- Email: [email protected]
The development of spindle inclusions of Choristoneura fumiferana (Lepidoptera: Tortricidae) infected with entomopox virus
JOURNAL
OF INVERTEBRATE
The Development (Lepidoptera:
PATHOMGY
23, 325-332 (1974)
of Spindle Tortricidae)
Inclusions infected
of Choristoneura with Entomopox
fumiferana Virus’
F. T. BIRD Insect
Pathology
Research Institute, Service, P. 0. Box
Department 490, Sault Ste.
Received
September
of the Environment, Marie, Ontario, Canada
Canadinn
Forestry
21, 19?3
Spindle-shaped crystals from 0.1 to 0.5 pm in length develop in the cytoplasm of cells infected with entomopox virus. They become enclosed in membranes and are occhrded with entomopox virions in the formation of polyhedra. Large protein masses that develop in the cytoplasm of infected cells appear to be the source of protein for both polyhedra and spindles.
INTR~DUOTI~N
Spindle-shaped inclusions are formed in several species of Coleoptera and Lepidoptera infected with entomopox virus (Weiser and Vago, 1966; Vago and Bergoin, 1968; Vago et al., 1968a,b; Meynadier et al., 1968 ; Goodwin and Filshie, 1969). These inclusions are large protein crystals averaging 5-10 pm in length, attaining a very large size of 20 x 13 pm in Demoda boron.esis (Vago et al., 1968a). They are generally delimited by a membrane that appears to be of cytoplasmic origin (Bergoin et al., 1968). Molecular lattice spacing of about 60 W occurs in the spindles formed in Melolontha melolontha and Figulus sp. (Bergoin et al., 1968). Spindle-shaped inclusions are also formed in Chorktoneura fumiferana infected with an entomopox virus. They were not identified as spindles in a preliminary note (Bird et al., 1971). Cunningham et al. (1973) reported them as spindles and found similar inclusions in C. con@tana. The spindles in C. fumifetana and C. confZictana differ from those previously described in that they are much smaller and are occluded along with entomopox virions in the ’ Contribution
No. 260.
formation of polyhedra. This paper describes the spindles and their development in C. fumiferana. MATERIALS
Third-instar spruce budworm larvae, reared at 22%, were infected with entomopox virus by allowing them to feed on artificial diet sprayed with an aqueous suspension of entomopox polyhedra (lo5 polyhedra/ml at 0.05 ml/mm2). Early stages of infection were obtained after 10-14 days, moderate infection after 14-20 days, and heavy infection after 20 days. Frozen sections (15 pm thick) were prepared of fat tissue pressed out of larvae that had been bisected. Using standard histological techniques (Humason, 1967)) sections were fixed in Bouin’s fluid and stained with Heidenhain’s iron hematoxylin and eosin. Protein masses were identified in sections exposed to Millon’s reaction (Gomori, 1952). For electron microscopy, fat tissue was immersed for 1 hr in glutaraldehyde in cacodylate buffer, pH 7.0, postfixed in 2% osmium tetroxide in Veronal acetate buffer, dehydrated in an ethanol series, embedded in Epon, sectioned, and stained with uranyl acetate and lead citrate.
325 Copyright 0 1974 by Academic Press, Inc. All rights-of reproduction in any-form reserved.
AND METHODS
326
F.
T.
RESULTS
Morphology
BIRD
and neither the spindle nor the virion disturbed the protein lattice of the polyhedron.
