Automated Video Tracking System Research Articles

Longitudinal quantitative assessment of TMEV-IDD-induced MS phenotypes in two inbred mouse strains using automated video tracking technology

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system affecting over 2.5 million people worldwide. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a murine model that reproduces the progressive form of MS and serves as a reference model for studying virus-induced demyelination.Certain mouse strains such as SJL are highly susceptible to this virus and serve as a prototype strain for studying TMEV infection. Other strains such as SWR are also susceptible, but their disease course following TMEV infection differs from SJL's.The quantification of motor and behavioral deficits following the induction of TMEV-IDD could help identify the differences between the two strains. Motor deficits have commonly been measured with the rotarod apparatus, but a multicomponent assessment tool has so far been lacking.For that purpose, we present a novel way of quantifying locomotor deficits, gait alterations and behavioral changes in this well-established mouse model of multiple sclerosis by employing automated video analysis technology (The PhenoTyper, Noldus Information Technology). We followed 12 SJL and 12 SWR female mice and their mock-infected counterparts over a period of 9 months following TMEV-IDD induction.We demonstrated that SJL and SWR mice both suffer significant gait alterations and reduced exploration following TMEV infection. However, SJL mice also display an earlier and more severe decline in spontaneous locomotion, especially in velocity, as well as in overall activity. Maintenance behaviors such as eating and grooming are not affected in either of the two strains. The system also showed differences in mock-infected mice from both strains, highlighting an age-related decline in spontaneous locomotion in the SJL strain, as opposed to hyperactivity in the SWR strain.Our study confirms that this automated video tracking system can reliably track the progression of TMEV-IDD for 9 months. We have also shown how this system can be utilized for longitudinal phenotyping in mice by describing useful parameters that quantify locomotion, gait and behavior.

The Effects of Prebiotics and Probiotics Following Antibiotic Exposure in a Mouse Model of Autism Spectrum Disorder: A Research Protocol

The human microbiota consists of 10-100 trillion symbiotic microbial cells critical for one’s digestive system, immune system and for managing neurological symptoms experienced in neurodevelopmental conditions. With early exposure to antibiotics, an individual’s microbiome composition is negatively affected by reducing the diversity of microbial species found in the microbiome and can lead to an imbalance in the Gut-Brain-Axis (GBA). While the direct relationship between the GBA and neurodevelopmental functioning is still unclear, evidence suggests that individuals with a disruptive microbiome and an imbalanced GBA have an increased risk of developing neurodevelopmental disorders, specifically autism spectrum disorder (ASD). To improve microbiome diversity, exposure to a high prebiotic and probiotic diet in the early stages of life can reintroduce beneficial bacteria back into the microbiome and improve microbial diversity. Pre- and pro-biotics can improve microbiome diversity and restore balance to the GBA. With the introduction of a prebiotic and probiotic diet and a balanced GBA, there is a possibility to reduce the severity of ASD symptoms. By reducing the severity of ASD symptoms, the quality of life of those with severe ASD can potentially be improved allowing them to maintain functional independence. This research protocol intends to utilize an automated video tracking system and three-chambered social approach to evaluate the behavioural symptoms in BTBR strain mice which exhibit symptoms before and after administration of pre- and probiotics following antibiotic exposure.

Unveiling the molecular mechanisms and developmental consequences of mercury (Hg) toxicity in zebrafish embryo-larvae: A comprehensive approach

