Increasing the levels of the essential trace elements Se, Zn, Cu and Mn in rotifers (Brachionus plicatilis) used as live feed

Rotifers are a common first feeding diet for rearing marine fish larvae. However, the levels of Mn, Cu, Zn, Se and iodine found in rotifers are low and may be insufficient to meet larval fish requirements. This study investigates increasing the concentration of Mn, Cu, Zn, Se and iodine simultaneously in rotifers (Brachionus plicatilis) in both short term enrichments (3 h) or during batch cultures (6 days), using either organically bound or inorganic mineral sources. This study demonstrates that rotifers can simultaneously be produced with Mn, Cu, Zn and Se concentrations up to and higher than the known requirements of fish, while increasing the level of iodine in rotifers was ineffective at the concentrations tested. To produce rotifers with copepod levels of Mn, Cu, Zn and Se, only 6% of a commercial rotifer enrichment diet had to be replaced with organically bound minerals, leaving a large percentage of the rotifer diet free to deliver other important nutrients such as lipid and proteins. Rotifers enriched to copepod mineral levels and stored for 18 h retained 75–110% of their Se, Zn and Mn and 50% of their Cu. Overall, increasing rotifer mineral levels appears to be most effective when the mineral is available in an insoluble and hence ingestible form.publishedVersio

Redox regulation in Atlantic cod (Gadus morhua) embryos developing under normal and heat-stressed conditions

With regard to predicted oceanic warming, we studied the effects of heat stress on the redox system during embryonic development of Atlantic cod (Gadus morhua), with emphasis on the glutathione balance, activities of key antioxidant enzymes, and their mRNA levels. The embryos were incubated at optimal temperature for development (6 °C) or slightly above the threshold temperature (10 °C). The regulation of all the redox-related parameters measured at optimum development was highly dynamic and complex, indicating the importance of both maternal and zygotic contributions to maintaining redox equilibrium. Development at 10 °C caused a significantly higher mortality at the blastula and early gastrula stages, indicating severe stress. Measures of the glutathione redox couple showed a significantly more reduced state in embryos at 10 °C compared to 6 °C at the post-gastrula stages. Mean normalized expression of nrf2, trxred, g6pd, gclc, nox1, CuZnsod, and mt in embryos kept at 10 °C revealed stage-specific significantly reduced mRNA levels. Activities of antioxidant enzymes changed both during ontogenesis and in response to temperature, but did not correlate with mRNA levels. As the embryos need a tightly regulated redox environment to coordinate between growth and differentiation, these findings suggest that the altered redox balance might participate in inducing phenotypic changes caused by elevated temperature.publishedVersio

Iodine nutrition and toxicity in Atlantic cod (Gadus morhua) larvae

-Copepods as feed promote better growth and development in marine fish larvae than rotifers. However, unlike rotifers, copepods contain several minerals such as iodine (I), at potentially toxic levels. Iodine is an essential trace element and both under and over supply of I can inhibit the production of the I containing thyroid hormones. It is unknown whether marine fish larvae require copepod levels of I or if mechanisms are present that prevent I toxicity. In this study, larval Atlantic cod (Gadus morhua) were fed rotifers enriched to intermediate (26 mg I kg-1 dry weight; MI group) or copepod (129 mg I kg-1 DW; HI group) I levels and compared to cod larvae fed control rotifers (0.6 mg I kg-1 DW). Larval I concentrations were increased by 3 (MI) and 7 (HI) fold compared to controls during the rotifer feeding period. No differences in growth were observed, but the HI diet increased thyroid follicle colloid to epithelium ratios, and affected the essential element concentrations of larvae compared to the other groups. The thyroid follicle morphology in the HI larvae is typical of colloid goitre, a condition resulting from excessive I intake, even though whole body I levels were below those found previously in copepod fed cod larvae. This is the first observation of dietary induced I toxicity in fish, and suggests I toxicity may be determined to a greater extent by bioavailability and nutrient interactions than by total body I concentrations in fish larvae. Rotifers with 0.6 mg I kg-1 DW appeared sufficient to prevent gross signs of I deficiency in cod larvae reared with continuous water exchange, while modelling of cod larvae versus rotifer I levels suggests that optimum I levels in rotifers for cod larvae is 3.5 mg I kg-1 DW

