Fathead minnows learn to recognize predator odour when exposed to concentrations of artificial alarm pheromone below their behavioural-response threshold

Hypoxanthine-3-N-oxide (H3NO) has been identified as the putative alarm pheromone of ostariophysan fishes. Previously we demonstrated a population-specific minimum behavioural-response threshold in fathead minnows (Pimephales promelas) to a H3NO concentration of approximately 0.4 nM. Minnows may, however, perceive low concentrations of H3NO as a predation threat, even though they do not exhibit an overt behavioural response. We conducted a series of laboratory trials to test the hypothesis that minnows can detect the alarm pheromone at concentrations below the minimum behavioural-response threshold. We exposed predator-naive fathead minnows to H3NO at concentrations ranging from 0.4 to 0.05 nM paired with the odour of a novel predator (yellow perch, Perca flavescens) or distilled water paired with perch odour. We observed significant increases in antipredator behaviour (increased shoal cohesion, movement towards the substrate, a reduction in feeding, and an increase in the occurrence of dashing and freezing behaviour) in shoals of minnows exposed to a combined cue of 0.4 nM H3NO and perch odour (compared with a distilled-water control), but not by shoals exposed to lower concentrations of H3NO paired with perch odour or those exposed to distilled water paired with perch odour. When exposed to perch odour alone 4 days later, minnows initially conditioned to H3NO at concentrations of 0.4-0.1 nM exhibited significant increases in antipredator behaviour. These data demonstrate that minnows attend to the alarm pheromone at concentrations below the minimum behavioural-response threshold and are able to acquire the ability to recognize a novel predator even though they do not exhibit an overt behavioural response.