of Spindles and V&ions
Polyhedra’ formed in C. fumiferana infected with entomopox virus contain both spindles and entomopox virions (Fig. 1). The spindles are protein crystals, fusiform in longitudinal sections, from 0.1 to 0.5 pm in length. The virions3 about 0.35 X 0.28 pm, have large dumbbell-shaped cores and each virion is surrounded by a margin of low density. These margins might represent artifacts from shrinkage during processing, or by a disorganization of the crystalline lattice in the vicinity of the occluded virions as suggested by Bergoin and Dales (1971). To the author, however, they appear to be due to the delicate tubular projections of the outer membrane of the virions described by Bergoin and Dales (1971), which give the virions of C. fumiferana and other insect and animal viruses their mulberry appearance. The margins have therefore been considered as part of the virions for purposes of measurement in this paper but will be the subject of further investigation. Such wide margins do not surround the spindles although the spindles frequently are surrounded by a narrow margin of low density which because of the uneven outline appears to be an artifact (Fig. 2). Measurements of the protein molecular lattices of polyhedron protein and of an occluded spindle (Fig. 2) gave 54 A spacings for the polyhedron protein and 58 A spacings for the spindle protein. The patterns of the lattices of the two proteins were oriented in different directions as expected from a random positioning of the spindles a These are not polyhedra in the geometric sense, but, in structure and function they appear analogous to polyhedral inclusions of nuclear and cytoplasmic polyhedrosis infections in insects. The term polyhedra has therefore been retained in agreement with Stoltz and Summers (1972). ’ The average size of 4000 x 3000 A given in a previous report (Bird ct ah, 1971) included both spindles and virions.
Development
of Spindles
The first symptom of infection by entomopox virus observed in squash preparations of fat tissue under the phase-contrast light microscope is a swelling of the nuclei. Polyhedra then appear in the cytoplasm. Examination of sectioned and stained tissue has shown that, before polyhedron formation occurs, large masses of protein, which gave a positive Millon’s reaction, appear in the cytoplasm (Fig. 3). As the polyhedra increase in number and size, the protein masses decrease and finally disappear, evidence that the protein is associated with polyhedron formation. Spheres of protein from 2 to 5 pm in diameter are scattered throughout the cytoplasm of fat cells of healthy mature C. fumiferana larvae. These are identified as albuminoid granules described in electron microscope studies by Walker (1965) in the lepidopteran Philosamia Cynthia ricini during larval-pupal metamorphosis. Similar but much larger (2-10 pm in diameter) and much more numerous protein granules are formed in C. fumiferana infected with entomopox virus. These granules (Fig. 4, cell A) undergo a process of degradation during which fragments break or budd off (Fig. 4, cell B). These fragments appear to be the spindles (Fig. 5). Entomopox virions appear in the cell at about the same time as the spindles. Although not easily identified in Fig. 4, cell B, they are shown along with the spindles in Fig. 5. Polyhedron formation, during which both spindles and virions become occluded by proteins, occurs at a time when the original protein granules have greatly diminished (Fig. 4, cell C) . Each spindle becomes enclosed within a membrane (Fig. 5). Occasionally two or more spindles are enclosed hy a single membrane. Membrane-enclosed spindles and virions are occluded by protein to form
SPINDLE
INCLUSIONS
FIGS. 1-7. Magnification scale FIG. 1. Section of a polyhedron
IN
Choristoneura
on each photograph containing spindles
a polyhedron (Fig. 6). Polyhedron formation involving only spindles occurs (Fig. 7)) but this is rare. An examination of electron
is in micrometers. (S) and virions
327
(V)
micrographs of sections through 60 polyhedra gave a count of 1 spindle to 1.4 virions.
328
F.
Section of polyhedron of molecular lattices. FIQ. 3. Frozen section of fat in the cytoplasm of virus-infected iron hematoxylin and eosin). FIG.
2.
containing
T.
BIRD
spindle
(S)
and
virion
(VI.
masses Bouin’s
(PM) fixative
Arrows
indicate
direction
tissue showing large cells and polyhedra
protein (P).
which develop (Heidenhain’s
SPINDLE
INCLUSIONS
IN
ChOTistO?ZeuR-Z
FIG. 4. Section of fat tissue infected with entomopox (A) with numerous large spherical protein granules, (B) of degradation during which fragments (possibly spindles) ment of polyhedra (P).
virus showing 3 adjacent cells: protein granules in advanced stages break or bud off, and (C) develop-
F.
T.
BIRD
5. Part of a protein granule formed in a virus-infected in process of budding off, spindles enclosed by membranes and mitochondrion (M). FIG. 6. Polyhedron formation during which membrane-enclosed (V) are occluded by protein. FIG.
(S) (V),
fat cell showing (SM), entomopox spindles
(SM)
and
spindle virions virione
SPINDLE
FIG. virions.
7.
