Multifractal fluctuations in zebrafish (Danio rerio) polarization time series.

In this work, we study the polarization time series obtained from experimental observation of a group of zebrafish (Danio rerio) confined in a circular tank. The complex dynamics of the individual trajectory evolution lead to the appearance of multiple characteristic scales. Employing the Multifractal Detrended Fluctuation Analysis (MF-DFA), we found distinct behaviors according to the parameters used. The polarization time series are multifractal at low fish densities and their average scales with ρ - 1 / 4 . On the other hand, they tend to be monofractal, and their average scales with ρ - 1 / 2 for high fish densities. These two regimes overlap at critical density ρ c , suggesting the existence of a phase transition separating them. We also observed that for low densities, the polarization velocity shows a non-Gaussian behavior with heavy tails associated with long-range correlation and becomes Gaussian for high densities, presenting an uncorrelated regime.

Pebble to the Metal: A Boulder Approach to Enrichment for Danio rerio.

Zebrafish are an established and widely used animal model, yet there is limited understanding of their welfare needs. Despite an increasing number of studies on zebrafish enrichment, in-tank environmental enrichment remains unpopular among researchers. This is due to perceived concerns over health/hygiene when it comes to introducing enrichment into the tank, although actual evidence for this is sparse. To accommodate this belief, regardless of veracity, we tested the potential benefits of enrichments presented outside the tank. Thus, we investigated the preferences and physiological stress of zebrafish with pictures of pebbles placed underneath the tank. We hypothesized that zebrafish would show a preference for enriched environments and have lower stress levels than barren housed zebrafish. In our first experiment, we housed zebrafish in a standard rack system and recorded their preference for visual access to a pebble picture, with two positive controls: visual access to conspecifics, and group housing. Using a crossover repeated-measures factorial design, we tested if the preference for visual access to pebbles was as strong as the preference for social contact. Zebrafish showed a strong preference for visual access to pebbles, equivalent to that for conspecifics. Then, in a second experiment, tank water cortisol was measured to assess chronic stress levels of zebrafish housed with or without a pebble picture under their tank, with group housing as a positive control. Cortisol levels were significantly reduced in zebrafish housed with pebble pictures, as were cortisol levels in group housed zebrafish. In fact, single housed zebrafish with pebble pictures showed the same cortisol levels as group housed zebrafish without pebble pictures. Thus, the use of an under-tank pebble picture was as beneficial as being group housed, effectively compensating for the stress of single housing. Pebble picture enrichment had an additive effect with group housing, where group housed zebrafish with pebble pictures had the lowest cortisol levels of any treatment group.

Aging in zebrafish is associated with reduced locomotor activity and strain dependent changes in bottom dwelling and thigmotaxis.

Aging is associated with a wide range of physiological and behavioral changes in many species. Zebrafish, like humans, rodents, and birds, exhibits gradual senescence, and thus may be a useful model organism for identifying evolutionarily conserved mechanisms related to aging. Here, we compared behavior in the novel tank test of young (6-month-old) and middle aged (12-month-old) zebrafish from two strains (TL and TU) and both sexes. We find that this modest age difference results in a reduction in locomotor activity in male fish. We also found that background strain modulated the effects of age on predator avoidance behaviors related to anxiety: older female TL fish increased bottom dwelling whereas older male TU fish decreased thigmotaxis. Although there were no consistent effects of age on either short-term (within session) or long-term (next day) habituation to the novel tank, strain affected the habituation response. TL fish tended to increase their distance from the bottom of the tank whereas TU fish had no changes in bottom distance but instead tended to increase thigmotaxis. Our findings support the use of zebrafish for the study of how age affects locomotion and how genetics interacts with age and sex to alter exploratory and emotional behaviors in response to novelty.

Teaching Fin Regeneration Using a Dominant Negative Receptor.

