Toxicity evaluation of neonicotinoids to earthworm (Eisenia fetida) behaviors by a novel locomotion tracking assay.

Pesticides are substances used for controlling, preventing, and repelling pests in agriculture. Among them, neonicotinoids have become the fastest-growing class of insecticides because of their efficiency in targeting pests. They work by strongly binding to nicotinic acetylcholine receptors (nAChRs) in the central nervous system of insects, leading to receptor blockage, paralysis, and death. Despite their selectivity for insects, these substances may be hazardous to non-target creatures, including earthworms. Although earthworms may be invasive in some regions like north America, they contribute to the development of soil structure, water management, nutrient cycling, pollution remediation, and cultural services, positively impacting the environment, particularly in the soil ecosystem. Thus, this study aimed to develop a novel earthworm behavior assay since behavior is a sensitive marker for toxicity assay, and demonstrated its application in evaluating the toxicity of various neonicotinoids. Here, we exposed Eisenia fetida to 1 and 10 ppb of eight neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram pestanal, thiacloprid, thiametoxam, and sulfoxaflor) for 3 days to observe their behavior toxicities. Overall, all of the neonicotinoids decreased their locomotion, showed by a reduction of average speed by 24.94-68.63% and increment in freezing time movement ratio by 1.51-4.25 times, and altered their movement orientation and complexity, indicated by the decrement in the fractal dimension value by 24-70%. Moreover, some of the neonicotinoids, which were acetamiprid, dinotefuran, imidacloprid, nitenpyram, and sulfoxaflor, could even alter their exploratory behaviors, which was shown by the increment in the time spent in the center area value by 6.94-12.99 times. Furthermore, based on the PCA and heatmap clustering results, thiametoxam was found as the neonicotinoid that possessed the least pronounced behavior toxicity effects among the tested pesticides since these neonicotinoid-treated groups in both concentrations were grouped in the same major cluster with the control group. Finally, molecular docking was also conducted to examine neonicotinoids' possible binding mechanism to Acetylcholine Binding Protein (AChBP), which is responsible for neurotransmission. The molecular docking result confirmed that each of the neonicotinoids has a relatively high binding energy with AChBP, with the lowest binding energy was possessed by thiametoxam, which consistent with its relatively low behavior toxicities. Thus, these molecular docking results might hint at the possible mechanism behind the observed behavior alterations. To sum up, the present study demonstrated that all of the neonicotinoids altered the earthworm behaviors which might be due to their ability to bind with some specific neurotransmitters and the current findings give insights into the toxicities of neonicotinoids to the environment, especially animals in a soil ecosystem.

Comparison of the psychoactive activity of four primary Areca nut alkaloids in zebrafish by behavioral approach and molecular docking.

Areca palm nut (Areca catechu) has been listed as one of the most addictive substances, along with tobacco, alcohol, and caffeine. It belongs to the family Arecaceae and is widely used in Asia. Areca nut contains seven psychoactive alkaloids; however, the effects of these alkaloids on behaviors are rarely to be addressed in zebrafish. Therefore, this study aims to compare the psychoactive and potential adverse effects of four primary alkaloids (arecoline, arecaidine, guvacine, and guvacoline) isolated from areca nut on zebrafish. We found that four alkaloids induced hyperactivity-like behaviors in zebrafish larvae. Cooperating the results with the previous study, molecular docking scores suggested these alkaloids might bind to multiple muscarinic acetylcholine receptors (mAChRs), and various best binding modes were shown. According to the adult zebrafish behavioral test, arecoline was found to slightly increase the locomotor activity and caused tightening shoaling formations of adult zebrafish. Meanwhile, zebrafish exposed to arecaidine have reduced aggressiveness and conspecific social interaction. Similar to arecaidine, guvacoline treatment also caused abnormalities in zebrafish social behaviors. Furthermore, the fish displayed abnormal exploratory behaviors after being exposed to guvacoline. Interestingly, altered fear response behaviors were only displayed by guvacine-treated fish besides their lower locomotor activity. Based on the results of molecular docking, we hypothesize that the behavior alterations might be a consequence of the interaction between alkaloids and multiple mAChRs in the nervous system. In summary, our study found that each alkaloid specifically affects adult zebrafish behaviors.

