1a. Objectives (from AD-416):
The long-term objectives of this project involve the application of new microscopy technologies for the identification and management of agricultural pests and pathogens. The Beltsville Agricultural Research Center (BARC) Electron and Confocal Microscopy Unit (ECMU) serves the research projects of the ARS that require electron and confocal microscopy data necessary to achieve their specific research objectives. The ECMU will use standard protocols as well as develop new technologies and methodologies as needed to meet the needs of its clientele. Over the next 5 years we will focus on the following objectives: 1. Develop new techniques and methodologies in microscopy that generate high-resolution images of biological specimens more efficiently and effectively. [NP303, C1, PS1] 2. Apply novel microscopy approaches to facilitate the systematic identification and characterization of plant pathogens and pests, alone or with their hosts. [NP303, C1, PS1]
1b. Approach (from AD-416):
The Electron and Confocal Microscopy Unit (ECMU), housed on the BARC campus, performs collaborative research with a diverse group of ARS scientists needing microscopic imaging to validate their research hypotheses. The facility is equipped with state-of-the-art electron microscopes [transmission (TEM) and scanning (SEM)], confocal laser scanning microscope (CLSM), wide-field fluorescence and bright field microscope, and a digital video microscope. TEMs and SEMs can discern the internal and external structures of plants, animals, microbes, and materials at high resolution and at magnifications far exceeding those of light microscopes. Structures can be photographed with great depth of field and in stereo revealing their true three dimensional (3D) structures. The Confocal Laser Scanning Microscope (CLSM), a microscope that uses specific wavelengths of light produced by lasers to excite fluorescent compounds, has the ability to optically (non-destructively) slice through specimens and identify fluorescently labeled tissues, proteins, organisms, cells, etc. The ECMU staff, using software, interactively reconstructs the slices to produce 3D renderings. Techniques that will be used include: critical-point drying apparatus, sputter coating devices, glow discharger , carbon and other metal evaporation systems, freeze-etching equipment , ultra-microtomes, centrifuges, a freeze substitution system, stereo microscopes, TEM prep microwave system, vacuum oven, incubators , 60” and 40 “ large screen monitors, computer equipment for image storage, digitization, printing, and associated software as well as conventional laboratory equipment. Members of the ECMU are responsible for training all personnel on the proper use and maintenance of the microscopes and equipment within the facility. The final result is dramatic, high-resolution, digitally-achievable images of many of the most important pests and pathogens affecting agriculture.
3. Progress Report:
The first objective of this project is to develop new techniques and methodologies in microscopy that generate high-resolution images of biological specimens more efficiently and effectively. Several microscopy techniques including scanning (SEM) and transmission electron microscopy (TEM) as well as confocal laser scanning microscopy (CLSM) and fluorescent microscopy (FL) were used to prove that Varroa destructor, the single greatest driver of the global honey bee colony losses, feeds on adult bees and primarily on fat bodies not hemolymph (bee’s blood). Four separate TEM studies were conducted on Sarcocystis, a fatal disease-causing parasite, in the Western Brown rat, White-nosed coati, elk and red-tailed hawk. Additional progress was made on sixty different projects where the Electron and Confocal Microscopy Unit collaborated with scientists to describe 25 new species of mites, to explain the unique feeding system of a family of small worm-like mites, and to optimize a new rapid microwave procedure for fixing and embedding plant tissue for TEM studies. The Unit also used SEM and TEM to study Baculoviruses that infect insects; to study 3 chiggers found in Brazil, one which causes dermatitis in humans, and another that potentially carries Rickettsia that infects birds; and to develop a CLSM technique for obtaining 3D renderings of agriculturally important mites and converting them into 3D printed models. The second objective of this project is to apply novel microscopy approaches to facilitate the systematic identification and characterization of plant pathogens and pests, alone or with their hosts. The Unit developed a fluorescent molecular probe that was used with FL and CLSM to visualize the downy mildew fungus on leaves and stems of Impatiens. The Unit inspected over 50 rose varieties from 12 different states and the District of Columbia and identified 3 species of very small microscopic 4 legged mites on roses. One of these mites, the rose bud mite, carries the rose rosette virus that kills rose bushes within a year and is devastating to the rose industry.