Rising in popularity as a model organism in the classroom, zebrafish have numerous characteristics that make them ideal for teaching. In this study, we describe an experiment that helps students better understand the concept of tissue regeneration and the genes that control it. This experiment utilizes a dominant negative transgene for fgfr1 and allows students to observe the consequences of its activation. The first part of the laboratory is hands-on, and includes details of the amputation of caudal fins, heat shocking, general fish care, and visual observations. Over the course of a week, students observed the differences between the activated and unactivated transgene in the zebrafish. The second part was literature based, in which students tried to determine which gene is responsible for inhibiting regeneration. This encouraged students to sharpen their skills of deductive reasoning and critical thinking as they conduct research based on the information they receive about dominant negative receptors and transgenes. Having both a hands-on and critical thinking component in the laboratory helped synthesize the learning goals and allowed students to actively participate.

A 3D-Printed and Freely Available Device to Measure the Zebrafish Optokinetic Response Before and After Injury.

Zebrafish eyes are anatomically similar to humans and have a higher percentage of cone photoreceptors more akin to humans than most rodent models, making them a beneficial model organism for studying vision. However, zebrafish are different in that they can regenerate their optic nerve after injury, which most other animals cannot. Vision in zebrafish and many other vertebrate animals, including humans, can be accessed using the optokinetic response (OKR), which is an innate eye movement that occurs when tracking an object. Because fish cannot use an eye chart, we utilize the OKR that is present in virtually all vertebrates to determine if a zebrafish has vision. To this end, we have developed an inexpensive OKR setup that uses 3D-printed and off-the-shelf parts. This setup has been designed and used by undergraduate researchers and is also scalable to a classroom laboratory setup. We demonstrate that this setup is fully functional for assessing the OKR, and we use it to illustrate the return of the OKR following optic nerve injury in adult zebrafish.

Neurotoxicity of tetramethylammonium ion on larval and juvenile zebrafish: Effects on neurobehaviors and multiple biomarkers.

Tetramethylammonium hydroxide (TMAH) is an important compound that utilized and released by the rapidly expanding semiconductor industry, which could hardly be removed by the conventional wastewater treatment techniques. As a cholinergic agonist, the tetramethylammonium ion (TMA+) has been reported to induce toxicity to muscular and respiratory systems of mammals and human, however the toxicity on aquatic biota remains poorly known. We investigated the neurotoxic effects of TMA+ exposure on zebrafish, based on neurobehavior tests and a series of biomarkers. Significant inhibitions on the swimming distance of zebrafish larvae were observed when the exposure level exceeded 50 mg/L, and significant alterations on swimming path angles (straight and deflective movements) occurred even at 10 mg/L. The tested neurobehavioral endpoints of zebrafish larvae were significantly positively correlated with reactive oxygen species (ROS) and malondialdehyde (MDA), significantly negatively related with the activities of antioxidant enzymes, but not significantly correlated with the level of acetylcholinesterase (AChE). Such relationship indicates that the observed neurotoxic effects on swimming behavior of zebrafish larvae is mainly driven by oxidative stress, rather than the alterations of neurotransmitter. At the highest exposure concentration (200 mg/L), TMA+ evoked more severe toxicity on zebrafish juveniles, showing significantly stronger elevation on the MDA activity, and greater inhibitions on the activities of antioxidant enzymes and AChE, suggesting juveniles were more susceptible to TMA+ exposure than larval zebrafish.

Behavioral toxicity and neurotoxic mechanisms of PLA-PBAT biodegradable microplastics in zebrafish.

Escalation of ecological concern due to biodegradable plastics has attracted the attention of many contemporary researchers. This study searched to investigate the acute and sub-chronic toxicity of polylactic acid (PLA) and polybutyleneadipate-co-terephthalate (PLA-PBAT) bio-microplastics on 3-month-old zebrafish to elucidate their potential toxic mechanisms. Acute toxicity assessments revealed 96 h-LC50 value of 12.69 mg/L for PLA-PBAT. Sub-chronic exposure of over 21 days revealed deviations in critical behavioral patterns and physiological indicators. In treated groups, weight gain and specific growth rates were significantly lower than those obtained for the control group, such that high doses induced significant reductions in total organ coefficient (p < 0.05). A positive correlation was observed between zebrafish mortality and increased doses. Detailed behavioral evaluations revealed a dose-dependent decrease in the speed and range of swimming, along with modifications in shoaling behavior, anxiety-like responses, and avoidance behaviors. Brain tissues transcriptomic analyses revealed the molecular responses underlying sub-chronic exposure to PLA-PBAT. Totally 702 DEGs and 5 KEGG pathways were significantly identified in low-dose group, with the top 2 significant pathways being ribosome pathway and cytokine-cytokine receptor interaction pathway. Totally 650 DEGs and 5 KEGG pathways were significantly identified in medium-dose group, with the top 2 significant pathways being Herpes simplex virus 1 infection pathway and complement and coagulation cascades pathway. Totally 1778 DEGs and 16 KEGG pathways were significantly identified in high-dose group, with the top 2 significant pathways being metabolism of xenobiotics by cytochrome P450 and drug metabolism - cytochrome P450 pathway. Most significantly enriched pathways are associated with immune responses. The validation of key gene in cytokine-cytokine receptor interaction pathway also confirmed its high correlation with behavioral indicators. These results indicate that PLA-PBAT is likely to cause behavioral abnormalities in zebrafish by triggering immune dysregulation in the brain.

