Aerobic and anaerobic metabolism for the zebrafish, Danio rerio, reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development

In order to verify the influence of chronic and acute ambient oxygen levels from egg to adult stage of the zebrafish, in vivo oxygen consumption (MO2), critical tensions of oxygen (Pcrit), heart rate (fH) and total body lactate concentration (Lc) were determined for Danio rerio (Hamilton, 1822) raised at 28 °C under normoxic (7.5 mgO2.L-1 or 80 mm.Hg-1) and hypoxic conditions (4.3 mgO2.L-1) and exposed to acute hypoxia during different developmental stages. Our findings confirmed that very early stages do not respond effectively to ambient acute hypoxia. However, after the stage corresponding to the age of 30 days, D. rerio was able to respond to acute hypoxia through effective physiological mechanisms involving aerobic and anaerobic metabolism. Such responses were more efficient for the fishes reared under hypoxia which showed that D. rerio survival capability increased during acclimation to mild hypoxia. Measurements of body mass and length showed that moderate hypoxia did not affect growth significantly until the fish reached the stage of 60 days. Moreover, a growth delay was verified for the hypoxic-reared animals. Also, the D. rerio eggs-to-larvae survival varied from 87.7 to 62.4% in animals reared under normoxia and mild hypoxia, respectively. However, the surviving animals raised under moderated hypoxia showed a better aptitude to regulate aerobic and anaerobic capacities when exposed to acute hypoxia.

Toxicity screening of Diclofenac, Propranolol, Sertraline and Simvastatin using Danio rerio and Paracentrotus lividus embryo bioassays

Early life-stage bioassays have been used as an alternative to short-term adult toxicity tests since they are cost-effective. A single couple can produce hundreds or thousands of embryos and hence can be used as a simple high-throughput approach in toxicity studies. In the present study, zebrafish and sea urchin embryo bioassays were used to test the toxicity of four pharmaceuticals belonging to different therapeutic classes: diclofenac, propranolol, simvastatin and sertraline. Simvastatin was the most toxic tested compound for zebrafish embryo, followed by diclofenac. Sertraline was the most toxic drug to sea urchin embryos, inducing development abnormalities at the ng/L range. Overall, our results highlight the potential of sea urchin embryo bioassay as a promising and sensitive approach for the high-throughput methods to test the toxicity of new chemicals, including pharmaceuticals, and identify several drugs that should go through more detailed toxicity assays.

Estudio de la expresión de Stxbp3, Stxbp6 y Claudin F en la formación del lumen en el oído interno del Pez Cebra, Danio rerio

Durante los últimos años se ha fomentado la investigación mediante el pez cebra como modelo biológico gracias a las considerables ventajas que ofrece respecto a modelos utilizados habitualmente. Una de las aplicaciones más destacadas de este modelo es en el estudio de las células sensoriales en el oído interno, ya que tienen un gran parecido con las células sensoriales de los humanos. Gracias a la facilidad de visualización y estudio de estas células en el pez cebra, se han podido llevar a cabo numerosas investigaciones sobre enfermedades que afectan a este órgano sensorial, así como la sordera. No obstante, para poder analizar todas las estructuras y células que forman parte del oído interno, es importante entender la morfogénesis de este órgano. Este proyecto se basa en el estudio de la morfogénesis del oído interno, concretamente, en la formación de lumen, una estructura que se forma en el oído interno en estadios tempranos del embrión, y que a partir de la cual se forman las demás estructuras que constituirán el oído interno. Para poder entender la formación del lumen en estadios tempranos del embrión, es necesario la caracterización de proteínas que participen en este proceso. Por lo que el objetivo principal de este proyecto es el estudio de la de la expresión de los genes Stxbp3, Stxbp6 y Claudin F en la apertura del lumen en el oído interno del pez cebra.

Natural preference of zebrafish (Danio rerio) for a dark environment

The zebrafish (Danio rerio) has been used as a model in neuroscience but knowledge about its behavior is limited. The aim of this study was to determine the preference of this fish species for a dark or light environment. Initially we used a place preference test and in a second experiment we applied an exit latency test. A two-chamber aquarium was used for the preference test. The aquarium consisted of a black chamber and a white chamber. In the first experiment the animal was placed in the aquarium and the time spent in the two compartments was recorded for 10 min. More time was spent in the black compartment (Wilcoxon matched-pairs signed-rank test, T = 7, N1 = N2 = 18, P = 0.0001). In the second experiment the animal was placed in the black or white compartment and the time it took to go from the initial compartment to the opposite one was recorded. The test lasted a maximum of 10 min. The results showed that the animal spent more time to go from the black to the white compartment (Mann-Whitney rank sum test, T = 48, N1 = 9, N2 = 8, P<0.0230). These data suggest that this fish species has a natural preference for a dark environment and this characteristic can be very useful for the development of new behavioral paradigms for fish.

Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis

The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tiete River Basin (Sao Paulo, Brazil)

The Tiete River and its tributary Pinheiros River receive a highly complex organic and inorganic pollutants load from sanitary sewage and industrial sources, as well as agricultural and agroindustrial activities. The aim of the present study was to evaluate the embryotoxic and teratogenic effects of sediments from selected locations in the Tiete River Basin by means of the sediment contact embryo toxicity assay with Danio rerio, in order to provide a comprehensive and realistic insight into the bioavailable hazard potential of these sediment samples. Lethal and sub-lethal effects were recorded, and high embryo toxicity could be found in the samples not only in the vicinity of the megacity Sao Paulo (Billings reservoir and Pinheiros River samples), but also downstream (in the reservoirs Barra Bonita, Promissao and Tres Irmaos). Results confirm that most toxicity is due to the discharges of the metropolitan area of Sao Paulo. However, they also indicate additional sources of pollutants along the river course, probably from industrial, agricultural and agroindustrial residues, which contribute to the degradation of each area. The sediment contact fish embryo test showed to be powerful tool to detect embryo toxicity in sediments, not only by being a sensitive method, but also for taking into account bioavailability. This test provides an ecological highly realistic and relevant exposure scenario, and should therefore be added in ecotoxicological sediment quality assessments. (C) 2011 Elsevier Inc. All rights reserved.

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: A basis for neurotoxicology

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure. (C) 2010 Elsevier Inc. All rights reserved.

Avaliação ecotoxicológica de efluentes industriais utilizando Danio rerio Hamilton-Buchanan, 1822 (TELEOSTEI, CYPRINIDAE)

The industrial effluents are one of the main sources of water pollution. For an appropriate characterization and control of their discharges, the most efficient strategy is the integrated use of chemical, physical and ecotoxicological analyses. The aims of this study were to asses the efficiency of the treatment plant of a textile industry performing acute toxicity tests and physical-chemical analyses of the effluents before and after the treatment, besides evaluate the toxicity of the effluents of the Treatment System of Liquids Effluents (Sistema de Tratamento de Efluentes Líquidos - SITEL) of Distrito Industrial de Natal (DIN) and some of their physical-chemical variables. The species used in the ecotoxicological tests was the fish Danio rerio. The results showed that the treatment plant reduced significantly (around 50%) the toxicity of the raw textile effluent in only three of the seven tests but, in general, it promoted the reduction of the physical-chemical parameters analyzed. The toxicity and the physical-chemical factors of the effluents of SITEL of DIN varied among the tests and show the importance of monitoring their discharges in the Potengi river, one of the most important rivers of the Rio Grande do Norte state

Investigação e avaliação da toxicidade aguda dos agrotóxicos mais utilizados no cinturão verde da Grande Natal (RN, Brasil) para o peixe-zebra (Danio rerio Hamilton Buchanan, 1822, Teleostei, Cyprinidae

The objectives of this research were to investigate the agrotoxic most used in the Gramorezinho region in the green belt of Natal, and to evaluate the acute toxicity of these, based on the LC50-48h values estimated in tests for Danio rerio, internationally used as test organism. The acute toxicity tests were performed under laboratory conditions, according to standardized methods (ABNT/NBR/15088/04) for this species. The LC50-48h estimated to Tamaron BR was 352.89 mg.L-1, which characterizes that as practically non-toxic, according to toxicological classes cited by Zucker. For Decis 25EC, the LC50-48h estimated was 0.0004156 mg.L-1 (4.156 X 10-4 mg.L-1), which classifies it as highly toxic to this species

Avaliação da toxicidade aguda do inseticida metomil e o seu efeito sobre a atividade da acetilcolinesterase do peixe Danio rerio