An Update Report on the Biosafety and Potential Toxicity of Fullerene-Based Nanomaterials toward Aquatic Animals.

Fullerene molecules are composed of carbon in the form of a hollow sphere, tube, or ellipsoid. Since their discovery in 1985, they have gained a lot of attention in many science fields. The unique carbon cage structure of fullerene provides immense scope for derivatization, rendering potential for various industrial applications. Thus, the prospective applications of fullerenes have led to assorted fullerene derivatives. In addition, their unique chemical structure also eases them to be synthesized through various kinds of conjugating techniques, where fullerene can be located either on the backbone or the branch chain. In this review, we have compiled the toxicity and biosafety aspects of fullerene in aquatic organisms since the frequent use of fullerene is likely to come in contact and interact with the aquatic environment and aquatic organisms. According to the current understanding, waterborne exposure to fullerene-based nanomaterials indeed triggers toxicities at cellular, organic, molecular, and neurobehavioral levels.

Interspecies Behavioral Variability of Medaka Fish Assessed by Comparative Phenomics.

Recently, medaka has been used as a model organism in various research fields. However, even though it possesses several advantages over zebrafish, fewer studies were done in medaka compared to zebrafish, especially with regard to its behavior. Thus, to provide more information regarding its behavior and to demonstrate the behavioral differences between several species of medaka, we compared the behavioral performance and biomarker expression in the brain between four medaka fishes, Oryzias latipes, Oryzias dancena, Oryzias woworae, and Oryzias sinensis. We found that each medaka species explicitly exhibited different behaviors to each other, which might be related to the different basal levels of several biomarkers. Furthermore, by phenomics and genomic-based clustering, the differences between these medaka fishes were further investigated. Here, the phenomic-based clustering was based on the behavior results, while the genomic-based clustering was based on the sequence of the nd2 gene. As we expected, both clusterings showed some resemblances to each other in terms of the interspecies relationship between medaka and zebrafish. However, this similarity was not displayed by both clusterings in the medaka interspecies comparisons. Therefore, these results suggest a re-interpretation of several prior studies in comparative biology. We hope that these results contribute to the growing database of medaka fish phenotypes and provide one of the foundations for future phenomics studies of medaka fish.

Pharmaceutical Assessment Suggests Locomotion Hyperactivity in Zebrafish Triggered by Arecoline Might Be Associated with Multiple Muscarinic Acetylcholine Receptors Activation.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

Exploiting the Freshwater Shrimp Neocaridina denticulata as Aquatic Invertebrate Model to Evaluate Nontargeted Pesticide Induced Toxicity by Investigating Physiologic and Biochemical Parameters.

As a nicotinoid neurotoxic insecticide, imidacloprid (IMI) works by disrupting nerve transmission via nicotinic acetylcholine receptor (nAChR). Although IMI is specifically targeting insects, nontarget animals such as the freshwater shrimp, Neocaridina denticulata, could also be affected, thus causing adverse effects on the aquatic environment. To investigate IMI toxicity on nontarget organisms like N. denticulata, their physiology (locomotor activity, heartbeat, and gill ventilation) and biochemical factors (oxidative stress, energy metabolism) after IMI exposure were examined. IMI exposure at various concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 ppm) to shrimp after 24, 48, 72 h led to dramatic reduction of locomotor activity even at low concentrations. Meanwhile, IMI exposure after 92 h caused reduced heartbeat and gill ventilation at high concentrations. Biochemical assays were performed to investigate oxidative stress and energy metabolism. Interestingly, locomotion immobilization and cardiac activity were rescued after acetylcholine administration. Through molecular docking, IMI demonstrated high binding affinity to nAChR. Thus, locomotor activity and heartbeat in shrimp after IMI exposure may be caused by nAChR blockade and not alterations caused by oxidative stress and energy metabolism. To summarize, N. denticulata serves as an excellent and sensitive aquatic invertebrate model to conduct pesticide toxicity assays that encompass physiologic and biochemical examinations.

