Progress 06/03/04 to 03/08/07
Outputs
Progress Report Objectives (from AD-416) 1) Process samples and materials for all of the electron microscopy needs of researchers at the Beltsville Agricultural Research Center (BARC) and other collaborators and facilitate the imaging of experimental materials using an array of electron microscopy techniques. 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. 3) Provide computer support, image digitization and production of publication quality images for researchers utilizing photographs generated through electron microscopy. 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 (EM Unit) 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 EM Unit 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 EM Unit, 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. Accomplishments The Beltsville Electron Microscopy Unit provided Electron Microscopy support for numerous research projects. Numerous research projects at the Beltsville Agricultural Research Center and at other USDA, ARS locations; at other Federal Agencies and at universities require electron microscopy expertise. The goals of these projects are wide ranging including the protection of plants and animals from various pathogens; taxonomic characterization of microbes, insects and mites; structural definition of food products and byproducts; the determination of the water content of winter snow packs; the characterization of microbe behavior through freeze immobilization; the description of olfactory sensoria, the localization of water, heavy metal and other compounds in cells, and the characterization of healthy and infected plant and animal tissues. New techniques for low temperature scanning electron microscopy were developed in close coordination with collaborators and their needs to visualize samples under specific conditions in response to the requests of over 30 collaborators from 16 ARS laboratories and the National Aeronautics and Space Administration, through the use of scanning and transmission electron microscopy of insects, mites, nematodes, parasites, bacteria, food products and other agriculturally important materials. Using low temperature scanning electron microscopy, pests and pathogens were preserved in natural behavioral positions, allowing researchers to study host pathogen relationships, thereby increasing their ability to control these organisms. These methodological improvements have impacted the fields of entomology, acarology, nematology and zoology, allowing researchers to rapidly identify and study organisms of major agricultural importance. Technology Transfer Number of Newspaper Articles,Presentations for NonScience Audiences: 2
Impacts
(N/A)
Publications
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Progress 10/01/05 to 09/30/06
Outputs
Progress 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? Solving many agricultural problems involving environmental science, food quality, plant and animal diseases, cell biology, and pest identification requires detailed microscopic examination by transmission and scanning electron microscopes. Because few individual research laboratories have the facilities and personnel to support and maintain this equipment, centralization of support is a very effective means of providing electron microscopy broadly within an organization. This project supports the Beltsville Electron Microscopy Unit, which provides high quality and timely research support and collaboration with ARS scientists. A large number of significant problems reduce American agricultural productivity by billions of dollars each year. Solving these problems often requires a variety
of technical approaches, with imaging techniques being among the most powerful. The images and results provided by the Electron Microscopy (EM) Unit have aided in the completion of many high priority ARS projects that have increased the productivity and sustainability of U. S. agriculture. This project is assigned (70%) to National Program 303, Plant Diseases and (30%) to National Program 304, Crop Protection and Quanantine. A large component of the electron microscopy support provided by the Electron Microscopy Unit focuses on projects involving plant-parasitic nematodes and other biotic pests of plants, such as arthropods that are carriers of plant diseases. However, the EM Unit provides broad support that is utilized within many National Programs. 2. List by year the currently approved milestones (indicators of research progress) Objective 1 Year 1-5 (FY 2004-2008) In each of the five years, the EM Unit expects participation in approximately 40 research projects at BARC. For
