2004 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? There has been significant loss of genetic diversity in the honey bee population of the United States due to the introduction of two parasitic mites, Varroa destructor and Acarapis woodi, and the development of antibiotic resistance by the bacteria that causes American foulbrood disease. The long term solutions for these problems, along with the improvement of economically important honey bee traits, depend on selective breeding. The technology to preserve honey bee germplasm, both semen and embryos, will provide an important tool to maintain the genetic variability necessary for effective selection by both scientists and commercial breeders. High quality genetic stock is necessary to maintain healthy honey bee colonies for pollination and honey production. This project has three specific goals: 1. optimize cryopreservation technology and develop non-frozen storage methodology for semen; 2. understand the genetics and physiology of natural sperm storage by the queen; and 3. develop cryopreservation of honey bee embryos. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY04) No milestones. Year 2 (FY05) Develop new semen quality assays for evaluation of honey bee semen. Develop optimum diluents (extenders) for non-frozen and cryopreserved honey bee semen. Develop non-frozen semen storage as an alternative system. Determine the source tissues of antioxidant genes that are important to natural sperm storage. Develop a system for permeabilization of embryo membranes. Year 3 (FY06) Determine the optimum cryoprotectant agent (CPA) for honey bee semen. Identify new sperm storage genes from the honey bee genome sequences. Year 4 (FY07) Determine the optimum freeze/thaw cycle for cryopreservation of honey bee semen. Develop embryo vitrification/cytoskeletal stabilization protocols. Determine the optimum freeze/thaw cycle for cryopreservation of honey bee embryos. Modify laboratory techniques for rearing cryopreserved larvae for queen production. Year 5 (FY08) Evaluate the functional capacity of stored semen using artificial insemination (AI) of queens. Characterize and bank desirable honey bee semen. Using gene function assays, determine the function of newly identified genes. Confirm that queens reared from cryopreserved embryos can be artificially inseminated and produce normal offspring. Characterize and bank desirable honey bee embryos. 3.Milestones: A. List the milestones (from the list in Question #2) that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. None of the milestones are scheduled to be completed in FY04. 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 milestone? Year 2 (FY05) Develop new semen quality assays for evaluation of honey bee semen including metabolic, motility and fertilizing capability assays. These assays will allow breeders to choose highly effective drones for semen storage. Develop optimum diluents (extenders) for non-frozen and cryopreserved honey bee semen. Extend the biochemical description of semen so that naturally occurring components can be included in an extender, and determine the best osmolality and pH levels. Using the best extenders for each storage protocol will produce the maximum survival of spermatozoa. Develop non-frozen semen storage as an alternative system, including time frames and conditions of storage, and the development of naturally occurring components as additives to extend shelf life. The impact of a non-frozen system will be practical short-term storage for queen breeders that requires little specialized equipment. Determine the source tissues of antioxidant genes that are important to natural sperm storage. Use additional biological samples to screen for antioxidant sources in bees and refine RNA-level tests of these sources to use for determining mechanisms behind sperm mortality. Identification of the tissue sources will help our understanding of the biological control of natural sperm storage. Develop a system for permeabilization of embryo membranes, with an emphasis of testing several enzymes that create pores in membranes. Also improve diluents and washing/rearing equipment for treated eggs. Successful completion of this process is necessary to developing a cryopreservation technology for embryos. Year 3 (FY06) Determine the optimum cryoprotectant agent (CPA) for honey bee semen by comparing alternatives to previously used DMSO, including the optimum concentration to be used, using sperm viability assays. Determine the possible impact of the chosen CPAs on artificially inseminated queens. The impact of this research will be a CPA that will allow for maximum sperm survival and not harm queens during AI. Identify new sperm storage genes from the honey bee genome sequences. Search the honey bee genome for genes involved with both sperm storage by queens and the production and viable transport of sperm, based on known genes from other organisms, especially fruit flies. Identification of the genes related to semen production and storage will allow us to understand the natural process and improve semen storage technology. Year 4 (FY07) Determine the optimum freeze/thaw cycle for cryopreservation of honey bee semen using DMSO as the CPA. Compare slow cool and freeze cycles as used with mammals with flash freezing, the technique successfully used on fruit fly embryos. Verify that the freeze/thaw cycle works for the optimum cryoprotectant. Develop practical systems and commercial contacts for beekeepers to accomplish frozen storage. The impact of this research will be to maximize the survival of frozen sperm and to facilitate the transfer of the technology to the commercial sector. Develop embryo vitrification/cytoskeletal stabilization protocols. Evaluate the effectiveness of several penetrating and non-penetrating CPAs and skeletal stabilizing compounds for use with freezing honey bee embryos. Identification of an effective CPA and stabilizing compounds will make honey bee embryo cryopreservation possible. Determine the optimum freeze/thaw cycle for cryopreservation of honey bee embryos., based on fruit fly work and the best cycle for use semen. Optimizing the temperature cycle will impact the maximum survival levels for embryo cryopreservation. Modify laboratory techniques for rearing cryopreserved larvae for queen production. Develop a washing setup for embryos that can be used for incubator rearing as well, and then verify that incubator reared larvae will be accepted by colonies to rear queens. Because stored embryos are very delicate, they must be reared to the one day larval stage in the laboratory. The impact is that honey bee colonies must accept these larvae as normal to mesh with the existing commercial queen rearing technology. 