Schreckstoff: It takes two to panic.

Schreckstoff (fear substance) is an alarm signal released by injured fish that induces a fear response. Its chemical nature has long been debated. A new study finds that zebrafish Schreckstoff is composed of at least three components, two of which elicit the fear response only in combination.

Bacterial accumulation in intestinal folds induced by physical and biological factors.

BACKGROUND: The gut microbiota, vital for host health, influences metabolism, immune function, and development. Understanding the dynamic processes of bacterial accumulation within the gut is crucial, as it is closely related to immune responses, antibiotic resistance, and colorectal cancer. We investigated Escherichia coli behavior and distribution in zebrafish larval intestines, focusing on the gut microenvironment. RESULTS: We discovered that E. coli spread was considerably suppressed within the intestinal folds, leading to a strong physical accumulation in the folds. Moreover, a higher concentration of E. coli on the dorsal side than on the ventral side was observed. Our in vitro microfluidic experiments and theoretical analysis revealed that the overall distribution of E. coli in the intestines was established by a combination of physical factor and bacterial taxis. CONCLUSIONS: Our findings provide valuable insight into how the intestinal microenvironment affects bacterial motility and accumulation, enhancing our understanding of the behavioral and ecological dynamics of the intestinal microbiota.

Mixture of pesticides based on dimethylamine and imidacloprid affects locomotion of adult zebrafish.

Numerous chemical compounds are found in aquatic environments; among them are pesticides. Pesticides are widely used worldwide, and this use has progressively increased in recent decades, resulting in the accumulation of potentially toxic compounds in surface waters. Dimethylamine-based herbicides (DBH) and imidacloprid-based insecticides (IBI) have low soil absorption and high water solubility, facilitating the arrival of these compounds in aquatic environments. In this study, our objective was to analyze whether two pesticides, DBH and IBI at environmentally relevant concentrations of 320 µg/L for each compound, and their mixtures impact the behavioral and endocrine parameters of adult zebrafish, verifying the effect of pesticides on exploratory behavior and social and analyzing hormonal parameters related to stress. Acute exposure to the mixture of pesticides reduced fish locomotion. Pesticides alone and in combination did not affect cortisol levels in exposed animals. Pesticides, when tested together, can cause different effects on non-target organisms, and the evaluation of mixtures of these compounds is extremely important.

Plants buffer some of the effects of a pair of cadmium-exposed zebrafish on the un-exposed majority.

Certain individuals have a disproportionate effect on group responses. Characteristics may include susceptibility to pollutants, such as cadmium (Cd), a potent trace metal. Here, we show how a pair of Cd-exposed individuals can impact the behavior of unexposed groups. We used behavioral assessments to characterize the extent of the effects of the Cd-exposed individuals on group boldness, cohesion, foraging, activity, and responses to plants. We found that groups with a pair of Cd-exposed fish remained closer to novel stimuli and plants than did groups with untreated (control) fish. The presence of plants reduced Cd-induced differences in shoal cohesion and delays feeding in male shoals. Shoals with Cd- and water-treated fish were equally active. The results suggest that fish acutely exposed to environmentally relevant Cd concentrations can have profound effects on the un-exposed majority. However, the presence of plants may mitigate the effects of contaminants on some aspects of social behavior.