purposes of this report, milestones are indicated as major research collaborations in which the EM Unit participates. These include the following: Obtain EM data on immunocytochemical labeling of (Erwinia maxima) mobile sporozoites in support of the Animal Parasite Diseases Laboratory, USDA, BARC. Obtain EM data on (Varroa jacobsoni) mite immuno virus in support of the Bee Research Laboratory, USDA, BARC. Obtain EM data on imunocytochemical labeling of (Neospora caninum) bradyzoites in support of the Animal Parasitic Diseases Laboratory, USDA, BARC. Obtain EM data on Tagetitoxin produced from (Pseudomonas syringae) to control Canada Thistle in support of the Sustainable Agricultural Systems Laboratory, USDA, BARC. Obtain EM data on fungi lethal to Formosan termites in support of USDA, Formosan Subterranean Termite Research Unit, (SARC), New Orleans, LA. EM data will be collected on (Trichoderma sp). fungi and their effects on Cacoa in support of the Alternative Crops and Systems
Laboratory, USDA, BARC. Obtain EM data on nickel accumulation in phytoaccumulating plants in support of the Animal Manure and By-Products Laboratory, USDA, BARC. Low temperature SEM of microarthropods from La Selva, Costa Rica for the Systematic Entomology Laboratory, USDA, BARC. Low temperature SEM observation of newly discovered mite species (Eutetranychus, Brevipalpus, Astigmata and Eriophyid) mites for the Systematic Entomology Laboratory, USDA, BARC. Low temperature SEM of bed bugs (Cimex lectularius) for the Insect Control and Research Inc., Baltimore, MD. Electron microscopy for location of the causal agent in citrus plants, thought to be a virus for the Plant Germplasm Quarantine Office, USDA, BARC. Photographic documentation of unknown Potex type virus in Perennial Rye Grass for the Fruit Laboratory, USDA, BARC. Thin sectioning of antennal segments and spermatheca of the Coffee Berry Borer Beetle (Hypothenemus hampel), adults and larvae, a major beetle pest of Coffee beans
for the Insect Biocontrol Laboratory, USDA, BARC. Observe the tergal gland, dorsal aorta, spermatheca and antennal segments of the Formosan termite (Coptotermes formosanus) by transmission electron microscopy (TEM) for the USDA, ARS, Formosan Termite Research Unit, New Orleans. Cell and surface structure of mineral hyperaccumulating plants. (Alyssum Sp.,) nickel accumulator for the Animal Manure & By Products Laboratory, USDA, BARC. Electron microscopy to find casual agent in cranberry flower causing funky flower, thought to be a virus for the Fruit Laboratory, USDA, BARC. Room temperature and low temperature SEM of newly invasive insect species (Halyomorpha halys and Euschistus tristigmus) with parasitoids (Eucalyptus flava, Gymnosoma & Trichopoda sp.), and other eggs for the Chemicals Affecting Insect Behavior Laboratory, USDA, BARC. Scanning Electron Microscopy of (Chrysopidae sp.) Lacewings to locate the pheromone glands for the Chemicals Affecting Insect Behavior Laboratory,
USDA, BARC. Electron microscopy of (Neospora caninum) for the Animal Parasitic Diseases Laboratory, USDA, BARC. Ultrastructure preservation and comparison of bacteroids of soybean roots for the Soybean Genomics and Improvement Laboratory, USDA, BARC. Room temperature SEM of stress fractures in chicken feather composite biomaterials for the Environmental Quality Laboratory, USDA, BARC. Low Temperature Scanning Electron Microscopy of Soybean cotyledons and Begonia leaves for the University of Toledo group from the Plant Science Research Group and the Sustainable Agricultural Systems Laboratory, USDA, BARC. Low Temperature Scanning Electron Microscopy of the surface of Alfalfa plant leaf surfaces, Wisfal and R-3 for the Soybean Genomics and Improvement Laboratory, USDA, BARC. Low Temperature Scanning Electron Microscopy of nematodes PS2123 and PS2017 Lawnscape for the Nematology Laboratory, USDA, BARC. Transmission electron microscopy and Low Temperature SEM of Rust fungus pustules on
