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.

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.

Cardiovascular Performance Measurement in Water Fleas by Utilizing High-Speed Videography and ImageJ Software and Its Application for Pesticide Toxicity Assessment.

Water fleas are a good model for ecotoxicity studies, and were proposed for this purpose by the United States Environmental Protection Agency, due to their easy culture, body transparency, and high sensitivity to chemical pollution. Cardiovascular function parameters are usually used as an indicator of toxicity evaluation. However, due to the nature of the heart and blood flow, and the speed of the heartbeat, it is difficult to perform precise heartbeat and blood flow measurements with a low level of bias. In addition, the other cardiovascular parameters, including stroke volume, cardiac output, fractional shortening, and ejection fraction, have seldom been carefully addressed in previous studies. In this paper, high-speed videography and ImageJ-based methods were adopted to analyze cardiovascular function in water fleas. The heartbeat and blood flow for three water flea species, Daphnia magna, Daphnia silimis, and Moina sp., were captured by high-speed videography and analyzed using open-source ImageJ software. We found the heartbeat is species-dependent but not size-dependent in water fleas. Among the three water fleas tested, D. magna was identified as having the most robust heartbeat and blood flow rate, and is therefore suitable for the ecotoxicity test. Moreover, by calculating the diameter of the heart, we succeeded in measuring other cardiovascular parameters. D. magna were challenged with temperature changes and a pesticide (imidacloprid) to analyze variations in its cardiovascular function. We found that the heartbeat of D. magna was temperature-dependent, since the heartbeat was increasing with temperature. A similar result was shown in the cardiac output parameter. We also observed that the heartbeat, cardiac output, and heartbeat regularity are significantly reduced when exposed to imidacloprid at a low dose of 1 ppb (parts per billion). The blood flow rate, stroke volume, ejection fraction, and fractional shortening, on the contrary, did not display significant changes. In conclusion, in this study, we report a simple, highly accurate, and cost-effective method to perform physiological and toxicological assessments in water fleas.

Ecotoxicity Assessment of Fe3O4 Magnetic Nanoparticle Exposure in Adult Zebrafish at an Environmental Pertinent Concentration by Behavioral and Biochemical Testing.

Magnetic Nanoparticles (MNPs) are widely being investigated as novel promising multifunctional agents, specifically in the fields of development for theranostics, electronics, waste water treatment, cosmetics, and energy storage devices. Unique, superior, and indispensable properties of magnetization, heat transfer, and melting temperature make MNPs emerge in the field of therapeutics in future healthcare industries. However, MNPs ecotoxicity as well as behavioral toxicity is still unexplored. Ecotoxicity analysis may assist investigate MNPs uptake mechanism and its influence on bioavailability under a given set of environmental factors, which can be followed to investigate the biomagnification of MNPs in the environment and health risk possessed by them in an ecological food chain. In this study, we attempted to determine the behavioral changes in zebrafishes at low (1 ppm) or high (10 ppm) concentration levels of Fe3O4 MNPs. The synthesized Fe3O4 MNPs sized at 15 nm were characterized by the transmission electron microscope (TEM), the superconducting quantum interference device (SQUID) magnetometer, and the multiple behavior tests for novel tank, mirror biting, conspecific social interaction, shoaling, circadian rhythm, and short-term memory of zebrafish under MNPs chronic exposure were demonstrated. Low concentration MNP exposure did not trigger alteration for majority behavioral and biochemical tests in adult zebrafish. However, tight shoal groups were observed at a high concentration of MNPs exposure along with a modest reduction in fish exploratory behavior and a significant reduction in conspecific social interaction behavior. By using enzyme-linked immunosorbent assays (ELISA), we found a high dose of MNPs exposure significantly elevated cortisol, acetylcholine, and catalase levels while reducing serotonin, acetylcholine esterase, and dopamine levels in the brain. Our data demonstrates chronic MNPs exposure at an environmentally-relevant dose is relatively safe by supporting evidence from an array of behavioral and biochemical tests. This combinational approach using behavioral and biochemical tests would be helpful for understanding the MNPs association with anticipated colloids and particles effecting bioavailability and uptake into cells and organisms.

Establishing simple image-based methods and a cost-effective instrument for toxicity assessment on circadian rhythm dysregulation in fish.

Analysis of circadian rhythm behavior alteration in fish for toxicity assessment usually requires expensive commercial equipment and laborious and complicated tweaking. Here, we report a simple setup that consists of a custom-made light box equipped with white and 940 nm light-emitting diode (LED) light strips as light sources, where the locomotion activities of zebrafish or catfish are captured using an infrared-sensitive coupled charged device (CCD). The whole setup was housed in a temperature-controlled incubator to isolate external noise and to maintain consistent experimental conditions. The video recording and light triggering were synchronized using Total Recorder, a recording scheduling software. By using the setup mentioned above and open source software such as ImageJ or idTracker, the locomotion activities of diurnal (e.g. zebrafish) and nocturnal (e.g. catfish) fish during day and night cycles can be quantitatively analyzed. We used simple image-based methods and a cost-effective instrument to assess the circadian rhythm of multiple fish species, as well as other parameters such as age, ambient temperature and chemical toxicology with high precision and reproducibility. In conclusion, the instrument setting and analysis methods established in this study provide a reliable and easy entry point for toxicity assessment on circadian rhythm dysregulation in fish.

The aquatic animals' transcriptome resource for comparative functional analysis.