Associations Between Behavioral Effects of Bisphenol A and DNA Methylation in Zebrafish Embryos

Endocrine-disrupting contaminants have been associated with aberrant changes in epigenetic pathways in animals. In this study, zebrafish embryos were exposed bisphenol A (BPA) to search for associations between behavior and epigenetic mechanisms in fish. For concentration-dependent responses, embryos were exposed to a range of BPA concentrations (0.1 nM to 30 μM). Embryos were analyzed for locomotor activity at 3-, 4-, and 5-days post fertilization (dpf) in response to changing light conditions. Based on concentration-dependent effects on behavior and gene expression, 10 μM BPA [from 24 to 96 hours post fertilization (hpf)] was used for a whole-genome bisulfite sequencing (WGBS) study searching for genome-wide impacts on DNA methylation. Over the examined concentration ranges, hyperactivity was demonstrated for exposures to 0.001 μM BPA in comparison to embryos exposed to lower or higher BPA concentrations. Transcriptional analysis showed significant effects at >0.01 μM BPA for two genes related to DNA methylation (dnmt1, cbs). BPA exposure did not significantly affect global DNA methylation, but 20,474 differentially methylated (DM) sites in 4,873 genes were identified by WGBS analysis. Most DM sites were identified within gene bodies. The genes with the most DM sites were all protocadherin 2 gamma subfamily genes, related to axon targeting, synaptic development and neuronal survival. KEGG pathways most significantly affected by BPA exposure were phosphatidylinositol signaling system, followed by VEGF and MAPK signaling pathways. This study shows that BPA can affect zebrafish embryo swimming activity at very low concentrations as well as affecting numerous methylated sites in genes which are overrepresented in functionally relevant metabolic pathways. In conclusion, altered methylation patterns of genes associated with nervous system development might lead to abnormal swimming activity.Associations Between Behavioral Effects of Bisphenol A and DNA Methylation in Zebrafish EmbryospublishedVersio

The effects of selenium on methylmercury toxicity in zebrafish

Methylmercury (MeHg) is a potent neurotoxicant that remains a global concern. Selenium (Se) is an important micronutrient which is able to decrease MeHg toxicity, although the underlying mechanisms for this protection remain unclear. One hypothesis is that Se-mediated protection against MeHg toxicity occurs due to the function of Se containing proteins, termed selenoproteins. Many selenoproteins play key roles in maintaining both cellular and extracellular redox balance, and it is believed that via these roles, selenoproteins reduce MeHg-induced oxidative stress. A second hypothesis is that MeHg toxicity reduces the availability of Se for selenoprotein synthesis. Additional Se can thus be beneficial as it increases the availability of Se for selenoprotein production, reducing MeHg-induced disruption of the selenoproteome. These two hypotheses are linked, as they share a common theme suggesting that functional selenoproteins are key factors in reducing MeHg toxicity. The aim of this thesis was to explore how dietary Se reduces MeHg toxicity. As Se status affects MeHg toxicity, an initial study aimed to identify the Se requirements of zebrafish (Paper I). Juvenile zebrafish were fed diets with increasing levels of Se. The Se requirements were then assessed primarily from the response of growth and the mRNA expression and activity of a key Se-dependent protein, glutathione peroxidase (GPX). The second step of this thesis was to assess how changes in Se status affect MeHg induced toxicity both at the whole organism level (Paper II) and then at the molecular level, with a focus on the mRNA expression of selenoprotein coding genes (Paper III). To do this, female zebrafish were exposed to requirement (from Paper I) or elevated levels of dietary Se alone or in combination with elevated levels of dietary MeHg in a 2×2 factorial experimental design. These diets were fed to fish (F0 generation) for a five month period, during which the fish were crossed against untreated male fish to produce a maternal transfer exposed F1 generation. The effects of these diets on growth, survival, element composition and reproductive outcomes were explored in the adult generation (Paper II). The F1 generation were analysed during the embryonic stage to examine the underlying mechanisms of the Se×MeHg interactions on the expression of selenoprotein coding genes during development 7 (Paper III). The 30 selenoprotein coding genes analysed cover most of the selenoprotein families; including several members of the gpx, thioredoxin reductase, iodothyronine deiodinase and methionine sulfoxide reductase families, along with selenophosphate synthetase 2, selenoprotein h, j-p, t, w, 15, fep15 and fam213aa. As functional selenoprotein levels determine the Se-mediated antioxidant response, and the primary site of MeHg toxicity is the central nervous system, the total GPX activity and locomotor activity were also analysed in the F1 generation. The Se requirements of zebrafish were found to be 0.3 mg Se/kg DM based on growth, similar to other fish species. Meanwhile, maximum GPX activity did not correspond to zebrafish Se requirements, a controversial finding (Paper I). Elevated dietary Se reduced MeHg-induced decreases in growth and survival of adult fish, as found previously for vertebrates. However, elevated Se and MeHg had a synergistic negative affect on reproductive outcomes, such as embryo survival (Paper II), a novel finding in fish. Analyses of the mRNA expressions of selenoprotein coding genes demonstrated that only a subset of these genes were affected by MeHg. The affected genes coded for selenoproteins primarily from antioxidant pathways, and were downregulated by elevated MeHg. Meanwhile, MeHg also decreased GPX activity and induced larval hypoactivity. Elevated Se prevented the MeHg-induced downregulation for most of the affected genes. However, elevated Se only partially prevented the MeHg-induced decreases in GPX activity and larval locomotor activity (Paper III). As MeHg primarily affected antioxidant selenoproteins, which are also affected by Se deficiency, the response of selenoproteins to Se deficiency were then analysed in zebrafish embryos from parents fed diets deficient or replete in Se (Thesis Supp. Material). Considerable overlap was observed between the antioxidant selenoprotein genes downregulated by Se deficiency and those downregulated by MeHg toxicity. Overall mRNA downregulation of antioxidant selenoprotein genes by both MeHg toxicity and Se deficiency were prevented by elevating the Se status, suggesting that MeHg regulates the selenotranscriptome mainly via Se status, and that Se deficiency is a factor in MeHg toxicity

