Photobiomodulation Reduces the Cytokine Storm Syndrome Associated with COVID-19 in the Zebrafish Model.

Although the exact mechanism of the pathogenesis of coronavirus SARS-CoV-2 (COVID-19) is not fully understood, oxidative stress and the release of pro-inflammatory cytokines have been highlighted as playing a vital role in the pathogenesis of the disease. In this sense, alternative treatments are needed to reduce the level of inflammation caused by COVID-19. Therefore, this study aimed to investigate the potential effect of red photobiomodulation (PBM) as an attractive therapy to downregulate the cytokine storm caused by COVID-19 in a zebrafish model. RT-qPCR analyses and protein-protein interaction prediction among SARS-CoV-2 and Danio rerio proteins showed that recombinant Spike protein (rSpike) was responsible for generating systemic inflammatory processes with significantly increased levels of pro-inflammatory (il1b, il6, tnfa, and nfkbiab), oxidative stress (romo1) and energy metabolism (slc2a1a and coa1) mRNA markers, with a pattern similar to those observed in COVID-19 cases in humans. On the other hand, PBM treatment was able to decrease the mRNA levels of these pro-inflammatory and oxidative stress markers compared with rSpike in various tissues, promoting an anti-inflammatory response. Conversely, PBM promotes cellular and tissue repair of injured tissues and significantly increases the survival rate of rSpike-inoculated individuals. Additionally, metabolomics analysis showed that the most-impacted metabolic pathways between PBM and the rSpike treated groups were related to steroid metabolism, immune system, and lipid metabolism. Together, our findings suggest that the inflammatory process is an incisive feature of COVID-19 and red PBM can be used as a novel therapeutic agent for COVID-19 by regulating the inflammatory response. Nevertheless, the need for more clinical trials remains, and there is a significant gap to overcome before clinical trials can commence.

Plasma proteome responses in zebrafish following λ-carrageenan-Induced inflammation are mediated by PMN leukocytes and correlate highly with their human counterparts.

Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.

Sound stimulus effects on dusky damselfish behavior and cognition.

Anthropogenic noises are widespread and affect marine wildlife. Despite the growing knowledge on noise pollution in the marine environment, its effects on fish cognition are scarce. Here, we investigated the effects of sound exposure on anxiety-like behavior and memory retention on dusky damselfish Stegastes fuscus. The animals were trained in a conditioned place aversion task, and exposed to two daily sessions of music at intensities of 60-70 dBA or 90-100 dBA, while the control group was kept at 42-46 dBA (no music) for five days. After that, fish were tested in the novel tank paradigm and tested for the memory of the aversive task. In the novel tank, animals exposed to sound spent more time still and decreased the distance from the bottom of the tank. Animals also spent more time on the aversive side of the conditioning tank. These results suggest that anthropogenic noise applied through high-intensity music can increase anxiety and decrease memory retention in S. fuscus, suggesting the deleterious potential of noise for reef species.

Toxicity of spike fragments SARS-CoV-2 S protein for zebrafish: A tool to study its hazardous for human health?

Despite the significant increase in the generation of SARS-CoV-2 contaminated domestic and hospital wastewater, little is known about the ecotoxicological effects of the virus or its structural components in freshwater vertebrates. In this context, this study evaluated the deleterious effects caused by SARS-CoV-2 Spike protein on the health of Danio rerio, zebrafish. We demonstrated, for the first time, that zebrafish injected with fragment 16 to 165 (rSpike), which corresponds to the N-terminal portion of the protein, presented mortalities and adverse effects on liver, kidney, ovary and brain tissues. The conserved genetic homology between zebrafish and humans might be one of the reasons for the intense toxic effects followed inflammatory reaction from the immune system of zebrafish to rSpike which provoked damage to organs in a similar pattern as happen in severe cases of COVID-19 in humans, and, resulted in 78,6% of survival rate in female adults during the first seven days. The application of spike protein in zebrafish was highly toxic that is suitable for future studies to gather valuable information about ecotoxicological impacts, as well as vaccine responses and therapeutic approaches in human medicine. Therefore, besides representing an important tool to assess the harmful effects of SARS-CoV-2 in the aquatic environment, we present the zebrafish as an animal model for translational COVID-19 research.

