Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

A role for dopamine in control of the hypoxic ventilatory response via D2 receptors in the zebrafish gill.

Dopamine is a neurotransmitter involved in oxygen sensing and control of reflex hyperventilation. In aquatic vertebrates, oxygen sensing occurs in the gills via chemoreceptive neuroepithelial cells (NECs), but a mechanism for dopamine in autonomic control of ventilation has not been defined. We used immunohistochemistry and confocal microscopy to map the distribution of tyrosine hydroxylase (TH), an enzyme necessary for dopamine synthesis, in the gills of zebrafish. TH was found in nerve fibers of the gill filaments and respiratory lamellae. We further identified dopamine active transporter (dat) and vesicular monoamine transporter (vmat2) expression in neurons of the gill filaments using transgenic lines. Moreover, TH- and dat-positive nerve fibers innervated NECs. In chemical screening assays, domperidone, a D2 receptor antagonist, increased ventilation frequency in zebrafish larvae in a dose-dependent manner. When larvae were confronted with acute hypoxia, the D2 agonist, quinpirole, abolished the hyperventilatory response. Quantitative polymerase chain reaction confirmed expression of drd2a and drd2b (genes encoding D2 receptors) in the gills, and their relative abundance decreased following acclimation to hypoxia for 48 h. We localized D2 receptor immunoreactivity to NECs in the efferent gill filament epithelium, and a novel cell type in the afferent filament epithelium. We provide evidence for the synthesis and storage of dopamine by sensory nerve terminals that innervate NECs. We further suggest that D2 receptors on presynaptic NECs provide a feedback mechanism that attenuates the chemoreceptor response to hypoxia. Our studies suggest that a fundamental, modulatory role for dopamine in oxygen sensing arose early in vertebrate evolution.

Exploring transcriptional and post-transcriptional epigenetic regulation of crf and 11ßhsd2 in rainbow trout brain during chronic social stress.

Using dominance hierarchies in juvenile rainbow trout (Oncorhynchus mykiss) as a model of chronic social stress in fish, we explored whether epigenetic transcriptional and post-transcriptional mechanisms are involved in the gene expression of corticotropin-releasing factor (crf) and 11ß-hydroxysteroid dehydrogenase (11ßhsd2), key factors involved in the regulation of the endocrine stress axis response. In juvenile rainbow trout pairs, subordinate individuals display sustained elevation of circulating cortisol concentrations. Cortisol production is controlled by the hypothalamic-pituitary-interrenal (HPI) axis in fish and initiated by CRF release from the preoptic area (POA). Given that crf is modulated during chronic social stress, and that such stress has been implicated in the epigenetic regulation of crf in other taxa, we probed a role for epigenetic regulation of crf transcript abundance in chronically stressed rainbow trout. We also investigated the regulation of the cortisol-metabolising enzyme 11ßhsd2 in the POA, which is upregulated in subordinates. The potential involvement of DNA methylation and microRNAs (miRNAs) in the regulation of crf transcript abundance was investigated during social stress in the POA of fish, as was the potential involvement of miRNAs in 11ßhsd2 regulation. Although transcript abundances of crf were elevated in subordinate fish after 4 days, DNA methylation profiles within putative promoter sequences upstream of the crf gene were not significantly affected by chronic stress. An inverse relationship between crf and its predicted posttranscriptional regulator miR-103a-3p in the POA suggests that miRNAs may be involved in mediating the effects of chronic social stress on key components of the endocrine stress axis.

Lactate signaling and fuel selection in rainbow trout: mobilization of energy reserves.

