The nano-bio interactions of rare-earth doped BaF2 nanophosphors shape the developmental processes of zebrafish.

Nanoparticles with biomedical applications should be evaluated for their biocompatibility. Rare-earth doped nanoparticles with unique spectral properties are superior in vivo optical probes in comparison with quantum dots and organic dyes, however, studies describing their nano-bio interactions are still limited. Here, we have evaluated the nano-bio interactions of green-synthesized, phase-pure BaF2 nanoparticles doped with rare-earth (RE3+ = Ce3+/Tb3+) ions using larval zebrafish. We found that zebrafish can tolerate a wide concentration range of these nanoparticles, as the maximal lethality was observed at very high concentrations (more than 200 mg L-1) upon five days of continuous exposure. At a concentration of 10 mg L-1, at which Zn2+, Ti4+ and Ag+ nanoparticles are reported to be lethal to developing zebrafish, continuous exposure to our nanoparticles for four days produced no developmental anomalies, craniofacial defects, cardiac toxicity or behavioural abnormalities in the developing zebrafish larvae. We have also found that the doping of rare-earth ions has no major effect on these biomarkers. Interestingly, the function of acetylcholinesterase (AChE) and the cellular metabolic activity of whole zebrafish larvae remained unchanged, even during continuous exposure to these nanoparticles at 150 mg L-1 for four days; however, severe developmental toxicities were evident at this high concentration. Based on these results, we can conclude that the biocompatibility of rare-earth doped nanoparticles is concentration dependent. Not all biomarkers are sensitive to these nanoparticles. The high concentration-dependent toxicity occurs through a mechanism distinct from changes in the metabolic or AChE activity. The significance of these findings lies in using these nanoparticles for bioimaging applications and biomarker studies, especially for prolonged exposure times.

Triclosan alters adult zebrafish behavior and targets acetylcholinesterase activity and expression.

Triclosan is widely used in consumer products as an antimicrobial agent. Epidemiological studies have reported the association of triclosan with adverse birth outcomes. The toxic effects of triclosan on the developing stages of zebrafish are reported, however, its role as behavioral modifier is limited. In the present study, adult zebrafish were exposed to triclosan (0.3 and 0.6 mg/L) for 48 h and the exploratory behavior was analyzed using ZebraTrack. Triclosan exposed group showed significantly reduced locomotion concomitant with increased freezing duration. They also showed erratic movements suggesting that triclosan induced anxiety-like behavior in adult zebrafish. Next, we tested the hypothesis that the anxiety-like behavior is linked to altered acetylcholinesterase activity. We found that the triclosan exposure decreased acetylcholinesterase activity in the brain and skeletal muscle but acetylcholinesterase (ache) gene was significantly down-regulated only in the skeletal muscle of the adult zebrafish exposed to triclosan. In addition, we also observed a down-regulation of myelin basic protein (mbp) gene in the skeletal muscle of adult zebrafish treated with triclosan. Thus, our data indicates that even short exposure of triclosan is potent enough to induce behavioral anomalies in adult zebrafish that appear to involve acetylcholinesterase and other structural proteins especially in the skeletal muscle.

Quantitative assessment of cypermethrin induced behavioural and biochemical anomalies in adult zebrafish.

Cypermethrin is one of the top five pesticides used globally. Although the effect of cypermethrin on the embryonic stages of zebrafish is well characterized, its toxic effect on the behaviour of adult zebrafish is largely unknown. Here we used videogram and automated tracking approach to quantitatively assess behavioural toxicity induced by the short exposure of cypermethrin to adult zebrafish. We observed that cypermethrin at 25 ppb level induced behavioural toxicity in adult zebrafish. Motor activity of the treated group was significantly retarded which affected their overall exploratory behaviour including their visit to the central arena of the open-field test. Furthermore, the treated group showed erratic movements (covered less distance per unit time) without affecting their angle based behavioural endpoints. In contrast to the control group, the cypermethrin exposed group showed frequent freezing behaviour. However, their freezing episodes were characterized by constant drift-like movement caused by the loss of their voluntary control over the motor coordination. These behavioural changes are similar to typical anxiety-like behaviour. Though, cypermethrin exposure at ppb level for just half an hour was sufficient to induce behavioural toxicity, it failed to alter brain superoxide dismutase and acetylcholine esterase enzyme activity. Our data indicates that acute short-term exposure of cypermethrin induces behavioural anomalies in adult zebrafish through a mechanism distinct from alteration of brain superoxide dismutase and the acetylcholine esterase activity.

ZebraPace: An Open-Source Method for Cardiac-Rhythm Estimation in Untethered Zebrafish Larvae.

