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Electron microscopy and recovery of rice transitory yellowing virus from its leafhopper vector, Nephotettix cincticeps
47,483-486
VIROLOGY
(1972)
Short E!ectron Microscopy
and Recovery Leafhopper
Communications of Rice Transitory
Vector,
Nephofeffix
In the transmission experiments, Chiu and Jean (3) and Chiu et al.. (4) demonstrated that rice transitory yel1owin.g was transmitted by two species of green rice leafhoppers, Nephotettix apicalis Motsch. and N. cincticeps Uhler and indicated the possibility of multiplication of t,he virus in its insect vectors. Recen.tly, the authors described numerous bullet-shaped virus particles in leaf tissues of rice plants infected with rice transitory yellowing virus (RTYV) (1, 6). Therefore, it seemed feasible to search for RTYV in the salivary gland, gut, and other organs of insect vectors. The young nymphs of virus-free colonies of N. cincticeps were first allowed an acquisition feeding of 48 hours on RTYV-infected rice’ plants and subsequently transferred t#o Panicum crusgalli var. submutica which is immune to RTYV infection. For testing the presence of RTYV in various organs of leafhopper vectors, the salivary gland, gut, Malpighian tubules, an.d fat body of viruliferous leafhoppers were dissected out in Mitsuhashi’s culture medium (5). Thirty of each organ were transferred to rough-surface hollow slides cont,aining 0.03 ml of culture medium and then crushed and homogenized at 4’. The homogenates were centrifuged in a capillary for 10 minutes at 3000 rpm in a climcal centrifuge at 4’. The supernatant fluids Tvere injected into young virus-free leafhopper nymphs in a cold room at S” using fine glass n.eedles. The injected insects were fed on. P. crwsgalli var. suhmutica 10 days to allow completion of the virusincubation period. The surviving insects were confined on healt#hy rice seedlings to determine their infectivity. Samplings of t,he virus-exposed insects after feeding on. source plants were made at 483 Copyright
0
197’2 by Academic
Press.
Inc.
Yellowing
Virus from its
cincficeps
5-day int,ervals by the same’ procedures described previously (7, 8). As shown in Table 1, four of eight plants inoculated by the insects injected with extracts from salivary gland transmitted the virus after an incubationperiod of 9-16 days. When the insects were injected with gut extracts, all plants inoculated (8/S) were infected. The results show the virus is present in these organs. Insects in.jected with extracts from Malpighian tubules and fat body did not transmit the virus. Electron microscopic observat#ions of ultrathin sections revealed that salivary gland sampled at 25 and 30 days from the start of acquisition feeding contained bullet-shaped particles similar to those found in diseased leaves infected with RTYV. These virus particles were found only in the vacuolate part,s associated with the membrane structures in the lobes of the salivary gland (Fig. 1). Occasionally, electronlucent sac-like structures at the blunt end of the virus particles were seen (Fig. 1, arrows). These structures had been observed previously in diseased tissues of rice plants where they were closely connected to the outer membranes of the virus part#icles (2). The virus particles in salivary gland have an average length of 216 nm and are 92 nm in width. Tubular structures were located in nuclei (Fig. 2) and the cytoplasm of the epidermal cells of the intesbine. Their structures differed from the virus particles found in salivary gland. Their length, 1100-2000 nm, were 5-10 times longer than those of typical virus particles. However, transverse sections of the tubular structures showed a threelayered struct,ure, as indicated in Fig. 3, similar to the particles found in the diseased tissues of rice leaves (4). The particles varied
484
SHORT
COMMUNICATIONS TABLE
TESTS
FOR THE PRESENCE
.\ND
~---------------
Virus source” Salivary gland Gut Malpighian tubule Fat hod?
OF
KTTV F.\T
1
IN SALIURY GL.ANDS, GUT, BODIES OF LE~~FHOPPERS
M\LPIGHIIN
TUBULES,
No. of insects injected
Survivors after 10 days
No. of insects per plant
No. of plants inoculated
No. of diseased plants
30 30 30 30
16 1; 18
2 2 2
8 8 9
4* gb 0
li
2
8
L’
(1The organs were dissected from the vectors 2 days after acquisition feeding and maintained 1’. cr~rrgnlli var. shnutica for 3 weeks t.heu used as SOllrCe of inoculum into virus-free leafhoppers. b All inoculated plants became infected 9-16 days after injected insects began feeding on healthy seedling.
