Determination of volatile compounds that elicit removal of diseased brood by hygienic honey bees.

Determination of volatile compounds that elicit removal of diseased brood by hygienic honey bees.

Summary

Honey bees are susceptible to a number of diseases and parasitic mites, which are commonly treated with antibiotics and pesticides in commercial apiaries. In addition to the dangers of introducing chemicals into our environment, the widespread use of chemical treatments has caused the pathogens and parasites to develop resistance to them. Beekeepers are quickly exhausting chemical options to treat their bees. However, there are bees that can control the presence of diseases and mites naturally. These adult bees, termed hygienic, reduce the pathogen load by removing diseased and parasitized brood from the hive. Previous research suggests that hygienic bees respond to olfactory cues coming from the abnormal brood. This project was designed to investigate the chemical profiles of chalkbrood infected larvae and to confirm that hygienic behavior is a response to olfactory cues.

Objectives/Performance Targets

1. Determine differences between the volatile chemical composition of healthy honey bee larvae and those that are infected with the fungal disease chalkbrood.
2. Determine which compounds unique to chalkbrood infected larvae are detected by honey bees.
3. Compare the ability of hygienic and non-hygienic bees to learn compounds unique to chalkbrood infected larvae.
4. Develop a bioassay that involves introducing synthetic compounds of diseased brood into the field colonies to elicit the removal behavior.

Accomplishments/Milestones

Objective 1: I used gas chromatography – mass spectrometry to obtain chemical profiles of healthy fifth instar larvae, fifth instar larvae that showed early signs of chalkbrood infection and larvae that were completely overcome with the disease (mummies). This work showed there are volatile compounds given off by diseased larvae that are not present in healthy larvae.

Objective 2: To determine which compounds unique to chalkbrood are detected by adult bees, I used gas chromatography coupled with electroantennographic detection. Using volatiles from early stage chalkbrood infected larvae, I was able to show there are three compounds unique to diseased larvae that elicit a physiological response from adult honey bee antennae; phenethyl acetate, phenyl ethanol and benzyl alcohol.

Objective 3: I used the associative conditioning method of proboscis extension reflex to demonstrate that there are no statistically significant differences between hygienic and non-hygienic bees in their ability to learn phenethyl acetate, phenyl ethanol and benzyl alcohol at two concentrations, 10-4 and 10-16. I also used a mixture of the three compounds at the two concentrations. Non-hygienic bees learned the mixture at 10-16 better than hygienic bees.

Objective 4: I used a bioassay to elicit hygienic behavior in field colonies. I used a picospritzer to puff 0.5ul of phenethyl acetate, phenyl ethanol and benzyl alcohol, a mixture of the three compounds or water onto 25 fifth instar larvae. I repeated this treatment on twelve colonies with varying levels of hygienic behavior. Regression analysis showed a significant relationship between the level of hygienic behavior of the colony and the removal of larvae treated with phenethyl acetate and the mixture.

Impacts and Contributions/Outcomes

This research provides valuable insights into the mechanisms behind hygienic behavior. It verifies that hygienic behavior is a behavioral response to olfactory cues. Furthermore, it shows that all bees, regardless of the level of hygienic behavior of the colony, can learn the three compounds that bees detect in chalkbrood but only phenethyl acetate and the mixture of the compounds elicited the behavior in the field.

Currently, the field method used to test for hygienic behavior is called a freeze-killed brood assay. The removal of freeze-killed brood is generally correlated with the removal of diseased and parasitized brood, but it would be better to have an assay that is more realistic without having to challenge the colonies with disease. The freeze-killed brood assay requires killing brood, is time consuming, and may involve the use of dangerous materials (i.e. liquid nitrogen). The bioassay used here may be developed into a commercially available assay that uses the same olfactory cues the bees detect in diseased brood to improve breeding programs for hygienic behavior. By making it easier for beekeepers to test their colonies, I hope to encourage increased use of hygienic honey bees and decreased use of chemicals for the treatment of disease.