Differences in honeybee queen pheromones revealed by LC-MS/MS: Reassessing the honest signal hypothesis.

The honest signal hypothesis posits that social insect queens emit pheromonal signals that convey information about fecundity, which workers use to make decisions around investing in direct or indirect fitness. We used liquid chromatography-tandem mass spectrometry to measure honeybee (Apis mellifera) queen retinue pheromone (QRP) in relation to age, laying status, and acceptance using a protocol that enables concurrent metabolomic and lipidomic analyses. Older queens produced higher levels of the QRP components 9-R-hydroxydec-2(E)-enoic acid (9(R)-HDA), linolenic acid (LEA), and 4-hydroxy-3-methoxyphenylethanol (HVA) compared to younger queens, with HVA also correlating with ovary mass. However, ovary mass was not an influential metric for worker decision-making around queen acceptance; therefore, the relationship between HVA and ovary mass is merely an "honest signal" of a non-influential metric. Parallel metabolomic and lipidomic analyses showed that samples cluster according to queen age and mating status, but not ovary mass, revealing many other physiological changes occurring in the queen's early life.