Gene Expression In Rainbow Trout Research Articles

Modulation of humoral immunological and antioxidant responses and gut bacterial community and gene expression in rainbow trout, Oncorhynchus mykiss, by dietary lactic acid supplementation

The present study was conducted to find the effects of dietary lactic acid (LA) supplementation on growth performance, hematological characteristics, humoral antioxidant and immunological factors, intestinal gene expression and bacteriological parameters in rainbow trout, Oncorhynchus mykiss. The fish (104 ± 0.90 g) were randomly distributed in twelve tanks (150 L water) as four treatments, receiving diets supplemented with 0, 5, 10, and 20 g/kg LA for 70 days. At the end of the feeding trial, no significant differences were observed in the fish growth performance and survival among the treatments. Blood/plasma red blood cell (5–20 g/kg LA), white blood cell (10 g/kg LA), hematocrit (5 and 10 g/kg LA), hemoglobin (5 and 10 g/kg LA), superoxide dismutase (10 g/kg LA), glutathione peroxidase (10 g/kg LA), total antioxidant capacity (10 g/kg LA), lysozyme (5–20 g/kg LA), and alternative complement activity (10 g/kg LA) significantly increased, as malondialdehyde (10 g/kg LA) significantly decreased in the LA-treated fish. Dietary LA supplementation induced no significant changes in the intestinal defensin gene expression, but significantly up-regulated the expression of toll-like receptor-3 (5–20 g/kg LA), Mx-9 (5–20 g/kg LA), and heat shock protein-70 (5 g/kg LA) genes; the highest expressions were observed at 5 g/kg LA supplementation. Dietary LA supplementation significantly increased total bacterial count (5 and 10 g/kg LA), Lactobacillus sp. (5–20 g/kg LA), and Bacteroides sp. (5–20 g/kg LA), but decreased Streptococcus iniae (5–20 g/kg LA) in the fish intestine; the highest Lactobacillus sp. And Bacteroides sp. Were observed in 10 and 5 g/kg LA supplementation, respectively. Overall, dietary LA supplementation, although has no significant effects on the fish growth performance, is beneficial to improve humoral antioxidant parameters, humoral and intestinal immunological parameters and intestinal microflora. According to the results, dietary 5–10 g/kg LA is recommended for the formulation of rainbow trout feed.

Iron supplementation in plant-based aquafeed: Effects on growth performance, tissue composition, iron-related serum parameters and gene expression in rainbow trout (Oncorhynchus mykiss)

This study evaluated the effects of iron supplementation in plant-based aquafeed on growth performances, feed intake, nutrient composition, digestibility, iron-related serum parameters and gene expression in juvenile rainbow trout (Oncorhynchus mykiss). Triplicate groups of fish were fed for 60 days using incremental levels of iron in a ferrous sulphate heptahydrate form, as follows: 0 (group Fe0); 150 (group Fe150); 300 (group Fe300); 600 (group Fe600) mg iron/kg feed, corresponding to actual iron levels of 132, 273, 403, 741 mg iron/kg feed, respectively. As a further control, a fifth group was fed a commercial feed (CMF; 253 mg iron/kg feed). Growth performances showed that the highest specific growth rate (SGR), condition factor (CF), protein efficacy ratio (PER), feed intake (FI) and lowest feed conversion rate (FCR) were found in Fe150, Fe300 and CMF groups. Final weight and weight gain (WG) were significantly higher in CMF and Fe150 groups, followed by the Fe300, Fe600 and Fe0 groups (P < 0.05). Apparent digestibility coefficients (ADCs) of protein, lipid, ash and iron were over 90% in Fe150 and Fe300 groups, while significantly lower ADCs were observed in Fe0 and Fe600 groups. Intestinal histomorphological measurements of muscular thickness (muscularis externa; ME, inner circular layer; ICL, outer longitudinal layer; OLL) were higher in CMF and Fe150 groups, while villus length was generally similar among groups, with the exception of the Fe0 group. Serum iron levels were significantly higher in Fe150 and Fe300 groups, while total iron-binding capacity (TIBC) was highest in the Fe150 group. No significant differences were observed in either serum ferritin or liver ferritin gene expression among groups, although hepcidin gene expression was highest in the Fe300 group followed by the Fe150 group. Transferrin gene expression was the lowest in the Fe0 group. Appetite-related Leptin A1 gene expression was not affected by iron addition, but Leptin A2 was up-regulated in all groups except for the Fe0 group. In summary, the study showed that the addition of 150 mg/kg feed of iron (equivalent to ~270 mg/kg feed total) improved growth performances and feed utilization in rainbow trout, which aligns well with the iron levels in current commercial feeds.

