4-Methylbenzylidene camphor induced neurobehavioral toxicity in zebrafish (Danio rerio) embryos.

4-Methylbenzylidene camphor (4-MBC) is a widely used organic UV filter in personal care products. Extensive use of 4-MBC and its frequent detection in aquatic ecosystems defile the biota with muscular and neuronal impairments. This study investigates the neurobehavioral toxicity of 4-MBC using Danio rerio as a model organism. Embryos were exposed semi-statically to 4-MBC at 5, 50, and 500 µg/L concentrations for 10-day post fertilization (dpf). Embryos exhibited a significant thigmotaxis and decreased startle touch response with altered expression of nervous system mRNA transcripts on 5 & 10 dpf. Compared to the sham-exposed group, 4-MBC treated larvae exhibited changes in the expression of shha, ngn1, mbp, elavl3, α1-tubulin, syn2a, and gap43 genes. Since ngn1 induction is mediated by shh signaling during sensory neuron specification, the elevated protein expression of NGN1 indicates 4-MBC interference in the sonic hedgehog signaling pathway. This leads to sensory neuron impairment and function such as 'sense' as evident from reduced touch response. In addition, larval brain histology with a reduced number of cells in the Purkinje layer emblazing the defunct motor coordination. Predictive toxicity study also showed a higher affinity of 4-MBC to modeled Shh protein. Thus, the findings of the present work highlighted that 4-MBC is potential to induce developmental neurotoxicity at both behavioral and molecular functional perspectives, and developing D. rerio larvae could be considered as a suitable alternate animal model to assess the neurological dysfunction of organic UV filters.

State-dependent, visually guided behaviors in the nudibranch Berghia stephanieae.

Nudibranch mollusks have structurally simple eyes whose behavioral roles have not been established. We tested the effects of visual stimuli on the behavior of the nudibranch Berghia stephanieae under different food and hunger conditions. In an arena that was half-shaded, animals spent most of their time in the dark, where they also decreased their speed and made more changes in heading. These behavioral differences between the light and dark were less evident in uniformly illuminated or darkened arenas, suggesting that they were not caused by the level of illumination. Berghia stephanieae responded to distant visual targets; animals approached a black stripe that was at least 15 deg wide on a white background. They did not approach a stripe that was lighter than the background but approached a stripe that was isoluminant with the background, suggesting the detection of spatial information. Animals traveled in convoluted paths in a featureless arena but straightened their paths when a visual target was present even if they did not approach it, suggesting that visual cues were used for navigation. Individuals were less responsive to visual stimuli when food deprived or in the presence of food odor. Thus, B. stephanieae exhibits visually guided behaviors that are influenced by odors and hunger state.

Comparative Observation and Analysis of Preference Behavior Based on Three Types of Taxes and Locomotor Activity in the Goldfish, Carassius auratus.

Psychophysiological studies in vertebrates have focused on taxes as indicators of behavioral change. Actually, a considerable number of studies about anxiety-like and anti-anxiety-like behaviors involving geotaxis, scototaxis, and thigmotaxis have been conducted on fish. However, few analyses considering these behaviors based on taxes in fish have been conducted. Here, using goldfish, we measured the time spent in the bright or dark area of a horizontally long rectangular tank (HLRT), in the upper or lower area of a vertically long rectangular tank (VLRT), and in the central or edge area of a circular tank (CT), respectively, for the first 30 min and the last 30 min in a 3-h period after fish had been introduced to tanks. Dark, lower, and edge preference behaviors were observed for the first 30 min in all tanks. While dark and edge preference behaviors were maintained even for the last 30 min, the lower preference was lost. Swimming distance and the number of area crossings in each tank were also compared between the first 30 min and the last 30 min. Both decreased significantly or tended to decrease in the last 30 min in the HLRT and the CT, but no change was observed in the VLRT. These results suggest that, in goldfish, preference behavior is stable for a short time, and that environmental habituation may depend on the shape of the tank and the elapsed time.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Exposure of zebrafish embryos to sodium propionate disrupts circadian behavior and glucose metabolism-related development.

Sodium propionate is widely used as a preservative in food. The widespread use of preservatives is known to cause both environmental and public health problems. This study aimed to investigate the effects of sodium propionate on the developmental behavior and glucose metabolism of zebrafish. Our results showed that sodium propionate had no significant effect on the embryonic morphological development of zebrafish embryos but changed the head eye area. Then we found sodium propionate disturbed the thigmotaxis behavior, impaired neural development. Moreover, changes in clock gene expression disrupted the circadian rhythm of zebrafish. Circadian genes regulated insulin sensitivity and secretion in various tissues. Then our results showed that the disorder of circadian rhythm in zebrafish affected glucose metabolism and insulin resistance, which damaged the development of retina. Therefore, the safety of propionate should be further evaluated.

