Leonardo José Gil Barcellos

Unpredictable chronic stress model in zebrafish (Danio rerio): Behavioral and physiological responses

Zebrafish (Danio rerio) have emerged as a promising model organism to study development, toxicology, pharmacology, and neuroscience, among other areas. Despite the increasing number of studies using zebrafish, behavioral studies with this species are still elementary when compared to rodents. The aim of this study was to develop a model of unpredictable chronic stress (UCS) in zebrafish. We evaluated the effects of UCS protocol during 7 or 14 days on behavioral and physiological parameters. The effects of stress were evaluated in relation to anxiety and exploratory behavior, memory, expression of corticotrophin-releasing factor (CRF) and glucocorticoid receptor (GR), and cortisol levels. As expected, UCS protocol increased the anxiety levels, impaired cognitive function, and increased CRF while decreased GR expression. Moreover, zebrafish submitted to 7 or 14 days of UCS protocol presented increased cortisol levels. The protocol developed here is a complementary model for studying the neurobiology and the effects of chronic stress in behavioral and physiological parameters. In addition, this protocol is less time consuming than standard rodent models commonly used to study chronic stress. These results confirm UCS in zebrafish as an adequate model to preclinical studies of stress, although further studies are warranted to determine its predictive validity.

Nursery rearing of jundiá, Rhamdia quelen (Quoy & Gaimard) in cages: cage type, stocking density and stress response to confinement

Abstract The use of net cages allows the exploration of some water bodies, without usual aquacultural techniques for earthen ponds. Several advantages are proposed for the use of cages, however some disadvantages, as the higher possibility of stress and diseases are known. Growth parameters, survival rate and stress response of hatchery-bred Jundia ( Rhamdia quelen , Quoy & Gaimard) fry reared at different cage types and densities in net cages suspended in pond were evaluated. The first experiment compared fry reared at the circular and cubic-shaped cages at a density of 100 fry/m 3 . Fish held in cubic cages were heavier than the fish held in circular cages, presented higher weight gain and daily weight gain and a better food conversion. The survival rates were similar between fish raised in both types of cages. In the second experiment, cubic cages were stocked with 100, 200 and 300 fry/m 3 . Fish held in cages with lower density were heavier than the ones held at higher densities, presented higher weight gain and daily weight gain. Food conversion and survival rates were similar between different fish densities. According to the results presented in this study, the growth of jundia in cubic-shaped cages was density dependent. In terms of weight parameters, the most effective stocking density of jundia was 100 fry/m 3 that reaches 63.74±3.69 g of body weight. However, if the desirable weight was from 30 to 40 g, the density of 300 fry/m 3 is most effective because the fingerling production increases three times. In experiment 3, in all sample days, the serum cortisol levels were higher than the pre-stock and basal levels. The 80 days of cage confinement were not enough for adaptation of fingerlings to cage environment. The maintenance of high cortisol concentrations after 80 days of experimental period suggests the presence of typical chronic stress response that might have some detrimental effects over fish growth. Taken together, the data presented herein suggests that the improvement of rearing conditions (e.g. water quality, food quality, cage design, culture management, etc.) capable to minimize the stress response, may affect positively the growth performance in jundia during the nursery period.

Assessment of oxidative stress in Rhamdia quelen exposed to agrichemicals.