Effect of different dietary zinc sources on seminal plasma enzymatic activity, antioxidant, and immune-related gene expression in rainbow trout (Oncorhynchus mykiss)

Effect of different dietary zinc sources on seminal plasma enzymatic activity, antioxidant, and immune-related gene expression in rainbow trout (Oncorhynchus mykiss)

Effects of dietary leucine levels on growth performance, hematobiochemical parameters, liver profile, intestinal enzyme activities and target of rapamycin signalling pathway related gene expression in rainbow trout, Oncorhynchus mykiss fingerlings

A study was designed to investigate the influence of leucine (Leu) levels on growth performance, enzyme activities and relative expression of TOR and 4E-BP genes in rainbow trout, Oncorhynchus mykiss (average initial body weight: 1.60 ± 0.25 g/fish; average initial length 5.20 ± 0.30 cm/fish). Six isonitrogenous (450 g/kg) and energetic (20.90 kJ 100 g−1, gross energy) experimental diets in gradation of 2.5 g/kg of dry diets were formulated to contain graded Leu levels (10.0, 12.5, 15.0, 17.5, 20.0 and 22.5 g/kg, drymatter basis). 20 fish were stocked in triplicate groups in 75 L trough connected with flow-through system (2–2.5 L/min) and were fed twice daily (09:00 and 17:00 hours) at 5% BW day−1. Quadratic regression analysis of live weight gain (LWG%), feed conversion ratio (FCR), protein efficiency ratio (PER) and body protein deposition (BPD) data indicated Leu requirement at 17.70, 17.42, 17.25 and 17.57 g/kg dry diet, respectively. Highest (p < .05) protein, lowest moisture and intermediate fat contents were noted at 17.5 g/kg Leu fed diet. Significant variation in haematological parameters was also observed. While no significant (p ˃ .05) differences in plasma total protein, cholesterol, triglycerides, albumin and globulin were noted. However, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), glucose, urea and uric acid levels produced significant differences. Except amylase, higher enzymatic activities were observed at 17.5 g/kg Leu diet. Higher relative expression level of target of rapamycin (TOR) and eukaryotic translation initiation factor 4E-binding protein (4E-BP) mRNA was observed at 17.5 and 15.0 g/kg Leu fed diets. Based on the above data, it is recommended that 17.45 g/kg Leu, corresponding to 38.77 g/kg of dietary protein would be useful for optimum growth and also regulates relative gene expression of TOR and 4E-BP signalling pathway.

Nutritional regulation of gene expression and enzyme activity of phosphoenolpyruvate carboxykinase in the hepatic gluconeogenesis pathway in golden pompano (Trachinotus ovatus)

Phosphoenolpyruvate carboxykinase (PEPCK) is known to exist as mitochondrial (PEPCK-M) and cytosolic isoforms (PEPCK-C). They are involved in glucose production and play vital roles in regulating glucose homeostasis. This study aimed to (1) clone and characterize PEPCK-C cDNA from golden pompano fish (Trachinotus ovatus), which are known to have poor utilization of dietary carbohydrates, and (2) analyze the regulation of its expression by varying dietary carbohydrate-to-lipid ratios and nutritional status. A full length golden pompano PEPCK-C cDNA fragment of 2,652 bp was cloned, which contains an open reading frame of 1,875 bp encoding 624 amino acids and shows a high homology to cobia (Rachycentron canadum) PEPCK-C sequence (92% similarity). The analysis of tissue distributions of PEPCK-C mRNA showed that a high abundance of PEPCK-C was expressed in golden pompano liver, followed by kidney; while lower expression levels were found in the heart and intestine. In liver, PEPCK is under the regulation of dietary composition and nutritional status at the enzymatic and molecular levels. Our results indicate that PEPCK-C plays a modulating role in the adaptation of hepatic gluconeogenesis to different nutrient conditions. Thus, the absence of molecular inhibition of gluconeogenic PEPCK gene expression in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), which can at least partially account for their poor utilization of dietary carbohydrate, is specific to these species and is not observed in golden pompano. Further studies that examine, at the enzymatic and molecular levels, other key enzymes involved in hepatic glucose metabolism are necessary to understand the poor utilization of carbohydrates in golden pompano.