dry bean plants from South and Central America for the Vegetable Lab, USDA, BARC. Low Temperature SEM and TEM of Potato stolon and tuber to determine causal agent of Zebra Chip in potato for the Molecular Plant Pathology Laboratory, USDA, BARC. Low temperature SEM of the, Diaprepes Root Weevil (Diaprepes abbreviatus) antennae for the Chemicals Affecting Insect Behavior Laboratory, USDA, BARC. Low temperature SEM of three ages of bacteria samples (Chromobacter sp.) bacteria grown on culture media for the Insect Biocontrol Laboratory, USDA, BARC. Electron microscopy of meat exposed to bacteria (Listeria, E. coli and Salmonella sp.) then (HDP) hydrodynamic pressure treated for the Food Technology and Safety Laboratory, USDA, BARC. Low temperature SEM of Tiger mosquito eggs laid on moist germination paper for the Chemicals Affecting Insects Laboratory, USDA, BARC. Low temperature SEM of the potential nematode killing fungi (Macrosporium drechsleri) and (Monacrasporium ellipsosporum) with
(Panagrellus sp.) nematode for the Nematology Laboratory, USDA, BARC. Low temperature SEM of snow crystals collected by the University of Puget Sound, Tacoma, Washington. Low temperature SEM of Sweet Potato White Fly (Bemisia tabaci) and parasitoid Hymenoptera for the Tel Aviv University and the Insect Biocontrol Laboratory, USDA, BARC. Low temperature SEM of teliospores of the Smut fungi (Microbotryum silybum 04-046) and (Microbotryum sp. cf. cardui) for measurements of the spore characteristics for the ForeignWeed Research Unit, Fort Detrick, MD, USDA, Low temperature SEM of fungal cultures of (Monacrosporium ellipsosporum and Monacrosporium dreschsleri) and captured nematodes Panagrellus (sp.) for the Nematology Laboratory, USDA, BARC and the National Institute of Agricultural Science and Technology, Korea. Room temperature SEM of sand samples to observe grain surfaces for the Animal Manure and By-Products Laboratory, USDA, BARC. Low temperature SEM of invasive mite (Raoiella
indica) on the lower surface of Coconut Palm leaves (Cocos nucifera) collected on the island of Saint Lucia on 9/15/2005 by the Systematic Entomology Laboratory, USDA, BARC. Immunocytochemical labeling of (Neospora caninum, Cryptosporidia parvum, Elmeria acervulina, Elmeria tenella) merozoite and sporozoite forms for the Animal Parasitic Diseases Laboratory, USDA, BARC. Transmission electron microscopy of negative stained plant virus, (Cucumber Mosaic Virus) for the Microbiology and Plant Pathology Laboratory, USDA, BARC. Transmission electron microscopy of Sunflower seedlings infected with (Pseudomonas syringae PV tagetis) for the Sustainable Agricultural Systems Laboratory, USDA, BARC. Ultrastructure comparison of the gut and peritrophic membrane of the Diamondback moth (Plutella xylostella) after BT treatment for the Insect Biocontrol Laboratory. USDA, BARC. Research was conducted in response to the requests of over 40 collaborators from 25 ARS laboratories and NASA, universities
and private industry. Research support was provided through the scanning and transmission electron microscopy of insects, mites, nematodes, parasites, bacteria, food products and other agriculturally important materials. Objective 2 Year 1 (FY 2004) Complete development of the technique, which combines complementary freeze-fracturing with low temperature SEM and X-ray microanalysis for in support of the Animal Manure and By-Products Laboratory, USDA, BARC. Modifications to the newly acquired S-4700 scanning electron microscope will be completed as well as modifications to the Polaron/Quorum cryostage and cryopreparation chamber. Modification and design of specimen holder devises for low temperature scanning electron microscopy. Quorum/PolaronPolar Prep 2000 Cryo Transfer System. Year 2 (FY 2005) Specimen holder redesign in collaboration with will with Energy Beam Sciences Corporation to be completed. Year 5 (FY 2008) Design and construction of the Insect Freezing Chamber Devise will
begin in 2004 and continue through 2008. Objective 3 Year 1-5 (FY 2004-2008) Computer support in microscopy image analysis, colorization and manuscript preparation and presentation will be completed for various projects as required. 4a List the single most significant research accomplishment during FY 2006. Taxonomic Descrption of a New Invasive Mite Species: At the request of APHIS, the curator of the National Mite Collection was sent to Saint Lucia and Trinidad to collect samples of an invasive mite responsible for massive destruction of a variety of plant species, including coconut palms and bananas in the Caribbean. Microscopic documentation of key anatomical characters of this newly invasive pest, (Raoiella indica), was undertaken utilizing low temperature scanning electron microscopy. Taxonomic descriptions, based on electron microscopy were produced to aid in identification of this pest in advance of its expected movement in the near future into the Southern United States.
