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.

Evaluation of sub-chronic toxicity of melamine via systematic or oral delivery in adult zebrafish based on behavioral endpoints.

Melamine-tainted products have been found in the market and raised issues about food safety. Recent studies done in rodents and humans demonstrated the toxicities of melamine, especially in causing kidney damage and bladder stone formation. However, very few studies assessed its behavior toxicity in organisms, including fish. Therefore, in this study, the researchers aim to determine whether sub-chronic exposure to melamine via oral and systematic administration could induce behavioral abnormality in zebrafish. After 14 days of systematic exposure to melamine at doses of 0.1 and 10 ppm levels, zebrafish were subjected to multiple behavioral assays. Results from both exposure routes showed that melamine indeed slightly increased fish locomotion and altered their exploratory behaviors in the novel tank assay. Furthermore, tightened shoaling formation was also displayed by the treated fish in the waterborne exposure group. However, melamine exposure did not cause any obvious alterations in fish behaviors during other behavioral tests. In addition, in comparison with previously published data on the behavior toxicities of several solvents in zebrafish, our phenomic analysis suggests the relatively low behavior toxicities of melamine via either systematic exposure or oral administration to zebrafish compared to those solvents. Nevertheless, our data indicate that the potential neurotoxicity of chronic low-dose melamine should not be ignored.

Assessments of carbon nanotubes toxicities in zebrafish larvae using multiple physiological and molecular endpoints.

In recent years, carbon nanotubes (CNTs) have become one of the most promising materials for the technology industry. However, due to the extensive usage of these materials, they may be released into the environment, and cause toxicities to the organism. Here, their acute toxicities in zebrafish embryos and larvae were evaluated by using various assessments that may provide us with a novel perspective on their effects on aquatic animals. Before conducting the toxicity assessments, the CNTs were characterized as multiwall carbon nanotubes (MWCNTs) functionalized with hydroxyl and carboxyl groups, which improved their solubility and dispersibility. Based on the results, abnormalities in zebrafish behaviors were observed in the exposed groups, indicated by a reduction in tail coiling frequency and alterations in the locomotion as the response toward photo and vibration stimuli that might be due to the disruption in the neuromodulatory system and the formation of reactive oxygen species (ROS) by MWCNTs. Next, based on the respiratory rate assay, exposed larvae consumed more oxygen, which may be due to the injuries in the larval gill by the MWCNTs. Finally, even though no irregularity was observed in the exposed larval cardiac rhythm, abnormalities were shown in their cardiac physiology and blood flow with significant downregulation in several cardiac development-related gene expressions. To sum up, although the following studies are necessary to understand the exact mechanism of their toxicity, the current study demonstrated the environmental implications of MWCNTs in particularly low concentrations and short-term exposure, especially to aquatic organisms.

Evaluation of Tacrolimus' Adverse Effects on Zebrafish in Larval and Adult Stages by Using Multiple Physiological and Behavioral Endpoints.

Tacrolimus (FK506) is a common immunosuppressant that is used in organ transplantation. However, despite its importance in medical applications, it is prone to adverse side effects. While some studies have demonstrated its toxicities to humans and various animal models, very few studies have addressed this issue in aquatic organisms, especially zebrafish. Here, we assessed the adverse effects of acute and chronic exposure to tacrolimus in relatively low doses in zebrafish in both larval and adult stages, respectively. Based on the results, although tacrolimus did not cause any cardiotoxicity and respiratory toxicity toward zebrafish larvae, it affected their locomotor activity performance in light-dark locomotion tests. Meanwhile, tacrolimus was also found to slightly affect the behavior performance, shoaling formation, circadian rhythm locomotor activity, and color preference of adult zebrafish in a dose-dependent manner. In addition, alterations in the cognitive performance of the fish were also displayed by the treated fish, indicated by a loss of short-term memory. To help elucidate the toxicity mechanism of tacrolimus, molecular docking was conducted to calculate the strength of the binding interaction between tacrolimus to human FKBP12. The results showed a relatively normal binding affinity, indicating that this interaction might only partly contribute to the observed alterations. Nevertheless, the current research could help clinicians and researchers to further understand the toxicology of tacrolimus, especially to zebrafish, thus highlighting the importance of considering the toxicity of tacrolimus prior to its usage.