Effect of stocking density on growth performance, plasma biochemistry and muscle gene expression in rainbow trout (Oncorhynchus mykiss)

The present study investigated in young-of-the-year rainbow trout, the effects of stocking density on growth parameters, plasma biochemistry and the expression of muscle stress- and growth-related genes including heat shock protein 70 (HSP70), insulin-like growth factor-1(IGF-1), myosin heavy chain (MHC), myostatin-1a (MSTN-1a) and follistatin (FST). Animals were exposed to three different stocking densities: 12, 24 and 44 kg/m3, termed as low (LD), moderate (MD) and high (HD) density treatments, respectively, in an open culture system for 60 days. Also, on day 40, the culture density of a group of fish were reduced from 44 to 12 kg/m3 (reduced density; RD treatment) for a further period of 20 days. The results showed significant changes in growth parameters, so that the final weight and condition factor were significantly decreased and feed conversion ratio was increased in HD compared to LD treatments. On the other hand, no significant alterations were found in fin, hepatosomatic and viscerosomatic indices as well as plasma glucose, total protein, triglyceride, cholesterol and cortisol contents among different experimental groups. The expression of HSP70 was upregulated in HD compared to LD on days 20, 40 and 60, being the expression of MHC decreased and MSTN-1a increased significantly on day 60 only. The expression of IGF-1 and FST did not show any significant variations among treatments. After 20 days of RD treatment, no significant changes were observed in growth parameters between RD and HD, being final weight and condition factor lower and feed conversion ratio higher than LD values. In addition, the expression of HSP70 and MSTN-1a was significantly downregulated, and MHC was upregulated in RD groups compared to HD ones. In conclusion, increase of stocking density, reduced the growth and changed the expression of stress- and growth-related genes in muscle. Finally, the 20-day period of RD treatment could not recover the fish to LD conditions and a longer treatment period or, alternatively, experience of adjusted stocking densities would probably be required for a full recovery.

Dietary olive leaf (Olea europea L.) extract alters some immune gene expression levels and disease resistance to Yersinia ruckeri infection in rainbow trout Oncorhynchus mykiss

The aim for the present study was to investigate the effects of olive leaf (Olea europea L.) extract (OLE) on the control of Yersinia ruckeri infection in rainbow (Oncorhynchus mykiss) trout and to assess the impact on the expression of immune-related genes in the spleen and serum biochemical parameters of rainbow trout. Five experimental diets were prepared by adding 0.0%, 0.1%, 0.25%, 0.50% and 1.0% of OLE. Each diet was fed to triplicate groups of fish (mean body weight 51.22 ± 3.04 g) twice a day (at 09:00 and 17:00 h) for 60 days. The dietary supplementation of OLE did not affect growth performance and feed utilization (P > 0.05). Major changes due to graded levels of OLE in the diets were observed in blood biochemical parameters (P < 0.05). TNFα, IL1-β and IL-8 gene expressions were significanlty up-regulated in OLE 0.1% group compared with others (P < 0.05). Also, diet supplemented with OLE reduced mortality in rainbow trout fed with OLE 0.1% added diet. Present study suggests that OLE especially at 0.1% added feed may effectivelly enhance the serum biochemical parameters, survival rate and immune gene expression in rainbow trout.

