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

High-glucose feeding of gilthead seabream (Sparus aurata) larvae: effects on molecular and metabolic pathways

Nutritional programming has begun to arouse interest as a novel tool to alter specific metabolic pathways or functions in farmed animals. The aim of the present study was to explore the potential of early glucose stimuli to induce changes in nutrient metabolism of gilthead seabream. Nutritional conditioning was performed by delivering glucose-rich feed at three distinct recurrent periods of larval feeding regime: during first-feeding with rotifers (3 days after hatching, DAH) and mid-feeding with Artemia metanauplii (20DAH) and the beginning of inert diet feeding (30DAH), called the Recurrent treatment (REC). As opposed, the control treatment (CTRL) did not experience any glucose stimuli. At post-larval stage (from 50 to 60DAH), both treatments were challenged with a high-carbohydrate diet (50%). The immediate response to the early stimuli was assessed through gene expression of metabolic markers and by nutrient metabolism using [C-14] tracers. Each dietary stimulus induced metabolic changes on REC larvae, shown by altered expression of some genes, including those involved in glycolysis, and by a different pattern of glucose utilization. However, none of the molecular adaptations (except G6PDH gene) were persistent in the viscera and muscle of challenged post-larvae from REC group. In contrast, the glucose metabolism of challenged REC post-larvae revealed a shift towards a higher catabolism and lower glucose retention in tissues, compared to the CTRL group, suggesting an improvement of glucose oxidation pathways. In addition, the REC group showed a higher bio-conversion of glucose into lipids, indicating enhanced hepatic lipogenesis. The early stimuli did not affect the relative retention or use of amino acids or the growth and survival of challenged fish, up to 60DAH. In summary, although not substantiated at a molecular level, our data reveal that a recurrent high-glucose stimulus during larval stages affects the short-term modulation of pathways for glucose utilization in gilthead seabream. (C) 2015 Elsevier B.V. All rights reserved

Weaning of Senegalese sole (Solea senegalensis) postlarvae to an inert diet with a co-feeding regime | Destete de postlarvas del lenguado senegalés (Solea senegalensis) a una dieta inerte utilizando un régimen de coalimentación

The objective of this study was to evaluate the weaning success of Senegalese sole, Solea senegalensis, postlarvae when cofed a compound diet, by means of the analysis of growth parameters (relative growth rate [RGR] and condition factor [K]), survival, protein and lipid content. Total ammonia in the rearing water was also measured. The experiment lasted for 46 days (36 to 82 days posthatching). One group was fed only with enriched Artemia sp. metanauplii (Artemia treatment), whereas in another group, the Artemia sp. metanauplii were gradually replaced by the International Council for the Exploration of the Sea (ICES) diet over a period of 39 days (ICES treatment). Postlarvae were sampled on rearing days 0, 9, 23, 30, 39 and 46 for growth and biochemical analysis. Water was sampled on rearing day 30 during a 24-h cycle in both treatments. Postlarvae from the ICES treatment were weaned, though by the end of the experiment the Artemia treatment exhibited significantly higher values for growth, RGR, K and survival rates (P <0.05). No significant differences were observed concerning total lipid content. Protein content was significantly higher for the Artemia treatment after 46 days of rearing (P < 0.05). The total ammonia nitrogen concentration in the water increased after each meal and remained below a harmless level for postlarvae.El objetivo de este estudio consistió en evaluar el éxito del destete de postlarvas del lenguado senegalés, Solea senegalensis, utilizando un régimen de coalimentación. El estudio se realizó mediante el análisis de parámetros de crecimiento (tasa de crecimiento relativa [RGR] y factor de condición [K]), supervivencia y contenido de proteínas y lípidos. La concentración del amonio total en el agua de cultivo también fue analizada. El experimento se realizó durante 46 días (de 36 a 82 días posteclosión). Un grupo de postlarvas se alimentó únicamente con metanauplios de Artemia sp. enriquecidos (tratamiento con Artemia), mientras que en otro grupo los metanauplius de Artemia sp. fueron gradualmente substituidos por la dieta del International Council for the Exploration of the Sea (tratamiento ICES) durante un periodo de 39 días. Las postlarvas se muestrearon durante los días 0, 9, 23, 30, 39 y 46 de cultivo para el estudio de crecimiento y el análisis bioquímico Las muestras de agua se tomaron el día 30 de cultivo durante un ciclo de 24 h en ambos tratamientos. Las postlarvas del tratamiento ICES fueron destetadas, aunque al final del experimento se registraron valores significativamente mayores en relación al crecimiento, RGR, K y tasas de supervivencia (P < 0.05) en el tratamiento con Artemia. No se observaron diferencias significativas en el contenido total de lípidos. El contenido de proteínas fue significativamente mayor en el tratamiento con Artemia a los 46 días de cultivo (P < 0.05). La concentracion de amonio total en el agua se incrementó después de cada toma de alimento pero se mantuvo por debajo del nivel tóxico para las postlarvas de peces marinos