Auditory environmental enrichment prevents anxiety-like behavior, but not cortisol responses, evoked by 24-h social isolation in zebrafish.

The zebrafish (Danio rerio) is widely used as a promising translational model organism for studying various brain disorders. Zebrafish are also commonly used in behavioral and drug screening assays utilizing individually tested (socially isolated) fish. Various sounds represent important exogenous factors that may affect fish behavior. Mounting evidence shows that musical/auditory environmental enrichment can improve welfare of laboratory animals, including fishes. Here, we show that auditory environmental enrichment mitigates anxiogenic-like effects caused by acute 24-h social isolation in adult zebrafish. Thus, auditory environmental enrichment may offer an inexpensive, feasible and simple tool to improve welfare of zebrafish stocks in laboratory facilities, reduce unwanted procedural stress, lower non-specific behavioral variance and, hence, collectively improve zebrafish data reliability and reproducibility.

Effects of different doses of eugenol on plasma cortisol levels and the quality of fresh and frozen-thawed sperm in South American catfish (Rhamdia quelen).

The production of captive fish is only possible through artificial reproduction, but manipulation is a known stressor stimulus. Thus, the objective of the present study was to evaluate the effects of different eugenol concentrations (0, 30, 40, 50 and 60 mg/L) during reproductive management of Rhamdia quelen. Seventy-five mature male R. quelen were randomly distributed among the five treatments, and blood samples were collected at the time of semen collection to measure plasma cortisol. The following parameters were evaluated in the fresh semen samples: motility, motility duration, concentration and fertilization rate. The following parameters were evaluated in the frozen semen samples: motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality. The animals anesthetized with eugenol at concentrations of 40 and 50 mg/L had lower levels of plasma cortisol (88.4 and 83.3 ng/mL, respectively) than the control (147.1 ng/mL). For fresh semen, the control treatment presented the highest rate and time of motility but differed (P < 0.05) only from the animals treated with 60 mg/L eugenol. For the cryopreserved semen the highest rates and motility time were observed in the control treatment and in the animals anesthetized with 40 mg/L eugenol, differing (P < 0.05) from anesthetized animals with 50 and 60 mg/L. Mitochondrial functionality was higher in fish anesthetized with 30 mg/L eugenol differing only for animals anesthetized with 60 mg/L. There was no difference between treatments for sperm concentration and fertilization rate of fresh semen. There were no differences (P > 0.05) between treatments in the parameters of membrane integrity, DNA integrity and% of normal spermatozoa after thawing of the cryopreserved semen samples. The use of 30, 40 and 50 mg/L eugenol maintained the seminal quality of the fresh semen, and the quality of the thawed semen was maintained with 30 and 40 mg/L eugenol. These results show that stress reduction can be reconciled with reproductive management without compromising reproductive performance.

Feeding regimen modulates zebrafish behavior.

Here we show that the feeding regimen modulates zebrafish (Danio rerio) behavior. With regard to the time elapsed between feeding and behavioral evaluation, fish fed 3 h before behavioral evaluation in the novel tank test (NTT) showed decreased activity and a trend toward an anxiolytic reaction (increased use of the upper section of the aquarium) in comparison to fish fed 0.5, 6, 12, 24 or 48 h before testing, although differences were not statistically significant for all comparisons. Activity and use of the upper section of the aquarium did not differ significantly among the other treatments. Regarding feeding frequency, fish fed once a day showed higher anxiety-like behavior (decreased use of the upper section of the aquarium) in comparison to fish fed twice a day, but feeding four or six times per day or only every second day did not result in differences from feeding twice a day. Feeding frequency had no effect on activity level. Metabolically, fish fed once a day presented decreased levels of glucose and glycogen and increased lactate when compared to the regular feeding (fish fed twice a day), suggesting that feeding regimen may modulate carbohydrate metabolism. Mechanistically, we suggest that the metabolic changes caused by the feeding regimen may induce behavioral changes. Our results suggest that the high variability of the results among different laboratories might be related to different feeding protocols. Therefore, if issues pertaining to the feeding regimen are not considered during experiments with zebrafish, erroneous interpretations of datasets may occur.

