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

Final Report for GNC07-083

Project Type: Graduate Student
Funds awarded in 2007: $10,000.00
Projected End Date: 12/31/2008
Grant Recipient: University of Minnesota
Region: North Central
State: Minnesota
Graduate Student:
Faculty Advisor:
Marla Spivak
University of Minnesota
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Project Information


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 and confirmed with field bioassays that hygienic behavior is a response to olfactory cues.


I investigated the chemical stimuli that individual honey bees detect in diseased larvae that initiate hygienic behavior. Hygienic behavior is defined as the detection and removal of diseased larvae from the nest (Rothenbuhler 1964). Hygienic behavior has been recognized as an important natural mechanism of disease resistance in honey bee colonies since the late 1930’s (Woodrow and Holst, 1942). One of the most important aspects of hygienic behavior is that individual bees must detect and respond to appropriate stimuli from the diseased larvae early in the progression of the infection, before the pathogen becomes infectious. In this way, the quick and efficient detection and removal of the diseased larvae by many bees prevent disease transmission throughout the colony.

Specifically, we investigated the hygienic response of bees to the fungal pathogen, Ascosphaera apis, which causes chalkbrood disease in larvae. Chalkbrood infection begins in the gut of a larva and slowly grows outward until it penetrates the cuticle before pupation begins. At this point, rapid growth and sporulation of the fungus occurs (Gilliam, 1997). If adult bees remove infected larvae after the fungus has penetrated the cuticle, spores are ingested by adult bees and fed back to healthy larvae in the bee food. The uninfected larvae ingest the spores and the infection perpetuates. Therefore, for hygienic behavior to be effective against chalkbrood, the adult bees must remove the infected larva before the fungus has penetrated the cuticle.

I tested 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.

Project Objectives:

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. Develop a bioassay that involves introducing synthetic compounds of diseased brood into field colonies to directly test if hygienic behavior can be elicited by the compounds alone.


Materials and methods:

The breeding program for colonies that display hygienic behavior began in 1993 at the University of Minnesota. Rapid-hygienic and slow-hygienic lines of bees (sensu Wilson-Rich et al., 2009) were selected from colonies derived from the Italian subspecies Apis mellifera ligustica. Colonies were initially selected based on their ability to survive winter in Minnesota, their ability to increase in population rapidly in spring, to produce large amounts of honey, and to be gentle (not sting) during normal management. If the colonies displayed the above set of traits they were then tested for hygienic behavior, using a freeze-killed brood assay (Spivak and Reuter, 1998), which is an indirect measure of a colony’s hygienic behavior. The removal of freeze-killed brood is correlated with the removal rates of diseased brood; colonies that uncap and remove approximately 160 freeze-killed pupae within 24 hr will tend to remove diseased brood rapidly. Colonies that exhibited the most rapid removal rates were chosen to raise queens for the next generation. Daughter queens were then instrumentally inseminated with sperm from drones from other rapid-hygienic colonies. The criterion for choosing rapid-hygienic colonies for breeding is an arbitrary cutoff established at 95% removal of freeze killed brood within 24 hr.

Volatile Collection
Volatiles were collected from bees for two purposes. The first purpose identified the changes in volatiles between healthy and diseased brood. Three types of larvae were used, including:
a) healthy fifth instar larvae,
b) fifth instar larvae in the early stage of chalkbrood infection, and
c) larvae fully overcome with fungal mycelia (mummies).
Each type of larvae was removed from the comb with sterile forceps and placed in quick-fit glass aeration chambers. The disease was confirmed by fungal growth through the larvae after the volatile collections were completed. Collections were determined to be from healthy larvae if no larvae showed clinical symptoms of disease at the conclusion of the volatile collection period. Each collection consisted of either
a) 25 healthy fifth instar larvae,
b) 25 early-stage infection larvae, or
c) 75 mummies.
For use with the electroantennography, the second collection of volatiles employed a large quantity of healthy larvae or diseased larvae placed in a larger aeration chamber.

For each experiment, the volatile collection apparatus was constructed as follows: charcoal-filtered and humidified air at 0.5 L/min was passed over the larvae and then through pre-conditioned Super-Q adsorbent traps for 24 hr at room temperature. Each trap was eluted with 150 l methylene chloride forced through the absorbent with filtered nitrogen gas. The elutions stored at -70°C until used.