1. Varroa destructor is a mite that feeds on European honey bees. The Varroa destructor mite is the biggest single cause of the world-wide colony loss of honey bees. Past research always led scientists to believe that the mite fed on a bee’s blood. That was until collaborate studies conducted by ARS researchers at Beltsville, Maryland, and the University of Maryland, College Park, Maryland, showed that the mite primarily feeds on a bee’s body fat and not its blood. Two different microscopy methods, low temperature scanning electron microscopy and transmission electron microscopy, revealed feeding damage in the abdominal membrane of honey bees. The images provided conclusive evidence that Varroa mites feed on the fat bodies of adult bees. These findings generated a paradigm shift in the understanding of how, where, and what the Varroa mite eats and provides a fresh path for developing strategies to control this global threat to honey bees.
2. Three microscopic mites have been discovered on roses. Three microscopic four-legged mites have been identified on roses, one of which is known to carry a virus causing rose rosette disease. Various microscopy techniques were used by ARS researchers at Beltsville, Maryland, to inspect rose samples from 12 states and the District of Columbia as part of a collaborative research project involving state universities, federal agencies, and private rose organizations. Mites were found on enclosed vegetative buds, on leaves, and inside the flower buds near the location where seeds are produced. High resolution scanning electron microscopy images of the three rose mites have been published in scientific journals, in extension bulletins, and web pages on rose rosette disease for use by researchers, extension agents and growers of roses interested in diagnosing rose rosette disease.
Bauchan, G.R., Otero-Colina, G., Hammond, J., Jordan, R.L., Ochoa, R. 2019. Rose rosette disease: It all started with a small mite. Acta Horticulturae. 1232(33):227-232. https://doi.org/10.17660/ActaHortic.2019.1232.33.
Zhang, M., Chu, Y., Mowery, J.D., Konkel, B., Konkel, B., Galli, S., Theos, A., Golestaneh, N. 2018. PGC-1a repression and high fat diet induce age-related macular degeneration-like phenotypes in mice. Disease Models and Mechanisms. https://doi.org/10.1242/dmm.032698.
Dubey, J.P., Trupkiewicz, J.G., Verma, S.K., Mowery, J.D., Adedoyin, G., Georoff, T., Grigg, M. 2017. Atypical fatal sarcocystosis associated with Sarcocystis neurona in a white-nosed coati (Nasua narica molaris). Veterinary Parasitology. 247:80-84. https://doi.org/10.1016/j.vetpar.2017.10.003.
Rezende, J.M., Lofego, A., Bauchan, G.R., Ochoa, R. 2018. New Ceratotarsonemus species from the Amazon forest in Brazil (Prostigmata; Tarsonemidae). Zootaxa. 4483(2):271-294.
Byrne, D.H., Klein, P., Hall, C., Windham, M., Ochoa-Corona, F., Olson, J., Paret, M., Babu, M., Knox, G., Jordan, R.L., Hammond, J., Ong, K., Ochoa, R., Bauchan, G.R., Evans, T., Windham, A., Hale, F., Palma, M.A., Ribera, L., Pemberton, H.B. 2019. Combating rose rosette disease US national project. Acta Horticulturae. 1232:203-212. https://doi.org/10.17660/ActaHortic.2019.1232.30.
Murata, F., Cerqueira-Cezar, C., Thompson, P., Tewari, K., Mowery, J.D., Verma, S., Rosenthal, B.M., Sharma, R., Dubey, J.P. 2018. Sarcocystis cymruensis: Discovery in Western hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: Redescription, molecular characterization, transmission to IFN-gamma gene knockout mice via sporocysts. Parasitology Research. 117:1195–1204. https://doi.org/10.1007/s00436-018-5799-5
Ramsey, S., Ochoa, R., Bauchan, G.R., Gulbronson, C., Mowery, J., Cohen, A., Lim, D., Joklik, J., Cierco, J., Ellis, J., Hathorne, D.J., Vanengelsdorp, D. 2019. Varroa destructor (Varroidae) feed on honey bee fat body tissue not hemolymph. Proceedings of the National Academy of Sciences. 116(5):1792-1801.
Beard, J.J., Ochoa, R., Bauchan, G.R., Dowling, A.G. 2018. Raoiella (Trombidiformes: Tenuipalpidae) of the world. Zootaxa. 4501(1):1-302.
