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Polyhedra without virions in a vertically transmitted nuclear polyhedrosis virus
JOURNAL
OF INVERTEBRATE
PATHOLOGY
60, 53-58 (19%)
Polyhedra without
Virions in a Vertically Transmitted Polyhedrosis Virus’
Nuclear
J. R. FUXA Department
of
Entomology, Louisiana
Agricultural Experiment Station, Louisiana Baton Rouge, Louisiana 70803
State University Agricultuml
Center,
E. H. WEIDNER Department
of
Zoology and Physiology, Louisiana
State University, Baton Rouge, Louisiana
70803
AND
A. R. RICHTER Department
of
Entomology, Louisiana
Agricultural Experiment Station, Louisiana Baton Rouge, Louisiana 70803
State University Agricultural
Center,
Received May 30, 1991; accepted October 18, 1991
Polyhedra were observed in larvae, pupae, and adults
The Spodoptera frugiperda nuclear polyhedrosis virus (NPV) can be transmitted vertically, from parents to offspring. Horizontal transmission by environmental contamination and ingestion is the primary method of transmission of S. frugiperda NPV (Fuxa and 1 Approved for publication by the Director of the Louisiana cultural Experiment Station as manuscript 91-17-5282.
Agri-
Geaghan, 1983). However, Kuno (1979) observed 1.6% infection of S. frugiperda larvae whose parents survived exposure to the NPV. Fuxa and Richter (1991) confirmed Kuno’s observation of vertical transmission of S. frugiperda NPV; the rate of infection in the progeny Cfi generation) was dependent on the instar in which the parents survived exposure to the virus. Fuxa and Richter (1991) also were able to select for a strain of S. frugiperda NPV with a significantly greater rate of vertical transmission (up to 24% infection in the fi generation) than that found with the wild NPV isolate. Certain phenomena associated with vertical transmission of S. frugiperda NPV raised questions about exploitation of this mechanism in enhancing microbial control. The NPV clearly caused sublethal infections in S. frugiperdu (Fuxa and Richter, 1991). Not only did certain insects survive exposure to transmit NPV to their progeny, but also the case fatality rate in the fi insects was less than 50%. Some of the infected fi insects died as larvae or pupae and some became deformed adults, but most survived to become normal adults. If the S. frugiperda NPV strain selected for increased vertical transmission is to be used in microbial control, it is important to learn about the infectivity and other characteristics of the polyhedra found in lethal and sublethal infections in the fi insects. The original purpose of this study was to determine the infectivity of the polyhedra isolated from various stages of fi S. frugiperda after vertical transmission of the NPV. After the initial results indicated differences in infectivity, a second purpose was to determine the nature of the polyhedra.
53 0022-2011/92
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
54
FUXA. WEIDNER, MATERIALS
AND METHODS
The S. frugiperda colony originated from larvae collected from corn near Hammond, Louisiana, in 1985, and the S. frugiperda NPV was isolated from S. frugiperda larvae collected from corn near Isabela, Puerto Rico, in 1984 (the “wild” Puerto Rican strain of the NPV). In previous research, this Puerto Rican NPV was vertically transmitted in S. frugiperda, and virus isolated from pupae in the fi generation became the “selected” Puerto Rican strain (Fuxa and Richter, 1991). The S. frugiperda larvae were reared by standard techniques on artificial diet (Greene et al., 1976), and adults mated and oviposited in 3.8-liter ice cream cartons with a cheesecloth oviposition substrate and a beer-honey suspension for feeding (Fuxa and Richter, 1991). The viruses and their polyhedral inclusion bodies were produced in S. frugiperda larvae, purified by homogenization and density gradient centrifugation (31-54% CsCl), counted with a Petroff-Hauser counting chamber, and stored at -4°C until use. The NPV was vertically transmitted in S. frugiperda according to previous procedures (Fuxa and Richter, 1991). The median lethal concentration of NPV was fed by diet-surface contamination to either third or fifth instars of S. frugiperda, and the survivors were reared on diet and mated. Random samples of eggs (fi generation) were collected and reared through the adult stage. Tissue samples from larvae and pupae that died in the fi generation were smeared on slides and examined for polyhedra under phase-contrast microscopy. Most infections in the fi generation