JOURNAL
OF INVERTEBRATE
A Nuclear
25,
PATHOLOGY
141-143
(1975)
Polyhedrosis Virus of Be/ha (Lepidoptera : Noctuidae)
The biology of Bellura gortynoides, the yellow water lily borer, has been under investigation in two ponds in Tippecanoe County, Indiana, since 1972. The aquatic larvae of this noctuid moth feed exclusively in the leaves and petioles of the yellow water lily, Nuphar advena. Examination of larvae collected from petiole tunnels revealed that some were infected with a polyhedrosis virus. The pathogen was identified as a nuclear polyhedrosis virus (NPV) of the multiploid virion type. The B. gortynoides larvae were reared on a semisynthetic medium with a pinto bean base in 1 oz plastic cups and inoculated at fourth instar per OS with a suspension of isolated from field-collected polyhedra larvae. Normal and infected larvae were examined in smear preparations or prepared for light microscopy and stained with modified azan stain according to the procedure of J. J. Hamm (J. Invertbr. Pathol. 8, 125-126, 1966). Pellets of polyhedra were prepared for electron microscopy by fixation in 6% glutaraldehyde followed by 1% osmium tetroxide, both buffered with 0.1 M cacodylate, pH 7.4. After prestaining with 0.5% uranyl acetate, pellets were dehydrated with ethanol and embedded in epoxy (A. R. Spurr, J. Ultravtruct. Res. 26, 31.-43, 1969). Thin sections were placed on carbon-coated membrane grids, stained with Reynold’s lead citrate and examined with a Philips EM-201 electron microscope calibrated according to R. Luftig (J. Ultrastruct. Res. 20, 91 102, 1967) with catalase crystals. Hemolymph and tissue homogenates of larvae were also examined in preparations applied directly to grids and negatively stained with 3% ammonium molybdate, pH 6.5. Larvae inoculated with polyhedra showed characteristic NPV symptoms. The normal
gortynoides
olive-brown coloration turned to pale green within 3 days after inoculation; by 5 days larvae were dark and lethargic. When the delicate integument was ruptured the liquified body contents were released. Infection was confirmed by light microscopy as evidenced by polyhedra accumulation in nuclei of the hypodermis, fat body, tracheal matrix, sarcolemma, neurilemma, hemocytes, and hindgut. No other region of the gut or other tissues exhibited polyhedra formation. The primary sites of polyhedra accumulation were the fat body, hypodermis, and tracheal matrix. When compared to the normal larval tissues (Fig. 1) these tissues showed hypertrophy and hyperplasia with the latter being most pronounced in the hypodermis (Fig. 2). Numerous polyhedra, free virions, and nucleocapsids were observed in negatively stained homogenates of NPV-infected larvae. In thin sections (Figs. 3, 4), the NPV exhibited typical polyhedra ranging in size from 2 to 5 pm that occluded virions of the multiploid type (J. E. Dahlberg and E. H. Simon, Virology 38, 666-678, 1969). The number of nucleocapsids within an envelope varied from one to ten, with the multiple assemblages being the more predominant. The mean size of 14 nucleocapsids was 33 x 250 nm, with a range of 31-36 x 245-260 nm. The crystalline lattice of the polyhedral protein, measured on lattice lines with 90” intersections, had a periodicity of 69 ,& (Fig. 5). Although polyhedra with virions (Fig. 6) were the most predominant type, polyhedra without occluded virions were also found (Fig. 7). These exhibited the same protein crystalline lattice and periodicity. The multiploid virion NPV of B. gortynoides is a new pathogen for this species. Its structure is similar to the NPV’s described by G. H. Bergold (J. Znvertebr. 141
Cop)rlght Printed
t 1975 b) Academic in the United States.
Press,
Inc.
,411 rlphts
of reproduclton
in any
form
reserved.
FIG. 1. and midgut FIG. 2. mulation in FIG. 3. FIG. 4. FIG. 5. FIG. 6. FIG. 7.
Transverse section of normal larva showing the hypodermis (H), fat body (FB), trachael matrix lumen (ML). x 64. Transverse section of NPV-infected larva exhibiting hypertrophy and hyperplasia with polyhedra the nuclei of infected cells. x 64. Electron micrograph of the predominant size and type of polyhedra isolated from infected larvae. A typical polyhedron showing single (S) and multiple enveloped (M) nucleocapsids. Crystalline lattice structure of the protein matrix. Multiploid virion polyhedron exhibiting the randomly occluded virions of various assemblages. Polyhedron lacking occluded virions.