Chemical Analysis
To identify the disease-related volatiles, Samples were analyzed by gas chromatography and the chromatograms were compared from the healthy, early stage infected and fully diseased brood. Volatile compounds were confirmed by comparison of their chromatographic retention times and mass spectra with commercially available standards analyzed on the same instrument. To confirm detection by bees of these identified compounds, gas chromatography linked electroantennal detection (GC-EAD) was used for the diseased-brood samples, the healthy larvae samples and the standards. The effluent was split to allow simultaneous recording of the flame ionization detector (FID) and the antennal response (EAD). Electroantennogram recordings were obtained from excised antennae of bees that were age 15-20 days old as this is the age of bee that would be performing hygienic behavior (Arathi et al., 2000). Three source colonies used for the GC-EAD tests were from the University of Minnesota breeding program and were deemed greater than 95% hygienic based on freeze killed brood tests.

Field Bioassays:
I. Topical Applications. The 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. The assays were conducted in August, 2007. Twelve colonies were chosen for the bioassay based on the freeze-kill removal method. A comb containing larvae was removed from each of the twelve colonies and the position of 25 fifth instar larvae on the comb was marked on a transparency overlaying the comb. A Picospritzer II (General Valve Corporation) was used to dispense 0.5 l of each treatment compound, onto healthy larvae. Six treatments were applied to separate groups of 25 healthy larvae. The treatment compounds were benzyl alcohol, 2-phenylethanol (phenethyl acetate) and deionized water. The sixth treatment was 0.5 l of a mixture of equal parts benzyl alcohol, 2-phenylethanol, and phenethyl acetate. The comb was then returned to the colony from which it was taken. At 4 and 24 hr the transparency was replaced on the frame and the number of larvae that the adult bees had removed was recorded.

II. Paraffin larval dummies. The assay utilizing paraffin larval dummies 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. Paraffin larval dummies were made by first melting paraffin wax at 60-65oC. A 10-2 dilution of methyl linolenate was made with the wax. Methyl linolenate is one of several compounds present in brood pheromone of fifth instar larvae. When this compound is added to a paraffin wax dummy and placed in a larval cell, adult bees cap the cell containing the dummy (Le Conte, 1990). The addition of chalkbrood volatile compounds to wax dummies containing brood pheromone would result in a decrease in capping of these dummies if the adult bees detect the disease compounds. The response threshold model can be tested by measuring the differential abilities of adult bees to detect these chalkbrood compounds at variable concentrations.

From this mixture of wax and methyl linolenate a total of eight treatment compound /concentrations were made: phenethyl acetate at 10-2 ml/ml and 10-9 ml/ml, 2-phenylethanol at 10-2 ml/ml and 10-9 ml/ml, benzyl alcohol at 10-2 ml/ml and 10-9 ml/ml and a mixture of the previous three compounds at 10-2 ml/ml and 10-9 ml/ml. Additional treatments were made of methyl linolenate alone and paraffin wax without the addition of methyl linolenate or disease compounds. The liquid wax containing each treatment was poured into plastic drinking straws and allowed to cool and harden. The straw was then cut away from the wax and the wax cut into 25 mm sections to create larval dummies.

The assays were conducted in August 2008. Six colonies that displayed varying degrees of hygienic behavior, as determined through freeze-killed brood assays, were chosen for this bioassay. A comb containing fifth instar larvae was removed from the colony. Combs with fifth instar larvae were chosen so that the larval dummies would be in context with actual fifth instars that were about to be capped with wax by adult bees. Twenty larval dummies of each treatment were placed into empty cells and their location was marked on a transparency. No more than two treatments were tested on a single comb at a time. The combs were then returned to the colony and the number of dummies the bees capped with wax was recorded after 24 hr.

Statistical Analysis
Binomial regressions were performed to compare the proportion of healthy topically treated larvae removed or paraffin dummies that were left uncapped in response to each treatment with the degree of hygienic behavior of the colony as a covariate. The delta method was used to calculate the student’s t value for contrasts between treatments with differences declared at  =0.05. Analyses were done with Arc (Cook and Weisberg, 1999).

Research results and discussion:

Chemical Analysis
Three peaks associated with diseased brood were non-detectable in the extractions of the healthy brood, appeared in the early stage diseased larvae and increased in the fully diseased larvae. Identified and confirmed by GC/MS, these compounds included phenethyl acetate, 2-phenylethanol and benzyl alcohol. Via GC-EAD, bee antennae responded in a consistent and repetitive pattern from the volatiles of larvae that were in the early stages of chalkbrood infection.

