Department of Plant and Microbial Biology, 251 Koshland Hall, Berkeley, CA 94720-3102, USA1
DuPont Agricultural Products, Stine-Haskell Research Center, PO Box 30, Elkton Road, Newark, DE 19714, USA2
Author for correspondence: Jan Washburn. Fax +1 510 642 4995. e-mail janwash{at}nature.berkeley.edu
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Abstract |
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Introduction |
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Helicoverpa zea S nucleopolyhedrovirus (HzSNPV) was the first of several NPV species to be marketed commercially as a microbial pesticide in the 1970s. HzSNPV, however, was not a commercial success, and most production was halted in 1982 (Benz, 1986 ). The failure of HzSNPV to compete successfully with chemical control agents can be attributed to several factors, including its limited host range and the variability in its efficiency at inducing mortality, even within permissive pest species (Black et al., 1997
). HzSNPV is the most effective baculovirus known for killing larval heliothines (Noctuidae), including Heliothis virescens and Helicoverpa zea, two species that are major economic pests of corn, cotton, tobacco and other crops (King & Coleman, 1989
). By contrast, HzSNPV is relatively non-infectious to Trichoplusia ni, another noctuid pest that is highly susceptible to Autographa californica M nucleopolyhedrovirus (AcMNPV), the best studied of the NPVs (Black et al., 1997
).
Results from early studies on the dosemortality relationships of HzSNPV with its host species suggested that five of the seven major heliothine pests (i.e. Heliothis spp., Helicoverpa spp.) are similarly susceptible to fatal infection (e.g. Allen & Ignoffo, 1969 ; Granados & Williams, 1986
). Data from some comparative studies with H. virescens and H. zea larvae, however, showed that H. virescens larvae were 30 to 50% more susceptible than H. zea, regardless of which of the two host species was the source of the viral inoculum (Ignoffo 1963
, 1973
). Part of this discrepancy in the relative permissiveness among species may have been due to developmental resistance, the phenomenon whereby host larvae become progressively more resistant to fatal infection as they age within and among instars (Stairs, 1965
; Allen & Ignoffo, 1969
). While information documenting the temporal acquisition of resistance within instars is lacking for most HzSNPV hosts (cf. Heliothis punctiger; Teakle et al., 1986
), increasing resistance among instars with increasing age has been reported following oral inoculation of several heliothines (e.g. Ignoffo & Couch, 1981
; Zou & Young, 1994
). Further, studies by Forschler et al. (1992)
showed that the susceptibility of H. zea larvae to fatal infection by HzSNPV was modulated by the source of larval food; specifically, third instar larvae fed on cotton showed significantly lower mortality levels than those fed on either tomato or artificial diet. These authors concluded that host-plant foliar constituents affected the midgut environment of the host, resulting in a reduction in polyhedrosis disease (see also Hoover et al., 1998
, 2000
). Such studies demonstrate that a precise understanding of the effects of diet and host developmental stage is necessary in order to elucidate the true nature of larval susceptibility to fatal infection by HzSNPV.
In the study described here, we quantified mortality levels for three noctuid species following oral and intrahaemocoelic microinjection of precisely defined developmental cohorts of fourth instars with various dosages of an HzSNPV recombinant, HzSNPV-hsp70/lacZ, containing a -galactosidase reporter gene driven by the Drosophila hsp70 promoter. Using the lacZ gene product as a marker for infection, we also elucidated the early events of pathogenesis and compared the kinetics of primary and secondary infection for two permissive heliothine hosts, H. zea and H. virescens. Because all of our test larvae were carefully matched with regard to age (i.e. hours post-moult), and all were fed on the same semisynthetic diet, we were able to examine in detail important interspecific differences in response to virus challenge. Finally, we quantified the effects of the optical brightener M2R on HzSNPV-induced mortality in developmental cohorts of the two heliothines. Prior studies have demonstrated that M2R enhances the performance of several NPV species by reducing the time to death and increasing the level of mortality when orally administered to larvae in combination with occlusions (e.g. Shapiro, 1992
; Hamm & Shapiro, 1992
; Shapiro & Dougherty, 1994
). Results from our laboratory also have shown that M2R blocks permissive noctuid larvae from sloughing midgut cells infected with AcMNPV (Washburn et al., 1998
), and one of our objectives in the current study was to determine if M2R has the same effect on HzSNPV infections in vivo.
