Water and suspended sediment runoff from vineyard watersheds affecting the behavior and physiology of zebrafish.

Viticulture plays an important role in generating income for small farms globally. Historically, vineyards use large quantities of phytosanitary products, such as Bordeaux mixture [Ca(OH)2 + CuSO4], to control plant diseases. These products result in the accumulation of copper (Cu) in the soil and increases the risk of transfer to water bodies. Thus, it is important to evaluate whether the presence of Cu-bearing particles in water is toxic to aquatic fauna. This study conducted chemical, mineralogical, and particle size evaluations on water samples and sediments collected from a watershed predominantly cultivated with old vineyards. The proportion of Cu-rich nanoparticles (<10 nm) in the sediment was ~27%. We exposed zebrafish to different dilutions of water and sediment samples that collected directly from the study site (downstream river) under laboratory conditions. Then, we evaluated their exploratory behavior and the stress-related endocrine parameter, whole-body cortisol. We also carried out two experiments in which zebrafish were exposed to Cu. First, we determined the median lethal concentration (LC50-96 h) of Cu and then assessed whether Cu exposure results in effects similar to those associated with exposure to the water and sediment samples collected from the study site. The water and sediment samples directly impacted the exploratory behavior of zebrafish, showing clear anxiety-like behavioral phenotype and stress in terms of cortisol increase (during the second rain event). The Cu exposure did not mimic the same behavioral changes triggered by the water and sediment samples, although it had caused similar stress in the fish. Our results highlight that even at low concentrations, the water and sediment samples from vineyard watershed runoff were able to induce behavioral and endocrine changes that may harm the ecological balance of an aquatic environment.

Stress responses to conspecific visual cues of predation risk in zebrafish.

Chemical communication relating to predation risk is a trait common among fish species. Prey fish under threat of predation can signal risk to conspecific fish, which then exhibit defensive responses. Fish also assess predation risk by visual cues and change their behavior accordingly. Here, we explored whether these behavioral changes act as visual alarm signals to conspecific fish that are not initially under risk. We show that shoals of zebrafish (Danio rerio) visually exposed to a predator display antipredator behaviors. In addition, these defensive maneuvers trigger antipredator reactions in conspecifics and, concomitantly, stimulate the hypothalamus-pituitary-interrenal axis, leading to cortisol increase. Thus, we conclude that zebrafish defensive behaviors act as visual alarm cues that induce antipredator and stress response in conspecific fish.

α-Methyltyrosine, a tyrosine hydroxylase inhibitor, decreases stress response in zebrafish (Danio rerio).

In this article, we show that the tyrosine hydroxylase inhibitor α-Methyl-l-tyrosine (AMPT) decreased the responsiveness of the zebrafish stress axis to an acute stressful challenge. These effects were specific for responses to stimulation, since unstimulated (basal) cortisol levels were not altered by AMPT. Moreover, AMPT decreased the stress response 15min after stimulation, but not after that time period. To our knowledge, this is the first report about the effects of AMPT on the neuroendocrine axis of adult zebrafish in acute stress responses. Overall, these results suggest a mechanism of catecholamine-glucocorticoid interplay in neuroendocrine responses of fish, pointing an interesting avenue for physiological research, as well as an important endpoint that can be disrupted by environmental contamination. Further experiments will unravel the mechanisms by which AMPT blocked the cortisol response.

Waterborne aripiprazole blunts the stress response in zebrafish.

Here we provide, at least to our knowledge, the first evidence that aripiprazole (APPZ) in the water blunts the stress response of exposed fish in a concentration ten times lower than the concentration detected in the environment. Although the mechanism of APPZ in the neuroendocrine axis is not yet determined, our results highlight that the presence of APPZ residues in the environment may interfere with the stress responses in fish. Since an adequate stress response is crucial to restore fish homeostasis after stressors, fish with impaired stress response may have trouble to cope with natural and/or imposed stressors with consequences to their welfare and survival.

Fish Aversion and Attraction to Selected Agrichemicals.

In agriculture intensive areas, fishponds and natural water bodies located in close proximity to these fields receive water with variable amounts of agrichemicals. Consequently, toxic compounds reach nontarget organisms. For instance, aquatic organisms can be exposed to tebuconazole-based fungicides (TBF), glyphosate-based herbicides (GBH), and atrazine-based herbicides (ABH) that are potentially dangerous, which motivates the following question: Are these agrichemicals attractant or aversive to fish? To answer this question, adult zebrafish were tested in a chamber that allows fish to escape from or seek a lane of contaminated water. This attraction and aversion paradigm was evaluated with zebrafish in the presence of an acute contamination with these compounds. We showed that only GBH was aversive to fish, whereas ABH and TBF caused neither attraction nor aversion for zebrafish. Thus, these chemicals do not impose an extra toxic risk by being an attractant for fish, although TBF and ABH can be more deleterious, because they induce no aversive response. Because the uptake and bioaccumulation of chemicals in fish seems to be time- and dose-dependent, a fish that remains longer in the presence of these substances tends to absorb higher concentrations than one that escapes from contaminated sites.

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.

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.

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.

Death-associated odors induce stress in zebrafish.