Lactate is now recognized as a regulator of fuel selection in mammals because it inhibits lipolysis by binding to the hydroxycarboxylic acid receptor 1 (HCAR1). The goals of this study were to quantify the effects of exogenous lactate on: 1) lipolytic rate or rate of appearance of glycerol in the circulation (Ra glycerol) and hepatic glucose production (Ra glucose), and 2) key tissue proteins involved in lactate signaling, glucose transport, glycolysis, gluconeogenesis, lipolysis, and ß-oxidation in rainbow trout. Measurements of fuel mobilization kinetics show that lactate does not affect lipolysis as it does in mammals (Ra glycerol remains at 7.3 ± 0.5 µmol·kg-1·min-1), but strongly reduces hepatic glucose production (16.4 ± 2.0 to 8.9 ± 1.2 µmol·kg-1·min-1). This reduction is likely induced by decreasing gluconeogenic flux through the inhibition of cytosolic phosphoenolpyruvate carboxykinase (Pck1, alternatively called Pepck1; 60% and 24% declines in gene expression and protein level, respectively). It is also caused by lactate substituting for glucose as a fuel in all tissues except white muscle that increases glut4a expression and has limited capacity for monocarboxylate transporter (Mct)-mediated lactate import. We conclude that lipolysis is not affected by hyperlactatemia because trout show no activation of autocrine Hcar1 signaling (gene expression of the receptor is unchanged or even repressed in red muscle). Lactate regulates fuel mobilization via Pck1-mediated suppression of gluconeogenesis and by replacing glucose as a fuel. This study highlights important functional differences in the Hcar1 signaling system between fish and mammals for the regulation of fuel selection.

Knock-out of vasotocin reduces reproductive success in female zebrafish, Danio rerio.

The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F2α, a hormone involved in ovarian maturation, egg release and regulation of female courtship behavior in some cyprinids. However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species.

Variation in habitat use and its consequences for mercury exposure in two Eastern Ontario bat species, Myotis lucifugus and Eptesicus fuscus.

The St. Lawrence River in Eastern Ontario, Canada, has been a designated an area of concern due to past industrial contamination of sediment in some areas and transport of mercury from tributaries. Previous research using bats as sentinel species identified elevated concentrations of total mercury (THg) in fur of local bats and species-specific variation between little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus). Here, we investigated the mercury exposure pathways for these two species by testing the hypothesis that diet variation, particularly the reliance on aquatic over terrestrial insects, is a determinant of local bat mercury concentrations. We analyzed THg concentration and stable isotope ratios of δ15N and δ13C in fur of little and big brown bats, and in aquatic and terrestrial insects. Big brown bats, especially males, accumulated significantly higher THg concentrations in their fur compared to little brown bats. However, this difference was not related to diet because big brown bats consumed terrestrial insects, which were lower in mercury than aquatic insects, the primary prey for little brown bats. We also evaluated whether fur THg concentrations translate into molecular changes in tissues linked to (methyl)mercury toxicity by quantifying tissue changes in global DNA methylation and mitochondrial DNA abundance. No significant changes in DNA molecular markers were observed in relation to fur THg concentration, suggesting mercury exposure to local bats did not impact molecular level changes at the DNA level. Higher mercury in bats was not associated with local aquatic contamination or genotoxicity in this study area.

Effects of PFOS, F-53B and OBS on locomotor behaviour, the dopaminergic system and mitochondrial function in developing zebrafish (Danio rerio).

Perfluorooctanesulfonic acid (PFOS) has widely been reported to persist in the environment and to elicit neurotoxicological effects in wildlife and humans. Following the restriction of PFOS use, 6:2 chlorinated polyfluorinated ether sulfonate (F-53B) and sodium p-perfluorous nonenoxybenzene sulfonate (OBS) have emerged as novel PFOS alternatives and have been detected in the environment. However, knowledge on the toxicological effects of these alternatives remains scarce. Using developing transgenic Tg(dat:eGFP) zebrafish, we evaluated the consequences of exposure to 0, 0.1 and 1 mg/l PFOS, F-53B and OBS on the dopaminergic system, locomotor behaviour and mitochondrial function. All compounds generally reduced locomotor activity under light conditions irrespective of exposure concentration. Exposure to OBS (at all concentrations), as well as PFOS and F-53B (at 1 mg/l), significantly reduced subpallial dopaminergic neuron abundance. PFOS also significantly reduced dat and pink1 expression irrespective of exposure concentration, while F-53B and OBS tended to reduce mitochondrial pink1 and fis1 expression across concentrations without reaching statistical significance. Mitochondrial function, in the form of reduced oxygen consumption rate and marginally inhibited ATP-linked oxygen consumption rate, was affected only in response to 1 mg/l PFOS. Together, PFOS and the emerging contaminants F-53B and OBS inhibit locomotion at similar concentrations, a finding correlated with decreased dopaminergic neuron numbers in the subpallium and decreased expression of pink1. These findings are relevant to wildlife and human health, as they suggest that PFOS as well as replacement compounds affect locomotion likely in part by negatively impacting the dopamine system.