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Methods |
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Virus preparation and quantification.
HzSNPV-hsp70/lacZ (Elcar or HzSNPV-Ignoff parental strain) was used for all of the experiments described herein. This construct contains all of the wild-type viral genes from the parental strain and the Escherichia coli -galactosidase gene driven by the Drosophila hsp70 promoter. The HzSNPV transfer vector was constructed by restriction digestion of the HzSNPV-Ignoff genomic DNA with XbaI, and EcoRV. A 7·2 kb restriction fragment containing the polyhedrin gene was isolated and cloned into a pBluescript vector. The EcoRI restriction site from this DNA fragment at 845 bp upstream of the polyhedrin ATG start site was further modified into NotI and AscI sites. All foreign gene fragments were cloned into this HzSNPV transfer vector by using these two NotI and AscI rare cutter sites. The hsp70/lacZ gene fragment was isolated from pAcDZ1 (Zuidema et al., 1990
) as a 3·75 kb XbaIBamHI fragment and cloned into a modified pBluescript SKII(+) vector (with KnpI replaced by AscI). Subsequently, the hsp70/lacZ cassette was isolated as a NotIAscI fragment from the modified pBluescript SKII(+) vector and inserted into the HzSNPV transfer vector using the unique NotI and AscI sites. Liposome-mediated cotransfection was performed on BCIRL-HV-AMI cells (McIntosh et al., 1981
) to generate recombinant HzSNPV. HzSNPV-hsp70/lacZ were plaque purified by blue colour selection using X-Gal substrate. The HzSNPV-hsp70/lacZ clone was restriction analysed (data not shown) to ensure the purity of the isolate. Temporal expression of lacZ from this recombinant is coincident with the expression of early viral genes in cell culture. Moreover, following injection of HzSNPV-hsp70/lacZ BV into the haemocoel, lacZ expression was observed in haemocytes of both H. virescens and H. zea larvae at 2 h post-inoculation (p.i.) (J. O. Washburn & L. E. Volkman, unpublished results).
In bioassays with H. virescens and H. zea larvae, we found that HzSNPV-hsp70/lacZ produced mortalities comparable to its parental wide-type strain when administered orally with the same number of occlusions (J. O. Washburn & L. E. Volkman, unpublished results). Occlusions of HzSNPV-hsp70/lacZ were isolated from liquefied cadavers of H. virescens and partially purified by centrifugation on sucrose gradients (Summers & Smith, 1987 ). Subsequently, the occlusions were pelleted, resuspended in water and then diluted in a neutrally buoyant glycerolwater solution (3:2, v/v); occlusion dilutions used in this study were stored at 4 °C in the dark and quantified using a haemocytometer (minimum of five counts for each inoculum).
HzSNPV-hsp70/lacZ BV was harvested from the haemolymph of H. virescens larvae 72 h after oral inoculation of fourth instars with occlusions. Larvae were anaesthetized and surface-sterilized with 70% ethanol and then bled completely from a cut proleg onto a sheet of Parafilm placed on ice. Haemolymph samples from several infected larvae were then pooled and centrifuged at 1000 g for 5 min at 4 °C to pellet the haemocytes. We discarded the haemocytes and added Graces Insect Cell Culture Medium (Gibco BRL) to the haemolymph in order to make a dilution series of BV for intrahaemocoelic inoculations. BV dosages are not reported in plaque forming units here because we found that inocula that generated mortalities as great as 20% in larval heliothines did not produce any plaques in BCIRL-HV-AM1 cells (McIntosh et al., 1981 ); this cell line is derived from H. virescens and routinely used to amplify HzSNPV in vitro (e.g. Kasman & Volkman, 2000
).