Field Bioassays:
I. Topical applications. Colonies with higher degrees of hygienic behavior removed significantly more treated larvae at 4 hr (t = 4.844, df = 54, P < 0.001) and 24 hr (t = 8.917, df = 54; P 0.05).
At four hr, most larvae treated with 2-phenylethanol, benzyl alcohol, the mixture and water were not removed. However, significantly more larvae were removed that were treated with phenethyl acetate compared to all other treatments.

At 24 hr, all compounds tested were removed to some degree. Significantly more larvae were removed that were treated with phenethyl acetate compared to 2-phenylethanol, benzyl alcohol, the mixture and water. The mixture of compounds did not elicit increased removal of the larvae, but significantly more larvae treated with a mixture of the compounds were removed compared to larvae treated with 2-phenylethanol, benzyl alcohol and water.

II. Paraffin larval dummies. Bees were unable to physically remove the paraffin dummies from the cell at nest temperatures (32-34oC) as the paraffin became soft and sticky. Therefore in this assay when brood pheromone was added to the paraffin dummy, if the bees placed a wax capping over the dummy it was an indication that the adult bees accepted the 5th instar as normal. When both brood pheromone and disease volatile(s) were added to the paraffin, if the bees did not cap the dummies with wax, it corresponded to higher levels of detection of the disease volatiles and therefore of hygienic behavior.

None of the colonies capped paraffin dummies in which no brood pheromone or diseased larval compounds were added. The addition of the brood pheromone, methyl linolenate, to the paraffin dummies resulted in a significant increase in capping. Capping of brood pheromone dummies ranged from 70% to 95%.

As predicted, when dummies were treated with both brood pheromone and one or a mixture of the disease volatiles, the bees in all colonies capped significantly fewer dummies treated with higher concentrations (10-2 ml/ml) of the diseased larvae compounds compared to dummies treated with lower concentrations (10-9 ml/ml) for all compounds; phenethyl acetate (t = 4.437, df = 9, P < 0.001), 2-phenylethanol (t = 9.778, df = 9, P < 0.001), benzyl alcohol (t = 7.388, df = 9, P < 0.001) and the mixture (t = 3.138, df = 9, P = 0.001).

There was no significant interaction between treatment and hygienic behavior (all P > 0.05) (again, colonies of all levels of hygienic behavior responded similarly to all treatments). Colonies with higher degrees of hygienic behavior capped significantly fewer larval dummies (P < 0.001).

At the higher concentration (10-2 ml/ml) there was no significant difference between the numbers of dummies capped that were treated with phenethyl acetate, the mixture and paraffin alone. Phenethyl acetate and the mixture were both capped significantly less than 2-phenylethanol and benzyl alcohol. All compounds at the 10-2 ml/ml concentration were capped significantly less than the brood pheromone alone, methyl linolenate.

At the lower concentration (10-9 ml/ml) phenethyl acetate and the mixture were both capped significantly less than methyl linolenate, 2-phenylethanol and benzyl alcohol. There was no significant difference between the numbers of dummies capped that were treated with methyl linolenate and 2-phenylethanol, between methyl linolenate and benzyl alcohol or between 2-phenylethanol and benzyl alcohol. There was not a significant difference between phenethyl acetate and the mixture.

This is the first study to identify volatile compounds present in diseased honey bee larvae that elicit hygienic behavior by honey bees. Three volatile compounds collected from larvae infected with the fungal pathogen, Ascosphaera apis, were not detected in healthy brood, and subsequently shown to be antennally active. Our bioassays showed that one of the three compounds, phenethyl acetate, was the key volatile that elicited appropriate behavioral responses by bees in large field colonies.

Our field bioassays confirmed two hypotheses, previously tested only in laboratory assays (Arathi and Spivak, 2001; Spivak et al., 2003): 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.
Colonies that contain a majority of bees with high olfactory sensitivity respond quickly to low concentration stimuli associated with diseased brood, while colonies of bees with lower sensitivity take longer to respond, allowing expression and transmission of clinical symptoms.

Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

This research has been submitted for publication to the Journal of Chemical Ecology: “Odorants that induce hygienic behavior in honey bees: identification of volatile compounds in chalkbrood-infected honey bee larvae.” Authors: Jodi A. I. Swanson, Baldwyn Torto, Stephen A. Kells, Karen A. Mesce, James H. Tumlinson and Marla M. Spivak.

Jodi Swanson defended her Master’s thesis in November 2008 titled: Volatile Compounds from Chalkbrood Infected Larvae Elicit Honey Bee (Apis mellifera L.) Hygienic Behavior.

Jodi Swanson gave oral presentations titled “Determination of volatile compounds that elicit removal of diseased brood by hygienic honey bees” at the following venues:
• Minnesota Hobby Beekeepers Association. St. Paul, MN. April 2008.
• American Beekeeping Federation. Sacramento, CA. January 2008.
• Entomological Society of America Annual Meeting. San Diego, CA. December 2007.
• Minnesota Hobby Beekeepers Association. St. Paul, MN. April 2007.
• Minnesota Honey Producers Association. St. Cloud, MN. December 2006.

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).

Project Outcomes

Project outcomes:

The results of this study have a practical application for beekeepers and important implications for the health of honey bees. It is critical that the beekeeping industry reduce their reliance on chemical treatments for diseases and parasitic 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. Colonies that rapidly remove freeze-killed brood tend to be behaviorally resistant to two brood pathogens, chalkbrood (A. apis), and American foulbrood (Paenibacullus larvae) (Spivak and Reuter, 2001). Colonies that rapidly remove freeze-killed brood also tend to detect remove pupae infested with the parasitic mite, Varroa destructor (Spivak, 1996). However, the correspondence between the removal of freeze-killed brood and removal of diseased or parasitized brood is not perfect. Also, colonies selected for hygienic behavior still require treatments to control V. destructor as the bees are not able to detect and remove sufficient infested pupae at high mite infestations (Spivak and Reuter, 1999, 2001; Ibrahim et al, 2007). While hygienic behavior is a generalized response to the presence of diseased, parasitized, and abnormal pupae, it is necessary to have a more specific assay to selectively breed colonies for resistance to a particular pathogen or parasite. The methods employed in this study could be modified into a commercially available test that beekeepers can use to test for hygienic behavior in their colonies.

Economic Analysis

Honey bees are a crucial component to sustaining the agricultural industry in the United States. They are important pollinators of vegetable and seed crops (e.g., alfalfa, sunflowers, vine crops), commercial fruits (e.g., apples, cranberries, blueberries, strawberries), and home fruit and vegetable gardens. Varroa mites are largely responsible for the decimation of feral honey bee colonies in the United States. Therefore, human maintenance of honey bee colonies is necessary for our agricultural systems to continue current levels of production. The costs associated with controlling diseases and parasites have become prohibitive for many beekeepers resulting in a national decline in the number of commercial and hobby beekeepers.

A commercial beekeeper with 2500 colonies currently spends $15,000-$30,000 yearly to treat his/her colonies with ApiGuard® ($6.00/treatment, 2 treatments/yr/colony), ApiLife Var® ($5.67/treatment, 2 treatments/yr/colony) or Mite Away II® ($3.00/treatment, 2 treatments/yr/colony) to control Varroa mites. 2500 colonies is an average number of colonies necessary to sustain a livelihood. On an aggregate basis, Minnesota beekeepers spend approximately $990,000 each year on these pesticides alone. When antibiotics are added in to prevent the spread of bee diseases (Tylan costs $625/2500 colonies) these costs increase. A sustainable alternative to pesticides and antibiotics is the use of hygienic honey bees. An efficient and comparably inexpensive test for hygienic behavior will increase beekeeper use of these bees, which will in turn increase the number of colonies an individual can afford to maintain.

Farmer Adoption

Employees and students of the University of Minnesota bee lab are currently demonstrating the paraffin larval dummy assay with beekeepers in California to test for hygienic behavior in their colonies. The response to date has been positive. We are planning to demonstrate this method to beekeepers in Minnesota, North Dakota, Texas and Florida during the summer of 2009.

Without the costs associated with pathogen prevention and treatment, farmers we may see an increase in the number of farmers who implement their own beekeeping operations for the pollination of their crops.


Areas needing additional study

The experiments performed here could be modified to focus on the specific stimuli that elicit detection and removal of larvae infected with the American foulbrood pathogen and pupae infested with V. desitructor, furthering efforts to breed bees for resistance to these economically important problems


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Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.