- Animals: bees
- Animal Production: Beekeeping
Honey bees are susceptible to a number of diseases and parasitic mites, which are commonly treated with antibiotics and pesticides in commercial apiaries. Hygienic honey bees are those that detect, uncap and remove diseased brood from the colony before it reaches an infectious stage, thus these bees are able to reduce disease transmission. Previous studies suggest this is a behavioral response to olfactory cues. This study aims to determine the chemical mechanisms behind this stimulus through the following objectives. Objective 1: Determine differences between the chemical profiles of chalkbrood-infected and healthy honey bee larvae. Objective 2: Determine the compounds unique to disease brood that are detected by adult honey bees. Objective 3: Confirm that hygienic behavior is a response to olfactory stimulus through the development of bioassay in which synthetic compounds of disease odors are introduced into the colony. By using bees bred for hygienic behavior, beekeepers are able to reduce or discontinue their reliance on chemical treatments. The long-term goal of this project is to aid in the development of a commercially available field test that will allow beekeepers to determine if their honey bee colonies are hygienic and select these colonies for breeding purposes.
Project objectives from proposal:
The long-term goal of this project is to aid in the development of a commercially available field test which will allow beekeepers to determine if their honey bee colonies are hygienic and select these colonies for breeding purposes. Hygienic honey bees are those that detect, uncap and remove diseased brood from the colony before it reaches an infectious stage, thus these bees are able to reduce disease transmission. By using bees bred for hygienic behavior, beekeepers are able to reduce or discontinue their reliance on chemical treatments.
The short-term goals of this project are:
1) to determine differences between the volatile chemical composition of healthy honey bee larvae and those that are infected with the fungal disease chalkbrood, and
2) to determine which of these compounds are detected by honey bees.
The intermediate-term goal of this project is to confirm that hygienic behavior is triggered by olfactory cues by developing a bioassay that involves introducing synthetic compounds of diseased brood into the hive to elicit the removal behavior.
Context, Background and Rationale:
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 (Spivak, 1996; Spivak and Reuter, 2001a, b; Ibhrahim et al., in press). Research in M. Spivak’s lab suggests that hygienic bees respond to olfactory cues coming from the abnormal brood (Masterman et al., 2001; Spivak et al., 2003).
Currently, the field method used to test for hygienic behavior is called a freeze-killed brood assay (Spivak and Reuter, 2005). This assay can be accomplished in one of two ways. A piece of comb containing pupae (“sealed brood”) may be cut from the frame and frozen for 24 hours. The comb is then replaced into the frame and returned to the colony. Alternatively, a hollow cylinder may be placed on a section of comb containing sealed brood. Liquid nitrogen is then poured into the cylinder, killing all brood within. The frame is then returned to the hive. For either method the frame is checked after 48 hours and the amount of dead brood removed is recorded. Hygienic colonies will remove the dead brood within 24 hours, whereas non-hygienic bees will not (Spivak, 2005 SARE fact sheet). The most hygienic colonies are selected for breeding purposes.
The removal of freeze-killed brood is generally correlated with the removal of diseased and parasitized brood, but it would be better to have a test that is more realistic without having to challenge the colonies with disease. The freeze-killed brood test requires killing brood, is time consuming, and may involve the use of dangerous materials (i.e. liquid nitrogen). My goal is to develop a more accurate assay that uses the same olfactory cues the bees detect in diseased brood to improve breeding programs for hygienic behavior.
The results of this study will increase our knowledge of the mechanisms behind hygienic behavior and offer beekeepers a safer, more efficient way of testing their colonies for the trait. 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.