Increasing the levels of the essential trace elements Se, Zn, Cu and Mn in rotifers (Brachionus plicatilis) used as live feed

Rotifers are a common first feeding diet for rearing marine fish larvae. However, the levels of Mn, Cu, Zn, Se and iodine found in rotifers are low and may be insufficient to meet larval fish requirements. This study investigates increasing the concentration of Mn, Cu, Zn, Se and iodine simultaneously in rotifers (Brachionus plicatilis) in both short term enrichments (3 h) or during batch cultures (6 days), using either organically bound or inorganic mineral sources. This study demonstrates that rotifers can simultaneously be produced with Mn, Cu, Zn and Se concentrations up to and higher than the known requirements of fish, while increasing the level of iodine in rotifers was ineffective at the concentrations tested. To produce rotifers with copepod levels of Mn, Cu, Zn and Se, only 6% of a commercial rotifer enrichment diet had to be replaced with organically bound minerals, leaving a large percentage of the rotifer diet free to deliver other important nutrients such as lipid and proteins. Rotifers enriched to copepod mineral levels and stored for 18 h retained 75–110% of their Se, Zn and Mn and 50% of their Cu. Overall, increasing rotifer mineral levels appears to be most effective when the mineral is available in an insoluble and hence ingestible form

Redox regulation in Atlantic cod (Gadus morhua) embryos developing under normal and heat-stressed conditions

With regard to predicted oceanic warming, we studied the effects of heat stress on the redox system during embryonic development of Atlantic cod (Gadus morhua), with emphasis on the glutathione balance, activities of key antioxidant enzymes, and their mRNA levels. The embryos were incubated at optimal temperature for development (6 °C) or slightly above the threshold temperature (10 °C). The regulation of all the redox-related parameters measured at optimum development was highly dynamic and complex, indicating the importance of both maternal and zygotic contributions to maintaining redox equilibrium. Development at 10 °C caused a significantly higher mortality at the blastula and early gastrula stages, indicating severe stress. Measures of the glutathione redox couple showed a significantly more reduced state in embryos at 10 °C compared to 6 °C at the post-gastrula stages. Mean normalized expression of nrf2, trxred, g6pd, gclc, nox1, CuZnsod, and mt in embryos kept at 10 °C revealed stage-specific significantly reduced mRNA levels. Activities of antioxidant enzymes changed both during ontogenesis and in response to temperature, but did not correlate with mRNA levels. As the embryos need a tightly regulated redox environment to coordinate between growth and differentiation, these findings suggest that the altered redox balance might participate in inducing phenotypic changes caused by elevated temperature

