Alison McAfee is a PhD candidate working under the supervision of Dr. Leonard Foster at the University of British Columbia. Not satisfied with investigating the molecular nature of bacteria, yeast, or mammalian cells, Alison is attempting to deduce the molecular mechanism behind a type of social immunity in honey bees. We sat down with Alison to discuss her work, and learn about the ups and downs of working with such an unusual model organism.
First things first: Why honey bees?
Good question! Honey bees are critically important for our food security. While it’s not only honey bees that drive pollination – other bees are involved as well – they are a huge component of it. The demands for pollination services are growing at a faster rate than the population of colonies, so anything we can do to improve their health and vitality is worthwhile.
What aspect of honey bee biology are you interested in studying?
In our lab we study host-pathogen interactions, including bee diseases and immune responses. I’ve chosen to study a few proteins that honey bees express in their antennae, which correlate with something we call social immunity. Bees have innate immunity like virtually all animals, but they lack conventional adaptive immunity. The thought is that they make up for that lack of immunity by working together as a team, which we call social immunity. I study a social immunity trait called hygienic behaviour, which is when the worker bees sense sick, parasitized, or diseased brood, and remove them from the colony. It’s a form of quarantine. By taking that diseased individual away, they reduce transmission to healthy individuals in the hive.
How much was previously known about the molecular mechanism behind hygienic behaviour?
Hygienic behaviour is actually divided into the activity of “uncappers”, which are the bees that smell the diseased brood, and the “removers”, which are thought to be a different group of bees. I’m interested in studying the mechanism by which the uncappers sense the diseased brood, which we know surprisingly little about.
Researchers previously found that hygienic bees produce significantly more octopamine – which is a molecule that enhances their sense of smell – compared to non-hygienic bees. They also found that when non-hygienic bees were fed octopamine, they were better able to smell disease odours. But when they did the same thing to hygienic bees, no change was observed, likely because they’re already maxed out in terms of their sensitivity. So we know that the mechanism behind the behaviour is tightly linked the sense of smell.
There have also been a number of differential expression studies that have been performed between hygienic bees and normal bees. But what people have not done is taken one of those genes that is differentially expressed, and manipulated the expression to look at the effect on behaviour. That exact experiment is actually my ultimate goal. I’ve been trying to get RNAi knockdown to work for some of these olfactory proteins for over three years without success. RNAi has been successfully performed in other organs of honey bees, but I’m trying to do it in their antennae which is proving to be a very big challenge.
What are some of your most interesting findings?
The thing that I’m the most excited about right now is kind of a hypothesis in progress related to the roles of the different odours that are emitted by dead bees. I’ve done gas chromatography-mass spectrometry (GCMS) comparing the odour profiles between dead and live pupae, and two of the strongest molecules that are found predominantly in dead pupae are oleic acid and beta-ocimene. Oleic acid is a well known inducer of hygienic behaviour in other social insects, but its so viscous and oily that it doesn’t really emit an airborne smell. We’ve tested puffing a bolus of air over a piece of filter paper that is coated with oleic acid, so that any airborne molecules travel to an antennae at the other end. We measured the neuron activity in the antennae, but we found that there’s no stimulation despite it being a known necromone in other social insects.
Beta-ocimene is a brood pheromone, which we know from previous research, increases the attention the worker gives the cells emitting the molecule. It’s normally a sort of “feed me!” signal that the larvae emits. But if diseased brood also emit this molecule, then you can imagine it might be playing a role in hygienic behaviour as well, by acting as an attention signal for a bee that is in distress. So I think the two odours are working together. Beta-ocimene is the volatile, further-reaching signal that attracts the attention of the uncappers, an once they are within physical contact with the pupae they can sense the oleic acid, which signals the hygienic behaviour. So I think there’s an attention signal, followed by a signal that dictates behaviour.
Do you have any ideas how these odours are working on a mechanistic level?
It’s not exactly known, but we have a pretty good idea. Some of the proteins that correlate with hygienic behaviour are Odorant Binding Proteins (OBPs). Our lab has previously performed a ligand binding assay with recombinant OBPs and various ligands. Oleic acid had the strongest binding of all the ligands that were tested. This was done before I’d performed my GCMS experiments where I’d identified oleic acid as being a compound that is emitted in high abundance from dead bees.
In general OBPs are a pretty well conserved class of proteins. We think they bind odours at the pores of the antennae. These are the interface between the air and the sensillum lymph fluid, which is a liquid that occupies the space between the cuticle and the actual olfactory neurons. So, somehow hydrophobic airborne molecules need to get from the pore to the receptor on the neuron. The proposed function of OBPs is that they bind these molecules at the pore and transport them to the receptor, where they stimulate the olfactory neurons.
What are the biggest challenges with working with bees?
Definitely a lack of traditional molecular biology tools. For instance, having to spend three years to get RNAi to work in antennae, because no one has done it before! Plus you can’t just order a transgenic bee with gene x knocked out in only one part of it. There’s only one publication ever published that describes how to make transgenic bees, and that was in 2014. It makes them a very challenging organism to work with.
Thank you so much for taking the time to talk with us, Alison! We wish you the best of luck with your future research!