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Occurrence of viruslike particles in midgut epithelial cells of the large elm bark beetle, scolytus scolytus
JOURNAL OF INVERTEBRATE PATHOLOGY 29, 373--381 (1977)
Occurrence of Viruslike Particles in Midgut Epithelial Cells of the Large Elm Bark Beetle, Scolytus scolytus MARGARET K.
ARNOLD AND
G.
BARSON 1
NERC, Unit of Invertebrate Virology, 5 South Parks Road, Oxford, England Received October 18, 1976 Virus-like particles have been observed in the midgut cells of two larvae of the large elm bark beetle, Scolytus scolytus, treated with virus extracted from infected rhinoceros beetles, Oryctes rhinoceros. The structure of these particles has been described and compared with similar structures found in the midgut cells of the yellow mealworm, Tenebrio molitor. Observations suggest these particles have features in common with insect baculoviruses; they may also represent a variant form of Oryctes virus. There was no significant difference in mortality between larvae treated with Oryctes virus and untreated controls.
INTRODUCTION
MATERIALS AND METHODS
An attempt was made to infect larvae of the large elm bark beetle, Scolytus scolytus, with the baculovirus of the rhinoceros beetle, Oryctes rhinoceros. This work formed part of an investigation to identify potential pathogens ofS. scolytus in view of its importance as one of the vectors of Dutch Elm disease in Britain. Two kinds of virus-like particles were observed in the midgut cells. In 2 out of 29 larvae treated with Oryctes virus, virus-like particles were found which appeared to have a marked resemblance to the flexuous rod-shaped structures observed in nuclei of midgut cells of another member of the Coleoptera, Tenebrio molitor, and described by Devauchelle and Vago (1969), Devauchelle (1970), and Thomas and Gouranton (1975). The morphology of the particles found in S. scolytus is described in this paper for comparison. No such particles were observed in specimens not treated with Oryctes virus, but, in one of these, a second type of particle, spherical in shape and about 28 nm in diameter,
Fourth- and fifth-instar S. scolytus larvae were removed from elm bark collected near Farnham, Surrey, United Kingdom. Eighty larvae were immersed, each for 10 sec, in a suspension of Oryctes virus and then were placed individually in plastic containers. Forty larvae were treated similarly with distilled water as controls. The larvae were incubated at 20°C. At daily intervals, the midguts were removed from three treated larvae and one control and were prepared for electron microscopy by double fixation in 5% glutaraldehyde followed by 2% osmium tetroxide, using Trager's (1935) B medium for the diluent and for the intermediate washes. After dehydration through a graded ethanol series, the specimens were embedded in Epikote, sectioned, and stained in 2% uranyl acetate followed by lead citrate (Reynolds, 1963). They were examined on an AE 1 EM6B at 60 kV. Originally, 41 larvae were examined. Later, another 14 larvae were processed for electron microscopy immediately after collection from a further supply of elm bark from the same area in Surrey. Infection ofS. scolytus larvae by Oryctes virus, as described before, was attempted
was seen. 1 Present address: M.A.F.F. Pest Infestation Control Laboratory, London Road, Slough, Berks, England.
373 Copyright © 1977by AcademicPress. inc. All rightsof reproductionin any formreserved.
ISSN 0022-2011
374
A R N O L D A N D BARSON
FIG. 1. Infected nucleus with partially disintegrated membrane. Bar = 2.5/zm. Fl~. 2. Healthy cells adjoining an infected one. Bar = 2.5/zm.
