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 by beekeepers. The widespread use of chemical treatments has resulted in the contamination of hive products and chemical resistance by pathogens and parasites. Bees bred for hygienic behavior 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 investigated the chemical profiles of chalkbrood infected larvae to confirm with field bioassays 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 field colonies to directly test two hypotheses: 1) hygienic behavior of honey bees can be elicited in field colonies by experimental application of appropriate olfactory stimuli; and 2) hygienic behavior is based on a threshold response in which colonies containing bees with the highest olfactory sensitivity to diseased brood initiate the hygienic response more quickly as compared to colonies containing bees with a lower olfactory sensitivity.

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: I used gas chromatography coupled with electroantennographic detection to determine which compounds, unique to chalkbrood, are detected by adult bees. 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.

Objective 4: I developed two bioassays to test hygienic behavior in field colonies. A topical application assay tested the hypothesis that hygienic behavior of honey bees can be elicited in field colonies by experimental application of appropriate olfactory stimuli to healthy larvae. Twelve colonies that displayed varying degrees of hygienic behavior were chosen for the bioassay. 0.5 μl of each treatment compound (benzyl alcohol, phenyl ethanol, phenethyl acetate, a mixture of the previous three, and deionized water) was applied onto healthy larvae. Colonies with higher degrees of hygienic behavior removed significantly more treated larvae at 4 hours and 24 hours. At both four and 24 hours, significantly more larvae were removed that were treated with phenethyl acetate compared to all other treatments.

A second bioassay tested the hypothesis that hygienic behavior is based on a threshold response in which bees with the highest olfactory sensitivity to diseased brood initiate the hygienic response more quickly as compared to bees with a lower olfactory sensitivity. Larval paraffin dummies were made by melting paraffin wax and adding methyl linolenate at a 10-2 ml/ ml concentration. Methyl linolenate is one of several compounds present in brood pheromone of fifth instar larvae that, when added to a paraffin wax dummy and placed in a larval cell, cause adult bees to cap the cell containing the dummy. From this mixture of wax and methyl linolenate a total of eight treatment compound /concentrations were added of high (10-2 ml/ml) and low (10-9 ml/ml) concentrations of phenethyl acetate, phenyl ethanol, benzyl alcohol and a mixture of the three. Control treatments were made of methyl linolenate alone and paraffin wax without the addition of methyl linolenate or disease compounds. When both brood pheromone and disease volatile(s) were added to the paraffin, bees from rapid-hygienic colonies did not cap the dummies with wax; apparently they were able to detect the disease volatiles over the brood pheromone and did not cap the dummy with wax. Bees with less olfactory sensitivity (slow-hygienic colonies) were not able to detect the disease volatiles over the brood pheromone, and capped the paraffin dummy. There was a significant correlation between the level of hygienic behavior of the tested colonies and the lack of capping of the treated paraffin dummies. Therefore, the lack of capping in this assay was an indirect but very sensitive measure of the level of hygienic behavior of the tested colonies.

Impacts and Contributions/Outcomes

This research provides valuable insights into the mechanisms behind hygienic behavior. This is the first study to identify volatile compounds present in diseased honey bee larvae that elicit hygienic behavior by honey bees.

Our field bioassays confirmed two hypotheses:
1) hygienic behavior of honey bees is elicited by olfactory stimuli; and
2) the expression of hygienic behavior depends on the olfactory response threshold of individual bees within the colony. Furthermore, this research showed that one of the three compounds, phenethyl acetate, was the key volatile that elicited appropriate behavioral responses by bees in large field colonies.

The results of this study have an important practical application for beekeepers. It is critical that the beekeeping industry reduce their reliance on chemical treatments for diseases and mites due to the risk of contaminating hive products with residue, and the development of resistance to the treatments by the pathogen and parasites. The most sustainable solution is to selectively breed bees for resistance to diseases and mites. To date, hygienic behavior is one of the few resistance mechanisms that has a simple field assay that beekeepers can employ called a freeze-killed brood assay. A commercially available assay could then be developed using the same olfactory cues the bees detect in diseased brood furthering our efforts to breed bees that display hygienic behavior against them. This could have important implications for the health of honey bees, the beekeeping and agricultural industries and in turn ensure the future of an ample human food supply.

This information has been presented at numerous outlets. Ms. Swanson presented talks at two Minnesota Hobby Beekeepers Association monthly meetings, Minnesota Honey Producers Association 2007 annual meeting, the American Beekeeping Federation 2008 annual meeting and Entomological Society of America 2007 annual meeting. Marla Spivak presented these findings to over 25 different professional and public meetings of beekeepers, scientists and the general public in 11 states across the US including 12 talks to groups within MN. She also presented in Peru, Chile, Argentina, and Nordic-Baltic countries (including Norway, Denmark, Sweden, Finland, Estonia, Latvia, Lithuania).