Polyhedron
formation
INCLUSIONS
involving
1>1scuss10n-
7 ‘here must be a very close relationship
beti ween the polyhedron protein and the spirIdle protein. They appear to be derived
IN
(A)
only
Choristoneura
spindles
and
(B)
both
spindles
and
from the same starting material, that of the spindle appearing during degradation of the protein, that of the polyhedron after II lost of the original protein has disappeared
332
F.
T.
Bergold (1963) stated that a definite but unknown selective process takes place during inclusion body formation whereby only complete virions of polyhedrosis viruses are occluded. The occlusion of the spindles along with entomopox virions in the formation of polyhedra is the first time in the ,study of insect viruses that any other component except virus has been found to be occluded. A careful study is therefore indicated to determine the origins of the outer membrane of the virion and delimiting membrane of the spindle. ACKNOWLEDGMENTS Grateful achnowledgment is extended to Mrs. D. Higginson for technical assistance and to members of the Insect Pathology Research Institute for their assistance in the preparation of this manuscript. REFERENCES M. AND DALES, S. 1971. Comparative observations on poxviruses of invertebrates and vertebrates. In “Comparative Virology.” (Y. Maramorosch and E. Kurstak, eds.), pp. 169-205. Academic Press, New York. BERGOIN, M., DEVAUCHELLE, G., DUTHIOT, J., AND VAGO, C. 1968. Etude au microscope dlectronique des inclusions de la virose $ fuseaux des Colkopteres. C. R. Acad. Sci., 266, 2126-2128. BERGOIN, M., DEVAUCHELLE, G., AND VAGO, C. 1969. Electron microscopy study of the pox-like virus of Melolontha melolontha L. (Coleoptera, Scarabeidae). Arch. Ges. Virusforsch., 28, 285-302. BERGOLD, G. H. 1963. The molecular structure of some insect virus inclusion bodies. J. Ultrastruck Res., 8, 360-378. BIRD, F. T., SANDERS, C. J., AND BURKE, J. M. 1971. A newly discovered virus disease of the BERCOIN,
BIRD
spruce budworm, Choristoeura biennis (Lepidoptera: Tortricidae). J. Znvertebr. Pathol., 18, 159-161. CUNNINGHAM, J. C., BURKE, J. M., AND ARIF, B. M. 1973. An Entomopoxvirus found in populations of the large aspen tortrix Choristoneura conflictana Wlk. Lepidoptera: Tortricidae) in Ontario. Can. Entomol., 105, 767-773. GOMORI, G. 1952. “Microscopic Histochemistry” Univ. Chicago Press, Chicago, Illinois. GOODWIN, R. H., AND FILSHIRE, B. K. 1969. Morphology and development of an occluded virus from the black-soil scarab, Othnonius batesi. J. Znvertebr. Pathol., 13, 317-329. HUMASON, G. L. 1967. “Animal Tissue Techniques.” Freeman, San Francisco, California. MEYNADIER, J. F., VAGO, C., DUTHIOT, J. L., AND BRES, N. 1968. Une virose b inclusions ovoides chew un Lepidoptkre. Ann Epiphyt., 19, 703-166. STOLTZ, D. B., AND SUMMERS, M. D. 1972. Observations on the morphogenesis and structure of a nemocytic poxvirus in the midge Chironomous attenuatus. J. Ultrastruct. Res., 40, 581-598. VAGO, C., AND BERGOIN, M. 1968. Visuses of invertebrates. Advan. Virus Res., 13, 247-303. VAGO, C., AMARGIER, A., HURPIN, B., MEYNADIER, G., AND DUTHIOT, J. L. 1968a. Virose zt fuseau d’un Scarab&de d’Amkrique du Sud. Entomophaga, 13, 373-376. VAGO, C., MONSARRAT, P., DUTHIOT, J. L., AMARGIER, A., MEYNADIER, G., AND VAN WAEREBEKE, D. 1968b. Nouvelle virose a fuseaux observee chez un Lucanide (Coleoptera) de Madagascar. C. R. Acad. Sci. Ser., D. 266, 1621~1623. WALKER, P. A. 1966. An electron microscope study of the fat body of the moth Philosamia during growth and metamorphosis. J. Insect. Physiol., 12, 1008-1009. WEISER, J., AND VAGO, C. 1966. A newly described virus of the winter moth, Operophtera brumata Hubner (Lepidoptera: Geometridae). J. Znvertebr. Pathol.. 8, 314-319.