VIRUS-LIKE PARTICLES IN SCOL YTUS SCOLYTUS
on three separate occasions. Assessments of mortality were made daily after each attempt. RESULTS No clearly recognizable evidence of infection by the Oryctes virus was seen in any of the treated specimens examined, but virus-like particles having a morphology different from that of Oryctes virus (Payne, 1974) were seen in two specimens in which Oryctes virus infection had been attempted. These particles strongly resemble those described by Devauchelle (1970) and Thomas and Gouranton (1975) in T. molitor adults. The particles were found in the columnar cells of the midgut epithelium ofS. scolytus, frequently in the nucleus and sometimes in the cytoplasm. Apparently healthy cells could often be found adjoining those containing particles (Figs. 1, 2). The particles are pleomorphic and variable in dimension. They consist of an inner electron-dense flexuous rod surrounded by a space and enclosed, usually only loosely, within a trilaminar unit membrane or envelope structure. In section, the rod may appear to be straight or may be looped round within this membrane (Fig. 3). The complete length of a rod is rarely visible in section, but the longest ones observed measure about 485 nm. In cross-section (Fig. 4), the dense rod is 22 nm in diameter, and the rod surrounded by the membrane has a total diameter of about 65 nm. One or more of these enveloped flexuous rods apparently becomes encased within an electron-dense shell, which is again surrounded by an outer limiting unit membrane (Fig. 5). The electron-dense shell may be closely adpressed to the innermost layer of the outer unit membrane, so that the trilaminar structure of the membrane is not clearly resolved. Alternatively, the shell could be formed by direct thickening of the inner layer of the trilaminar membrane, though this seems
375
less likely (Fig. 6). In longitudinal section, the ovoid bodies thus formed have an average length and width of 314 and 173 nm, respectively. Crystals as described in the infected nuclei of T. molitor (Thomas and Gouranton, 1975) were not found in S. scolytus. However, bodies within infected nuclei, similar in appearance to areas described in T. molitor nuclei as "amorphous zones" (Thomas and Gouranton, 1975), were observed. These bodies have a granular appearance, and, sometimes, membrane material is visible within them (Fig. 7). Fibrillar material is also evident in affected nuclei (Fig. 8), while in the cytoplasm close to affected nuclei, there seems to be extensive accumulation of microtubules (Fig. 9). When the infection is visible within a more or less intact nucleus (Fig. 10), peripheral densely staining regions occur (Fig. 11), reminiscent of the virogenic stroma observed in cells infected with nuclear-polyhedrosis viruses. The nuclear membrane appears to disintegrate during infection (Fig. 1), and, occasionally, groups of enveloped flexuous rods were observed in the cytoplasm, contained within large vesicles (Fig. 12). Enveloped rods also were seen free throughout an extensively vacuolated area of cytoplasm close to the basement membrane (Fig. 13), a situation reminiscent of nuclear-polyhedrosis viruses in gut columnar cells of Lepidoptera, while in Fig. 14, the ovoid bodies can be seen in cytoplasm close to the microvillar surface of a columnar epithelial cell. No virus-like particles were seen in the fat body tissue of either of the two larvae in whose midgut cells the pleomorphic particles occurred or in nine other specimens of fat body examined. There was no significant difference in mortality between larvae treated with Oryctes virus and untreated control larvae. Particles resembling a quite different type of virus were seen in the midgut epithelial
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ARNOLD AND BARSON
FIG. 3. Flexuous rods looped within their loosely fitting unit membrane envelopes. Bar = 100 nm. FIG. 4. Cross-section of a rod within its envelope, encased by the electron-dense shell of the ovoid body and its outer limiting membrane. Bar = 100 nm. FIG. 5. Ovoid bodies in longitudinal section. Bar = 100 nm.
I
FIG. 6. Diagrammatic representation of the possible structure of the ovoid body containing the loosely enveloped flexuous rod: (a) the electron-dense shell closely adpressed to the innermost layer of the outer trilaminar membrane or (b) the electron-dense shell as a direct thickening of the inner layer of the trilaminar membrane. FIG. 7. Granular body or nucleolus in an infected columnar cell nucleus. Note membranous material within the nucleolus. Bar = 500 nm. FIG. 8. FibriUar material in an infected nucleus. Bar = 500 nm. FIG. 9. Microtubules in cytoplasm close to the membrane of an infected nucleus. Bar = 500 nm. 377
378
ARNOLD AND BARSON
FIG. 10. Infected nucleus containing several areas of virogenic stroma, a granular body, and many enveloped particles. Bar = 2.5/xm. FIG. 11. Virogenic stroma with differentiating enveloped rods. Bar = 1 #m. FIG. 12. Vesicles containing groups of enveloped rods in the cytoplasm. Bar = 500 nm.
V I R U S - L I K E PARTICLES IN SCOLYTUS SCOLYTUS
379
FIG. 13. Vacuolated region of cytoplasm containing enveloped rods near the basement membrane of a columnar epithelial cell. Bar = 250 nm. FIG. 14. Ovoid bodies in the cytoplasm close to the microvillar surface of a columnar epithelial cell. Bar = 500 nm. FIG. 15. Spherical particles, approximately 28 nm in diameter, within a vesicle in the cytoplasm of a midgut epithelial cell. Bar - 1 /zm.
