Wolbachia is an intracellular symbiont that causes reproductive disorders in many insects. Its presence in the leafhoppers Hishimonoides sellatiformis and Hishimonus sellatus, vectors of mulberry dwarf-Phytoplasma, was confirmed by the PCR analysis of 16S rDNA, ftsZ and wsp. Sequencing of cloned PCR products revealed that two Wolbachia strains coexist in both leafhoppers. The phylogenetic analysis of wsp revealed that these strains belong in novel positions in the B-group of Wolbachia. These strains were detected by PCR and/or PCR-RFLP in all of the tested non-genital organs including salivary glands, as well as in the tested genital organs of Hishimonoides sellatiformis. In addition, Wolbachia-like organisms were observed by electron microscopy in all PCR-positive organs. We discuss the possible horizontal transmission of Wolbachia via mulberry trees.

The presence of mulberry dwarf (MD) phytoplasmas in organs of the inoculative vector insects Hishimonoides sellatiformis and Hishimonus sellatus was determined by means of electron microscopy (EM) and polymerase chain reaction (PCR) assays. Many MD phytoplasmas were detected in genital organs as well as in the intestines, salivary glands, brains, fat bodies, and thoracic ganglia of Hishimonoides sellatiformis, but only in the intestine and salivary glands of Hishimonus sellatus. Many phytoplasmas with characteristic morphology were observed via EM in ovaries, seminal receptacles, and testes, and they were further identified by PCR assays with group I-specific primers. In addition, the organisms were detected by direct or nested PCR assays in eggs (head pigmentation stage of embryos) laid on mulberry shoots by inoculative leafhoppers and in the newly hatched nymphs from these eggs. These findings indicate that transovarial transmission of MD phytoplasmas occurs in Hishimonoides sellatiformis.

The mode of action by which entomopoxvirus (EPV) spindles, proteinaceous crystalline bodies produced by EPVs, enhance EPV infection has not been clarified. We fed Anomala cuprea EPV (AcEPV) spindles to host insects; subsequent scanning electron microscopy revealed the disruption of the peritrophic membranes (PMs) of these insects. The PM is reportedly a barrier against the infection of some insects by viruses. Quantitative PCR of AcEPV DNA in the ectoperitrophic area revealed that PM disruption facilitated the passage of EPVs through the PM toward the initial infection site, the midgut epithelium. These results indicate that EPV spindles enhance infection by EPVs by disrupting the PM in the host insects. Fusolin is almost exclusively the constituent protein of the spindles and is the enhancing factor of the infectivity of nucleopolyhedroviruses (NPVs) and possibly that of EPVs. Spheroid is another type of proteinaceous crystalline structure produced by EPVs. Pseudaletia separata EPV (PsEPV) spheroids reportedly contain considerable amounts of fusolin and enhance NPV infection. We assessed the ability of AcEPV spheroids to enhance EPV infectivity and their effect on the PM and carried out immunological experiments; these experiments showed that AcEPV spheroids contain little or no fusolin and are biologically inactive, in contrasts to the situation in PsEPV.Entomopoxviruses (EPVs) are poxviruses that infect insects of orders such as Coleoptera, Lepidoptera, Orthoptera, and Diptera. Many EPVs form two types of proteinaceous crystalline bodies: spheroids and spindles. The spheroid contains virions, whereas the spindle does not; thus, spheroids are agents of infection. The major constituent proteins of the spheroid and spindle are spheroidin and fusolin, respectively, and these two are the most abundant proteins among those produced by EPVs. Peroral administration of the spindles of a coleopteran EPV, Anomala cuprea EPV (AcEPV), to Bombyx mori and Spilosoma imparilis, lepidopteran hosts of two species of nucleopolyhedroviruses (NPVs), enhances the infectivity of not only the occluded NPVs of these two species (16, 19) but also of a nonoccluded NPV of B. mori (4), although AcEPV itself cannot infect these two insects. However, these are artificial phenomena that occur by experimental combination of the spindles with NPVs; these insects on plants rarely ingest AcEPV spindles that are present in soils, together with the NPVs. Also, this mechanism of enhanced infectivity of NPVs is considered to be worthless for the survival of AcEPVs that cannot infect the insects. NPVs and EPVs are taxonomically distant DNA insect viruses. NPVs belong to the family Baculoviridae, whereas EPVs belong to the Poxviridae. However, AcEPV spindles also enhance the infectivity of AcEPV in its host insect, the larva of the cupreous chafer A. cuprea, strongly suggesting that the natural biological function of EPV spindles is to enhance the infectivity of EPVs (15,20). (17) showed that the peritrophic membrane (PM) of B. mori larvae was disinte...

Distribution patterns of mulberry dwarf (MD) phytoplasma were investigated in several organs of dwarfdiseased mulberry trees using direct and nested polymerase chain reactions and electron microscopy. MD phytoplasma was detected in the root of all MD-diseased trees collected from overwintering to before sprouting in the cold district; however, it was not always found in winter buds of the same trees. On the other hand, MD phytoplasma was also detected frequently in reproductive organs, such as flowers (female and bisexual flowerets), fruits (catkins), and seed coats, from a single diseased tree, although the distribution patterns were not uniform. In addition, MD phytoplasma was verified to move into roots from ground plant organs after an artificial inoculation test using the leafhopper Hishimonoides sellatiformis as a vector.

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