OF INVERTEBRATE
The Development (Lepidoptera:
PATHOMGY
23, 325-332 (1974)
of Spindle Tortricidae)
Inclusions infected
of Choristoneura with Entomopox
fumiferana Virus’
F. T. BIRD Insect
Pathology
Research Institute, Service, P. 0. Box
Department 490, Sault Ste.
Received
September
of the Environment, Marie, Ontario, Canada
Canadinn
Forestry
21, 19?3
Spindle-shaped crystals from 0.1 to 0.5 pm in length develop in the cytoplasm of cells infected with entomopox virus. They become enclosed in membranes and are occhrded with entomopox virions in the formation of polyhedra. Large protein masses that develop in the cytoplasm of infected cells appear to be the source of protein for both polyhedra and spindles.
INTR~DUOTI~N
Spindle-shaped inclusions are formed in several species of Coleoptera and Lepidoptera infected with entomopox virus (Weiser and Vago, 1966; Vago and Bergoin, 1968; Vago et al., 1968a,b; Meynadier et al., 1968 ; Goodwin and Filshie, 1969). These inclusions are large protein crystals averaging 5-10 pm in length, attaining a very large size of 20 x 13 pm in Demoda boron.esis (Vago et al., 1968a). They are generally delimited by a membrane that appears to be of cytoplasmic origin (Bergoin et al., 1968). Molecular lattice spacing of about 60 W occurs in the spindles formed in Melolontha melolontha and Figulus sp. (Bergoin et al., 1968). Spindle-shaped inclusions are also formed in Chorktoneura fumiferana infected with an entomopox virus. They were not identified as spindles in a preliminary note (Bird et al., 1971). Cunningham et al. (1973) reported them as spindles and found similar inclusions in C. con@tana. The spindles in C. fumifetana and C. confZictana differ from those previously described in that they are much smaller and are occluded along with entomopox virions in the ’ Contribution
No. 260.
formation of polyhedra. This paper describes the spindles and their development in C. fumiferana. MATERIALS
Third-instar spruce budworm larvae, reared at 22%, were infected with entomopox virus by allowing them to feed on artificial diet sprayed with an aqueous suspension of entomopox polyhedra (lo5 polyhedra/ml at 0.05 ml/mm2). Early stages of infection were obtained after 10-14 days, moderate infection after 14-20 days, and heavy infection after 20 days. Frozen sections (15 pm thick) were prepared of fat tissue pressed out of larvae that had been bisected. Using standard histological techniques (Humason, 1967)) sections were fixed in Bouin’s fluid and stained with Heidenhain’s iron hematoxylin and eosin. Protein masses were identified in sections exposed to Millon’s reaction (Gomori, 1952). For electron microscopy, fat tissue was immersed for 1 hr in glutaraldehyde in cacodylate buffer, pH 7.0, postfixed in 2% osmium tetroxide in Veronal acetate buffer, dehydrated in an ethanol series, embedded in Epon, sectioned, and stained with uranyl acetate and lead citrate.
325 Copyright 0 1974 by Academic Press, Inc. All rights-of reproduction in any-form reserved.
AND METHODS
326
F.
T.
RESULTS
Morphology
BIRD
and neither the spindle nor the virion disturbed the protein lattice of the polyhedron.
of Spindles and V&ions
Polyhedra’ formed in C. fumiferana infected with entomopox virus contain both spindles and entomopox virions (Fig. 1). The spindles are protein crystals, fusiform in longitudinal sections, from 0.1 to 0.5 pm in length. The virions3 about 0.35 X 0.28 pm, have large dumbbell-shaped cores and each virion is surrounded by a margin of low density. These margins might represent artifacts from shrinkage during processing, or by a disorganization of the crystalline lattice in the vicinity of the occluded virions as suggested by Bergoin and Dales (1971). To the author, however, they appear to be due to the delicate tubular projections of the outer membrane of the virions described by Bergoin and Dales (1971), which give the virions of C. fumiferana and other insect and animal viruses their mulberry appearance. The margins have therefore been considered as part of the virions for purposes of measurement in this paper but will be the subject of further investigation. Such wide margins do not surround the spindles although the spindles frequently are surrounded by a narrow margin of low density which because of the uneven outline appears to be an artifact (Fig. 2). Measurements of the protein molecular lattices of polyhedron protein and of an occluded spindle (Fig. 2) gave 54 A spacings for the polyhedron protein and 58 A spacings for the spindle protein. The patterns of the lattices of the two proteins were oriented in different directions as expected from a random positioning of the spindles a These are not polyhedra in the geometric sense, but, in structure and function they appear analogous to polyhedral inclusions of nuclear and cytoplasmic polyhedrosis infections in insects. The term polyhedra has therefore been retained in agreement with Stoltz and Summers (1972). ’ The average size of 4000 x 3000 A given in a previous report (Bird ct ah, 1971) included both spindles and virions.