The effects of auditory enrichment on zebrafish behavior and physiology.

Environmental enrichment is widely used to improve welfare and behavioral performance of animal species. It ensures housing of laboratory animals in environments with space and complexity that enable the expression of their normal behavioral repertoire. Auditory enrichment by exposure to classical music decreases abnormal behaviors and endocrine stress responses in humans, non-humans primates, and rodents. However, little is known about the role of auditory enrichment in laboratory zebrafish. Given the growing importance of zebrafish for neuroscience research, such studies become critical. To examine whether auditory enrichment by classical music can affect fish behavior and physiology, we exposed adult zebrafish to 2 h of Vivaldi's music (65-75 dB) twice daily, for 15 days. Overall, zebrafish exposed to such auditory stimuli were less anxious in the novel tank test and less active, calmer in the light-dark test, also affecting zebrafish physiological (immune) biomarkers, decreasing peripheral levels of pro-inflammatory cytokines and increasing the activity of some CNS genes, without overt effects on whole-body cortisol levels. In summary, we report that twice-daily exposure to continuous musical sounds may provide benefits over the ongoing 50-55 dB background noise of equipment in the laboratory setting. Overall, our results support utilizing auditory enrichment in laboratory zebrafish to reduce stress and improve welfare in this experimental aquatic organism.

Aloysia triphylla in the zebrafish food: effects on physiology, behavior, and growth performance.

Dietary supplements are commonly used by animals and humans and play key roles in diverse systems, such as the immune and reproductive systems, and in metabolism. Essential oils (EOs), which are natural substances, have potential for use in food supplementation; however, their effects on organisms remain to be elucidated. Here, we examine the effects of dietary Aloysia triphylla EO supplementation on zebrafish behavior, metabolism, stress response, and growth performance. We show that fish fed diets containing A. triphylla EO presented an anxiolytic response, with reduced exploratory activity and oxygen consumption; no changes were observed in neuroendocrine stress axis functioning and growth was not impaired. Taken together, these results suggest that the A. triphylla EO supplementation is a strong candidate for use in feed, since it ensures fish welfare (anxiolytic behavior) with decreased oxygen consumption. This makes it suitable for use in high-density production systems without causing damage to the neuroendocrine stress axis and without growth performance being impaired.

Environmental enrichment modulates the response to chronic stress in zebrafish.

Several studies have shown that manipulations to the housing environment modulate susceptibility to stress in laboratory animals, mainly in rodents. Environmental enrichment (EE) is one such manipulation that promotes neuroprotection and neurogenesis, besides affecting behaviors such as drug self-administration. Zebrafish are a popular and useful animal model for behavioral neuroscience studies; however, studies evaluating the impact of housing conditions in this species are scarce. In this study, we verified the effects of EE on behavioral (novel tank test) and biochemical [cortisol and reactive oxygen species (ROS)] parameters in zebrafish submitted to unpredictable chronic stress (UCS). Consistent with our previous findings, UCS increased anxiety-like behavior, cortisol and ROS levels in zebrafish. EE for 21 or 28 days attenuated the effects induced by UCS on behavior and cortisol, and prevented the effects on ROS levels. Our findings reinforce the idea that EE exerts neuromodulatory effects across species, reducing vulnerability to stress and its biochemical impact. Also, these results indicate that zebrafish is a suitable model animal to study the behavioral effects and neurobiological mechanisms related to EE.

Modulation of Cortisol Responses to an Acute Stressor in Zebrafish Visually Exposed to Heterospecific Fish During Development.

Studies regarding predator-prey relationships have often focused on prey assessment and the responses to predation risk, but few have verified the relationship in the presence or absence of visual contact with a predator (e.g., tiger oscar, Astronotus ocellatus) or a nonpredator (e.g., goldfish, Carassius auratus) during the developmental phase, which could alter several physiological and neuroendocrine mechanisms in adulthood. Herein, we determined responses to physical (chasing with a net) and biological stressors (visualization to predator) in adult zebrafish raised in visual contact with a predator or nonpredator fish. We demonstrated that adult naive zebrafish show a more intense cortisol stress response than fish housed in visual contact with the stimulus fish (predator or nonpredator) when larvae, and that this alteration is related with movement specificity of the stimulus fish.