Behavioural effects of early-life exposure to parabens in zebrafish larvae.

Parabens are classified as endocrine disrupting chemicals due to their ability to activate several nuclear receptors causing changes in hormones-dependent signalling pathways. Central nervous system of developing organisms is particularly vulnerable to changes in hormonal pathways, which could lead to altered brain function, abnormal behaviour and even diseases later in life. The aim of the present study was to investigate the effects of exposure to butylparaben (BuP), ethylparaben (EtP) and methylparaben (MeP) during early development on nervous system using zebrafish larvae's behavioural models. Zebrafish were exposed until 4 days post fertilization (dpf) to three concentrations of each paraben chosen considering the environmentally realistic concentrations of human exposure and the benchmark-dose lower bound calculated for zebrafish larvae (BuP: 5, 50 and 500 µg/L; EtP: 50, 500 and 5000 µg/L; MeP: 100, 1000 and 10,000 µg/L). Activity in novel and in familiar environment, thigmotaxis, visual startle response and photic synchronization of the behavioural circadian rhythms were analysed at 4, 5 and 6 dpf. Zebrafish larvae exposed to BuP 500 µg/L and EtP 5000 µg/L revealed increased anxiety-like behaviour in novel environment. Larvae treated with 500 µg/L of BuP showed reduced activity in familiar and marginally in unfamiliar environment, and larvae exposed to 5000 µg/L of EtP exhibited hyperactivity in familiar environment. Parabens exposure did not influence the visual startle response and the photic synchronization of circadian rhythms in zebrafish larvae. This research highlighted as the exposure to parabens has the potential to interfere with behavioural development of zebrafish.

Deletion of c16orf45 in zebrafish results in a low fertilization rate and increased thigmotaxis.

c16orf45 is located at 16p13.11, an important locus related to neurodevelopmental diseases. Clinical studies have demonstrated that c16orf45 is associated with various neurodevelopmental diseases. To further elucidate the effect of c16orf45 on neural development, we constructed a zebrafish model with a stably inherited c16orf45 deletion via CRISPR/Cas9 technology. We found that deletion of c16orf45 significantly reduced the zebrafish fertilization rate, and both females and males showed reduced fertility. Meanwhile, the homozygous c16orf45 knockout zebrafish showed a developmental delay at 24 hr postfertilization (hpf). However, morphological changes were not apparent after 2 days postfertilization (dpf). Notably, the results of behavioral experiments revealed increased thigmotaxis in c16orf45-/- zebrafish at 2 months. In conclusion, these findings demonstrate that c16orf45 plays an important role in nervous system and reproductive system.

Wall following in Xenopus laevis is barrier-driven.

The tendency of animals to follow boundaries within their environment can serve as a strategy for spatial learning or defensive behaviour. We examined whether Xenopus laevis tadpoles and froglets employ such a strategy by characterizing their swimming pattern in a square tank with shallow water. Trajectories obtained from video recordings were analysed for proximity to the nearest wall. With the exception of young larvae, the vast majority of animals (both tadpoles and froglets) spent a disproportionately large amount of time near the wall. The total distance covered was not a confounding factor, but animals were stronger wall followers in smaller tanks. Wall following was also not influenced by whether the surrounding walls of the tank were black or white, illuminated by infrared light, or by the presence or absence of tentacles. When given a choice in a convex tank to swim straight and leave the wall or turn to follow the wall, the animals consistently left the wall, indicating that wall following in X. laevis is barrier-driven. This implies that wall following behaviour in Xenopus derives from constraints imposed by the environment (or the experimenter) and is unlikely a strategy for spatial learning or safety seeking.

Testing the parasite mass burden effect on alteration of host behaviour in the Schistocephalus-stickleback system.

