2006 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? Why does it matter? Throughout the U.S. beekeeping industry, significant losses of colonies and revenue are caused by diseases, parasites, pesticides, and other factors. Research conducted in the laboratory and field is necessary in order to understand and limit the damage caused by the parasites varroa and small hive beetle, by the bacterial diseases American foulbrood and chalkbrood, by Africanized honey bees, and by pesticides used either within the hive or on crops visited by bees. Fluvalinate- and coumaphos-resistant varroa have been documented across the U.S. This is a serious problem because varroa is considered by U.S. beekeepers as the most serious parasite attacking honey bees in their operations and in the wild. The small hive beetle continues to infest bee colonies throughout the entire eastern U.S., it has recently been found in Texas, and it will likely spread further westward. Pesticide exposure to bees, both within the hive and in crops they visit during foraging, is a continuing and serious problem for the majority of beekeepers who provide bees for pollination. The combined losses inflicted by parasites, diseases, and pesticides have put in jeopardy the livelihood of many beekeepers, and have resulted in shortages of pollinating bees for commercial agricultural crops. The molecular basis for insecticide resistance in bees is lacking and needs definition. Genomic work on the honey bee is needed to facilitate major advances in identifying toxins associated with bee diseases. The long distance transport of honey bees for pollination is a major source of physiological stress, in addition to that caused by pests, diseases, and pesticides. Project work addresses all of the above research needs, and will result in development of holistic approaches for a complete integrated pest management scheme to optimize colony health. Honey bee bacterial and fungal pathogens are responsible for substantial economic losses to the beekeeping industry. Research conducted in our Molecular Laboratory continues in order to understand host-pathogen interactions, molecular mechanisms that are involved in honey bee innate immune responses to microbial pathogens, and identification of virulence factors (including toxins) produced by the entomopathogenic bacteria Paenibacillus larvae, and their role in establishing the honey bee disease American foulbrood (AFB). Sequencing of bacterial and fungal genomes of the major honey bee pathogens would provide the first opportunity for interactive whole genome analysis of an insect host (Apis mellifera) and its natural microbial pathogens. Antibiotics are routinely used to control microbial pathogens in honey bees, but rapid development of antibiotic-resistant microbial strains and growing concern for residues in honey bee products prompted the need to investigate the use of alternate compounds with antimicrobial activity. The over-use of antibiotics in food production has triggered resistance in human and livestock bacterial pathogens. The conventional antibiotic oxytetracycline (OTC) remains the only registered antibiotic to control AFB in honey bees, but the development of the OTC-resistant bacterial strains lessens its efficacy. The antibiotic Tylosin was found to control OTC-resistant bacterial species. It is highly effective against gram-positive bacterial isolates, but has no inhibitory effect on fungal bee pathogens. There is no chemotherapeutic agent registered in the U.S. for use against chalkbrood disease. Given the failure of conventional antibiotics to control the full spectrum of bee diseases and concern for residues in honey bee products, the search for natural products will provide an avenue for safe and effective solutions to this problem. The project contributes to the Bees and Pollination component of the Crop Production National Program (NP 305) by reducing the impact of hive pests, thus increasing crop pollination and honey production. The project contributes to the Pest Control Technologies, and Integrated Pest Management Systems and Areawide Suppression Programs components of NP 304 (Crop Protection and Quarantine) by developing new pest control products and techniques that can be utilized by all beekeepers. To facilitate achievement of project objectives, Specific Cooperative Agreements are in place with Texas A&M University, the Baylor College of Medicine, and the University of Georgia. This project is supported, in part, by outside funds via Trust Agreements with Vita, Ltd. and Wellmark Intl.; these funds are facilitating research progress at an accelerated pace. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2004): 1. Establish colonies of comparative bee lines. 2. Evaluate biorational varroa compounds in standard bee lines. 3. Evaluate IPM tactics on pollination. 