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?
Honey bee colonies are threatened by numerous parasites, pathogens, and pests, including Varroa and tracheal mites, bacterial diseases like American foulbrood (AFB) and an assortment of bee viruses, all of which affect colony well being. Using a combination of chemical, cultural and genetic methods, the Bee Research Laboratory is investigating these threats and developing integrated pest management strategies to deal with them in a safe and environmentally acceptable manner. This project has three specific goals over the next five years:.
1)Investigate honey bee pathogens and mitigate their impact on bee colonies;.
2)improve honey bee colony health through the management of parasitic mites; and.
3)identify and investigate factors that negatively impact queen longevity and durability and develop strategies to mitigate their effect.

2.List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY2004) Conduct in vitro laboratory screening of new antibiotics for their ability to inhibit the bacterium that causes American foulbrood disease of honey bees.

Develop analytical assay methods for selected antibiotics.

Modernize our bee disease diagnosis record keeping.

Provide expert diagnosis of honey bee diseases to our customers/stakeholders.

Year 2 (FY2005) Investigate the seasonal incidence of honey bee viruses at the colony level.

Determine the toxicity of selected antibiotics when applied to honey bee colonies and the ability of specific antibiotics to control American foulbrood disease.

Screen potential compounds for their ability to inhibit and control the parasitic protozoan Nosema.

Characterize genomic libraries of honey bees to identify genes involved in the immune response.

Optimize the use of formic acid gel to control parasitic mites of honey bees.

Year 3 (FY2006) Develop an alternative assay for assessing antiprotozoal compounds that do not rely on live honey bees.

Screen the honey bee genome using microarrays for genes involved in the response to disease.

Determine the effects of miticide residues on queen bee performance.

Year 4 (FY2007) Determine the role of the parasitic mite Varroa in the transmission of honey bee viruses.

Conduct and evaluate breeding experiments to verify the contribution of specific genes to their ability to help bees fight disease.

Evaluate drone (male) bee trapping techniques as a form of mite control.

Conduct etiological studies on the effect of Nosema on queen bees.

Year 5 (FY2008) Develop a honey bee cell line that can be used for virus propagation. Collect and submit all necessary data required for FDA registration of antibiotics.

Evaluate field colonies for genetic variation in their responses to disease.

Test and evaluate combined control strategies (formic acid gel, drone trapping, resistant queens) to control parasitic mites of honey bees.

Assess the effect of miticides and Nosema on queen performance and investigate Nosema-induced queen supersedure (replacement) studies.

3.Milestones:
A. List the milestones (from the list in Question #2) that were scheduled to be addressed in FY04. How many milestones did you fully or substantially meet in FY2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so.

All milestones for FY04 were completed.

Conduct in vitro laboratory screening of new antibiotics for their ability to inhibit the bacterium that causes American foulbrood disease of honey bees.

Develop analytical assay methods for selected antibiotics.

Modernize our bee disease diagnosis record keeping.

Provide expert diagnosis of honey bee diseases to our customers/stakeholders. This milestone is "service-oriented" and is done year after year. Therefore, we will not address it under Question 3B.

B. List the milestones (from the list in Question #2) that you expect to address over the next 3 years (FY 2005, 2006, 2007). What do you expect to accomplish, year by year, over the next 3 years under each of the milestones?

FY2005 Investigate the seasonal incidence of honey bee viruses at the colony level. By comparing and monitoring honey bee colonies with varying levels of virus with uninfected colonies, we hope to determine the impact of virus infection on colony survivability. This will allow us to determine the economic impact of viral infections.

Determine the toxicity of selected antibiotics when applied to honey bee colonies and the ability of specific antibiotics to control American foulbrood disease. The impact of this research will be the identification of antibiotics that are both safe to bees and can control a devastating bacterial disease affecting immature bees.

Screen potential compounds for their ability to inhibit and control the parasitic protozoan Nosema. There is only one compound currently registered for Nosema control and manufacturing problem may result in its removal form the market. The impact of our research will be to identify alternative compounds for Nosema control.

Characterize genomic libraries of honey bees to identify genes involved in the immune response. These genes may help fight disease and thereby contribute to future bee breeding programs.

