Zebrafish Adjust Their Behavior in Response to an Interactive Robotic Predator

Zebrafish (Danio rerio) constitutes a valuable experimental species for the study of the biological determinants of emotional responses, such as fear and anxiety. Fear-related test paradigms traditionally entail the interaction between focal subjects and live predators, which may show inconsistent behavior throughout the experiment. To address this technical challenge, robotic stimuli are now frequently integrated in behavioral studies, yielding repeatable, customizable, and controllable experimental conditions. While most of the research has focused on open-loop control where robotic stimuli are preprogrammed to execute a priori known actions, recent work has explored the possibility of two-way interactions between robotic stimuli and live subjects. Here, we demonstrate a “closed-loop control” system to investigate fear response of zebrafish in which the response of the robotic stimulus is determined in real-time through a finite-state Markov chain constructed from independent observations on the interactions between zebrafish and their predator. Specifically, we designed a 3D-printed robotic replica of the zebrafish allopatric predator red tiger Oscar fish (Astronotus ocellatus), instrumented to interact in real-time with live subjects. We investigated the role of closed-loop control in modulating fear response in zebrafish through the analysis of the focal fish ethogram and the information-theoretic quantification of the interaction between the subject and the replica. Our results indicate that closed-loop control elicits consistent fear response in zebrafish and that zebrafish quickly adjust their behavior to avoid the predator's attacks. The augmented degree of interactivity afforded by the Markov-chain-dependent actuation of the replica constitutes a fundamental advancement in the study of animal-robot interactions and offers a new means for the development of experimental paradigms to study fear

Zebrafish Adjust Their Behavior in Response to an Interactive Robotic Predator

Zebrafish (Danio rerio) constitutes a valuable experimental species for the study of the biological determinants of emotional responses, such as fear and anxiety. Fear-related test paradigms traditionally entail the interaction between focal subjects and live predators, which may show inconsistent behavior throughout the experiment. To address this technical challenge, robotic stimuli are now frequently integrated in behavioral studies, yielding repeatable, customizable, and controllable experimental conditions. While most of the research has focused on open-loop control where robotic stimuli are preprogrammed to execute a priori known actions, recent work has explored the possibility of two-way interactions between robotic stimuli and live subjects. Here, we demonstrate a "closed-loop control" system to investigate fear response of zebrafish in which the response of the robotic stimulus is determined in real-time through a finite-state Markov chain constructed from independent observations on the interactions between zebrafish and their predator. Specifically, we designed a 3D-printed robotic replica of the zebrafish allopatric predator red tiger Oscar fish (Astronotus ocellatus), instrumented to interact in real-time with live subjects. We investigated the role of closed-loop control in modulating fear response in zebrafish through the analysis of the focal fish ethogram and the information-theoretic quantification of the interaction between the subject and the replica. Our results indicate that closed-loop control elicits consistent fear response in zebrafish and that zebrafish quickly adjust their behavior to avoid the predator's attacks. The augmented degree of interactivity afforded by the Markov-chain-dependent actuation of the replica constitutes a fundamental advancement in the study of animal-robot interactions and offers a new means for the development of experimental paradigms to study fear

Collective responses of a large mackerel school depend on the size and speed of a robotic fish but not on tail motion

So far, actuated fish models have been used to study animal interactions in small-scale controlled experiments. This study, conducted in a semi-controlled setting, investigates robot5interactions with a large wild-caught marine fish school (∼3000 individuals) in their natural social environment. Two towed fish robots were used to decouple size, tail motion and speed in a series of sea-cage experiments. Using high-resolution imaging sonar and sonar-video blind scoring, we monitored and classified the school's collective reaction towards the fish robots as attraction or avoidance. We found that two key releasers—the size and the speed of the robotic fish—were responsible for triggering either evasive reactions or following responses. At the same time, we found fish reactions to the tail motion to be insignificant. The fish evaded a fast-moving robot even if it was small. However, mackerels following propensity was greater towards a slow small robot. When moving slowly, the larger robot triggered significantly more avoidance responses than a small robot. Our results suggest that the collective responses of a large school exposed to a robotic fish could be manipulated by tuning two principal releasers—size and speed. These results can help to design experimental methods for in situ observations of wild fish schools or to develop underwater robots for guiding and interacting with free-ranging aggregated aquatic organisms.This work was financed by the Norwegian Research Council (grant 204229/F20) and Estonian Government Target Financing (grant SF0140018s12). JCC was partially supported by a grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism, operated by Universidad Complutense de Madrid. We are grateful to A. Totland for his technical help. The animal collection was approved by The Royal Norwegian Ministry of Fisheries, and the experiment was approved by the Norwegian Animal Research Authority. The Institute of Marine Research is permitted to conduct experiments at the Austevoll aquaculture facility by the Norwegian Biological Resource Committee and the Norwegian Animal Research Committee (Forsøksdyrutvalget)

