Eusocial insects live in teeming societies with thousands of their kin. In this crowded environment, workers combat disease by removing or burying their dead or diseased nestmates. For honey bees, we found that hygienic brood-removal behavior is triggered by two odorants – β-ocimene and oleic acid – which are released from brood upon freeze-killing. β-ocimene is a co-opted pheromone that normally signals larval food-begging, whereas oleic acid is a conserved necromone across arthropod taxa. Interestingly, the odorant blend can induce hygienic behavior more consistently than either odorant alone. We suggest that the volatile β-ocimene flags hygienic workers’ attention, while oleic acid is the death cue, triggering removal. Bees with high hygienicity detect and remove brood with these odorants faster than bees with low hygienicity, and both molecules are strong ligands for hygienic behavior-associated odorant binding proteins (OBP16 and OBP18). Odorants that induce low levels of hygienic behavior, however, are weak ligands for these OBPs. We are therefore beginning to paint a picture of the molecular mechanism behind this complex behavior, using odorants associated with freeze-killed brood as a model.

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.

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 freezekilled 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.Honey bees (Apis mellifera L.) face many challenges, but disease is perhaps the most significant 1 and hygienic behaviour (HB) is an important method of disease control [2][3][4][5][6] . Bees perform HB by detecting, uncapping and removing diseased brood from the colony to reduce pathogen load, thereby improving disease resistance and leading to higher survival rates when challenged with Varroa destructor, American foulbrood and chalkbrood [2][3][4][5][6][7] . The most well-established method of selecting for hygienic bees is the freeze-killed brood (FKB) assay, in which patches of brood are frozen with liquid nitrogen, returned to the hive and evaluated after 24 h. The HB score is defined as the fraction of dead pupae that have been detected and removed 4,7 . Colonies that perform well in this test also have improved outcomes when challenged with real diseases, allowing the FKB assay to be an effective tool for selective breeding 7 . There is a large body of evidence suggesting that hygienic bees identify diseased brood through olfactory cues [8][9][10][11][12][13][14] and that they are more sensitive to and better at discriminating between them 8,14 . The antennae, bees' main olfactory organ 15 , have been shown to play a pivotal role in HB with multiple independent research groups identifying significantly differentially expressed antennal genes in hygienic versus non-hygienic bees, as well as strong antennal biomarkers for selective breeding 9,13,[16][17][18] . Odorant binding protein (OBPs) aid odour detection and are consistently upregulated in hygienic bees' antennae. However, relatively little is known about precisely what odours the bees are detecti...