are sublethal (Fuxa and Richter, 19911, so random samples of live adults were sacrificed and similarly examined for polyhedra. Adults were counted as being infected only if the polyhedra satisfied three criteria at the light microscope level: dissolution in NaOH; nonstainability with Sudan III; and lack of light transmission through crossed, polarizing filters (Thomas, 1974). The infectivity of vertically transmitted NPV was tested. Polyhedra were isolated from insects of the appropriate stage from the fi generation; the polyhedra from one insect were used to treat the insects in one replicate of each treatment in the experiment. The dead insect was homogenized and filtered through cheesecloth; there was no further purification of polyhedra due to the relatively small amounts available. All polyhedral suspensions (treatments) were diluted with sterile distilled water to 4 x lo6 polyhedra/ml and fed to first instars by a larval drinking technique (Hughes and Wood, 1981; Mitchell and Smith, 1985). There were 34 replicates per treatment, and for each treatment NPV was fed to 26-48 insects/replicate. The data were analyzed by analysis of variance (SAS Institute, 1987), and means were separated by Duncan’s (1955) multiple range test. Polyhedra in certain adults were tested to determine whether they were truly viral in origin by use of a
AND RICHTER
monoclonal antibody in conjunction with fluorescence microscopy. The monoclonal antibody (clone 1003) was formed against polyhedrin protein from the Heliothis zea NPV but reacts with polyhedrin from several baculoviruses, including that from S. frugiperda NPV (Huang et al., 1985). Polyhedra were tested from four infected fi adult females whose parents survived exposure to wild NPV as third instars; these polyhedra were noninfectious (Table 1). Polyhedra from an infected larva in the parental generation were tested as a positive control, and a tissue suspension from an uninfected insect was tested as a negative control. The monoclonal anti-polyhedrin antibody reaction was visualized by indirect double immunolabeling. The suspected polyhedra or insect tissue, which were placed in a water droplet on a microscope slide without purification, were immersed in primary antibody at a concentration of 50 pg/ml for 30 min. After three washes in neutral-pH phosphate-buffered saline (PBS), they were incubated in a second antibody conjugate with fluorescein isothiocyanate (anti-mouse) at 30.5 kg/ml. After staining, samples were mounted with PBS/30% glycerol. Photographs were taken on Tri-X film (Kodak) or Ektachrome 400 and developed with D-76. Infectious and noninfectious polyhedra from fi female adults were examined by electron microscopy. TABLE 1 Infectivity of Polyhedra Isolated from Different Stages of fi S. frugiperda Whose Parents Survived Exposure to NPV Source of polyhedra” Parental instar exposed to NPV
NPV isolateb
Stage of f, insect
Controld 3 3 3 5 5 5 5
Wild Wild Wild None” Selected Selected Selected Selected
Larvae Pupae Female adults Female adults Larvae Pupae Female adults Male adults
Percentage of infection’ No. (SE) treated 0 85.8 87.8 0 0 98.3 68.9 11.6 0
a(O) d (8.23) d (1.11) a(01 a (0) e (1.67) c (2.14) b (2.94) a (01
90 148 90 120 120 122 119 117 116
Note. The parental insects were exposed to different NPV isolates at different ages. “Polyhedra appeared to be viral polyhedral inclusion bodies in phase microscopy; they dissolved in NaOH, did not stain with Sudan III, and did not transmit double-polarized light. b The “wild” NPV was isolated from S. frug@erda collected in Puerto Rico; the “selected” isolate was selected from the wild Puerto Rico NPV population for an increased rate of vertical transmission (Fuxa and Richter, 1991). ’ Test larvae treated in first instar with 4 x lo6 PIB/ml. Means followed by the same letter were not significantly different (P < 0.05, ANOVA, Duncan’s multiple range test). d Control for current experiment. Test insects were not exposed to any polyhedra. e Polyhedra isolated from insect whose parents had no history of exposure to NPV.
EMPTY POLYHEDRA
IN VERTICALLY
FIG. 1.
Polyhedron containing virions from female adult of S. whose parents survived exposure to S. fiugiperdu NPV as fifth instars. Polyhedra in these females were infectious to first instars. X77,500.
TRANSMITTED
NPV
frugiperdu
FIG. 2. Polyhedron from female adult whose parents survived exposure to S. frugiperdu NPV as third instars. Polyhedra in these females were not infectious to first instars. x 25,480.