Antidepressant Screening Demonstrated Non-Monotonic Responses to Amitriptyline, Amoxapine and Sertraline in Locomotor Activity Assay in Larval Zebrafish.

Antidepressants are well-known drugs to treat depression and major depressive disorder for humans. However, the misuse and abuse of antidepressants keep increasing with several side effects reported. The aim of this study was to assess the potential adverse effects of 18 antidepressants by monitoring zebrafish larval locomotor activity performance based on the total distance traveled, burst movement count, and total rotation count at four dark-light intercalated phases. In general, zebrafish larvae displayed sedative effects after antidepressant exposure by showing a significant reduction in all of the locomotor activity-related endpoints. However, three antidepressants i.e., amitriptyline, amoxapine, and sertraline were able to trigger a significantly high locomotor activity in zebrafish larvae during the light cycle. These differences might be due to the pharmacologic differences among the antidepressants. In addition, since each antidepressant possesses a different dosage range from the other, overdoses of these antidepressants might also be the causes of these differences. Furthermore, based on these results, a further study was conducted to observe the effect of these three antidepressants in lower concentrations. From the results, biphasic effects in terms of zebrafish larval locomotor activity were demonstrated by these drugs. Even though further studies are still required to validate the mechanism, these findings indicate that these antidepressants might share a common mechanism responsible for their effects on zebrafish larval locomotor activity although there were some differences in potency of these effects.

Comparison of the chronic toxicities of graphene and graphene oxide toward adult zebrafish by using biochemical and phenomic approaches.

Graphene (GR) and graphene oxide (GO) are widely being used as promising candidates for biomedical applications, as well as for bio-sensing, drug delivery, and anticancer therapy. However, their undesirable side effects make it necessary to assess further the toxicity and safety of using these materials. The main objective of the current study was to investigate the toxicities of GR and GO in predicted environmental relevant concentrations in adult zebrafish (Danio rerio), particularly on their behaviors, and conducted biochemical assays to elucidate the possible mechanism that underlies their toxicities. Zebrafish was chronically (∼14 days) exposed to two different doses of GR (0.1 and 0.5 ppm) or GO (0.1 and 1 ppm). At 14 ± 1 days, a battery of behavioral tests was conducted, followed by enzyme-linked immunosorbent assays (ELISA) test on the following day to inspect the alterations in antioxidant activity, oxidative stress, and neurotransmitters in the treated zebrafish brain. An alteration in predator avoidance behavior was observed in all treated groups, while GR-treated fish exhibited abnormal exploratory behavior. Furthermore, altered locomotor activity was displayed by most of the treated groups, except for the high concentration of the GR group. From the ELISA results, we discovered a high concentration of GR exposure significantly decreased several neurotransmitters and cortisol levels. Meanwhile, elevated reactive oxygen species (ROS) were displayed by the group treated with low and high doses of GR and GO, respectively. These significant changes would possibly affect zebrafish behaviors and might suggest the potential toxicity from GR and GO exposures. To sum up, the present study presented new evidence for the effects of GR and GO in zebrafish behavioral dysregulation. We hope these assessments can contribute to our understanding of graphene and graphene oxide biosafety.

Duplicated dnmt3aa and dnmt3ab DNA Methyltransferase Genes Play Essential and Non-Overlapped Functions on Modulating Behavioral Control in Zebrafish.

