Submissive behaviour is affected by group size in a social fish.

For social groups to form and be stable over time, animals must develop strategies to cope with conflict among group members. Animals may behave submissively either by fleeing from an aggressor, or by signalling submission. The use of these two submissive responses may vary depending on the social and ecological context. Group size is a key aspect of social context for group living animals, as individuals in smaller groups may respond to aggression differently than those from larger groups. Here, we examine the relationship between group size and submissive behaviour in a cooperatively breeding fish, the daffodil cichlid (Neolamprologus pulcher). We found that subordinate fish showed similar levels of submission signals in response to dominant aggression in larger and smaller groups, however, subordinates from larger groups were less likely to flee from dominant aggression than those in smaller groups. Subordinates in larger groups also showed more digging behaviour which may be also used to avoid conflict with the dominant group members. Our data show that social context affects submissive behaviour in a cooperatively breeding fish.

Heme Scavenging and Delivery: The Role of Human Serum Albumin.

Heme is the reactive center of several metal-based proteins that are involved in multiple biological processes. However, free heme, defined as the labile heme pool, has toxic properties that are derived from its hydrophobic nature and the Fe-atom. Therefore, the heme concentration must be tightly controlled to maintain cellular homeostasis and to avoid pathological conditions. Therefore, different systems have been developed to scavenge either Hb (i.e., haptoglobin (Hp)) or the free heme (i.e., high-density lipoproteins (HDL), low-density lipoproteins (LDL), hemopexin (Hx), and human serum albumin (HSA)). In the first seconds after heme appearance in the plasma, more than 80% of the heme binds to HDL and LDL, and only the remaining 20% binds to Hx and HSA. Then, HSA slowly removes most of the heme from HDL and LDL, and finally, heme transits to Hx, which releases it into hepatic parenchymal cells. The Hx:heme or HSA:heme complexes are internalized via endocytosis mediated by the CD91 and CD71 receptors, respectively. As heme constitutes a major iron source for pathogens, bacteria have evolved hemophores that can extract and uptake heme from host proteins, including HSA:heme. Here, the molecular mechanisms underlying heme scavenging and delivery from HSA are reviewed. Moreover, the relevance of HSA in disease states associated with increased heme plasma concentrations are discussed.

Head up displays are a submission signal in the group-living daffodil cichlid.

Dominance hierarchies can reduce conflict within social groups and agonistic signals can help to establish and maintain these hierarchies. Behaviours produced by subordinates in response to aggression are often assumed to function as signals of submission, however, these behaviours may serve other purposes, for example, defence or escape. For a behaviour to act as a submission signal, the receiver must respond by reducing their likelihood of further aggression towards the signaller. In the current study, we examine the receiver response to a putative signal of submission, the head up display, within established social groups of the cooperatively breeding fish, the daffodil cichlid (Neolamprologus pulcher). We found that when subordinate signallers produce the head up display in response to aggression from the breeder male, he exhibited a longer latency to behave aggressively towards that individual again. We also report that head up displays are rarely produced without being elicited by aggression, and the number of head up displays correlates with the amount of aggression received. Our results demonstrate that the head up display is used as a signal of submission in the daffodil cichlid and provide insight into intragroup communication in an emerging model system for the study of social behaviour.

Social environment modulates anxiogenic effects of caffeine in zebrafish.

Zebrafish has recently become a species of choice in a number of preclinical studies to examine whether and how psychoactive compounds modulate individual and social behaviors. Here, we sought to contribute an improved understanding of the effects of caffeine, a well-known anxiogenic compound, on the swimming activity and the collective response of zebrafish. To investigate how the social environment influences individual response to acute caffeine treatment, we measured the behavior of a caffeine-treated subject swimming in isolation or in the presence of a group of untreated conspecifics. The experimental paradigm used a recently developed automatic tracking system to extract individual trajectories of zebrafish and preserve their identities over time. Our results indicate that caffeine reduces the swimming activity of fish tested in isolation, and that this effect is mitigated by the presence of untreated conspecifics. The collective response is also influenced by caffeine exposure at low doses, whereby caffeine-treated subjects may act as group leaders by taking anticipatory turning manoeuvres that are followed by the rest of the group. While anxiogenic effects of caffeine could explain the reduction in the swimming activity, the observed variation in the collective response is likely related to the interplay between anxiogenic and psychostimulant properties of caffeine.

The Tagging Procedure of Visible Implant Elastomers Influences Zebrafish Individual and Social Behavior.

One commonly used method to preserve individual identity in the study of social behavior of zebrafish is through silicone-based visible implant elastomers (VIEs), which represent a safe and durable tagging procedure. While the effects of VIE tagging on welfare and general health have been addressed in detail, whether this procedure influences social behavior remains unclear. In this study, we compared individual and group behaviors exhibited by shoals composed of three individuals: two nontagged and one (focal subject) that was either nontagged (control condition) or sham-, purple-, blue-, or yellow tagged. Traditional behavioral parameters of activity, shoaling, and schooling (speed, polarization, and interindividual distances), along with an information-theoretic measure of social interaction (transfer entropy), were used to study the effect of tagging. Our findings indicate that tagging procedure per se significantly increased individual speed of the tagged subjects and of the group. The tagging procedure also altered the level of interaction between individuals, measured by transfer entropy. Conversely, tagging procedure did not influence shoaling and schooling tendencies. These findings suggest that VIE tagging may elicit some level of stress, which may affect some behavioral responses more than others. We recommend use of alternative methods such as multitracking systems when possible.

Closed-loop control of zebrafish behaviour in three dimensions using a robotic stimulus.