Sea Cucumber Viscera Contains Novel Non-Holostane-Type Glycoside Toxins that Possess a Putative Chemical Defense Function.

Sea cucumbers frequently expel their guts in response to predators and an aversive environment, a behavior perceived as releasing repellents involved in chemical defense mechanisms. To investigate the chemical nature of the repellent, the viscera of stressed sea cucumbers (Apostichopus japonicus) in the Yellow Sea of China were collected and chemically analyzed. Two novel non-holostane triterpene glycosides were isolated, and the chemical structures were elucidated as 3ꞵ-O-[ꞵ-D-glucopyranosyl-(1→2)-ꞵ-D-xylopyranosyl]-(20S)-hydroxylanosta-7,25-diene-18(16)-lactone (1) and 3ꞵ-O-[ꞵ-D-quinovopyranosyl-(1→2)-ꞵ-D-xylopyranosyl]-(20S)-hydroxylanosta-7,25-diene-18(16)-lactone (2) by spectroscopic and mass-spectrometric analyses, exemplifying a triterpene glycoside constituent of an oligosaccharide containing two sugar-units and a non-holostane aglycone. Zebrafish embryos were exposed to various doses of 1 and 2 from 4 to 96 hpf. Compound 1 exposure showed 96 h-LC50 41.5 µM and an increased zebrafish mortality rates in roughly in a dose- and time-dependent manner. Compound 2, with different sugar substitution, exhibited no mortality and moderate teratogenic toxicity with a 96 h-EC50 of 173.5 µM. Zebrafish embryos exhibited teratogenic effects, such as reduced hatchability and total body length. The study found that triterpene saponin from A. japonicus viscera had acute toxicity in zebrafish embryos, indicating a potential chemical defense role in the marine ecosystem.

Biotoxicity responses of zebrafish in environmentally relevant concentration of di (2-ethylhexyl) phthalate.

As an emerging environmental contaminant, di (2-ethylhexyl) phthalate (DEHP) is widely present in the aquatic environment, however, the effects and underlying mechanisms of DEHP on the aquatic organisms are poorly understood. This study systematically investigated the ecotoxicity induced by chronic exposure to environmental relevant concentrations of DEHP (0.03 mg/L, 0.1 mg/L, and 0.3 mg/L) on zebrafish brain. Results indicated that DEHP exposure significantly increased the levels of ROS and disturbance of the antioxidant enzymes activities in the brain, which may further enhance lipid peroxidation and DNA damage. Furthermore, acetylcholinesterase activity was first stimulated and inhibited by exposure to DEHP, and the antioxidant and apoptosis related genes were mainly upregulated. Risk assessment indicated that the ecotoxicity of DEHP on the zebrafish showed an "enhancement-reduction" trend as the exposure time was prolonged. Overall, these results provided new insights and useful information to ecological risk assessment and environmental management of DEHP pollution.

One-trial odour recognition learning and its underlying brain areas in the zebrafish.

Distinguishing familiar from novel stimuli is critical in many animals' activities, and procedures based on this ability are among the most exploited in translational research in rodents. However, recognition learning and the underlying brain substrates remain unclear outside a few mammalian species. Here, we investigated one-trial recognition learning for olfactory stimuli in a teleost fish using a behavioural and molecular approach. With our behavioural analysis, we found that zebrafish can learn to recognise a novel odour after a single encounter and then, discriminate between this odour and a different one provided that the molecular structure of the cues is relatively differentiated. Subsequently, by expression analysis of immediate early genes in the main brain areas, we found that the telencephalon was activated when zebrafish encountered a familiar odour, whereas the hypothalamus and the optic tectum were activated in response to the novel odour. Overall, this study provided evidence of single-trial spontaneous learning of novel odours in a teleost fish and the presence of multiple neural substrates involved in the process. These findings are promising for the development of zebrafish models to investigate cognitive functions.

Dimethyl phthalate induced cardiovascular developmental toxicity in zebrafish embryos by regulating MAPK and calcium signaling pathways.