Mercury (Hg) is a global contaminant affecting aquatic ecosystems' health. Chronic exposure to Hg has shown that the normal development of zebrafish embryo-larvae is affected. However, the molecular mechanisms behind the toxicity of Hg on fish embryonic development are still poorly understood. This work aimed to investigate the effects of Hg exposure on zebrafish embryo-larvae using a combined approach at individual (mortality, embryo development and locomotor behavior) and biochemical (neurotoxicity and oxidative stress enzymatic activities and protein phosphatase expression) levels. The Fish Embryo Toxicity assay followed the Organization for Economic Cooperation and Development Guideline 236 and used a concentration range between 13 and 401 μg Hg/L. Lethal and developmental endpoints were examined at 24, 48, 72 and 96 hpf. Biochemical markers, including Acetylcholinesterase (AChE), Catalase (CAT), Glutathione Reductase (GR), and Glutathione-S-Transferase (GST) activities and, for the first time, the expression of the protein phosphatase 1 gamma (PP1γ) was assessed after 24, 48, 72 and 96 h of exposure to 10 and 100 μg Hg/L. The behavioral effects of a sublethal range of Hg (from 0.8 to 13 μg Hg/L) were assessed using an automated video tracking system at 120 hpf. Several developmental abnormalities on zebrafish embryos and larvae, including pericardial edema, spin and tail deformities and reduced rate of consumption of the yolk sac, were found after exposure to Hg (LC50 at 96 hpf of 139 μg Hg/L) with EC50 values for total malformations ranging from 22 to 264 μg Hg/L. After 96 hpf, no significant effects were observed in the CAT and GR activities. However, an increase in the GST activity in a concentration and time-dependent manner was found, denoting possible stress-related adaptation of zebrafish embryos to deleterious effects of Hg exposure. The AchE activity showed a response pattern in line with the behavioral responses. At the lowest concentration tested, no significant effects were found for the AChE activity, whereas a decrease in AChE activity was observed at 100 μg Hg/L, suggesting that exposure to Hg induced neurotoxic effects in zebrafish embryos which in turn may explain the lack of equilibrium found in this study (EC50 at 96 hpf of 83 μg Hg/L). Moreover, a decrease in the PP1γ expression was found after 96 h of exposure to 10 and 100 μg Hg/L. Thus, we suggest that Hg may be an inhibitor of PP1γ in zebrafish embryos-larvae and thus, along with the alterations in the enzymatic activity of GST, explain some of the developmental malformations observed, as well as the lack of equilibrium. Hence, in this study, we propose the use of PP1 expression, in combination with apical and biochemical endpoints, as a precursor for assessing Hg's toxic mechanism on embryonic development.

Locomotion behavior of juveniles of the freshwater pearl mussel Margaritifera margaritifera: A new non-invasive tool for the evaluation of stress effects

The lack of knowledge about the sensitivity of the endangered freshwater pearl mussel (FWPM) Margaritifera margaritifera to environmental pollution and the rapid decline of its populations in Europe, have led to the need of developing non-destructive experimental protocols in order to assess the impact of such pollution. This species has a complex life cycle and the early life stages are considered the most sensitive. This study deals with the development of a methodology for the assessment of juvenile mussels' locomotor behavior using an automated video tracking system. Different parameters were determined such as the duration of the video recording and light exposure as a stimulus during the experiment. Locomotion behavior pattern of juveniles was assessed in control condition and also following exposure to sodium chloride as a positive control in order to validate the experimental protocol developed in this study. Results showed that juveniles locomotion behavior was stimulated under light exposure. Moreover, exposure to sublethal concentrations of sodium chloride (0.8 and 1.2 g/L) for 24 h was found to decrease juveniles’ locomotion by almost three-times, thus validating our experimental methodology. This study allowed to provide a new tool for the assessment of stress condition impacts on the juveniles of the endangered FWPM, highlighting the interest of such non-destructive biomarker of health for protected species. Consequently, this will help in the improvement of our knowledge on M. margaritifera sensitivity to environmental pollution.

