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? Honey bees are vital to US agriculture because of their value as pollinators of important crops such as almonds, apples, and squash. Honey has less economic value than the billions of dollars attributed to pollination activities by bees, yet honey provides the major source of income for most beekeepers. Both pollination and honey production require healthy colonies of bees, and honey bee populations in the USA are under constant attack by Varroa destructor, an external parasite of the honey bee. This parasite is the number one problem in the beekeeping industry in the USA and worldwide. Until the late 1960's, this mite was restricted to Southeast Asia as a parasite of Apis cerana, the Asian hive bee. It now has worldwide distribution. If a susceptible colony of bees is not protected with miticide, it dies within two years. Because of chemical contamination issues, cost of treating with miticides, and the ability of these mites to develop resistance to miticides, our long-term solution is to breed honey bees for resistance to varroa. The primary goal of this research is to control the parasitic mite (Varroa destructor) by breeding honey bees for resistance. Our approaches to this problem are as follows: 1. Continue work with suppressing mite reproduction, the SMR trait. To improve breeding strategies, we will (1) determine how many genes are involved in the SMR trait (our preliminary data suggest that there are two); (2) determine if the SMR trait is associated with other characteristics of bees; (3) establish the SMR trait into inbred lines; and (4) combine these inbred lines into productive hybrids that are virtually free of mites. 2. Complement the SMR trait by developing another varroa-resistance trait, percent mites in brood (P-MIB). Although we have demonstrated that we can achieve almost total resistance to varroa mites with only the SMR trait, it is not prudent to rely on one mechanism for mite resistance. P-MIB is the percentage of a mite population that resides in brood cells. The remaining mites in a bee colony are phoretic on adult bees. Although this trait has been associated with lower mite populations and we have already shown it to be a heritable trait of the honey bee, it has not been subjected to selective breeding. Our goal is to select bees for low P-MIB and thereby prolong the time that a mite spends on adult bees. Our approach is to isolate, intensify, and evaluate P-MIB or possibly another mite-resistance trait that affects mites outside the brood cell. 3. Combine multiple traits. We will combine the SMR trait with other mite-resistance traits and determine if the mite-resistance traits are independent, if they are compatible when bred into the same bee or the same colony, and if their interactions affect mite resistance. 4. Pollination component. Because pollination is the primary economic benefit derived from the beekeeping industry, we will evaluate pollination-related foraging performance of new bee stocks to help judge the suitability of the bees as commercial pollinators. Newly identified foraging attributes may be used in breeding programs; foraging detriments will be corrected by management or breeding. Customers, products, and impact. We expect to produce additional mite-resistance traits of the honey bee and to improve the SMR (suppressed mite reproduction) trait that we released to the bee industry in 2001. New traits will be offered to the beekeeping industry as well as new hybrid combinations of the SMR trait and the SMR trait in combination with other mite-resistance traits. When free-mated to unselected drones (standard procedure for commercial queen rearing), queens with two mite-resistance traits may be more resistant to mites or less variable in their level of resistance than queens with the SMR trait alone. Also, with two mite-resistance traits in each queen that we supply to queen breeders, we will double the rate at which mite-resistance genes are added to our commercial and feral populations of honey bees. Our immediate customers are commercial queen producers who will propagate and sell queen bees from the genetic material that we provide to them. Our ultimate customers are beekeepers (commercial, sideline, and hobbyist) who purchase mite-resistance queens from queen producers. Those beekeepers will benefit from having stocks of honey bees that are productive and resistant to mites. Our distribution of mite-resistance traits should help beekeepers and other bee scientists select their bees for mite-resistance traits, and the propagation and selection of these beneficial genes should increase levels of mite-resistance genes in our nationwide population of honey bees. Increased frequency of mite-resistance genes will make it easier to find and produce mite-resistant bees and should hasten the return of our feral population of bees. Feral honey bees not only provide free pollination service to gardeners and backyard fruit growers, but their countless numbers and adaptation to every corner of our nation make them our most valuable resource for honey bee germplasm. 