Dimethyl phthalate (DMP), the lowest-molecular-weight phthalate ester (PAE), is one of the most commonly detected persistent organic pollutants in the environment, but its toxic effects, especially cardiovascular developmental toxicity, are largely unknown. In this study, zebrafish embryos were exposed to sublethal concentrations of DMP from 4 to 96 hpf. Our results showed that DMP treatment induced yolk retention, pericardial edema, and swim bladder deficiency, as well as increased SV-BA distance and decreased heart rate, stroke volume, ventricular axis shortening rate and ejection fraction. In addition, oxidative stress and apoptosis were found to be highly involved in this process. The results of transcriptome sequencing and mRNA expression of related genes indicated that MAPK and calcium signaling pathways were perturbed by DMP. These findings have the potential to provide new insights into the potential developmental toxicity and cardiovascular disease risk of DMP.

Toxic effects and comparison of common amino antioxidants (AAOs) in the environment on zebrafish: A comprehensive analysis based on cells, embryos, and adult fish.

The ubiquity of amino antioxidants (AAOs) in the environment has attracted increasing attention, given their potential toxicity. This investigation represents a pioneering effort, systematically scrutinizing the toxicological effects of four distinct AAOs across the developmental spectrum of zebrafish, encompassing embryonic, larvae, and adult stages. The results indicate that four types of AAO exhibit varying degrees of cell proliferation toxicity. Although environmentally relevant concentrations of AAOs exhibit a comparatively circumscribed impact on zebrafish embryo development, heightened concentrations (300 µg/L) of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and N-isopropyl-N'-phenyl-p-phenylenediamine (IPPD) distinctly evoke developmental toxicity. Behavioral analysis results indicate that at concentrations of 20 and 300 µg/L, the majority of AAOs significantly reduced the swimming speed and activity of larvae. Moreover, each AAO triggers the generation of reactive oxygen species (ROS) in larvae, instigating diverse levels of oxidative stress. The study delineates parallel toxicological patterns in zebrafish exposed to 300 µg/L of 6PPD and IPPD, thereby establishing a comparable toxicity profile. The comprehensive toxicity effects among the four AAOs is as follows: IPPD >6PPD > N-Phenyl-1-naphthylamine (PANA) > diphenylamine (DPA). These findings not only enrich our comprehension of the potential hazards associated with AAOs but also provide data support for structure-based toxicity prediction models.

Mechanistic interplay of dual environmental stressors: Bisphenol-A and cadmium-induced ovarian follicular damage and hepatocyte dysfunction in vivo.

This study investigates the individual and combined toxic effects of Bisphenol A (BPA) and Cadmium (Cd) in zebrafish, recognizing the complex mixture of pollutants organisms encounter in their natural environment. Examining developmental, neurobehavioral, reproductive, and physiological aspects, the study reveals significant adverse effects, particularly in combined exposures. Zebrafish embryos exposed to BPA + Cd exhibit synergistically increased mortality, delayed hatching, and morphological abnormalities, emphasizing the heightened toxicity of the combination. Prolonged exposure until 10 days post-fertilization underscores enduring effects on embryonic development. BPA and Cd induce oxidative stress, as evidenced by increased production of reactive oxygen species and lipid peroxidation. This oxidative stress disrupts cellular functions, affecting lipid metabolism and immune response. Adult zebrafish exposed to BPA and Cd for 40 days display compromised neurobehavioral functions, altered antioxidant defenses, and increased oxidative stress, suggesting potential neurotoxicity. Additionally, disruptions in ovarian follicle maturation and skeletal abnormalities indicate reproductive and skeletal impacts. Histological analysis reveals significant liver damage, emphasizing the synergistic hepatotoxicity of BPA and Cd. Molecular assessments further demonstrate compromised cellular defense mechanisms, synaptic function, and elevated cellular stress and inflammation-related gene expression in response to combined exposures. Bioaccumulation analysis highlights differential tissue accumulation patterns. In conclusion, this study provides comprehensive insights into the multifaceted toxicological effects of BPA and Cd in zebrafish, raising concerns about potential adverse impacts on environmental ecosystems and human health.

'Environmental standard limit concentration' arsenic exposure is associated with anxiety, depression, and autism-like changes in early-life stage zebrafish.