The polyhedra originated from four infected fi adult females: noninfectious polyhedra from two females whose parents survived exposure to wild NPV as third instars and infectious polyhedra from two females whose parents survived exposure to the selected NPV as fifth instars (Table 1). Polyhedra and associated tissues were fixed in cold 2% formaldehyde or 2% glutaraldehyde in 0.1 M cacodylate buffer for 15 min. Samples were washed and incubated in the monoclonal antipolyhedrin antibody diluted 1:20 in PBS. After 30-60 min incubation in primary antibody and four washes in PBS, samples were immersed in peroxidase conjugated with protein A (EY Laboratories, San Mateo, CA) (No. HP-02) diluted in 1:20 in PBS. Samples were refixed in 1% glutaraldehyde for 30 min and washed in cacodylate buffer. After rinsing in Tris buffer, samples were developed for 10 min with diaminobenzidine (Polysciences, Inc., Warring-ton, PA) and fixed in 1% osmium tetroxide. Sections of Epon-embedded material were examined without further staining on a JEOL 100 CX electron microscope.
were infectious to first instar S. frugiperda (Table 1). Polyhedra isolated from dead fi larvae and pupae were infectious to first instars. The polyhedra isolated from fi adults were not infectious except for those from female adults whose parents survived exposure to the selected NPV in the fifth instar. However, the polyhedra from this group of female adults infected a significantly lower percentage of bioassay larvae than the polyhedra from larvae or pupae. The percentage of infection from the four replicates of this treatment (fifth instar exposure, selected NPV, fi female adults) ranged from 3.3-16.7%, indicating that there was at least some infectious virus in each of the four fi females. Electron microscopy and the monoclonal antipolyhedrin in conjunction with fluorescence microscopy helped explain the infectiousness of polyhedra from fi insects. The infectious polyhedra from fi female adults whose parents survived exposure to the selected NPV in the fifth instar (Table 1) clearly contained typical, multiply embedded virions (Fig. 1). The noninfectious polyhedra from the fi female adults whose parents survived exposure to the wild NPV in the third instar (Table 1) had evidence of the periodicity typical of NPV polyhedral inclusion bodies (not visible), but
RESULTS
Certain of the polyhedra that passed the light microscope criteria for viral polyhedral inclusion bodies
56
FUXA, WEIDNER,
AND RICHTER
FIG. 3. Noninfectious polyhedra immunolabeled with a monoclonal antipolyhedrin, examined by fluorescence microscopy. The polyhedra came from a female adult whose parents survived exposure to S. frugiperdu NPV as third instars. x 1,500.
these crystals did not contain virions (Fig. 2). Furthermore, these noninfectious polyhedra from three females from the latter group clearly were fluorescent when treated with conjugated monoclonal antipolyhedrin (Fig. 31, closely resembling polyhedral inclusion bodies in the positive control (Fig. 4). Polyhedra from one other such female tested negatively with the conjugated antibody. DISCUSSION
The results clearly demonstrated that the vertically transmitted SfNPV causes infections in the fi genera-
tion, where it can produce polyhedra that do not occlude virions. In addition to passing three light microscope tests for NPV, the noninfectious polyhedra fluoresced when treated with the monoclonal, conjugated antibody, which is a definitive test. Many of the noninfectious polyhedra had an unusual shape (Fig. 2), which is very similar to that of the PIB of a variant of NPV of Lambdina fiscellaria fiscellaria (Cunningham, 1970). Empty polyhedra have been observed previously, mostly in cell culture. Polyhedra of Lymantria dispar NPV with a reduced number or complete lack of virions resulted when gypsy moth cells were grown in a serum-
EMPTY POLYHEDRA
IN VERTICALLY
TRANSMITTED
NPV
57
FIG. 4. Infectious polyhedra immunolabeled with a monoclonal antipolyhedrin, examined by fluorescence microscopy. The polyhedra came from a larva which had ingested and become infected by the S. fiugiperdn NPV. x 1,500.
