Hygienic worker bees have a demanding job. They are key players in the hive’s social immune system, detecting and removing sick and dying brood before the pathogens and parasites they harbor can spread. Evolutionarily, this makes sense; hygienic behavior probably evolved because in the face of brood diseases, the hygienic colonies were more likely to fight off the disease and survive. But from the individual bee’s perspective, what exactly is going on? How are they able to tell who’s sick and who’s healthy? We are closer than ever to zeroing in on the link between the genes hygienic honey bees express and their ability to detect dead brood. But it turns out, we might be untangling just one of many potential ways that bees can be hygienic.
Scientists have been studying hygienic behavior since at least the 1960’s, when WC Rothenbuhler, at Iowa State University, started working out the genetic basis of this trait.1 By observing how different inbred lines of honey bees responded to dead brood (back then, they killed the brood with cyanide gas – now, we use liquid nitrogen, which is much safer), Rothenbuhler deduced that there were probably two genes controlling the trait – one gene for uncapping, and one for removing.
About 40 years later, Marla Spivak showed that this model was too simplistic.2 In reality, there are at least seven locations on the genome that control the trait – probably many more – suggesting that hygienic behavior is more complex than Rothenbuhler ever imagined. Spivak’s foundational work also showed that bees’ sense of smell is indispensable for completing hygienic tasks.
In the March issue of American Bee Journal,3 I described two odorants emitted from dead brood which we think are involved in hygienic behavior – β-ocimene and oleic acid. At the time, I had not actually done any behavioral experiments – we just knew that these were strong signals coming from dead pupae, and the roles these chemicals play in other contexts lends them probable cause. Hungry larvae emit β-ocimene like a volatile flag, waving to attract the attention of workers and alert them that the larva is starving. Oleic acid, on the other hand, induces necrophoretic behavior (i.e., transport of corpses away from the colony) in ants and termites, and avoidance behavior in cockroaches and crickets. Together, this led me to hypothesize that β-ocimene probably attracts hygienic workers, while oleic acid is the determinant death cue. Now, I have behavioral data to back that up.4
To be clear, these odorants are not likely responsible for all hygienic behavior – just that which is induced by freeze-killed brood. Other diseases and parasites (chalkbrood, American foulbrood, and Varroa destructor) could easily stimulate hygienic behavior with different odorants, and some work by Spivak and Olav Rueppell suggests that is the case. Studying freeze-killed brood, though, is a useful model to begin with, because it’s a simpler system than one that involves real pathogens. It’s also much safer than cyanide.
We started with a quick and dirty behavioral test. We simply uncapped brood cells and added small amounts of the different odorants with a pipette (including a control treatment of hexane), replaced the frame and came back 3 hours later to see how many were removed. The hexane treatment acted as a reference point to compare β-ocimene and oleic acid to, since we know that it’s bad at inducing hygienic behavior. It’s important to include treatments like this because even the simple act of uncapping a cell and adding something – anything – can induce low levels of hygienic behavior, even if it’s not a death or disease odor. In total, we uncapped about 3,000 brood cells spread over 10 different colonies, with each cell cap individually picked away with tweezers (I’m very grateful to have had the diligent help of another student that summer!).
The results? Hygienic colonies indeed removed β-ocimene- and oleic acid-treated brood more often than hexane-treated brood, and they were better at removing brood than non-hygienic colonies. These results told us we were on the right track, but it’s arguably not a very realistic experiment. In reality, workers need to sense the odor through the wax cap, without being in direct contact with the brood. What’s more, since oleic acid is so oily, I doubted that it could become sufficiently airborne for its odor to penetrate the cap in the first place.
I wanted a better behavioral test. One that allowed me to add odorants to a brood cell without breaking its integrity. Without uncapping, without puncturing the wax, and without harming the pupa inside. I was stumped. And when I’m stumped, I talk to Heather.
Heather Higo has been in the business of bee science since before my own supervisor – now a full professor at the University of British Columbia – had even finished his undergrad. She’s also one of the leaders for the selective breeding project I wrote about in the article “Breeding a better bee: Three social immunity traits, one massive experiment.”5 In 2016, she received the Fred Rathje Memorial Award – a prestigious recognition for her contributions to improving the Canadian beekeeping industry. Not surprisingly, Heather instantly came up with a solution to my odor-introduction problem. “Can’t you just use a Jenter set?” she asked, as if wondering why I hadn’t thought of that before.
In case you’re not familiar, a Jenter set is a contraption normally meant for queen rearing. It includes square queen cages, complete with a….