An Overview towards Zebrafish Larvae as a Model for Ocular Diseases.

Despite the obvious morphological differences in the visual system, zebrafish share a similar architecture and components of the same embryonic origin as humans. The zebrafish retina has the same layered structure and cell types with similar metabolic and phototransduction support as humans, and is functional 72 h after fertilization, allowing tests of visual function to be performed. The zebrafish genomic database supports genetic mapping studies as well as gene editing, both of which are useful in the ophthalmological field. It is possible to model ocular disorders in zebrafish, as well as inherited retinal diseases or congenital or acquired malformations. Several approaches allow the evaluation of local pathological processes derived from systemic disorders, such as chemical exposure to produce retinal hypoxia or glucose exposure to produce hyperglycemia, mimicking retinopathy of prematurity or diabetic retinopathy, respectively. The pathogenesis of ocular infections, autoimmune diseases, or aging can also be assessed in zebrafish larvae, and the preserved cellular and molecular immune mechanisms can be assessed. Finally, the zebrafish model for the study of the pathologies of the visual system complements certain deficiencies in experimental models of mammals since the regeneration of the zebrafish retina is a valuable tool for the study of degenerative processes and the discovery of new drugs and therapies.

Revisiting Retinal Degeneration Hallmarks: Insights from Molecular Markers and Therapy Perspectives.

Visual impairment and blindness are a growing public health problem as they reduce the life quality of millions of people. The management and treatment of these diseases represent scientific and therapeutic challenges because different cellular and molecular actors involved in the pathophysiology are still being identified. Visual system components, particularly retinal cells, are extremely sensitive to genetic or metabolic alterations, and immune responses activated by local insults contribute to biological events, culminating in vision loss and irreversible blindness. Several ocular diseases are linked to retinal cell loss, and some of them, such as retinitis pigmentosa, age-related macular degeneration, glaucoma, and diabetic retinopathy, are characterized by pathophysiological hallmarks that represent possibilities to study and develop novel treatments for retinal cell degeneration. Here, we present a compilation of revisited information on retinal degeneration, including pathophysiological and molecular features and biochemical hallmarks, and possible research directions for novel treatments to assist as a guide for innovative research. The knowledge expansion upon the mechanistic bases of the pathobiology of eye diseases, including information on complex interactions of genetic predisposition, chronic inflammation, and environmental and aging-related factors, will prompt the identification of new therapeutic strategies.

Zebrafish Larvae Behavior Models as a Tool for Drug Screenings and Pre-Clinical Trials: A Review.

To discover new molecules or review the biological activity and toxicity of therapeutic substances, drug development, and research relies on robust biological systems to obtain reliable results. Phenotype-based screenings can transpose the organism's compensatory pathways by adopting multi-target strategies for treating complex diseases, and zebrafish emerged as an important model for biomedical research and drug screenings. Zebrafish's clear correlation between neuro-anatomical and physiological features and behavior is very similar to that verified in mammals, enabling the construction of reliable and relevant experimental models for neurological disorders research. Zebrafish presents highly conserved physiological pathways that are found in higher vertebrates, including mammals, along with a robust behavioral repertoire. Moreover, it is very sensitive to pharmacological/environmental manipulations, and these behavioral phenotypes are detected in both larvae and adults. These advantages align with the 3Rs concept and qualify the zebrafish as a powerful tool for drug screenings and pre-clinical trials. This review highlights important behavioral domains studied in zebrafish larvae and their neurotransmitter systems and summarizes currently used techniques to evaluate and quantify zebrafish larvae behavior in laboratory studies.

Pesticides and Their Impairing Effects on Epithelial Barrier Integrity, Dysbiosis, Disruption of the AhR Signaling Pathway and Development of Immune-Mediated Inflammatory Diseases.

The environmental and occupational risk we confront from agricultural chemicals increases as their presence in natural habitats rises to hazardous levels, building a major part of the exposome. This is of particular concern in low- and middle-income countries, such as Brazil, known as a leading producer of agricultural commodities and consumer of pesticides. As long as public policies continue to encourage the indiscriminate use of pesticides and governments continue to support this strategy instead of endorsing sustainable agricultural alternatives, the environmental burden that damages epithelial barriers will continue to grow. Chronic exposure to environmental contaminants in early life can affect crucial barrier tissue, such as skin epithelium, airways, and intestine, causing increased permeability, leaking, dysbiosis, and inflammation, with serious implications for metabolism and homeostasis. This vicious cycle of exposure to environmental factors and the consequent damage to the epithelial barrier has been associated with an increase in immune-mediated chronic inflammatory diseases. Understanding how the harmful effects of pesticides on the epithelial barrier impact cellular interactions mediated by endogenous sensors that coordinate a successful immune system represents a crucial challenge. In line with the epithelial barrier hypothesis, this narrative review reports the available evidence on the effects of pesticides on epithelial barrier integrity, dysbiosis, AhR signaling, and the consequent development of immune-mediated inflammatory diseases.

Glyphosate- and Fipronil-Based Agrochemicals and Their Mixtures Change Zebrafish Behavior.