DNA methylation plays several roles in regulating neuronal proliferation, differentiation, and physiological functions. The major de novo methyltransferase, DNMT3, controls the DNA methylation pattern in neurons according to environmental stimulations and behavioral regulations. Previous studies demonstrated that knockout of Dnmt3 induced mouse anxiety; however, controversial results showed that activation of Dnmt3 causes anxiolytic behavior. Thus, an alternative animal model to clarify Dnmt3 on modulating behavior is crucial. Therefore, we aimed to establish a zebrafish (Danio rerio) model to clarify the function of dnmt3 on fish behavior by behavioral endpoint analyses. We evaluated the behaviors of the wild type, dnmt3aa, and dnmt3ab knockout (KO) fish by the novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, color preference, and short-term memory tests. The results indicated that the dnmt3aa KO fish possessed abnormal exploratory behaviors and less fear response to the predator. On the other hand, dnmt3ab KO fish displayed less aggression, fear response to the predator, and interests to interact with their conspecifics, loosen shoaling formation, and dysregulated color preference index ranking. Furthermore, both knockout fishes showed higher locomotion activity during the night cycle, which is a sign of anxiety. However, changes in some neurotransmitter levels were observed in the mutant fishes. Lastly, whole-genome DNA methylation sequencing demonstrates a potential network of Dnmt3a proteins that is responsive to behavioral alterations. To sum up, the results suggested that the dnmt3aa KO or dnmt3ab KO fish display anxiety symptoms, which supported the idea that Dnmt3 modulates the function involved in emotional control, social interaction, and cognition.

Evaluation of the Adverse Effects of Chronic Exposure to Donepezil (An Acetylcholinesterase Inhibitor) in Adult Zebrafish by Behavioral and Biochemical Assessments.

Donepezil (DPZ) is an acetylcholinesterase inhibitor used for the clinical treatment of mild cognitive impairment. However, DPZ has been reported to have adverse effects, including causing abnormal cardiac rhythm, insomnia, vomiting, and muscle cramps. However, the existence of these effects in subjects without Dementia is unknown. In this study, we use zebrafish to conduct a deeper analysis of the potential adverse effects of DPZ on the short-term memory and behaviors of normal zebrafish by performing multiple behavioral and biochemical assays. Adult zebrafish were exposed to 1 ppm and 2.5 ppm of DPZ. From the results, DPZ caused a slight improvement in the short-term memory of zebrafish and induced significant elevation in aggressiveness, while the novel tank and shoaling tests revealed anxiolytic-like behavior to be caused by DPZ. Furthermore, zebrafish circadian locomotor activity displayed a higher reduction of locomotion and abnormal movement orientation in both low- and high-dose groups, compared to the control group. Biomarker assays revealed that these alterations were associated with an elevation of oxytocin and a reduction of cortisol levels in the brain. Moreover, the significant increases in reactive oxygen species (ROS) and malondialdehyde (MDA) levels in muscle tissue suggest DPZ exposure induced muscle tissue oxidative stress and muscle weakness, which may underlie the locomotor activity impairment. In conclusion, we show, for the first time, that chronic waterborne exposure to DPZ can severely induce adverse effects on normal zebrafish in a dose-dependent manner. These unexpected adverse effects on behavioral alteration should be carefully addressed in future studies considering DPZ conducted on zebrafish or other animals.

Which Zebrafish Strains Are More Suitable to Perform Behavioral Studies? A Comprehensive Comparison by Phenomic Approach.

Wild-type (WT) zebrafish are commonly used in behavioral tests, however, the term WT corresponds to many different strains, such as AB, Tübingen long fin (TL), and Wild Indian Karyotype (WIK). Since these strains are widely used, there has to be at least one study to demonstrate the behavioral differences between them. In our study, six zebrafish strains were used, which are AB, absolute, TL, golden, pet store-purchased (PET), and WIK zebrafishes. The behavior of these fishes was tested in a set of behavioral tests, including novel tank, mirror-biting, predator avoidance, social interaction, and shoaling tests. From the results, the differences were observed for all behavioral tests, and each strain displayed particular behavior depending on the tests. In addition, from the heatmap and PCA (principal component analysis) results, two major clusters were displayed, separating the AB and TL zebrafishes with other strains in another cluster. Furthermore, after the coefficient of variation of each strain in every behavioral test was calculated, the AB and TL zebrafishes were found to possess a low percentage of the coefficient of variation, highlighting the strong reproducibility and the robustness of the behaviors tested in both fishes. Each zebrafish strain tested in this experiment showed specifically different behaviors from each other, thus, strain-specific zebrafish behavior should be considered when designing experiments using zebrafish behavior.

