Bacterial arsenite oxidation at the molecular level

According to the WHO, arsenic is one of the top 10 chemical contaminants in drinking-water worldwide and affects more than 140 million people. The arsenite oxidising enzyme (Aio), from microorganisms Rhizobium sp. NT-26 (NT-26_Aio) and Alcaligenes faecalis (A.f._Aio), and their final electron acceptors – cytochrome c552 (NT-26_cytc552) and azurin (A.f._azu), respectively – are currently being studied for their use as biosensors and in bioremediation processes. Both Aio enzymes share high structural similarity (948 matching residues with an r.m.s.d. of 1.84 Å for Cα atoms) and are composed of a large subunit (AioA) which contains a molybdenum centre and a [3Fe-4S] cluster, and a small subunit (AioB) that possess a Rieske [2Fe-2S] cluster. Aiming to elucidate the catalysis mechanism of the enzymes, and their electron transfer to the final electron acceptors, a combination of expression and purification of the proteins, crystallisation, structural analysis, enzyme kinetics and affinity tests were conducted. A 1.84 Å resolution structure of A.f._Aio in complex with a substrate analogue - SbV oxoanion - was determined using molecular replacement (PDB: IG8K). Additionally, a previously obtained 1.89 Å resolution structure of NT-26_Aio, containing a SbIII oxoanion near the active site, was investigated and used for comparison. Analysis of bond lengths and geometry of the ligands at the Mo active site suggests that both crystallized enzymes reveal different reaction intermediates, corresponding to different stages of the mechanism. The specific activity of two active site mutants of NT-26_Aio – D169A and E453A – determined by UV-Vis spectroscopy, revealed that these only uphold 46 and 8% of the WT enzyme efficiency, respectively. This information, together with structural analysis, strongly suggest that both amino acid residues play an essential role in substrate orientation through a complex network of hydrogen-bonds

Amino acid metabolism in gilthead seabream is affected by the dietary protein to energy ratios

The dietary protein to energy ratio (P/E) has proven to influence protein utilization and/or growth in several fish species. This study intended to unravel the bioavailability and metabolic fate of lysine and methionine in gilthead seabream (Sparus aurata) juveniles fed plant diets with different P/E ratios. Seabream juveniles were fed two isonitrogenous diets (45% crude protein) differing in crude lipids (20 and 14%): LowP/E (P/E ratio=20.0 mg protein kJ-1) and HighP/E (P/E ratio=21.4 mg protein kJ-1). After three weeks, fish (11.6 +/- 4.3 g) were tube-fed the respective diet labelled with C-14-protein (L-amino acid mixture), C-14-lysine, or C-14-methionine. Protein, lysine, and methionine utilization were determined based on the proportion of C-14-amino acid evacuated, retained in the free or protein-bound fraction of liver and muscle, or catabolized. This study revealed that a decrease in P/E ratio resulted in lower amino acid evacuation (p < 0.05), contributing to a more efficient amino acid uptake. Results indicate that amino acids are retained as protein in the liver and not only temporarily available in the free pool. The amount of free amino acids retained in the muscle of LowP/E fed fish was significantly higher than in HighP/E fish (p < 0.05) due to a simultaneous higher retention of lysine and methionine, without affecting the overall protein retention. Methionine catabolism was significantly lower than lysine or protein independently of the P/E ratio (p < 0.05), reinforcing that this amino acid is preferentially spared for metabolic functions and not used as energy source. In contrast, increasing the dietary P/E ratio decreased lysine catabolism and increased its availability for growth. The bioavailability and metabolism of individual amino acids should be considered when optimizing P/E ratios in diets for gilthead seabream juveniles. Formulating diets with optimum P/E ratios will improve diet utilization and fish performance.info:eu-repo/semantics/publishedVersio

Antemortem versus postmortem methods for detection of betanodavirus in Senegalese sole (Solea senegalensis)

The suitability of nested reverse transcription polymerase chain reaction (nRT-PCR) to detect betanodavirus in blood samples from naturally infected Senegalese sole (Solea senegalensis) was evaluated in comparison with other diagnostic methods. Results indicated that histologic examination of brain lesions could be regarded as the most consistent indicator of nodavirus infection in this species. The nRT-PCR showed low to moderate levels of detection; the best values were obtained in brain samples followed by blood samples. Inoculation of SSN-1 and SAF-1 cells with fish samples did not cause cytopathic effect, although virus was detected by reverse transcription polymerase chain reaction in approximately 25% of the SSN-1 inoculated wells. The efficiency of detection of the viral genome was dramatically increased by the use of nRTPCR, reaching 90.6% of positives in brain samples and 84.4% in blood samples. The sensitivity and the negative predictive value of nRT-PCR in blood samples were slightly lower than those obtained using brain samples. Nevertheless, it is suggested that the advantage of being able to perform diagnosis on live fish adequately counterbalances the slightly lower sensitivity of nRT-PCR on blood samples. This technique is proposed as a useful tool, not only for the selection of nodavirus-free breeders but also to check the fish status during ongrowing