Bioassay and time-course experiments.
For bioassays and time-course experiments, HzSNPV-hsp70/lacZ occlusion suspensions (in 1·0 µl aliquots) were inoculated orally into the anterior midguts of newly moulted (i.e. within 15 min of shedding the third instar cuticle) or 16 h±15 min post-moult fourth instar larvae (hereafter designated 40 and 416, respectively). Inoculum was delivered via a 1 ml syringe fitted with a 32 gauge blunt-tip needle mounted onto a microapplicator (Burkard). For bioassays using BV, a 32 gauge sharp-tip needle was inserted through the planta of one of the prolegs of 24±6 h post-moult fourth instar larvae, and a 1·0 µl suspension of virus was delivered directly into the haemocoel as described by Engelhard et al. (1994) . After inoculation, larvae from all experiments were maintained in a growth chamber at 28±2 °C under constant illumination in individual 25 ml plastic cups containing diet (Stoneville).
To quantify developmental resistance within instars, and to examine the effects of M2R on the establishment and severity of infection, we compared mortality levels of H. zea and H. virescens following inoculation of 40 and 416 cohorts with a solution containing 12 occlusions of HzSNPV-hsp70/lacZ in the presence or absence of 1% (w/v) M2R (see Washburn et al., 1998 ). In this experiment, we also determined the number of viral foci per larva by sacrificing additional insects (n=32 per treatment) at 24 h p.i. and processing their tissues for the lacZ signal. Viral foci numbers among treatments were compared with one-way ANOVA, and a Tukey range test (p<0·05) was used to discriminate means.
For time-course experiments, we chose a dosage of 15 occlusions of HzSNPV-hsp70/lacZ; in five replicated bioassays, this dosage yielded average mortalities of 92 and 84% following inoculation of 40 larvae of H. zea and H. virescens, respectively (H. zea; range=81100%; H. virescens, range=7594%). At various times during the first 24 h p.i., cohorts consisting of 2633 larvae of each species were sacrificed, and their midgut and associated tissues were removed and processed for the lacZ gene product as previously described (Engelhard et al., 1994 ; Washburn et al., 1995
). These tissue preparations were then examined for the blue reporter signal with stereo (1050x) and/or compound microscopy (100480x) to quantify foci of infection and to identify infected cell types (Washburn et al., 1995
). An additional cohort of 32 insects was inoculated and retained as an internal control to check mortality levels. In these studies, we focused specifically on the timing and identity of the first host cells expressing lacZ and on the initiation of systemic infection within the hosts haemocoel. Several time-course experiments with similar results were conducted using each heliothine; data used for the analyses of HzSNPV-hsp70/lacZ pathogenesis presented here were generated from two such experiments in which the tissues from 246 and 298 H. zea and H. virescens, respectively, were examined. The parameters of primary and secondary infection kinetics were evaluated by least squares regression.
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Results |
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Following inoculation, the temporal rise in the average number of viral foci within larvae reflects the rate at which HzSNPV-hsp70/lacZ primary infections became detectable. In cohorts of both H. zea and H. virescens, foci numbers increased in a robust linear fashion (i.e. r2=0·94 and 0·96, respectively) between 4 and 20 h p.i. (Fig. 4). Average foci numbers within species were not significantly different for cohorts sampled 20 and 24 h p.i. (data not shown). The slopes of the regression lines describing the rise in foci numbers during the first 20 h p.i. were 4·1 and 1·7 for H. zea and H. virescens, respectively. The ratio of these slopes shows that HzSNPV established primary infection of H. zea midgut cells at a rate 2·4 times greater than in the same cellular targets in H. virescens. Moreover, at 20 h p.i., the time when maximum foci numbers were achieved in both hosts, lacZ-positive H. zea supported an average of 63·8 foci, compared to only 28·9 for H. virescens (Fig. 4
). Thus, at the same dosage, the ODV of HzSNPV-hsp70/lacZ established twice as many foci at more than twice the rate in 40-inoculated larvae of H. zea.