ARNOLD AND BARSON
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cells of one of the control larvae. These were spherical, had a diameter of about 28 nm, and occurred extensively within large vesicles in the cytoplasm (Fig. 15). Attempts to isolate the two types of particles described here using trituration and differential centrifugation, both from other larvae of the sample in which the affected larvae occurred and from further samples collected at the same location, have been unsuccessful so far.
DISCUSSION The measurements of the pleomorphic particles observed in S. scolytus are approximately the same as those reported for the particles seen in T. molitor. The morphological similarity of the agents in the two insect species suggests that they are very closely related. Based on the evidence obtained by Thomas and Gouranton (1975), it seems likely that these particles are viruses. Our observations suggest that the particles have features in common with the baculoviruses of insects, and such an interpretation supports that of Thomas and Gouranton (1975). By analogy with the morphogenesis known for certain insect baculoviruses (Harrap, 1972), one can postulate a possible sequence of events in the replication of these particles. The appearance of fibrillar and membranous material in the nucleus apparently represents the earliest visible indication of infection. Some nuclei exhibiting these features also contain peripheral areas of dense chromatin from which rods, usually enveloped, seem to be differentiating. The flexuous rods, presumably nucleocapsids, seem to acquire the loosely fitting envelope within the intact nucleus. Many of the enveloped rods then become encapsulated within shells to form the ovoid bodies. The nuclear membrane of this enlarged nucleus ruptures and disintegrates, releasing enveloped rods and ovoid bodies into
the cytoplasm. It is, perhaps, attractive to suppose that the enveloped rods move through the cytoplasm to infect the nuclei of neighboring cells, and that the ovoid bodies function in a role similar to that of the polyhedra or capsules of other baculoviruses, having no further activity until reaching another host insect. Further information on the properties of these virus-like particles will have to await their isolation and culture from infected elm bark beetles. All efforts to achieve this in our laboratory so far have been abortive. In the absence of such additional data, the morphological similarity of the pleomorphic particles from S. scolytus to those from T. molitor is their most striking feature. Despite the lack of proof of infectivity, it is tempting to consider these particles as viruses having some morphological similarity to baculoviruses, while exhibiting other quite unique structural features. We consider these particles to be present in the S. scolytus larvae probably quite independently of our attempts to infect them with Oryctes virus, which in its natural host is easily distinguishable morphologically. However, it is conceivable that the particles represent a variant form of Oryctes virus, because, in moth (Spodoptera frugiperda) cell cultures, Oryctes virus was found to replicate in a manner morphologically different from that seen in vivo (Kelly, 1976).
ACKNOWLEDGMENTS We are grateful to Elizabeth Morton and Linda Spencer for their expert technical assistance and to Dr. K. A. Harrap and Dr. C. C. Payne for their helpful advice and discussion.
REFERENCES DEVAUCHELLE, G., AND VAGO, C. 1969. Presence de particules d'allure virale dans les noyaux des cellules de l'intestin moyen du Col6opt6re Tenebrio molitor (L.) C. R. Acad. Sci. (Paris), 269, 1142-1144. DEVAUCHELLE, G. 1970. Inclusions cristallines et particules d'aUure virale dans les noyaux des cellules
VIRUS-LIKE PARTICLES IN SCOLYTUS SCOLYTUS de l'intestin moyen du Col6opt6re Tenebrio molitor (L.) J. Ultrastruct. Res., 33, 263-277. H A R ~ e , K. A. 1972. The structure of nuclear polyhedrosis viruses. III. Virus assembly. Virology, 50, 133-139. KELLY, D. C. 1976. "Oryctes" virus replication: Electron microscopic observations on infected moth and mosquito cells. Virology, 69, 596-606. PAYNE, C. C. 1974. The isolation and characterization of a virus from Oryctes rhinoceros. J. Gen. Virol., 25, 105-116.
381
REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol., 17, 208-212. THOMAS, D., AND GOURANTON, J. 1975. Development of virus-like particles in the crystal-containing nuclei of the midgut cells of Tenebrio molitor. J. Invertebr. Pathol., 25, 159-169. TRAGER, W. 1935. Cultivation of the virus of grasserie in silkworm tissue cultures. J. Exp. Med., 61, 501-513.