Method Standardization for Conducting Innate Color Preference Studies in Different Zebrafish Strains.

The zebrafish has a tetrachromatic vision that is able to distinguish ultraviolet (UV) and visible wavelengths. Recently, zebrafish color preferences have gained much attention because of the easy setup of the instrument and its usefulness to screen behavior-linked stimuli. However, several published papers dealing with zebrafish color preferences have contradicting results that underscore the importance of method standardization in this field. Different laboratories may report different results because of variations in light source, color intensity, and other parameters such as age, gender, container size, and strain of fish. In this study, we aim to standardize the color preference test in zebrafish by measuring light source position, light intensity, gender, age, animal size to space ratio, and animal strain. Our results showed that color preferences for zebrafish are affected by light position, age, strain, and social interaction of the fish, but not affected by fish gender. We validated that ethanol can significantly induce color preference alteration in zebrafish which may be related to anxiety and depression. We also explored the potential use of the optimized method to examine color preference ranking and index differences in various zebrafish strains and species, such as the tiger barb and glass catfish. In conclusion, zebrafish color preference screening is a powerful tool for high-throughput neuropharmacological applications and the standardized protocol established in this study provides a useful reference for the zebrafish research community.

Toxicity Studies on Graphene-Based Nanomaterials in Aquatic Organisms: Current Understanding.

Graphene and its oxide are nanomaterials considered currently to be very promising because of their great potential applications in various industries. The exceptional physiochemical properties of graphene, particularly thermal conductivity, electron mobility, high surface area, and mechanical strength, promise development of novel or enhanced technologies in industries. The diverse applications of graphene and graphene oxide (GO) include energy storage, sensors, generators, light processing, electronics, and targeted drug delivery. However, the extensive use and exposure to graphene and GO might pose a great threat to living organisms and ultimately to human health. The toxicity data of graphene and GO is still insufficient to point out its side effects to different living organisms. Their accumulation in the aquatic environment might create complex problems in aquatic food chains and aquatic habitats leading to debilitating health effects in humans. The potential toxic effects of graphene and GO are not fully understood. However, they have been reported to cause agglomeration, long-term persistence, and toxic effects penetrating cell membrane and interacting with cellular components. In this review paper, we have primarily focused on the toxic effects of graphene and GO caused on aquatic invertebrates and fish (cell line and organisms). Here, we aim to point out the current understanding and knowledge gaps of graphene and GO toxicity.

Systematical exploration of the common solvent toxicity at whole organism level by behavioral phenomics in adult zebrafish.

Common solvents are frequently used as carriers to dissolve chemicals with a hydrophobic property that is extensively applied in the industrial and biomedical fields. In this study, we aimed to systematically study the sub-chronic effect of ten common solvents at low concentration exposure in adult zebrafish and perform neurobehavioral assessments for mechanistic exploration. After exposed to ten common solvents, including methanol, ethanol (EtOH), dimethyl sulfoxide (DMSO), isopropanol, acetone, polyethylene glycol-400 (PEG-400), glycerol, butanol, pentane, and tetrahydrofuran for continuous 10 day at 0.1% concentration level, adult zebrafish were subjected to perform a serial of behavioral tests, such as novel tank, mirror biting, predator avoidance, social interaction and shoaling. Later, 20 behavioral endpoints obtained from these five tests were transformed into a scoring matrix. Principal component analysis (PCA) and hierarchy clustering were performed to evaluate and compare the zebrafish behavior profiling. By using this phenomic approach, we were able to systematically evaluate the toxicity of the common solvents in zebrafish at a neurobehavioral level for the first time and found each common solvent-induced unique behavioral alteration to produce fingerprint-like patterns in hierarchy clustering and heatmap analysis. Among all tested common solvents, acetone and PEG-400 displayed better biocompatibility and less toxicity since they triggered less behavioral and biochemical alterations while methanol and DMSO caused severe behavior alterations in zebrafish after chronic exposure of these solvents. We conclude the behavioral phenomic approach conducted in this study providing a powerful tool to a systematical exploration of the common solvent toxicity at the whole organism level.