Effect of insecticides on locomotory behaviour and enzyme activity of Spodoptera litura (Fabricius) populations from Punjab

Aim: The variations in locomotory behaviour and enzyme activity, i.e., acetylcholinesterase and general esterases of Spodoptera litura Fabricius larval populations collected from different regions of Punjab, i.e., Amritsar, Kapurthala and Ludhiana were investigated in the laboratory prior exposure to different insecticides. Methodology: The test larvae were exposed to sublethal doses of different insecticides via surface and food treatment. For locomotory behaviour, they were allowed to move on treated surface and their movements were recorded with an automated video tracking system, Ethovision while on other hand food treatment was opted for observing the influence on enzyme activity. Results: The pronounced variations were observed in the locomotor behaviour of S. litura upon surface exposure of different insecticides. The larva travelled more distance with higher speed and larger turn angle on insecticide-treated surfaces than the untreated ones, while on comparison of different larval populations, the Amritsar population larvae travelled less distance with low speed and maximum turnings. The decrease in acetylcholinesterase and general esterase activity was recorded in treated larvae than untreated ones. The influence of emamectin benzoate was recorded more on locomotion and enzyme activity of S. litura as compared to the remaining insecticides and the Amritsar population was least affected. Interpretation: The sublethal doses of insecticides influenced the overall behavioural physiology of S. litura and this biological parameter could be useful in designing the management strategies for S. litura. Key words: Acetylcholinesterase, Esterases, Insecticides, Locomotory behaviour, S. litura

Caffeine-induced bradycardia, death, and anxiety-like behavior in zebrafish larvae

Caffeine, a major ingredient of coffee and other drinks, causes minimal health problems. However, excessive caffeine intake may induce emotional and cardiac events. Studies on zebrafish larvae are considered useful for understanding drug poisoning because they can be used for multiple simulations. The locomotion (swim distance) of zebrafish larvae exposed to varying concentrations of caffeine was measured using a light–dark test and an automatic video-tracking system. Mortality and heart rates were assessed 4 and 24 h after caffeine exposure and 24 h after caffeine removal. The heart and survival rates were reduced by exposure to caffeine of > 300 mg/L after 24 h in a dose-dependent manner; this was attenuated by caffeine removal at 4 h. Caffeine reduced the heart rate and survival considerably at 1000 mg/L, supporting the notion of caffeine-induced cardiac arrest resulting from bradycardia. The locomotion of zebrafish larvae during six alternating cycles of light–dark exposure was slower during light periods and faster during dark periods; 100–300 mg/L caffeine increased and decreased locomotion during light and dark cycles, respectively, with high inter-cycle reproducibility. Given the anxiogenic effect of caffeine and light preference of zebrafish larvae, caffeine-induced changes in light–dark cycles could provide a new reliable marker for anxiety. Therefore, the “light–dark locomotion test” employed in this study is valuable for research on anxiogenic or anxiolytic substances. The test may allow simultaneous and automatic analysis of the locomotion of many fish under multiple conditions, thereby enabling evaluation of dose–response effects of caffeine.

Developing a two-dimensional landscape model of opportunities for penetrative passing in association football – Stage I

ABSTRACT This study investigated a method for modelling a landscape of opportunities for penetrative passing completed on the ground by ball carriers in association football. Analysis of video footage of competitive, professional football performance was undertaken, identifying a sample (n = 20) of attacking sub-phases of gameplay which ended in a penetrative pass being made between defenders to a receiver. Players’ relative co-positioning during performance was modelled using bi-dimensional x and y coordinates of each player recorded at 25 fps. Data on player movements during competitive interactions were captured using an automatic video tracking system, recording player co-locations emerging over time, as well as current and estimated running velocities. Results revealed that the half spaces between the midfield and both sidelines were the key locations on field providing most affordances for penetrating passes in the competitive performance sample analysed. Due to the dynamics of players’ co-adaptive performance behaviours, it was expected that opportunities for penetrative passing by ball carriers would not display a homogeneous space-time spread across the entire field. Results agreed with these expectations, showing how a landscape of opportunities for penetrative passing might be specified by information emerging from continuous player interactions in competitive performance.

Effects of tenuifolin on rest/wake behaviour in zebrafish.