This work aligns with National Program 304, Crop Protection and Quarantine as part of Research Component A: Insects and Mites - Identification and Classification of Insects and Mites. 5. Describe the major accomplishments to date and their predicted or actual impact. The project entitled: Using Electron Microscopy Technology to Solve Agricultural Problems was approved on May 04, 2004. Over the past year, the Soybean Genomics and Improvement Laboratory-Electron Microscopy Unit provided support for numerous research projects in response to the requests of over 30 collaborators from 16 ARS laboratories, NASA, the Centers for Disease Control through the use of scanning and transmission electron microscopy of insects, mites, nematodes, parasites, bacteria, food products and other agriculturally important materials. Using low temperature scanning electron microscopy, pests and pathogens were preserved in natural behavioral positions, allowing researchers to study host pathogen
relationships, thereby increasing their ability to control these organisms. The service of the EM Unit is unique, in that it efficiently provides high quality images, as well as a genuine collaboration with ARS scientists to solve problems that may require technical innovations. The goals of these projects are wide ranging including the protection of plants and animals from various pathogens; taxonomic characterization of microbes, insects and mites; structural definition of food products and byproducts; the determination of the water content of winter snow packs; the characterization of microbe behavior through freeze immobilization; the description of olfactory sensoria, the localization of water, heavy metal and other compounds in cells, and the characterization of healthy and infected plant and animal tissues. New techniques for low temperature scanning electron microscopy have been developed in close coordination with collaborators and their needs to visualize samples under
specific conditions. The impact of this is widespread, allowing for increased resolution of structural features used for accurate identification and the study of the association of organisms with the host material. These methodological improvements have impacted the fields of entomology, acarology, nematology and zoology, allowing researchers to rapidly identify and study organisms of major agricultural importance. During the past year the Beltsville Electron Microscopy Unit has resulted in coauthorship on 10 peer-reviewed journal articles and proceedings in scientific meetings, photographs used in numerous articles (with or w/o acknowledgement), popular articles and books using photographs, one journal using a photograph as cover photo, several presentations of photographs used on television, video and extensive website usage. These accomplishments align with National Program 303, Plant Diseases; and National Program 304, Crop Protection and Quarantine. 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 Electron Microscopy Unit provides research and technical support to collaborating scientists within the USDA, NASA and Centers for Disease Control. The low-temperature techniques developed by the Unit are at the leading edge of this technology and have proven useful in projects as diverse as snow pack research with NASA and systematics research with mites within ARS by preserving and visualizing previously unseen taxonomic features. The innovations of the Unit have been demonstrated to manufacturers of low-temperature equipment and to users of this technology in government, industry and university laboratories. 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). 1. Photos in Popular Articles A chance (Discovery) of snow. Cover photo and article for Annual science supplement section of the World Book Encyclopedia. Yearbook. pp. 29-39, Science Year. 2005. Photo selected as featured picture representing one of the finest images on Wikipedia. September 2005. Under a microscope: Seeing is truly believing. The Washington Times. LIFE/Science & Technology. 24(286):B1 & B4, October 13, 2005. 2. Stories about EM Unit Research in the Popular Press Up Close and Personal: Frosted Flakes and Theyre Great. The Sun Magazine. pp.46. August 29, 2005. 3. Presentations to Organizations NASA course entitled: The History of Winter for 22 teachers from 12 states, held in Lake Placid, Feb. 12-17th 2006. In association withTeacher-as-Scientist (TAS) Professional Development for NASA Explorer Schools from the Goddard Space Flight Center. (Code N project funded by the director of
NASA) Eric Erbe invited to assist, present 3 talks and participate in videotaping and demonstrations. Conducted a laboratory tour and presented snow and ice collection techniques to Paul Pregont, Expedition Director of GoNorth!-Adventure Learning for the K-12 Classroom for use in upcoming expedition to traverse North Alaska and collect snow crystals for Low Temperature SEM in the Spring of 2006 Received best paper award at the Western Snow Conference for paper entitled: Wergin, W.P., Rango, A., Foster, J., Erbe, E.F. Pooley, C. 2006. Observations of snow and ice crystals with low temperature scanning electron microscopy (review). Western Snow Conference 2006. Santa Fe, New Mexico.
Impacts
(N/A)
Publications
- Handoo, Z.A., Skantar, A.M., Carta, L.K., Erbe, E.F. 2005. Morphological and molecular characterization of a new root-knot nematode, meloidogyne thailandica n.sp. (nematoda: meloidogynidae), parasitizing ginger (zingiber spp.). Journal of Nematology. 37(3):343-353.