Entropy and Fractal Techniques for Monitoring Fish Behaviour and Welfare in Aquacultural Precision Fish Farming—A Review

In a non-linear system, such as a biological system, the change of the output (e.g., behaviour) is not proportional to the change of the input (e.g., exposure to stressors). In addition, biological systems also change over time, i.e., they are dynamic. Non-linear dynamical analyses of biological systems have revealed hidden structures and patterns of behaviour that are not discernible by classical methods. Entropy analyses can quantify their degree of predictability and the directionality of individual interactions, while fractal dimension (FD) analyses can expose patterns of behaviour within apparently random ones. The incorporation of these techniques into the architecture of precision fish farming (PFF) and intelligent aquaculture (IA) is becoming increasingly necessary to understand and predict the evolution of the status of farmed fish. This review summarizes recent works on the application of entropy and FD techniques to selected individual and collective fish behaviours influenced by the number of fish, tagging, pain, preying/feed search, fear/anxiety (and its modulation) and positive emotional contagion (the social contagion of positive emotions). Furthermore, it presents an investigation of collective and individual interactions in shoals, an exposure of the dynamics of inter-individual relationships and hierarchies, and the identification of individuals in groups. While most of the works have been carried out using model species, we believe that they have clear applications in PFF. The review ends by describing some of the major challenges in the field, two of which are, unsurprisingly, the acquisition of high-quality, reliable raw data and the construction of large, reliable databases of non-linear behavioural data for different species and farming conditions.The work was supported by the Spanish MINECO (Grant RTC-2014–2837-2- “SELATUN: Minimización de la problemática del mercurio del atún y valorización del atún como alimento saludable, Programa Retos-Colaboración 2014”. The funding source had no involvement in the preparation of this manuscript

Responsive robotic prey reveal how predators adapt to predictability in escape tactics

To increase their chances of survival, prey often behave unpredictably when escaping from predators. However, the response of predators to, and hence the effectiveness of, such tactics is unknown. We programmed interactive prey to flee from an approaching fish predator (the blue acara, Andinoacara pulcher) using real-time computer vision and two-wheeled robots that controlled the prey’s movements via magnets. This allowed us to manipulate the prey’s initial escape direction and how predictable it was between successive trials with the same individual predator. When repeatedly exposed to predictable prey, the predators adjusted their behavior before the prey even began to escape: prey programmed to escape directly away were approached more rapidly than prey escaping at an acute angle. These faster approach speeds compensated for a longer time needed to capture such prey during the subsequent pursuit phase. By contrast, when attacking unpredictable prey, the predators adopted intermediate approach speeds and were not sensitive to the prey’s escape angle but instead showed greater acceleration during the pursuit. Collectively, these behavioral responses resulted in the prey’s predictability having no net effect on the time taken to capture prey, suggesting that unpredictable escape behavior may be advantageous to prey in fewer circumstances than originally thought. Rather than minimizing capture times, the predators in our study appear to instead adjust their behavior to maintain an adequate level of performance during prey capture

Inferring causal relationships in zebrafish-robot interactions through transfer entropy: a small lure to catch a big fish