Color Perspectives in Aquatic Explorations: Unveiling Innate Color Preferences and Psychoactive Responses in Freshwater Crayfish.

Color preference assay is a test for an animal's innate and adaptive response to differentiate colors and can be used as an endpoint for psychoactive activity evaluation. Several color preference test methods in aquatic animals that can be used to perform behavioral screening have been established. However, the color preference test conditions have yet to be extensively studied and standardized in aquatic invertebrates. This study aimed to replicate and optimize the previously published method to evaluate the potential color preference in freshwater crayfish based on four different approaches: species, life stages, sex, and pharmaceutical exposure. Using the optimized setup, two crayfish species display color preferences to some specific colors. P. clarkii displays more dominant color preference behavior than C. quadricarinatus in terms of color preference ranking and index. P. clarkii prefers the red color compared to other colors (red > green > blue > yellow), while C. quadricarinatus dislikes yellow compared to other colors (blue = green = red > yellow). Since P. clarkii has a more obvious color index ranking and several advantages compared to C. quadricarinatus, we conducted further tests using P. clarkii as an animal model. In the juvenile and adult stages of P. clarkii, they prefer red and avoid yellow. However, the juvenile one did not display a strong color preference like the adult one. Different sex of crayfish displayed no significant differences in their color preference responses. In addition, we also evaluated the potential effect of the antidepressant sertraline on color preference in P. clarkii and found that waterborne antidepressant exposure can significantly alter their color preference. This fundamental information collected from this study supports the crayfish color preference test as a good behavioral test to address environmental pollution.

The anti-platelet drug cilostazol enhances heart rate and interrenal steroidogenesis and exerts a scant effect on innate immune responses in zebrafish.

RATIONALE: Cilostazol, an anti-platelet phosphodiesterase-3 inhibitor used for the treatment of intermittent claudication, is known for its pleiotropic effects on platelets, endothelial cells and smooth muscle cells. However, how cilostazol impacts the endocrine system and the injury-induced inflammatory processes remains unclear. METHODS: We used the zebrafish, a simple transparent model that demonstrates rapid development and a strong regenerative ability, to test whether cilostazol influences heart rate, steroidogenesis, and the temporal and dosage effects of cilostazol on innate immune cells during tissue damage and repair. RESULTS: While dosages of cilostazol from 10 to 100 µM did not induce any noticeable morphological abnormality in the embryonic and larval zebrafish, the heart rate was increased as measured by ImageJ TSA method. Moreover, adrenal/interrenal steroidogenesis in larval zebrafish, analyzed by whole-mount 3ß-Hsd enzymatic activity and cortisol ELISA assays, was significantly enhanced. During embryonic fin amputation and regeneration, cilostazol treatments led to a subtle yet significant effect on reducing the aggregation of Mpx-expressing neutrophil at the lesion site, but did not affect the immediate injury-induced recruitment and retention of Mpeg1-expressing macrophages. CONCLUSIONS: Our results indicate that cilostazol has a significant effect on the heart rate and the growth as well as endocrine function of steroidogenic tissue; with a limited effect on the migration of innate immune cells during tissue damage and repair.

A comprehensive painkillers screening by assessing zebrafish behaviors after caudal fin amputation.

Recently, the usage of zebrafish for pain studies has increased in the past years, especially due to its robust pain-stimulated behaviors. Fin amputation has been demonstrated to induce a noxious response in zebrafish. However, based on the prior study, although lidocaine, the most used painkiller in zebrafish, has been shown to ameliorate amputated zebrafish behaviors, it still causes some prolonged effects. Therefore, alternative painkillers are always needed to improve the treatment quality of fin-amputated zebrafish. Here, the effects of several analgesics in recovering zebrafish behaviors post-fin amputation were evaluated. From the results, five painkillers were found to have potentially beneficial effects on amputated fish behaviors. Overall, these results aligned with their binding energy level to target proteins of COX-1 and COX-2. Later, based on their sub-chronic effects on zebrafish survivability, indomethacin, and diclofenac were further studied. This combination showed a prominent effect in recovering zebrafish behaviors when administered orally or through waterborne exposure, even with lower concentrations. Next, based on the ELISA in zebrafish brain tissue, although some changes were found in the treated group, no statistical differences were observed in most of the tested biomarkers. However, since heatmap clustering showed a similar pattern between biochemical and behavior endpoints, the minor changes in each biomarker may be sufficient in changing the fish behaviors.

