João Gabriel Santos da Rosa

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.

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.

Responsiveness of the interrenal tissue of Jundiá (Rhamdia quelen) to an in vivo ACTH test following acute exposure to sublethal concentrations of agrichemicals.

As in many aquatic environments, pollution is a widespread problem in Southern Brazil. In our previous work, we demonstrated that sublethal contamination with some agrichemicals impairs the capacity of fishes to elevate cortisol levels in response to an additional acute stressor. In earlier experiments, the experimental design did not allow us to conclude where this effect occurs. In the present work, we used the adrenocorticotropic hormone (ACTH) challenge test to help us identify if the impairment occur in the interrenal tissue. For this purpose, five experiments were conducted, each with one specific agrichemical (methyl-parathion, atrazine+simazine, atrazine, tebuconazole, and glyphosate) in sublethal concentrations of 16.6% of the LC(50-96h), as previously determined. Fish were subjected to the ACTH challenge test protocol as follows: group 1, were non-injected and maintained as the specific control group; group 2 received an injection of the vehicle alone (the saline group); and group 3 receive an injection of ACTH. One hour later, blood samples were taken from the caudal plexus, using sterile syringes. In all specific control groups, the injection of ACTH induced a strong rise in plasma cortisol, compared with the fish injected only with the vehicle and the non-injected group. Fish exposed to methyl-parathion and tebuconazole did not elevate cortisol in response to the ACTH injection, with values significantly lower than the control fish. Fish exposed to sublethal concentrations of atrazine+simazine, atrazine, and glyphosate showed a rise in plasma cortisol very similar to the control fish. We conclude that the ACTH challenge test revealed that R. quelen exposed to sublethal concentrations of tebuconazole and methyl-parathion had a reduced ability to elevate plasma cortisol in response to an intraperitoneal (i.p.) injection of exogenous ACTH, indicating that the interrenal tissue is the site of the impairment within the HPI axis. These ACTH challenge tests also revealed that the impairment of the cortisol response verified in fish exposed to atrazine+simazine and glyphosate, as shown in our previous work, seems to be related to steps of cortisol secretion in higher levels within the HPI axis.

Alcohol impairs predation risk response and communication in zebrafish.

The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.

Divergent time course of cortisol response to stress in fish of different ages.

This study investigated differences in the cortisol response of fish at different developmental stages after exposure to an acute stressor. Three experiments using 126 fish each were performed using 3 different age groups of jundiá (Rhamdia quelen): fingerlings at 60 days of age, juveniles at 180 days, and adults at 360 days. In each experiment, the fish in each group were randomly distributed into either a handled experimental group or a non-handled control group. The handled group was then exposed to an acute stressor for measurement of cortisol concentrations at 5, 15, 30, 60, and 240 min after exposure and subsequent comparison of cortisol concentrations with those of the non-handled groups at the 6 sampling times. Between the experimental and control groups, the results revealed that the handled fish in each of the 3 age groups had higher cortisol concentrations compared to the non-handled fish in the equivalent age group. Among the age groups of the handled fish, the results revealed that the fingerlings and juveniles attained peak cortisol concentrations within 5 to 30 min after stressor exposure whereas the adults attained peak concentrations 60 min after exposure. This finding has important implications for the design of research into stress and welfare among fish at different developmental stages.

Neonatal handling prevents anxiety-like symptoms in rats exposed to chronic mild stress: Behavioral and oxidative parameters

This study investigated the influence of neonatal handling on behavioral and biochemical consequences of chronic mild stress (CMS) in adulthood. Male rat pups were submitted to daily tactile stimulation (TS) or maternal separation (MS), from postnatal day 1 (PND1) to postnatal day 21 (PND21), for 10 min/day. In adulthood, half the number of animals were exposed to CMS for 3 weeks and submitted to behavioral testing, including sucrose preference (SP), elevated plus maze (EPM), and defensive burying tasks (DBTs), followed by biochemical assessments. CMS reduced SP, increased anxiety in EPM and DBT, and increased adrenal weight. In addition, CMS decreased plasma vitamin C (VIT C) levels and increased protein carbonyl (PC) levels, catalase (CAT) activity in hippocampus and cortex, and superoxide dismutase (SOD) levels in cortex. In contrast, both forms of neonatal handling were able to prevent reduction in SP, anxiety behavior in DBT, and CMS-induced adrenal weight increase. Furthermore, they were also able to prevent plasma VIT C reduction, hippocampal PC levels increase, CAT activity increase in hippocampus and cortex, and SOD levels increase in cortex following CMS. Only TS was able to prevent CMS-induced anxiety symptoms in EPM and PC levels in cortex. Taken together, these findings show the protective role of neonatal handling, especially TS, which may enhance ability to cope with stressful situations in adulthood.

Acute exposure to waterborne psychoactive drugs attract zebrafish.

Psychotropic medications are widely used, and their prescription has increased worldwide, consequently increasing their presence in aquatic environments. Therefore, aquatic organisms can be exposed to psychotropic drugs that may be potentially dangerous, raising the question of whether these drugs are attractive or aversive to fish. To answer this question, adult zebrafish were tested in a chamber that allows the fish to escape or seek a lane of contaminated water. These attraction and aversion paradigms were evaluated by exposing the zebrafish to the presence of acute contamination with these compounds. The zebrafish were attracted by certain concentrations of diazepam, fluoxetine, risperidone and buspirone, which were most likely detected by olfaction, because this behavior was absent in anosmic fish. These findings suggest that despite their deleterious effects, certain psychoactive drugs attract fish.

My stress, our stress: blunted cortisol response to stress in isolated housed zebrafish.

Here, we show that individually housed zebrafish presented a reduced cortisol response to an acute stressor (persecution with a pen net for 120 s) compared to zebrafish housed in groups of 10. We hypothesized that the cortisol response to stress was reduced in individually housed zebrafish because they depend solely on their own perceptions of the stressor, whereas among grouped zebrafish, the stress response might be augmented by chemical and/or behavioral cues from the other members of the shoal. This hypothesis was based on previous described chemical communication of stress in fish as well on individual variation in stressor perception and potential individual differences in fish personality.

Agrichemicals chronically inhibit the cortisol response to stress in fish.

We studied the stress response of Rhamdia quelen fingerlings at 45, 90, 135 and 180 d following acute exposure to agrichemicals. Herein, we report the novel observation that acute exposure of fingerling-aged fish to a methyl parathion-based insecticide (MPBI) and to a tebuconazole-based fungicide (TBF) induced chronic inhibition of the stress response. In contrast, fish exposed to an atrazine-simazine-based herbicide (ASBH) recovered the stress response on day 45, and fish exposed to a glyphosate-based herbicide (GBH) did not present stress response inhibition. Additionally, fish exposed to MPBI, GBH and ASBH showed lower survival rates and attained lower final weights. In the case of TBF, the presence of the stressful stimulus more strongly influenced the changes in the performance parameters than did the agrichemical exposure itself. An impairment of the cortisol response may seriously hamper the adaptive response and the ability to promote the necessary metabolic and ionic adjustments to respond to environmental stress.

Waterborne Risperidone Decreases Stress Response in Zebrafish.

The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.