Due to the proximity of crop and fish culture areas, some agrichemicals that could be harmful for fish could enter into fishponds by different ways, such as by leaching through rain. Rhamdia quelen (Teleostei) were exposed to sublethal concentrations of methyl parathion (MP), a glyphosate based herbicide (Gly), and tebuconazole (Teb). The liver of R. quelen exposed to MP and Teb showed enhanced levels of thiobarbituric acid reactive substances (TBARS), higher than in the control fish (56% and 59%, respectively). In contrast, Gly did not alter the TBARS generation. The protein carbonyl content increased only in fish exposed to Teb. Fish exposed to the three agrichemicals showed a significant decrease of catalase activity (52%, 48%, and 67%, respectively) and increased glutathione-S-transferase (57%, 46%, and 160%, respectively) activity. Fish exposed to MP, Gly, and Teb showed higher reduced glutathione (151%, 472%, and 130%, respectively, when compared with the control levels) and ascorbic acid concentrations (121%, 102%, and 184%, respectively),while the non-protein thiol content increased only in R. quelen exposed to tebuconazole. Fish exposed to MP and Teb showed several pathological changes in the liver, including hepatocyte degeneration and bile stagnation. The present work reports for the first time the toxicity of the pesticide MP and the fungicide Teb in R. quelen, and as in other works, suggests the relatively lower liver toxicity of Gly for fish. The data presented herein demonstrate that sublethal concentrations of MP and Teb cause changes in oxidative stress parameters as well as hepatic cell injuries in R. quelen, and that these parameters have the potential to be developed as bioindicators of exposure to these agrichemicals.

Chronic exposure to sub-lethal concentration of a glyphosate-based herbicide alters hormone profiles and affects reproduction of female Jundiá (Rhamdia quelen).

This work was carried out to verify the effect of a glyphosate-based herbicide on Jundiá hormones (cortisol, 17β-estradiol and testosterone), oocyte and swim-up fry production. Earthen ponds containing Jundiá females were contaminated with glyphosate (3.6mg/L); blood samples were collected from eight females from each treatment immediately before, or at 1, 10, 20, 30 and 40 days following contamination. A typical post-stress rise in cortisol levels was observed at the 20th and 40th days following exposure to glyphosate. At the 40th day, 17β-estradiol was decreased in the exposed females. A similar number of oocytes were stripped out from females from both groups; however, a lower number of viable swim-up fry were obtained from the herbicide exposed females, which also had a higher liver-somatic index (LSI). The results indicate that the presence of glyphosate in water was deleterious to Rhamdia quelen reproduction, altering steroid profiles and egg viability.

Acute toxicity test of agricultural pesticides on silver catfish (Rhamdia quelen) fingerlings

Toxicity risks of agricultural pesticides to fishes are pivotal. Currently, many questions remain unsolved regarding to the toxicity of commonly used pesticides to silver catfish (Rhamdia quelen), a South American catfish. The present studies have been designed to investigate the acute toxicity and the lethal concentration (LC50) of four herbicides, two fungicides and two insecticides to silver catfish fingerlings. All experiments were carried out in triplicates, in a static bioassay system, using commercially available pesticides. The data was analyzed through the Trimmed Spearman-Karber method available from the Environmental Protection Agency. The 96hLC50 and 95% lower and upper confidence limits, respectively, for the following pesticides were determined: glyphosate (7.3mg L-1; 6.5-8.3), atrazine (10.2mg L-1; 9.1-11.5), atrazine+simazine (10.5mg L-1; 8.9-12.4), mesotrione (532.0mg L-1; 476.5-594), tebuconazole (5.3mg L-1; 4.9-5.7), methylparathion (4.8mg L-1; 4.3-5.3), strobulurin and triazol (9.9mg L-1; 8.7-11.2). Diflubenzuron was also tested and caused no fish mortality up to 1g L-1. The toxic concentration of these pesticides to silver catfish fingerlings fell above the concentration used for application in the field and, except following accidental application or misplacing of empty recipients, it should not cause fish mortality. Nonetheless, the data obtained will be useful to study the long-term effect of these products on the hematological, biochemical, hormonal and immunological parameters of silver catfish and related fish species in South Brazil.

Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC(50-96 h)) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms.

