Effects of oral Bt-maize (MON810) exposure on growth and health parameters in normal and sensitised Atlantic salmon, Salmo salar L.

Responses to GM maize Bt-maize, MON810) expressing Cry1Ab protein from the soil bacterium Bacillus thuringiensis (Bt) in diets for both normal and immune-sensitised (with soyabean meal (SBM)-induced enteropathy) post-smolt Atlantic salmon were investigated following 33 and 97 d of exposure. Triplicate tanks of salmon were fed one of four diets, all containing 20% whole-kernel meal maize, either Bt-maize or its near-isogenic maternal line, without or with 15% extracted SBM inclusion. The fish fed Bt-maize utilised the feed less efficiently, as revealed by lower protein and mineral digestibilities and lower lipid and energy retention efficiencies. Higher intestinal weight, as well as increased interferon-γ and decreased sodium-glucose co-transporter mRNA expression, and a transient increase in T-helper cell presence, as measured by cluster of differentiation 4 (CD4) protein in the distal intestine (DI), may partly explain the lower nutrient digestibilities and retentions. The Bt-maize seemed to potentiate oxidative cellular stress in the DI of immune-sensitised fish, as indicated by increases in superoxide dismutase and heat shock protein 70 mRNA expression. The data suggest that Cry1Ab protein or other antigens in Bt-maize have local immunogenic effects in salmon DI. No systemic immune responses could be detected, as indicated by haematology, differential leucocyte counts, plasma clinical chemistry, as well as absence of Cry1Ab-specific antibodies and Cry1Ab protein in plasma. The responses to Bt-maize observed in the present study differed from results from earlier studies in salmon and other animals fed the same event Bt-maize. Longer-term experiments and more in-depth studies on intestinal physiology and immune responses are needed to evaluate health implications.

Cross-generational feeding of Bt (Bacillus thuringiensis)-maize to zebrafish (Danio rerio) showed no adverse effects on the parental or offspring generations.

In the present study, zebrafish (Danio rerio) were fed casein/gelatin-based diets containing either 19% Bt (Bacillus thuringiensis)-maize or its parental non-Bt (nBt)-maize control for two generations (F0: sixty fish; F1: forty-two to seventy fish per treatment). The study focused on growth and reproductive performance, liver CuZn superoxide dismutase (SOD) enzyme activity, gene transcript levels targeting important cellular pathways in the liver and mid-intestine, histomorphological evaluation of the intestine, differential leucocyte counts, offspring larva swimming activity and global DNA methylation in offspring embryos. No significant effects were observed in the parental generation. The offspring were either fed the same diets as those fed to their parents (Bt-Bt or nBt-nBt) or switched from the Bt diet to the nBt diet (Bt-nBt). The Bt-Bt offspring exhibited a significantly higher body mass increase, specific growth rate and feed utilisation than fish fed the nBt-nBt diet and/or fish fed the Bt-nBt diet. Liver and mid-intestinal gene transcript levels of CuZn SOD were significantly higher in fish fed the nBt-nBt diet than in those fed the Bt-Bt diet. Liver gene transcript levels of caspase 6 were significantly lower for the nBt-nBt group than for the Bt-Bt group. Overall, enhanced growth performance was observed in fish fed the Bt diet for two generations than in those fed the nBt diet for one and two generations. Effects observed on gene biomarkers for oxidative stress and the cell cycle (apoptosis) may be related to the contamination of nBt-maize with fumonisin B1 and aflatoxin B1. In conclusion, it is suggested that Bt-maize is as safe and nutritious as its nBt control when fed to zebrafish for two generations.

Are apparent negative effects of feeding GM MON810 maize to Atlantic salmon, Salmo salar, caused by confounding factors?