Lithium prevents scopolamine-induced memory impairment in zebrafish.

We examined whether lithium carbonate (Li2CO3, 100mg/L) is able to prevent memory impairment induced by scopolamine. Moreover, we evaluated the effects of lithium on anxiety-like behavior and acetylcholinesterase activity in adult zebrafish. We demonstrated that lithium prevents the memory impairment induced by scopolamine, decreases exploration and increases the activity of acetylcholinesterase in zebrafish. Collectively, this contributes to a better understanding of the pharmacology of lithium, its interaction with cholinergic neurotransmission, and its possible application to therapeutic treatments aimed at improving cognition.

Divergent effect of fluoxetine on the response to physical or chemical stressors in zebrafish.

Fluoxetine is a selective serotonin reuptake inhibitor that increases serotonin concentration in the central nervous system and modulates various systems, including the control of sympathetic outflow and the hypothalamus-pituitary-adrenal. However, it is not yet established whether fluoxetine can modulate the responses to stressors stimulants (physical or chemical) that trigger cortisol response in zebrafish. We demonstrate that fluoxetine blunts the response to physical stress, but not to chemical stress.

Gender differences in aggression and cortisol levels in zebrafish subjected to unpredictable chronic stress.

Chronic stress may cause physical, behavioral and neuropsychiatric changes, affecting the health condition of an individual. Aggression is a universal behavior with great relevance on human and animal social systems. Despite studies showing the influence of chronic stress on aggression, the effects of unpredictable chronic stress (UCS) on aggressive behavior in male and female zebrafish remain unknown. Thus, the aim of this study was to evaluate the effects of UCS on the aggressive behavior and cortisol levels in adult zebrafish of both sexes. Our results showed that UCS increased aggression in males, but not in females, which displayed more aggressive behavior at baseline than control males. Increased whole-body cortisol levels were observed in stressed males; however, no differences were found between female groups. In conclusion, we reported for the first time gender differences on behavioral parameters and cortisol levels in response to UCS in zebrafish. These results highlight the relevance of studying behavioral and physiological parameters in both sexes separately.

Effects of ZnSO4-induced peripheral anosmia on zebrafish behavior and physiology.

Olfaction plays a key role in modulating behavioral and physiological responses of various animal species, including fishes. Olfactory deficits can be induced in fish experimentally, and utilized to examine the role of olfaction in their normal and pathological behaviors. Here, we examine whether experimental anosmia, evoked by ZnSO4 in adult zebrafish can be associated with behavioral and/or physiological responses. We show that experimental ZnSO4-induced anosmia caused acute, but not prolonged, anxiogenic-like effects on zebrafish behavior tested in the novel tank test. The procedure also elevated whole-body cortisol levels in zebrafish. Moreover, ZnSO4 treatment, but not sham, produced damage to olfactory epithelium, inducing overt basal cell vacuolization and intercellular edema. The loss of olfaction, assessed by the fish food preference behavior in the aquatic Y-maze, was present 1h, but not 24h, after the treatment. Collectively, this suggests that transient experimental anosmia by ZnSO4 modulates zebrafish behavior and olfaction, which can be used to evoke and assess their stress-related anxiety-like states.

Behavioral responses of zebrafish depend on the type of threatening chemical cues.

In fish, defensive reactions are induced by different chemical cues that emanate from sense-related stresses [physical, chemical, and visual (visual contact with predator)] or food stresses (acute fasting and chronic food restriction). Using a shuttle box with a two-chamber unmixed laminar flow that allowed fish to remain or flee from a chemical cue, we showed that the avoidance response depended on the type of the chemical cue. We show that zebrafish (Danio rerio) retreated from water conditioned with chemical cues released by chemically or physically stressed fish and acutely fasted fish, but not from water with cues from fish experiencing visual contact with predatory fish or fish suffering from chronic food restriction. Our data reinforced the hypothesis that fish use a combination of information and the context of the situation to determine their evasion strategy.