In the field of animal behavior, effective methods to apprehend causal relationships that underlie the interactions between animals are in dire need. How to identify a leader in a group of social animals or quantify the mutual response of predator and prey are exemplary questions that would benefit from an improved understanding of causality. Information theory offers a potent framework to objectively infer cause-and-effect relationships from raw experimental data, in the form of behavioral observations or individual trajectory tracks. In this targeted review, we summarize recent advances in the application of the information-theoretic concept of transfer entropy to animal interactions. First, we offer an introduction to the theory of transfer entropy, keeping a balance between fundamentals and practical implementation. Then, we focus on animal-robot experiments as a means for the validation of the use of transfer entropy to measure causal relationships. We explore a test battery of robotics-based protocols designed for studying zebrafish social behavior and fear response. Grounded in experimental evidence, we demonstrate the potential of transfer entropy to assist in the detection and quantification of causal relationships in animal interactions. The proposed robotics-based platforms offer versatile, controllable, and customizable stimuli to generate a priori known cause-and-effect relationships, which would not be feasible with live stimuli. We conclude the paper with an outlook on possible applications of transfer entropy to study group behavior and clarify the determinants of leadership in social animals

Animal-Robot Interactions: Electrocommunication, Sensory Ecology, and Group Dynamics in a Mormyrid Weakly Electric Fish