Insomnia is a common sleep disorder with a high prevalence and substantial adverse consequences. There is growing interest in identifying novel therapeutics from herbal medicine. Tenuifolin is a major constituent of the well-known anti-insomnia herb Radix Polygala. The present study investigated the neural activity in response to tenuifolin during rest/wake behaviour in zebrafish and identified the potential biological signalling pathways involved. An automatic video tracking system was used to monitor the behavioural response of zebrafish larvae for 24 h after treatment with tenuifolin. In total, six rest/wake parameters were measured and visualized with a behavioural fingerprint. Time series analysis was conducted by averaging the total rest and waking activity in 10 min intervals. A correlation analysis was performed between tenuifolin and well-known compounds to analyse the underlying biological signalling pathways. Reverse transcription-quantitative PCR was also performed to detect the effects of tenuifolin on the transcription of interesting genes associated with the signalling pathways that were potentially involved. The present results suggested tenuifolin significantly increased the total rest time during the dark phase, with a slight effect on the waking activity in zebrafish larvae. This behavioural phenotype induced by tenuifolin is similar to that of selective serotonin reuptake inhibitors and gamma-aminobutyric acid (GABA) agonists. Furthermore, the expression levels of GABA transporter 1 were significantly increased after tenuifolin treatment. No significant difference was determined in other associated genes in untreated control and tenuifolin-treated larvae. The present results suggested that tenuifolin caused sleep-promoting activity in zebrafish and that these effects may be mediated by the serotoninergic systems and the GABAergic systems.

A pilot study on assessment of locomotor behavior using a video tracking system in minipigs.

Pigs are often selected for large animal models including for neuroscience and behavioral research, because their anatomy and biochemistry are similar to those of humans. However, behavioral assessments, in combination with objective long-term monitoring, is difficult. In this study, we introduced an automated video tracking system which was previously used in rodent studies, for use with pig models. Locomotor behaviors (total distance, number of zone transitions, and velocity) were evaluated and their changes were validated by different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration methods and dosing regimens. Three minipigs (23–29 kg) received subcutaneous or intravenous MPTP, either 1 or 3 times per week. Immediately after MPTP injection, the minipigs remained in a corner and exhibited reduced trajectory. In addition, the total distance travelled, number of zone transitions, and velocity were greatly reduced at every MPTP administration in all the minipigs, accompanying to increased resting time. However, the MPTP-induced symptoms were reversed when MPTP administration was terminated. In conclusion, this automated video-tracking system was able to monitor long-term locomotor activity and differentiate detailed alterations in large animals. It has the advantages of being easy to use, higher resolution, less effort, and more delicate tracking. Additionally, as our method can be applied to the animals’ home pen, no habituation is needed.

Behavioural patterns and functional responses of a generalist predator revealed using automated video tracking.

Tracking predator-prey interactions using automated image recording systems provides insights into novel patterns and mechanisms of predator-prey dynamics, thus these systems have the potential to evaluate biological control agents effectively. Using an automated video tracking system, we aimed to quantify the behavioural patterns of a generalist predator Harmonia axyridis in response to changing prey density. The effect of intraspecific interactions on foraging efficiency was evaluated. In addition, functional response parameters were compared between the observations and model predictions. The associated behavioural component of prey consumption by H. axyridis was modified by prey density, especially for dual predator trials. Both individual and paired H. axyridis exhibited type II functional responses and a consistent cycle of behaviour. Interestingly, intra-specific interference did not affect overall prey consumption. Divergence between estimated and calculated functional response parameters was observed, which might due to the difficulty of separating foraging and non-foraging activity. Interference interactions between H. axyridis conspecifics might alter their foraging patterns; however, the outcome of prey consumption was not affected by this behaviour. In conclusion, automated video tracking systems may be used to expose the detailed foraging behaviour of predators and could be used to evaluate a wide range of natural enemies. © 2019 Society of Chemical Industry.