free medium (Goodwin and Adams, 1980). In another study, empty polyhedra of Autographa californica NPV were produced in armyworm cells grown in different media. There was no correlation between formation of abnormal polyhedra and the type of medium, but free virions were observed by electron microscopy in cells containing empty polyhedra (Vaughn et al., 1991). Also, cubic polyhedra produced by an A. californica NPV mutant often do not occlude virions (Carstens et al., 1986). Polyhedra without virions have been observed in uiuo in one study. A mixture of normal and empty polyhedra was observed in Trichoplusia ni infected with NPV (Summers and Arnott, 1969). The
empty polyhedra had a variety of shapes, all of which differed from that of normal polyhedra-containing virions. The authors also concluded that virus particles do not become occluded if they do not acquire an outer membrane. The nature of the polyhedra in the fi generation has implications for viral transmission and microbial control. The infectious polyhedra occur in the f1 larvae and pupae, which also are the fi stages that exhibit overt, lethal infections (Fuxa and Richter, 1991). In nature, the larvae are the feeding stage and are in the best situation, feeding on the host plant, to spread the disease horizontally when they die and disintegrate. On
58
FUXA, WEIDNER,
the other hand, it might be advantageous to the NPV to be in a noninfectious, relatively innocuous state in infected adults, which are specialized for movement and reproduction. The viral infections in adults are not lethal, and the virus may have evolved to be transported by the adults. If these nonlethally infected adults oviposit on tall, fast-growing host plants (e.g., corn) that are not readily contaminated from the viral soil reservoir, the fi larvae that die and release infectious polyhedra provide a mechanism for foci of infections to form in host populations on those taller plants. The observations of empty polyhedra in adults raises the question of latent infections in the S. frugiperdu NPV system. A demonstration of latency requires that the pathogen in the host be in a noninfective and nonreplicative state, without causing overt disease, and that the pathogen be transformed to an infective and replicative state when the host is stressed (adapted from Tanada and Fuxa, 1987). While latency of viral infections of insects probably is widespread, definitive proof of all the requirements in one insect-virus system is lacking, except perhaps in one case not involving a NPV (Tanada and Fuxa, 1987). The nonlethal infections with empty polyhedra in our fi adults may indicate latent infection by NPV, which should be further investigated. Since polyhedrin is a late viral protein (Volkman and Knudson, 1986), it is possible that there were infectious, nonoccluded virions in the infected adults. However, we did not observe nonoceluded virions in the limited field of view of the electron microscope, and homogenates of adult tissues containing empty polyhedra were not infectious (Table 1). Thus, it is possible that there were no infectious, nonoccluded virions present in the adults. It is possible that the type of viral transmission advantageous to epizootiology is attuned to, or even triggered by, changes in the host cells, perhaps during metamorphosis. As the insect approaches the adult stage, a change in the quality of the infected cells may induce the virus to produce nonlethal, vertically transmitted, possibly latent infections (characterized by mostly empty polyhedra) just at the time in the host life cycle when viral transport becomes more epizootiologically important than horizontal transmission. ACKNOWLEDGMENTS The authors thank Dr. C. Kawanishi (EPA, Research Triangle Park, North Carolina) for providing the monoclonal antibody and Dr. D. P. Pashley (Louisiana State University) for providing the insects from which the viruses were isolated.
AND RICHTER REFERENCES Carstens, E. B., Krebs, A., and Gallerneault, C. E. 1986. Identification of an amino acid essential to the normal assembly of Autogrupha caZifornicu nuclear polyhedrosis virus polyhedra. J. ViFOl. 58, 684688. Cunningham, J. C. 1970. Strains of nuclear polyhedrosis viruses displaying different inclusion body shapes. J. Invertebr. Pathol. 16, 293300. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics 11, l-42. Fuxa, J. R., and Geaghan, J. P. 1983. Multiple-regression analysis of factors affecting prevalence of nuclear polyhedrosis virus in spodopteru frugiperda (Lepidoptera: Noctuidae) populations. Environ. Entomol. 12, 311316. Fuxa, J. R., and Richter, A. R. 1991. Selection for an increased rate of vertical transmission of Spodopteru fiugiperdu (Lepidoptera: Noctuidae) nuclear polyhedrosis virus. Environ. EntomoZ. 