Robotics is continuously being integrated in animal behaviour studies to create customizable, controllable, and repeatable stimuli. However, few systems have capitalized on recent breakthroughs in computer vision and real-time control to enable a two-way interaction between the animal and the robot. Here, we present a "closed-loop control" system to investigate the behaviour of zebrafish, a popular animal model in preclinical studies. The system allows for actuating a biologically-inspired 3D-printed replica in a 3D workspace, in response to the behaviour of a zebrafish. We demonstrate the role of closed-loop control in modulating the response of zebrafish, across a range of behavioural and information-theoretic measures. Our results suggest that closed-loop control could enhance the degree of biomimicry of the replica, by increasing the attraction of live subjects and their interaction with the stimulus. Interactive experiments hold promise to advance our understanding of zebrafish, offering new means for high throughput behavioural phenotyping.

Information theory and robotics meet to study predator-prey interactions.

Transfer entropy holds promise to advance our understanding of animal behavior, by affording the identification of causal relationships that underlie animal interactions. A critical step toward the reliable implementation of this powerful information-theoretic concept entails the design of experiments in which causal relationships could be systematically controlled. Here, we put forward a robotics-based experimental approach to test the validity of transfer entropy in the study of predator-prey interactions. We investigate the behavioral response of zebrafish to a fear-evoking robotic stimulus, designed after the morpho-physiology of the red tiger oscar and actuated along preprogrammed trajectories. From the time series of the positions of the zebrafish and the robotic stimulus, we demonstrate that transfer entropy correctly identifies the influence of the stimulus on the focal subject. Building on this evidence, we apply transfer entropy to study the interactions between zebrafish and a live red tiger oscar. The analysis of transfer entropy reveals a change in the direction of the information flow, suggesting a mutual influence between the predator and the prey, where the predator adapts its strategy as a function of the movement of the prey, which, in turn, adjusts its escape as a function of the predator motion. Through the integration of information theory and robotics, this study posits a new approach to study predator-prey interactions in freshwater fish.

Three-dimensional scoring of zebrafish behavior unveils biological phenomena hidden by two-dimensional analyses.

The study of zebrafish behavior represents a cornerstone upon which basic researchers promise to advance knowledge in life sciences. Although zebrafish swim in a three-dimensional (3D) space, their behavior in the lab is almost exclusively scored in two dimensions, whereby zebrafish are recorded using a single camera providing 2D videos. Whether this dimensional reduction preserves the reliability of data has not been addressed. Here we show that, compared to a 3D observation, 2D data are flawed by over-reporting and under-reporting of locomotory differences. Specifically, we first reconstructed 3D trajectories through the integration of synchronous information derived from two cameras, and then compared them with the original 2D views in classical experimental paradigms assessing shoaling tendency, fear, anxiety, and general locomotion. Our results suggest that traditional behavioral scoring of individual zebrafish performed in 2D may undermine data integrity, thereby requiring a general reconsideration of scoring zebrafish behavior to incorporate a 3D approach. We then demonstrate that, compared to 2D, a 3D approach requires a reduced number of subjects to achieve the same degree of validity. We anticipate these findings to largely benefit animal welfare by reducing the number of experimental subjects, without affecting statistical power.

How different is a 3D-printed replica from a conspecific in the eyes of a zebrafish?

Robotics is emerging as a promising tool for aiding research on animal behavior. The possibility of generating customizable, controllable, and standardized robotic stimuli has been demonstrated through a number of behavioral assays, involving vertebrates and invertebrates. However, the specific appraisal of the nature of robotic stimuli is currently lacking. Here, we attempt to evaluate this aspect in zebrafish, through a within-subject design in which experimental subjects are faced with three experimental conditions. In the first test, we investigated sociability by measuring zebrafish response to a conspecific separated by a one-way glass. In the second test, we studied zebrafish behavior in response to a 3D-printed zebrafish replica actuated along realistic trajectories through a novel four-degree-of-freedom robotic platform. Last, we investigated fear responses in a shelter-seeking test. In agreement with our expectations, zebrafish exhibited an equivalent preference for live and robotic stimuli, and the degree of preference for the robotic replica correlated negatively with the individual propensity to seek shelter. The equivalent preference for the replica and conspecific suggests that the appraisal of the target stimuli is analogous. The preliminary evidence of a correlation between behavioral responses across tests points to the readability of robotics-based approaches to investigate interindividual differences.

Zebrafish response to a robotic replica in three dimensions.

As zebrafish emerge as a species of choice for the investigation of biological processes, a number of experimental protocols are being developed to study their social behaviour. While live stimuli may elicit varying response in focal subjects owing to idiosyncrasies, tiredness and circadian rhythms, video stimuli suffer from the absence of physical input and rely only on two-dimensional projections. Robotics has been recently proposed as an alternative approach to generate physical, customizable, effective and consistent stimuli for behavioural phenotyping. Here, we contribute to this field of investigation through a novel four-degree-of-freedom robotics-based platform to manoeuvre a biologically inspired three-dimensionally printed replica. The platform enables three-dimensional motions as well as body oscillations to mimic zebrafish locomotion. In a series of experiments, we demonstrate the differential role of the visual stimuli associated with the biologically inspired replica and its three-dimensional motion. Three-dimensional tracking and information-theoretic tools are complemented to quantify the interaction between zebrafish and the robotic stimulus. Live subjects displayed a robust attraction towards the moving replica, and such attraction was lost when controlling for its visual appearance or motion. This effort is expected to aid zebrafish behavioural phenotyping, by offering a novel approach to generate physical stimuli moving in three dimensions.