Modulation of Immune-Related Gene Expressions in Zebrafish (Danio rerio) by Dietary Purslane (Portulaca oleracea) Extract.

To promote fish's immunity against pathogens in the aquaculture industry, fish dietary fortification with additives or compounds has increasingly attracted attention. In the present study, zebrafish (Danio rerio) was used as an animal model to investigate the effects of purslane, Portulaca oleracea, extract (PE) on the relative expression level of some immune-related genes. A total of 300 zebrafish were randomly divided into four treatment groups and fed for 8 weeks with the basal diets supplemented with 0.5, 1, 1.5, and 2% of PE. The control group was fed with a basal diet without PE. At the end of 8 weeks, the mRNA expression levels of interleukin 1-beta (IL-1ß), interleukin 10 (IL-10), transforming growth factor-beta (TGF-ß), tumor necrosis factor-alpha (TNF-α), superoxide dismutase (SOD), and lysozyme (LYZ) in the fish were evaluated. The results showed that the mRNA expression level of IL-1ß was significantly upregulated in the fish fed with 1 and 2% PE compared to the control group (p < 0.05). Moreover, the evaluation of the mRNA expression level of TGF-ß was significantly increased in a dose-dependent manner in the 1.5 and 2% fed groups compared to the control group (p < 0.05). However, the IL-10 was significantly downregulated in all treated groups compared to the control group (p < 0.05). The expression of the TNF-α gene was not affected amongst all groups by the inclusion of PE in the zebrafish diet (p > 0.05). Based on the results, the diet supplemented with 1.5 and 2% PE significantly upregulated the mRNA expression levels of LYZ and SOD, respectively, compared to the control group (p < 0.05). In conclusion, dietary inclusion of PE may result in beneficial effects on some immune responses via upregulation of some immune genes in zebrafish.

Toxicity of Zinc at sublethal exposure to rainbow trout (Oncorhynchus mykiss) / Toxicidad de zinc por exposición subletal en trucha arco iris (Oncorhynchus mykiss) / Toxicidade por exposição ao zinco em trutas arco-íris (Oncorhynchus mykiss)

Abstract Zinc (Zn), can alter different physiological processes in fish such as growth and serum parameters. The objective of this study was to investigate the toxicity of Zn on sublethal exposure to rainbow trout (Oncorhnychus mykiss). Fish exposed to higher levels of Zn grew slower than fish exposed to lower levels of Zn. Weight gain and SGR (Specific Growth Rate) decreased linearly as the Zn level in the water increased. The CF (Condition factor) of fish grown in water with a high concentration of Zn also decreased significantly compared to the low concentration of Zn, whereas the Food Conversion Ratio (FCR) increased with the concentration of Zn in water. Both AST (Aspartate Transaminase) and ALT (Alanine Transaminase) activities were significantly altered by Zn and showed a linear pattern and decreased after 30 days, while ALP (Alkaline Phosphatase) levels showed a remarkable increase from a mean level of control. This study suggests that biochemical parameters of growth and serum could be used as important and sensitive biomarkers to monitor heavy metal toxicity on fish health status.

Resumen TZinc (Zn), puede alterar diferentes procesos fisiológicos en peces, tales como, el crecimiento y los parámetros séricos. El objetivo de este estudio fue investigar la toxicidad de Zn en la exposición subletal de la trucha arco iris (Oncorhnychus mykiss). Los peces expuestos a niveles más altos de Zn crecieron más lentamente que los peces expuestos a niveles más bajos de Zn. El aumento de peso y la SGR (tasa de crecimiento específica) disminuyeron linealmente a medida que aumentó el nivel de Zn en el agua. El CF (factor de condición) de los peces cultivados en agua con una alta concentración de Zn, también disminuyó significativamente en comparación con la baja concentración de Zn, mientras que el índice de conversión de alimentos (FCR) aumentó con la concentración de Zn en el agua. Las actividades AST (Aspartate Transaminase) y ALT (Alanine Transaminase) fueron significativamente alteradas por el Zn y mostraron un patrón lineal y disminuyeron después de 30 días, mientras que los niveles de ALP (Fosfatasa Alcalina) mostraron un aumento notable desde un nivel medio de control. Este estudio sugiere que los parámetros bioquímicos de crecimiento y suero podrían usarse como biomarcadores importantes y sensibles para controlar la toxicidad de metales pesados en el estado de salud de los peces.