The present study was conducted to follow up on apparent differences in growth, relative organ sizes, cellular stress and immune function in Atlantic salmon fed feed containing GM Bacillus thuringiensis maize compared with feed containing the non-modified parental maize line. Gene expression profiling on the distal intestinal segment and liver was performed by microarray, and selected genes were followed up by quantitative PCR (qPCR). In the liver, qPCR revealed some differentially regulated genes, including up-regulation of gelsolin precursor, down-regulation of ferritin heavy subunit and a tendency towards down-regulation of metallothionein (MT)-B. This, combined with the up-regulation of anti-apoptotic protein NR13 and similar tendencies for ferritin heavy chain and MT-A and -B in the distal intestine, suggests changes in cellular stress/antioxidant status. This corresponds well with and strengthens previous findings in these fish. To exclude possible confounding factors, the maize ingredients were analysed for mycotoxins and metabolites. The GM maize contained 90 µg/kg of deoxynivalenol (DON), while the non-GM maize was below the detection limit. Differences were also observed in the metabolite profiles of the two maize varieties, some of which seemed connected to the mycotoxin level. The effects on salmon observed in the present and previous studies correspond relatively well with the effects of DON as reported in the literature for other production animals, but knowledge regarding effects and harmful dose levels in fish is scarce. Thus, it is difficult to conclude whether the observed effects are caused by the DON level or by some other aspect of the GM maize ingredient.

Zebrafish ( Danio rerio) as a model for investigating the safety of GM feed ingredients (soya and maize); performance, stress response and uptake of dietary DNA sequences.

A 20-d zebrafish (Danio rerio) feeding trial, in which a near doubling of fish weight was achieved, was conducted with GM feed ingredients to evaluate feed intake, growth, stress response and uptake of dietary DNA. A partial aim of the study was to assess zebrafish as a model organism in GM safety assessments. Roundup Ready soya (RRS), YieldGard Bt maize (MON810) and their non-modified, maternal, near-isogenic lines were used in a 2 x 2 factorial design. Soya variety and maize variety were the main factors, both with two levels; non-GM and GM. Compared with fish fed non-GM maize, those fed GM maize exhibited significantly better growth, had lower mRNA transcription levels of superoxide dismutase (SOD)-1 and a tendency (non-significant) towards lower transcription of heat shock protein 70 in liver. Sex of the fish and soya variety had significant interaction effects on total RNA yield from the whole liver and transcription of SOD-1, suggesting that some diet component affecting males and females differently was present in different levels in the GM and the non-GM soya used in the present study. Dietary DNA sequences were detected in all of the organs analysed, but not all of the samples. Soya and maize rubisco (non-transgenic, multicopy genes) were most frequently detected, while MON810 transgenic DNA fragments were detected in some samples and RRS fragments were not detected. In conclusion, zebrafish shows promise as a model for this application.

Recommendations for dietary level of micro-minerals and vitamin D3 to Atlantic salmon (Salmo salar) parr and post-smolt when fed low fish meal diets.

Atlantic salmon (Salmo salar) feeds have changed drastically in their composition from being predominantly marine-based to plant-based. This has altered the dietary supply and availability of micro-nutrients to Atlantic salmon. The impact of graded inclusion levels of a nutrient package (NP) comprising of 25 different micro-nutrients were studied in Atlantic salmon parr in freshwater (Trial 1) and post-smolts in seawater (Trial 2). In brief, the NP was included from 0 to 400%, where 100% corresponded to the recommendation by the National Research Council, 2011. Micro-nutrients, namely Zn, Mn, Se, Cu, Fe, Co, I and vitamin D3 were included in the NP with the objective of (re)evaluating the dietary need to meet the requirement of Atlantic salmon parr and post-smolt, when fed low fish meal, plant ingredient-based diets. Responses in apparent availability coefficient (AAC), whole body and vertebrae mineral concentrations, and retention were analysed. AAC of Cu, Mn, Se and Zn responded in a quadratic fashion with an increase in NP from 0 to 400% in freshwater parr; AAC could not be measured in post-smolt salmon. The whole-body concentration of Zn, Se, Co and I in Atlantic salmon parr were significantly affected by increasing NP inclusion; the same was observed for Zn, Se and Co in post-smolt Atlantic salmon. Vertebrae mineral concentration as the response criterion was non-responsive in parr; whereas, in post-smolt, Co had a linear increase, while Zn and Se showed a non-linear increase upon 0 to 400 NP inclusion. Zinc concentration and activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) in vertebrae indicated increased bone resorption in post-smolt Atlantic salmon; TRAP activity increased linearly with NP inclusion in post-smolt, but not in parr. Significant correlations between Zn and Se were observed in AAC and vertebral concentrations, indicating an interaction in intestinal uptake and vertebral deposition. Overall, Atlantic salmon parr held in freshwater were able to satisfy the requirement for the trace minerals Zn, Mn, Se, Cu, and Fe through supply from 100-150 NP, corresponding to 101-132, 47-63, 0.6-0.8, 12-16 and 150-166 mg kg -1, respectively; for iodine, dietary supply from 150-200 NP, corresponding to 0.7-1.6 mg kg-1, was required. In the seawater, Atlantic salmon post-smolt, in general, required micro-minerals and vitamin D3 levels as supplied through 150-200 NP, corresponding to 140-177, Zn; 61-67, Mn; 0.9-1, Se; 14-16, Cu; and vitamin D3, 0.06-0.09 mg kg -1 to fulfil the requirement, except for Cu which was satisfied at 100-150 NP, equivalent to 13-14 mg kg -1 diet.