Occurrence of Viruslike Particles in Midgut Epithelial Cells of the Large Elm Bark Beetle, Scolytus scolytus MARGARET K.
ARNOLD AND
G.
BARSON 1
NERC, Unit of Invertebrate Virology, 5 South Parks Road, Oxford, England Received October 18, 1976 Virus-like particles have been observed in the midgut cells of two larvae of the large elm bark beetle, Scolytus scolytus, treated with virus extracted from infected rhinoceros beetles, Oryctes rhinoceros. The structure of these particles has been described and compared with similar structures found in the midgut cells of the yellow mealworm, Tenebrio molitor. Observations suggest these particles have features in common with insect baculoviruses; they may also represent a variant form of Oryctes virus. There was no significant difference in mortality between larvae treated with Oryctes virus and untreated controls.
INTRODUCTION
MATERIALS AND METHODS
An attempt was made to infect larvae of the large elm bark beetle, Scolytus scolytus, with the baculovirus of the rhinoceros beetle, Oryctes rhinoceros. This work formed part of an investigation to identify potential pathogens ofS. scolytus in view of its importance as one of the vectors of Dutch Elm disease in Britain. Two kinds of virus-like particles were observed in the midgut cells. In 2 out of 29 larvae treated with Oryctes virus, virus-like particles were found which appeared to have a marked resemblance to the flexuous rod-shaped structures observed in nuclei of midgut cells of another member of the Coleoptera, Tenebrio molitor, and described by Devauchelle and Vago (1969), Devauchelle (1970), and Thomas and Gouranton (1975). The morphology of the particles found in S. scolytus is described in this paper for comparison. No such particles were observed in specimens not treated with Oryctes virus, but, in one of these, a second type of particle, spherical in shape and about 28 nm in diameter,
Fourth- and fifth-instar S. scolytus larvae were removed from elm bark collected near Farnham, Surrey, United Kingdom. Eighty larvae were immersed, each for 10 sec, in a suspension of Oryctes virus and then were placed individually in plastic containers. Forty larvae were treated similarly with distilled water as controls. The larvae were incubated at 20°C. At daily intervals, the midguts were removed from three treated larvae and one control and were prepared for electron microscopy by double fixation in 5% glutaraldehyde followed by 2% osmium tetroxide, using Trager's (1935) B medium for the diluent and for the intermediate washes. After dehydration through a graded ethanol series, the specimens were embedded in Epikote, sectioned, and stained in 2% uranyl acetate followed by lead citrate (Reynolds, 1963). They were examined on an AE 1 EM6B at 60 kV. Originally, 41 larvae were examined. Later, another 14 larvae were processed for electron microscopy immediately after collection from a further supply of elm bark from the same area in Surrey. Infection ofS. scolytus larvae by Oryctes virus, as described before, was attempted
was seen. 1 Present address: M.A.F.F. Pest Infestation Control Laboratory, London Road, Slough, Berks, England.
373 Copyright © 1977by AcademicPress. inc. All rightsof reproductionin any formreserved.
ISSN 0022-2011
374
A R N O L D A N D BARSON
FIG. 1. Infected nucleus with partially disintegrated membrane. Bar = 2.5/zm. Fl~. 2. Healthy cells adjoining an infected one. Bar = 2.5/zm.
