Abstract
Hygienic behaviour (HB) is a social immunity trait in honey bees (Apis mellifera L.) whereby workers detect, uncap and remove unhealthy brood, improving disease resistance in the colony. This is clearly economically valuable; however, the molecular mechanism behind it is not well understood. The freeze-killed brood (FKB) assay is the conventional method of HB selection, so we compared odour profiles of FKB and live brood to find candidate HB-inducing odours. Surprisingly, we found that significantly more brood pheromone (β-ocimene) was released from FKB. β-ocimene abundance also positively correlated with HB, suggesting there could be a brood effect contributing to overall hygiene. Furthermore, we found that β-ocimene stimulated worker antennae in a dose-dependent manner, with the left antennae responding significantly stronger than right antennae in hygienic bees, but not in non-hygienic bees. Five other unidentifiable compounds were differentially emitted from FKB which could also be important for HB. We also compared odour profiles of Varroa-infested brood to healthy brood and found an overall interactive effect between developmental stage and infestation, but specific odours did not drive these differences. Overall, the data we present here is an important foundation on which to build our understanding the molecular mechanism behind this complex behaviour.
Highlights
We found that a well-known brood pheromone, β-ocimene, was strongly emitted from freeze-killed brood (FKB) and this pattern positively correlates with hygienic behaviour (HB) score
We identified one compound, oleic acid, which was consistently released in higher amounts in FKB, across colonies and across developmental stages
We found that hygienic bees, but not non-hygienic bees, elicit a dose-dependent lateralized olfactory response to β-ocimene
Summary
Honey bee colonies were kept at three separate locations in Greater Vancouver, BC, Canada. Honey bee pupae with no visible signs of disease were collected from colonies by carefully uncapping cells and removing pupae with clean stainless steel forceps. Age was determined based on eye and cuticle pigment using the following relationships: white-eyed = 12–13 d, pink-eyed = 14–15 d, purple-eyed white body = 16 d and purple-eyed tan body = 17–18 d. Eye and cuticle pigment was matched exactly so that each bee in each age group was at the same developmental stage. Pupae were placed in clean glass vials, removing any wax debris and avoiding abrasions or cuticle indentations. After the 15 min freeze, all pupae were completely solid and brittle so there is no doubt that they were mortally frozen
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