Location: Systematic Entomology Laboratory
2022 Annual Report
Objectives
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]
Approach
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.
Progress Report
This is the final report for 8042-22000-305-000D. For further information report for project 8042-32000-325-000D (BRIDGE).
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.
Progress was made on thirty-two different microscopy projects where studies were initiated, continued, and finalized by the staff in the Electron and Confocal Microscopy Unit (ECMU) by collaborating with USDA-ARS, university, and international scientists. Some of the projects are highlighted.
In order to optimize the efficient generation of high-resolution images, continuous improvement have been made to a new, cutting-edge Cryo-Scanning Electron Microscope (Cryo-SEM). New solenoid valves were obtained and installed in the Hitachi Cryo-SEM to improve the performance of the high vacuum gate valves, and airflow to the vacuum insulated lines has been improved to prevent condensation and ice formation on the nitrogen gas lines. Additionally, a new compressor was installed into the water chiller of the Hitachi Transmission Electron Microscope (TEM) to facilitate improve functionality.
A new and improved funnel system for the collection of soil mites was developed, which we have name the Gaylen-Berlase Funnel. This new modified Berlese funnel was developed by ECMU staff utilizing off-the-shelf components which optimizes the temperature and humidity levels inside the apparatus by featuring a mild heat source, sufficiently separated from the sample to prevent overheating and killing the mites before they can escape down the funnel. The Gaylen-Berlase funnel represents a significant innovation for the collection of soil mites. A 3D model of the apparatus has been generated and published and is expected to drastically increase the number of mite specimens which are available for high resolution electron microscopy studies.
New techniques were developed for imaging bacteria biofilms on stainless steel plates to simulate the growth of bacteria biofilms on the metal surface of industrial produce processing plants. 1cm x1cm stainless steel plates were inoculated with carrot extract allowing bacteria biofilms to grow on the surface of the metal. The metal plates were frozen in liquid nitrogen and imaged by CryoSEM representing the first method for SEM imaging of these delicate bacteria biofilms on stainless steel plates. This technique will allow researchers to compare the growth of different bacteria biofilms and allow for a comparison of the effectiveness of various sanitizers.
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 host.
Novel microscopy approaches were developed for imaging scale insects. Scale insects have extremely soft and fragile bodies that are prone to desiccation the moment they are removed from their ethanol storage solution. This presents a major challenge for SEM imaging because specimens must be completely dried before observation in an SEM. Additionally scale insect are covered in a dense layer of wax that complete covers the exterior of their bodies and occludes the SEM observation of fine morphological structures. Thus a new technique was developed for clearing the wax off the bodies of scale insects which also provides added rigidity so that the body of the scale insects resist desiccation long enough to be mounted for SEM. Additionally a new technique was developed using a porous mesh filter which allows for the wet scale insect to be mounted into a drop of alcohol on an SEM stub and properly positioned; before wicking away the alcohol from below the mesh filter. This new technique has drastically improved the ability to image these scale insects, resulting in stunning, high-resolution images and enabling the morphological description of a new species and providing an improved understating of the wax that covers the cuticle these insects.
Novel Cryo-SEM studies have been completed to describe three new nematode species of the genera Longidorus, Hemicyclosphoram and Tylenchus. In the case of Tylenchus, new methods were developed for imaging old specimens that had been stored on collection slides. Various solvents were utilized to the remove the mounting media and to rehydrate the specimens for optimal Cryo-SEM imaging. ECMU researchers prepared line drawings of these three species for the new species descriptions.
A major project has been initiated to study the beech leaf disease, which is an emerging disease caused by a plant-parasitic nematode threatening beech trees across the U.S. ECMU staff have generated CryoSEM, TEM, and Confocal images of the nematodes and are working to fully characterize the nematode and the symptomatic leaf tissue, in partnership with the Mycology and Nematology Genetic Diversity and Biology Laboratory.
