1a.Objectives (from AD-416)
The long-term objectives of this project involve the application of electron microscopy for the observation of a wide range of sample materials of importance to research projects whose goals include the protection of plants, animals, and humans from various pathogens and parasites. The Beltsville Agricultural Research Center (BARC) Electron Microscopy Unit (EMU) serves research projects at BARC that require electron microscopy data, for achieving their specific research objectives. The EMU will use standard electron microscopy protocols as well as develop new techniques and methodologies as needed to meet the needs of its clientele. Over the next 5 years we will focus on the following objectives,.
1)design and develop new techniques, preparative methodologies, specimen holders, and equipment required for the identification, characterization, and classification of plant pathogens and other pests,.
2)develop new techniques and methodologies in electron microscopy, specific to individual research projects, for achieving previously unobtainable data and improvement of the quality of results, and.
3)develop digital imaging and provide computer assistance and graphics for use in publications and periodicals.

1b.Approach (from AD-416)
Sample materials including healthy and diseased plant materials, mites, insects, fungi, viral pathogens, nematode pests, animal and human parasites, non-biological materials, food products, snow and ice crystals and numerous other materials will be imaged at the BARC Electron Microscopy Unit (EMU) using a range of electron microscopy techniques and instrumentation. In some instances it is not possible to meet the specific and diverse specimen preparation needs of a range of plant, animal, microbe, and other research projects using standard approaches. In such cases, procedures, devices, and methods will be developed and/or adapted in consultation with individual researchers to produce optimal results. The EMU has computer equipment, servers, data storage devices, DVD, CD and slide writers, scanners and printers, and associated software for the production of quality photographs and publication ready documents and files. Utilizing this equipment and the IT skills available in the EMU, unique applications of computer technology will be used for colorization of black and white electron microscope images of mites and other biological tissues with true colors as seen using light microscopy. Training will also be provided to staff of collaborating scientists to assist in creating and enhancing images.

3.Progress Report
More than 400 scientists are working on research projects at the Beltsville Agricultural Research Center and many of them have an occasional or frequent need to visualize materials using electron and confocal microscopy provided by the Electron and Confocal Microscopy Unit. There were 40 different projects which we provided support which include: characterization of viruses in bees, Freesia, tobacco, yeast, cabbage looper; bacteria in spinach, lettuce, apple, and bees and fungi on bean, rose, and sugar cane rust, switchgrass smut, Phytophthora as a biocontrol; animal parasites (Giardia, yeast tachyozoites); 3 genera of nematodes, insects (mosquitoes); and 9 genera of mites, localization of transformation products in tobacco, spinach, eggplant, Freesia, and soybeans; characterization of healthy and infected plants and chicken tissues; and food safety issues including: bacterial contamination of tomatoes, spinach, lettuce and cyptosporidium in spinach, lettuce, and apples. The techniques required to prepare and visualize this diversity of experimental materials are time consuming, tedious and required significant experience and skill to accomplish. In addition, there was a continuous need for the development of new techniques and the modification of existing techniques for the preparation of the ever-changing spectrum of sample materials.

4.Accomplishments
1. A plant mite, Aceria mangiferae, was shown to carry spores for the fungus, Fusarium mangiferae, a disease on mango. Mango trees which were not yielding fruit were found to be infected with a fungus and also a mite. Scanning electron microscopy studies showed that the mites found on mango can spread the spores of the fungus which causes a proliferation of vegetative buds on the terminal branches of mango, thus, reducing fruit production. Pest control of the mites may reduce or prevent the spread of the disease.

2. Cryptosporidium oocysts have been found inside plant stomata. Cryptosporidium parvum is an animal parasite found in contaminated water. Fluorescently labeled Cryptosporidium parvum oocysts were found using laser confocal microscopy near and inside plant stomata, the breathing holes of plants. This could be a hiding place for this animal parasite which could potentially be a food safety issue for fresh cut leafy vegetables.

3. Morphological, molecular and phylogenetic studies were conducted on the microsporidian parasite, Nosema ceranae. Microsporidian parasites were found to attack honey bees which reduces honey production. Ultra-structural studies using the transmission electron microscope as well as molecular markers confirmed that this new parasite was a member of the genus Nosema which are pests of honey bees. Control of this new pest can improve the health of Honey bees and help increase honey production.

4. Immunolabeling of Giardia lamblia trophozoites identified the location of a molecule on the ventral disk of the disease-causing developmental stage of the parasite. Giardia lamblia is an intestinal parasite of humans, dogs, cats, birds, cows, and sheep. Transmission electron microscopy studies using immunolabeling showed that the ventral disk of the parasite is the primary structure responsible for attachment of G. lamblia trophozoites to host intestinal cells. Blockage of this binding site may prevent the disease.

6.Technology Transfer

Number of Web Sites Managed

2

Review Publications
Chen, Y., Evans, J.D., Murphy, C.A., Gutell, R., Lee, J., Zuker, M., Gundersen, D.E., Pettis, J.S. 2009. Morphological, molecular, and phylogenetic characterization of Nosema cerana, a microsporidian parasite isolated from the European honey bee, Apis mellifera. Journal of Eukaryotic Microbiology. 56(2):142-147.