The mammalian insulin antagonist S961 does not exhibit insulin receptor antagonism in rainbow trout in vivo.

Due to their reported 'glucose-intolerant' phenotype, rainbow trout have been the focus of comparative studies probing underlying endocrine mechanisms at the organismal, tissue and molecular level. A particular focus has been placed on the investigation of the comparative role of insulin, an important glucoregulatory hormone, and its interaction with macronutrients. A limiting factor in the comparative investigation of insulin is the current lack of reliable assays to quantify circulating mature and thus bioactive insulin. To circumvent this limitation, tissue-specific responsiveness to postprandial or exogenous insulin has been quantified at the level of post-translational modifications of cell signalling proteins. These studies revealed that the insulin responsiveness of these proteins and their post-translational modifications are evolutionarily highly conserved and thus provide useful and quantifiable proxy indices to investigate insulin function in rainbow trout. While the involvement of specific branches of the intracellular insulin signalling pathway (e.g., mTor) in rainbow trout glucoregulation have been successfully probed through pharmacological approaches, it would be useful to have a functionally validated insulin receptor antagonist to characterize the glucoregulatory role of the insulin receptor pathway in its entirety for this species. Here, we report two separate in vivo experiments to test the ability of the mammalian insulin receptor antagonist, S961, to efficiently block insulin signalling in liver and muscle in response to endogenously released insulin and to exogenously infused bovine insulin. We found that, irrespective of the experimental treatment or dose, activation of the insulin pathway in liver and muscle was not inhibited by S961, showing that its antagonistic effect does not extend to rainbow trout.

Reproductive roles of the vasopressin/oxytocin neuropeptide family in teleost fishes.

The vertebrate nonapeptide families arginine vasopressin (AVP) and oxytocin (OXT) are considered to have evolved from a single vasopressin-like peptide present in invertebrates and termed arginine vasotocin in early vertebrate evolution. Unprecedented genome sequence availability has more recently allowed new insight into the evolution of nonapeptides and especially their receptor families in the context of whole genome duplications. In bony fish, nonapeptide homologues of AVP termed arginine vasotocin (Avp) and an OXT family peptide (Oxt) originally termed isotocin have been characterized. While reproductive roles of both nonapeptide families have historically been studied in several vertebrates, their roles in teleost reproduction remain much less understood. Taking advantage of novel genome resources and associated technological advances such as genetic modifications in fish models, we here critically review the current state of knowledge regarding the roles of nonapeptide systems in teleost reproduction. We further discuss sources of plasticity of the conserved nonapeptide systems in the context of diverse reproductive phenotypes observed in teleost fishes. Given the dual roles of preoptic area (POA) synthesized Avp and Oxt as neuromodulators and endocrine/paracrine factors, we focus on known roles of both peptides on reproductive behaviour and the regulation of the hypothalamic-pituitary-gonadal axis. Emphasis is placed on the identification of a gonadal nonapeptide system that plays critical roles in both steroidogenesis and gamete maturation. We conclude by highlighting key research gaps including a call for translational studies linking new mechanistic understanding of nonapeptide regulated physiology in the context of aquaculture, conservation biology and ecotoxicology.

Social status-dependent regulation and function of the somatotropic axis in juvenile rainbow trout.

Juvenile rainbow trout (Oncorhynchus mykiss) develop social hierarchies when competing for resources in a constrained environment. Among the physiological consequences of social status are changes in organismal energy metabolism, which generally favour anabolic pathways in dominant fish and catabolic pathways in subordinate fish. The somatotropic axis is an important regulator of metabolism and growth that could be involved in mediating metabolic changes in response to social status in juvenile rainbow trout. Here we used juvenile trout housed either in dyads or individually (sham controls) to determine whether social status changes indices of somatotropic axis function. Although pituitary growth hormone expression (gh1 and gh2) did not differ among groups, circulating growth hormone (GH) increased ∼12-fold in subordinate fish compared to sham and dominant fish. Social status caused consistent differential expression of GH receptor paralogues in liver and muscle, two principal target tissues of GH. Compared to dominant and/or sham fish, ghra paralogue expression (ghra1 and ghra2) was lower, while ghrb1 expression was higher in subordinate fish. Across tissues, ghra paralogue expression was generally positively correlated with expression of insulin growth factors (igf1, igf2), while ghrb1 expression was positively correlated with transcript abundance of hormone sensitive lipase (hsl1). Because igf and hsl expression are subject to context-dependent GH control in rainbow trout, these results suggest that increased circulating GH in conjunction with differential expression of ghr paralogues may translate into prioritization of downstream catabolic lipolytic pathways in subordinate rainbow trout. These findings support a social context-dependent role for GH signalling in mediating metabolic changes in juvenile rainbow trout.