BACKGROUND: Aquatic animals have great economic and ecological importance. Among them, non-model organisms have been studied regarding eco-toxicity, stress biology, and environmental adaptation. Due to recent advances in next-generation sequencing techniques, large amounts of RNA-seq data for aquatic animals are publicly available. However, currently there is no comprehensive resource exist for the analysis, unification, and integration of these datasets. This study utilizes computational approaches to build a new resource of transcriptomic maps for aquatic animals. This aquatic animal transcriptome map database dbATM provides de novo assembly of transcriptome, gene annotation and comparative analysis of more than twenty aquatic organisms without draft genome. RESULTS: To improve the assembly quality, three computational tools (Trinity, Oases and SOAPdenovo-Trans) were employed to enhance individual transcriptome assembly, and CAP3 and CD-HIT-EST software were then used to merge these three assembled transcriptomes. In addition, functional annotation analysis provides valuable clues to gene characteristics, including full-length transcript coding regions, conserved domains, gene ontology and KEGG pathways. Furthermore, all aquatic animal genes are essential for comparative genomics tasks such as constructing homologous gene groups and blast databases and phylogenetic analysis. CONCLUSION: In conclusion, we establish a resource for non model organism aquatic animals, which is great economic and ecological importance and provide transcriptomic information including functional annotation and comparative transcriptome analysis. The database is now publically accessible through the URL http://dbATM.mbc.nctu.edu.tw/ .

A rapid assessment for predicting drug-induced hepatotoxicity using zebrafish.

INTRODUCTION: Zebrafish have been used as a model to access drug-induced hepatotoxicity. However, individual differences occur in the liver development of zebrafish. METHODS: We used a transgenic line of zebrafish that expressed enhanced green fluorescent protein (EGFP) in the liver and then used a calculation of the liver index area, a potentially new endpoint of hepatotoxicity, to evaluate drug-induced liver injury. To further validate the reliability of the liver area index as a quick evaluation of zebrafish liver function damage, the liver area index level was correlated with hepatic transaminase activities using the Pearson correlation coefficient and confirmed by histopathology. RESULTS: Zebrafish larvae treated with high doses of the known mammalian hepatotoxic drugs carbaryl, isoniazide, and pyrazinamide showed significantly decreased liver area index levels, which are suggestive of liver injury and correspond with the higher alanine transaminase (ALT) and aspartate transaminase (AST) activities and histological liver alterations. The results showed a significant negative correlation between the degree of liver injury and the liver area index level. DISCUSSION: Our data support the use of the liver area index as a reliable and comparable indicator to screen hepatotoxic agents using the zebrafish model.

The complete chloroplast genome of Gracilariopsis lemaneiformis, an important economic red alga of the family Gracilariaceae.

The complete chloroplast DNA (cpDNA) of a famous red alga of the family Gracilariaceae, Gracilariopsis lemaneiformis, was deduced by using next-generation sequencing and de novo assembly technology. The complete cpDNA of G. lemaneiformis consists of 182 505 bp and encodes 230 unique genes consisting 204 protein-coding genes (PCGs), 21 transfer RNA genes, 3 ribosomal RNA genes, 1 transfer-messenger RNA genes and 1 non-coding RNA genes. Among 204 PCGs, ccsA gene is interrupted by a intron. Unlike the typical quadripartite structure (a pair of inverted repeats separated by the small single-copy and large single-copy units) of cpDNA in higher plants, the complete cpDNA of G. lemaneiformis is very compact, containing no inverted repeat and just one copy of rRNA gene cluster consisting of 16S, 23S and 5S rRNA genes. The genic regions account for 83.7% of whole cpDNA genome, and the G + C content of the cpDNA was 27.4%. The low G + C content of G. lemaneiformis cpDNA is largely contributed by high A + T content in the PCGs and non-coding regions. A phylogenetic analysis of the 15 complete cpDNA from rhodophyta shows that G. lemaneiformis is closely related to macroalga Gracilaria salicornia. The complete cpDNA of G. lemaneiformis provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for rhodophyta.

The complete chloroplast genome of Wakame (Undaria pinnatifida), an important economic macroalga of the family Alariaceae.

We decoded the complete chloroplast DNA (cpDNA) sequence of the Wakame (Undaria pinnatifida), an important economic macroalga of the family Alariaceae, by using next-generation sequencing technology. The genome consists of 130 336 bp containing a pair of inverted repeats (IRs) of 4790 bp, which was separated by a large single-copy region and a small single-copy region of 77 821 and 42 934 bp, respectively. The genic regions account for 77.7% of whole cpDNA, and the GC content of the cpDNA was 30.6%. The U. pinnatifida cpDNA encodes 153 unigenes (129 protein-coding genes, 3 rRNA genes and 21 tRNA genes). There are 1 PCG (rpl33) and 1 tRNA genes (trnL) containing an intron. A phylogenetic analysis of the four complete cpDNA from Phaeophyceae showed that U. pinnatifida is closely related to Saccharina japonica with high bootstrap value supported. The complete cpDNA of U. pinnatifida provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for brown algae.

[Zebrafish research in Taiwan].

The initiation time for zebrafish research in Taiwan was around 1996 and experienced eight years (1996-2003) to establish the fundamental base. From 2004 onwards, zebrafish research project and population in Taiwan is exponentially increasing to a total number of 83 independent research laboratories at this moment. Three major research topics of developmental biology, human disease, and biotechnology are conducted by zebrafish community in Taiwan. Up to now, 342 scientific refereed papers have been published by Taiwan researchers. By 2010, two zebrafish stock centers of TZCAS and ZeTH have been established to provide fish resources to zebrafish community. By the fully support of two zebrafish stock centers, attending physicians and experts in the fields of engineering and bioinformatics are working together with zebrafish people and obtained fruitful results in zebrafish researches in recent years.