Methomyl (Lannate®) is an insecticide from the carbamate group, frequently used in pest control in various types of crops. This compound works inhibiting the activity of the enzyme acetylcholinesterase. The use of physicochemical and ecotoxicological analysis is the most efficient strategy for the correct characterization and control of residues of metomil. The main objectives of this study were to evaluate the acute toxicity of methomyl in 96 hours of exposure and, through a sublethal assay of 5 hours, to assess its effect on the activity of acetylcholinesterase present in brain and squeletic dorsal muscle of the Danio rerio fish. The results showed that the LC50-96 found to D. rerio was 3.4 mg/L and it was found through the average of four definitive tests. In vitro assays were used to test the inhibitory action of methomyl directly over soluble AChE, extracted from the squeletic dorsal muscle, with maximum inhibition of 68.57% to the insecticide concentrations of 0.2 mg/L. In sublethal tests with D. rerio, inhibitory effect of methomyl was found over the soluble form of AChE in the squeletic dorsal muscle, both in one and five hours of fish exposure to the insecticide. In both period, the average values of inhibition were around 61%. In the same condition, no significant inhibitory effect of methomyl soluble and membrane AChE of the D. rerio was observed in the 0.42, 0.85, 1.70 and 2.50 mg/L concentrations and in both times of fish exposure

Whole-body cortisol increases after direct and visual contact with a predator in zebrafish, Danio rerio

Ornamental fish may be severely affected by a stressful environment. Stressors impair the immune response, reproduction and growth rate; thus, the identification of possible stressors will aid to improve the overall quality of ornamental fish. The aim of this study was to determine whole-body cortisol of adult zebrafish, Danio rerio, following visual or direct contact with a predator species. Zebrafish were distributed in three groups: the first group, which consisted of zebrafish reared completely isolated of the predator, was considered the negative control; the second group, in which the predator, Parachromis managuensis was stocked together with zebrafish, was considered the positive control; the third group consisted of zebrafish stocked in a glass aquarium, with direct visual contact with the predator. The mean whole-body cortisol concentration in zebrafish from the negative control was 6.78 +/- 1.12 ng g(-1), a concentration statistically lower than that found in zebrafish having visual contact with the predator (9.26 +/- 0.88 ng g(-1)) which, in turn, was statistically lower than the mean whole-body cortisol of the positive control group (12.35 +/- 1.59 ng g(-1)). The higher whole-body cortisol concentration found in fish from the positive control can be attributed to the detection, by the zebrafish, of relevant risk situations that may involve a combination of chemical, olfactory and visual cues. One of the functions of elevated cortisol is to mobilize energy from body resources to cope with stress. The elevation of whole-body cortisol in fish subjected to visual contact with the predator involves only the visual cue in the recognition of predation risk. We hypothesized that the zebrafish could recognize predator characteristics in P managuensis, such as length, shape, color and behavior. Nonetheless, the elevation of whole-body cortisol in zebrafish suggested that the visual contact of the predator may elicit a stress response in prey fish. This assertion has a strong practical application concerning the species distribution in ornamental fish markets in which prey species should not be allowed to see predator species. Minimizing visual contact between prey and predator fish may improve the quality, viability and welfare of small fish in ornamental fish markets. (c) 2007 Elsevier B.V. All rights reserved.

A comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Spatio-temporal distribution of fatty acid-binding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio)

We have determined the structure of the fatty acid-binding protein 6 (fabp6) gene and the tissue-specific distribution of its transcripts in embryos, larvae and adult zebrafish (Danio rerio). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns. The coding region of the gene and expressed sequence tags code for a polypeptide of 131 amino acids (14 kDa, pI 6.59). The putative zebrafish Fabp6 protein shared greatest sequence identity with human FABP6 (55.3%) compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps. Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade with the mammalian FABP6s. The zebrafish fabp6 gene was assigned to linkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome 11. Zebrafish fabp6 transcripts were first detected in the distal region of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7-day-old larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RT-PCR in RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue), but not in RNA from skin, brain, gill, eye or muscle. In situ hybridization of a fabp6 riboprobe to adult zebrafish sections revealed intense hybridization signals in the adrenal homolog of the kidney and the distal region of the intestine, and to a lesser extent in ovary and liver, a transcript distribution that is similar, but not identical, to that seen for the mammalian FABP6/Fabp6 gene. © 2008 The Authors.

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio)

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides. © 2012 Elsevier Ltd. All rights reserved.

Estudo do efeito de metais em esterases de zebrafish (Danio rerio)

Pós-graduação em Química - IBILCE