Eishiro Shikata

Electron microscopic evidence for the systemic invasion of an insect host by a plant pathogenic virus

Abstract Ultrathin sections were prepared of viruliferous Agallia constricta Van Duzee, a leafhopper vector and host of the plant pathogenic wound-tumor virus (WTV), in an attempt to localize the virus within tissues and cells of various anthropod organs. Earlier infectivity and serological tests had revealed the presence of virus in some insect organs and tissues, without permitting precise localization. In the present study, excised fat body tissues, gut, Malpighian tubules, and salivary glands, as well as whole abdomens, were fixed and embedded in methacrylate or in epoxy resin. Alternating thick and ultrathin sections were made with a Porter-Blum microtome and stained with uranyl acetate and lead. For light microscopy, the thick sections were stained by Delafields hematoxylin and eosin. Thin sections, dried on carbon-coated collodion or Formvar film, were examined by a Siemens Elmiskop I. Largest accumulations of WTV, often in the form of microcrystals, were found in the fat body tissues; crystalline arrangements were also seen in muscle cells. Virus clusters were observed in defined cytoplasmic structures within the Malpighian tubules, mycetome, trachea, and epidermis. Only one or two lobes of the salivary gland contained scattered virus particles not associated with defined cytoplasmic structures. The findings point to the fat body as the most likely site of virus multiplication. The occurrence of virus accumulations in almost all organs of A. constricta is interpreted as direct evidence for systemic invasion of this arthropod host. Lack of a sizable accumulation of WTV in the salivary gland may explain the erratic and poor transmission record of the insect vector. This finding is in contrast with findings made with the very efficient vector of rice dwarf virus, in which the lobes of the salivary gland contain large masses of virus particles.

Electron microscopy of wound tumor virus assembly sites in insect vectors and plants

Abstract Thin sections were prepared for electron microscopy from Agallia constricta insect vectors and from Trifolium incarnatum and Melilotus alba plants infected with wound tumor virus. Insects acquired the virus by feeding on wound tumor diseased plants. Electron microscopy of thin sections of various organs of viruliferous insects indicated that the virus is assembled in accumulations of electron-dense aggregates, designated viroplasms, formed in the cytoplasm of fat body, muscle, midgut, Malpighian tubules, trachea, salivary gland, and epidermal cells. The presence of viroplasms in the cells of the mycetome was not ascertained. The viroplasms are considered the sites of virus assembly. Careful examination of infected plants during early stages of disease revealed the formation of viroplasms in the root tumor cell cytoplasm and in the cytoplasm of cells of enlarged leaf veinlets. In plant cell sections prepared shortly after the appearance of first signs of the wound tumor disease, mature particles were found first at the periphery of the viroplasmic zone. A few days later virions were detected in increasing number inside the viroplasmic area. These morphological findings indicate that wound tumor virus is assembled in plant and insect host cells in viroplasms, formed within the cytoplasmic matrix proper. The similarity of wound tumor viroplasms with those of several viruses infecting higher animals, and the significance of the observations in the elucidation of the precise foci of virus assembly in cells and organs of plant and invertebrate animal hosts of the virus are discussed.

Electron microscopy of pea enation mosaic virus in plants and aphid vectors

Abstract Ultrathin sections were prepared from tissues of pea plants infected with pea enation mosaic virus and from pea aphid vectors of this virus. Infected plant tissues and various organs of the insect vector were excised, fixed with glutaraldehyde-osmic acid, embedded in epoxy resin, sectioned, stained with uranyl acetate and lead, and examined with a Siemens Elmiskop I in an attempt to locate the position of virions in cells. Numerous spherical particles, about 28 mμ in diameter, were found in the cytoplasm and central vacuoles of necrotic and nonnecrotic pea leaf and pod enations, but not in the mitochondria. Similar particles were detected in the fat body and gut lumen of viruliferous pea aphids. Dip preparations of sap from diseased plants also contained particles of the same size and shape. No similar particles were found in healthy pea tissues, in virus-free pea aphids, or in dip preparations from healthy plant material. The spherical particles probably represent the virions of pea enation mosaic virus. Unusual formations of particles were observed in vacuolated structures of plant cells. In rare instances virus microcrystals were found in the cytoplasm of plant cells. Pea enation mosaic virus is the first circulative aphid-borne virus localized by electron microscopy in plants and insects in situ .

Electron microscopy of pea enation mosaic virus in plant cell nuclei

Abstract Ultrathin sections from chlorotic lesions of diseased pea leaves and pea pods with pea enation mosaic were prepared for electron microscopy. In necrotic and non-necrotic cells, large accumulations of virions were found in nuclei. These accumulations were greater than those in the cytoplasm and were observed in cells obtained during earliest stages of disease. From these observations it was concluded that pea enation mosaic virus invades, and then multiplies in, cell nuclei, from which it penetrates into the cytoplasm of the infected cell. Mitochondria were devoid of virions. The diameter of the virions in ultrathin sections of the cytoplasm and of nuclei was approximately 28 mμ. Virions appeared hexagonal in shape under high magnification. Pea enation mosaic virus is the first plant-pathogenic virus of cubical symmetry observed by electron microscopy in nuclei of sectioned plant cells.