Expression patterns of lacZ within host tissues from two separate time-course experiments for each heliothine are shown in Fig. 5. The cellular composition of viral foci revealed several important features of HzSNPV pathogenesis in H. zea and H. virescens larvae. First, between 16 and 20 h p.i. in both hosts, there was a sharp decline in the proportions of viral foci restricted to the midgut epithelium and a corresponding rise in the proportion of foci containing one or more tracheal cells. This demonstrates that by 20 h p.i. most primary infections had been established by ODV and had already transmitted BV to tracheal cells within the hosts haemocoel. Second, at both time-points and in both species, we found that some foci consisted exclusively of infected tracheal epidermal cells. Because tracheal cells are not in direct contact with the midgut lumen and, therefore, must be infected by BV generated from midgut cells (see Washburn et al., 1998
), we know that the underlying primary cellular targets were sloughed previously from these foci. In both experiments, the proportions of viral foci restricted to the tracheal epidermis of H. zea larvae at 20 h p.i. were approximately twice that of H. virescens (Fig. 5
), suggesting that the midgut cells of H. zea that were infected by HzSNPV ODV were sloughed at twice the rate.
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Discussion |
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Teakle et al. (1986) reported declining mortalities for cohorts of Heliothis punctiger orally inoculated with HzSNPV progressively later within each of the larval instars. Results from our oral bioassays confirmed that both H. zea and H. virescens also acquired strong developmental resistance to mortal infection by HzSNPV-hsp70/lacZ as they aged within the fourth instar, and they provided evidence that the severity of acquired resistance was greater in H. zea. In paired bioassays in which we orally administered the same numbers of HzSNPV-hsp70/lacZ occlusions to newly moulted fourth instar larvae of the two species, the final mortalities for H. zea cohorts were significantly greater than those of H. virescens. In contrast, the two species were equally susceptible to fatal infection when larvae were inoculated 16 h after the moult, showing a greater relative loss in mortality for H. zea as they aged. Larvae from 40-inoculated cohorts of H. zea contained more than twice as many viral foci as H. virescens at 16 (Fig. 4
) and 24 h p.i. (Table 1
), whereas for 416-inoculated cohorts H. virescens supported significantly more foci than H. zea at 24 h p.i. (Table 1
). Moreover, at a dosage of 12 occlusions, the number of viral foci per larva at 24 h p.i. from 416-inoculated cohorts of H. zea was reduced by 89% relative to 40-inoculated cohorts; the reduction in foci for the same two developmental cohorts of H. virescens was only 59% (Table 1
).
The cellular composition of viral foci revealed important features of HzSNPV pathogenesis in H. zea and H. virescens that helps to explain these patterns in mortality and foci numbers. By 20 h p.i., the proportions of midgut-restricted foci within infected larvae of both species were declining (Fig. 5), suggesting that the wave of primary infection resulting from the pulse of inoculum was almost over. This conclusion also is supported by the observation that at 20 h p.i. the proportions of lacZ-positive larvae of the two species were already predictive of their respective final mortalities (Fig. 3
). Our foci data also suggested that H. zea larvae were shedding infected columnar cells from the midgut epithelium much more rapidly than H. virescens at 20 h p.i. (Fig. 5
). Because the great majority of hosts of both species already supported systemic infections by 20 h p.i. (Figs 3
and 6
), sloughing was not an effective defensive strategy for clearing HzSNPV infections in either host when they were inoculated immediately after moulting to the fourth instar. The enhanced sloughing response of H. zea, however, could explain the lower rate (i.e. efficiency) at which individual ODV-infected midgut cells transmitted BV to tracheal cells (Fig. 6A
). For 416-inoculated cohorts, the dramatic reduction in foci numbers at 24 h p.i., relative to 40-inoculated cohorts of both hosts, may be attributable to age-related increases in sloughing rates that were species specific (Table 1
). A greater sloughing response by H. zea, for example, would explain the shift we observed in relative foci numbers in H. zea and H. virescens when we compared developmental cohorts. Thus, while both hosts exhibited strong developmental resistance to fatal infection by HzSNPV during the first 16 h of the fourth instar, resistance was greater for H. zea and correlated with a reduced ability of the virus either to establish and/or maintain primary midgut infections in the older larvae.
