific, thereby validating our approach. Many others, however, were novel candidates, demonstrating the usefulness of our strategy and of such tissue-specific resources. This new sequence information, acquired using NGS 454-Titanium technology, deeply enriched our current knowledge of the expressed genes in rainbow trout through the identification of an increased number of tissue-specific sequences. This identification allowed a precise cDNA tissue repertoire to be characterized in several important rainbow trout tissues. The rainbow trout contig browser can be accessed at the following publicly available web site (http://www.sigenae.org/).

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater.

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.

Molecular profiling of the gilthead sea bream (Sparus aurata L.) response to chronic exposure to the myxosporean parasite Enteromyxum leei.

The aim of the present work was to investigate the transcriptome response of gilthead sea bream (Sparus aurata) after challenge with the myxosporean Enteromyxum leei, a wide-spread enteric parasite causing heavy economic losses in Mediterranean sparid farms. This parasite causes severe desquamative enteritis which usually leads to death of the fish, and there are no preventative or curative treatments for this enteromyxosis. After 113 days of exposure to parasite-contaminated effluent, fish were classified into three cohorts: control fish not exposed to parasite, those that were exposed and infected, and those that were exposed but not infected. In order to detect target genes that may be candidates for infective status or resistance, a cDNA microarray containing 18,490 cDNA clones enriched in genes differentially expressed after infection was hybridised with head kidney and intestine samples. In infected fish, 371 and 373 genes were differentially regulated at the >1.5-fold level in intestine and head kidney respectively, whereas in non-infected fish 175 and 501 genes were differentially regulated in these tissues, respectively. A global marked gene down-regulation was evident in infected fish, mainly in genes involved in the immune and acute phase response particularly complement and mannose binding lectin. Microarray analysis demonstrated a complex interplay between host and/or parasite derived proteases and protease inhibitors, apoptosis, cell proliferation and antioxidant defence genes in exposed fish. In the head kidney of non-infected fish a marked depression of genes involved in the acute phase response was evident. By contrast, in the intestine of non-infected fish, interferon-stimulated and MHC class II genes involved in antigen processing and presentation were up-regulated, possibly indicating that an active immune response at the local level is important to avoid infection with or proliferation of the parasite.

Generation of fluorescent zebrafish to study endocrine disruption and potential crosstalk between thyroid hormone and corticosteroids.

Several environmental chemicals disrupt thyroid function, a key regulator of normal development involved in many physiological processes in fish. We studied the effects of such chemicals in vivo using transient transgenic zebrafish (Danio rerio), expressing Green Fluorescent Protein (GFP) under the control of a TH/bZIP promoter from Xenopus laevis. Exposure to thyroid hormone (T3) at 10(-8)M increased GFP fluorescence in F0 embryos and larvae. Transient transgenic embryos were exposed to a T3 signaling agonist (TRIAC) or antagonists (NH(3) or NaClO(4)), or to the endocrine disruptor Bisphenol A (BPA). When tested alone, TRIAC increased fluorescence, confirming the specificity of our model. Exposure to NH(3) or NaClO(4) decreased fluorescence, reflecting inhibition of thyroid function. When tested alone, BPA did not modify fluorescence, but when tested with T3, it significantly reduced T3-induced fluorescence, suggesting disruption of the thyroid function by BPA. The expression of genes involved in the TH axis (TR-alpha, TR-beta, TSH) and the corticoid axis (GR and MR) was followed by q-PCR after T3 or BPA exposure (24 or 48h) and at different developmental stages (0, 1, or 5 days post-fertilization). Expression of TR-alpha, TR-beta, and TSH genes increased after 48h T3 exposure in 1-day-old larvae. When tested alone, BPA only slightly affected gene expression. When applied with T3, BPA decreased expression of all candidate genes in 1-day-old embryos compared to the T3 treated group, in agreement with data obtained with the TH/bZIP-eGFP zebrafish model. Finally, we show that T3 exposure leads to up-regulation of MR and GR genes. This study provides a new rapid diagnostic tool for characterizing the disrupting effects of toxicants on thyroid function and suggests possible crosstalk between the TR and Corticoid Signaling system.

Detection of QTL with effects on osmoregulation capacities in the rainbow trout (Oncorhynchus mykiss).

BACKGROUND: There is increasing evidence that the ability to adapt to seawater in teleost fish is modulated by genetic factors. Most studies have involved the comparison of species or strains and little is known about the genetic architecture of the trait. To address this question, we searched for QTL affecting osmoregulation capacities after transfer to saline water in a nonmigratory captive-bred population of rainbow trout. RESULTS: A QTL design (5 full-sib families, about 200 F2 progeny each) was produced from a cross between F0 grand-parents previously selected during two generations for a high or a low cortisol response after a standardized confinement stress. When fish were about 18 months old (near 204 g body weight), individual progeny were submitted to two successive hyper-osmotic challenges (30 ppt salinity) 14 days apart. Plasma chloride and sodium concentrations were recorded 24 h after each transfer. After the second challenge, fish were sacrificed and a gill index (weight of total gill arches corrected for body weight) was recorded. The genome scan was performed with 196 microsatellites and 85 SNP markers. Unitrait and multiple-trait QTL analyses were carried out on the whole dataset (5 families) through interval mapping methods with the QTLMap software. For post-challenge plasma ion concentrations, significant QTL (P < 0.05) were found on six different linkage groups and highly suggestive ones (P < 0.10) on two additional linkage groups. Most QTL affected concentrations of both chloride and sodium during both challenges, but some were specific to either chloride (2 QTL) or sodium (1 QTL) concentrations. Six QTL (4 significant, 2 suggestive) affecting gill index were discovered. Two were specific to the trait, while the others were also identified as QTL for post-challenge ion concentrations. Altogether, allelic effects were consistent for QTL affecting chloride and sodium concentrations but inconsistent for QTL affecting ion concentrations and gill morphology. There was no systematic lineage effect (grand-parental origin of QTL alleles) on the recorded traits. CONCLUSIONS: For the first time, genomic loci associated with effects on major physiological components of osmotic adaptation to seawater in a nonmigratory fish were revealed. The results pave the way for further deciphering of the complex regulatory mechanisms underlying seawater adaptation and genes involved in osmoregulatory physiology in rainbow trout and other euryhaline fishes.

Effects of iron on rainbow trout gill cells in primary culture.

This study investigated the effects of iron in the form of iron sulphate (FeSO(4)·7H(2)O), over the range 0.01-1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO(4) did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO(4) reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron.

Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation.

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.