Epigenetic and post-transcriptional repression support metabolic suppression in chronically hypoxic goldfish.

Goldfish enter a hypometabolic state to survive chronic hypoxia. We recently described tissue-specific contributions of membrane lipid composition remodeling and mitochondrial function to metabolic suppression across different goldfish tissues. However, the molecular and especially epigenetic foundations of hypoxia tolerance in goldfish under metabolic suppression are not well understood. Here we show that components of the molecular oxygen-sensing machinery are robustly activated across tissues irrespective of hypoxia duration. Induction of gene expression of enzymes involved in DNA methylation turnover and microRNA biogenesis suggest a role for epigenetic transcriptional and post-transcriptional suppression of gene expression in the hypoxia-acclimated brain. Conversely, mechanistic target of rapamycin-dependent translational machinery activity is not reduced in liver and white muscle, suggesting this pathway does not contribute to lowering cellular energy expenditure. Finally, molecular evidence supports previously reported chronic hypoxia-dependent changes in membrane cholesterol, lipid metabolism and mitochondrial function via changes in transcripts involved in cholesterol biosynthesis, ß-oxidation, and mitochondrial fusion in multiple tissues. Overall, this study shows that chronic hypoxia robustly induces expression of oxygen-sensing machinery across tissues, induces repressive transcriptional and post-transcriptional epigenetic marks especially in the chronic hypoxia-acclimated brain and supports a role for membrane remodeling and mitochondrial function and dynamics in promoting metabolic suppression.

Metabolic Consequences of Developmental Exposure to Polystyrene Nanoplastics, the Flame Retardant BDE-47 and Their Combination in Zebrafish.

Single-use plastic production is higher now than ever before. Much of this plastic is released into aquatic environments, where it is eventually weathered into smaller nanoscale plastics. In addition to potential direct biological effects, nanoplastics may also modulate the biological effects of hydrophobic persistent organic legacy contaminants (POPs) that absorb to their surfaces. In this study, we test the hypothesis that developmental exposure (0-7 dpf) of zebrafish to the emerging contaminant polystyrene (PS) nanoplastics (⌀100 nm; 2.5 or 25 ppb), or to environmental levels of the legacy contaminant and flame retardant 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47; 10 ppt), disrupt organismal energy metabolism. We also test the hypothesis that co-exposure leads to increased metabolic disruption. The uptake of nanoplastics in developing zebrafish was validated using fluorescence microscopy. To address metabolic consequences at the organismal and molecular level, metabolic phenotyping assays and metabolic gene expression analysis were used. Both PS and BDE-47 affected organismal metabolism alone and in combination. Individually, PS and BDE-47 exposure increased feeding and oxygen consumption rates. PS exposure also elicited complex effects on locomotor behaviour with increased long-distance and decreased short-distance movements. Co-exposure of PS and BDE-47 significantly increased feeding and oxygen consumption rates compared to control and individual compounds alone, suggesting additive or synergistic effects on energy balance, which was further supported by reduced neutral lipid reserves. Conversely, molecular gene expression data pointed to a negative interaction, as co-exposure of high PS generally abolished the induction of gene expression in response to BDE-47. Our results demonstrate that co-exposure to emerging nanoplastic contaminants and legacy contaminants results in cumulative metabolic disruption in early development in a fish model relevant to eco- and human toxicology.

A cross-species comparative approach to assessing multi- and transgenerational effects of endocrine disrupting chemicals.

A wide range of chemicals have been identified as endocrine disrupting chemicals (EDCs) in vertebrate species. Most studies of EDCs have focused on exposure of both male and female adults to these chemicals; however, there is clear evidence that EDCs have dramatic effects when mature or developing gametes are exposed, and consequently are associated with in multigenerational and transgenerational effects. Several publications have reviewed such actions of EDCs in subgroups of species, e.g., fish or rodents. In this review, we take a holistic approach synthesizing knowledge of the effects of EDCs across vertebrate species, including fish, anurans, birds, and mammals, and discuss the potential mechanism(s) mediating such multi- and transgenerational effects. We also propose a series of recommendations aimed at moving the field forward in a structured and coherent manner.