Arsenic is a worldwide environmental pollutant that can impair human health. Previous studies have identified mental disorders induced by arsenic, but the environmental exposure concentrations in the early life stages associated with these disorders are poorly understood. In the present study, early-life stage zebrafish were used to explore the effects on mental disorders under 'environmental standard limit concentrations' arsenic exposures of 5, 10, 50, 150, and 500 µg/L. The results showed that arsenic exposure at these concentrations changed the locomotor behavior in larval zebrafish and was further associated with anxiety, depression, and autism-like behavior in both larval and juvenile zebrafish. Changes were noted at benchmark dose limit (BMDL) concentrations as low as 0.81 µg/L. Transcriptomics showed that immediate early genes (IEGs) fosab, egr1, egr2a, ier2b, egr3, and jund were decreased after arsenic exposure in larval and juvenile zebrafish. Nervous system impairment and anxiety, depression, and autism-like behaviors in early-life stage zebrafish at 'environmental standard limit concentrations' may be attributed to the downregulation of IEGs. These findings in zebrafish provided new experimental support for an arsenic toxicity threshold for mental disorders, and they suggest that low levels of environmental chemicals may be causative developmental factors for mental disorders.

Transgenerational effects of extracts containing Microcystin-LR exposure on reproductive toxicity and offspring growth inhibition in a model organism zebrafish.

Cyanobacteria cell lysates release numerous toxic substances (e.g., cyanotoxins) into the water, posing a serious threat to human health and aquatic ecosystems. Microcystins (MCs) are among the most abundant cyanotoxins in the cell lysates, with microcystin-LR (MC-LR) being one of the most common and highly toxic congeners. In this study, zebrafish (Danio rerio) were exposed to different levels MC-LR that from extracts of Microcystis aeruginosa. Changes in the MC-LR accumulations, organ coefficients, and antioxidant enzyme activities in the zebrafish were analyzed. Transgenerational reproductive toxicity of MC-LR in the maternal and paternal generations was further investigated, as well as the influences of extracts containing MC-LR exposures of the F1 on the growth of zebrafish. The study found that high levels of MC-LR could be detected in the major organs of adult zebrafish, particularly in spleen. Notably, concentration of MC-LR in the spermary was significantly higher than that in the ovarium. MC-LR could induce oxidative damage by affecting the activities of catalase and superoxide dismutase. Inherited from F0, MC-LR led to impaired development in the F1 generation. Difference in offspring survival rates could be observed in the groups with different MC-LR levels of maternal and paternal exposures. This study reveals transgenerational effects of MC-LR on the reproductive toxicity and offspring growth inhibition to the aquatic organisms, which should be emphasized in the future ecological risk assessment.

Polystyrene microplastics mitigate lead-induced neurotoxicity by reducing heavy metal uptake in zebrafish larvae.

The combined pollution of lead (Pb) and polystyrene microplastics (PS-MPs) is common in aquatic environments. However, the combined neurotoxicity of these two pollutants is still poorly understood. In this study, zebrafish (Danio rerio) larvae were used to assess the combined neurotoxicity and mechanism of Pb and PS-MPs at environmentally relevant concentrations. The results showed that Pb (10 µg/L) induced abnormal behavior including significantly reduced movement distance, maximum acceleration, and average velocity (P < 0.05) along with altered expression of neurodevelopment-related genes (gap43 and α1-tubulin) (P < 0.05). PS-MPs (25 µg/L, 250 µg/L; diameter at 25 µm) co-exposure not only significantly reduced the concentration of Pb in the exposed solution (P < 0.01), but also decreased the uptake of Pb by downregulating the divalent metal transporter 1 gene (dmt1) (P < 0.01), thereby alleviating Pb-induced neurotoxicity. However, to demonstrate that PS-MPs alleviate the neurotoxicity of Pb by reducing Pb uptake, upregulation of dmt1 by addition of deferoxamine (DFO, an efficient iron chelator, 100 µM) significantly increased the Pb uptake and exacerbated neurotoxicity in zebrafish. In summary, our results demonstrated that PS-MPs alleviate Pb neurotoxicity by downregulating the mRNA level of dmt1 and decreasing the Pb uptake. This study provides a new insight into the combined neurotoxicity and underlying mechanisms of PS-MPs and Pb on zebrafish.