- Jenkins, M., Murphy, C.A., Trout, J.M., Fayer, R. 2006. An improved electron microscopic technique for the immunolabeling of Cryptosporidium parvum oocysts. Journal of Parasitology. 92:403-405.
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Progress 10/01/04 to 09/30/05
Outputs
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? Solving many agricultural problems involving environmental science, food quality, plant and animal diseases, cell biology, and pest identification requires detailed microscopic examination by transmission and scanning electron microscopes. Because few individual research laboratories have the facilities and personnel to support and maintain this equipment, centralization of support is a very effective means of providing electron microscopy broadly within an organization. This project supports the Beltsville Electron Microscopy Unit, which provides high quality and timely research support and collaboration with ARS scientists. A large number of significant problems reduce American agricultural productivity by billions of dollars each year. Solving these problems often requires a variety of technical
approaches, with imaging techniques being among the most powerful. The images and results provided by the Electron Microscopy Unit have aided in the completion of many high priority ARS projects that have increased the productivity and sustainability of U.S. agriculture. This project is assigned (70%) to National Program 303, Plant Diseases and (30%) to National Program 304. A large component of the electron microscopy support provided by the Electron Microscopy Unit focuses on projects involving plant-parasitic nematodes and other biotic pests of plants, such as arthropods that are carriers of plant diseases. However, the Electron Microscope Unit provides broad support that is utilized within many National Programs. 2. List the milestones (indicators of progress) from your Project Plan. Year 1-5 (FY 2004-2008) 1. In each of the next five years (2003-2008), participation by the EM Unit in approximately 40 research projects at BARC is expected. For purposes of this report,
milestones are indicated as major research collaborations in which the EM Unit plans to participate over the next five years. These include the following: Anatomical and structural data on mite and insect materials from the curator of the National Mite Collection and the USDA Systematic Entomology Laboratory (SEL), Plant Sciences Institute (PSI), (BARC) will be accumulated. Scanning electron microscopic data will be obtained on samples from the Systematic Botany and Mycology Laboratory (SBML), Plant Sciences Institute (PSI), (BARC). The collection of snow and ice samples in cooperation with NASA will continue with the preparation and observation of collected and stored samples from the Cold Land Processes Experiment 2003. Both LTSEM (low temperature scanning electron microscopy) and TEM (transmission electron microscopy) data will be obtained for identification of nematodes and fungal antagonists of nematodes in support of the Nematology Laboratory (NL), Plant Sciences Institute
(PSI), (BARC). Electron microscopy data will be obtained in support of the US National Parasite Collection and the Animal Parasitic Diseases Laboratory (APDL), Animal and Natural Resources Institute (ANRI), (BARC). EM data will be collected on newly described species of Chromobacter sp. for potential control of Colorado potato beetle larvae, adult corn rootworm and diamond back moth larvae. Insect Biocontrol Laboratory (IBL), Plant Sciences Institute (PSI), (BARC.) EM data will be collected on Trichoderma sp. fungi and their effects on Cacao in support Alternative Crops and Systems Laboratory (ACSL), Plant Sciences Institute (PSI), (BARC.) Obtain EM data on immunocytochemical labeling of Erwinia maxima- mobile sporozoites, Animal Parasite Diseases Laboratory, Animal and Natural Resources Institute (ANRI), (BARC). Obtain EM data on Varroa jacobsoni mite immuno virus, Bee Research Laboratory (BRL), Plant Sciences Institute (PSI), (BARC). Obtain EM data on imunocytochemical labeling of
Neospora caninum bradyzoites, Animal Parasitic Diseases Laboratory (APDL), Animal and Natural Resources Institute (ANRI), (BARC). Obtain EM data on Tagetitoxin produced from Pseudomonas syringae to control Canada Thistle, Sustainable Agricultural Systems Laboratory (SASL) , Animal and Natural Resources Institute (ANRI), (BARC). Obtain EM data on fungi lethal to Formosan termites, (USDA) Formosan Subterranean Termite Research Unit, (SARC), New Orleans, LA. Obtain EM data on nickel accumulation in phytoaccumulating plants, Animal Manure and By-Products Laboratory (AMBL), Animal and Natural Resources Institute (ANRI), (BARC). (Add additional research projects which will be carried out in the next year or 2005) Objective 2 Year 1 (FY 2004) Complete development of the technique which combines complementary freeze-fracturing with low temperature SEM and X-ray microanalysis, Animal Manure and By-Products Laboratory (AMBL), Animal and Natural Resources Institute (ANRI), (BARC). Modifications