Diet affects the redox system in developing Atlantic cod(Gadus morhua) larvae

The growth and development of marine fish larvae fed copepods is superior to those fed rotifers, but the underlying molecular reasons for this are unclear. In the following study we compared the effects of such diets on redox regulation pathways during development of Atlantic cod (Gadus morhua) larvae. Cod larvae were fed a control diet of copepods or the typical rotifer/Artemia diet commonly used in commercial marine fish hatcheries, from first feeding until after metamorphosis. The oxidised and reduced glutathione levels, the redox potential, and the mRNA expression of 100 genes in redox system pathways were then compared between treatments during larval development. We found that rotifer/Artemia-fed cod larvae had lower levels of oxidised glutathione, a more reduced redox potential, and altered expression of approximately half of the redox system genes when compared to copepod-fed larvae. This rotifer/Artemia diet-induced differential regulation of the redox system was greatest during periods of suboptimal growth. Upregulation of the oxidative stress response transcription factor, nrf2, and NRF2 target genes in rotifer/Artemia fed larvae suggest this diet induced an NRF2-mediated oxidative stress response. Overall, the data demonstrate that nutritional intake plays a role in regulating the redox system in developing fish larvae. This may be a factor in dietary-induced differences observed in larval growth

Iodine nutrition and toxicity in Atlantic cod (Gadus morhua) larvae

Copepods as feed promote better growth and development in marine fish larvae than rotifers. However, unlike rotifers, copepods contain several minerals such as iodine (I), at potentially toxic levels. Iodine is an essential trace element and both under and over supply of I can inhibit the production of the I containing thyroid hormones. It is unknown whether marine fish larvae require copepod levels of I or if mechanisms are present that prevent I toxicity. In this study, larval Atlantic cod (Gadus morhua) were fed rotifers enriched to intermediate (26 mg I kg-1 dry weight; MI group) or copepod (129 mg I kg-1 DW; HI group) I levels and compared to cod larvae fed control rotifers (0.6 mg I kg-1 DW). Larval I concentrations were increased by 3 (MI) and 7 (HI) fold compared to controls during the rotifer feeding period. No differences in growth were observed, but the HI diet increased thyroid follicle colloid to epithelium ratios, and affected the essential element concentrations of larvae compared to the other groups. The thyroid follicle morphology in the HI larvae is typical of colloid goitre, a condition resulting from excessive I intake, even though whole body I levels were below those found previously in copepod fed cod larvae. This is the first observation of dietary induced I toxicity in fish, and suggests I toxicity may be determined to a greater extent by bioavailability and nutrient interactions than by total body I concentrations in fish larvae. Rotifers with 0.6 mg I kg-1 DW appeared sufficient to prevent gross signs of I deficiency in cod larvae reared with continuous water exchange, while modelling of cod larvae versus rotifer I levels suggests that optimum I levels in rotifers for cod larvae is 3.5 mg I kg-1 DW

Methylmercury Induced Neurotoxicity and the Influence of Selenium in the Brains of Adult Zebrafish (Danio rerio)

The neurotoxicity of methylmercury (MeHg) is well characterised, and the ameliorating effects of selenium have been described. However, little is known about the molecular mechanisms behind this contaminant-nutrient interaction. We investigated the influence of selenium (as selenomethionine, SeMet) and MeHg on mercury accumulation and protein expression in the brain of adult zebrafish (Danio rerio). Fish were fed diets containing elevated levels of MeHg and/or SeMet in a 2 × 2 full factorial design for eight weeks. Mercury concentrations were highest in the brain tissue of MeHg-exposed fish compared to the controls, whereas lower levels of mercury were found in the brain of zebrafish fed both MeHg and SeMet compared with the fish fed MeHg alone. The expression levels of proteins associated with gap junction signalling, oxidative phosphorylation, and mitochondrial dysfunction were significantly (p < 0.05) altered in the brain of zebrafish after exposure to MeHg and SeMet alone or in combination. Analysis of upstream regulators indicated that these changes were linked to the mammalian target of rapamycin (mTOR) pathways, which were activated by MeHg and inhibited by SeMet, possibly through a reactive oxygen species mediated differential activation of RICTOR, the rapamycin-insensitive binding partner of mTOR