VIRUS-LIKE PARTICLES IN SCOL YTUS SCOLYTUS
on three separate occasions. Assessments of mortality were made daily after each attempt. RESULTS No clearly recognizable evidence of infection by the Oryctes virus was seen in any of the treated specimens examined, but virus-like particles having a morphology different from that of Oryctes virus (Payne, 1974) were seen in two specimens in which Oryctes virus infection had been attempted. These particles strongly resemble those described by Devauchelle (1970) and Thomas and Gouranton (1975) in T. molitor adults. The particles were found in the columnar cells of the midgut epithelium ofS. scolytus, frequently in the nucleus and sometimes in the cytoplasm. Apparently healthy cells could often be found adjoining those containing particles (Figs. 1, 2). The particles are pleomorphic and variable in dimension. They consist of an inner electron-dense flexuous rod surrounded by a space and enclosed, usually only loosely, within a trilaminar unit membrane or envelope structure. In section, the rod may appear to be straight or may be looped round within this membrane (Fig. 3). The complete length of a rod is rarely visible in section, but the longest ones observed measure about 485 nm. In cross-section (Fig. 4), the dense rod is 22 nm in diameter, and the rod surrounded by the membrane has a total diameter of about 65 nm. One or more of these enveloped flexuous rods apparently becomes encased within an electron-dense shell, which is again surrounded by an outer limiting unit membrane (Fig. 5). The electron-dense shell may be closely adpressed to the innermost layer of the outer unit membrane, so that the trilaminar structure of the membrane is not clearly resolved. Alternatively, the shell could be formed by direct thickening of the inner layer of the trilaminar membrane, though this seems
375
less likely (Fig. 6). In longitudinal section, the ovoid bodies thus formed have an average length and width of 314 and 173 nm, respectively. Crystals as described in the infected nuclei of T. molitor (Thomas and Gouranton, 1975) were not found in S. scolytus. However, bodies within infected nuclei, similar in appearance to areas described in T. molitor nuclei as "amorphous zones" (Thomas and Gouranton, 1975), were observed. These bodies have a granular appearance, and, sometimes, membrane material is visible within them (Fig. 7). Fibrillar material is also evident in affected nuclei (Fig. 8), while in the cytoplasm close to affected nuclei, there seems to be extensive accumulation of microtubules (Fig. 9). When the infection is visible within a more or less intact nucleus (Fig. 10), peripheral densely staining regions occur (Fig. 11), reminiscent of the virogenic stroma observed in cells infected with nuclear-polyhedrosis viruses. The nuclear membrane appears to disintegrate during infection (Fig. 1), and, occasionally, groups of enveloped flexuous rods were observed in the cytoplasm, contained within large vesicles (Fig. 12). Enveloped rods also were seen free throughout an extensively vacuolated area of cytoplasm close to the basement membrane (Fig. 13), a situation reminiscent of nuclear-polyhedrosis viruses in gut columnar cells of Lepidoptera, while in Fig. 14, the ovoid bodies can be seen in cytoplasm close to the microvillar surface of a columnar epithelial cell. No virus-like particles were seen in the fat body tissue of either of the two larvae in whose midgut cells the pleomorphic particles occurred or in nine other specimens of fat body examined. There was no significant difference in mortality between larvae treated with Oryctes virus and untreated control larvae. Particles resembling a quite different type of virus were seen in the midgut epithelial
376
ARNOLD AND BARSON
FIG. 3. Flexuous rods looped within their loosely fitting unit membrane envelopes. Bar = 100 nm. FIG. 4. Cross-section of a rod within its envelope, encased by the electron-dense shell of the ovoid body and its outer limiting membrane. Bar = 100 nm. FIG. 5. Ovoid bodies in longitudinal section. Bar = 100 nm.
I
FIG. 6. Diagrammatic representation of the possible structure of the ovoid body containing the loosely enveloped flexuous rod: (a) the electron-dense shell closely adpressed to the innermost layer of the outer trilaminar membrane or (b) the electron-dense shell as a direct thickening of the inner layer of the trilaminar membrane. FIG. 7. Granular body or nucleolus in an infected columnar cell nucleus. Note membranous material within the nucleolus. Bar = 500 nm. FIG. 8. FibriUar material in an infected nucleus. Bar = 500 nm. FIG. 9. Microtubules in cytoplasm close to the membrane of an infected nucleus. Bar = 500 nm. 377
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ARNOLD AND BARSON
FIG. 10. Infected nucleus containing several areas of virogenic stroma, a granular body, and many enveloped particles. Bar = 2.5/xm. FIG. 11. Virogenic stroma with differentiating enveloped rods. Bar = 1 #m. FIG. 12. Vesicles containing groups of enveloped rods in the cytoplasm. Bar = 500 nm.
V I R U S - L I K E PARTICLES IN SCOLYTUS SCOLYTUS
379
FIG. 13. Vacuolated region of cytoplasm containing enveloped rods near the basement membrane of a columnar epithelial cell. Bar = 250 nm. FIG. 14. Ovoid bodies in the cytoplasm close to the microvillar surface of a columnar epithelial cell. Bar = 500 nm. FIG. 15. Spherical particles, approximately 28 nm in diameter, within a vesicle in the cytoplasm of a midgut epithelial cell. Bar - 1 /zm.