to the newly acquired S-4700 scanning electron microscope will be completed as well as modifications to the Polaron/Quorum cryostage and cryopreparation chamber. Year 2 (FY 2005) 2. Specimen holder redesign in collaboration with will with Energy Beam Sciences Corporation to be completed. Year 1-5 3. Design and construction of the Insect Freezing Chamber Device will begin in 2004 and continue through 2008. Objective 3 Year 1-5 (FY 2004-2008) 4. Computer support in microscopy image analysis, colorization and manuscript preparation and presentation will be completed for various projects as required. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Participation by the EM Unit in approximately 40 research projects at BARC is expected each year. Milestone Fully Met 2. Specimen holder redesign in collaboration with will with Energy Beam Sciences Corporation to be
completed. Milestone Fully Met 3. Design and construction of the Insect Freezing Chamber Device will begin in 2004 and continue through 2008. Milestone Fully Met 4. Computer support in microscopy image analysis, colorization and manuscript preparation and presentation will be completed for various projects as required. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Emphasis of the project is to provide, the more than 400 scientists at BARC, the ability to use the electron microscopy skills and hardware of the Beltsville Electron Microscopy (EM) Unit to generate structural information pertaining to their individual research projects. Many of these projects have the need for microscopic observation of materials in order to understand some facet of structure and how it relates to function, however costly and highly
specialized equipment often makes these data difficult to obtain. Microscopy work continues on all of the projects listed, with new projects added continually as research needs of the more than 20 collaborating laboratories warrant. Over 80 projects have been undertaken over the past 5 years and this trend should be an indication of future usage. Objective 1 Year 3-5 (FY 2006-2008) 1. Participation by the EM Unit in approximately 40 research projects at BARC is expected each year. These will include the following: Low Temperature Scanning Electron Microscopy(LTSEM) of Pyemotes mite for the Systematic Entomology Laboratory. LTSEM of the Bed Bug Cimex lectularius for the Insect Control & Research, Inc. LTSEM of Microarthropods from Costa Rica. LTSEM of insects newly invasive species Halyomorpha halys and Euschistus tristigmus with parasitoids Eucalyptus flava, Gymnosoma and other eggs for the Chemicals Affecting Insect Behavior Lab. LTSEM of Wheat Curl Mites, A. tosichella and A.
tulipae for the University of Nebraska, Panhandle Research & Extension Center. Room Temperature (RT) Scanning Electron Microscopy (SEM) of biomaterials SIS, PGA Mesh and Collagen-Chitosan beads for the Growth Biology Laboratory. RT-SEM of Chrysopidae sp. Lacewings to locate the pheremone glands for the Chemicals Affecting Insect Behavior Laboratory. Transmission electron microscopy (TEM) thin sectioning of QPX parasite of clams to assist in determining the taxonomic relationships to other parasitic organisms. Referred to the EMU by NPS for the State University of New York, Marine Sciences Research Center, Stony Brook, NY. TEM of negative stained plant virus for the Molecular Plant Pathology Laboratory. RT and LTSEM of dead and living insects and eggs of Halyomorpha halys with parasitoid eggs, (Gymnosoma sp. & Trichopoda sp.). RTSEM of stress fractures in chicken feather composite biomaterials for the Environmental Quality Laboratory. LTSEM of Soybean cotyledons and Begonia leaves for
the University of Toledo group from the Plant Science Research Group. Low Temperature Scanning Electron Microscopy of the surface of Alfalfa plant leaf surfaces, Wisfal and R-3 for the Soybean Genomics and Improvement Laboratory. Low Temperature Scanning Electron Microscopy of nematodes PS2123 and PS2017 Lawnscape for the Nematology Laboratory. Low Temperature Scanning Electron Microscopy and TEM of Potato stolon and tuber infected with Zebra Chip bacteria for the Molecular Plant Pathology Laboratory. LTSEM of insect (Diaprepes abbreviatus) Diaprepes Root Weevil antennae for the Chemicals Affecting Insect Behavior Laboratory and the National Program Staff. LTSEM of snow crystals collected from Washington State maritime snow pack for The University of Puget Sound, Department of Chemistry, Tacoma, WA. LTSEM of bacterial samplesthree ages of Chromobacter grown on culture media for the Insect Biocontrol Laboratory. LTSEM of mosquito eggs laid on moist germination paper -Tiger Mosquito