Link between elevated locomotor activity and the spike bursting of antennal thermosensitive neurons in the carabid beetle Pterostichus oblongopunctatus

Many aspects of ectothermic life are affected by external temperature conditions. Therefore, thermosensation and thermoregulation are crucial for survival of ground dwelling carabid beetles. Research in sensory physiology with Pterostichus oblongopunctatus has shown change in the reaction of antennal thermoreceptor neurons at temperatures above the P. oblongopunctatus’s preferred temperature. From 25 °C, these neurons in dome shaped sensilla start to change from regular impulse firing to firing in bursts. We hypothesized that temperature-dependent impulse bursts are involved in P. oblongopunctatus’s behavioural thermoregulation. Behavioural experiments were carried out on the arena inside an environmental test chamber, and in two different temperature conditions – one with linearly increasing temperature, and the other with constant temperature. The temperature was increased from 10 °C to 40 °C linearly during 50 min on first case and with 5 degree steps on second case with duration 50 min each step. Locomotion parameters (velocity and travelled distance) were measured using an automated video tracking system. We observed both low and high activity zones in P. oblongopunctatus. The threshold temperature for changing between zones occurred was at 25.8 °C. This indicates a link between spike burst of antennal thermoreceptor neurons and behavioural thermoregulation in P. oblongopunctatus.

Automatic analysis of treadmill running to estimate times to fatigue and exhaustion in rodents.

IntroductionThe determination of fatigue and exhaustion in experimental animals is complicated by the subjective nature of the measurement. Typically, it requires an observer to watch exercising animals, e.g. rats running on the treadmill, and to identify the time of the event. In this study, we hypothesized that automatic analysis of the time-averaged position of a rat on a treadmill could be an objective way for estimating times to fatigue and exhaustion. To test this hypothesis, we compared these times measured by a human observer to the results of an automated video tracking system.MethodsRats, previously familiarized to running on the treadmill, ran at a fixed speed with zero incline, until exhaustion. The experiments were performed at either room temperature (24 °C) or in a hot environment (32 °C). Each experiment was video recorded. A trained observer estimated the times to fatigue and exhaustion. Then, video tracking software was used to determine the position of the animals on the treadmill belt. The times to fatigue and exhaustion were determined, based on the position on the treadmill using predefined criteria.ResultsManual scores and the average position on the treadmill had significant correlation. Both the observer and the automated video tracking determined that exercise in a hot environment, compared with the exercise at room temperature, results in shorter times to exhaustion and fatigue. Also, estimates of times made by the observer and the automated video tracking were not statistically different from each other.DiscussionA similarity between the estimates of times to fatigue and exhaustion made by the observer and the automated technique suggests that video tracking of rodents running on a treadmill can be used to determine both parameters in experimental studies. Video tracking technique allows for a more objective measure and would allow for an increased performance in experimentation. The Supplemental information to this manuscript contains an Excel file, which includes the code in Virtual Basic with freeware license, to process and visualize running data and automatically estimate the times to fatigue and exhaustion. Instructions for the software are also included.

Newly developed method for mouse olfactory behavior tests using an automatic video tracking system

ObjectiveThe mouse is the most popular animal model in olfactory research. Behavior tests with odorants are essential for determining olfactory phenotype. To the best of our knowledge, the mouse olfactory behavior test has not been standardized, making the results vulnerable to inter-observer variation. We sought to develop a new mouse olfactory behavior test assessed by an automatic video tracking system with minimal inter-observer variation. MethodsA video-tracking system was used to automatically track mouse behavior in standard breeding cages with C57BL/6N mice. We tested two odorants (peanut butter for the preference test, 2MB acid for the avoidance test) and distilled water (for a control). Mouse behavior was recorded for 3min and analyzed. For the preference test, investigation time was measured. For the avoidance test, time spent in sectors away from the odorant zone was measured. To confirm our experimental settings, we also evaluated an anosmia mouse model prepared with intranasal administration of ZnSO4. ResultsAll strains of mice showed reproducible behavior patterns of preference or avoidance for the odorants. The anosmia mouse model, as expected, failed to show an olfactory ability for preference or avoidance, and this was well-matched by histologic changes caused by the ZnSO4 treatment. The automatic video tracking system successfully tracked and automatically calculated mouse behavior with good reproducibility. ConclusionOur olfactory behavior test offers a simple and accurate method to evaluate olfactory function in mice. This test can be utilized as a possible standard method to search for features of olfactory phenotypes in mice.