Environmental contamination caused by the human occupancy and economic activities that generate a wide range of contaminated effluents that reach natural water resources, is a current reality. Residues of agrichemicals used in plant production were detected in different environments and in different countries. Among these agrochemicals, we studied a glyphosate-based herbicide (GBH), a fipronil-based insecticide (FBI), and their mixtures (GBH + FBI). Zebrafish exposed to 3 and 5 mg/L of GBH spend more time in the top zone and less time in the bottom zone. Fish exposed to 0.009 and 0.018 mg/L of FBI spent less time in the bottom zone, whereas zebrafish exposed to the three GBH + FBI mixtures spend more time in the top zone compared with unexposed control fish. This clear anxiolytic pattern, in an environmental context, can directly impair the ability of fish to avoid or evade predators. We concluded that both glyphosate-based herbicide and fipronil-based insecticide and their mixtures alter zebrafish behavior, which may result in significant repercussions on the maintenance of the species as well as on the food chain and the ecosystem.

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.

Impaired brain StAR and HSP70 gene expression in zebrafish exposed to Methyl-Parathion based insecticide.

Fish production ponds and natural water body areas located in close proximity to agricultural fields receive water with variable amounts of agrochemicals, and consequently, compounds that produce adverse effects may reach nontarget organisms. The aim of this study was to investigate whether waterborne methyl-parathion-based insecticide (MPBI) affected gene expression patterns of brain glucocorticoid receptor (GR), steroidogenic acute regulatory protein (StAR), and heat shock protein 70 (hsp70) in adult zebrafish (Danio rerio) exposed to this chemical for 96 h. Treated fish exposed to MPBI-contaminated water showed an inhibition of brain StAR and hsp70 gene expression. Data demonstrated that MPBI produced a decrease brain StAR and hsp70 gene expression.

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.

Recapitulation of Retinal Damage in Zebrafish Larvae Infected with Zika Virus.

Zebrafish are increasingly being utilized as a model to investigate infectious diseases and to advance the understanding of pathogen-host interactions. Here, we take advantage of the zebrafish to recapitulate congenital ZIKV infection and, for the first time, demonstrate that it can be used to model infection and reinfection and monitor anti-viral and inflammatory immune responses, as well as brain growth and eye abnormalities during embryonic development. By injecting a Brazilian strain of ZIKV into the yolk sac of one-cell stage embryos, we confirmed that, after 72 h, ZIKV successfully infected larvae, and the physical condition of the virus-infected hosts included gross morphological changes in surviving embryos (84%), with a reduction in larval head size and retinal damage characterized by increased thickness of the lens and inner nuclear layer. Changes in locomotor activity and the inability to perceive visual stimuli are a result of changes in retinal morphology caused by ZIKV. Furthermore, we demonstrated the ability of ZIKV to replicate in zebrafish larvae and infect new healthy larvae, impairing their visual and neurological functions. These data reinforce the deleterious activity of ZIKV in the brain and visual structures and establish the zebrafish as a model to study the molecular mechanisms involved in the pathology of the virus.

17-α-Ethinylestradiol modulates endocrine and behavioral responses to stress in zebrafish.

The synthetic estrogen, 17-α-ethinylestradiol (EE2), present in contraceptive pills, is an endocrine-disrupting chemical (EDC) that can be found in the aquatic environment. We examined the impacts of EE2 on zebrafish behavioral and physiological responses through the novel tank test (NTT), which measures anxiety-like behavior; the mirror-induced aggression (MIA) test, which measures aggressiveness; and the social preference test (SPT), which measures social cohesion. The steroid hormone levels were also measured. Here, we show that exposure to EE2 impairs stress responses by regulating the levels of specific hormones and eliciting an anxiolytic response, increasing aggression, and reducing social preference in zebrafish. In nature, these changes in behavior compromise reproduction and anti-predator behaviors, which, in turn, affects species survival. The maintenance of an intact behavioral repertoire in zebrafish is essential for their survival. Thus, our results point to the danger of environmental contamination with EE2 as it may alter the dynamics of the prey-predator relationship.

Psychotropic in the environment: risperidone residues affect the behavior of fish larvae.

The ability to avoid and escape from predators are clearly relevant behaviors from the ecological perspective and directly interfere with the survival of organisms. Detected in the aquatic environment, risperidone can alter the behavior of exposed species. Considering the risk of exposure in the early stages of life, we exposed zebrafish embryos to risperidone during the first 5 days of life. Risperidone caused hyperactivity in exposed larvae, which in an environmental context, the animals may be more vulnerable to predation due to greater visibility or less perception of risk areas.

Just Keep Swimming: Neuroendocrine, Metabolic, and Behavioral Changes After a Forced Swimming Test in Zebrafish.

In this study, we show that an adaptation of the spinning test can be used as a model to study the exercise-exhaustion-recovery paradigm in fish. This forced swimming test promotes a wide range of changes in the hypothalamus-pituitary-interrenal axis functioning, intermediary metabolism, as well in fish behavior at both exercise and recovery periods. Our results pointed that this adapted spinning test can be considered a valuable tool for evaluating drugs and contaminant effects on exercised fish. This can be a suitable protocol both to environmental-to evaluate contaminants that act in fish energy mobilization and recovery after stressors-and translational perspectives-effects of drugs on exercised or stressed humans.

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.

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.

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.

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.

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.

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.