Fig. 1: Experimental design and recording methods.

a Trial design. Day 1: males were paired as competitors and placed into an arena separated by a mesh barrier indicated by a dashed line (Mesh 1). The mesh barrier was removed and males entered into an aggressive contest (Fight) concluding in winning or losing males. Day 2: each male was placed into an empty arena (Empty). Day 3: males were placed back into the mesh arena with the same (familiar) male competitor from the first trial (Mesh 2). Day 4: each male was exposed to one of 4 possible treatments of aliquoted male urine of 3 possible identities (self, familiar and unfamiliar) into two urine-marked zones. The 4 treatment groups: self-self, self-familiar male, self-unfamiliar male, familiar male-unfamiliar male. The familiar male stimulus is the urine of a male’s paired competitor. b A thermal snapshot of a Mesh trial and the regions of interest (ROIs: Wall vs. Barrier) used to score urine mark depositions and track space use. The dashed line indicates the mesh barrier separating the two males. The solid lines depict ROIs males can traverse through on their side of the barrier. Urine marks are hot (orange-pink: close to the body temperature) on a cool (dark blue) ambient substrate (filter paper) temperature. c A thermal snapshot of an Empty trial (Day 2) with the ROIs used for scoring (Corners vs. Center) indicated with solid lines. The same ROIs were to score Empty (Day 2) and Marked (Day 4) trials. An example track of the mouse’s trajectory two seconds before and after its current location is shown (light turquoise). d, e An example urine blot of an Empty trial imaged under UV light (D), and the processed inverted urine blot image (E: black spots: urine marks). f Density plot depicting the temporal distribution of all thermally detected urine marks across all trials. g Box and violin plot of the total number of urine marks detected across trials using thermal imaging and UV blot imaging recording methods (boxplot midline: median, box limits: upper and lower quartiles, whiskers: 1.5× interquartile range, points: outliers). A linear mixed model was used to model the relationship between recording method and the total urine marks detected (M1: Table S1). An analysis of variance was used to test for the overall effect of recording method (significance code: NS p > 0.05).