Mormyrid weakly electric fish possess a specialized electrosensory system. During the process of active electrolocation, these animals perceive self-generated electric organ dis-charges (EOD) and are thereby able to detect objects in their nearby environment. The EOD is a short, biphasic pulse, which is simultaneously used to communicate with conspe-cifics. There are two principles according to which information exchange occurs during electrocommunication. The waveform of the EOD constitutes a relatively stable identity marker that signals species, gender, and status of an individual. In contrast, the temporal sequence of inter-discharge intervals (IDI) is highly variable and encodes context-specific information. Modifications of IDI-duration not only alter the instantaneous discharge fre-quency but also enable the generation of specific signaling patterns and interactive dis-charge sequences. One such interactive discharge behavior is the so-called echo response, during which a fish responds with a constant latency of only a few milliseconds to the EOD of a conspecific. Animals can synchronize their signaling sequences by mutually generating echoes to each other's signals over a coherent period. Although active electrolocation and electrocommunication are mediated by different types of electroreceptor organs and neural pathways, an unambiguous assignment of electromotor behavior to only one of the two functions is often problematic. In this thesis, the significance of IDI-based signaling sequences during motor and electro-motor interactions of the mormyrid fish Mormyrus rume proboscirostris were investigated. To this end, different electrical playback sequences of species-specific EODs were generated via mobile fish dummies, and the motor and electromotor responses of live fish were analyzed. In Part One of this thesis, electrocommunication strategies of the fish were analyzed, and particularly the functions of double pulses, discharge regularizations, and echo responses were examined in an adaptive context. Double pulses were classified as an aggressive mo-tivation signal, whereas regularizations may have a communicative function during the early stages of the sequential assessment of a potential opponent. In this context, discharge synchronization by means of echo responses may enable a mutual assessment for the net benefit of both contestants. Because echo responses occur in various behavioral contexts, and artificial echoes of the dummy evoked increased echoing by the fish, it was hypothesized that the echo response serves a more general purpose by enabling mutual allocation of social attention between two fish. In Part Two of this thesis, a biomimetic robotic fish was designed to investigate the senso-ry basis on which fish followed the dummy. It was shown that electrical playback signals induced following-behavior in live fish, whereas biomimetic motility patterns had no ef-fect. By subsequently reducing the mobile dummy to only the electric signaling sequence from the perspective of the fish, it could be shown that passive perception of electrical communication signals is also involved in mediating the spatial coordination of social in-teractions. This passive perception is likely mediated by the same electroreceptor organs that are used during electrocommunication. The EOD can therefore be considered to be an essential social stimulus that makes it possible to integrate a dummy into a group of weak-ly electric fish as an artificial conspecific. The influence of an interactively signaling mobile dummy fish on small groups of up to four individuals was investigated in Part Three of this thesis. Typical schooling behavior was a rare occurrence in this context. However, EOD-synchronizations through mutual echo responses between two fish, or between a fish and the interactive dummy, were fre-quently observed during social interactions in small groups. Motor interactions during synchronization episodes supported the hypothesis that mormyrids may use discharge synchronizations between individuals to allocate social attention, and the echo response may thus adopt a particularly useful function during communication in groups.Schwach elektrische Fisch aus der Familie der Mormyriden verfügen über ein spezialisier-tes elektrosensorisches Sinnessystem. In einem Prozess, der als aktive Elektroortung be-zeichnet wird, sind diese Tiere in der Lage, selbstgenerierte elektrische Organentladungen (EOD) wahrzunehmen, und dadurch Objekte in ihrer unmittelbaren Nähe zu detektieren. Das EOD ist ein kurzer bipolarer Puls, der gleichzeitig auch zur Kommunikation mit Artge-nossen dient. Informationsaustausch während der Elektrokommunikation basiert auf zwei verschiedenen Prinzipien: Die Wellenform des EOD stellt einen relativ konstanten Identi-tätsmarker dar, der beispielsweise Art, Geschlecht und Status eines Individuums signali-siert. Die zeitliche Abfolge der Intervalle zwischen den EODs ist hingegen höchst variabel und kodiert kontextspezifische Information. Durch Modifikation der Intervalldauer ändert sich nicht nur die Entladungsfrequenz, sondern es können auch spezifische Signalmuster und interaktive Entladungssequenzen generiert werden. Ein interaktives Entladungsver-halten stellt beispielsweise die Echoantwort dar, bei der ein Fisch mit einer konstanten Latenz von wenigen Millisekunden auf das EOD eines Artgenossen reagiert. Zwei Tiere können ihre Entladungssequenzen synchronisieren, indem sie ihre Signale über einen kohärenten Zeitraum gegenseitig mit Echos beantworten. Obwohl aktive Elektroortung und Elektrokommunikation über unterschiedliche Rezeptororgansysteme und neuronale Pfade vermittelt werden, ist eine eindeutige Zuordnung der elektromotorischen Verhal-tensäußerungen der Fische zu nur einer der beiden Funktionen oft problematisch. In der vorliegenden Arbeit wurde die Bedeutung intervallbasierter EOD-Sequenzen für motorische und elektromotorische Interaktionen des Mormyriden Mormyrus rume proboscirostris erforscht. Hierzu wurden verschiedene elektrische Playbacksequenzen artspezifischer EODs generiert und durch mobile Fischattrappen wiedergegeben. Die mo-torischen und elektromotorischen Verhaltensreaktionen der Fische wurden analysiert. Im ersten Teil der Arbeit wurden Elektrokommunikationsstrategien der Fische analysiert und die adaptive Funktion insbesondere von Doppelpulsen, Entladungsregularisierungen und Echoantworten untersucht. Doppelpulse wurden als aggressives Motivationssignal kategorisiert, wohingegen die Kommunikationsfunktion von Regularisierungen im gegen-seitigen Einschätzen zu Beginn einer kompetitiven Begegnung zu liegen scheint. Entla-dungssynchronisation durch gegenseitige Echoantworten kann dabei eine Einschätzung des Gegenübers zum Vorteil beider Parteien erleichtern. Da Echoantworten in verschiede-nen Verhaltenssituationen auftreten und artifizielle Echoantworten der Attrappe vermehrt zu Echos vonseiten der Fische führten, wurde postuliert, dass die Echoantwort eine generellere Funktion bei der Fokussierung gegenseitiger sozialer Aufmerksamkeit über-nehmen kann. Im zweiten Teil der Arbeit wurde ein biomimetischer Fischroboter konstruiert, um zu untersuchen, auf welcher sensorischen Grundlage die Fische der Attrappe folgen. Es konnte gezeigt werden, dass elektrische Playbacksignale, nicht aber biomimetische Bewe-gungsmuster, Folgeverhalten der Fische induzieren. In einem weiteren Schritt konnte durch die Reduktion der Attrappe auf die elektrischen Signalsequenzen aus der Perspektive der Versuchsfische gezeigt werden, dass passive Wahrnehmung elektrischer Kommu-nikationssignale auch bei der räumlichen Koordination sozialer Interaktionen von Bedeu-tung ist. Dies wird mutmaßlich über die gleichen Rezeptororgane vermittelt, die auch für die Elektrokommunikation verantwortlich sind. Das EOD kann daher als ein soziales Signal betrachtet werden, das es ermöglicht, eine Attrappe als künstlichen Artgenossen in eine Gruppe schwach elektrischer Fische zu integrieren. Der Einfluss einer elektrisch interaktiven mobilen Fischattrappe auf kleine Gruppen von bis zu vier Individuen wurde im dritten Teil der Arbeit getestet. Typisches Schwarmver-halten konnte in diesem Zusammenhang nur selten beobachtet werden. In kleinen Gruppen kam es während sozialer Interaktionen jedoch häufig zu EOD-Synchronisationen durch Echoantworten zwischen zwei Fischen, oder zwischen einem Fisch und der interaktiven Attrappe. Motorische Verhaltensinteraktionen im Zeitraum dieser Synchronisationen stützen die Hypothese, dass Mormyriden durch elektrische Entladungssynchronisation soziale Aufmerksamkeit zwischen Individuen herstellen können, und die Echoantwort somit besonders in Gruppen eine nützliche Kommunikationsfunktion übernehmen kann