Optimize the use of formic acid gel to control parasitic mites of honey bees. We plan to optimize the treatment regime for a previously developed formic acid gel. The impact will be an improved method of controlling parasitic mites of honey bees.

FY2006 Develop an alternative assay for assessing antiprotozoal compounds that do not rely on live honey bees. The requirement for newly-emerged bees in screening experiments places seasonal restrictions on research. The development of in vitro assay systems will allow year-round research on the identification of new compounds to control Nosema disease of honey bees.

Screen the honey bee genome using microarrays for genes involved in the response to disease. The identification of additional genes that are involved in the honey bee's response to disease will impact our ability to determine the underlying reasons why some bees are more successful in fighting disease than others. Determine the effects of miticide residues on queen bee performance. We will evaluate the impact of pesticides on the health of honey bee queens, by determining rearing, mating, and oviposition success. Impact is to increase queen longevity and durability, thereby eliminating the need to replace queens more frequently than expected. FY 2007 Determine the role of the parasitic mite Varroa in the transmission of honey bee viruses. We plan to examine vertical transmission of bee viruses and compare transmission efficiency to that of mite (horizontal) transmission, in an effort to determine the contribution of each to the overall epidemiology of viral disease. Impact is to make colony management recommendations to reduce the impact of viral diseases of honey bees.

Conduct and evaluate breeding experiments to verify the contribution of specific genes to their ability to help bees fight disease. Functional genetic assays will allow a bee breeding program that incorporates honey bees with heightened natural defenses toward disease agents. Develop drone (male) bee trapping techniques as a form of mite control. We plan to develop and refine a non-chemical control strategy for parasitic mites that relies upon removal of brood bees that selectively attract mites. Impact is a mite control strategy that does not rely on chemicals.

Conduct etiological studies to determine the effect of Nosema infection on queen bees. We plan to artificially infect newly-emerged queens with the parasitic protozoan Nosema and subsequently compare mating success and egg-laying with uninfected individuals. Impact is to assess the contribution of Nosema to queen failure.

4.What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY2004: Detection of deformed wing virus in the U. S. Scientists at the Bee Research Laboratory in Beltsville MD detected deformed wing virus in honey bees and in parasitic mites of honey bees. This virus has never been detected in the U. S. and may provide an answer to previously unexplained honey bee colony losses. Using molecular techniques, scientists detected this virus in different stages of honey bees, including adult bees with deformed wings, normal-appearing adult bees, eggs, larvae, and pupae. The detection of virus in normal-appearing adults suggests that virus titers may have to reach certain levels before the infection results in deformed wings and has a subsequent negative economic impact. An additional outcome is that the detection of virus in bee stages not associated with mite parasitism suggests there are alternate ways for the virus to spread within colonies.

B. Other Significant Accomplishment(s), if any: Development of methodologies to detect multiple virus infections Scientists at the Bee Research Laboratory in Beltsville MD have developed a laboratory method that allows the simultaneous detection of multiple bee viruses in a single honey bee. Honey bees can often suffer from multiple virus infections and it is not possible to diagnose these infections in the field since many of the viruses produce no apparent symptoms. Using molecular techniques, scientists designed, developed and tested an accurate procedure for detecting six honey bee viruses in a single reaction. This method is quick, accurate and cost-effective, and can be used to isolate and quarantine infected colonies to prevent the spread of disease. The method can also be used by regulatory agencies to determine the virus status of bee colonies from countries interested in exporting bees to the United States.

Honey bee disease genomics A scientist at the Bee Research Laboratory has used genomic approaches to identify novel components in the honey bee immune response and has shown how bees regulate gene expression when facing disease agents. A Bee Research Laboratory scientist also serves on the Advisory Committee for the Honey Bee Genome Project and is coordinating efforts to screen this genome for genes important to honey bee health. These efforts will ensure that the Genome Project provides results that improve both honey bee management and honey bee breeding.