for the Chemicals Affecting Insects Behavior Laboratory. LTSEM of Sweet Potato White Fly (Bemisia tabaci) and parasitoid Hymenoptera for Tel Aviv University, Israel and visiting scientist at Insect Biocontrol laboratory. In addition, research will be conducted to: Image healthy and diseased plant and animal materials as well as materials produced from their byproducts such as chicken feather fiber composites. Images of various insect, mite, nematode, fungal, parasite, bacterial species will be acquired to assist in identification and function of observable structural features. Image the associations of organisms with their host plant or animal, symbiotic or biocontrol organisms and disease causing agents. Images of collected snow samples will be acquired to add to the snow structure data base in association with the Cold Land Processes Experiment 2003 and NASA (HOW project). Objective 2 Year 3-5(FY 2006-2008) Design and construction of the Insect Freezing Chamber Device will begin in
2004 and continue through 2008. The initial prototype will be constructed in 2005 with subsequent testing and redesign as needed in 2006 and 2007. Continued research on the combination of complementary freeze-fracturing with low temperature SEM and X-ray microanalysis for the Animal Manure & By-Products Laboratory (AMBL), Animal and Natural Resources Institute (ANRI), BARC. Objective 3 Year 3-5 (FY 2006-2008) Computer support in microscopy image analysis, colorization and manuscript preparation and presentation will be continuous through the duration of the five year period. 4a What was the single most significant accomplishment this past year? Developed a fixation and embedding technique for Cryptosporidium parvum oocysts that avoids poor resolution and distortion of oocyst wall for subsequent immunolabeling with specific antibodies. Cryptosporidium oocysts were immersed in fixative, plunged into liquid nitrogen and allowed to thaw to room temperature. This procedure allows the
fixative to penetrate the oocyst wall and preserve the ultrastructure much more effectively than previous methods. This work was done in collaboration with the Animal Parasitic Diseases Laboratory, USDA, ARS, Beltsville, MD. The technique is a major advance in the study of this protozoan parasite and has application to proteomics and genomics where localization of specific proteins inside the parasite is required. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The project entitled: Using Electron Microscopy Technology to Solve Agricultural Problems was approved on May, 04, 2004. Over the past year, the Soybean Genomics and Improvement Laboratory-Electron Microscopy Unit provided support for numerous research projects in response to the requests of over 30 collaborators from 16 ARS laboratories, NASA, the Centers for Disease Control through the use of scanning and transmission electron microscopy of insects, mites,
nematodes, parasites, bacteria, food products and other agriculturally important materials. Using low temperature scanning electron microscopy, pests and pathogens were preserved in natural behavioral positions, allowing researchers to study host pathogen relationships, thereby increasing their ability to control these organisms. The service of the Unit is unique, in that it efficiently provides high quality images, as well as a genuine collaboration with ARS scientists to solve problems that may require technical innovations. The goals of these projects are wide ranging including the protection of plants and animals from various pathogens; taxonomic characterization of microbes, insects and mites; structural definition of food products and byproducts; the determination of the water content of winter snowpacks; the characterization of microbe behavior through freeze immobilization; the description of olfactory sensoria, the localization of water, heavy metal and other compounds in