Characterisation of ZBTB46 and DC-SCRIPT/ZNF366 in rainbow trout, transcription factors potentially involved in dendritic cell maturation and activation in fish

ZBTB46 and DC-SCRIPT/ZNF366 are two zinc finger transcription factors that play important roles in regulating differentiation of dendritic cells in mammals. In this study, the ZBTB46 and DC-SCRIPT/ZNF366 homologues were identified in rainbow trout Oncorhynchus mykiss and their expression analysed in vivo and in vitro. As transcription factors, they are well conserved in sequence, genomic organisation and gene synteny. Their expression was differentially modulated by bacterial and viral PAMPs in the monocyte/macrophage-like cell line RTS-11, in primary head kidney (HK) macrophages, and in HK macrophages cultured with IL-4/13A. In the RTS-11 cells and primary HK macrophages, all the ZBTB46 and DC-SCRIPT/ZNF366 homologues were down-regulated by interferon gamma (type II IFN) but unaffected by IFN2 (type I IFN), administered as recombinant proteins to cell cultures. In fish gills, infection with amoebae (Paramoebae perurans) resulted in reduction of ZBTB46 and DC-SCRIPT/ZNF366 expression in Atlantic salmon Salmo salar, whilst infection with Yersinia ruckeri induced gene expression in rainbow trout.

Development of a mucosal adjuvant for fish vaccination

Vaccines rely on the acquired immune system and prime an animal to a particular antigen so that on subsequent encounter it is recognised and quickly destroyed. Most fish vaccines were developed in the 1990s, and three routes of administration are used; immersion and injection for priming, and oral for boosting (when needed). Injection vaccines were developed as a consequence of the need for an adjuvant in the formulation and have been very successful. However, they do have some drawbacks and it is timely to re-examine the potential for mucosally delivered vaccines that include adjuvants. Polyethyleneimine (PEI) is a polycation with potent mucosal and systemic adjuvant activity. PEI triggers both T and B cell responses in mammals by activating the innate and adaptive arms of the immune response. In this study varying doses of PEI were used to assess the adjuvant effect of PEI when used with a commercial immersion vaccine to Yersinia ruckeri, the causative agent of Enteric Redmouth Disease. We analysed immune gene expression in rainbow trout (Oncorhynchus mykiss) at days 1, 3, 7 and 10 post-vaccination, as well as serum antibody levels at weeks 6, 8 and 10. We found a trend towards enhanced durability of the antibody response across a dosage range of PEI but there was no evidence of enhanced pro-inflammatory gene expression in mucosal tissues (gills, skin) consistent with findings in mammals. This study helps refine the dose of PEI that is suitable for use in fish and serves as the basis of future experiments we plan to optimise further immersion delivery of fish vaccines.

The longevity of the antimicrobial response in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) supplemented diet

This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout (Oncorhynchus mykiss) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1–14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups.The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes.

Cloning and characterization of the proximal promoter region of rainbow trout (Oncorhynchus mykiss) interleukin-6 gene

Interleukin-6 (IL6) is a pleiotropic cytokine with important immunoregulatory functions. Its expression is inducible in immune cells and tissues of several fish species. We also found that IL6 mRNA abundance was significantly increased in spleen, liver, and gill of rainbow trout after experimental infection with Aeromonas salmonicida. Genomic DNA sequences of IL6 orthologs from three salmonid species revealed a conserved exon/intron structure and a high overall nucleotide identity of >88%. To uncover key mechanisms regulating IL6 expression in salmonid fish, we amplified a fragment of the proximal IL6 promoter from rainbow trout and identified in-silico conserved binding sites for NF-κB and CEBP. The activity of this IL6 promoter fragment was analyzed in the established human embryonic kidney line HEK-293. Luciferase- and GFP-based reporter systems revealed that the proximal IL6 promoter is activated by Escherichia coli. Essentially, both reporter systems proved that NF-κB p50, but not NF-κB p65 or CEBP, activates the IL6 promoter fragment. Truncation of this fragment caused a significant decrease in IL6 promoter activation. This characterization of the proximal promoter of the IL6-encoding gene provides basic knowledge about the IL6 gene expression in rainbow trout.