4.What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: A scientist at the Bee Research Laboratory has shown that honey bee queens inseminated with low viability (>50% live) semen will function as well as normally inseminated queens for at least one summer season. Previous work showed that queens with this level of viable sperm would store sperm effectively and produce normal amounts of worker brood (fertilized eggs) for three weeks. Queens were inseminated with various levels of fresh and freeze-killed semen (25% live to 100% live sperm) and compared for the amount of worker vs. drone (unfertilized eggs) offspring produced. Large amounts of worker larvae are necessary for successful queen production. Queen breeders using stored semen to reconstruct selected stocks would normally expect to use an inseminated queen as a queen mother for virgins and drones for only one summer. B. Other Significant Accomplishments, if any: Scientists of the Bee Research Laboratory have used molecular genetic techniques to identify the spermatheca and spermathecal gland of queens as the key source for the antioxidant enzyme, catalase, and have shown a rapid increase in production of this protein upon queen mating. They have also shown an overproduction of genes encoding this protein in sperm-producing tissues. Reproductive tissues from virgin, newly mated and older laying queens, and drones of various ages, were compared for levels of expression of several antioxidant genes and for a well-expressed housekeeping gene. This information can help explain some failures by queens to store sperm effectively, and will assist in the development of germplasm storage technology. Use of naturally occurring compounds/enzymes should increase the viability of stored sperm such that this technology can be profitably used by scientific and commercial queen breeders. C. Significant Activities that Support Special Target Populations: None. D. Progress Report: None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This research project started 9/10/2003 and replaced Project Number 1272-21000-182-00D, Preservation of Honey Bee Germplasm. The work and accomplishments of the previous project are: In cooperation with the Germplasm and Gamete Physiology Lab, Beltsville, MD, a dual fluorescent staining technique to assess sperm viability was developed for use with honey bee semen. The impact of this achievement is that researchers now can utilize a direct method to assess semen viability rather than the lengthy process of artificially inseminating queens. A scientist of the Bee Research Laboratory demonstrated that queen bees artificially inseminated with semen of greater than 46% viability were able to produce normal patterns of offspring. This result set a lower limit for acceptable levels of sperm survival during storage. Semen stored at room temperature was still viable enough for use with AI at 6 months and an improved method for pooling semen from many drones was developed using slower centrifugation, 82 g for 20-30 minutes, yielding semen of high viability. The impact of this research is that beekeepers producing queens may store semen for several months and use semen that has less than 100% viability in breeding programs. Techniques were developed for: a. the collection of honey-bee eggs that allows removal from the hive with little mortality; b. the removal of the outer protective layer of the egg in preparation for preservation by freezing; and c. an in incubator system for the hatching of eggs that results in close to 100% survival. The impact of this work is that each of these steps is necessary for collection and preparation of embryos for frozen storage and the rearing of stored embryos for use by beekeepers rearing queens for sale to others in the industry. Optimum conditions for all stages of handling stored embryos will ensure the maximum output from a process that normally has limited survival. By isolating RNA found in queen storage organs and semen, two scientists at the Bee Research Laboratory, Beltsville, MD, have identified three oxygen-protective enzymes that are at high levels in mated queens and mature drones. At least one of these enzymes could increase the survival of sperm stored at room temperature or by deep freezing, thus impacting our ability to keep high quality semen beyond the lifetime of queens or drones. 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? The dual fluorescent staining assay for direct evaluation of semen viability is now available to other scientists. Several laboratories have consulted with a Bee Research Laboratory scientist on applications of this technology. The major limitation is the requirement for a light source on the microscope with the proper fluorescent wave lengths. Scientists at the Bee Research Laboratory were contacted to preserve the genotype of the queen providing drones for sequencing the honey bee genome. Non-frozen stored semen from this queen's drones was used to produce brother-sister artificial inseminations with her daughters. Granddaughters have been provided to other scientists and to a commercial breeder, and have been naturally mated at the Bee Research Laboratory as drone sources for future work. Of specific interest are possible crosses of known single gene genotypes to the sequenced background. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Invited presentation on laboratory research to the American Beekeeping Federation, Jacksonville, Fl, Jan 14, 2004, Perkiomen Valley Apiaries, Obelisk, PA. May 1, 2004, and Lehigh Valley Beekeeper¿s Association., May 20, 2004. Allentown, PA Presentation to the Apiary Inspector's Workshop, Beltsville, MD. March 2, 2004. Article in the Baltimore Sun, June 14, 2004 on germplasm preservation and artificial insemination and its use in a project on honey bee immune responses. Article called "Honeybee sex gene discovered" in Nature (Aug. 18, 2003). Review Publications Collins, A.M., Evans, J.D., Williams, V.P. 2004. Antioxidant gene expression and sperm storage in the honey bee apis mellifera. Insect Biochemistry and Molecular Biology. 13:141-146. Collins, A.M. 2004. Variation in time of egg hatch by the honey bee, apis mellifera (hymenoptera: apidae). Annals of the Entomological Society of America. 97(1): 140-146. Collins, A.M. 2003. Effective viability threshold for preserved honey bee semen. Reproduction, Fertility and Development. 16:166. Evans, J.D., Shearman, D., Oldroyd, B. 2004. Molecular genetics of honey bee sex determination. Trends in Ecology and Evolution. 19(1):1-3.