Weaning of Senegalese sole (Solea senegalensis) postlarvae to an inert diet with a co-feeding regime

The objective of this study was to evaluate the weaning success of Senegalese sole, Solea senegalensis, postlarvae when co-fed a compound diet, by means of the analysis of growth parameters (relative growth rate [RGR] and condition factor [K]), survival, protein and lipid content. Total ammonia in the rearing water was also measured. The experiment lasted for 46 days (36 to 82 days posthatching). One group was fed only with enriched Artemia sp. metanauplii (Artemia treatment), whereas in another group, the Artemia sp. metanauplii were gradually replaced by the International Council for the Exploration of the Sea (ICES) diet over a period of 39 days (ICES treatment). Postlarvae were sampled on rearing days 0, 9, 23, 30, 39 and 46 for growth and biochemical analysis. Water was sampled on rearing day 30 during a 24-h cycle in both treatments. Postlarvae from the ICES treatment were weaned, though by the end of the experiment the Artemia treatment exhibited significantly higher values for growth, RGR, K and survival rates (P < 0.05). No significant differences were observed concerning total lipid content. Protein content was significantly higher for the Artemia treatment after 46 days of rearing (P < 0.05). The total ammonia nitrogen concentration in the water increased after each meal and remained below a harmless level for postlarvae.info:eu-repo/semantics/publishedVersio

Daily feeding and protein metabolism rhythms in Senegalese sole post-larvae

Fish hatcheries must adapt larval feeding protocols to feeding behavior and metabolism patterns to obtain more efficient feed utilization. Fish larvae exhibit daily ingesting rhythms rather than ingesting food continuously throughout the day. The aim of this study was to determine the daily patterns of feed intake, protein digestibility, protein retention and catabolism in Senegalese sole post-larvae (Solea senegalensis; 33 days post-hatching) using C-14-labeled Artemia protein and incubation in metabolic chambers. Sole post-larvae were fed at 09: 00, 15: 00, 21: 00, 03: 00 and 09: 00+1 day; and those fed at 09: 00, 21: 00, 03: 00 and 09: 00+1 day showed significantly higher feed intake than post-larvae fed at 15: 00 h (P=0.000). Digestibility and evacuation rate of ingested protein did not change during the whole cycle (P=0.114); however, post-larvae fed at 21: 00 and 03: 00 h showed the significantly highest protein retention efficiency and lowest catabolism (P=0.002). Therefore, results confirm the existence of daily rhythmicity in feeding activity and in the utilization of the ingested nutrients in Senegalese sole post-larvae.Fundacao para a Ciencia e a Tecnologia (FCT; Portugal) [CCMAR/Multi/04326/2013, 310305/FEP/71, IF/00482/2014/CP1217/CT0005]; PROMAR Program; Fundo Regional para a Ciencia e Tecnologia (FEDER); Ministerio de Economia y Competitividad (MINECO; Spain) by project EFISHDIGEST [AGL2014-52888-R]; FEDER/European Region Development Fund (ERDF); Ministerio de Economia y Competitividad (Spain) [BES-2012-051956]; European Social Fund under the Operational Programme for the Enhancement of Human Potentialinfo:eu-repo/semantics/publishedVersio

Improving growth performance of Senegalese sole postlarvae

Tese dout., Aquacultura, Universidade do Algarve, 200

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

Daily oxygen consumption rhythms of Senegalese sole solea senegalensis (Kaup, 1858) juveniles

Senegalese sole is known to be a species with pronounced nocturnal feeding behaviour. However, as for most fish species, there is a lack of knowledge concerning the influence of such biological rhythmon metabolic rate. The aim of this study was to determine whether individual variation in routine and fed metabolic rate was affected by daily light–dark rhythms in juveniles of Senegalese sole. The individual oxygen consumption measurements in Senegalese sole juveniles were determined by flow-through respirometry, at fasted conditions and after the fish were fed a single meal, the meal time started at 0930 h and fish fed ad libitum for 30 min. The measurements were made during 22 h, of which 8 h was in the light and 14 h in the dark, and started immediately after transfer to the respiratory chambers at 1100 h. The results suggest an influence of light–dark cycles in routine metabolism. It was observed that oxygen consumption increased during the dark phase in fasted fish (FAST) but was higher during the light phase in fed fish (FEED). However, when feed is provided during the light phase, juveniles are capable of shifting oxygen consumption rhythms to respond to the energetic demands of digestion and growth. These results suggest that routine metabolism varies according to the species natural habits as Senegalese sole is known to be nocturnal. The findings of this study underline the importance of understanding the biological rhythms of the species under study before metabolic data are interpreted

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