2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? More honey bee colonies are needed now by U.S. agriculture than ever before to pollinate crops. However, fewer colonies are available. Modern beekeeping places extraordinary stress on honey bees from overcrowded apiaries, frequent movement of colonies for pollination, and challenges from diseases and parasitic mites. Another major problem facing the U.S. Beekeeping Industry is the Africanization of European honey bees. Colonies from across the U.S. are moved to pollinate more than $15 billion worth of crops each year. Some enter areas with feral African populations (e.g., California for fruit and nut pollination). If colonies are moved into regions with African honey bees, they could become Africanized. When colonies are returned to their home apiaries, Africanized bees could be spread to non-Africanized regions of the U.S. The project has three specific goals:. 1)creating an artificial diet that is comparable in nutritional value to pollen,. 2)developing methods to control Varroa population growth in honey bee colonies and. 3)preventing the invasion of European colonies by swarms of African bees. A minor project that we are conducting in cooperation with the USDA, APHIS, PPQ, CPHST, Soil Inhabiting Pest Laboratory, Gulfport, MS is to test the effects of fire ant repellents on honey bee colony foraging behavior and mortality. Fire ants nest beneath bee equipment and can be spread when colonies are moved between states for pollination. In addition to testing repellents, we also are developing bait stations that can be used for inspection of honey bee colonies while in transit. The device will enable inspectors to determine quickly whether fire ants are on equipment being moved from a fire ant infested area to one that is free of fire ants. Accomplishments related to fire ant repellents and baits are under Subordinate projects. The research falls within National Program 305 (Crop Production) Component 3 Section B (Bee Management and Pollination). Specifically, the project addresses the goals to:. 1)identify nutritional deficiencies that contribute to colony decline during pollination and develop/improve supplemental feeds, and. 2)determine factors that promote interbreeding and colony takeovers by African bees. Developing new methods to control Varroa mites falls within the goals of Bees and Pollination Section A. (Pest Management). Attaining our Objectives will benefit beekeepers, queen breeders, and growers producing crops that require honey bee pollination for seed/fruit set. An artificial diet will help insure that colonies remain populous when flowering plants are not in bloom and while hives are transported for pollination. New methods to control Varroa will reduce the dependency on chemical miticides. Preventing European colony invasion by African swarms will help to prevent the spread of African bees and insure that pure European colonies are available for pollination. Scientists will benefit from the information we will generate on honey bee nutrition and population interactions between honey bees and Varroa mites. 2.List the milestones (indicators of progress) from your Project Plan. Sub-Objective 1.1: Develop patty diet Year 1 (2005) *Determine components needed for patty diet and test formulations for texture and stability. *Collect pollen from colonies to make pollen patties for feeding studies. *Identify phagostimulants in pollen *Write manuscript on nutritional needs of honey bees Year 2 (2006) *Begin feeding trials with different formulations of diet to determine those most palatable to bees. *Conduct brood survival, longevity, and hemolymph protein analyses on colonies fed artificial diets. *Include compounds that are phagostimulants in diet mixtures to determine if they increase palatability. *Write manuscript on factors that stimulate and deter feeding. Year 3 (2007) *Add phagostimulants to the diet to determine if it causes bees to consume more diet. *Determine if and how long colonies can rear brood when fed exclusively on the patty diet. *Conduct brood survival, longevity, and hemolymph protein analyses on colonies fed artificial diets. *Write manuscript on the effects of diet on brood rearing. Year 4 (2008 *Continue tests to determine if and how long colonies can rear brood when fed exclusively on the patty diet containing phagostimulants. *Conduct tests on longevity of adult workers fed the patty diet while as larvae and as adults. *Conduct brood survival, longevity, and hemolymph protein analyses on colonies fed artificial diets. *Apply for possible patent for diet Year 5 (2009) *Continue longevity tests. *Begin testing of patty diets in commercial beekeeping operations. *Write manuscripts on feeding patty Sub-Objective 1.2: Identify methods to incorporate miticides into liquid and solid artificial diets to control Varroa mites. Year 1 (2005) *Begin choice tests with essential oils in the diet at various concentrations using adult worker bees. Year 2 (2006) *Track oils fed in the liquid diet to adult bees, nurse bees, and larvae. *Measure cell infestation and reproductive rates of the mites. *Begin choice tests with essential oils in the patty diet at various concentrations using adult worker bees. Year 3 (2007) *Continue to measure cell infestation and reproductive rates of mites. *Write manuscript on the effects of essential oils fed in diets on mite reproduction. Year 4 (2008) *Determine minimum amounts of essential oils needed to reduce mite populations. *Write manuscript on the effects of essential oils on host