Waterborne Exposure of Paclobutrazol at Environmental Relevant Concentration Induce Locomotion Hyperactivity in Larvae and Anxiolytic Exploratory Behavior in Adult Zebrafish.

The available arable land is unable to fulfill the food production need of rapidly the exponentially growing human population in the world. Pesticides are one of those different measures taken to meet this demand. As a plant growth regulator to block gibberellin, paclobutrazol (PBZ) is used excessively throughout the world to promote early fruit setting, and to increase seed setting which might be harmful because PBZ is a very stable compound; therefore, it can bioaccumulate into the food chain of an ecosystem. In the present study, we discovered unexpected effects of PBZ on zebrafish larvae and adult behaviors by challenging them with low dose exposure. Zebrafish larvae aged 4 days post-fertilization (dpf) were exposed for 24 h at 10 µg/L (0.01 ppm) and 100 µg/L (0.1 ppm) of PBZ, respectively, and adults were incubated at 100 µg/L (0.1 ppm) and 1000 µg/L (1 ppm) concentrations of PBZ, respectively, for fourteen days. After incubation, the locomotor activity, burst, and rotation movement for the larvae; and multiple behavioral tests such as novel tank exploration, mirror biting, shoaling, predator avoidance, and social interaction for adult zebrafish were evaluated. Brain tissues of the adult fish were dissected and subjected to biochemical analyses of the antioxidant response, oxidative stress, superoxide dismutase (SOD), and neurotransmitter levels. Zebrafish larvae exposed to PBZ exhibited locomotion hyperactivity with a high burst movement and swimming pattern. In adult zebrafish, PBZ resulted in anxiolytic exploratory behavior, while no significant results were found in social interaction, shoal making, and predator avoidance behaviors. Interestingly, high dose PBZ exposure significantly compromised the innate aggressive behavior of the adult fish. Biochemical assays for oxidative stress, antioxidant response, and superoxide dismutase (SOD) showed significant reductions in their relative contents. In conclusion, for the first time, our behavior assays revealed that chronic PBZ exposure induced behavioral alterations in both larvae and the adult zebrafish. Because PBZ is a widely-used plant growth regulator, we suggest that it is necessary to conduct more thorough tests for its biosafety and bioaccumulation.

Surface Modification of Magnetic Nanoparticles by Carbon-Coating Can Increase Its Biosafety: Evidences from Biochemical and Neurobehavioral Tests in Zebrafish.

Recently, magnetic nanoparticles (MNPs) have gained much attention in the field of biomedical engineering for therapeutic as well as diagnostic purposes. Carbon magnetic nanoparticles (C-MNPs) are a class of MNPs categorized as organic nanoparticles. C-MNPs have been under considerable interest in studying in various applications such as magnetic resonance imaging, photothermal therapy, and intracellular transportof drugs. Research work is still largely in progress for testing the efficacy of C-MNPs on the theranostics platform in cellular studies and animal models. In this study, we evaluated the neurobehavioral toxicity parameters on the adult zebrafish (Danio rerio) at either low (1 ppm) or high (10 ppm) concentration level of C-MNPs over a period of two weeks by waterborne exposure. The physical properties of the synthesized C-MNPs were characterized by transmission electron microscopy, Raman, and XRD spectrum characterization. Multiple behavior tests for the novel tank, mirror biting, predator avoidance, conspecific social interaction, shoaling, and analysis of biochemical markers were also conducted to elucidate the corresponding mechanism. Our data demonstrate the waterborne exposure of C-MNPs is less toxic than the uncoated MNPs since neither low nor high concentration C-MNPs elicit toxicity response in behavioral and biochemical tests in adult zebrafish. The approach combining biochemical and neurobehavioral approaches would be helpful for understanding C-MNPs association affecting the bioavailability, biosafety, interaction, and uptake of these C-MNPs in the living organism.