142
(TM), accu-
143
NOTES
Pathol. 5, 111-128, 1963) that exhibited “virus bundles” and to the multiploid virion NPV isolated from the noctuid Rachiplusia ou by C. Y. Kawanishi and J. D. Paschke (J. Invertebr. Pathol. 16, 89-92, 1970). Its multiploid structure conforms to the characteristics exhibited by multiploid viruses as defined and comprehensively reviewed by E. H. Simon (Progr. Med. Viral. 14, 36-67, 1972). ACKNOWLEDGMENTS The authors thank Drs. Chandler for their support
J. D. Paschke and L. and advice during this
investigation and Ms. Donell Litzenberg for her technical assistance. This research was supported in part by U.S. Public Health Service Research Grant Al-09972 and NDEA Fellowship 70-019322. STANLEY
R. WEBB ELI
Laboratory of Insect Pathology Department of Entomology Purdue University West Lafayette, Indiana 47907
Received May 6,1974
LEVINE
OF INVERTEBRATE
A Nuclear
25,
PATHOLOGY
141-143
(1975)
Polyhedrosis Virus of Be/ha (Lepidoptera : Noctuidae)
The biology of Bellura gortynoides, the yellow water lily borer, has been under investigation in two ponds in Tippecanoe County, Indiana, since 1972. The aquatic larvae of this noctuid moth feed exclusively in the leaves and petioles of the yellow water lily, Nuphar advena. Examination of larvae collected from petiole tunnels revealed that some were infected with a polyhedrosis virus. The pathogen was identified as a nuclear polyhedrosis virus (NPV) of the multiploid virion type. The B. gortynoides larvae were reared on a semisynthetic medium with a pinto bean base in 1 oz plastic cups and inoculated at fourth instar per OS with a suspension of isolated from field-collected polyhedra larvae. Normal and infected larvae were examined in smear preparations or prepared for light microscopy and stained with modified azan stain according to the procedure of J. J. Hamm (J. Invertbr. Pathol. 8, 125-126, 1966). Pellets of polyhedra were prepared for electron microscopy by fixation in 6% glutaraldehyde followed by 1% osmium tetroxide, both buffered with 0.1 M cacodylate, pH 7.4. After prestaining with 0.5% uranyl acetate, pellets were dehydrated with ethanol and embedded in epoxy (A. R. Spurr, J. Ultravtruct. Res. 26, 31.-43, 1969). Thin sections were placed on carbon-coated membrane grids, stained with Reynold’s lead citrate and examined with a Philips EM-201 electron microscope calibrated according to R. Luftig (J. Ultrastruct. Res. 20, 91 102, 1967) with catalase crystals. Hemolymph and tissue homogenates of larvae were also examined in preparations applied directly to grids and negatively stained with 3% ammonium molybdate, pH 6.5. Larvae inoculated with polyhedra showed characteristic NPV symptoms. The normal
gortynoides
olive-brown coloration turned to pale green within 3 days after inoculation; by 5 days larvae were dark and lethargic. When the delicate integument was ruptured the liquified body contents were released. Infection was confirmed by light microscopy as evidenced by polyhedra accumulation in nuclei of the hypodermis, fat body, tracheal matrix, sarcolemma, neurilemma, hemocytes, and hindgut. No other region of the gut or other tissues exhibited polyhedra formation. The primary sites of polyhedra accumulation were the fat body, hypodermis, and tracheal matrix. When compared to the normal larval tissues (Fig. 1) these tissues showed hypertrophy and hyperplasia with the latter being most pronounced in the hypodermis (Fig. 2). Numerous polyhedra, free virions, and nucleocapsids were observed in negatively stained homogenates of NPV-infected larvae. In thin sections (Figs. 3, 4), the NPV exhibited typical polyhedra ranging in size from 2 to 5 pm that occluded virions of the multiploid type (J. E. Dahlberg and E. H. Simon, Virology 38, 666-678, 1969). The number of nucleocapsids within an envelope varied from one to ten, with the multiple assemblages being the more predominant. The mean size of 14 nucleocapsids was 33 x 250 nm, with a range of 31-36 x 245-260 nm. The crystalline lattice of the polyhedral protein, measured on lattice lines with 90” intersections, had a periodicity of 69 ,& (Fig. 5). Although polyhedra with virions (Fig. 6) were the most predominant type, polyhedra without occluded virions were also found (Fig. 7). These exhibited the same protein crystalline lattice and periodicity. The multiploid virion NPV of B. gortynoides is a new pathogen for this species. Its structure is similar to the NPV’s described by G. H. Bergold (J. Znvertebr. 141
Cop)rlght Printed
t 1975 b) Academic in the United States.
Press,
Inc.
,411 rlphts
of reproduclton
in any
form
reserved.
FIG. 1. and midgut FIG. 2. mulation in FIG. 3. FIG. 4. FIG. 5. FIG. 6. FIG. 7.
Transverse section of normal larva showing the hypodermis (H), fat body (FB), trachael matrix lumen (ML). x 64. Transverse section of NPV-infected larva exhibiting hypertrophy and hyperplasia with polyhedra the nuclei of infected cells. x 64. Electron micrograph of the predominant size and type of polyhedra isolated from infected larvae. A typical polyhedron showing single (S) and multiple enveloped (M) nucleocapsids. Crystalline lattice structure of the protein matrix. Multiploid virion polyhedron exhibiting the randomly occluded virions of various assemblages. Polyhedron lacking occluded virions.
142
(TM), accu-
143
NOTES
Pathol. 5, 111-128, 1963) that exhibited “virus bundles” and to the multiploid virion NPV isolated from the noctuid Rachiplusia ou by C. Y. Kawanishi and J. D. Paschke (J. Invertebr. Pathol. 16, 89-92, 1970). Its multiploid structure conforms to the characteristics exhibited by multiploid viruses as defined and comprehensively reviewed by E. H. Simon (Progr. Med. Viral. 14, 36-67, 1972). ACKNOWLEDGMENTS The authors thank Drs. Chandler for their support
J. D. Paschke and L. and advice during this
investigation and Ms. Donell Litzenberg for her technical assistance. This research was supported in part by U.S. Public Health Service Research Grant Al-09972 and NDEA Fellowship 70-019322. STANLEY
R. WEBB ELI
Laboratory of Insect Pathology Department of Entomology Purdue University West Lafayette, Indiana 47907
Received May 6,1974
LEVINE