Using DeepLabCut for markerless cardiac physiology and toxicity estimation in water fleas (Daphnia magna).

Daphnia magna is one species of water flea that has been used for a long time for ecotoxicity studies. In addition, Daphnia has a myogenic heart that is very useful for cardiotoxicity studies. Previous attempts to calculate the cardiac parameter endpoints in Daphnia suffer from the drawback of tedious operation and high variation due to manual counting errors. Even the previous method that utilized deep learning to help the process suffer from either overestimation of parameters or the need for specialized equipment to perform the analysis. In this study, we utilized DeepLabCut software previously used for animal pose tracking and demonstrated that ResNet_152 was the best fit for training the network. The trained network also showed comparable results with ImageJ and Kymograph, which was mostly done manually. In addition to that, several macro scripts in either Excel or Python format were developed to help summarize the data for faster analysis. The trained network was then challenged to analyze the potential cardiotoxicity of imidacloprid and pendimethalin in D. magna, and it showed that both pesticides cause alteration in their cardiac performance. Overall, this method provides a simple and automatic method to analyze the cardiac performance of Daphnia by utilizing DeepLabCut. The method proposed in this paper can contribute greatly to scientists conducting fast and accurate cardiotoxicity measurements when using Daphnia as a model.

Using crayfish behavior assay as a simple and sensitive model to evaluate potential adverse effects of water pollution: Emphasis on antidepressants.

The freshwater crayfish, Procambarus clarkii is an excellent aquatic animal model that is highly adaptable and tolerant. P. clarkii is widely used as a toxicity model to study various pharmaceutical exposure. This animal model has complex behavioral traits and is considered sensitive to environmental changes, making it an excellent candidate to study psychoactive drugs based on a behavioral approach. However, up to now, most behavioral studies on crayfish use manual observation and scoring that require panelists. In this study, we aim to develop an automation pipeline to analyze crayfish behavior automatically. We use a deep-learning approach to label body parts in multiple crayfish, and based on the trajectory results, the intra- or inter-individual crayfish were calculated. Reliable and fast results of several behavior endpoints in multiple crayfish were retrieved. We then validated the detection performance of numerous crayfish in specific gender groups (male-male and female-female). Based on the result, the male crayfish displayed significantly higher aggression than females. We also tested the antidepressant exposure on this animal model to evaluate the psychoactive effects of this drug. As male crayfish display more distinct agonistic behavior than females, we exposed them to sertraline (SRT) 1 ppb for 7 and 14 days. It was revealed that sertraline was able to alter several behavioral endpoints in crayfish. Significant increases in extend claw ratio, total distance moved, average speed, and rapid movement were displayed in sertraline-exposed crayfish but decreased interaction time and longest interaction time. In addition, SRT 14 days exposure could atler the aggressiveness and bold behavior In the present method, DeepLabCut (DLC) has been utilized to analyze the locomotion behavior of multiple crayfish. This established method provides rapid and accurate ecotoxicity measurements using freshwater crayfish, which beneficient and applicable for environmental research.

The Effect of the Pyrethroid Pesticide Fenpropathrin on the Cardiac Performance of Zebrafish and the Potential Mechanism of Toxicity.