Development
of Spindles
The first symptom of infection by entomopox virus observed in squash preparations of fat tissue under the phase-contrast light microscope is a swelling of the nuclei. Polyhedra then appear in the cytoplasm. Examination of sectioned and stained tissue has shown that, before polyhedron formation occurs, large masses of protein, which gave a positive Millon’s reaction, appear in the cytoplasm (Fig. 3). As the polyhedra increase in number and size, the protein masses decrease and finally disappear, evidence that the protein is associated with polyhedron formation. Spheres of protein from 2 to 5 pm in diameter are scattered throughout the cytoplasm of fat cells of healthy mature C. fumiferana larvae. These are identified as albuminoid granules described in electron microscope studies by Walker (1965) in the lepidopteran Philosamia Cynthia ricini during larval-pupal metamorphosis. Similar but much larger (2-10 pm in diameter) and much more numerous protein granules are formed in C. fumiferana infected with entomopox virus. These granules (Fig. 4, cell A) undergo a process of degradation during which fragments break or budd off (Fig. 4, cell B). These fragments appear to be the spindles (Fig. 5). Entomopox virions appear in the cell at about the same time as the spindles. Although not easily identified in Fig. 4, cell B, they are shown along with the spindles in Fig. 5. Polyhedron formation, during which both spindles and virions become occluded by proteins, occurs at a time when the original protein granules have greatly diminished (Fig. 4, cell C) . Each spindle becomes enclosed within a membrane (Fig. 5). Occasionally two or more spindles are enclosed hy a single membrane. Membrane-enclosed spindles and virions are occluded by protein to form
SPINDLE
INCLUSIONS
FIGS. 1-7. Magnification scale FIG. 1. Section of a polyhedron
IN
Choristoneura
on each photograph containing spindles
a polyhedron (Fig. 6). Polyhedron formation involving only spindles occurs (Fig. 7)) but this is rare. An examination of electron
is in micrometers. (S) and virions
327
(V)
micrographs of sections through 60 polyhedra gave a count of 1 spindle to 1.4 virions.
328
F.
Section of polyhedron of molecular lattices. FIQ. 3. Frozen section of fat in the cytoplasm of virus-infected iron hematoxylin and eosin). FIG.
2.
containing
T.
BIRD
spindle
(S)
and
virion
(VI.
masses Bouin’s
(PM) fixative
Arrows
indicate
direction
tissue showing large cells and polyhedra
protein (P).
which develop (Heidenhain’s
SPINDLE
INCLUSIONS
IN
ChOTistO?ZeuR-Z
FIG. 4. Section of fat tissue infected with entomopox (A) with numerous large spherical protein granules, (B) of degradation during which fragments (possibly spindles) ment of polyhedra (P).
virus showing 3 adjacent cells: protein granules in advanced stages break or bud off, and (C) develop-
F.
T.
BIRD
5. Part of a protein granule formed in a virus-infected in process of budding off, spindles enclosed by membranes and mitochondrion (M). FIG. 6. Polyhedron formation during which membrane-enclosed (V) are occluded by protein. FIG.
(S) (V),
fat cell showing (SM), entomopox spindles
(SM)
and
spindle virions virione
SPINDLE
FIG. virions.
7.
Polyhedron
formation
INCLUSIONS
involving
1>1scuss10n-
7 ‘here must be a very close relationship
beti ween the polyhedron protein and the spirIdle protein. They appear to be derived
IN
(A)
only
Choristoneura
spindles
and
(B)
both
spindles
and
from the same starting material, that of the spindle appearing during degradation of the protein, that of the polyhedron after II lost of the original protein has disappeared
332
F.