Alanine alters the carbohydrate metabolism of rainbow trout: glucose flux and cell signaling.

In rainbow trout, dietary carbohydrates are poorly metabolized compared with other macronutrients. One prevalent hypothesis suggests that high dietary amino acid levels could contribute to the poor utilization of carbohydrates in trout. In mammals, alanine is considered an important gluconeogenic precursor, but has recently been found to stimulate AMP-activated protein kinase (AMPK) to reduce glucose levels. In trout, the effect of alanine on glucose flux is unknown. The goal of this study was to determine the effects of 4 h exogenous alanine infusion on glucose metabolism in rainbow trout. Glucose flux, and the rate of glucose appearance (Ra) and disposal (Rd) were measured in vivo. Key glycolytic and gluconeogenic enzyme expression and activity, and cell signaling molecules relevant to glucose metabolism were assessed in the liver and muscle. The results show that alanine inhibits glucose Ra (from 13.2±2.5 to 7.3±1.6 µmol kg-1 min-1) and Rd (from 13.2±2.5 to 7.4±1.5 µmol kg-1 min-1) and the slight mismatch between Ra and Rd caused a reduction in glycemia, similar to the effects of insulin in trout. The reduction in glucose Rd can be partially explained by a reduction in glut4b expression in red muscle. In contrast to mammals, trout alanine-dependent glucose-lowering effects did not involve hepatic AMPK activation, suggesting a different mechanistic basis. Interestingly, protein kinase B (AKT) activation increased only in muscle, similar to effects observed in insulin-infused trout. We speculate that alanine-dependent effects were probably mediated through stimulation of insulin secretion, which could indirectly promote alanine oxidation to provide the needed energy.

Transgenerational effects of polychlorinated biphenyls: 2. Hypothalamic gene expression in rats†.

Polychlorinated biphenyls (PCBs) are endocrine-disrupting chemicals (EDCs) with well-established effects on reproduction and behavior in developmentally-exposed (F1) individuals. Because of evidence for transgenerational effects of EDCs on the neuroendocrine control of reproductive physiology, we tested the hypothesis that prenatal PCB exposure leads to unique hypothalamic gene-expression profiles in three generations. Pregnant Sprague-Dawley rats were treated on gestational days 16 and 18 with the PCB mixture Aroclor 1221 (A1221), vehicle (3% DMSO in sesame oil), or estradiol benzoate (EB, 50 µg/kg), the latter a positive control for estrogenic effects of A1221. Maternal- and paternal-lineage F2 and F3 generations were bred using untreated partners. The anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), involved in the hypothalamic control of reproduction, were dissected from F1 to F3 females and males, RNA extracted, and gene expression measured in a qPCR array. We detected unique gene-expression profiles in each generation, which were sex- and lineage-specific. In the AVPV, treatment significantly changed 10, 25, and 11 transcripts in F1, F2, and F3 generations, whereas 10, 1, and 12 transcripts were changed in these generations in the ARC. In the F1 AVPV and ARC, most affected transcripts were decreased by A1221. In the F2 AVPV, most effects of A1221 were observed in females of the maternal lineage, whereas only Pomc expression changed in the F2 ARC (by EB). The F3 AVPV and ARC were mainly affected by EB. It is notable that results in one generation do not predict results in another, and that lineage was a major determinant in results. Thus, transient prenatal exposure of F1 rats to A1221 or EB can alter hypothalamic gene expression across three generations in a sex- and lineage-dependent manner, leading to the conclusion that the legacy of PCBs continues for generations.

Comparative epigenetics in animal physiology: An emerging frontier.