Fatty Acid‐Specific Alterations in Leptin, PPARα, and CPT‐1 Gene Expression in the Rainbow Trout

It is known that fatty acids (FA) regulate lipid metabolism by modulating the expression of numerous genes. In order to gain a better understanding of the effect of individual FA on lipid metabolism related genes in rainbow trout (Oncorhynchus mykiss), an in vitro time-course study was implemented where twelve individual FA (butyric 4:0; caprylic 8:0; palmitic (PAM) 16:0; stearic (STA) 18:0; palmitoleic16:1n-7; oleic 18:1n-9; 11-cis-eicosenoic 20:1n-9; linoleic (LNA) 18:2n-6; α-linolenic (ALA) 18:3n-3; eicosapentenoic (EPA) 20:5n-3; docosahexaenoic (DHA) 22:6n-3; arachidonic (ARA) 20:4n-6) were incubated in rainbow trout liver slices. The effect of FA administration over time was evaluated on the expression of leptin, PPARα and CPT-1 (lipid oxidative related genes). Leptin mRNA expression was down regulated by saturated fatty acids (SFA) and LNA, and was up regulated by monounsaturated fatty acids (MUFA) and long chain PUFA, whilst STA and ALA had no effect. PPARα and CPT-1mRNA expression were up regulated by SFA, MUFA, ALA, ARA and DHA; and down regulated by LNA and EPA. These results suggest that there are individual and specific FA induced modifications of leptin, PPARα and CPT-1 gene expression in rainbow trout, and it is envisaged that such results may provide highly valuable information for future practical applications in fish nutrition.

Effects of excretory/secretory products from Anisakis simplex (Nematoda) on immune gene expression in rainbow trout (Oncorhynchus mykiss)

Excretory/secretory (ES) products are molecules produced by parasitic nematodes, including larval Anisakis simplex, a parasite occurring in numerous marine fish hosts. The effects of these substances on host physiology have not been fully described. The present work elucidates the influence of ES substances on the fish immune system by measuring immune gene expression in spleen and liver of rainbow trout (Oncorhynchus mykiss) injected intraperitoneally with ES products isolated from A. simplex third stage larvae. The overall gene expression profile of exposed fish showed a generalized down-regulation of the immune genes tested, suggesting a role of ES proteins in immunomodulation. We also tested the enzymatic activity of the ES proteins and found that lipase, esterase/lipase, valine and cysteine arylamidases, naphthol-AS-BI-phosphohydrolase and α-galactosidase activities were present in the ES solution. This type of hydrolytic enzyme activity may play a role in nematode penetration of host tissue. In addition, based on the notion that A. simplex ES products may have an immune-depressive effect (by minimizing immune gene expression) it could also be suggested that worm enzymes directly target host immune molecules which would add to a decreased host immune response and increased worm survival.

Differential gene expression in rainbow trout (Oncorhynchus mykiss) liver and ovary after exposure to zearalenone

Zearalenone (ZEA) is a mycotoxin of worldwide occurrence, and it has been shown to produce numerous adverse effects in both laboratory and domestic animals. However, regardless of recent achievements, the molecular mechanisms underlying ZEA toxicity remain elusive, and little is known about transcriptome changes of fish cells in response to ZEA occurrence. In the present study, differential display PCR was used to generate a unique cDNA fingerprint of differentially expressed transcripts in the liver and ovary of juvenile rainbow trout after either 24, 72, or 168h of intraperitoneal exposure to ZEA (10mg/kg of body mass). From a total of 59 isolated cDNA bands (ESTs), 5 could be confirmed with Real-Time qPCR and their nucleotide sequences were identified as mRNAs of: acty (β-centractin), the cytoskeleton structural element; bccip, responsible for DNA repair and cell cycle control; enoa (α-enolase), encoding enzyme of the glycolysis process; proc (protein C), that takes part in the blood coagulation process; and frih, encoding the heavy chain of ferritin, the protein complex important for iron storage. Further qPCR analysis of the confirmed ESTs expression profiles revealed significant mRNA level alterations in both tissues of exposed fish during the 168h study. The results revealed a complex network of genes associated with different biological processes that may be engaged in the cellular response to ZEA exposure, i.e. blood coagulation or iron-storage processes.