Suppressed swimming activity in Zebrafish (Danio rerio) exposed to 1,4,5-oxadithiepane, a sulphur mustard degradation product

This rapid communication presents novel data on the ecotoxicity of the cyclic sulphur mustard degradation compound, 1,4,5-oxadithiepane. Groups of adult male Zebrafish (Danio rerio) were exposed to 1,4,5-oxadithiepane at 40.3 ± 2.9 μg L-1, 133.3 ± 6.7 μg L-1 and 1533.3 ± 266.7 μg L-1, respectively. After 14 days of exposure, fitness and lethality were accessed and the swimming behaviour of the Zebrafish was measured by an automated video tracking system. The average swimming velocity was decreased significantly from 40.4 ± 2.5 mm s-1 in the control group to 31.8 ± 1.6 mm s-1 in the low dose. The low and high doses demonstrated suppressed average velocities, partly due to elevated immobile periods (rest time). Thus, at 40.3 ± 2.9 μg L-1 and 1533.3 ± 266.7 μg L-1 both parameters were significantly different from the control group. No adverse effects were found at the 1533.3 ± 266.7 μg L-1 treatment level and no lethality, associated with the toxic properties of 1,4,5-oxadithiepane, was observed during the 14 days of exposure suggesting a 14 days No-Observed-Effect-Concentration above 1533.3 μg L-1.

Differential Effects of Sex Pheromone Compounds on Adult Female Sea Lamprey (Petromyzon marinus) Locomotor Patterns.

Synchronization of male and female locomotor activity plays a critical role in ensuring reproductive success, especially in semelparous species. The goal of this study was to elucidate the effects of individual chemical signals, or pheromones, on the locomotor activity in the sea lamprey (Petromyzon marinus). In their native habitat, adult preovulated females (POF) and ovulated females (OF) are exposed to sex pheromone compounds that are released from spermiated males and attract females to nests during their migration and spawning periods. In this study, locomotor activity of individual POF and OF was measured hourly in controlled laboratory conditions using an automated video-tracking system. Differences in the activity between a baseline day (no treatment exposure) and a treatment day (sex pheromone compound or control exposure) were examined for daytime and nighttime periods. Results showed that different pheromone compound treatments affected both POF and OF sea lamprey (p < 0.05) but in different ways. Spermiated male washings (SMW) and one of its main components, 7α,12α,24-trihydroxy-5α-cholan-3-one 24 sulfate (3kPZS), decreased activity of POF during the nighttime. SMW also reduced activity in POF during the daytime. In contrast, SMW increased activity of OF during the daytime, and an additional compound found in SMW, petromyzonol sulfate (PZS), decreased the activity during the nighttime. In addition, we examined factors that allowed us to infer the overall locomotor patterns. SMW increased the maximum hourly activity during the daytime, decreased the maximum hourly activity during the nighttime, and reduced the percentage of nocturnal activity in OF. Our findings suggest that adult females have evolved to respond to different male compounds in regards to their locomotor activity before and after final maturation. This is a rare example of how species-wide chemosensory stimuli can affect not only the amounts of activity but also the overall locomotor pattern in a vertebrate species.

