|Ochoa, Ronald - Ron|
|PALEVSKY, ERIC - Newe Ya'Ar Research Center|
Submitted to: Microscopy and Microanalysis
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/24/2020
Publication Date: N/A
Interpretive Summary: Plant-parasitic nematodes are microscopic worms found in soil. The nematodes cause plant disease and crop loss. In nature, there are predatory mites that naturally feed on the nematodes, and scientists are hopeful that these mites could be used to reduce the populations of parasitic nematodes in the soil. Because the mites and nematodes are so small, it is necessary to use an electron microscope to see them. But first the nematodes and mites need to be frozen to maintain the shape of their body structures. Several techniques have been developed to prepare the specimins prior to freezing. In this research, a new system for feeding nematodes to predatory mites was developed to enable the observation of mite feeding. This application is expected to better enable scientists and biologists at federal agencies, universities and private instiutions to study the behavior of mites with the hope of reducing nematode disease on plants.
Technical Abstract: Phytoparasitic nematodes or plant-parasitic nematodes are tiny, slender roundworms; typically between 15 µm to 35 µm wide and 250 µm to 12 mm in length. Most of the agriculturally important nematodes are parasites of plants which cause over $100 billion in global crop losses. The body is often ornamented with various ridges (annulations), lateral lines, bristle or other distinctive structures. Nematodes are often described as a tube within a tube due to the digestive system following from the mouth to the anus. Nematodes are typically extracted from soil, roots or other plant material utilizing Baermann funnels or gown from cultures and then mounted on a microscope slide with a coverslip. Observing nematodes using polarization or differential interference contrast microscopy (DIC) provides improved contrast for imaging external structures such as the oral lips and phasmids (secretory organs), number of annulations, shape and position of the vulva (female genital region) and spicules (male genital structures), location of anus, and shape of the tail. Since nematodes are translucent, internal structures can also be identified including the length of feeding stylet, the shape of the knobs at the base of the stylet, the position and shape of digestive and secretory organs, the reproductive organs of males and females as well as a set of measurement ratios called the De Man Indices. SEM imaging of nematodes started with the classical system or fixation, dehydration, critical point drying and sputter coating, however, due to the soft-bodied nature of the worms many shriveled and did not retain important diagnostic features. Low-temperature SEM (LT-SEM) images preserve the turgidity and the critical morphological structures required for identification of nematodes. We have developed several techniques for preparing nematodes for observation in the LT-SEM including picking up live nematodes with an eyelash and placing them on carbon sticky tabs, pipetting liquid cultures onto filter paper and then allowing them to dry briefly prior to contact freezing in liquid nitrogen. A high angel azimuth plate holder has been used to obtain lateral views of nematodes. Recently a feeding arena was designed to record videos of predatory mites feeding on nematodes prior to flash freezing, capturing the native feeding behavior for observation in the LT-SEM. Initial studies have also begun to observe internal structures of nematodes using a confocal laser scanning microscope. These modern microscopy techniques will be discussed as a multi-faceted system to observe these agriculturally important nematodes and linking behavior with their biology.