Living animals exploit information released from dead animals to conduct adaptive biological responses. For instance, a recently published study has shown that avoidance behavior is triggered by death-associated odors in zebrafish. Stress can clearly act as an adaptive response that allows an organism to deal with an imminent threat. However, it has not been demonstrated whether these chemical cues are stressful for fish. Here, we confirmed that dead zebrafish scents induce defensive behavior in live conspecifics. Additionally, we show for the first time in fish that these scents increase cortisol in conspecifics. To reach this conclusion, firstly, we exposed zebrafish to multi-sensorial cues (e.g., visual, tactile, chemical cues) from dead conspecifics that displayed defensive behaviors and increased cortisol. Also, when we limited zebrafish to chemical cues from dead conspecifics, similar responses arose. These responses coincide with the decaying destruction of epidermal cells, indicating that defensive and stress responses could take place as an effect of substances emanating from decaying flesh, as well as alarm substance released due to rupture of epidermal cells. Taken together, these results illustrate that living zebrafish utilize cues from dead conspecific to avoid or to cope with danger and ensure survival.

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.

Bee products prevent agrichemical-induced oxidative damage in fish.

In southern South America and other parts of the world, aquaculture is an activity that complements agriculture. Small amounts of agrichemicals can reach aquaculture ponds, which results in numerous problems caused by oxidative stress in non-target organisms. Substances that can prevent or reverse agrichemical-induced oxidative damage may be used to combat these effects. This study includes four experiments. In each experiment, 96 mixed-sex, 6-month-old Rhamdia quelen (118±15 g) were distributed into eight experimental groups: a control group that was not exposed to contaminated water, three groups that were exposed to various concentrations of bee products, three groups that were exposed to various concentrations of bee products plus tebuconazole (TEB; Folicur 200 CE™) and a group that was exposed to 0.88 mg L(-1) of TEB alone (corresponding to 16.6% of the 96-h LC50). We show that waterborne bee products, including royal jelly (RJ), honey (H), bee pollen (BP) and propolis (P), reversed the oxidative damage caused by exposure to TEB. These effects were likely caused by the high polyphenol contents of these bee-derived compounds. The most likely mechanism of action for the protective effects of bee products against tissue oxidation and the resultant damage is that the enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) are increased.

Repeated stressors do not provoke habituation or accumulation of the stress response in the catfish Rhamdia quelen

Fish repeatedly experience stressful situations under experimental and aquaculture conditions, even in their natural habitat. Fish submitted to sequential stressors can exhibit accumulation or habituation on its cortisol response. We posed a central question about the cortisol response profiles after exposure to successive acute stressors of a similar and different nature in Rhamdia quelen. We have shown that successive acute stressors delivered with 12-h, 48-h, and 1-week intervals provoked similar cortisol responses in juvenile R. quelen, without any habituation or accumulation. The cumulative stress response is more associated to short acute stressors with very short intervals of minutes to hours. In our work, we used an interval as short as 12h, and no cumulative response was found. However, if the length of time between stressors is of a day or week as used in our work the most common and an expected phenomenon is the attenuation of the response. Thus, also, the absence of both accumulation of the stress response and the expected habituation is an intriguing result. Our results show that R. quelen does not show habituation or accumulation in its stress responses to repeated stressors, as reported for other fish species.

Os peixes, tanto em condições de cultivo quanto em condições experimentais ou na natureza, podem experimentar situações de estresse. Peixes submetidos a estressores sequenciais podem apresentar acumulação de sua resposta de cortisol com aumento dos níveis desse hormônio em cada resposta sucessiva ou habituação da resposta do cortisol, com redução dos níveis de cortisol em cada resposta sequencial. Abordamos a questão central de como é o perfil de resposta do cortisol de R. quelen após a exposição a estressores agudos sucessivos de natureza semelhante e diferente. Os dados mostraram que sucessivos estressores agudos com intervalos de 12 e 48h e de uma semana, provocaram respostas de cortisol semelhantes em juvenis de R. quelen, sem qualquer habituação ou acumulação. No presente trabalho usamos intervalos tão curtos quanto 12h e não verificamos qualquer tipo de acumulação da resposta. No entanto, se o intervalo entre estressores forem de um dia ou semana, o fenômeno mais comum e esperado é o da atenuação da resposta. Assim, tanto a ausência de acumulação da resposta ao estresse quanto à ausência da habituação esperada são resultados interessantes. Nossos resultados mostraram que juvenis de R. quelen não mostram habituação ou acumulação em suas respostas de estresse a estressores repetidos, como relatado para outras espécies de peixes.

Fish age, instead of weight and size, as a determining factor for time course differences in cortisol response to stress.

In many vertebrate species, including humans, the developmental stage directly influences an organism's reactivity to stress. For instance, fishes appear to exhibit "stress insensitive" periods early in development, which contributes to important alterations in stress responses. This phenomenon raises the important question of whether size or age influences fish stress responses, as there may be large discrepancies in size at any stage of ontogeny. We therefore posed the following question: what key role does the age and/or weight/length of fish play in contributing to different stress levels? To address this question, we conducted 2 simple experiments to compare the cortisol response to stress in the fish Rhamdia quelen. In the first experiment, we compared the cortisol response of fish of 2 different age groups (3 vs. 12 months) with the same body size, whereas in the second experiment we compared 2 groups of the same age (3 months) but with different body sizes. The results showed a similar stress response in fish of the same age but different size, but a large difference in the stress response in fish of the same size but different age. Both tests indicate that age is the determining factor for the functioning of the hypothalamo-pituitary-interrenal (HPI) axis, and is probably related to the stage of maturation. This study makes a significant contribution to our knowledge of the stress, behavior, and welfare of fish of different age classes, primarily with respect to the timing of measurements and the accurate determination of fish age, regardless of size.

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.