FIG. 1. An ultrathin section of infective ,Vephotettir cincticeps shows virus part,icles vacuolate structure of a lobe of the salivary gland. The part,icles are cut longitudinally, transversely. Arrows show the sac-like structure at t.he blunt end of the particles.
indefinitely in diameter from 82 to 11-L nm. No bullet-shaped part,icles or t,ubular structures were found in the salivary gland and gut, of virus-free insects or in the insects sampled less than 20 days from the acqui&ion feeding. We also failed to detect such part,icles in the Malpighia,n tubules or fat body of the viruliferous inseck. The bullet-shnpcd particles observed in
on rice
scattered in the obliquely, and
t,he ultrat.hin sections of salivary gland of virulferous green rice leafhoppers were in accordance with the morphology of those found in tissues of RTYV-infested leaves. These parUes were detected only in vacuolate structures within the cytoplasm of the salivary gland. Those observations differed significant,ly from those of virus particles in the cells of diseased leaves, where t,he virus
FIG. 2. .kn ult,rathin section through the nucleus from the epithelial cell of the intestine from a viruliferous insect showing tubular structures which have been cut longitudinally, obliquely, and t.ransversely. T-tubular structures; N-nucleus; Nbl-nuclear membrane; BS-bacteroid symbiont.
FIG. 3. High magnification of a transverse of the structures is clearly visible.
section 485
of tubular
particles.
The three-layered
membrane
486
SHORT COMMUNICATIONS
appeared mostly around perinuclear
zones
CL).
Tran.smission experiments showed the presence of a lat,ent period when RTYV-free insects were fed on the virus source plants. Approximately 10 days were needed to complete transmission of virus (unpublished data). However, t.he electron microscopic observation failed to reveal any virus particles in t,he salivary gland of green rice leafhoppers which were fed on virus source plants for 5, 10, 15, or 20 days. Virus particles were found only in insects being fed on the plants for 25 and 30 days. The injection experiments (Table 1) showed a high recovery of the virus from gut tissues. However, electron microscopic observations failed to show the bullet-shaped partitles within gut tissues.
the Nat.ional Science Council, China, t.o Moh-jih Chen.
of
REFERENCES
1. CHEN, M. J., and SHIKATA, E. Plant Protect. ‘. S ’
4. 6
Bull. Taiw~an 10, 19-28 (1968). CHEN, M. J., and SHIK.~T.~, E., Virology 46, (1971), in press. CHIU, R. J., and JEAN, J. H., Symposium on Virus Diseases of the Rice Plant at the International Rice Research Institute, Philippines, April 25-28 (1967). CHIU, R. J., JEAN, J. H., CHEN, M. H., and Lo, T. C., Phytopathology 58,740-745 (1968). MITSUH.~SHI, J., Jap. J. Appl. Entomol. 2001.
9, 107-111 (1965).
6. SHIK.4TA, E., and CHEN, M. J., J. Viral.
3,
261-264 (1969). SHIK.ITA, E., and M.~RAMOROSCH, K., virology ‘. 27, 461-475 (1965). 8. SHIKATA, E., and ~I.~R.~~~oRoscH, K., J. Viral. 1, 1052-1073 (1967). MOH-JIHCHEN~ EISHIRO SHIKATA
ACKNOWLEDGMENTS The authors express their appreciation to Dr. Daiki Murayama of the Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo, Japan for his suggestions during the experiments, and Dr. Karl Maramorosch, Boyce Thompson Instit.ute, Yonkers, NY for reading this manuscript. The work was supported in part by a grant from
the Republic
Department of Botany Faculty of Agricultwe
Hokkaido University Sapporo Japan
Accepied October 18, 1971 1 Present address : Department of Plant Pathology, Chung-Hsing University, Taichung, Taiwan, China.