volatiles and mite host finding and reproductive behaviors. Sub-Objective 2.1: Identify volatile chemical cues from different lines of mite-susceptible and mite-resistant bee stock and from Varroa mites that might affect Varroa host or mate funding behaviors. Year 1 (2005) *Start colonies of various resistant and susceptible lines. Release Varroa into colonies. *Begin collecting volatiles from larvae and pupae *Write manuscript on volatile profiles from resistant and tolerant bee lines. Year 2 (2006) *Continue collection of volatiles from larvae, mites, and cells. *Test for seasonal differences in volatiles from samples. *Begin identification of compounds. *Write manuscript on seasonal changes in chemical profiles of bees and mites. Year 3 (2007) *Continue identification of compounds. Year 4 (2008) *Continue identification of compounds. Sub-Objective 2.2: Evaluate reproductive rates of mites when exposed to selected compounds isolated from Objective 2.1. Year 1 (2005) N/A Year 2 2006 N/A Year 3 (2007) *Begin behavior bioassays to identify compounds that influence mite reproduction. *Write manuscript on effects of compounds on mite reproductive behaviors Year 4 (2008) *Continue bioassays with single compounds and combinations. *Write manuscript on effects of compounds (various amounts and ratios) on mite reproduction. Year 5 (2009) *Continue bioassays with single compounds and combinations. manuscripts *Write on influence of various volatile compounds on mite reproduction rates. Sub-Objective 3.1: Determine effects of usurpation on worker-queen interactions. Year 1 (2005) *Establish European colonies. *Begin filming worker-queen interactions as usurpations occur. *Refine sampling procedures as needed. *Collect data from films. Year 2 (2006) *Continue filming and data collection of worker-queen interactions during usurpation events. *Write manuscript on nest usurpation behaviors. Year 3 (2007) *If necessary, continue filming and data collection. *Write manuscripts of worker-queen interactions during usurpation events. Year 4 (2008) N/A Year 5 (2009) N/A Sub-Objective 3.2: Characterize pheromone profiles of invading bees Year 1 (2005) *Sample volatiles from swarm and host colony bees. *Adjust sampling procedures if needed. *Begin compound identification. *Write manuscript on differences in volatiles from colonies depending upon their state. Year 2 (2006) *Continue sampling volatiles from swarm and host colony bees. *Continue compound identification. Year 3 (2007) *Continue sampling volatiles from swarm and host colony bees. *Continue compound identification. *Begin analysis to determine if volatiles from swarm workers change as usurpation progresses. Year 4 (2008) *Continue analysis of the changes in volatiles. *Continue compound identification. *Write manuscripts on the role of volatiles in the usurpation of honey bee Year 5 (2009) N/A 4a.What was the single most significant accomplishment this past year? Beekeepers need an artificial diet to feed bees during times when flowering plants are unavailable and brood rearing in the colony declines. In collaboration with a CRADA partner, we addressed the need for an artificial diet by creating a liquid protein diet. Bees fed the liquid diet maintain hemolymph protein levels that are significantly higher than bees fed pollen or other commercial diets currently available and live as long as those fed with pollen. A patent was filed for the diet formulation, and commercial production should begin within a year. The diet will have tremendous impact on both the beekeeping industry and the production of crops requiring honey bee pollination because colonies that continuously rear brood have large populations and the ideal age structure for pollination. 4b.List other significant accomplishments, if any. Alternative Controls for Varroa: The most serious problem affecting the survival of honey bee colonies is Varroa mites, because they have become resistant to both miticides registered for their use. Dr. Diana Sammataro is developing a systemic treatment for Varroa using naturally occurring essential oils fed in a diet. We screened numerous essential oils for miticidal properties, and developed an emulsification procedure to mix the essential oils with a carrier that is fed to bee larvae. The first carrier we tried was a sugar syrup solution and while consumption of the syrup/oil mixture was high, very little oil was found in the larvae and the treatments had almost no impact on mite reproduction. We added the emulsified oils in our liquid protein diet, and it proved to be a compatible carrier and mite reproduction levels were significantly reduced. The impact of the research will be to provide several new control compounds for Varroa mites using the liquid protein diet as a delivery system. We found that a highly volatile compound (2-heptanone) made by bees in the hive during comb building is a very potent miticide. However, before it could be effective in controlling Varroa, a delivery system that maintained levels of 2-heptanone that could impact mites for 42 days needed to be developed. We have been conducting experiments with a new delivery system where 2-heptanone is microencapsulated in corn starch and coated with a mixture of sugar and wax. The bees tear the tablet apart and thus release the compound. In our pilot tests, certain formulations caused mite mortality rates that were comparable to commercially available miticides used on nonresistant populations. The impact of the research will be to provide a new method to control Varroa that is both biodegradable