Hygienic behavior was first recognized in the 1930’s when researchers, searching for natural mechanisms of disease resistance, observed adult bees removing diseased larvae. Since that time, it has been determined that this behavior confers resistance because the adult bees remove the diseased larvae before it reaches the infectious stage of the disease (Woodrow, 1942). Additionally, Rothenbuhler (1964) demonstrated that the behavior is a recessive trait genetically controlled by two recessive loci. Current studies, using modern molecular techniques, are testing the validity of Rothenbuhler’s findings (Lapidge et al., 2002; B. Oldroyd and P. Oxley, personal communication)
I am determining the chemical cues associated with the fungal disease chalkbrood Ascosphaera apis. I am using this disease as this research requires the use of diseased material and it is easy to extract this material from the colony. Chalkbrood was first discovered in the United States in 1968. By 1980, chalkbrood was observed in honey bee colonies in all 50 states (Heath, 1985). Chalkbrood is a heterothallic fungus meaning there are two strains which must come in contact for the fungus to form fruiting bodies (Gilliam, 1997). Spores are inadvertently fed to young larvae through the brood food deposited in the cells by adult bees. The fungal spores reside in the gut until the larvae is capped to begin pupation at which time sporulation occurs. The fungal mycelium and spores penetrate the gut wall and eventually the outer cuticle of the larvae. The larvae take on a characteristic mummified appearance. The disease is only transmissible after the spores have penetrated the outer cuticle. There is currently no commercially available treatment for chalkbrood. Hygienic behavior is a mechanism of resistance against chalkbrood because the bees remove the diseased brood before the spores penetrate the outer cuticle (Gilliam et al., 1983).
Approach, Activities, Methods and Inputs:
Objective 1: Determine differences between the chemical profiles of chalkbrood-infected and healthy honey bee larvae.
The volatile compounds released from honey bee larvae can be collected by placing the larvae in collection vials. Air is passed over the larvae and is drawn through a Super Q filter which absorbs the volatile compounds. The filter is then rinsed with dichloromethane. This rinse is analyzed by gas chromatography coupled with mass spectrometry (GC-MS). I have already completed this work with Drs. Baldwyn Torto and Jim Tumlinson at the USDA in Gainesville, Florida. I collected volatiles from both healthy larvae and those showing early stage symptoms of chalkbrood disease. I have analyzed the chromatograms and identified several compounds which are unique to diseased larvae including many alcohols and their byproducts.
Objective 2: Determine the compounds unique to disease brood that are detected by adult honey bees.
To determine which compounds are detected by the honey bees, I will be using gas chromatography (GC) coupled with electroantennographic detection (EAD). This is a common method of determining compounds that are biologically relevant to an insect. The rinse from the collection of volatiles is injected into the gas chromatograph. The solution is run through a glass column which separates the constituent compounds based on the chemical properties (i.e. polarity). After running through the column, the concentrations of the compounds are divided. A portion goes to a flame ionization detector which detects the compounds and measures their concentrations. The other portion is delivered to an excised insect antenna which is placed on a probe and connected to an amplifier. The probe-amplifier measures the difference in the electrical charge of the antenna as the compound (odor) is passed over it. The data generated from this technique consists of two graphs which are chronologically aligned so that a peak on the GC graph is lined up with the peak from the EAD corresponding to the insect’s response to that compound. I have started this work and tentatively identified three compounds that elicit responses from honey bees: benzyl alcohol, phenyl ethanol and phenyl ethyl acetate. I will verify this portion of the investigation in early summer 2007 and expect to have it completed by July.
Objective 3: Confirm that hygienic behavior is a response to olfactory stimuli.
This objective will be met by developing a bioassay involving introducing synthetic compounds (identified in Objectives 1 and 2) onto healthy brood to determine if the chemical cues alone will elicit removal behavior. I have begun developing this bioassay and am currently searching for a method of carrying the compounds and introducing them on to healthy brood so that the bees remove only those treatments that contain the diseased compounds. I will be continuing this work with chemists at the USDA in Florida during the winter and plan to complete it in Minnesota during the summer of 2007. Once this bioassay is successfully developed, it will be modified into a commercially available test that beekeepers can use to test the hygienic status of their colonies. They can then select those colonies that display hygienic behavior for breeding and expand their use of hygienic bees. A future study could expand this bioassay to include compounds produced by other honey bee diseases and parasitic mites.
I will present the results of my research at state and national beekeeping association meetings (e.g., MN Honey Producers, American Beekeeping Federation) and professional meetings (American Bee Research Conference, Entomological Society of America). I will also present the findings through extension activities of the Spivak lab such as beekeeping short courses and queen breeding courses. I plan to publish my findings in Journal of Chemical Ecology and bee research journals (e.g. Apidologie).
The success of this research will be based on beekeeper feedback on use of the end product (synthetic compound bioassay). This success will be evaluated by the number of sales of the product through beekeeping supply companies and advertisements in bee journals and supply catalogs.