Dual-fluorescence imaging and automated trophallaxis detection for studying multi-nutrient regulation in superorganisms

Abstract

Much like a single organism, colonies of social insects regulate their collective nutrition towards a specific intake of nutrients. Yet, the mechanisms behind this colony-level regulation are not fully understood. Although foraging behaviour in social insects has been studied extensively, not much is known on its relation with feeding processes that occur within the nest. In the nest, food is commonly transferred between individuals in numerous oral feeding interactions (trophallaxis). Studies on the properties of these trophallactic networks have so far been limited by: (a) the difficulty to non-intrusively measure food inside individual ants, let alone its nutritional composition, and (b) the meticulous manual labour involved in detecting trophallactic events. Our dual-fluorescence imaging set-up is designed to track two food sources, each labelled with a different fluorophore, as they are disseminated throughout a freely behaving colony of individually tagged ants. Additionally, our image-based deep learning algorithm for automatic detection of ant trophallaxis events efficiently yields a detailed record of all food-transfer interactions. Using a series of calibration experiments, we demonstrate the reliability of our measurements. We then exemplify the capabilities of our new method by tracking food dissemination in a colony of Camponotus sanctus ants supplied with two nutritionally distinct food sources. We follow the amount of each food type in the crop of each ant in the colony. Our data reveal the path in nutrient space that the colony took to reach its final nutrient intake, the contributions of the different forager ants to this path and the dynamic nutritional states of all ants in the colony. We further demonstrate the potential applicability of dual-fluorescence food imaging to other insect species. Our system provides access to data that were previously unavailable and is crucial for a complete description of nutrient regulation by social insects. More broadly, the ability to simultaneously and efficiently track two material flows in a behaving colony provides new opportunities for tackling many open and emerging questions on topics other than nutrition, such as disease spread and social regulation of colony development.