ECMU staff has collaborated with USNA researchers and Texas A&M University to study the Rose Rosette Disease cause in part by a virus associated with the Eriophyid mite. An Incoming Fund Request has been submitted for a Reimbursable Cooperative Agreement with Texas A&M University, funded the ECMU will receive $75,333.33 in funding to help characterize the mites and rose and virus association as part of a larger project to develop resistant rose varieties.
A new mite chamber was developed for studying the feeding mechanism of predatory mites. This novel mite chamber contains the mites in a small area with a transparent cover that allow the mites to be observed under a stereomicroscope. Mites can then be observed feeding on provisioned nematodes and can be instantaneously frozen with liquid nitrogen at the precise moment when a mite is feeding on a nematode, enabling the frozen mite to be transferred into a Cryo-SEM. This novel procedure has enabled the mites feeding mechanisms to be observed in stunning high resolution for the first time. A 3D model of the mite chamber has been created and which is expected to enable a range of use cases and application for the study of other insect specimens in addition to mites. This microscopy research was critical to understand the feeding behavior and feeding mechanisms of soil predatory mite and had led to two research publications in FY22.
Accomplishments
Review Publications
Wei, W., Inaba, J., Zhao, Y., Mowery, J.D., Hammond, R. 2022. Phytoplasma infection blocks starch breakdown and triggers autophagic degradation of chloroplasts, leading to premature leaf senescence, sucrose reallocation, and spatiotemporal redistribution of phytohormones. International Journal of Molecular Sciences. 23(3):1810. https://doi.org/10.3390/ijms23031810.
Rueda-Ramirez, D., Santos, J.C., Young, M., Mowery, J.D., Ochoa, R., Palevsky, E. 2022. Utilizing an integrated taxonomy approach for the description of a new species of Gamasellodes (Mesostigmata: Ascidae). Systematic and Applied Acarology. 27(2):165-180. https://doi.org/10.11158/saa.27.2.2.
Di Palma, A., Bauchan, G.R., Beard, J.J., Ochoa, R., Seeman, O., Kitajima, E.W. 2022. Ultrastructure and functional morphology of the mouthparts in Raoiella mites (Tetranychoidea: Tenuipalpidae): How they use the cheliceral stylets during feeding. Systematic and Applied Acarology. 27(2):346-367. https://doi.org/10.11158/saa.27.2.9.
Sousa, A.G., Rezende, J.M., Lofego, A., Ochoa, R., Bauchan, G.R., Gulbronson, C., Oliveira, A.R. 2022. New species and records of Metatarsonemus (Acari: Tarsonemidae) from Central and South Americas. Systematic and Applied Acarology. 27(2):381-398. https://doi.org/10.11158/saa.27.2.11.
Fife, A., Carrillo, D., Knox, G., Fanny, I., Kishore, D., Roy, A., Ochoa, R., Bauchan, G.R., Paret, M., Xavier, M. 2021. First report of the Brevipalpus-transmitted (Trombidiformes: Tenuipalpidae) virus, Orchid fleck virus (Mononegavirales: Rhabdoviridae) infecting three ornamentals in Florida. Integrated Pest Management. 12(1):43;1-6. https://doi.org/10.1093/jipm/pmab035.
Huanca, J., De Giosa, M., Bauchan, G.R., Evans, G.A., Ochoa, R. 2021. First record of Cenopalpus wainsteini [Trombidiformes: Tetranychoidea: Tenuipalpidae] in the Americas and a description of the symptoms it causes on pines in Peru. Neotropical Entomology. 50(6):1-12. https://doi.org/10.1007/s13744-021-00926-7.
Rueda-Ramírez, D., Carta, L.K., Mowery, J.D., Bauchan, G.R., Ochoa, R., Young, M., Palevsky, E. 2022. In memory of Gary Bauchan: Integrated taxonomy of soil predatory mites in farming systems. Systematic and Applied Acarology. 27(2):181–208. https://doi.org/10.11158/saa.27.2.3.
