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Research Project: The Role of Dietary Essential Fatty Acids in Honey Bee Hygienic Behavior

Location: Carl Hayden Bee Research Center

Project Number: 2022-21000-022-004-I
Project Type: Interagency Reimbursable Agreement

Start Date: Sep 1, 2020
End Date: Aug 31, 2022

Honey bees are critical to US agriculture, but are experiencing declines due to factors including pesticides, disease, mites, and insufficient forage. Lack of suitable nutritional resources is a major problem facing honey bee health. Bees forage on a variety of plants that provide differing nutrient levels, and increased land use intensification combined with unpredictable weather patterns has negatively impacted plant diversity and availability. However, the negative effects of nutritional stress pale in comparison with the stresses that honey bee colonies incur due to the parasitic Varroa mite. Adequate Varroa control has proven elusive and beekeepers must rely on multiple costly chemical treatments each year for their colonies to persist. Non-chemical treatments are largely ineffective on their own, but can be part of an IPM-like control strategy when used in combination. Nonetheless, mites persist and colonies keep dying from them, and the desire for additional sustainable strategies for mite control remains. "Hygienic" genotypes control mite populations in the hive by locating and uncapping cells containing diseased brood and remove them. Olfaction is a critical part of the hygienic phenotype – hygienic bees are highly sensitive to the odor of diseased brood and respond accordingly. Beyond their increased sensitivity to such odors, the mechanism underlying this behavior is unknown. For example, the brains of hygienic bees may develop differently or process olfactory inputs more readily than unselected lines. Our first objective is therefore to examine the underlying neural mechanisms generating hygienic behavior. With this information, we can then further develop strategies for manipulating the growth, development, or activity of these brain regions in honey bees. Beyond simply understanding how brain regions contribute to Varroa control, our next two objectives seek to increase the occurrence of this beneficial behavior in the hive. We hypothesize that targeted nutrition will serve this purpose. Recent work suggests that fatty acids influence bee behavior – honey bees foraging on pollen diets low in omega fatty acids were better able to learn from olfactory cues than those fed diets low in these fatty acids. This work has interesting parallels with earlier studies of hygienic behavior, where hygienic bees are more sensitive to brood odors than unselected non-hygienic bees. The proximate mechanisms explaining the connection between fatty acids and behavior are not yet clear, but because fatty acids influence olfaction and olfaction is so critical for hygienic behavior, this leaves a great opportunity to test whether dietary fatty acids can influence hygienic behavior. We will first test whether fatty acids influence the development and function of brain regions important for hygienic behavior documented as part of our first objective. Next, we will test whether dietary fatty acids influence the actual hygienic behavior, an important mechanism for controlling disease and Varroa levels in the hive.

Experiment 1: Does fatty acid consumption affect hygienic behavior? Honey bee colonies selected for hygienic behavior will be placed in flight cages with controlled access to artificial diets. Colonies of hygienic bees will be reared from the Minnesota Hygienic Line (MHL) queens, which have been artificially selected for high hygienic behavior. I will compare an unselected colony (UC) with the MHL under two artificial diets, a standard commercial diet and supplemented diet. The control group will be fed a standard commercial diet that lacks fatty acids and treatment groups will have the fatty acids, omega-3 and omega-6 supplemented to their diet. Foragers will be collected from both groups and brought into the laboratory to test detection and learning acquisition to diseased brood odors. Proboscis extension reflex (PER) discrimination conditioning will be used to assess detection of diseased brood and healthy brood odors. Diseased brood odors will be derived from diseased pupae removed from an infected colony with chalk brood. Prediction: Bees supplemented with fatty acids should have higher learning acquisition to diseased brood odors. Bees deficient in fatty acids should also have lower learning acquisition to diseased brood odors. Fatty acid supplementation should increase learning acquisition the UC group, with comparable learning curves to the commercial diet MHL group. Experiment 2: Does nutrition affect brain olfactory processing of MHL and UC bees? Foragers will be collected from flight cages in a similar fashion to experiment 1. Using electrophysiology and histology, the neural differences in early olfactory processing between the MHL and UC groups fed supplemented or commercial diets will be examined. Electrophysiology: Using extracellular electrophysiology, I will measure antennal lobe responses to brood odors in MHL and UC fed a standard commercial or fatty acid supplemented diets. We will open the head capsule of live bees and insert a tetrode into the antennal lobe of the brain. With the tetrode inserted, odors from diseased brood and healthy brood will be administered and resulting electrical responses of the antennal lobe will be measured. Histology: Whole brains from MHL and UC bees fed standard commercial or fatty acid supplemented diets will be dissected and fixed in 4% paraformaldehyde. Fixed brains will be sectioned and stained, revealing critical parts of the brain involved in olfactory processing such as the antennal lobes and lip of the mushroom bodies. Prediction: MHL bees should have increased antennal lobe activity to diseased brood odors versus the UC group. Further, MHL bees may invest more in olfactory regions of the brain. Because fatty acids contribute to increased olfaction, there may be increased plasticity in olfactory portions of the brain in bees fed a fatty acid rich diet.