VIROLOGY
(1972)
Short E!ectron Microscopy
and Recovery Leafhopper
Communications of Rice Transitory
Vector,
Nephofeffix
In the transmission experiments, Chiu and Jean (3) and Chiu et al.. (4) demonstrated that rice transitory yel1owin.g was transmitted by two species of green rice leafhoppers, Nephotettix apicalis Motsch. and N. cincticeps Uhler and indicated the possibility of multiplication of t,he virus in its insect vectors. Recen.tly, the authors described numerous bullet-shaped virus particles in leaf tissues of rice plants infected with rice transitory yellowing virus (RTYV) (1, 6). Therefore, it seemed feasible to search for RTYV in the salivary gland, gut, and other organs of insect vectors. The young nymphs of virus-free colonies of N. cincticeps were first allowed an acquisition feeding of 48 hours on RTYV-infected rice’ plants and subsequently transferred t#o Panicum crusgalli var. submutica which is immune to RTYV infection. For testing the presence of RTYV in various organs of leafhopper vectors, the salivary gland, gut, Malpighian tubules, an.d fat body of viruliferous leafhoppers were dissected out in Mitsuhashi’s culture medium (5). Thirty of each organ were transferred to rough-surface hollow slides cont,aining 0.03 ml of culture medium and then crushed and homogenized at 4’. The homogenates were centrifuged in a capillary for 10 minutes at 3000 rpm in a climcal centrifuge at 4’. The supernatant fluids Tvere injected into young virus-free leafhopper nymphs in a cold room at S” using fine glass n.eedles. The injected insects were fed on. P. crwsgalli var. suhmutica 10 days to allow completion of the virusincubation period. The surviving insects were confined on healt#hy rice seedlings to determine their infectivity. Samplings of t,he virus-exposed insects after feeding on. source plants were made at 483 Copyright
0
197’2 by Academic
Press.
Inc.
Yellowing
Virus from its
cincficeps
5-day int,ervals by the same’ procedures described previously (7, 8). As shown in Table 1, four of eight plants inoculated by the insects injected with extracts from salivary gland transmitted the virus after an incubationperiod of 9-16 days. When the insects were injected with gut extracts, all plants inoculated (8/S) were infected. The results show the virus is present in these organs. Insects in.jected with extracts from Malpighian tubules and fat body did not transmit the virus. Electron microscopic observat#ions of ultrathin sections revealed that salivary gland sampled at 25 and 30 days from the start of acquisition feeding contained bullet-shaped particles similar to those found in diseased leaves infected with RTYV. These virus particles were found only in the vacuolate part,s associated with the membrane structures in the lobes of the salivary gland (Fig. 1). Occasionally, electronlucent sac-like structures at the blunt end of the virus particles were seen (Fig. 1, arrows). These structures had been observed previously in diseased tissues of rice plants where they were closely connected to the outer membranes of the virus part#icles (2). The virus particles in salivary gland have an average length of 216 nm and are 92 nm in width. Tubular structures were located in nuclei (Fig. 2) and the cytoplasm of the epidermal cells of the intesbine. Their structures differed from the virus particles found in salivary gland. Their length, 1100-2000 nm, were 5-10 times longer than those of typical virus particles. However, transverse sections of the tubular structures showed a threelayered struct,ure, as indicated in Fig. 3, similar to the particles found in the diseased tissues of rice leaves (4). The particles varied
484
SHORT
COMMUNICATIONS TABLE
TESTS
FOR THE PRESENCE
.\ND
~---------------
Virus source” Salivary gland Gut Malpighian tubule Fat hod?
OF
KTTV F.\T
1
IN SALIURY GL.ANDS, GUT, BODIES OF LE~~FHOPPERS
M\LPIGHIIN
TUBULES,
No. of insects injected
Survivors after 10 days
No. of insects per plant
No. of plants inoculated
No. of diseased plants
30 30 30 30
16 1; 18
2 2 2
8 8 9
4* gb 0
li
2
8
L’
(1The organs were dissected from the vectors 2 days after acquisition feeding and maintained 1’. cr~rrgnlli var. shnutica for 3 weeks t.heu used as SOllrCe of inoculum into virus-free leafhoppers. b All inoculated plants became infected 9-16 days after injected insects began feeding on healthy seedling.