Fenpropathrin, a pyrethroid insecticide, has been widely used for many years in agricultural fields. It works by disturbing the voltage-gated sodium channel, leading to paralysis and the death of the target animal. While past studies have focused on neurodegeneration following fenpropathrin poisoning in humans, relatively few pieces of research have examined its effect on other peripheral organs. This study successfully investigated the potential toxicity of fenpropathrin on the cardiovascular system using zebrafish as an animal model. Zebrafish larvae exposed to varying doses of fenpropathrin underwent an evaluation of cardiac physiology by measuring the heart rate, stroke volume, cardiac output, and shortening fraction. The blood flow velocity and the dorsal aorta diameter were also measured to assess the impact of fenpropathrin exposure on the vascular system. Furthermore, molecular docking was performed to evaluate the pesticide binding affinity to various proteins associated with the cardiovascular system, revealing the potential mechanism of the fenpropathrin cardiotoxic effect. The findings demonstrated a significant dose-dependent increase in the heart rate stroke volume, cardiac output, shortening fraction, and ejection fraction of zebrafish larvae after 24 h of acute treatment with fenpropathrin. Additionally, zebrafish treated at a concentration of 1 ppm exhibited significantly larger blood vessels in diameter and an increased blood flow velocity compared to the control group. According to molecular docking, fenpropathrin showed a high affinity for various voltage-gated sodium channels like scn1lab, cacna1sb, and clcn3. Finally, from the results, we found that fenpropathrin caused cardiomegaly, which may have been induced by the voltage-gated sodium channel disruption. This study highlights the significant disruption of fenpropathrin in the cardiovascular system and emphasizes the need for further research on the health implications of this pesticide.

Deep Learning-Based Automatic Duckweed Counting Using StarDist and Its Application on Measuring Growth Inhibition Potential of Rare Earth Elements as Contaminants of Emerging Concerns.

In recent years, there have been efforts to utilize surface water as a power source, material, and food. However, these efforts are impeded due to the vast amounts of contaminants and emerging contaminants introduced by anthropogenic activities. Herbicides such as Glyphosate and Glufosinate are commonly known to contaminate surface water through agricultural industries. In contrast, some emerging contaminants, such as rare earth elements, have started to enter the surface water from the production and waste of electronic products. Duckweeds are angiosperms from the Lemnaceae family and have been used for toxicity tests in aquatic environments, mainly those from the genus Lemna, and have been approved by OECD. In this study, we used duckweed from the genus Wolffia, which is smaller and considered a good indicator of metal pollutants in the aquatic environment. The growth rate of duckweed is the most common endpoint in observing pollutant toxicity. In order to observe and mark the fronds automatically, we used StarDist, a machine learning-based tool. StarDist is available as a plugin in ImageJ, simplifying and assisting the counting process. Python also helps arrange, manage, and calculate the inhibition percentage after duckweeds are exposed to contaminants. The toxicity test results showed Dysprosium to be the most toxic, with an IC50 value of 14.6 ppm, and Samarium as the least toxic, with an IC50 value of 279.4 ppm. In summary, we can provide a workflow for automatic frond counting using StarDist integrated with ImageJ and Python to simplify the detection, counting, data management, and calculation process.

Healing Wounds Efficiently with Biomimetic Soft Matter: Injectable Self-Healing Neutral Glycol Chitosan/Dibenzaldehyde-Terminated Poly(ethylene glycol) Hydrogel with Inherent Antibacterial Properties.

The high prevalence of acquiring skin wounds, along with the emergence of antibiotic-resistant strains that lead to infections, impose a threat to the physical, mental, and socioeconomic health of society. Among the wide array of wound dressings developed, hydrogels are regarded as a biomimetic soft matter of choice owing to their ability to provide a moist environment ideal for healing. Herein, neutral glycol chitosan (GC) was cross-linked via imine bonds with varying concentrations of dibenzaldehyde-terminated polyethylene glycol (DP) to give glycol chitosan/dibenzaldehyde-terminated polyethylene glycol hydrogels (GC/DP). These dynamic Schiff base linkages (absorption peak at 1638 cm-1) within the hydrogel structure endowed their ability to recover from damage as characterized by high-low strain exposure in continuous step strain rheology. Along with their good injectability and biodegradability, the hydrogels exhibited remarkable inhibition against E. coli, P. aeruginosa, and S. aureus. GC/DP hydrogels demonstrated high LC50 values in vivo using zebrafish embryos as a model system due to their relative biocompatibility and a remarkable 93.4 ± 0.88% wound contraction at 30-dpw against 49.1 ± 3.40% of the control. To the best of our knowledge, this is the first study that developed injectable glycol chitosan/dibenzaldehyde-terminated polyethylene glycol self-healing hydrogels for application in wound healing with intrinsic bacteriostatic properties against the three bacteria.

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.