20,603609. Goodwin, R. H., and Adams, J. R. 1980. Nutrient factors influencing viral replication in serum-free insect cell line culture. In “Invertebrate Systems In Vitro” (E. Kurstak, K. Maramorosch, and A. Diibendorfer, Eds.1, pp. 493-509. ElsevierNorth-Holland Biomedical, Amsterdam. Greene, G. L., Leppla, N. C., and Dickerson, W. A. 1976. Velvetbean caterpillar: A rearing procedure and artificial medium. J. Econ. Entomol. 69, 487-488. Huang, Y.-S., Hu, P. C., and Kawanishi, C. Y. 1985. Monoclonal antibodies identify conserved epitopes on the polyhedrin of Heliothis zea nuclear polyhedrosis virus. Virology 143, 380-391. Hughes, P. R., and Wood, H. A. 1981. A synchronous peroral technique for the bioassay of insect viruses. J. znvertebr. Pathol. 37, 154-159. Kuno, G. 1979. A nuclear-polyhedrosis virus of Spodopteru frugiperdu isolated in Puerto Rico. J. Agric. Univ. P. R. 63, 162-169. Mitchell, F. L., and Smith, J. W. 1985. Pathology and bioassays of the lesser cornstalk borer (Elasmopalpw IignoseZlus) entomopoxvirus. J. znvertebr. Pathol. 45, 75-80. SAS Institute. 1987. “SASSTAT” Guide for Personal Computers,” 6th Ed. SAS Institute, Cary, North Carolina. Summers, M. D., and H. J. Arnott. 1969. Ultrastructural studies on inclusion formation and virus occlusion in nuclear polyhedrosis and granulosis virus-infected cells of Trichopkuia ni (Htibner). J. Uhastructure Res. 28, 462-480. Tanada, Y., and Fuxa, J. R. 1987. The pathogen population. In “Epizootiology of Insect Diseases” (J. R. Fuxa and Y. Tanada, Eds.), pp. 113-157. Wiley, New York. Thomas, G. M. 1974. Diagnostic techniques. In “Insect Diseases. Vol. 1” (G. E. Cantwell, Ed.), pp. l-48. Dekker, New York. Vaughn, J. L., Fan, F., Dougherty, E. M., Adams, J. R., Guzo, D., and McClintock, J. T. 1991. The use of commercial serum replacements in media for the in vitro replication of nuclear polyhedrosis virus. J. Invertebr. Pathol. 58, 1297-304. Volkman, L. E., and Knudson, D. L. 1986. In vitro replication of baculoviruses. In “The Biology of Baculoviruses” (R. R. Granados and B. A. Federici, Eds.), Vol. 1, pp. 109-127. CRC Press, Boca Raton, FL.
OF INVERTEBRATE
PATHOLOGY
60, 53-58 (19%)
Polyhedra without
Virions in a Vertically Transmitted Polyhedrosis Virus’
Nuclear
J. R. FUXA Department
of
Entomology, Louisiana
Agricultural Experiment Station, Louisiana Baton Rouge, Louisiana 70803
State University Agricultuml
Center,
E. H. WEIDNER Department
of
Zoology and Physiology, Louisiana
State University, Baton Rouge, Louisiana
70803
AND
A. R. RICHTER Department
of
Entomology, Louisiana
Agricultural Experiment Station, Louisiana Baton Rouge, Louisiana 70803
State University Agricultural
Center,
Received May 30, 1991; accepted October 18, 1991
Polyhedra were observed in larvae, pupae, and adults
The Spodoptera frugiperda nuclear polyhedrosis virus (NPV) can be transmitted vertically, from parents to offspring. Horizontal transmission by environmental contamination and ingestion is the primary method of transmission of S. frugiperda NPV (Fuxa and 1 Approved for publication by the Director of the Louisiana cultural Experiment Station as manuscript 91-17-5282.
Agri-
Geaghan, 1983). However, Kuno (1979) observed 1.6% infection of S. frugiperda larvae whose parents survived exposure to the NPV. Fuxa and Richter (1991) confirmed Kuno’s observation of vertical transmission of S. frugiperda NPV; the rate of infection in the progeny Cfi generation) was dependent on the instar in which the parents survived exposure to the virus. Fuxa and Richter (1991) also were able to select for a strain of S. frugiperda NPV with a significantly greater rate of vertical transmission (up to 24% infection in the fi generation) than that found with the wild NPV isolate. Certain phenomena associated with vertical transmission of S. frugiperda NPV raised questions about exploitation of this mechanism in enhancing microbial control. The NPV clearly caused sublethal infections in S. frugiperdu (Fuxa and Richter, 1991). Not only did certain insects survive exposure to transmit NPV to their progeny, but also the case fatality rate in the fi insects was less than 50%. Some of the infected fi insects died as larvae or pupae and some became deformed adults, but most survived to become normal adults. If the S. frugiperda NPV strain selected for increased vertical transmission is to be used in microbial control, it is important to learn about the infectivity and other characteristics of the polyhedra found in lethal and sublethal infections in the fi insects. The original purpose of this study was to determine the infectivity of the polyhedra isolated from various stages of fi S. frugiperda after vertical transmission of the NPV. After the initial results indicated differences in infectivity, a second purpose was to determine the nature of the polyhedra.