Zou & Young (1994) reported that addition of the optical brightener Tinopal LPW (a formulation of M2R) to the surface of the larval diet increased mortality by HzSNPV for some, but not all, instars of H. virescens. In our hands, administration of a pulse of 1% M2R during oral inoculation of fourth instar H. virescens and H. zea with HzSNPV occlusions 16 h after moulting resulted in an increase in final mortalities, and in H. zea an increase in the number of primary midgut infections (Table 1
). By comparison, the effects of M2R on viral foci and mortality following inoculation of newly moulted hosts were much less. These results provide a useful caution for investigators to control for host age within an instar when evaluating the enhancing qualities of optical brighteners. Significantly, the age-specific effects of M2R revealed in this study are similar to those we previously reported for AcMNPV pathogenesis in permissive hosts. Results from our studies with both pathogens are consistent with the hypothesis that M2R improves baculovirus efficacy by blocking host insects from sloughing primary cellular targets of infection from the midgut epithelium (Washburn et al., 1998
; cf. Wang & Granados, 2000
). Age-dependent interactions between baculoviruses and their hosts also may explain, in part, why earlier investigations employing feeding assays and larvae of varying ages produced conflicting results on the relative susceptibility of H. zea and H. virescens to mortal infection by HzSNPV (e.g. Ignoffo, 1963
, 1973
; Allen & Ignoffo, 1969
; Granados & Williams, 1986
). For both host species, however, the interaction of HzSNPV ODV with primary cellular targets within the midgut epithelium is key to the outcome of infection and an important process underlying the phenomenon of developmental resistance (see also Engelhard & Volkman, 1995
).
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Acknowledgments |
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References |
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Benz, G. A. (1986). Introduction: historical perspectives. In The Biology of Baculoviruses , pp. 1-37. Edited by R. R. Granados & B. A. Federici. Boca Raton:CRC Press.
Black, B. C., Brennan, L. A., Dierks, P. M. & Gard, I. E. (1997). Commercialization of baculoviral insecticides. In The Baculoviruses , pp. 341-388. Edited by L. K. Miller. New York:Plenum Press.
Engelhard, E. K. & Volkman, L. E. (1995). Developmental resistance within fourth instar Trichoplusia ni orally inoculated with Autographa californica M nuclear polyhedrosis virus. Virology 209, 384-389.[Medline]
Engelhard, E. K., Kam-Morgan, L. N. W., Washburn, J. O. & Volkman, L. E. (1994). The insect tracheal system: a conduit for the systemic spread of Autographa californica M nuclear polyhedrosis virus. Proceedings of the National Academy of Sciences, USA 91, 3224-3227.[Abstract]
Forschler, B. T., Young, S. Y. & Felton, G. W. (1992). Diet and the susceptibility of Helicoverpa zea (Noctuidae: Lepidoptera) to a nuclear polyhedrosis virus. Environmental Entomology 21, 1220-1223.
Granados, R. R. & Williams, K. A. (1986). In vivo infection and replication of baculoviruses. In The Biology of Baculoviruses , pp. 89-108. Edited by R. R. Granados & B. A. Federici. Boca Raton:CRC Press.
Hamm, J. J. & Shapiro, M. (1992). Infectivity of fall armyworm (Lepidoptera: Noctuidae) nuclear polyhedrovirus enhanced by a fluorescent brightener. Journal of Economic Entomology 85, 2149-2152.
Hoover, K., Yee, J. L., Schultz, C. M., Hammock, B. D., Rocke, D. M. & Duffy, S. S. (1998). Effects of plant identity and chemical constituents on the efficacy of a baculovirus against Heliothis virescens. Journal of Chemical Ecology 24, 221-225.