ARNOLD AND BARSON
380
cells of one of the control larvae. These were spherical, had a diameter of about 28 nm, and occurred extensively within large vesicles in the cytoplasm (Fig. 15). Attempts to isolate the two types of particles described here using trituration and differential centrifugation, both from other larvae of the sample in which the affected larvae occurred and from further samples collected at the same location, have been unsuccessful so far.
DISCUSSION The measurements of the pleomorphic particles observed in S. scolytus are approximately the same as those reported for the particles seen in T. molitor. The morphological similarity of the agents in the two insect species suggests that they are very closely related. Based on the evidence obtained by Thomas and Gouranton (1975), it seems likely that these particles are viruses. Our observations suggest that the particles have features in common with the baculoviruses of insects, and such an interpretation supports that of Thomas and Gouranton (1975). By analogy with the morphogenesis known for certain insect baculoviruses (Harrap, 1972), one can postulate a possible sequence of events in the replication of these particles. The appearance of fibrillar and membranous material in the nucleus apparently represents the earliest visible indication of infection. Some nuclei exhibiting these features also contain peripheral areas of dense chromatin from which rods, usually enveloped, seem to be differentiating. The flexuous rods, presumably nucleocapsids, seem to acquire the loosely fitting envelope within the intact nucleus. Many of the enveloped rods then become encapsulated within shells to form the ovoid bodies. The nuclear membrane of this enlarged nucleus ruptures and disintegrates, releasing enveloped rods and ovoid bodies into
the cytoplasm. It is, perhaps, attractive to suppose that the enveloped rods move through the cytoplasm to infect the nuclei of neighboring cells, and that the ovoid bodies function in a role similar to that of the polyhedra or capsules of other baculoviruses, having no further activity until reaching another host insect. Further information on the properties of these virus-like particles will have to await their isolation and culture from infected elm bark beetles. All efforts to achieve this in our laboratory so far have been abortive. In the absence of such additional data, the morphological similarity of the pleomorphic particles from S. scolytus to those from T. molitor is their most striking feature. Despite the lack of proof of infectivity, it is tempting to consider these particles as viruses having some morphological similarity to baculoviruses, while exhibiting other quite unique structural features. We consider these particles to be present in the S. scolytus larvae probably quite independently of our attempts to infect them with Oryctes virus, which in its natural host is easily distinguishable morphologically. However, it is conceivable that the particles represent a variant form of Oryctes virus, because, in moth (Spodoptera frugiperda) cell cultures, Oryctes virus was found to replicate in a manner morphologically different from that seen in vivo (Kelly, 1976).
ACKNOWLEDGMENTS We are grateful to Elizabeth Morton and Linda Spencer for their expert technical assistance and to Dr. K. A. Harrap and Dr. C. C. Payne for their helpful advice and discussion.
REFERENCES DEVAUCHELLE, G., AND VAGO, C. 1969. Presence de particules d'allure virale dans les noyaux des cellules de l'intestin moyen du Col6opt6re Tenebrio molitor (L.) C. R. Acad. Sci. (Paris), 269, 1142-1144. DEVAUCHELLE, G. 1970. Inclusions cristallines et particules d'aUure virale dans les noyaux des cellules
VIRUS-LIKE PARTICLES IN SCOLYTUS SCOLYTUS de l'intestin moyen du Col6opt6re Tenebrio molitor (L.) J. Ultrastruct. Res., 33, 263-277. H A R ~ e , K. A. 1972. The structure of nuclear polyhedrosis viruses. III. Virus assembly. Virology, 50, 133-139. KELLY, D. C. 1976. "Oryctes" virus replication: Electron microscopic observations on infected moth and mosquito cells. Virology, 69, 596-606. PAYNE, C. C. 1974. The isolation and characterization of a virus from Oryctes rhinoceros. J. Gen. Virol., 25, 105-116.
381
REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol., 17, 208-212. THOMAS, D., AND GOURANTON, J. 1975. Development of virus-like particles in the crystal-containing nuclei of the midgut cells of Tenebrio molitor. J. Invertebr. Pathol., 25, 159-169. TRAGER, W. 1935. Cultivation of the virus of grasserie in silkworm tissue cultures. J. Exp. Med., 61, 501-513.