Prevention of unpredictable chronic stress-related phenomena in zebrafish exposed to bromazepam, fluoxetine and nortriptyline.

RATIONALE: Several model organisms have been employed to study the impacts of stress on biological systems. Different models of unpredictable chronic stress (UCS) have been established in rodents; however, these protocols are expensive, long-lasting, and require a large physical structure. Our group has recently reported an UCS protocol in zebrafish with several advantages compared to rodent models. We observed that UCS induced behavioral, biochemical, and molecular changes similar to those observed in depressed patients, supporting the translational relevance of the protocol. OBJECTIVES: Considering that a pharmacological assessment is lacking in this zebrafish model, our aim was to evaluate the effects of anxiolytic (bromazepam) and antidepressant drugs (fluoxetine and nortriptyline) on behavioral (novel tank test), biochemical (whole-body cortisol), and molecular parameters (cox-2, tnf-α, il-6, and il-10 gene expression) in zebrafish subjected to UCS. RESULTS: We replicated previous data showing that UCS induces behavioral and neuroendocrine alterations in zebrafish, and we show for the first time that anxiolytic and antidepressant drugs are able to prevent such effects. Furthermore, we extended the molecular characterization of the model, revealing that UCS increases expression of the pro-inflammatory markers cox-2 and il-6, which was also prevented by the drugs tested. CONCLUSIONS: This study reinforces the use of zebrafish as a model organism to study the behavioral and physiological effects of stress. The UCS protocol may also serve as a screening tool for evaluating new drugs that can be used to treat psychiatric disorders with stress-related etiologies.

Environmental and Pharmacological Manipulations Blunt the Stress Response of Zebrafish in a Similar Manner.

Here we provide evidence that both pharmacological and environmental manipulations similarly blunt the cortisol release in response to an acute stressor in adult zebrafish. Different groups of fish were maintained isolated or group-housed in barren or enriched tanks, and then exposed or not to diazepam or fluoxetine. Acute stress increased cortisol levels in group-housed zebrafish maintained in barren environment. Single-housed zebrafish displayed a blunted cortisol response to stress. Environmental enrichment also blunted the stress response and this was observed in both isolated and group-housed fish. The same blunting effect was observed in zebrafish exposed to diazepam or fluoxetine. We highlighted environmental enrichment as an alternative and/or complimentary therapeutic for reducing stress and as a promoter of animal welfare.

Evaluating "anxiety" and social behavior in jundiá (Rhamdia quelen).

Jundiá (Rhamdia quelen) is a suitable species for aquaculture in regions of temperate or subtropical climate. This species has received great attention regarding several aspects of physiology as well as an organism to study the impact of environmental contaminations. However, experiments using validated and objective tests to evaluate the jundiá behavior are scarce. The effects of acute stress have been studied in other fish species, such as zebrafish (Danio rerio), however, the effects in jundiá are lacking. Thus, we evaluated the effects of acute stress (net chasing) on anxiety-like and social behavior in jundiá. For these purpose, all behavioral analyses were carried out using automated tracking software. We showed that the acute stress protocol increased cortisol levels and induced anxiogenic-like behavior in the novel tank test, and decreased social behavior in jundiá. The antidepressant fluoxetine was able to prevent the effects of acute stress on social behavior. Here we show a behavioral evaluation of Rhamdia quelen using consolidated tests and computerized analysis, which allows more measurable, reliable and comparable results.

The smell of "anxiety": Behavioral modulation by experimental anosmia in zebrafish.

Olfaction is strongly involved in the regulation of fish behavior, including reproductive, defensive, social and migration behaviors. In fish, anosmia (the lack of olfaction) can be induced experimentally, impairing their ability to respond to various olfactory stimuli. Here, we examine the effects of experimental lidocaine-induced anosmia on anxiety-like behavior and whole-body cortisol levels in adult zebrafish (Danio rerio). We show that experimentally-induced anosmia reduces anxiolytic-like behavioral effects of fluoxetine and seems to interact with anxiogenic effect of stress also paralleling cortisol responses in zebrafish. These findings provide first experimental evidence that temporary anosmia modulates anxiety-like behaviors and physiology in adult zebrafish.