Hoover, K., Washburn, J. O. & Volkman, L. E. (2000). Midgut-based resistance of Heliothis virescens to baculovirus infection mediated by phytochemicals in cotton. Journal of Insect Physiology 46, 999-1007.[Medline]
Ignoffo, C. M. (1963). Susceptibility of the first instar of the bollworm, Heliothis zea, and the tobacco budworm, Heliothis virescens, to Heliothis nuclear-polyhedrosis virus. Journal of Invertebrate Pathology 5, 187-195.
Ignoffo, C. M. (1973). Development of a viral insecticide: concept to commercialization. Experimental Parasitology 33, 380-406.[Medline]
Ignoffo, C. M. & Couch, T. L. (1981). The nucleopolyhedrosis virus of Heliothis species as a microbial insecticide. In Microbial Control of Pests and Plant Diseases , pp. 1970-1980. Edited by H. D. Burgess. New York:Academic Press.
Kasman, L. M. & Volkman, L. E. (2000). Filamentous actin is required for lepidopteran nucleopolyhedrovirus progeny production. Journal of General Virology 81, 1881-1888.
King, E. G. & Coleman, R. J. (1989). Potential for biological control of Heliothis species. Annual Review of Entomology 34, 53-75.
McIntosh, A. H., Andrews, P. A. & Ignoffo, C. M. (1981). Establishment of two continuous cell lines of Heliothis virescens (Lepidoptera: Noctuidae). In Vitro 17, 649-650.
Shapiro, M. (1992). Use of optical brighteners as radiation protection for gypsy moth (Lepidoptera: Lymantriidae) nuclear polyhedrovirus. Journal of Economic Entomology 85, 1682-1686.
Shapiro, M. & Dougherty, E. M. (1994). Enhancement in activity of homologous and heterologous viruses against the gypsy moth (Lepidoptera: Lymantriidae) by an optical brightener. Journal of Economic Entomology 87, 361-365.
Stairs, G. R. (1965). Quantitative differences in susceptibility to nuclearpolyhedrosis virus among larval instars of the forest tent caterpillar, Malacosoma disstria (Hubner). Journal of Invertebrate Pathology 7, 427-429.
Summers, M. D. & Smith, G. E. (1987). A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agricultural Experiment Station Bulletin no. 1555.
Teakle, R. E., Jensen, J. M. & Giles, J. E. (1986). Age-related susceptibility of Heliothis punctiger to a commercial formulation of nuclear polyhedrosis virus. Journal of Invertebrate Pathology 47, 82-92.
Wang, P. & Granados, R. R. (2000). Calcoflour disrupts the midgut defense system in insects. Insect Biochemistry and Molecular Biology 30, 135-143.[Medline]
Washburn, J. O., Kirkpatrick, B. A. & Volkman, L. E. (1995). Comparative pathogenesis of Autographa californica M nuclear polyhedrosis virus in larvae of Trichoplusia ni and Heliothis virescens. Virology 209, 561-568.[Medline]
Washburn, J. O., Kirkpatrick, B. A., Haas-Stapleton, E. & Volkman, L. E. (1998). Evidence that the stilbene-derived optical brightener M2R enhances Autographa californica M nucleopolyhedrovirus infection of Trichoplusia ni and Heliothis virescens by preventing sloughing of infected midgut epithelial cells. Biological Control 11, 58-69.
Zou, Y. & Young, S. Y. (1994). Enhancement of nuclear polyhedrosis virus activity in larval pests of lepidoptera by a stilbene fluorescent brightener. Journal of Entomological Science 29, 130-133.
Zuidema, D., Schouten, A., Usmany, M., Maule, A. J., Belsham, G. J., Roosien, J., Klinge-Roode, E. C., van Lent, J. W. M. & Vlak, J. M. (1990). Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector. Journal of General Virology 71, 2201-2209.[Abstract]
Received 20 November 2000;
accepted 8 March 2001.