Honeybees possess a structurally diverse and functionally redundant set of queen pheromones

Abstract

Queen pheromones, which signal the presence of a fertile queen and induce workers to remain sterile, play a key role in regulating reproductive division of labour in insect societies. In the honeybee, volatiles produced by the queen's mandibular glands have been argued to act as the primary sterility-inducing pheromones. This contrasts with evidence from other groups of social insects, where specific queen-characteristic hydrocarbons present on the cuticle act as conserved queen signals. This led us to hypothesize that honeybee queens might also employ cuticular pheromones to stop workers from reproducing. Here, we support this hypothesis with the results of bioassays with synthetic blends of queen-characteristic alkenes, esters and carboxylic acids. We show that all these compound classes suppress worker ovary development, and that one of the blends of esters that we used was as effective as the queen mandibular pheromone (QMP) mix. Furthermore, we demonstrate that the two main QMP compounds 9-ODA and 9-HDA tested individually were as effective as the blend of all four major QMP compounds, suggesting considerable signal redundancy. Possible adaptive reasons for the observed complexity of the honeybee queen signal mix are discussed.

Keywords: Apis mellifera; honeybees; pheromones; reproduction; social insects.

Figure 1.

Queen-characteristic cuticular compounds in honeybees identified based on GC–MS analysis. Using z-scores calculated on the log₁₀ transformed relative peak areas of each compound, the heatmap shows that compounds clearly cluster in three groups characteristic for workers, egg-laying queens and virgin queens, respectively. The compounds highlighted in blue and purple are esters and alkenes that showed the largest differences between queens and workers (Cohen's d > 2.5 and relative peak area in queens greater than 1%, cf. table 1 and electronic supplementary material, table S1 and figure S1) and were tested as synthetic blends for queen pheromone bioactivity in our bioassays. (Online version in colour.)

Figure 2.

Bioassays reveal a large panel of novel bioactive honeybee queen pheromones. The synthetic blends of queen-characteristic cuticular esters and alkenes (cf. Figure 1; electronic supplementary material, figure S1) as well as tergal gland esters and carboxylic acids (table 1) all significantly inhibit worker ovary development in queenless worker groups compared to the solvent-only control. The tergal gland compounds were as effective at inhibiting worker ovary development as the total queen mandibular pheromone mix QMP and its two major constituents 9-ODA and 9-HDA, while the cuticular esters, cuticular alkenes and tergal gland acids were as effective as the two other major components of the total QMP mix, HOB and HVA. The columns and whiskers show the marginal predicted means of a binomial GLMM plus the 95% confidence bounds. Significances are indicated by asterisks (n = total number of workers dissected across each of the five replicate groups per treatment, see electronic supplementary material, table S2 for details). (Online version in colour.)