Behavioural toxicity assessment of silver ions and nanoparticles on zebrafish using a locomotion profiling approach

Zebrafish (Danio rerio) is not only a widely used species in the Fish Embryo Toxicity (FET) test but also an emerging model in behavioural ecotoxicology. By using automatic behaviour tracking technology, locomotion of developing zebrafish (ZF) larvae can be accurately recorded and potentially used in an ecotoxicological context to detect toxicant-induced behavioural alterations. In this study, we explored if and how quantitative locomotion data can be used for sub-lethal toxicity testing within the FET framework. We exposed ZF embryos to silver ions and nanoparticles, which previously have been reported to cause neurodevelopmental toxicity and behavioural retardation in early-life stages of ZF. Exposure to a broad range of silver (Ag+ and AgNPs) concentrations was conducted, and developmental toxicity was assessed using FET criteria. For behavioural toxicity assessment, locomotion of exposed ZF eleutheroembryos (120hpf) was quantified according to a customised behavioural assay in an automatic video tracking system. A set of repeated episodes of dark/light stimulation were used to artificially stress ZF and evoke photo-motor responses, which were consequently utilized for locomotion profiling. Our locomotion-based behaviour profiling approach consisted of (1) dose-response ranking for multiple and single locomotion variables; (2) quantitative assessment of locomotion structure; and (3) analysis of ZF responsiveness to darkness stimulation. We documented that both silver forms caused adverse effects on development and inhibited hatchability and, most importantly, altered locomotion. High Ag+ and AgNPs exposures significantly suppressed locomotion and a clear shift in locomotion towards inactivity was reported. Additionally, we noted that low, environmentally relevant Ag+ concentrations may cause subordinate locomotive changes (hyperactivity) in developing fish. Overall, it was concluded that our locomotion-based behaviour-testing scheme can be used jointly with FET and can provide endpoints for sub-lethal toxicity. When combined with multivariate data analysis, this approach facilitated new insights for handling and analysis of data generated by automatized behavioural tracking systems.

BichiCAM, an Underwater Automated Video Tracking System for the Study of Migratory Dynamics of Benthic Diadromous Species in Streams

Conventional methods for surveying diadromous fish migration from marine coastal waters to freshwater habitats are mainly based on electrofishing, a non-optimal technique for the study of fish migrations in rivers, and fishermen catch data. Underwater video has been recognized for a long time as a good alternative, but those approaches usually require intensive labour for retrieving the information from the video sequences. To overcome these problems, an underwater video system specifically designed for field work (low-weight, low-cost and autonomous) named BichiCAM has been developed for automatically counting, measuring and tracking fish observed in video sequences. The efficiency and precision of the BichiCAM system were tested by filming Sicyopterus lagocephalus juveniles passing through the camera field of vision in the Saint-Etienne River, Reunion Island, Western Indian Ocean. The BichiCAM system accurately measured fish length of the observed individuals when lens distortion of the camera was corrected, and the error percentages on the measurements presented a standard deviation of 5.1% of the total length. The BichiCAM system provides a powerful tool that will not only facilitate research on migrating fishes and invertebrates' communities but also allow studies of the effectiveness of fishways associated to dams or the impacts of fishery activities. Copyright © 2015 John Wiley & Sons, Ltd.

Carbendazim exposure induces developmental, biochemical and behavioural disturbance in zebrafish embryos

Carbendazim is a widely used broad spectrum benzimidazole fungicide; however, its effects to non-target aquatic organisms are poorly studied. The aim of this study was to investigate the toxic effects of carbendazim to zebrafish early life stages at several levels of biological organization, including developmental, biochemical and behavioural levels. The embryo assay was done following the OECD guideline 236 and using a concentration range between 1.1 and 1.8mg/L. Lethal and developmental endpoints such as hatching, edemas, malformations, heart beat rate, body growth and delays were assessed in a 96h exposure. A sub-teratogenic range (from 0.16 to 500μg/L) was then used to assess effects at biochemical and behavioural levels. Biochemical markers included cholinesterase (ChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH) and catalase (CAT) and were assessed at 96h. The locomotor behaviour was assessed using an automated video tracking system at 120h.Carbendazim (96h-LC50 of 1.75mg/L) elicited several developmental anomalies in zebrafish embryos with EC50 values ranging from 0.85 to 1.6mg/L. ChE, GST and LDH activities were increased at concentrations equal or above 4μg/L. The locomotor assay showed to be extremely sensitive, detecting effects in time that larvae spent swimming at concentrations of 0.16μg/L and thus, being several orders of magnitude more sensitive that developmental parameters or lethality. These are ecological relevant concentrations and highlight the potential of behavioural endpoints as early warning signs for environmental stress. Further studies should focus on understanding how the behavioural disturbances measured in these types of studies translate into fitness impairment at the adult stage.