53 0022-2011/92
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
54
FUXA. WEIDNER, MATERIALS
AND METHODS
The S. frugiperda colony originated from larvae collected from corn near Hammond, Louisiana, in 1985, and the S. frugiperda NPV was isolated from S. frugiperda larvae collected from corn near Isabela, Puerto Rico, in 1984 (the “wild” Puerto Rican strain of the NPV). In previous research, this Puerto Rican NPV was vertically transmitted in S. frugiperda, and virus isolated from pupae in the fi generation became the “selected” Puerto Rican strain (Fuxa and Richter, 1991). The S. frugiperda larvae were reared by standard techniques on artificial diet (Greene et al., 1976), and adults mated and oviposited in 3.8-liter ice cream cartons with a cheesecloth oviposition substrate and a beer-honey suspension for feeding (Fuxa and Richter, 1991). The viruses and their polyhedral inclusion bodies were produced in S. frugiperda larvae, purified by homogenization and density gradient centrifugation (31-54% CsCl), counted with a Petroff-Hauser counting chamber, and stored at -4°C until use. The NPV was vertically transmitted in S. frugiperda according to previous procedures (Fuxa and Richter, 1991). The median lethal concentration of NPV was fed by diet-surface contamination to either third or fifth instars of S. frugiperda, and the survivors were reared on diet and mated. Random samples of eggs (fi generation) were collected and reared through the adult stage. Tissue samples from larvae and pupae that died in the fi generation were smeared on slides and examined for polyhedra under phase-contrast microscopy. Most infections in the fi generation are sublethal (Fuxa and Richter, 19911, so random samples of live adults were sacrificed and similarly examined for polyhedra. Adults were counted as being infected only if the polyhedra satisfied three criteria at the light microscope level: dissolution in NaOH; nonstainability with Sudan III; and lack of light transmission through crossed, polarizing filters (Thomas, 1974). The infectivity of vertically transmitted NPV was tested. Polyhedra were isolated from insects of the appropriate stage from the fi generation; the polyhedra from one insect were used to treat the insects in one replicate of each treatment in the experiment. The dead insect was homogenized and filtered through cheesecloth; there was no further purification of polyhedra due to the relatively small amounts available. All polyhedral suspensions (treatments) were diluted with sterile distilled water to 4 x lo6 polyhedra/ml and fed to first instars by a larval drinking technique (Hughes and Wood, 1981; Mitchell and Smith, 1985). There were 34 replicates per treatment, and for each treatment NPV was fed to 26-48 insects/replicate. The data were analyzed by analysis of variance (SAS Institute, 1987), and means were separated by Duncan’s (1955) multiple range test. Polyhedra in certain adults were tested to determine whether they were truly viral in origin by use of a
AND RICHTER
monoclonal antibody in conjunction with fluorescence microscopy. The monoclonal antibody (clone 1003) was formed against polyhedrin protein from the Heliothis zea NPV but reacts with polyhedrin from several baculoviruses, including that from S. frugiperda NPV (Huang et al., 1985). Polyhedra were tested from four infected fi adult females whose parents survived exposure to wild NPV as third instars; these polyhedra were noninfectious (Table 1). Polyhedra from an infected larva in the parental generation were tested as a positive control, and a tissue suspension from an uninfected insect was tested as a negative control. The monoclonal anti-polyhedrin antibody reaction was visualized by indirect double immunolabeling. The suspected polyhedra or insect tissue, which were placed in a water droplet on a microscope slide without purification, were immersed in primary antibody at a concentration of 50 pg/ml for 30 min. After three washes in neutral-pH phosphate-buffered saline (PBS), they were incubated in a second antibody conjugate with fluorescein isothiocyanate (anti-mouse) at 30.5 kg/ml. After staining, samples were mounted with PBS/30% glycerol. Photographs were taken on Tri-X film (Kodak) or Ektachrome 400 and developed with D-76. Infectious and noninfectious polyhedra from fi female adults were examined by electron microscopy. TABLE 1 Infectivity of Polyhedra Isolated from Different Stages of fi S. frugiperda Whose Parents Survived Exposure to NPV Source of polyhedra” Parental instar exposed to NPV
NPV isolateb
Stage of f, insect
Controld 3 3 3 5 5 5 5
Wild Wild Wild None” Selected Selected Selected Selected
Larvae Pupae Female adults Female adults Larvae Pupae Female adults Male adults
Percentage of infection’ No. (SE) treated 0 85.8 87.8 0 0 98.3 68.9 11.6 0
a(O) d (8.23) d (1.11) a(01 a (0) e (1.67) c (2.14) b (2.94) a (01
90 148 90 120 120 122 119 117 116
Note. The parental insects were exposed to different NPV isolates at different ages. “Polyhedra appeared to be viral polyhedral inclusion bodies in phase microscopy; they dissolved in NaOH, did not stain with Sudan III, and did not transmit double-polarized light. b The “wild” NPV was isolated from S. frug@erda collected in Puerto Rico; the “selected” isolate was selected from the wild Puerto Rico NPV population for an increased rate of vertical transmission (Fuxa and Richter, 1991). ’ Test larvae treated in first instar with 4 x lo6 PIB/ml. Means followed by the same letter were not significantly different (P < 0.05, ANOVA, Duncan’s multiple range test). d Control for current experiment. Test insects were not exposed to any polyhedra. e Polyhedra isolated from insect whose parents had no history of exposure to NPV.