A Novel Function of the Lysophosphatidic Acid Receptor 3 (LPAR3) Gene in Zebrafish on Modulating Anxiety, Circadian Rhythm Locomotor Activity, and Short-Term Memory.

Lysophosphatidic acid (LPA) is a small lysophospholipid molecule that activates multiple cellular functions through pathways with G-protein-coupled receptors. So far, six LPA receptors (LPAR1 to LPAR6) have been discovered and each one of them can connect to the downstream cell message-transmitting network. A previous study demonstrated that LPA receptors found in blood-producing stem cells can enhance erythropoietic processes through the activation of LPAR3. In the current study, newly discovered functions of LPAR3 were identified through extensive behavioral tests in lpar3 knockout (KO) zebrafish. It was found that the adult lpar3 KO zebrafish display an abnormal movement orientation and altered exploratory behavior compared to that of the control group in the three-dimensional locomotor and novel tank tests, respectively. Furthermore, consistent with those results, in the circadian rhythm locomotor activity test, the lpar3 KO zebrafish showed a lower level of angular velocity and average speed during the light cycles, indicating an hyperactivity-like behavior. In addition, the mutant fish also exhibited considerably higher locomotor activity during the dark cycle. Supporting those findings, this phenomenon was also displayed in the lpar3 KO zebrafish larvae. Furthermore, several important behavior alterations were also observed in the adult lpar3 KO fish, including a lower degree of aggression, less interest in conspecific social interaction, and looser shoal formation. However, there was no significant difference regarding the predator avoidance behavior between the mutant and the control fish. In addition, lpar3 KO zebrafish displayed memory deficiency in the passive avoidance test. These in vivo results support for the first time that the lpar3 gene plays a novel role in modulating behaviors of anxiety, aggression, social interaction, circadian rhythm locomotor activity, and memory retention in zebrafish.

Chronic Exposure to Low Concentration Lead Chloride-Induced Anxiety and Loss of Aggression and Memory in Zebrafish.

Lead and lead-derived compounds have been extensively utilized in industry, and their chronic toxicity towards aquatic animals has not been thoroughly addressed at a behavioral level. In this study, we assessed the risk of exposure to lead at a waterborne environmental concentration in adult zebrafish by behavioral and biochemical analyses. Nine tests, including three-dimension (3D) locomotion, novel tank exploration, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm locomotor activity, color preference, and a short-term memory test, were performed to assess the behavior of adult zebrafish after the exposure to 50 ppb PbCl2 for one month. The brain tissues were dissected and subjected to biochemical assays to measure the relative expression of stress biomarkers and neurotransmitters to elucidate the underlying mechanisms for behavioral alterations. The results of the behavioral tests showed that chronic exposure to lead could elevate the stress and anxiety levels characterized by elevated freezing and reduced exploratory behaviors. The chronic exposure to PbCl2 at a low concentration also induced a sharp reduction of aggressiveness and short-term memory. However, no significant change was found in predator avoidance, social interaction, shoaling, or color preference. The biochemical assays showed elevated cortisol and reduced serotonin and melatonin levels in the brain, thus, altering the behavior of the PbCl2-exposed zebrafish. In general, this study determined the potential ecotoxicity of long-term lead exposure in adult zebrafish through multiple behavioral assessments. The significant findings were that even at a low concentration, long-term exposure to lead could impair the memory and cause a decrease in the aggressiveness and exploratory activities of zebrafish, which may reduce their survival fitness.

Nanoplastics Cause Neurobehavioral Impairments, Reproductive and Oxidative Damages, and Biomarker Responses in Zebrafish: Throwing up Alarms of Wide Spread Health Risk of Exposure.

