Searching for the egg-marking signal in honeybees.

Behavioural bioassays have shown that worker honeybees can distinguish between worker-laid and queen-laid eggs. By eating worker-laid eggs, nest-mate workers prevent each other from reproducing, a behaviour known as worker policing. However, the recognition cue used by worker honeybees to discriminate between worker-laid and queen-laid eggs remains elusive. This study describes observations made and experiments conducted to elucidate the nature of the recognition cue. No visually apparent physical differences were found between worker-laid and queen-laid eggs using scanning electron micrographs (SEMs) magnified up to 2500 times, which indicates that the cue is probably chemical in nature. We confirmed that the signal probably resides on the queen-laid and not the worker-laid eggs, since a worker-laid egg is protected when placed in contact with a queen-laid egg. We conducted a series of standard egg-removal bioassays in queenright colonies using queen-laid and workerlaid eggs treated with a wide range of potential recognition chemicals, solvents, buffers or gland extracts. The aim was either to disrupt or remove the egg-marking signal from queen-laid eggs, or to add the signal to worker-laid eggs. Despite a comprehensive set of experiments, we were unable to alter the egg-marking signal on queen-laid eggs or transfer the signal from queen-laid eggs to worker-laid eggs. Furthermore, two candidate signals, esters from queens and eicosenol from workers, were shown not to be the cues used by workers. This indicates that the egg-marking signal in honeybees is remarkably robust and consists of a chemical or group of chemicals not previously associated with chemical signalling in social insects.