Resumo O TZinc (Zn) pode alterar diferentes processos fisiológicos em peixes, como parámetros de crescimento e soro. O objetivo deste estudo foi investigar a toxicidade do Zn na exposição subletal à truta arco-íris (Oncorhnychus mykiss). Os peixes expostos a níveis mais altos de Zn cresceram mais lentamente do que os peixes expostos a níveis mais baixos de Zn. O ganho de peso e a SGR (Taxa de Crescimento Específico) diminuíram linearmente à medida que o nível de Zn na água aumentou. O CF (fator de condição) de peixes cultivados em água com alta concentração de Zn também diminuiu significativamente em comparação com a baixa concentração de Zn, enquanto a tax on de conversão alimentar (FCR) aumentou com a concentração de Zn na água. As atividades de AST (Aspartate Transaminase) e ALT (Alanine Transaminase) foram significativamente alteradas pelo Zn e mostraram um padrão linear e diminuíram após 30 dias, enquanto os níveis de ALP (Fosfatase Alcalina) mostraram um aumento notável em relação ao nível médio de controle. Este estudo sugere que parâmetros bioquímicos de crescimento e soro podem ser usados como biomarcadores importantes e sensíveis para monitorar a toxicidade de metais pesados no estado de saúde dos peixes.

Effects of cadmium at sub-lethal concentration on growth and biochemical parameters in rainbow trout (Oncorhynchus mykiss).

Cadmium (Cd), as one of heavy metals and an environmental stressor, may alter many physiological processes like growth and serum parameters in fish. The main objective of this study was to determine the effects of cadmium at sub-lethal concentrations (1 and 3 µg/l) on growth and serum biochemical parameters including enzymes, i.e. alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), glucose, triglyceride, cholesterol and total protein in rainbow trout (Oncorhynchus mykiss). Trout were exposed to cadmium, and, at intervals of 1, 15, and 30 days, selected parameters were evaluated. Condition Factor (K), Specific Growth Rate (SGR) and Body Weight Gain (BWG) consistently decreased, while Food Conversion Ratio (FCR) increased at the end of experiment. Glucose was elevated in trout exposed to both Cd concentrations at day 15 and then returned to levels comparable to control fish. Triglyceride and cholesterol decreased transiently at day 15 and then increased at day 30. Total protein, AST, ALT and ALP increased linearly by time and Cd concentration. This investigation suggests that growth and serum biochemical parameters could be used as important and sensitive biomarkers in ecotoxicological studies concerning the effects of metal contamination and fish health.

Effects of dietary nucleotides supplementation on rainbow trout (Oncorhynchus mykiss) performance and acute stress response.

This experiment was conducted to examine the effect of dietary nucleotides (NT) on fish performance and acute stress response on fingerling rainbow trout (23 g ± 0.01, mean weight ± SEM). Five experimental diets according to different levels of supplemented nucleotides (0, 0.05, 0.1, 0.15, and 0.2%) were assayed on experimental fish for 8 weeks. Growth, hematological parameters (hematocrit, hemoglobin, erythrocyte, lymphocyte, and neutrophil count), serum proteins (globulin, albumin), and plasma enzymatic activity (alkaline phosphatase, ALP; aspartate transaminase, AST; lactate dehydrogenase, LDH; alanine transaminase, ALT) were assayed. At the end of feeding trial, fish fed the control and 0.2% diets were subjected to handling and crowding stress. Modulatory effects of nucleotides on acute stress response (cortisol and glucose) and plasma electrolytes (Na(+), Cl(-), K(+), and Ca(2+)) were studied. The percentage of body weight gain (WG) and feed efficiency (FE) of fish were better when the fish were fed 0.15-0.2% diets. Fish fed the nucleotide-supplemented diets tended to have lower levels of serum enzymes including ALP, AST, LDH, and ALT. Plasma cortisol levels of fish fed on 0.2% diet under handling and crowding stress were significantly lower than fish fed the control diet at all post-stress time intervals. In our study, fish fed nucleotide-supplemented diet had significantly lower concentrations of glucose compared to those fed the basal diet. The concentrations of sodium, chloride, calcium, and potassium of fish fed the control diet were significantly lower than in fish fed nucleotide-supplemented diet. Dietary nucleotides administration seems to promote growth and to enhance resistance against handling and crowding stress in fingerling rainbow trout.