Dietary protein source significantly alters growth performance, plasma variables and hepatic gene expression in rainbow trout (Oncorhynchus mykiss) fed amino acid balanced diets

The objective of the study was to evaluate the effect of dietary protein source on fish growth, nutrient utilization, plasma variables and hepatic gene expression in juvenile rainbow trout (Oncorhynchus mykiss). Fishmeal (FM) and soy protein isolate (SPI) were used as the main sources of protein in six isonitrogenous, isolipidic and isocaloric diets. The amino acid profiles of the diets were completely balanced to minimize differences between experimental treatments and formulated to contain increasing levels of branched-chain amino acids (BCAA) based upon dietary requirements for trout (NRC, 1993). Dietary protein source more consistently changed the measured variables while BCAA supplementation had an unexpected effect over whole body lipid content. Growth performance and protein retention efficiency were significantly reduced in fish fed SPI diets independently of BCAA supplementation. Total concentration of amino acids as well as circulating indispensable amino acids (IAA) were significantly elevated in the plasma of fish receiving SPI diets compared to fish fed FM diets. The change in IAA was large enough to increase (p<0.05) plasma IAA/DAA (DAA: dispensable amino acids) proportion even when the diets were formulated to have a ratio close to 1. Levels of circulating BCAA and alanine were also elevated in the fish fed SPI diets, possibly indicating a change in protein turnover. The use of SPI caused a reduction (p<0.05) in the hepatic expression levels of alanine amino transferase (alt1) and glutamine synthetase 2 (gls02), while an increase was observed for aspartate aminotransferase (got2), and asparagine synthetase (asns) compared with FM diets. Expression of the gene tor (target of rapamycin) declined over time for all treatments, while expression of a gene known to repress tor function, redd-1, was consistently higher in the liver of fish fed SPI diets. Glucose 6 phosphate dehydrogenase (g6pd) also showed a significantly higher expression in the liver of fish fed SPI diets but only at higher levels of BCAA supplementation.In summary dietary protein source has a significant effect over growth performance, body composition and hepatic gene expression in rainbow trout. We also identified for the first time in fish changes in the expression of redd-1, which may represent another regulatory point in the TOR cascade.

Global hepatic gene expression in rainbow trout exposed to sewage effluents: A comparison of different sewage treatment technologies

Effluents from sewage treatment plants contain a mixture of micropollutants with the potential of harming aquatic organisms. Thus, addition of advanced treatment techniques to complement existing conventional methods has been proposed. Some of the advanced techniques could, however, potentially produce additional compounds affecting exposed organisms by unknown modes of action. In the present study the aim was to improve our understanding of how exposure to different sewage effluents affects fish. This was achieved by explorative microarray and quantitative PCR analyses of hepatic gene expression, as well as relative organ sizes of rainbow trout exposed to different sewage effluents (conventionally treated, granular activated carbon, ozonation (5 or 15mg/L), 5mg/L ozone plus a moving bed biofilm reactor, or UV-light treatment in combination with hydrogen peroxide). Exposure to the conventionally treated effluent caused a significant increase in liver and heart somatic indexes, an effect removed by all other treatments. Genes connected to xenobiotic metabolism, including cytochrome p450 1A, were differentially expressed in the fish exposed to the conventionally treated effluents, though only effluent treatment with granular activated carbon or ozone at 15mg/L completely removed this response. The mRNA expression of heat shock protein 70kDa was induced in all three groups exposed to ozone-treated effluents, suggesting some form of added stress in these fish. The induction of estrogen-responsive genes in the fish exposed to the conventionally treated effluent was effectively reduced by all investigated advanced treatment technologies, although the moving bed biofilm reactor was least efficient. Taken together, granular activated carbon showed the highest potential of reducing responses in fish induced by exposure to sewage effluents.