Many parasites with complex life cycles modify the behaviour of their intermediate host, which has been proposed to increase transmission to their definitive host. This behavioural change could result from the parasite actively manipulating its host, but could also be explained by a mechanical effect, where the physical presence of the parasite affects host behaviour. We created an artificial internal parasite using silicone injections in the body cavity to test this mechanical effect hypothesis. We used the Schistocephalus solidus and threespine stickleback (Gasterosteus aculeatus) system, as this cestode can reach up to 92% of its fish host mass. Our results suggest that the mass burden brought by this macroparasite alone is not sufficient to cause behavioural changes in its host. Furthermore, our results show that wall-hugging (thigmotaxis), a measure of anxiety in vertebrates, is significantly reduced in Schistocephalus-infected sticklebacks, unveiling a new altered component of behaviour that may result from manipulation by this macroparasite.

Innate turning preference of leaf-cutting ants in the absence of external orientation cues.

Most ants use a combination of cues for orientation but how do ants find their way when all external cues are suppressed? Do they walk in a random way or are their movements spatially oriented? Here, we show for the first time that leaf-cutting ants (Acromyrmex lundii) have an innate preference for turning counter-clockwise (left) when external cues are precluded. We demonstrated this by allowing individual ants to run freely on the water surface of a newly developed treadmill. The surface tension supported medium-sized workers but effectively prevented ants from reaching the wall of the vessel, which was important to avoid wall-following behaviour (thigmotaxis). Most ants ran for minutes on the spot but also slowly turned counter-clockwise in the absence of visual cues. Reconstructing the effective path walked revealed a looping pattern which could be interpreted as a search strategy. A similar turning bias was shown for groups of ants in a symmetrical Y-maze where twice as many ants chose the left branch in the absence of optical cues. Wall-following behaviour was tested by inserting a coiled tube before the Y-fork. When ants traversed a left-coiled tube, more ants chose the left box and vice versa. Adding visual cues in the form of vertical black strips either outside the treadmill or on one branch of the Y-maze led to oriented walks towards the strips. It is suggested that both turning bias and wall following are employed as search strategies for an unknown environment which can be overridden by visual cues.

Effects of picrotoxin on zebrafish larvae behaviors: A comparison study with PTZ.

Larval zebrafish (Danio rerio) have been suggested as a high-throughput experimental animal model for epilepsy-related genetic and developmental studies. The behavioral manifestations in response to the seizure-inducing drugs picrotoxin (PTX) (1, 5, 25, 125, or 625µM) or pentylenetetrazole (PTZ) (1, 2, 4, 8, or 16mM) under light-dark conditions were studied using zebrafish larvae at 5days post-fertilization (dpf). Two behavioral parameters, locomotor activity and thigmotaxis behavior, were analyzed. We conclude that high concentrations of PTX treatment increased locomotion and thigmotaxis in 5 dpf zebrafish larvae under continuous illumination and the locomotion of PTX-treated zebrafish was decreased under the dark condition. High concentrations of PTX treatment also increased thigmotaxis (an indicator of increased anxiety levels) in zebrafish larvae under both continuous illumination and dark condition. PTZ treatment increased the locomotion of 5 dpf zebrafish larvae under continuous illumination. However, 2mM PTZ decreased locomotion, and high concentrations of PTZ decreased the locomotion of larvae under dark conditions. High concentrations of PTZ treatment also increased thigmotaxis in the zebrafish larvae under both continuous illumination and dark condition. Compared with PTZ, PTX leads to higher levels of movement under light conditions and lower levels of movement under dark condition. However, the level of thigmotaxis in the zebrafish larvae was similar between the two drug treatments.

Sex differences after environmental enrichment and physical exercise in rats when solving a navigation task.

The effects of early environmental enrichment (EE) and voluntary wheel running on the preference for using a landmark or pool geometry when solving a simple spatial task in adult male and female rats were assessed. After weaning, rats were housed in same-sex pairs in enriched or standard cages (EE and control groups) for two and a half months. Then the rats were trained in a triangular-shaped pool to find a hidden platform whose location was defined in terms of these two sources of information, a landmark outside the pool and a particular corner of the pool. As expected, enriched rats reached the platform faster than control animals, and males and females did not differ. Enriched rats also performed better on subsequent test trials without the platform with the cues individually presented (either pool geometry or landmark). However, on a preference test without the platform, a clear sex difference was found: Females spent more time in an area of the pool that corresponded to the landmark, whereas males spent more time in the distinctive corner of the pool. The present EE protocol did not alter females' preference for the landmark cue. The results agree with the claim that environmental enrichment is a consequence of a reduced anxiety response (measured by thigmotaxis) during cognitive testing. A possible implication of ancestral selection pressures is discussed.

VideoHacking: Automated Tracking and Quantification of Locomotor Behavior with Open Source Software and Off-the-Shelf Video Equipment.