Metabolic fate is defined by amino acid nature in gilthead seabream fed different diet formulations

The sustainability of the Aquaculture industry relies on optimising diets to promote nitrogen retention and maximise fish growth. The aim of this study was to assess how different dietary formulations influence the bioavailability and metabolic fate of distinct amino acids in gilthead seabream juveniles. Amino acids (lysine, tryptophan, and methionine) were selected based on their ketogenic and/or glucogenic nature. Seabream were fed practical diets with different protein (44 and 40%) and lipid contents (21 and 18%): 44P21L, 44P18L, 40P21L, and 40P18L. After three weeks of feeding, the fish were tube-fed the correspondent diet labelled with 14C-lysine, 14C-tryptophan, or 14C-methionine. The amino acid utilisation was determined based on the evacuation, retention in gut, liver, and muscle, and the catabolism of the tracer. The metabolic fate of amino acids was mainly determined by their nature. Tryptophan was significantly more evacuated than lysine or methionine, indicating a lower availability for metabolic purposes. Methionine was more retained in muscle, indicating its higher availability. Lysine was mainly catabolised, suggesting that catabolism is preferentially ketogenic, even when this amino acid is deficient in diets. This study underpins the importance of optimising diets considering the amino acids’ bioavailability and metabolic fate to maximise protein retention in fish.info:eu-repo/semantics/publishedVersio

Modulation of dietary protein to lipid ratios for gilthead seabream on-growing during summer temperature conditions

Gilthead seabream (Sparus aurata) tend to increase fat deposition during summer farming conditions in the Mediterranean, which may negatively affect productive performance and consumers' quality perception of the final product. Therefore, this study evaluated the impacts of protein to lipid ratios in low fishmeal/fish oil diets on growth performance, body composition, feed conversion and nutrient utilization of seabream on-grown during summer temperature conditions. The experimental diets contained low levels of fishmeal, fish oil, and crude protein (39%), differing in crude lipid content: 16% (MF diet) or 12% (LF diet). A growth trial was per-formed with seabream (initial weight: 100 & PLUSMN; 7 g) from August to October (water temperature: 23.1 & PLUSMN; 2.2 & DEG;C). A digestibility trial was also performed (at 23 & DEG;C). Key performance indicators, whole-body composition and ac-tivities of digestive enzymes were evaluated at the end of the experiment (64 days). Low dietary lipid levels negatively affected lipid, energy, and amino acid digestibility, and as a result, fish fed the LF diet presented higher nitrogen faecal losses. Still, the decrease in nutrient digestibility was not related to dietary effects on the digestive enzyme activities. The experimental diets did not compromise the activity of pancreatic, gastric, and intestinal digestive enzymes nor feed utilization, but a slight growth impairment was observed in fish fed the LF diet, probably due to the lower amino acid and lipid digestibility. However, a potential benefit of this dietary treatment towards reducing fat accumulation in seabream during summer was observed. Nevertheless, the environmental impact of the nitrogen losses during seabream on-growing should be considered when estimating the sustainability of the production. This study demonstrated that the optimisation of diet formulations should account for the environmental conditions, especially in Mediterranean aquaculture, so the economic and envi-ronmental impacts may be correctly evaluated towards a more sustainable fish production.info:eu-repo/semantics/publishedVersio

Molecular regulation of muscle development and growth in Senegalese sole larvae exposed to temperature fluctuations

Author's accepted version (post-print).NOTICE: this is the author’s version of a work that was accepted for publication in Aquaculture (2014). Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Aquaculture (2014), 432. doi: http://dx.doi.org/10.1016/j.aquaculture.2014.04.035.The Senegalese sole (Solea senegalensis) is a marine flatfish that is naturally exposed to high temperature fluctuations (12 - 28 ºC) in the wild, with a life cycle predominantly estuarine during larval and juvenile phases. Farming of this species has largely improved in the past years but marked fluctuations of temperature during production still contribute to variation on growth and muscle cellularity, particularly if they occur during early stages of development. Such thermal plasticity of muscle growth must arise through changes in a multitude of physiological and molecular pathways, in which epigenetic gene regulation is likely to play an essential role. In the present work, we review recent studies addressing molecular, physiological and morphological aspects of the thermal plasticity of somatic growth in Senegalese sole larvae and early juveniles, thus aiming to improve sole rearing in aquaculture production. The present study shows that temperature during specific time frames of ontogeny has both short- and long-term effects on growth and muscle cellularity of Senegalese sole. Nevertheless, Senegalese sole also seems to rapidly adapt to environmental temperature through a set of epigenetic mechanisms and physiological responses such as regulation of feed intake, even at early developmental stages