2.List the milestones (indicators of progress) from your Project Plan. For 2004: (a) Conduct a backcross experiment to determine the number of genes involved in the SMR trait and to determine if poor brood production is related to the SMR trait; (b) Test effects of screening and ventilation on mite-resistance of bees and the behavior of mites; (c) Make initial selection for a low percent mites in brood (P-MIB) trait; (d) Measure cotton yield increase with honey bees; (e) Measure effect of increased visits to rabbiteye blueberries; (f) Establish test and select four lines with the SMR trait and begin to establish inbred lines for hybrid breeding; (g) Obtain test lines; verify QTLs and develop markers For 2005: (a) Evaluate potential mechanisms to explain the P-MIB trait; (b) Test resistance of combined traits; (c) Measure cotton yield increase with honey bees; (d) Continue to test and select inbred lines and test hybrid combinations; (e) determine the physiological basis for ovary development in queens; (f) Make backcrosses, conduct chalkbrood resistance field trials, and develop markers For 2006: (a) Evaluate ability of bees with the P-MIB trait and without the SMR trait to control varroa; (b) Enhance the combined traits into breeder queens that will be economically desirable to beekeepers; (c) Assess foraging of SMR hybrid x commercial Italian stock; (d) Test SMR hybrid combinations and begin initial release of hybrids to industry; (e) Adapt artificial insemination procedures to enhance oviposition of queens; (f) Determine microsatellite patterns For 2007: (a) Determine the number of genes involved in a trait of the honey bee that affects P-MIB; (b) Initial release of the P-MIB trait to the beekeeping industry; (c) Assess SMR x Italian stock on early season crops; (d) Evaluate hybrid bees that have genes for both the SMR and P-MIB traits; (e) Finish DNA marker tests and compare with chalkbrood scores to locate QTLs and linked microsatellites. 3.Milestones: 3A. List milestones that were scheduled to be addressed in 2004. (a) Conduct backcross experiment to determine the number of genes involved in the SMR trait and to determine if poor brood production is related to the SMR trait. Completed. Data suggest that 2 genes are involved in the SMR trait and the SMR trait was not associated with poor brood production. Therefore, it should be possible to produce productive hybrids that are virtually free of varroa. (b) Test effects of screening and ventilation on mite resistance in bees and the behavior of mites. Completed. Screen instead of wood on the floor of a bee hive was related to significant increase in brood production and a significant drop in mite populations. (c) Make initial selection for a low percent mites in brood (P-MIB) trait. Work in progress. Bi-directional selection for P-MIB produced a low group (P-MIB = 35 ± 13 %; n = 6 queens) and a high group (P-MIB = 78 ± 14 %; n = 4 queens) of breeder queens. The second generation of test queens was produced by crossing daughter queens to drones from within the same phenotypic group (low P-MIB × low P-MIB and high P-MIB × high P-MIB). Equal numbers of queens from these two groups are currently being tested. Experiments should be finished by September 15, 2004, at which time we can determine if colonies selected for low and high P-MIB are different. (d) Measure cotton yield increase with honey bees. Postponed. Cotton was not available in a planting pattern that would allow yield measurements. If possible, the project will be run next season. (e) Measure effect of increased visits to rabbiteye blueberries. Field work complete. Data collection is finished but analyses are incomplete. (f) Establish test and select four lines with the SMR trait and begin to establish inbred lines for hybrid breeding. Active. Six lines have been tentatively chosen and are being evaluated. Two will be discarded. None is as yet very inbred. (g) Obtain test lines; verify QTLs and develop markers. Modified. Resistance to chalkbrood disease was identified as a more suitable trait for QTL identification, replacing pollen foraging QTLs. Susceptible and resistant parent colonies were identified in field test and over 250 candidate microsatellite DNA markers were chosen. 3B. Expected accomplishments under each milestone for the next 3 years. For 2005: (a) Evaluate potential mechanisms to explain the P-MIB trait. The attractiveness of bee larvae to varroa mites will be compared between the high and low phenotypes for P-MIB. Separate experiments will test for differences in the effects of adult worker bees from the two phenotypes on invasion rates of varroa mites into brood cells. Identification of the major resistance factor (larval or adult bee effect) could help improve our selection methods for the low P-MIB trait; (b) Test resistance of combined traits. In 2004 we began combining mite-resistance traits and by 2005 we expect to have established lines that have both traits at the 75% level; (c) Measure cotton yield increase with honey bees. Data showing a positive influence of honey bee pollination on production would potentially lead to benefits for both cotton growers and beekeepers. For growers, greater crop yields obviously are desirable. Beekeepers minimally would benefit from better relationships with cotton growers by access to this nectar source and consideration during insecticide application; (d) Continue