Differences in nervous system function can result in differences in behavioral output. Measurements of animal locomotion enable the quantification of these differences. Automated tracking of animal movement is less labor-intensive and bias-prone than direct observation, and allows for simultaneous analysis of multiple animals, high spatial and temporal resolution, and data collection over extended periods of time. Here, we present a new video-tracking system built on Python-based software that is free, open source, and cross-platform, and that can analyze video input from widely available video capture devices such as smartphone cameras and webcams. We validated this software through four tests on a variety of animal species, including larval and adult zebrafish (Danio rerio), Siberian dwarf hamsters (Phodopus sungorus), and wild birds. These tests highlight the capacity of our software for long-term data acquisition, parallel analysis of multiple animals, and application to animal species of different sizes and movement patterns. We applied the software to an analysis of the effects of ethanol on thigmotaxis (wall-hugging) behavior on adult zebrafish, and found that acute ethanol treatment decreased thigmotaxis behaviors without affecting overall amounts of motion. The open source nature of our software enables flexibility, customization, and scalability in behavioral analyses. Moreover, our system presents a free alternative to commercial video-tracking systems and is thus broadly applicable to a wide variety of educational settings and research programs.

Single stimulus learning in zebrafish larvae.

Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7dpf larvae and extended the finding to 5 and 6dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed.

Mechanical processing via passive dynamic properties of the cockroach antenna can facilitate control during rapid running.

The integration of information from dynamic sensory structures operating on a moving body is a challenge for locomoting animals and engineers seeking to design agile robots. As a tactile sensor is a physical linkage mediating mechanical interactions between body and environment, mechanical tuning of the sensor is critical for effective control. We determined the open-loop dynamics of a tactile sensor, specifically the antenna of the American cockroach, Periplaneta americana, an animal that escapes predators by using its antennae during rapid closed-loop tactilely mediated course control. Geometrical measurements and static bending experiments revealed an exponentially decreasing flexural stiffness (EI) from base to tip. Quasi-static experiments with a physical model support the hypothesis that a proximodistally decreasing EI can simplify control by increasing preview distance and allowing effective mapping to a putative control variable--body-to-wall distance--compared with an antenna with constant EI. We measured the free response at the tip of the antenna following step deflections and determined that the antenna rapidly damps large deflections: over 90% of the perturbation is rejected within the first cycle, corresponding to almost one stride period during high-speed running (~50 ms). An impulse-like perturbation near the tip revealed dynamics that were characteristic of an inelastic collision, keeping the antenna in contact with an object after impact. We contend that proximodistally decreasing stiffness, high damping and inelasticity simplify control during high-speed tactile tasks by increasing preview distance, providing a one-dimensional map between antennal bending and body-to-wall distance, and increasing the reliability of tactile information.

Ants show a leftward turning bias when exploring unknown nest sites.

Behavioural lateralization in invertebrates is an important field of study because it may provide insights into the early origins of lateralization seen in a diversity of organisms. Here, we present evidence for a leftward turning bias in Temnothorax albipennis ants exploring nest cavities and in branching mazes, where the bias is initially obscured by thigmotaxis (wall-following) behaviour. Forward travel with a consistent turning bias in either direction is an effective nest exploration method, and a simple decision-making heuristic to employ when faced with multiple directional choices. Replication of the same bias at the colony level would also reduce individual predation risk through aggregation effects, and may lead to a faster attainment of a quorum threshold for nest migration. We suggest the turning bias may be the result of an evolutionary interplay between vision, exploration and migration factors, promoted by the ants' eusociality.

Graphene oxide quantum dots (GOQDs) induce behavioral disorders via the disturbance of kynurenine pathway in zebrafish larvae.