FIG. 1. An ultrathin section of infective ,Vephotettir cincticeps shows virus part,icles vacuolate structure of a lobe of the salivary gland. The part,icles are cut longitudinally, transversely. Arrows show the sac-like structure at t.he blunt end of the particles.
indefinitely in diameter from 82 to 11-L nm. No bullet-shaped part,icles or t,ubular structures were found in the salivary gland and gut, of virus-free insects or in the insects sampled less than 20 days from the acqui&ion feeding. We also failed to detect such part,icles in the Malpighia,n tubules or fat body of the viruliferous inseck. The bullet-shnpcd particles observed in
on rice
scattered in the obliquely, and
t,he ultrat.hin sections of salivary gland of virulferous green rice leafhoppers were in accordance with the morphology of those found in tissues of RTYV-infested leaves. These parUes were detected only in vacuolate structures within the cytoplasm of the salivary gland. Those observations differed significant,ly from those of virus particles in the cells of diseased leaves, where t,he virus
FIG. 2. .kn ult,rathin section through the nucleus from the epithelial cell of the intestine from a viruliferous insect showing tubular structures which have been cut longitudinally, obliquely, and t.ransversely. T-tubular structures; N-nucleus; Nbl-nuclear membrane; BS-bacteroid symbiont.
FIG. 3. High magnification of a transverse of the structures is clearly visible.
section 485
of tubular
particles.
The three-layered
membrane
486
SHORT COMMUNICATIONS
appeared mostly around perinuclear
zones
CL).
Tran.smission experiments showed the presence of a lat,ent period when RTYV-free insects were fed on the virus source plants. Approximately 10 days were needed to complete transmission of virus (unpublished data). However, t.he electron microscopic observation failed to reveal any virus particles in t,he salivary gland of green rice leafhoppers which were fed on virus source plants for 5, 10, 15, or 20 days. Virus particles were found only in insects being fed on the plants for 25 and 30 days. The injection experiments (Table 1) showed a high recovery of the virus from gut tissues. However, electron microscopic observations failed to show the bullet-shaped partitles within gut tissues.
the Nat.ional Science Council, China, t.o Moh-jih Chen.
of
REFERENCES
1. CHEN, M. J., and SHIKATA, E. Plant Protect. ‘. S ’
4. 6
Bull. Taiw~an 10, 19-28 (1968). CHEN, M. J., and SHIK.~T.~, E., Virology 46, (1971), in press. CHIU, R. J., and JEAN, J. H., Symposium on Virus Diseases of the Rice Plant at the International Rice Research Institute, Philippines, April 25-28 (1967). CHIU, R. J., JEAN, J. H., CHEN, M. H., and Lo, T. C., Phytopathology 58,740-745 (1968). MITSUH.~SHI, J., Jap. J. Appl. Entomol. 2001.
9, 107-111 (1965).
6. SHIK.4TA, E., and CHEN, M. J., J. Viral.
3,
261-264 (1969). SHIK.ITA, E., and M.~RAMOROSCH, K., virology ‘. 27, 461-475 (1965). 8. SHIKATA, E., and ~I.~R.~~~oRoscH, K., J. Viral. 1, 1052-1073 (1967). MOH-JIHCHEN~ EISHIRO SHIKATA
ACKNOWLEDGMENTS The authors express their appreciation to Dr. Daiki Murayama of the Department of Botany, Faculty of Agriculture, Hokkaido University, Sapporo, Japan for his suggestions during the experiments, and Dr. Karl Maramorosch, Boyce Thompson Instit.ute, Yonkers, NY for reading this manuscript. The work was supported in part by a grant from
the Republic
Department of Botany Faculty of Agricultwe
Hokkaido University Sapporo Japan
Accepied October 18, 1971 1 Present address : Department of Plant Pathology, Chung-Hsing University, Taichung, Taiwan, China.