Dietary administration of nucleotides to enhance growth, humoral immune responses, and disease resistance of the rainbow trout (Oncorhynchus mykiss) fingerlings.

A feeding study was conducted to determine the effect of dietary nucleotides (NT) on growth performance, immune responses and resistance of rainbow trout fingerlings to Streptococcus iniae. A basal diet supplemented with 0 (control), 0.5, 1, 1.5 and 2 g NT kg(-1) to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish with initial average weight of approximately 23 g. After 8 weeks of feeding trial, fish fed diets with 1.5-2 g NT kg(-1) had the highest final weight and SGR, followed by groups fed the diets with 0.5-1 g NT kg(-1), and lowest in fish fed the NT-free control diet. The feed conversion ratio (FCR) was better when the fish were fed diet with 2 g NT kg(-1). The ACH(50) level and lysozyme activity in serum were found to be significantly (P < 0.05) greater in fish fed nucleotides at 1.5-2 g kg(-1). The fish fed with 2 g NT kg(-1) had higher IgM level. The challenge experiment showed that relative percent survival (RPS) increased significantly (P < 0.05) with increasing levels of nucleotides from 0.5 to 2 g kg(-1) and was lowest in the control group. We therefore recommend dietary nucleotide administration at 1.5-2 g kg(-1) to promote growth and enhance immunity and resistance against S. iniae.

Arsenic and mercury in commercially valuable fish species from the Persian Gulf: influence of season and habitat.

This study was undertaken to determine the concentrations of arsenic and mercury in edible muscle of pelagic (Scomberomorus commerson, Chirocentrus dorab, Sphyraena jello, Rachycentron conadum, Thunus tonggol), benthopelagic (Trichiurus lepturus, Caranx sem, Pampus argenteus, Parastromateus niger) and demersal (Nemipterus japonicas, Epinephelus coioides, Platycephalus indicus, Psettodes erumei, Pomadasys argenteus, Acanthopagrus latus) fish species from the Persian Gulf during summer and winter. The samples were analyzed by graphite furnace atomic absorption spectrometry (GFAAS) for arsenic and cold vapor atomic absorption spectrometry (CVAAS) for mercury after wet-ashing digestion. The results were expressed as µg/g of wet weight. The resulted range of contamination in fish species was between 0.156-0.834 µg/g for arsenic; and 0.120-0.527 µg/g for mercury. Also study revealed that seasonal variation influenced the concentration of the metals in the fish samples. The highest concentration of arsenic and mercury was found in Epinephelus coioides (1.011 µg/g) and Thunus tonggol (0.548 µg/g) in winter, respectively. Moreover, demersal fish species had the highest arsenic (0.481 µg/g) and mercury (0.312 µg/g) concentrations. Our study demonstrates that estimated daily and weekly intakes of arsenic and mercury via consumption of fish flesh were far below the PTDI and PTWI values established by FAO/WHO.

Early interactions of Flavobacterium psychrophilum with macrophages of rainbow trout Oncorhynchus mykiss.