cells, and the characterization of healthy and infected plant and animal tissues. New techniques for low temperature scanning electron microscopy have been developed in close coordination with collaborators and their needs to visualize samples under specific conditions. The impact of this is widespread, allowing for increased resolution of structural features used for accurate identification and the study of the association of organisms with the host material. These methodological improvements have impacted the fields of entomology, acarology, nematology and zoology, allowing researchers to rapidly identify and study organisms of major agricultural importance. During the past year the Beltsville Electron Microscopy Unit has resulted in coauthorship on 14 peer-reviewed journal articles and proceedings in scientific meetings, photographs used in numerous articles (with or w/o acknowledgement), popular articles and books using photographs, 1 journal using a photograph as cover photo,
several presentations of photographs used on television, video and extensive website usage. 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 Electron Microscopy Unit provides research and technical support to collaborating scientists within the USDA, NASA and Centers for Disease Control. The low-temperature techniques developed by the Unit are at the leading edge of this technology and have proven useful in projects as diverse as snow pack research with NASA and systematics research with mites within ARS by preserving and visualizing previously unseen taxonomic features. The innovations of the Unit have been demonstrated to manufacturers of low-temperature equipment and to users of this technology in government, industry and university
laboratories. 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). 1. Cover Photos in Journal and Popular Press Journal of Acarology Vol. 31 Number 1, March, 2005 (Cover photo). World Book Encyclopedia Yearbook 2005. 2. Photos in Popular Articles Picture published in Science et Vie Junior. November 2004, Number 182, pp. 6-7 La Photo Du Mois (photo of the month). Pictures published in article in Russian version of GeoFocus Magazine. No. 12, 2004- No. 1, Dec/Jan. 2004/5, pp. 18-23. Tests sanguins et allergologie lors d'obstruction reccurente des voies respiratoires. Practique Veterinaire Equine - Les Editions Du Point Veterinaire. Vol. 36, No. special, p 72, 2004. Stopping the Coffee Berry Borer from Boring into Profits. Agricultural Research Magazine. pp.10-11. Vol. 52. No. 11. November 2004. Mysterious bites blamed on mites. Houston Chronicle:
The State. Sunday January 9, 2005. Pictures used in: Roy- Architecture of Risk- Michigan Architecture Papers 11, page 34, 2004. Published by the University of Michigan, A. Alfred Taubman College of Architecture + Urban Planning. Pictures used in: Himmlisches Design. Geo Magazine. pp.42-52, February 2005. Skeletons on the outside. The Washington Times. Life/Science & Technology. Thursday, pp. B1-B4, February 10, 2005. CLPX 2003 Cold Land Processes Experiment Digital DVD-produced and published by NASA Goddard Space Flight Center, e:SPI.Education:special projects division. 2005. Detectives search for antimicrobial- resistant organisms. Agricultural Research Magazine. pp.18-19. Vol. 53. No. 3. March 2005. Einfach komplex- Bildbaume und Baumbilder in der Wissenschaft. Museum fur Gestalung publication. Zurich, Switzerland. 2005. 2. Stories about EM Unit Research in the Popular Press De la neige - Portfolio Materiaux. La Recherche. No. 381, pp.5 & 64-71. December 2004.
Impacts
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Publications
- Broadhurst, C.L., Chaney, R.L., Angle, J.S., Maugel, T.K., Erbe, E.F., Murphy, C.A. 2004. Simultaneous hyperaccumulation of nickel, manganese and calcium in alyssum leaf trichomes. Environmental Science and Technology. 38(21):5797-5802.
- Broadhurst, C.L., Chaney, R.L., Angle, J.S., Erbe, E.F., Maugel, T.K. 2004. Nickel localization and response to increasing ni soil levels in leaves of the ni hyperaccumulator alyssum murale "kotodesh". Plant and Soil Journal. 265(1-2):225-242.
- Jenkins, M.C., Soares, R., Murphy, C.A., Hemphill, A., O'Handley, R., Dubey, J.P. 2004. Localization of a 56 kda antigen that is present in multiple developmental stages of neospora caninum. Journal of Parasitology. 90:660-663.
- Carta, L.K., Erbe, E.F., Ochoa, R., Klepzig, K., Moser, J.C., Konrad, H., Kirisits, T. 2004. Nematode and mite associates of bark beetles on pine in Louisiana (USA) and on elm in Austria. J. Nematology 36: 310.
- Raina, A.K., Park, Y.I., Ingber, B.F., Murphy, C.A. 2005. Structure and developmental changes in the tergal glands of adult females of coptotermes formosanus (isoptera, rhinotermitidae). Arthropod Structure and Development 34: 509-517.
- Wergin, W.P., Erbe, E.R., Rango, A., Foster, J., Pooley, C. 2005. Low temperature scanning electron microscopy of artificial snow. Scanning 27(2) :63-64.
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