The emergence of graphene quantum dots (GQDs) expands the use of graphene derivatives in nanomedicine for its direct therapeutic applications in treating neurodegeneration, inflammation, metabolic dysfunction, and among others. Nevertheless, the biosafety assessment of GQDs remains deficient mostly because of the diverse surface characteristics of the nanoparticles. Our prior work demonstrated that GQDs can induce strong thigmotactic effects in zebrafish larvae over a wide range of concentrations, yet the underlying metabolic mechanisms remain largely unknown. In this study, we conducted a further exploration about graphene oxide quantum dots (GOQDs) for its potential neurotoxic effect on the behaviors of zebrafish larvae by combining neurotransmitter-targeted metabolomics with locomotion analysis. After continuous exposure to a concentration gradient of GOQDs (12.5 - 25 - 50 - 100 - 200 µg/mL) for 7 days, the thigmotactic activities of zebrafish larvae were observed across all exposure concentrations relative to the control group, while the basal locomotor activities, including distance moved and average velocity, were significantly changed by low concentrations of GOQDs. Targeted metabolomics was performed using zebrafish larvae at 7 days post-fertilization (dpf) that were exposed to 12.5 and 200 µg/mL, both of which were found to perturb the kynurenine pathway by regulating the levels of kynurenine, 3-hydroxyanthranilic acid (3-HAA), and quinolinic acid (QA). Furthermore, the thigmotaxis of larval fish induced by GOQDs during exposure could be counteracted by supplementing Ro-61-8048, an agonist acting on kynurenine 3-monooxygenase (KMO). In conclusion, our study establishes the involvement of the kynurenine pathway in GOQDs-induced thigmotaxis, which is independent of the transcriptional modulation of glutamate receptor families.

Chemical and Visual Cues as Modulators of the Stress Response to Social Isolation in the Marine Medaka, Oryzias melastigma.

The marine medaka is emerging as a potential behavioral model organism for ocean studies, namely on marine ecotoxicology. However, not much is known on the behavior of the species and behavioral assays lack standardization. This study assesses the marine medaka as a potential model for chemical communication. We investigated how short exposure to visual and chemical cues mediated the stress response to social isolation with the light/dark preference test (LDPT) and the open field test (OFT). After a 5-day isolation period, and 1 h before testing, isolated fish were randomly assigned to one of four groups: (1) placed in visual contact with conspecifics; (2) exposed to a flow of holding water from a group of conspecifics; (3) exposed to both visual and chemical cues from conspecifics; or (4) not exposed to any stimuli (controls). During the LDPT, the distance traveled and transitions between zones were more pronounced in animals exposed to the conspecific's chemical stimuli. The time spent in each area did not differ between the groups, but a clear preference for the bright area in all animals indicates robust phototaxis. During the OFT, animals exposed only to chemical cues initially traveled more than those exposed to visual or both stimuli, and displayed lower thigmotaxis. Taken together, results show that chemical cues play a significant role in exploratory behavior in this species and confirm the LDPT and OFT as suitable tests for investigating chemical communication in this species.

Prediction of sperm motion behavior in microfluidic channel using sperm swimming model.

Several investigations have recently been conducted using microfluidic channels to sort highly motile sperm and thereby increase the probability of fertilization. To further enhance the efficiency of sperm sorting, predicting sperm movement in microfluidic channels through simulation techniques could be beneficial. In this study, we constructed a sperm swimming model based on the concept of an agent-based model. This model allows analysis at the same spatio-temporal scale similar to microfluidic channels. Sperm movement was simplistically modeled as a random walk, utilizing the distribution of sperm velocity and deflection angle obtained from experimental data. We have developed a thigmotaxis model to describe the phenomenon where sperm near the wall exhibit a reduced tendency to move away from it. Additionally, we created a rheotaxis model, in which sperm reorient in the direction opposite to the flow depending on the shear rate. Using these models, we investigated sperm behaviors within a microchannel featuring a tapered area. The results reveal that sperm accumulate within the tapered area, leading to a significant increase in sperm concentration for specific flow velocity ranges in the microchannel. This model provides valuable information for predicting the effects of sperm sorting in various microfluidic channels.

Flour beetles prefer corners over walls and are slowed down with increasing habitat complexity.

Movement affects all key behaviours in which animals engage, including dispersal and habitat use. The red flour beetle, known as a cosmopolitan pest of stored products, was the subject of our study. We examined whether the beetles preferred corners, walls or open areas, and how turns or obstacles in corridors delayed the beetles' arrival at a target cell. Beetles spent significantly more time in corners than expected by chance, while they spent considerably less time in open areas than expected. However, no significant difference was observed between areas with two or three surrounding walls. This could be attributed to the beetles' stronger attraction to corners than crevices or the insufficient proximity of the third wall to the other two. Movement through the corridor was delayed by turns or obstacles, expressed in arrival probabilities, arrival times, time in the corridor or movement speed. Obstacles on the corridor's perimeter had a stronger effect on the beetle movement than those in the corridor's centre owing to the beetles' tendency to follow walls. The research is important also for applied purposes, such as better understanding beetle movement, how to delay their arrival to new patches, and where to place traps.