and leaves no residues in the wax or colony equipment. Determine factors leading to the usurpation of European honey bee (EHB) colonies by African bees: A major impediment to maintaining European colonies in Africanized habitats is that small swarms of African bees invade and usurp European colonies especially if they are queenless. Dr. David Tarpy has investigated the role that chemical signals produced by colonies and queens might have on the success of colony invasions. Dr. Tarpy determined that usurping queens do not have a volatile profile that differs grossly from that of non-usurping queens, thereby ruling out one hypothesis for how usurpation queens are able to safely enter the host nest. However, there is a change in the gestalt colony odor as a result of queen loss that might be a cue used by usurpation swarms to detect susceptible European colonies. The impact of this research is that by understanding the chemical cues and behaviors used by African bees to find and usurp vulnerable European colonies, tools and management practices could be developed that would prevent colony invasions. 4c.List any significant activities that support special target populations. None. 4d.Progress report. We discovered that 2-heptanone, a compound made by honey bees during comb building, is an excellent miticide against Varroa. However, 2-heptanone is very volatile. Designing a delivery system for 2-heptanone that would release the compound over a 42-day period in the hive at an effective dose that did not disrupt colony behavior was a major challenge. This year we established a collaboration with the chemists at Bioproducts Research Laboratory, Western Regional research Center, USDA-ARS, Albany, CA. We have developed a method to microencapsulate 2-heptanone so it does not disrupt colony behavior, and is released for 42 days at doses that effectively reduce mite populations. In addition, the delivery system is made from natural products (corn starch, sugar, and wax) and is completely biodegradable so no residues are left in the colony. The microencapsulation system we developed for 2-heptanone is also being used for other miticides we are testing such as essential oils. The oils are microencapsulated in corn starch, mixed in our liquid diet, and fed to bees. Our preliminary data indicate that there might be reduced Varroa feeding and reproduction in cells with larvae fed diet with essential oils. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. The customers for our research are beekeepers, growers that relay on honey bees for pollination and the scientific community. Our work that resulted in a liquid protein diet solves a major problem in the beekeeping and pollination industries; i.e., how to keep colony populations growing when there are insufficient amounts of natural pollen and nectar available. The studies were conducted under the Milestone 1 (Determine components needed for patty diet and test formulations for texture and stability) for Sub-Objective 1.1 (Develop patty diet) and fulfills the National Program 305 (Crop Production) Component 3 Section B (Bee Management and Pollination) goals to identify nutritional deficiencies that contribute to colony decline during pollination and develop/improve supplemental feeds. 2-heptanone is an effective miticide against Varroa and provides an alternative compound for mite control. Currently, Varroa is resistant to miticides registered for its use, so new compounds are urgently needed. The research was a previous subordinate project (5342-21000-012-01T, Cerexagri, Using natural products to control mites parasitizing honey bees) conducted under National Program 305 (Crop Production) Component 3 Section A (Pest Management): Developing new methods to control Varroa mites falls within the goals of Bees and Pollination Section A. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Manufacturing and distribution of the liquid protein diet is scheduled to begin in the Fall of 2005. Review Publications Schneider, S.S., Deeby, T., Gilley, D.C. and DeGrandi-Hoffman, G. Seasonal nest usurpation of European colonies by African swarms in Arizona, USA. Insect. Soc. 51:359-364. 2004. DeGrandi-Hoffman, G., Curry, R. Description and Validation of a Mathematical Model of Varroa Mite (Varroa destructor) and Honeybee (Apis mellifera L.) Population Dynamics. 2004. Internat. J. Acarol. 2004. Vol. 30, No. 3. Sammataro, D., Untalan, P., Guerro, F., Finley, J. The resistance of Varroa mites (Acari: Varroidae) to acaricides and the presence of esterase. 2005. Internat. J. Acarol. Vol. 31, No. 1. 67-74. Hanna, A., Schmidt, J. Effect of phagostimulants in artificial diets on honey bee feeding behavior. Southwestern Entomologist. 2004. Vol. 29, No. 4. 253-261. DeGrandi-Hoffman, G., Curry, R. The population dynamics of Varroa mites in honey bee colonies: Part I - The VARROAPOP program. Amer. Bee J. 2005. Vol. 145(7):592-595. DeGrandi-Hoffman, G., Curry. R. Simulated population dynamics of Varroa mites in honey bee colonies: Part II - What the VARROAPOP model reveals. Amer. Bee J. 2005. Vol. 145(8): 629-632. DeGrandi-Hoffman, G., Chambers, M., Hooper, J.E., Schneider, S.S. Description of an intermorph between a worker and queen in African honey bees Apis mellifera scutellata (Hymenoptera: Apidae). Ann. Entomol. Soc. Am. 2004. 97:1299-1305. Schneider, S.S., DeGrandi-Hoffman, G. Mechanisms that favor the continuity of the African honeybee genome in the Americas. Proc. 8th IBRA Intl. Conf. Tropical Bees and VI Encontro sobre Abelhas, Ribeirao Preto, SP, Brazil. 2004. pp. 232-240.