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? 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 worth more than $14 billion to US agriculture, and honey production, the primary income source for beekeepers. 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. Relevant to National Program 301, Plant, Microbial and Insect Genetic Resources, Genomics and Genetic Improvement, Problem Areas 1b, Conserving Genetic Resources, 2a, Genome Characterization and 3c, Genome Databases and Bioinformatics and to National Program 305, Bees and Pollination. 2.List by year the currently approved milestones (indicators of research progress) FY2004 No milestones. FY2005 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. FY2006 Determine the optimum cryoprotectant agent (CPA) for honey bee semen. Identify new sperm storage genes from the honey bee genome sequences. FY2007 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. FY2008 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. 4a.List the single most significant research accomplishment during FY 2006. Optimum cryoprotectant and extenders for cryopreserved honey bee semen. A scientist from the Bee Research Laboratory has determined that dimethyl sulfoxide (DMSO) is the most effective cryoprotectant agent for honey bee sperm, although ethylene glycol also works. Two extenders work equally well, a modified Kiev solution or commercial poultry extender. A freeze/thaw cycle has been developed that results in sperm viability of greater than 50%, the known threshold for useful semen. Impacts National Program 301, 1b Conserving Genetics Resources and Associated Information Efficiently and Effectively. 4b.List other significant research accomplishment(s), if any. Identification of proteins of reproductive tissues. A team from the Bee Research Laboratory and two other BARC/ANRI Laboratories have successfully purified proteins from virgin and queen reproductive tissues, the spermatheca (storage organ) and its associated gland. 129 different proteins were identified from one or both of the tissues, including a gene found in the immune set. Comparisons were made with several mammals and the fruit fly, as well as the honey bee genome project. These identifications will be further developed and included in the ongoing annotation of the honey bee, Apis mellifera, genome. Related to National Program 301, 2a, Genome Characterization. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. 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. Results apply to National Program 301, 1b Conserving Genetics Resources and Associated Information Efficiently and Effectively and NP 305, Component III, 2b Bee Management and Pollination. 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. A spectrophotometric assay for spermatheca contents was applied by a BRL scientist to demonstrate that semen stored at room temperature for 2 weeks or more resulted in reduced sperm storage by an artificially inseminated queen, although the viability of the sperm was high up to 8 weeks. A likely explanation of the failure of stored sperm to migrate to the queen’s storage organ is that all energy sources were exhausted during storage. A semen extender to supply energy to the sperm must be developed to make room temperature storage a viable alternative methodology. Additionally, the assay was used to determine that drone-laying queens (those that fail to fertilize eggs) may sometimes have sufficient stored sperm, but fail for other reasons. This is a new observation about these queens. 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? Several queen breeders in the US and Europe have contacted the Bee Research Laboratory for information about semen extenders and semen handling. Results of completed research have allowed us to provide significant useful information to these industry users. Several honey bee queen breeders are using room temperature storage of semen as a part of their approach to artificial insemination of queens. This allows for shipment of semen and for adjustment of semen collection and insemination to accommodate bad weather. 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. (NOTE: List your peer reviewed publications below). Presented the talk “Proteomics and genomics of honey bee sperm storage.” At the Honey Bee Molecular Workshop, July 30, 2006. Washington, D.C. Organized a symposium titled “Current Topics in Reproductive Physiology” and presented the talk “Sperm storage in Apis mellifera, proteomics, genomics and technology “, XV Congress, International Union for the Study of Social Insects, Aug. 1, 2006, Washington D.C. Collins, A. M. Honey bee germplasm preservation. Bee Craft. 88(6): 11-14. 2006. Review Publications Collins, A.M., Caperna, T.J., Williams, V.P., Garrett, W.M., Evans, J.D. 2006.Proteomic analyses of male contributions to honey bee sperm storage and mating. Insect Molecular Biology. 15(5):541-549. Collins, A.M., Pettis, J.S., Wilbanks, R., Feldlaufer, M.F. 2006. Survival and function of queens reared in beeswax containing coumaphos. American Bee Journal. 146(4):341-344. Collins, A.M., Mazur, P. 2006. Chill sensitivity of honey bee, apis mellifera, embryos. Cryobiology. 53:22-27.