The early interactions of a low and a highly virulent Flavobacterium psychrophilum strain with head kidney and spleen macrophages of rainbow trout Oncorhynchus mykiss were characterized. The highly virulent strain was killed 5.8 to 11 times less frequently than the low virulent strain. The head kidney macrophages showed a microbicidal activity approximately twice as high as that of the spleen macrophages. A 2- to 3-fold higher production of reactive oxygen species (ROS) was induced by the highly virulent strain than by the low virulent one. The head kidney macrophages produced approximately twice as much ROS as the spleen macrophages. The low virulent strain was killed approximately 10 times more frequently by H2O2 than was the highly virulent strain. In spleen macrophages, the highly virulent strain caused twice as much cytotoxic effects compared to the low virulent strain. In conclusion, virulence in F. psychrophilum appears to be correlated with higher O. mykiss macrophage cytotoxicity and resistance to ROS and, therefore, with enhanced resistance to bacterial killing. Moreover, due to lower ROS production, spleen macrophages have a lower antimicrobial action against F. psychrophilum, compared to head kidney macrophages and, thus, might form a 'safe site' in which bacteria can reside.

Development of a gut perfusion model as an alternative to the use of live fish.

An alternative fish model with the principal aim of studying the interaction between fish pathogens and the intestinal tissue was developed. The preparation consisted of an excised gut tractus from rainbow trout (Oncorhynchus mykiss), perfused through cannulation of the aorta intestinalis ventralis with filtered and heparinized Cortland+dextran 1% as the perfusion fluid. This perfusion fluid was delivered by means of a drip. The perfused gut tractus was suspended in a circular bath filled with Ringer solution, which was aerated and kept at a constant temperature of 12 degrees C. Unperfused gut placed in Ringer solution at the same temperature served as the negative control. Perfusion was effective in maintaining the gut in a healthy condition for at least 60 min with only slight oedema and sloughing of the epithelium. Conversely, the unperfused gut revealed excessive tissue degeneration and severe necrosis.

Association of Flavobacterium psychrophilum strains with intestinal explants of rainbow trout Oncorhynchus mykiss.

Flavobacterium psychrophilum is an important pathogen in rainbow trout Oncorhynchus mykiss. The portal of entry for F. psychrophilum is not well known. In this study, the role of the intestine as a colonization site for F. psychrophilum was determined. For this purpose, the ability of a high (Dubois) and a low (99/10A) virulence strain of F. psychrophilum to adhere to intestinal explants of rainbow trout was evaluated. After incubation, samples of the gut were examined bacteriologically, histologically and by electron microscopy. The number of gut-associated F. psychrophilum bacteria was significantly higher for the Dubois than for the 99/10A strain. Histological samples clearly showed numerous bacteria of the high virulence strain associated with the intestinal tissue as opposed to only a few bacteria of the low virulence strain. Additionally, extensive exfoliation of intestinal epithelium was noted after incubation with the high virulence strain, but less with the low virulence strain. These findings were confirmed using scanning electron microscopy and suggest that the intestinal epithelium might represent an important site for colonization of the F. psychrophilum strain and may act as a portal of entry for high virulence F. psychrophilum.

Adhesion of high and low virulence Flavobacterium psychrophilum strains to isolated gill arches of rainbow trout Oncorhynchus mykiss.

The ability of Flavobacterium psychrophilum to adhere to the gill tissue of rainbow trout Oncorhynchus mykiss was evaluated. A gill perfusion model was adopted, offering a number of advantages compared to other in vitro as well as in vivo models. A comparison between the adhesion capacity of a high and low virulence F. psychrophilum strain was made. Experiments were additionally carried out to assess the influence of water quality (organic material, nitrite) and temperature on the adhesion process of the bacterial cells. The high virulence strain attached more readily to the gill tissue than did the low virulence strain. Moreover, the adherence of the high virulence strain of F. psychrophilum was influenced by a number of factors. These were immersion of the gill arches in water to which organic material or nitrite were added, and elevated temperature. The former 2 increased the adhesion ability, while the latter had a negative influence on the adherence process.