Correction: Exploring Early Micronutrient Deficiencies in Rainbow Trout (Oncorhynchus mykiss) by Next-Generation Sequencing Technology - From Black Box to Functional Genomics.

[This corrects the article DOI: 10.1371/journal.pone.0069461.].

Antioxidant nutrition in Atlantic salmon (Salmo salar) parr and post-smolt, fed diets with high inclusion of plant ingredients and graded levels of micronutrients and selected amino acids.

The shift from marine to plant-based ingredients in fish feeds affects the dietary concentrations and bioavailability of micronutrients, amino acids and lipids and consequently warrants a re-evaluation of dietary nutrient recommendations. In the present study, an Atlantic salmon diet high in plant ingredients was supplemented with graded levels of nutrient premix (NP), containing selected amino acids, taurine, cholesterol, vitamins and minerals. This article presents the results on the antioxidant nutrients vitamin C, E and selenium (Se), and effects on tissue redox status. The feed ingredients appeared to contain sufficient levels of vitamin E and Se to cover the requirements to prevent clinical deficiency symptoms. The body levels of α-tocopherol (TOH) in parr and that of Se in parr and post-smolt showed a linear relationship with dietary concentration, while α-TOH in post-smolt seemed to be saturable with a breakpoint near 140 mg kg-1. Ascorbic acid (Asc) concentration in the basal feed was below the expected minimum requirement, but the experimental period was probably too short for the fish to develop visible deficiency symptoms. Asc was saturable in both parr and post-smolt whole body at dietary concentrations of 190 and 63-89 mg kg-1, respectively. Maximum whole body Asc concentration was approximately 40 mg kg-1 in parr and 14 mg kg-1 in post-smolt. Retention ranged from 41 to 10% in parr and from -206 to 12% in post-smolt with increasing NP supplementation. This indicates that the post-smolts had an extraordinarily high consumption of Asc. Analyses of glutathione (GSH) and glutathione disulphide (GSSG) concentrations and the calculated GSH based redox potentials in liver and muscle tissue, indicated only minor effects of diets on redox regulation. However, the post-smolt were more oxidized than the parr. This was supported by the high consumption of Asc and high expression of gpx1 and gpx3 in liver. Based on the present trials, the recommendations for supplementation of vitamin C and E in diets for Atlantic salmon are similar to current practices, e.g. 150 mg kg-1 of α-TOH and 190 mg kg-1 Asc which was the saturating concentration in parr. Higher concentrations than what would prevent clinical deficiency symptoms are necessary to protect fish against incidents of oxidative stress and to improve immune and stress responses. There were no indications that the Se requirement exceeded the current recommendation of 0.3 mg kg-1.