Behavioural responses of freshwater planarians after short-term exposure to the insecticide chlorantraniliprole

Recent advances in video tracking technologies provide the tools for a sensitive and reproducible analysis of invertebrate activity under stressful conditions nurturing the field of behavioural ecotoxicology. This study aimed to evaluate behavioural responses of the freshwater planarian Dugesia subtentaculata exposed to a model compound, chlorantraniliprole (CAP). This compound is an anthranilic diamide insecticide and due to its neurotoxic action can, at low concentrations, impair behaviour of exposed organisms. Behavioural endpoints measured included feeding and locomotor activities. Feeding responses were based on planarian predatory behaviour using Chironomus riparius larvae as prey. Locomotion was measured by the traditional planarian locomotor velocity (pLMV) assay and additionally using an automated video tracking system using a Zebrabox® (Viewpoint, France) device. While feeding and pLMV were significantly impaired at 131.7μg/L CAP, the video tracking system showed that total distance covered by planarians was significantly reduced at concentrations as low as 26.2μg/L CAP. Our results show that more advanced automated video recording systems can be used in the development of sensitive bioassays allowing a reliable, time- and cost-effective quantification of behaviour in aquatic invertebrates. Due to their ecological relevance, behavioural responses should not be disregarded in risk assessment strategies and we advocate the suitability of planarians as suitable organisms for behavioural ecotoxicological studies.

Disrupted reproductive behavior in unexposed female zebrafish (Danio rerio) paired with males exposed to low concentrations of 17α-ethinylestradiol (EE2)

Estrogenic substances, including the contraceptive pharmaceutical 17α-ethinylestradiol (EE2), pose a threat to aquatic wildlife causing endocrine disturbances of physiological processes and reproductive behavior. We have previously demonstrated that the reproductive behavior of male zebrafish, Danio rerio, remain intact after lifelong exposure to low concentrations of EE2 (0.05 and 0.5ngL−1), concentrations high enough to cause morphological alterations of the male phenotype. Despite normal courtship behavior, the reproductive output is suppressed when these males were paired with unexposed females. In this study, we include the female courtship behavior in the analyzes of EE2’s effects on behavior. Groups of male zebrafish were exposed to nominally 0, 1, 3 and 10ngL−1 of EE2 from hatching to adulthood and subsequently individually paired with an unexposed female. Eleven distinct elements of the reproductive behavior were then extracted and analyzed using an automated video tracking system. Subsequently, male brains were isolated and the expression of genes encoding estrogenic receptors ERα, ERβ1 and ERβ2, the androgen receptor AR, and the aromatase cyp19a1b were compared with the expression in brains of unexposed males. (In this article gene expression is taken synonymous to transcription, although it is acknowledged that it can also be regulated by, e.g., mRNA and protein stability, and translation). The results confirmed that the male reproductive behavior was unaffected by the EE2 treatments. Also, the expressions of genes encoding estrogen and androgen receptors were unaffected. Only the gene encoding aromatase was 0.6 fold down-regulated. In contrast, and most surprisingly, nearly all the elements in the female courtship behavior were significantly disturbed, despite the fact that these females had never been exposed to EE2; most likely elicited by differences in male morphology, pheromones or some other unrevealed mechanism.