For the assessment of cardiac function, heartbeat represents one key parameter. Current methods of heartbeat measurements in the zebrafish larvae usually require larval immobilization, fluorescent transgenic strains and a confocal microscope, costly commercial software for analysis, or strong programming skills if the software is open-source. Here, we present a simple yet powerful method of heartbeat analysis using untethered, unlabeled zebrafish larva using ImageJ (open-source software), which does not require programming skills. We named it as ZebraPace for Zebrafish Precise Algorithm for Cardiac-rhythm Estimation. ZebraPace works directly with AVI videos and requires no image processing steps. ZebraPace uses pixel intensity change in a grayscale video to count the number of beats. We have validated the ZebraPace method by pharmacological alterations of the heartbeat in zebrafish larvae of 48 and 72 hpf stages. We have also determined beat-to-beat interval, which relates to rhythmicity of heartbeat. The results obtained by using ZebraPace corroborates well with the heartbeat values previously reported for similarly aged larvae as determined by using specialized software. We believe that the ZebraPace method is simple, cost-effective, and easy to grasp as it involves fewer steps. It not only reduces the manual workload but also eliminates sample preparation time and researcher subjectivity.

Open-RAC: Open-Design, Recirculating and Auto-Cleaning Zebrafish Maintenance System.

Zebrafish is a vertebrate animal model. Their maintenance in large number under laboratory conditions is a daunting task. Commercially available recirculating zebrafish maintenance systems are used to efficiently handle the tasks of automatic sediment cleaning from zebrafish tanks with minimal waste of water. Due to their compact nature, they also ensure the maximal use of available lab space. However, the high costs of commercial systems present a limitation to researchers with limited funds. A cost-effective zebrafish maintenance system with major features offered by commercially available systems is highly desirable. Here, we describe a compact and recirculating zebrafish maintenance system. Our system is composed of cost-effective components, which are available in local markets and/or can be procured via online vendors. Depending on the expertise of end users, the system can be assembled in 2 days. The system is completely customizable as it offers geometry independent zebrafish tanks that are capable of auto-cleaning the sediments. Due to these features, we called our setup as Open-RAC (Open-design, Recirculating and Auto-Cleaning zebrafish maintenance system). Open-RAC is a cost-effective and viable alternative to the currently available zebrafish maintenance systems. Thus, we believe that the use of Open-RAC could promote the zebrafish research by removing the cost barrier for researchers.

A novel method for automated tracking and quantification of adult zebrafish behaviour during anxiety.

BACKGROUND: Behavioural neuroscience relies on software driven methods for behavioural assessment, but the field lacks cost-effective, robust, open source software for behavioural analysis. NEW METHOD: Here we propose a novel method which we called as ZebraTrack. It includes cost-effective imaging setup for distraction-free behavioural acquisition, automated tracking using open-source ImageJ software and workflow for extraction of behavioural endpoints. Our ImageJ algorithm is capable of providing control to users at key steps while maintaining automation in tracking without the need for the installation of external plugins. RESULTS: We have validated this method by testing novelty induced anxiety behaviour in adult zebrafish. Our results, in agreement with established findings, showed that during state-anxiety, zebrafish showed reduced distance travelled, increased thigmotaxis and freezing events. Furthermore, we proposed a method to represent both spatial and temporal distribution of choice-based behaviour which is currently not possible to represent using simple videograms. COMPARISON WITH EXISTING METHOD(S): ZebraTrack method is simple and economical, yet robust enough to give results comparable with those obtained from costly proprietary software like Ethovision XT. CONCLUSION: We have developed and validated a novel cost-effective method for behavioural analysis of adult zebrafish using open-source ImageJ software.

Designing and Testing of Self-Cleaning Recirculating Zebrafish Tanks.

Maintenance of large number of zebrafish in captive conditions is a daunting task. This can be eased by the use of recirculating racks with self-cleaning zebrafish tanks. Commercially available systems are costly, and compatibility of intercompany products has never been investigated. Although various cost-effective designs and methods of construction of custom-made recirculating zebrafish racks are available in literature, the design of self-cleaning zebrafish tanks is still not available. In this study, we report the design and method of construction of the self-cleaning unit, which can be fitted in any zebrafish tank. We validated the design by investigating sediment cleaning process in rectangular and cylindrical tank geometries using time lapse imaging. Our results suggest that for both tank geometries, the tanks fitted with self-cleaning unit provided superior sediment cleaning than the tanks fitted with overflow-drain unit. Although the self-cleaning unit could clean the sediment completely from both geometries over prolonged period, the cleaning of sediments was faster in the cylindrical tank than the rectangular tank. In conclusion, cost and efforts of zebrafish maintenance could be significantly reduced through the installation of our self-cleaning unit in any custom-made zebrafish tank.