Atlantic salmon (Salmo salar) require increased dietary levels of B-vitamins when fed diets with high inclusion of plant based ingredients.

Aiming to re-evaluate current recommendations for nutrient supplementations when Atlantic salmon are fed diets based on plant ingredients, two regression experiments, with parr and post-smolt, were conducted. A control diet was included to evaluate if ingredients supplied sufficient nutrients without any added nutrient package (NP). The nutrient package consisted of vitamins B, C, E, minerals, cholesterol, methionine, taurine and histidine. This paper focus on B-vitamins. In parr, growth, health and welfare parameters responded on NP additions, but this was not observed in the seawater stage. During three months of feeding, parr tripled their weight. Parr given diets added the NP above NRC (2011) showed improved protein retention, and reduced liver and viscera indices. Post-smolt fed the same diets during five months showed a doubling of weight, but did not respond to the variation in NP to the same extent as parr. Significant regressions were obtained in body compartments for several of the B-vitamins in the premix. Whole body biotin concentration was unaffected by micronutrient premix level, and mRNA expression of the enzymes dependent of biotin showed only weak increases with increased biotin. Muscle thiamine plateaued at a diet level similar to NRC (2011) recommendation in freshwater, and showed stable values independent on premix addition in seawater. The mRNA expression of the enzyme G6PDH (glucose-6-phosphate dehydrogenase) is sensitive to thiamine availability; results did not indicate any need to add thiamine above levels recommended for fish in general. Niacin showed a steady increase in whole body concentrations as feed niacin increased. Muscle riboflavin peaked at a diet level of 12.4 mg kg-1. Sufficient riboflavin is important to avoid e.g., development of cataract. Cataract was not registered to be any problem, neither in fresh- nor in seawater. Cobalamin (B 12) in muscle and liver was saturated at 0.17 mg kg-1 diet. Muscle pyridoxine showed a dose-dependent level in muscle, and peaked around 10 mg kg -1 diet. White muscle ASAT (asparagine amino transferase) activity steadily increased, with indications of stable values when dietary pyridoxine was around 10-16 mg kg -1 diet. Pantothenic acid increased in gill tissue up to a level of 5.5 mg kg -1 soft gill tissue; at a dietary level of 22 mg kg-1. Improved performance, and coverage of metabolic need for niacin was at a dietary level of 66 mg kg -1, riboflavin 10-12 mg kg-1, pyridoxine 10 mg kg-1 and panthotenic acid 22 mg kg-1. Based on these results, recommended B-vitamin supplementation in plant based diets for Atlantic salmon should be adjusted.

Exploring early micronutrient deficiencies in rainbow Trout (Oncorhynchus mykiss) by next-generation sequencing technology--from black box to functional genomics.

This work studies final nutritional status and transcriptional responses of rainbow trout (Oncorhynchus mykiss Walbaum 1792) (28 g) after a 10 week feeding experiment designed to elucidate the effect of adding a vitamin and mineral premix on growth, health, and nutritional endpoints. Juvenile fish were fed a either a diet supplemented with a vitamin and mineral premix (Diet S) or the same diet without premix supplementation (Diet U). The analyzed micronutrient composition of diets differed accordingly. Pooled livers from 15 fish from each dietary group were used to create suppression subtractive hybridization (SSH) cDNA libraries that were sequenced with 454 FLX GS Titanium Technology. In total 552 812 reads were sequenced from the two cDNA libraries. Ingenuity pathway analysis (IPA) was then used to characterize the hepatic transcriptome of the two dietary groups of rainbow trout. In the present communication we discuss how selected micronutrients may affect the transcriptome at suboptimal status by directly impacting the cellular metabolism, functions, and structures, and by introducing respective compensatory mechanisms. Processes related to lipid metabolism, peptide hydrolysis, oxygen transportation, and growth development were mostly affected. Considering the transcriptomics data relative to changes in nutritional status from the feeding study and the background phenotypic outcome of growth performance and gill histopathology, the outcome of the transcriptional profiling are suggested to be mainly related to suboptimal pantothenic acid and vitamin C nutrition.