to test and select inbred lines and test hybrid combinations. The lines with the SMR trait are not yet highly inbred and will still respond to selection. Undesirable lines can be replaced or modified, and queens within each line will be selected on the basis of their compatibility with other lines, brood production, longevity, and beekeeping qualities (nonstinging and honey production). Four lines will emerge as the basis for the first SMR hybrid; (e) Determine the physiological basis for ovary development in queens. We will determine if a male factor can be added to insemination diluents to improve the performance of queens. Stimulation of ovary development could lead to an earlier onset of oviposition, and more rapid ovary development addresses many of the problems encountered during the early stages (first 6 weeks) of producing an artificially inseminated queen; (f) Make backcrosses, conduct chalkbrood resistance field trials, and develop markers. The results of the field testing of the backcross colonies will allow us to determine the variation in expression of chalkbrood symptoms in those colonies, and will enable us to assign phenotypes to the F1 parental drones. DNA will be extracted from the drones to analyze their microsatellite genotypes. For 2006: (a) Evaluate ability of bees with the P-MIB trait and without the SMR trait to control varroa. We will determine if the trait that reduces the rate at which mites enter brood cells (P-MIB) is able to control mite populations without the presence of the SMR trait; (b) Enhance the combined traits into breeder queens that will be economically desirable to beekeepers. We expect to have this combination ready for field-testing. We will find out if colonies of bees produced by queens with the combined trait are less variable and more resistant to mites than colonies produced by queens with the SMR trait alone when both groups of queens are free-mated with drones that have not been selected for resistance to mites; (c) Assess foraging of SMR hybrid x commercial Italian stock. Italian bees are used extensively in pollination operations because colonies tend to have large populations that grow early in the season and thus are ready for early blooming crops. Having a varroa-resistant Italian stock of honey bees would be a welcome addition in pollination oriented beekeeping nationwide; (d) Test SMR hybrid combinations and begin initial release of hybrids to industry. These hybrids should have 2 advantages over earlier releases of the SMR trait. They will have better beekeeping qualities, so they can be used alone in field colonies as artificially inseminated queens that are virtually free of varroa, or they can serve as SMR breeder queens, replacing previous queens that had been selected only for the SMR trait; (e) Adapt artificial insemination procedures to enhance oviposition of queens. The desired result is to improve the performance of artificially inseminated queens, making them comparable to naturally mated queens in their performance. In this way, specifically mated queens could be used by beekeepers in production colonies; (f) Determine microsatellite patterns. Micro-satellite genotypes will be determined from the DNA of each of the parental F1 drones. For 2007: (a) Determine the number of genes involved in a trait of the honey bee that affects P-MIB. Based on a backcross test involving 30 colonies, we will estimate the number of genes involved in the P-MIB trait. Although accuracy will be limited to 4 categories (1, 2, maybe 3, or more than 3 genes), this information will be useful when making plans to insert P-MIB genes into bee populations; (b) Initial release of the P-MIB trait to the beekeeping industry. Initial releases will be of two types, queens with only the P-MIB trait and queens that have both P-MIB and the SMR trait; (c) Assess SMR x Italian stock on early season crops. Demonstrating the usefulness on a crop of a varroa-resistant SMR x Italian honey bee would strengthen the concept of using genetically improved bees; (d) Evaluate hybrid bees that have genes for both SMR and P-MIB traits. We will determine if having both traits in a free-mated (outcrossed) queen is better than having only the SMR trait. At the present time, an average free-mated SMR queen retains an acceptable level of resistance to varroa, but there is considerable variation: some are highly susceptible and some are highly resistant. Even if multiple mite-resistance traits in a queen has no significant effect on mite populations but does reduce variability in an outcross, this would be useful because beekeepers could then predict that all of their free-mated queens have an effective level of resistance to varroa; (e) Finish DNA marker tests and compare with chalkbrood scores to locate QTLs and linked microsatellites. Genotyping will be completed for the chosen microsatellites, correlations will be calculated between genotypes and chalkbrood phenotypes, and QTLs will be located. By locating microsatellite DNA markers that are linked to genes controlling chalkbrood resistance (QTLs), it will be possible for bee breeders to select queens and drones for chalkbrood resistance (marker assisted breeding) before making the matings. 