A novel approach to quantifying inter-individual distance in insects

1Behaviour involving two or more individuals within the same species is known as social behaviour. Sexual dimorphisms and environmental cues as well as age, experience and social hierarchy shape social behaviour. The study of this complex behaviour, however, has one essential boundary condition: the distance between individuals. Because social signals (e.g. acoustic, visual or olfactory) have limited reach the inter-individual distance is crucial for the correct transmission of such signals. In this study we present a newly developed assay to study group behaviour and inter-individual distance in Drosophila melanogaster. This assay consists of a circular observation space flanked by two automatic release devices that allow flies to enter individually. By releasing the flies one at a time, the observer can study different features of (intra-)group behaviour with different group compositions. Importantly, the observer can control (manually or through automatisation) the increase of group size over time. Over the last decades insects became more relevant as convenient model organisms to study the neurophysiological and genetic basis of (human) neuro-developmental disorders. Drosophila models are used to study the genetic and neuronal underpinnings of a wide range of neurological disorders. In some cases the studies revealed alterations in social behaviour consistent with descriptions of behavioural symptoms in human disorders. Social behaviours in Drosophila are well-studied and include courtship, mating, aggression and group interactions. This setup will facilitate the analysis of these aspects of group interactions in Drosophila, allowing for a deeper understanding of the neuronal circuits and genetic factors involved in those behaviours. 2 Contribution to the fieldSocial behaviour pertains to the most sophisticated behavioural feats, as it involves multiple, interacting individuals. These complex interactions often conceal the underlying neuronal and ethological mechanisms. One of the most basal ethological mechanisms is the inter-individual distance, which resembles a perimeter in which each individual needs to formulate a response to the approach of others. We introduce a device that allows to test the inter-individual distance under consistent circumstances, by automating the entry time and direction of conspecifics into the arena. We can further observe the composition, dynamics and forming of larger animal groups as well as their separating. We can also observe how the individual distances alter during the process. Also other behaviours can be easily observed, e.g. aggression, courtship, homosexual courtship, etc. We successfully employed this approach in [1] and could discriminate the role of different neuroligins in social behaviour. We provide a detailed description including building plans and material lists for this social observation device. The system can be run in an automatic mode to ensure the consistency of experiments or in a manual mode to test animals under more flexible social situations. We provide multiple back lighting systems to test animals in the dark (infra-red LEDs) or in illuminated environments (vis. range LEDs). The system is fully automated and can be linked with a number of animal trackers (e.g. T-Rex, deeplabcut, LACE, etc.) via simple videography. We hope that our experimental setup augments the variety of behaviours testable in ethological setups (T-maze, water-mazes, operant conditing setups, etc) with social interaction and group formation.