Bassini-Silva, R., Jacinavicius, F.C., Maturano, R., Munoz-Leal, S., Ochoa, R., Bauchan, G.R., Labruna, M., Barros-Battesti, D.M. 2018. Blankaartia sinnamaryi (Floch & Fauran, 1956) (Trombidiformes: Trombiculidae) parasitizing birds in southeastern Brazil, with notes on Rickettsia detection. Revista Brasileira de Parasitologia Veterinaria. 27:354-362.
Bolton, S.J., Bauchan, G.R., Chetverikov, P.E., Ochoa, R., Klompen, H. 2018. A rudimentary sheath for the smallest of ‘biting’ chelicerae; A precursor to the stylet sheath of Eriophyoidea (Acariformes). International Journal of Acarology. 44(8):374-381.
Ramsey, S., Rulbronson, C., Mowery, J.D., Ochoa, R., Van Engledorp, D., Bauchan, G.R. 2018. A multi-microscopy approach to discover the feeding site and host tissue consumed by Varroa destructor on host honey bees. Microscopy and Microanalysis. 24(S1):1258-1259. https://doi.org/10.1017/S1431927618006773.
Gulbronson, C., Mowery, J.D., Pooley, C.D., Ochoa, R., Bolton, S., Bauchan, G.R. 2018. Three-dimensional printing of agriculturally important mites generated from confocal microscopy. Microscopy and Microanalysis. 24:1360-1361. https//doi.org/10.1017/S1431927618007286.
Mowery, J.D., Bauchan, G.R. 2018. Optimization of rapid microwave processing of botanical samples for transmission electron microscopy. Microscopy and Microanalysis. 24:1202-1203. https://doi.org/10/1017/S1431927618006499.
Harrison, R.L., Herniou, E.A., Jehle, J.A., Theilmann, D.A., Burand, J.P., Becnel, J.J., Krell, P.J., Van Oers, M.M., Mowery, J.D., Bauchan, G.R. 2018. ICTV virus taxonomy profile: Baculoviridae. Journal of General Virology. 99:1185-1186.
Otero-Colina, G., Ochoa, R., Amrine, J., Hammond, J., Jordan, R.L., Bauchan, G.R. 2019. Eriophyoid mites found on roses in the United States. Journal of Environmental Horticulture. 36(4):146-153.
Cezar-Cerqueira, C., Thompson, P., Murata, F., Mowery, J.D., Brown, J., Banfield, J., Rosenthal, B.M., Dubey, J.P. 2018. Histopathological, morphological, and molecular characterization of Sarcocystis species in elk (Cervus elaphus) from Pennsylvania, USA. Parasitology Research. 117:3245-3255. https://doi.org/10.1007/s00436-018-6024-2.
Salgado-Salazar, C., Bauchan, G.R., Wallace, E.C., Crouch, J.A. 2018. Detection and visualization of the impatiens downy mildew pathogen using fluorescence in situ hybridization (FISH). Plant Methods. 14:92. https://doi.org/10.1186/s13007-018-0362-z.
Harrison, R.L., Mowery, J.D., Bauchan, G.R., Theilmann, D.A., Erlandson, M.A. 2019. The complete genome sequence of a second alphabaculovirus from the true armyworm, Mythimna unipuncta: Implications for baculovirus phylogeny and host specificity. Virus Genes. 55:104-116.
Dubey, J.P., Cerqueira-Cezar, C., Murata, F., Mowery, J.D., Scott, D., Rosypal, A., Lindsay, D. 2019. Confirmation of Sarcocystis jamaicensis sarcocysts in IFN-gamma gene knock out mice orally inoculated with sporocysts from red-tailed hawk (Buteo jamaicensis). Journal of Parasitology. 105(1):143-145. https://doi.org/10.1645/18-148
Escasa, S.R., Harrison, R.L., Mowery, J.D., Bauchan, G.R., Cory, J.S. 2019. The complete genome sequence of an alphabaculovirus from Spodoptera exempta, an agricultural pest of major economic significance in Africa. PLoS One. 14(2):e0209937.
Castro, E.B., Ochoa, R., Bauchan, G.R., Feres, R. 2019. Two new species of Tenuipalpus sensu stricto (Acari; Tenuipalpidae) from Brazil, with a discussion on the ontogeny of leg setae. Zootaxa. 4540(1):178-210.