4.What were the most significant accomplishments this past year? 4A) Single most significant accomplishment during FY 2004 (one per Research (OOD) Project): Although the SMR (suppress mite reproduction) trait is very effective in controlling varroa, others and we had noticed that colonies with high levels of the SMR trait sometimes had poor brood production. If the SMR trait were related to poor brood production, we would need to alter our breeding strategy. However, based on results from a backcross of homozygous SMR queens to drones from a heterozygous queen, we found no relationship between the SMR trait and poor brood production or low bee populations. These results suggest that it should be possible to produce a hybrid bee that has good beekeeping qualities while it is virtually free of varroa mites. The study also suggested that two genes control the SMR trait. B) Other significant accomplishment(s), if any. Colonies with screen on the floor of their hive were compared with colonies with traditional wood on the floor of their hive. Results from two field tests in winter and summer in Louisiana showed that the colonies with screened floors produced more brood and had fewer varroa mites. Colonies with open screen floors also had a significantly lower percentage of their mite population residing in brood cells (low P-MIB). Although one cannot necessarily conclude that P-MIB caused lower mite populations, we can conclude that this is an example of an environmental factor that affects P-MIB and that we must control this factor as we select bees for the P-MIB trait. Queen honey bees normally mate during their first two weeks of life and then never mate again during a lifetime that is often as long as two years. This study determined that queen bees could be artificially reinseminated months after their first insemination and after months of laying eggs. DNA analyses of progeny by Dr. Pamela Gregory (now at the ARS bee lab in Weslaco but formerly on the Russian bee CRIS at this laboratory) confirmed that all queens produced progeny from a second insemination that was made after a queen had been laying eggs for several months. We concluded that reinsemination of queens is a potential procedure that can be used in bee breeding programs. C) Significant activities that support special target populations. None. D) Progress Report opportunity to submit additional programmatic information to your Area Office and NPS(optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This is the first report for this CRIS so all major accomplishments are listed above. 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? SMR queens were distributed for research work by scientists at Penn State University, the University of Minnesota, Texas A and M, the University of Florida, and the ARS bee lab in Tucson. A final shipment of SMR breeder queens was sent to Glenn Apiaries to conclude a two-year CRADA agreement that began in 2001. Glenn Apiaries continues to produce and distribute the SMR trait to the beekeeping industry. Scientists from this project addressed beekeepers at national meetings in Jacksonville, FL (American Beekeeping Federation, Jan 2004) and San Antonio, TX (American Honey Producer's Association, Jan. 2004), regional meetings in Hood River, OR (combined Oregon and Washington state meeting, Nov. 2003) and Nashville, TN (Heartland Apicultural Society, July 2004), foreign meetings in Limoges, France (French bee breeder's meeting, Nov. 2003) and Milton, Ontario (Ontario Beekeepers Assn., July 2003), and state beekeepers meetings in Alabama, Colorado, Louisiana, and Mississippi). We conducted a Laboratory Field Day for beekeepers on October 18, 2003, and hosted a group of 9 agriculture students from the Virgin Islands (a program at Alcorn State University that was coordinated by Clifford Peters, July 22, 2004). 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Peabody, E. (2004) SMR--This honey of a trait protects bees from deadly mites. Agricultural Research. 52(5):14-16. Peabody, E. (2004) The Ag. Research article was reprinted without change in the American Bee Journal. 144(7):544-545. Review Publications Harris, J.W., Villa, J.D., Danka, R.G. 2004. Environmental effects on the growth of varroa mite populations. Bee Culture. 132(5):23-25. Harris, J.W., Harbo, J.R. 2004. Selective breeding for honey bees with a low percentage of varroa mites in capped brood [abstract]. American Bee Journal. 144(5):405 Harbo, J.R., Harris, J.W. 2004. Effect of screen or wood bottomboards on populations of honey bees and varroa mites [abstract]. American Bee Journal. 144(5):404-405. Harris, J.W., Harbo, J.R., Villa, J.D., Danka, R.G. 2003. Variable population growth of varroa destructor (mesostigmata: varroidae) in colonies of honey bees (hymenoptera: apidae) during a 10-year period. Environmental Entomology/Population Ecology 32(6):1305-1312. Gregory, P.G., Rinderer, T.E., Harbo, J.R. 2004. Paternity of offspring from honey bee queens re-inseminated after producing worker brood [abstract]. American Bee Journal. 144(5(:404 Sampson, B.J., Danka, R.G., Stringer, S.J. 2004. Nectar robbery by bees [xylocopa virginica (l.) and apis mellifera l.] contributes to the pollination of rabbiteye blueberry. Journal of Economic Entomology. v.97 p. 735-740.