4. Establish package bee colonies in virgin equipment and foundation; expose subsets of selected acaricides/insecticides and evaluate colony vigor; analyze samples for production of stress proteins. 5. Describe honey bee microenvironment. 6. Evaluate pesticidal stress. Year 2 (FY 2005): 1. Establish new colonies. 2. Evaluate chalkbrood-resistant lines. 3. Evaluate IPM tactics of biorational compounds on bee pollination efficiency. 4. Describe honey bee microenvironment. 5. Evaluate pesticidal stress. 6. Assess colony vigor in susceptible vs. tolerant colonies infested with chalkbrood. 7. Investigate molecular mechanisms of honey bee resistance to the fungal pathogen A. apis. Year 3 (FY 2006): 1. Establish new bee colonies. 2. Provide data of pesticide efficacy, bee toxicity, and residues to EPA. 3. Establish small hive beetle study colonies. 4. Evaluate IPM tactics on small hive beetle. 5. Assess colony vigor in susceptible vs. tolerant colonies infested with chalkbrood. 6. Assess colony stress due to long-range transport of hives. 7. Establish apiaries for testing effects of transporting queen reproductive fitness. 8. Assess stress on queens due to long-range transport of hives. Year 4 (FY 2007): 1. Assess biorational compounds on chalkbrood-resistant bee lines. 2. Evaluate IPM tactics on small hive beetle. 3. Assess stress on queens due to long range transport of hives. 4. Transfer technology developed on chalkbrood and hive beetle control. 5. Povide data of pesticide efficacy, bee toxicity, and residues to EPA. Year 5 (FY 2008): 1. Complete studies ongoing from previous years. 2. Transfer technology to appropriate users. 3. Provide data of pesticide efficacy, bee toxicity, and residues to EPA: Additional full scale field trials in the U.S. to include residue trials in preparation for request of EPA registration for a marketable product. 4a.List the single most significant research accomplishment during FY 2006. Discovery of a New Honey Bee Immune-Related Gene Insect innate immunity involves a number of the intracellular signaling pathways, which control expression of the immune related molecules. Scientists at the Kika de la Garza Subtropical Agricultural Research Center have identified a number of genes from honey bee genome that are members of Toll and IMD intracellular signaling pathways; we cloned five Toll-related receptors from honey bee Apis mellifera and investigated their potential involvement in antimicrobial immune response. This work will allow development of a conceptual model of humoral immune responses in honey bees and development of a new drugs and methods to increase host resistance to microbial pathogens. (This project directly supports ARS National Program 305, Crop Production.) 4b.List other significant research accomplishment(s), if any. Discovery of RNA silencing (RNAi) in Honey Bees is Systemic Scientists at the Kika de la Garza Subtropical Agricultural Research Center have developed the in vivo challenge assay of the honey bee larvae with the microbial pathogens and modified the RNA interference assay (dsRNA) using honey bee larval injections and larval feeding with the RNA probes. As a culmination of several years of laboratory effort, we have discovered that RNA silencing (RNAi) in honey bee is systemic, by showing that feeding or soaking bees with a new class of regulatory compounds leads to silencing of gene expression in honey bee larvae and therefore, very specifically, changes bees' genetic response. This is significant because it identifies a very useful genetic mechanism that can be used by honey bee researchers to quickly turn off specific genes in an entire bee larvae in order to study their function. (This project falls under NP 305, Crop Production.) 4c.List significant activities that support special target populations. None 4d.Progress report. We have sequenced the genome of the important honey bee bacterial pathogen Paenibacillus larvae. This research was conducted under a Specific Agreement (58-6204-3-024) between USDA-ARS and Baylor College of Medicine Sequencing Center (BCM-HGSC). Our preliminary analysis of the P. larvae sequences resulted in identification of the bacterial genes that encode for various virulence factors (Zn-Metalloproteases, binary ADP-ribosylating toxins, Vegetative Insecticidal Toxins, and Ricin-type toxins) produced by this pathogen and potentially involved in host invasion, inhibition of the host immune system and host death. In collaboration with BCM-HGSC, we have isolated two mating types of honey bee fungal pathogen Ascosphaera apis, isolated genomic DNA and generated sequence data for the first mating type. Sequencing of the second fungal mating type is in progress. We found a wide variation in soluble protein levels in both newly emerged bees and bees sampled from broodnests in colonies pollinating