Atorvastatin up-regulate toxicologically relevant genes in rainbow trout gills

There are large and increasing discharges of statins into the aquatic environment. Statins are cholesterol-lowering pharmaceuticals, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, an enzyme in the cholesterol synthesis pathway. Earlier studies have shown that statins will affect the expression of a range of genes in mammalian tissues and this group of pharmaceuticals has also been shown to affect membrane transporters. Changes in gene expression and ion transport in aquatic organisms may have dramatic consequences for the individual. The aim of the present study was to clarify whether waterborne exposure to a selected statin, atorvastatin, would affect gene expression in rainbow trout (Oncorhynchus mykiss) gill or liver or ion regulation in gills. Juvenile rainbow trout were exposed to two atorvastatin acid and atorvastatin lactone concentrations for 7 days (nominal concentrations 200 ng L(-1) and 10 μg L(-1)). The exposures caused up-regulated gene expression in gill, not liver, and only at the lowest concentration. Genes involved in membrane transport (pgp, mrp1), oxidative stress response (sod, mt), apoptosis (bax) and biotransformation (sult2b) were differentially expressed whereas the expression of genes involved in cholesterol biosynthesis (hmgr, fdps) or peroxisomal proliferation (ppar) were not affected. There were no significant changes in gill Na(+)/K(+) ATPase activity following exposure to atorvastatin. The pattern of differentially expressed genes in rainbow trout gills differ from responses previously observed in mammalian tissues following statin exposure.

Analysis of stress-induced hepatic gene expression in rainbow trout ( Oncorhynchus mykiss) selected for high- and low-responsiveness to stress

The production and welfare of intensively reared fish would be improved by reducing stress responsiveness. One approach to achieving this goal is selective breeding utilising stress-responsive genes as direct genetic markers of the desirable trait. As a first step in this process, microarray analysis has been carried out on liver tissues of rainbow trout selectively bred for high (HR) or low (LR) responsiveness to a stressor. Microarray hybridizations provided gene expression profiles for pooled samples of fish confined for 6 h, 24 h and 168 h and for individual fish (168 h only). 161 genes were shown to be differentially regulated in HR and LR fish during confinement exposure and eight of these gene expression profiles were validated by quantitative PCR. Genes of particular interest included intelectin-2 precursor which showed greater than 100-fold higher expression in HR fish compared to LR fish irrespective of whether the fish were confined or not; interferon inducible transmembrane protein 3 which was differentially stress-induced between the two lines; and hepatic pro-opiomelanocortin B (POMC B) which was upregulated during stress in HR fish but downregulated in LR fish. All these offer potential as direct markers of low stress responsiveness in a marker-assisted selection scheme.

Effect of dietary supplements on cytokine gene expression in rainbow trout, Oncorhynchus mykiss (Walbaum)

Effect of dietary supplements on cytokine gene expression in rainbow trout, <i>Oncorhynchus mykiss</i> (Walbaum)

Real-time quantification of the immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus

Whether it is better to use viable or non-viable probionts in aquaculture is still a matter of debate. In this study, the molecular immunomodulation in rainbow trout Oncorhynchus mykiss induced by viable or killed forms of the probiont Lactobacillus rhamnosus JCM 1136 was investigated. Three forms of this probiont: (1) heat-killed (HK), (2) live spray (LI) and (3) freeze-dried (FD) were incorporated into a basal (control) diet for rainbow trout O. mykiss. The LI and FD diets are referred to as viable diets. A rearing trial, in triplicate, was conducted for 30 days, with the control and probiotic diets as treatments. The cytokine genes such as the tumour necrosis factor (TNF), transforming growth factor (TGF-β), interferon (IFN) and immune gene Immunoglobulin (Ig) found in tissues from the kidney and spleen were assessed for their expression pattern by real-time polymerase chain reaction. The tested immune genes were up-regulated in the treatment groups, sometimes even in many folds like in the case of the Ig gene. The TNF gene was found to be highly (P<0.05) up-regulated (5000-fold) in groups fed both viable forms (LI, FD). With regard to the TGF-β gene, the spleen of the HK and FD groups showed significant up-regulation of 20- and 30-folds respectively. The IFN gene was up-regulated (P<0.05) in all treatments, but more in the viable diet treatments. Kidney and spleen tissues showed similar expression patterns, i.e. all of these genes were up-regulated more with the viable diets that with the control, and in most cases, the viable diets induced a higher expression of the immune genes than the HK diet.