Alternative proteins for fish diets: implications beyond growth

Aquaculture has been challenged to find alternative ingredients to develop innovative feed formulations that foster a sustainable future growth. Given the most recent trends in fish feed formulation on the use of alternative protein sources to decrease the dependency of fishmeal, it is fundamental to evaluate the implications of this new paradigm for fish health and welfare. This work intends to comprehensively review the impacts of alternative and novel dietary protein sources on fish gut microbiota and health, stress and immune responses, disease resistance, and antioxidant capacity. The research results indicate that alternative protein sources, such as terrestrial plant proteins, rendered animal by-products, insect meals, micro- and macroalgae, and single cell proteins (e.g., yeasts), may negatively impact gut microbiota and health, thus affecting immune and stress responses. Nevertheless, some of the novel protein sources, such as insects and algae meals, have functional properties and may exert an immunostimulatory activity. Further research on the effects of novel protein sources, beyond growth, is clearly needed. The information gathered here is of utmost importance, in order to develop innovative diets that guarantee the production of healthy fish with high quality standards and optimised welfare conditions, thus contributing to a sustainable growth of the aquaculture industry.info:eu-repo/semantics/publishedVersio

Data on European seabass fed with methionine-enriched diets obtained through label free shotgun proteomics

This data article is associated with the research article "Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomics". Here it is described the data obtained from proteomic analysis of 36 European seabass juveniles (3 fish x 3 replicate tanks) after 18 days of feeding with experimental diets containing four inclusion levels of methionine (Met): 0.77%, 1%, 1.36% and 1.66% Met (w/w). We analysed this dataset and compared it with that obtained during the long-term feeding period i.e., 85 days. Fish liver proteins were digested with trypsin and purified peptides were analysed by LC-MS/MS. Proteins were identified with at least two peptides at 0.1% Decoy false discovery rate (FDR). In this dataset, we present the analysis of the differential abundant proteins (DAP) with significant differences across treatments after 18 days of feeding (One-Way ANOVA, p < 0.05). Treatment's comparisons were also performed between the 18- and 85-days feeding trials through Two-Way ANOVA (p < 0.05). MS/MS raw data are available via ProteomeXChange with identifiers PXD019610 and 10.6019/PXD019610 (18-days dataset); and PXD019622 and 10.6019/PXD019622 (85-days dataset). This dataset corresponds to fish sampled after 18-days of experimental trial and is made available to support the study conducted in the afore-mentioned article, by performing the analysis during a short-term period of feeding. The data presented may be further used in other nutritional studies e.g., addressing hepatic changes mediated by Met.ALG-01-0247-FEDER-3520/ UIDB/04326/2020/ IF/00482/2014/CP1217/CT0005info:eu-repo/semantics/publishedVersio

Fish processing and digestion affect parvalbumins detectability in Gilthead Seabream and European seabass

Consumption of aquatic food, including fish, accounts for 17% of animal protein intake. However, fish consumption might also result in several side-effects such as sneezing, swelling and anaphylaxis in sensitized consumers. Fish allergy is an immune reaction to allergenic proteins in the fish muscle, for instance parvalbumin (PV), considered the major fish allergen. In this study, we characterize PV in two economically important fish species for southern European aquaculture, namely gilthead seabream and European seabass, to understand its stability during in vitro digestion and fish processing. This information is crucial for future studies on the allergenicity of processed fish products. PVs were extracted from fish muscles, identified by mass spectrometry (MS), and detected by sandwich enzyme-linked immunosorbent assay (ELISA) after simulated digestion and various food processing treatments. Secondary structures were determined by circular dichroism (CD) after purification by anion exchange and gel filtration chromatography. In both species, PVs presented as α-helical and β-sheet structures, at room temperature, were shown to unfold at boiling temperatures. In European seabass, PV detectability decreased during the simulated digestion and after 240 min (intestinal phase) no detection was observed, while steaming showed a decrease (p < 0.05) in PVs detectability in comparison to raw muscle samples, for both species. Additionally, freezing (−20 °C) for up to 12 months continued to reduce the detectability of PV in tested processing techniques. We concluded that PVs from both species are susceptible to digestion and processing techniques such as steaming and freezing. Our study obtained preliminary results for further research on the allergenic potential of PV after digestion and processing