Cortisol response to acute stress in jundiá Rhamdia quelen acutely exposed to sub-lethal concentrations of agrichemicals

Exposure to agrichemicals can have deleterious effects on fish, such as disruption of the hypothalamus-pituitary-inter-renal axis (HPI) that could impair the ability of fish to respond to stressors. In this study, fingerlings of the teleost jundiá (Rhamdia quelen) were used to investigate the effects of the commonly used agrichemicals on the fish response to stress. Five common agrichemicals were tested: the fungicide - tebuconazole, the insecticide - methyl-parathion, and the herbicides - atrazine, atrazine+simazine, and glyphosate. Control fishes were not exposed to agrichemicals and standard stressors. In treatments 2-4, the fishes were exposed to sub-lethal concentrations (16.6%, 33.3%, and 50% of the LC(50)) of each agrichemical for 96 h, and at the end of this period, were subjected to an acute stress-handling stimulus by chasing them with a pen net. In treatments 5-7 (16.6%, 33.3%, and 50% of the LC(50)), the fishes were exposed to the same concentrations of the agrichemicals without stress stimulus. Treatment 8 consisted of jundiás not exposed to agrichemicals, but was subjected to an acute stress-handling stimulus. Jundiás exposed to methyl-parathion, atrazine+simazine, and glyphosate presented a decreased capacity in exhibiting an adequate response to cope with stress and in maintaining the homeostasis, with cortisol level lower than that in the control fish (P<0.01). In conclusion, the results of this study clearly demonstrate that the acute exposure to sub-lethal concentrations of methyl-parathion, atrazine+simazine, and glyphosate exert a deleterious effect on the cortisol response to an additional acute stressor in the jundiá fingerlings.

Exposure to sublethal concentration of glyphosate or atrazine-based herbicides alters the phagocytic function and increases the susceptibility of silver catfish fingerlings (Rhamdia quelen) to Aeromonas hydrophila challenge.

The resistance of fish to microorganisms challenge depends mainly on the efficacy of the immune response. Most studies on the natural immune response of fish have focused on the effect of diets and immunostimulants. Few studies correlated the presence of commonly used agrichemical and susceptibility to infection by aquatic microorganism. Thus, this study aimed to investigate the effect of glyphosate and atrazine-based herbicides on immune cell phagocytosis and susceptibility of silver catfish to Aeromonas hydrophila infection. Following exposure to sublethal concentrations of glyphosate or atrazine (10% of the LC(50-96 h)), a significant decrease in the number of intracelomatic cells and phagocytic index could be observed. In addition, silver catfish fingerlings exposed to glyphosate or atrazine were more susceptible to intracelomatic challenge with pathogenic A. hydrophila. Thus, the presence of these herbicides on the water alters the natural immune response to bacterial and possibly to other aquatic microorganism.

Diazepam and Fluoxetine Decrease the Stress Response in Zebrafish

The presence of pharmaceutical products in the aquatic environment has been reported in several studies. However, the impact of these drugs on living organisms is still uncharacterized. Here, we investigated the effects of acute exposure to either diazepam or fluoxetine on the stress response in Danio rerio. We showed that diazepam and fluoxetine inhibited the stress axis in zebrafish. Intermediate concentrations of diazepam suppressed the stress response as measured by cortisol levels, whereas fluoxetine inhibited cortisol increase at concentrations similar to those found in the environment. These data suggest that the presence of psychoactive drugs in aquatic ecosystems could cause neuroendocrine dysfunction in fish.

Chemical communication of handling stress in fish

We investigated whether juveniles of the nocturnal fish jundiá (Rhamdia quelen) and the diurnal fish Nile tilapia (Oreochromis niloticus) are able to chemically communicate stress to conspecifics. Groups of 8 fish were reared in tanks under recirculated water (water exchanged among all the tanks) for each species. Fish were handled in half of the tanks (stressor fish) and whole-body cortisol concentrations were compared among handled fish, non-handled fish exposed to water from the handled fish, and non-handled control fish held with no water communication. For each treatment cortisol concentrations were determined before exposure to the stressor (basal levels) and after 1, 2, 4, 8, and 24h. Basal levels of cortisol confirmed fish were unstressed in the beginning of the experiment. Cortisol was increased in the stressor fish 1h after handling. Fish receiving water from the stressor fish increased cortisol levels later (2h after the stressor fish were handled). As the isolated control group maintained cortisol levels unchanged throughout the experiment, we concluded that some chemical factor was released by the stressed fish in the water and thus stressed the conspecifics. This pattern was similar for both unrelated species, thus suggesting that this communication might have evolved earlier in fish and reinforcing the biological value of this kind of information.