Chemical cues are widely used in intraspecific and interspecific communication, either as substances deposited in the substrate or as molecules diffused in water or air. In tardigrades, an emerging microscopic study system, chemical communication and its role in reproduction are poorly known. Here, we assessed sex differences in the detection of (a) short-range diffusing signals and (b) deposited cue trails during the mate-searching behaviour of freely moving virgin male and female Macrobiotus polonicus. We tracked individual behaviour (a) in simultaneous double-choice chambers, where live conspecifics of each sex were presented in water and (b) of freely moving pairs on agar without water. We found that males, but not females, preferentially associated with opposite-sex individuals in trials conducted in water. In contrast, neither sex detected nor followed cues deposited on agar. In conclusion, our study suggests that mate discrimination and approach are male-specific traits and are limited to waterborne chemical cues. These results support the existence of Darwinian sex roles in pre-mating behaviour in an animal group with virtually non-existing sex differences in morphology or ecology.

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