Investigating Potential Cardiovascular Toxicity of Two Anti-Leukemia Drugs of Asciminib and Ponatinib in Zebrafish Embryos.

BCR-ABL, a fusion protein kinase, is a druggable target exclusively expressed in patients with chronic myeloid leukemia (CML). Several anti-leukemia medicines targeting this protein have been developed in recent years. However, therapeutic options are limited for CML patients bearing multiple BCR-ABL1 mutations. Ponatinib (PON), a potent tyrosinase inhibitor, was one of the approved drugs for managing BCR-ABL1 T315I mutant disease. However, treatment of patients with PON reported severe side effects related to cardiovascular events. Asciminib (ASC) was the first allosteric inhibitor approved to target the myristoyl pocket of BCR-ABL protein to inhibit protein activity. The different mechanism of inhibition opens the possibility of co-exposure with both medicines. Reports on cardiovascular side effects due to the combination use of PON + ASC in pre-clinical and clinical studies are minimal. Thus, this study aimed to observe the potential cardiovascular-related side effect after co-exposure to ASC and PON using zebrafish as an animal model. In this study, zebrafish were acutely exposed to both compounds. The cardiovascular physiology parameters and gene expression related to cardiovascular development were evaluated. We demonstrate that combining ASC with PON at no observed effect concentration (NOEC) did not cause any significant change in the cardiac performance parameter in zebrafish. However, a significant increase in nkx2.5 expression level and a substantial decrease in blood flow velocity were recorded, suggesting that combining these compounds at NOEC can cause mild cardiovascular-related side effects.

OpenBloodFlow: A User-Friendly OpenCV-Based Software Package for Blood Flow Velocity and Blood Cell Count Measurement for Fish Embryos.

The transparent appearance of fish embryos provides an excellent assessment feature for observing cardiovascular function in vivo. Previously, methods to conduct vascular function assessment were based on measuring blood-flow velocity using third-party software. In this study, we reported a simple software, free of costs and skills, called OpenBloodFlow, which can measure blood flow velocity and count blood cells in fish embryos for the first time. First, videos captured by high-speed CCD were processed for better image stabilization and contrast. Next, the optical flow of moving objects was extracted from the non-moving background in a frame-by-frame manner. Finally, blood flow velocity was calculated by the Gunner Farneback algorithm in Python. Data validation with zebrafish and medaka embryos in OpenBloodFlow was consistent with our previously published ImageJ-based method. We demonstrated consistent blood flow alterations by either OpenBloodFlow or ImageJ in the dorsal aorta of zebrafish embryos when exposed to either phenylhydrazine or ractopamine. In addition, we validated that OpenBloodFlow was able to conduct precise blood cell counting. In this study, we provide an easy and fully automatic programming for blood flow velocity calculation and blood cell counting that is useful for toxicology and pharmacology studies in fish.

Ractopamine at the Center of Decades-Long Scientific and Legal Disputes: A Lesson on Benefits, Safety Issues, and Conflicts.

Ractopamine (RAC) is a synthetic phenethanolamine, ß-adrenergic agonist used as a feed additive to develop leanness and increase feed conversion efficiency in different farm animals. While RAC has been authorized as a feed additive for pigs and cattle in a limited number of countries, a great majority of jurisdictions, including the European Union (EU), China, Russia, and Taiwan, have banned its use on safety grounds. RAC has been under long scientific and political discussion as a controversial antibiotic as a feed additive. Here, we will present significant information on RAC regarding its application, detection methods, conflicts, and legal divisions that play a major role in controversial deadlock and why this issue warrants the attention of scientists, agriculturists, environmentalists, and health advocates. In this review, we highlight the potential toxicities of RAC on aquatic animals to emphasize scientific evidence and reports on the potentially harmful effects of RAC on the aquatic environment and human health.

Acute and Chronic Effects of Fin Amputation on Behavior Performance of Adult Zebrafish in 3D Locomotion Test Assessed with Fractal Dimension and Entropy Analyses and Their Relationship to Fin Regeneration.