Zebrafish (Danio rerio) as a model for investigating dietary toxic effects of deoxynivalenol contamination in aquaculture feeds.

The effects of feeding six diets spiked with increasing levels of DON for 45 days to zebrafish (Danio rerio) on performance and liver gene biomarkers were investigated. In addition long term effects on fecundity, offspring larvae swimming activity and global DNA methylation in embryos were investigated. Zebrafish performance was not affected. Liver CYP1A mRNA levels were significantly higher in fish fed 2.0 ppm DON compared to the control group, 0.1, 0.5 and 1.5 ppm group. Gene transcripts of CuZn SOD and Cyclin G1 increased with increasing content of dietary DON. The percentage of 5-methylcytosine in embryos did not differ and was 7.0-7.1% across the groups. Fecundity showed a biphasic response pattern. Interestingly, fish fed 1.5 ppm DON had 22% higher fecundity compared to control. A trend towards increased larvae swimming activity was seen in the high DON group. Our data suggest that DON is detoxified in the liver through the phase 1 system resulting in a disturbance in the oxidative balance. We do not know if effects observed on fecundity and larvae swimming activity are attributed to a direct interaction of DON with the reproductive organ or secondary to the maternal/paternal liver oxidative imbalance.

Proteomic profiling of liver from Atlantic salmon (Salmo salar) fed genetically modified soy compared to the near-isogenic non-GM line.

The aim of this study was to investigate potential differences in liver protein expression of Atlantic salmon fed genetically modified (GM) Roundup Ready soy at a high inclusion level (25% inclusion, constituting 21% of crude protein in the diet) for 7 months or a compositionally similar non-GM diet. The liver was selected as the target organ due to its importance in the general metabolism, and 2D gel electrophoresis used as a screening tool. Samples from 12 individual fish from each diet group were evaluated. Of a total of 781 analysed protein spots, only 36 were significantly different by ANOVA (p < 0.05) in abundance between the diet groups. All these spots had low fold differences (1.2-1.6) and high false discovery rate (q = 0.44), indicating minor differences in liver protein synthesis between fish fed GM and non-GM soy. Additionally, low fold differences were observed. Four protein spots were analyzed by liquid chromatography tandem mass spectrometry and identified using a combination of online searches in NCBI and searches in an inhouse database containing salmonid expressed sequence tags and contigs. Follow-up on these proteins by real-time polymerase chain reaction did not identify differences at the transcriptional level.

A high retinol dietary intake increases its apical absorption by the proximal small intestine of juvenile sunshine bass (Morone chrysops x M. saxatilis).

The relationship between dietary intake and systemic availability of retinol is likely to be complex because although retinol is an essential nutrient, it is toxic at high levels. The present study determined whether rates of transapical retinol absorption are modulated so that availability is increased at low dietary levels, but decreased when dietary intake is excessive. Juvenile hybrid striped bass were fed for 6 wk diets with 568 (below), 1657 (approximating the requirement) and 40,244 (excessive) micro g/kg dry diet of trans retinol. Proximal small intestine segments were used to measure rates of retinol absorption and tissue concentrations. Initial and final body mass did not differ among groups; deficiency and toxicity symptoms were not observed. Uptake of tracer retinol was inhibited by unlabeled retinol, indicating the presence of saturable, carrier-mediated absorption. Increasing dietary levels of retinol increased the rates of absorption measured at 0.05 mmol/L [8.04 +/- 0.65; 15.2 +/- 1.53; 25.1 +/- 3.4 pmol/(mg. min) for below, approximating and exceeding the retinol requirement; P < 0.0001]; this resulted in higher tissue concentrations of all-trans retinol (0.21 +/- 0.03, 0.49 +/- 0.21 and 338 +/- 89 pmol/g; P < 0.0001) and dehydro-retinol (0.11 +/- 0.04, 0.91 +/- 0.04, and 454 +/- 109 pmol/g; P < 0.001). These findings suggest that the systemic availability of various dietary levels of retinol is modulated after transapical absorption.