Paint marking using CO2 anaesthetization does not affect exploratory and recruitment behaviours in the rock ant, Temnothorax rugatulus

The study of animal behaviour sometimes requires unique identification of individuals, especially in the study of social behaviours involving the interactions of multiple individuals. To this end, researchers have developed many different methods of marking individuals. For small animals like insects, paint marks are often applied to their bodies by anaesthetizing them using low temperature or carbon dioxide_. Despite this procedure being ubiquitous when studying social insects, the effect of paint and anaesthetics on their behaviour has not been well investigated, especially their effect on performance during a collective task. In our study, we investigate how paint marks and anaesthetics affect the movement and recruitment behaviours of the ant Temnothorax rugatulus in a house hunting context. We painted two thirds of colony members, half of them using CO₂ and the other half using low temperature as methods of anaesthetization, and left the one third unpainted as a control group. We then measured their exploratory behaviour prior to house hunting and their recruitment behaviours during house hunting. We found that neither paint marks nor anaesthetics reduce activity levels of these behaviours. However, low-temperature anaesthetized ants performed a higher number of recruitment behaviours than control ants. Because CO₂ anaesthetized ants performed all tasks at the same level as control ants, our data suggest that this is a good technique for paint marking ants, especially T. rugatulus. This is the first study empirically testing negative effects of paint marking on individual and collective outcomes in social insects. Our study represents an important step towards routine validation of individual identification methods used in the study of animal behaviour.