Methodology Description
The pairwise encounters were carried out with 2 adult individuals (aged from 4 to 8 months), either familiar or unfamiliar. For each of these categories, we carried out encounters between two males, two females or two individuals of different sex. Familiar encounters between males and females were carried out with unrelated breeding pairs of individuals housed in the same cage for at least 3 weeks and with no dependent offspring. Familiar encounters between individuals of the same sex were carried out with brothers or sisters housed together since birth. In this situation, the familiar individuals were also related. These first two situations mimic what happens in the wild, where the familiar individuals who share the same community nest are the parental pair and the siblings. Unfamiliar encounters (individuals of the same or different sex) were carried out with two individuals born and housed in two separate rooms, with no possibility of having seen, heard or smelled each other since birth. For each pairwise trial in each part of the experiment, the two individuals were taken out of their maintenance cage and isolated separately in two other cages, which were placed in separated acoustic chambers for the hour preceding the experiment. The mice were then introduced into an experimental cage placed in another acoustic chamber (SilentBox© with acoustic foam-covered walls). The experimental cage comprised two compartments separated by an opaque, acoustically insulated partition, meaning animals were not able to hear each other before the encounter (one compartment per mouse; Zolux Neo Panas XL cage, 71*41*48 cm). The partition was effective, as no USVs were recorded before the encounter. A trapdoor (with a 6 cm diameter round opening) allowed communication between the two compartments, which opening of the trapdoor was controlled from outside the acoustic chamber, avoiding any disturbance by human intervention. The cage floor was covered with 1 cm of clean bedding (Zolux Chambiose Nature). After a 20-minute habituation period, during which the mice remained in their respective compartments, the trapdoor was opened. Once one of the two mice had joined the other in its compartment, the two individuals remained together for 10 minutes, during which their vocal production was recorded.
Equipment Used
We recorded the ultrasonic vocalisations of the mice with an Avisoft© Bioacoustics CM16 microphone connected to a sound gate (Avisoft© Ultrasound Gate 416 H, sampling rate = 300 kHz). The microphone was placed above the centre of the experimental cage, at 55 cm above the cage floor.