The fin is known to play an important role in swimming for many adult fish, including zebrafish. Zebrafish fins consist of paired pectoral and pelvic with unpaired dorsal, anal, and caudal tail fins with specific functions in fish locomotion. However, there was no study comparing the behavior effects caused by the absence of each fin. We amputated each fin of zebrafish and evaluated their behavior performance in the 3D locomotion test using fractal dimension and entropy analyses. Afterward, the behavior recovery after the tail fin amputation was also evaluated, together with the fin regeneration process to study their relationship. Finally, we conducted a further study to confirm whether the observed behavior alterations were from pain elicited by fin amputation procedure or not by using lidocaine, a pain-relieving drug. Amputation in the caudal fin resulted in the most pronounced behavior alterations, especially in their movement complexity. Furthermore, we also found that their behavior was fully recovered before the caudal fin was fully regenerated, indicating that these behavioral changes were not majorly due to a mechanical change in tail length; instead, they may come from pain elicited from the fin amputation, since treatment with lidocaine could ameliorate the behavioral effects after the amputation procedure. However, lidocaine did not accelerate the behavior recovery process; instead, it caused the fishes to display some slight side effects. This study highlights the potential moderate severity of fin amputation in zebrafish and the importance of analgesia usage. However, side effects may occur and need to be considered since fin amputation is routinely conducted for various research, especially genomic screening.

Toxicity Assessment of an Anti-Cancer Drug of p-Toluene Sulfonamide in Zebrafish Larvae Based on Cardiovascular and Locomotion Activities.

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC50 was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca2+-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.

Using DeepLabCut as a Real-Time and Markerless Tool for Cardiac Physiology Assessment in Zebrafish.

DeepLabCut (DLC) is a deep learning-based tool initially invented for markerless pose estimation in mammals. In this study, we explored the possibility of adopting this tool for conducting markerless cardiac physiology assessment in an important aquatic toxicology model of zebrafish (Danio rerio). Initially, high-definition videography was applied to capture heartbeat information at a frame rate of 30 frames per second (fps). Next, 20 videos from different individuals were used to perform convolutional neural network training by labeling the heart chamber (ventricle) with eight landmarks. Using Residual Network (ResNet) 152, a neural network with 152 convolutional neural network layers with 500,000 iterations, we successfully obtained a trained model that can track the heart chamber in a real-time manner. Later, we validated DLC performance with the previously published ImageJ Time Series Analysis (TSA) and Kymograph (KYM) methods. We also evaluated DLC performance by challenging experimental animals with ethanol and ponatinib to induce cardiac abnormality and heartbeat irregularity. The results showed that DLC is more accurate than the TSA method in several parameters tested. The DLC-trained model also detected the ventricle of zebrafish embryos even in the occurrence of heart abnormalities, such as pericardial edema. We believe that this tool is beneficial for research studies, especially for cardiac physiology assessment in zebrafish embryos.

Evaluation of Locomotion Complexity in Zebrafish after Exposure to Twenty Antibiotics by Fractal Dimension and Entropy Analysis.

Antibiotics are extensively used in aquaculture to prevent bacterial infection and the spread of diseases. Some antibiotics have a relatively longer half-life in water and may induce some adverse effects on the targeted fish species. This study analyzed the potential adverse effects of antibiotics in zebrafish at the behavioral level by a phenomic approach. We conducted three-dimensional (3D) locomotion tracking for adult zebrafish after acute exposure to twenty different antibiotics at a concentration of 100 ppb for 10 days. Their locomotor complexity was analyzed and compared by fractal dimension and permutation entropy analysis. The dimensionality reduction method was performed by combining the data gathered from behavioral endpoints alteration. Principal component and hierarchical analysis conclude that three antibiotics: amoxicillin, trimethoprim, and tylosin, displayed unique characteristics. The effects of these three antibiotics at lower concentrations (1 and 10 ppb) were observed in a follow-up study. Based on the results, these antibiotics can trigger several behavioral alterations in adult zebrafish, even in low doses. Significant changes in locomotor behavioral activity, such as total distance activity, average speed, rapid movement time, angular velocity, time in top/bottom duration, and meandering movement are highly related to neurological motor impairments, anxiety levels, and stress responses were observed. This study provides evidence based on an in vivo experiment to support the idea that the usage of some antibiotics should be carefully addressed since they can induce a significant effect of behavioral alterations in fish.