1.13.4
TOMATO SPOTTED WILT TOSPOVIRUS MOVEMENT INTO THE SALIVARY GLANDS DURING THE DEVELOPMENT OF THRIPS, THRIPS SETOSUS, IS ASSOCIATED WITH THE TRANSMISSIBILITY

J OHNISHI1, D HOSOKAWA1, I FUJISAWA2 and S TSUDA3

1Tokyo University of Agriculture and Technology, Faculty of Agriculture, Tokyo, Japan; 2Hokuriku National Agricultural Experiment Station, Inada, Jouetu, Niigata, Japan; 3Plant Biotechnology Institute, Ibaraki Agricultural Center, Iwama, Nishi-Ibaraki, Ibaraki, Japan

Background and objective
TSWV is exclusively transmitted by several species of thrips in a unique persistent manner [1]. Thrips setosus is one of the insect vectors of TSWV, which has been found sporadically in various crops throughout Japan [2]. It is generally accepted that adult thrips transmit TSWV only if the virus is acquired in its larval stage. Previous studies by electron microscopy showed evidence that TSWV particles and proteins exist within cells of several organs of Frankliniella occidentalis. These ultrastructural observations are of great value in understanding the localization and distribution of the virus in the vector. However, it would be difficult to describe the temporal and spatial distribution of TSWV at the whole-insect level by electron microscopy. To define the relationship between virus distribution in T. setosus and its transmissibility, we have examined the localization of the virus in thrips at different developmental stages at the whole-insect level using immunofluorescence techniques.

Materials and methods
First instar larval T. setosus were allowed to feed on TSWV-infected leaves. The infected larvae were then transferred to TSWV-free leaves to complete their development to the adult stage. Infected first, second, pupal, and adult thrips were used for immunofluorescence using either antibody against nucleocapsid (N) or non-structural (NSs) protein, which is encoded by the antisense S-RNA segment of TSWV. Virus-free adult thrips were fed on infected leaves and then maintained on healthy leaves until 7 days after acquisition access. These adults were treated the same as adults infected at the larval stage. The accumulation of TSWV during thrips development from larvae to adult and in adult thrips after acquisition feeding was performed by the DAS-ELISA using anti-nucleocapsid monoclonal antibody.

Results and conclusions
Immunofluorescence of two antibodies, N or NSs, revealed significant differences in TSWV distribution in adult thrips, depending on whether acquired at the larval or adult stage. After virus acquisition at the larval stage, TSWV multiply rapidly in the midgut at the same stage, and disseminate into the salivary glands during pupation. The signal of TSWV in the midgut disappears during pupation and, after adult eclosion, TSWV is recognized only in the salivary glands of the adult. Virus accumulation was found to change dramatically during thrips development from larva to adult, first increasing in concentration during larval development, then decreasing during pupal and adult stages. It was observed that adult thrips could transmit TSWV only if the virus was acquired at the larval stage. In contrast, the immunolocalization of TSWV in adult acquisition was restricted to the midgut. Also, thrips that acquired TSWV in the adult stage were not able to transmit the virus. These results reveal that infection of the salivary glands is tightly associated with TSWV transmission by thrips. It is suggested that virus movement into this organ during development plays a crucial role in the transmission by viruliferous adult thrips.

References
1. German TL, Ullman DE, Moyer JW, 1992. Annual Reviews of Phytopathology 30, 315-348.
2. Tsuda S, Fujisawa I, Ohnishi J et al., 1996. Phytopathology 86, 1199-1203