almonds. We are currently evaluating possible interaction of nutrition and parasitism as a factor in colony collapse. A study comparing Australian versus U.S. honey bee colonies for pollinating almonds was conducted. These colonies were imported into the United States from Australia to make up for a projected deficit of colonies needed for almond pollination because of varroa mite control problems. We found that Australian four-pound package colonies established in late January 2006 performed about half as well as standard U.S. over-wintered colonies. Before we can begin to address the honey bee stress issues pertaining to long distance transport of honey bees for pollination, it is imperative that we ensure the colonies are healthy and not stressed prior to transportation. The almond industry requires colonies to be strong early in the season, thus many beekeepers have resorted to feeding their colonies with pre-collected pollen or artificial diet. We are studying the effect these practices have on long distance transport of honey bees. In a field study, four different protein diet treatments (fresh pollen, old pollen, and two commercial products, Bee Pro and Feed Bee) were used to study their effects on worker weight, longevity, and hemolymph protein levels. The three parameters studied produced consistent results, indicating that honey bees fed exclusively fresh pollen were as healthy or healthier than bees fed Feed Bee. Those fed Bee Pro or old pollen were the least healthy. In a laboratory cage experiment, there were four different treatments implemented to study the effects of worker consumption, worker weight, and hemolymph protein levels. Again, the parameters studied were consistent and indicated similar results as seen in the field study. Honey bees consumed Feed Bee as well as fresh pollen. Different types of sugars and quantities of sugars were analyzed in two commercially produced protein diets and from pollen collected by honey bees. More toxic sugars were found in the soy-based protein diet than in the pollen sample or Feed Bee. In a laboratory study, the moisture retention of two commercially produced protein diets (Feed Bee and Bee Pro) were compared to that of fresh frozen pollen. Feed Bee held moisture almost as well as fresh pollen. 5.Describe the major accomplishments to date and their predicted or actual impact. For the first time a new gene that encodes for Toll-related signal transducing receptor was identified in Hymenoptera. This work will allow development of a conceptual model of innate humoral immune responses in honey bees and development of new drugs and methods to increase host resistance to microbial pathogens. For the fist time we have shown systemic nature of the RNAi in honey bee and investigated the mechanism underlining this biological phenomenon. Sequences encoding for bacterial virulence factors (including toxins) have been identified from the genome of the honey bee pathogen Paenibacillus larvae. An understanding of bacterial toxins is relevant to understanding of the disease and the way to control it. Considering the recent emergence of antibiotic-resistant P. larvae bacterial strains, the identification of toxins produced by this pathogen is increasingly important and will potentially lead to development of the antimicrobial drugs that disrupt the process of the host invasion and prevent establishment of the disease. All accomplishments made under this project are fully consistent with relevant milestones listed in the Project Plan and with relevant research components as defined in the National Program 305 Action Plan. Accomplishments under this project contribute to the achievements of ARS Strategic Goal 3. 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? NONE 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Aronstein, K.A. 2006. Honey bee genomic research in Weslaco, Texas. Honey Producer's Conference, Houston, TX, January 12-13, 2006. Aronstein, K.A. 2006. Draft sequence of Ascosphaera apis genome. International Union for the Study of Social Insects Congress VX, Washington, DC, July 29 - August 4, 2006. Eischen, F.A. 2005. Controlling Varroa destructor with the fungus Metarhizium (and other products). North Dakota Beekeepers Meeting, Bismarck, ND, October 1-2, 2005. Eischen, F.A. 2006. Controlling Varroa destructor with Hivastan (fenpyroximate) in honey bee colonies. Western Apicultural Society Meeting, Dallas, TX, July 10-11, 2006. Review Publications Aronstein, K.A., Pankiw, T., Saldivar, E. 2006. SID-1 is implicated in systemic gene silencing in the honey bee. Journal of Apicultural Research. 45(1):20-24. Cabanillas, H.E., Elzen, P.J. 2006. Infectivity of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) against the small hive beetle Aethina tumida (Coleoptera: Nitidulidae). Journal of Apicultural Research. 45(1):49-50.