EMPTY POLYHEDRA
IN VERTICALLY
FIG. 1.
Polyhedron containing virions from female adult of S. whose parents survived exposure to S. fiugiperdu NPV as fifth instars. Polyhedra in these females were infectious to first instars. X77,500.
TRANSMITTED
NPV
frugiperdu
FIG. 2. Polyhedron from female adult whose parents survived exposure to S. frugiperdu NPV as third instars. Polyhedra in these females were not infectious to first instars. x 25,480.
The polyhedra originated from four infected fi adult females: noninfectious polyhedra from two females whose parents survived exposure to wild NPV as third instars and infectious polyhedra from two females whose parents survived exposure to the selected NPV as fifth instars (Table 1). Polyhedra and associated tissues were fixed in cold 2% formaldehyde or 2% glutaraldehyde in 0.1 M cacodylate buffer for 15 min. Samples were washed and incubated in the monoclonal antipolyhedrin antibody diluted 1:20 in PBS. After 30-60 min incubation in primary antibody and four washes in PBS, samples were immersed in peroxidase conjugated with protein A (EY Laboratories, San Mateo, CA) (No. HP-02) diluted in 1:20 in PBS. Samples were refixed in 1% glutaraldehyde for 30 min and washed in cacodylate buffer. After rinsing in Tris buffer, samples were developed for 10 min with diaminobenzidine (Polysciences, Inc., Warring-ton, PA) and fixed in 1% osmium tetroxide. Sections of Epon-embedded material were examined without further staining on a JEOL 100 CX electron microscope.
were infectious to first instar S. frugiperda (Table 1). Polyhedra isolated from dead fi larvae and pupae were infectious to first instars. The polyhedra isolated from fi adults were not infectious except for those from female adults whose parents survived exposure to the selected NPV in the fifth instar. However, the polyhedra from this group of female adults infected a significantly lower percentage of bioassay larvae than the polyhedra from larvae or pupae. The percentage of infection from the four replicates of this treatment (fifth instar exposure, selected NPV, fi female adults) ranged from 3.3-16.7%, indicating that there was at least some infectious virus in each of the four fi females. Electron microscopy and the monoclonal antipolyhedrin in conjunction with fluorescence microscopy helped explain the infectiousness of polyhedra from fi insects. The infectious polyhedra from fi female adults whose parents survived exposure to the selected NPV in the fifth instar (Table 1) clearly contained typical, multiply embedded virions (Fig. 1). The noninfectious polyhedra from the fi female adults whose parents survived exposure to the wild NPV in the third instar (Table 1) had evidence of the periodicity typical of NPV polyhedral inclusion bodies (not visible), but
RESULTS
Certain of the polyhedra that passed the light microscope criteria for viral polyhedral inclusion bodies
56
FUXA, WEIDNER,
AND RICHTER
FIG. 3. Noninfectious polyhedra immunolabeled with a monoclonal antipolyhedrin, examined by fluorescence microscopy. The polyhedra came from a female adult whose parents survived exposure to S. frugiperdu NPV as third instars. x 1,500.