Social Preference Tests in Zebrafish: A Systematic Review

The use of animal models in biology research continues to be necessary for the development of new technologies and medicines, and therefore crucial for enhancing human and animal health. In this context, the need to ensure the compliance of research with the principles Replacement, Reduction and Refinement (the 3 Rs), which underpin the ethical and human approach to husbandry and experimental design, has become a central issue. The zebrafish (Danio rerio) is becoming a widely used model in the field of behavioral neuroscience. In particular, studying zebrafish social preference, by observing how an individual fish interacts with conspecifics, may offer insights into several neuropsychiatric and neurodevelopmental disorders. The main aim of this review is to summarize principal factors affecting zebrafish behavior during social preference tests. We identified three categories of social research using zebrafish: studies carried out in untreated wild-type zebrafish, in pharmacologically treated wild-type zebrafish, and in genetically engineered fish. We suggest guidelines for standardizing social preference testing in the zebrafish model. The main advances gleaned from zebrafish social behavior testing are discussed, together with the relevance of this method to scientific research, including the study of behavioral disorders in humans. The authors stress the importance of adopting an ethical approach that considers the welfare of animals involved in experimental procedures. Ensuring a high standard of animal welfare is not only good for the animals, but also enhances the quality of our science

After 150 years of watching: is there a need for synthetic ethology?

The Darwinian idea of mental continuity is about 150 years old. Although nobody has strongly denied this evolutionary link, both conceptually and practically, relative slow advance has been made by ethology and comparative psychology to quantify mental evolution. Debates on the mechanistic interpretation of cognition often struggle with the same old issues (e.g., associationism vs cognitivism), and in general, experimental methods have made also relative slow progress since the introduction of the puzzle box. In this paper, we illustrate the prevailing issues using examples on ‘mental state attribution’ and ‘perspective taking” and argue that the situation could be improved by the introduction of novel methodological inventions and insights. We suggest that focusing on problem-solving skills and constructing artificial agents that aim to correspond and interact with biological ones, may help to understand the functioning of the mind. We urge the establishment of a novel approach, synthetic ethology, in which researchers take on a practical stance and construct artificial embodied minds relying of specific computational architectures the performance of which can be compared directly to biological agents

How aggressive interactions with biomimetic agents optimize reproductive performances in mass-reared males of the Mediterranean fruit fly

Mass-rearing procedures of insect species, often used in biological control and Sterile Insect Technique, can reduce the insects competitiveness in foraging, dispersal, and mating. The evocation of certain behaviours responsible to induce specific neuroendocrine products may restore or improve the competitiveness of mass-reared individuals. Herein, we used a mass-reared strain of Ceratitis capitata as model organism. C. capitata is a polyphagous pest exhibiting territorial displays that are closely related to its reproductive performance. We tested if the behaviour of C. capitata males could be altered by hybrid aggressive interactions with a conspecific-mimicking robotic fly, leading to more competitive individuals in subsequent mating events. Aggressive interactions with the robotic fly had a notable effect on subsequent courtship and mating sequences of males that performed longer courtship displays compared to naïve individuals. Furthermore, previous interactions with the robotic fly produced a higher mating success of males. Reproductive performances of C. capitata males may be improved by specific octopaminergic neurones activated during previous aggressive interactions with the robotic fly. This study adds fundamental knowledge on the potential role of specific neuro-behavioural processes in the ecology of tephritid species and paves the way to innovative biotechnological control methods based on robotics and bionics