Plastic pollution is a growing global emergency and it could serve as a geological indicator of the Anthropocene era. Microplastics are potentially more hazardous than macroplastics, as the former can permeate biological membranes. The toxicity of microplastic exposure on humans and aquatic organisms has been documented, but the toxicity and behavioral changes of nanoplastics (NPs) in mammals are scarce. In spite of their small size, nanoplastics have an enormous surface area, which bears the potential to bind even bigger amounts of toxic compounds in comparison to microplastics. Here, we used polystyrene nanoplastics (PS-NPs) (diameter size at ~70 nm) to investigate the neurobehavioral alterations, tissue distribution, accumulation, and specific health risk of nanoplastics in adult zebrafish. The results demonstrated that PS-NPs accumulated in gonads, intestine, liver, and brain with a tissue distribution pattern that was greatly dependent on the size and shape of the NPs particle. Importantly, an analysis of multiple behavior endpoints and different biochemical biomarkers evidenced that PS-NPs exposure induced disturbance of lipid and energy metabolism as well as oxidative stress and tissue accumulation. Pronounced behavior alterations in their locomotion activity, aggressiveness, shoal formation, and predator avoidance behavior were exhibited by the high concentration of the PS-NPs group, along with the dysregulated circadian rhythm locomotion activity after its chronic exposure. Moreover, several important neurotransmitter biomarkers for neurotoxicity investigation were significantly altered after one week of PS-NPs exposure and these significant changes may indicate the potential toxicity from PS-NPs exposure. In addition, after ~1-month incubation, the fluorescence spectroscopy results revealed the accumulation and distribution of PS-NPs across zebrafish tissues, especially in gonads, which would possibly further affect fish reproductive function. Overall, our results provided new evidence for the adverse consequences of PS-NPs-induced behavioral dysregulation and changes at the molecular level that eventually reduce the survival fitness of zebrafish in the ecosystem.

Behavioral Impairments and Oxidative Stress in the Brain, Muscle, and Gill Caused by Chronic Exposure of C70 Nanoparticles on Adult Zebrafish.

There is an imperative need to develop efficient whole-animal-based testing assays to determine the potential toxicity of engineered nanomaterials. While previous studies have demonstrated toxicity in lung and skin cells after C70 nanoparticles (NPs) exposure, the potential detrimental role of C70 NPs in neurobehavior is largely unaddressed. Here, we evaluated the chronic effects of C70 NPs exposure on behavior and alterations in biochemical responses in adult zebrafish. Two different exposure doses were used for this experiment: low dose (0.5 ppm) and high dose (1.5 ppm). Behavioral tests were performed after two weeks of exposure of C70 NPs. We found decreased locomotion, exploration, mirror biting, social interaction, and shoaling activities, as well as anxiety elevation and circadian rhythm locomotor activity impairment after ~2 weeks in the C70 NP-exposed fish. The results of biochemical assays reveal that following exposure of zebrafish to 1.5 ppm of C70 NPs, the activity of superoxide dismutase (SOD) in the brain and muscle tissues increased significantly. In addition, the concentration of reactive oxygen species (ROS) also increased from 2.95 ± 0.12 U/ug to 8.46 ± 0.25 U/ug and from 0.90 ± 0.03 U/ug to 3.53 ± 0.64 U/ug in the muscle and brain tissues, respectively. Furthermore, an increased level of cortisol was also observed in muscle and brain tissues, ranging from 17.95 ± 0.90 pg/ug to 23.95 ± 0.66 pg/ug and from 3.47 ± 0.13 pg/ug to 4.91 ± 0.51 pg/ug, respectively. Increment of Hif1-α level was also observed in both tissues. The elevation was ranging from 11.65 ± 0.54 pg/ug to 18.45 ± 1.00 pg/ug in the muscle tissue and from 4.26 ± 0.11 pg/ug to 6.86 ± 0.37 pg/ug in the brain tissue. Moreover, the content of DNA damage and inflammatory markers such as ssDNA, TNF-α, and IL-1ß were also increased substantially in the brain tissues. Significant changes in several biomarker levels, including catalase and malondialdehyde (MDA), were also observed in the gill tissues. Finally, we used a neurophenomic approach with a particular focus on environmental influences, which can also be easily adapted for other aquatic fish species, to assess the toxicity of metal and carbon-based nanoparticles. In summary, this is the first study to illustrate the adult zebrafish toxicity and the alterations in several neurobehavior parameters after zebrafish exposure to environmentally relevant amounts of C70 NPs.