these crystals did not contain virions (Fig. 2). Furthermore, these noninfectious polyhedra from three females from the latter group clearly were fluorescent when treated with conjugated monoclonal antipolyhedrin (Fig. 31, closely resembling polyhedral inclusion bodies in the positive control (Fig. 4). Polyhedra from one other such female tested negatively with the conjugated antibody. DISCUSSION
The results clearly demonstrated that the vertically transmitted SfNPV causes infections in the fi genera-
tion, where it can produce polyhedra that do not occlude virions. In addition to passing three light microscope tests for NPV, the noninfectious polyhedra fluoresced when treated with the monoclonal, conjugated antibody, which is a definitive test. Many of the noninfectious polyhedra had an unusual shape (Fig. 2), which is very similar to that of the PIB of a variant of NPV of Lambdina fiscellaria fiscellaria (Cunningham, 1970). Empty polyhedra have been observed previously, mostly in cell culture. Polyhedra of Lymantria dispar NPV with a reduced number or complete lack of virions resulted when gypsy moth cells were grown in a serum-
EMPTY POLYHEDRA
IN VERTICALLY
TRANSMITTED
NPV
57
FIG. 4. Infectious polyhedra immunolabeled with a monoclonal antipolyhedrin, examined by fluorescence microscopy. The polyhedra came from a larva which had ingested and become infected by the S. fiugiperdn NPV. x 1,500.
free medium (Goodwin and Adams, 1980). In another study, empty polyhedra of Autographa californica NPV were produced in armyworm cells grown in different media. There was no correlation between formation of abnormal polyhedra and the type of medium, but free virions were observed by electron microscopy in cells containing empty polyhedra (Vaughn et al., 1991). Also, cubic polyhedra produced by an A. californica NPV mutant often do not occlude virions (Carstens et al., 1986). Polyhedra without virions have been observed in uiuo in one study. A mixture of normal and empty polyhedra was observed in Trichoplusia ni infected with NPV (Summers and Arnott, 1969). The
empty polyhedra had a variety of shapes, all of which differed from that of normal polyhedra-containing virions. The authors also concluded that virus particles do not become occluded if they do not acquire an outer membrane. The nature of the polyhedra in the fi generation has implications for viral transmission and microbial control. The infectious polyhedra occur in the f1 larvae and pupae, which also are the fi stages that exhibit overt, lethal infections (Fuxa and Richter, 1991). In nature, the larvae are the feeding stage and are in the best situation, feeding on the host plant, to spread the disease horizontally when they die and disintegrate. On
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the other hand, it might be advantageous to the NPV to be in a noninfectious, relatively innocuous state in infected adults, which are specialized for movement and reproduction. The viral infections in adults are not lethal, and the virus may have evolved to be transported by the adults. If these nonlethally infected adults oviposit on tall, fast-growing host plants (e.g., corn) that are not readily contaminated from the viral soil reservoir, the fi larvae that die and release infectious polyhedra provide a mechanism for foci of infections to form in host populations on those taller plants. The observations of empty polyhedra in adults raises the question of latent infections in the S. frugiperdu NPV system. A demonstration of latency requires that the pathogen in the host be in a noninfective and nonreplicative state, without causing overt disease, and that the pathogen be transformed to an infective and replicative state when the host is stressed (adapted from Tanada and Fuxa, 1987). While latency of viral infections of insects probably is widespread, definitive proof of all the requirements in one insect-virus system is lacking, except perhaps in one case not involving a NPV (Tanada and Fuxa, 1987). The nonlethal infections with empty polyhedra in our fi adults may indicate latent infection by NPV, which should be further investigated. Since polyhedrin is a late viral protein (Volkman and Knudson, 1986), it is possible that there were infectious, nonoccluded virions in the infected adults. However, we did not observe nonoceluded virions in the limited field of view of the electron microscope, and homogenates of adult tissues containing empty polyhedra were not infectious (Table 1). Thus, it is possible that there were no infectious, nonoccluded virions present in the adults. It is possible that the type of viral transmission advantageous to epizootiology is attuned to, or even triggered by, changes in the host cells, perhaps during metamorphosis. As the insect approaches the adult stage, a change in the quality of the infected cells may induce the virus to produce nonlethal, vertically transmitted, possibly latent infections (characterized by mostly empty polyhedra) just at the time in the host life cycle when viral transport becomes more epizootiologically important than horizontal transmission. ACKNOWLEDGMENTS The authors thank Dr. C. Kawanishi (EPA, Research Triangle Park, North Carolina) for providing the monoclonal antibody and Dr. D. P. Pashley (Louisiana State University) for providing the insects from which the viruses were isolated.
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