The unprecedented access to annotated genomes now facilitates the investigation of the molecular basis of epigenetic phenomena in phenotypically diverse animals. In this critical review, we describe the roles of molecular epigenetic mechanisms in regulating mitotically and meiotically stable spatiotemporal gene expression, phenomena that provide the molecular foundation for the intra-, inter-, and trans-generational emergence of physiological phenotypes. By focusing principally on emerging comparative epigenetic roles of DNA-level and transcriptome-level epigenetic mark dynamics in the emergence of phenotypes, we highlight the relationship between evolutionary conservation and innovation of specific epigenetic pathways, and their interplay as a priority for future study. This comparative approach is expected to significantly advance our understanding of epigenetic phenomena, as animals show a diverse array of strategies to epigenetically modify physiological responses. Additionally, we review recent technological advances in the field of molecular epigenetics (single-cell epigenomics and transcriptomics and editing of epigenetic marks) in order to (1) investigate environmental and endogenous factor dependent epigenetic mark dynamics in an integrative manner; (2) functionally test the contribution of specific epigenetic marks for animal phenotypes via genome and transcript-editing tools. Finally, we describe advantages and limitations of emerging animal models, which under the Krogh principle, may be particularly useful in the advancement of comparative epigenomics and its potential translational applications in animal science, ecotoxicology, ecophysiology, climate change science and wild-life conservation, as well as organismal health.

Meta-analysis of differentially-regulated hepatic microRNAs identifies candidate post-transcriptional regulation networks of intermediary metabolism in rainbow trout.

MicroRNAs (miRNAs) are small non-coding RNAs which act as post-transcriptional regulators by decreasing targeted mRNA translation and stability. Principally targeting small 3' UTR elements of protein-coding mRNAs through complementary base-pairing, miRNAs are promiscuous regulators of the transcriptome. While potent roles for hepatic miRNAs in the regulation of energy metabolism have emerged in rodent models, comparative roles in other vertebrates remain largely unexplored. Indeed, while several miRNAs are deeply conserved among vertebrates, the acquisition of lineage- and species-specific miRNAs, as well as the rewiring between miRNA-mRNA target relationships beg the question of regulatory and functional conservation and innovation of miRNAs and their targets involved in energy metabolism. Here we provide a meta-analysis of differentially expressed hepatic miRNAs in rainbow trout, a scientifically and economically important teleost species with a 'glucose-intolerant' phenotype. Following exposure to nutritional and social context-dependent metabolic challenges, we analyzed differential miRNA expression from small-RNA-sequencing datasets generated with a consistent bioinformatics pipeline in conjunction with an in silico target prediction of metabolic transcripts and pathways. We provide evidence for evolutionary conserved (let-7, miRNA-27 family) and rewired (miRNA-30 family, miRNA-152, miRNA-722) miRNA-metabolic target gene networks in the context of the salmonid genome. These findings represent important first steps in our understanding of the comparative regulation and function of hepatic miRNAs in rainbow trout energy metabolism. We propose that the identified miRNA families should be prioritized for future comparative functional investigation in the context of hepatic energy- and glucose metabolism in rainbow trout.

Correction: Social status regulates the hepatic miRNAome in rainbow trout: Implications for posttranscriptional regulation of metabolic pathways.

[This corrects the article DOI: 10.1371/journal.pone.0217978.].

Acute and long-term metabolic consequences of early developmental Bisphenol A exposure in zebrafish (Danio rerio).

Bisphenol A (BPA) is an estrogenic contaminant linked to metabolic disruption. Developmental BPA exposure is of particular concern, as organizational effects may irreversibly disrupt metabolism at later life-stages. While BPA exposures in adult fish elicit metabolic perturbations similar to effects described in rodents, the metabolic effects of developmental BPA exposure in juvenile fish remain largely unknown. Following embryonic zebrafish exposure to BPA (0.1, 1 and 4 mg/L) and EE2 (10 ng/L) from 2 to 5 dpf, we assessed the metabolic phenotype in larvae (4-6 dpf) and juveniles (43-49 dpf) which had been divided into regular-fed and overfed groups at 29 dpf. Developmental BPA exposure in larvae dose-dependently reduced food-intake and locomotion and increased energy expenditure. Juveniles (29 dpf) exhibited a transient increase in body weight after developmental BPA exposure and persistent diet-dependent locomotion changes (43-49 dpf). At the molecular level, glucose and lipid metabolism-related transcript abundance clearly separated BPA exposed fish from controls and EE2 exposed fish at the larval stage, in juveniles on a regular diet and, to a lesser extent, in overfed juveniles. In general, the metabolic endpoints affected by BPA exposure were not mimicked by EE2 treatment. We conclude that developmental BPA exposure elicits acute metabolic effects in zebrafish larvae and fewer transient and persistent effects in juveniles and that these metabolic effects are largely independent of BPA's estrogenicity.