T.
Bergold (1963) stated that a definite but unknown selective process takes place during inclusion body formation whereby only complete virions of polyhedrosis viruses are occluded. The occlusion of the spindles along with entomopox virions in the formation of polyhedra is the first time in the ,study of insect viruses that any other component except virus has been found to be occluded. A careful study is therefore indicated to determine the origins of the outer membrane of the virion and delimiting membrane of the spindle. ACKNOWLEDGMENTS Grateful achnowledgment is extended to Mrs. D. Higginson for technical assistance and to members of the Insect Pathology Research Institute for their assistance in the preparation of this manuscript. REFERENCES M. AND DALES, S. 1971. Comparative observations on poxviruses of invertebrates and vertebrates. In “Comparative Virology.” (Y. Maramorosch and E. Kurstak, eds.), pp. 169-205. Academic Press, New York. BERGOIN, M., DEVAUCHELLE, G., DUTHIOT, J., AND VAGO, C. 1968. Etude au microscope dlectronique des inclusions de la virose $ fuseaux des Colkopteres. C. R. Acad. Sci., 266, 2126-2128. BERGOIN, M., DEVAUCHELLE, G., AND VAGO, C. 1969. Electron microscopy study of the pox-like virus of Melolontha melolontha L. (Coleoptera, Scarabeidae). Arch. Ges. Virusforsch., 28, 285-302. BERGOLD, G. H. 1963. The molecular structure of some insect virus inclusion bodies. J. Ultrastruck Res., 8, 360-378. BIRD, F. T., SANDERS, C. J., AND BURKE, J. M. 1971. A newly discovered virus disease of the BERCOIN,
BIRD
spruce budworm, Choristoeura biennis (Lepidoptera: Tortricidae). J. Znvertebr. Pathol., 18, 159-161. CUNNINGHAM, J. C., BURKE, J. M., AND ARIF, B. M. 1973. An Entomopoxvirus found in populations of the large aspen tortrix Choristoneura conflictana Wlk. Lepidoptera: Tortricidae) in Ontario. Can. Entomol., 105, 767-773. GOMORI, G. 1952. “Microscopic Histochemistry” Univ. Chicago Press, Chicago, Illinois. GOODWIN, R. H., AND FILSHIRE, B. K. 1969. Morphology and development of an occluded virus from the black-soil scarab, Othnonius batesi. J. Znvertebr. Pathol., 13, 317-329. HUMASON, G. L. 1967. “Animal Tissue Techniques.” Freeman, San Francisco, California. MEYNADIER, J. F., VAGO, C., DUTHIOT, J. L., AND BRES, N. 1968. Une virose b inclusions ovoides chew un Lepidoptkre. Ann Epiphyt., 19, 703-166. STOLTZ, D. B., AND SUMMERS, M. D. 1972. Observations on the morphogenesis and structure of a nemocytic poxvirus in the midge Chironomous attenuatus. J. Ultrastruct. Res., 40, 581-598. VAGO, C., AND BERGOIN, M. 1968. Visuses of invertebrates. Advan. Virus Res., 13, 247-303. VAGO, C., AMARGIER, A., HURPIN, B., MEYNADIER, G., AND DUTHIOT, J. L. 1968a. Virose zt fuseau d’un Scarab&de d’Amkrique du Sud. Entomophaga, 13, 373-376. VAGO, C., MONSARRAT, P., DUTHIOT, J. L., AMARGIER, A., MEYNADIER, G., AND VAN WAEREBEKE, D. 1968b. Nouvelle virose a fuseaux observee chez un Lucanide (Coleoptera) de Madagascar. C. R. Acad. Sci. Ser., D. 266, 1621~1623. WALKER, P. A. 1966. An electron microscope study of the fat body of the moth Philosamia during growth and metamorphosis. J. Insect. Physiol., 12, 1008-1009. WEISER, J., AND VAGO, C. 1966. A newly described virus of the winter moth, Operophtera brumata Hubner (Lepidoptera: Geometridae). J. Znvertebr. Pathol.. 8, 314-319.