apis mellifera, bee, hygienic behaviour - Alison McAfeeI am studying honey bees – specifically, a kind of disease resistance called hygienic behaviour. Hygienic worker bees detect sick or dying brood and remove them from the hive, protecting the healthy workers from catching disease. It’s a rather cut-throat form of quarantine, but it works.

This trait can be passed on from generation to generation, which means that we can selectively breed hygienic bees. A handful of genes that are involved in the sense of smell are tightly linked with the behaviour (we call these ‘biomarkers’; see the primary research article here) and my thesis revolves around finding the functional relationship between these genes and the behaviour. For example, we may know the genes are involved in smell, but what odours are the bees smelling? Without these genes, can they still smell? With more copies of the gene, do the bees get more hygienic? In the end, this project will provide a better understanding of the processes behind social disease resistance and may lead to new methods for maintaining healthy bees. An excerpt from my research proposal can be found below:

romancing the bee
A Spanish cave painting from the Mesolithic period

Humans have been intrigued with honey bees since the time of our early ancestors, and the economic value of their services is indisputable. Spanish cave paintings from 8,000 years ago even show humans collecting honey from wild bee hives [1]. Honey bees are only native to Europe, Africa and Asia, but since they became domesticated and intertwined with agriculture operations, they are now found on every habitable continent [2]. They provide us with a host of natural products – honey, wax, and propolis, to name a few – but most importantly, they are one of the primary means of crop pollination [3]. In the United States alone, honey bee pollination services are valued at over $14 billion annually [4].

Within the last decade, North American beekeepers have had unprecedented overwintering colony losses – approximately 30% annually [5] and up to 50% in some Canadian provinces [6] – in part due to the increasing threat of diseases and parasites [7]. While beekeepers can usually recover from such losses every year, it is economically costly to do so. Honey bees fight disease with a combination of innate immunity [8] and social immunity [9], which is a collection of grooming and hygiene behaviours that help maintain colony health. Hygienic behaviour (HB), in particular, is a desirable trait in honey bees because it significantly improves colony disease resistance without treating with synthetic chemicals [10].

HB is a highly heritable trait [11] that has long been thought to be closely tied to olfactory cues emitted by diseased brood [12]. In 2009, Swanson et al. found that bees in hygienic hives had heightened olfactory sensitivity and specificity to odorants produced from chalkbrood-infected larvae [13]. Swanson et al. also identified a volatile compound (phenethyl acetate) from chalkbrood that was sufficient to strongly induce HB even in relatively non-hygienic hives. Furthermore, a more recent study by Guarna et al. has identified a panel of antenna protein biomarkers for HB, two of which are odorant binding proteins (OBPs) [11]. These are promising targets to begin investigating the mechanism by which honey bees perceive diseased brood.

The honey bee’s antennae are the sites of peripheral odorant detection and are covered with small hair-like structures (sensilla), each of which houses the dendrites of five to thirty-five olfactory receptor neurons (ORNs; [14]).

Anatomy of a honey bee antenna. A) Diagram showing magnified sensilla trichodae and the underlying olfactory neurons. Bees also use other types of sensilla, including placodae (shield shaped) and basiconica (horn shaped). Diagrams were adapted from the Syntech user manual. B) A microscope image of a cross-section of a real antenna, showing the auxillary cell layer (where odorant binding proteins are produced) stained in blue. Image obtained from Danty et al. 1997.
Anatomy of a honey bee antenna. A) Diagram showing magnified sensilla trichodae and the underlying olfactory neurons. Bees also use other types of sensilla, including placodae (shield shaped) and basiconica (horn shaped). Diagrams were adapted from the Syntech user manual. B) A microscope image of a cross-section of a real antenna, showing the auxillary cell layer (where odorant binding proteins are produced) stained in blue. Image obtained from Danty et al. 1998.

Bundles of ORN axons project to the antennal lobe, where nerve signals are transmitted to other parts of the brain [15]. The antennal lobe itself is at the base of the antenna and is made up of 165 glomeruli, which is roughly the same number of predicted olfactory receptors (ORs), suggesting that each ORN expresses one OR and is joined to one glomerulus. ORs are located on the ORN membrane, and are in contact with the sensillium lymph fluid that occupies the space between the cuticular wall and the ORNs. It is through this fluid that OBPs are thought to aid in the transport of volatile odorants from the olfactory pores to the ORNs [16]. Very little is known about the specific roles of the different honey bee OBPs in odour discrimination and sensitivity. Furthermore, although it has been shown that OBPs are strongly correlated with HB [11], the functional relationship between these OBPs and performance of HB has not been previously demonstrated.

My hypothesis is that two specific OBPs are enhancing HB by increasing worker bees’ sensitivity to volatile compounds emitted by diseased or dying brood. I will begin my project by developing the tools to knock down gene expression of the proteins at both the individual level and at the scale of the hive in order to study the functions of these proteins. Following this, I will identify disease volatile mixtures that interact with the OBPs by comparing the neurological responses of OBP knock down bees relative to control bees. This will be complimented by colony-level experiments in which I will develop a brood comb odour impregnation assay to identify key odorants that are sufficient to induce HB in the hive. In addition, I will use the knock down methods I develop early on in conjunction with the key HB-inducing odorants I identify to determine if the OBPs are necessary for detection of these specific odorants. Finally, I will rear transgenic bees to facilitate OBP overexpression studies to determine if increasing levels of the OBP alone is sufficient to enhance HB. Together, the information gained by these experiments will provide a well-rounded view of the role these proteins play in a complex behaviour.

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