C. Significant Activities that Support Special Target Populations: none.

D. Progress Reports: none.

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
This research project started 09/10/2003 and replaced Project Number 1275-21000-143-00D, CONTROL OF PARASITIC MITES, PATHOGENS & OTHER PESTS OF HONEY BEES & IMPROVEMENT OF COLONY VIGOR. The work and accomplishments of the previous project are:

A formic acid gel treatment was developed to control parasitic mites of honey bees that offers near 100 percent control of the tracheal mite and also suppresses the level of Varroa mites. A patent was awarded (U.S. Patent 6,037,374 ¿ Composition and method for the control of parasitic mites of honey bees) and licensed. The gelled formic acid is being developed for commercial distribution to beekeepers, nationwide, to control parasitic mites of honey bees.

A modified hive bottom board/screened insert was developed as a physical barrier to prevent fallen Varroa mites from re-attaching to adult bees. Impact is a relatively inexpensive, non chemical method of mite control that can be used in an integrated pest management program.

A cultural control strategy that relies on circulating air to reduce humidity was developed to eliminate small hive beetle damage in honey combs awaiting extraction. Impact is a relatively inexpensive, non chemical control of small hive beetle in honey houses.

Genetic markers were developed for the small hive beetle that permitted scientists to determine both the source and point of introduction of this invasive honey bee pest. The impact is the ability to monitor the spread of this beetle.

Research was conducted to reduce the impact of a honey bee parasitic mite that is devastating honey bee colonies nationwide, by developed precise genetic markers that allow for the measurement of mite movement between and within colonies. Potential impact is designing non chemical mite control strategies based on mite population dynamics.

Research was conducted to reduce the impact of American foulbrood disease, a devastating bacterial infection of honey bees. Antibiotics were examined for efficacy in controlling the disease, and reports were submitted to FDA to gain approval for their use in bee colonies. Research was conducted to answer questions regarding the origin of antibiotic resistance in a honey bee bacterial pathogen. Scientists at the Bee Research Laboratory in Beltsville MD used molecular techniques to investigate over 100 bacterial strains for resistance or susceptibility to antibiotics. Specifically, they determined that bacterial resistance occurred over a wide geographic range and did not arise from a single source. Impact is that beekeepers and the beekeeping industry must carefully monitor all honey bee populations for disease and have infected colonies tested for bacterial resistance. Research was conducted to determine if chemicals used to control parasitic mites of honey bees negatively impacted development of honey bee queens. Scientists at the Bee Research Laboratory in Beltsville MD reared honey bee queens in beeswax containing known amounts of a pesticide used to control parasitic mites. Scientists determined the specific level of pesticide that would inhibit queen development. Impact is that careful monitoring of pesticide in beeswax is necessary to produce viable queen bees.

Research was conducted to evaluate the role of parasitic mites in transmitting honey bee viruses. Scientists at the Bee Research Laboratory in Beltsville MD demonstrated conclusively that parasitic mites of honey bees are capable of transmitting viruses from infected bees to uninfected bees. Using molecular techniques, researchers were able to calculate the transmission efficiency from mites to bees, and demonstrate that non infected mites can acquire virus by sharing the same cell with one or more infected mites. Impact is a better understanding of how bee viruses are spread between bees and emphasizes the importance of mite control.

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?
Information about a high density polyethylene (HDPE) plastic film that is suitable for a formic acid gel packaging has been transferred to the licensee.

Our effort to register two antibiotics for the control of American foulbrood disease of honey bees have been transferred to our customer groups through national meetings and popular articles. The actual technology should become available in FY2004. Constraints include lengthy review by FDA.

Sequence and expression data of honey bee genes have been registered in public access databases available to other scientists. The entire sequence of the honey bee genome and the bacterium that causes foulbrood disease will be available in FY2004.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work.
Invited presentations of laboratory research to the American Honey Producers Association and the American Beekeeping Federation, January 2004. Laboratory presentations to Apiary Inspectors Workshop, March 2004. Article entitled "Scientists Tap Bee Genome Map for Improved Traits" in February 2004 issue of Agricultural Research.

Review Publications
Chen, Y., Pettis, J.S., Evans, J.D., Feldlaufer, M.F. 2004. Molecular evidence for transmission of kashmir bee virus in honey bee colonies by ectoparasitic mite, varroa destructor. Apidologie. 35(4):441-448.