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Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in Pseudoplusia includens larvae
Biological Control 65 (2013) 101–108
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Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in Pseudoplusia includens larvae Aniska Chikhalya, Kimberly D. Stephens, James W. Archie, Eric J. Haas-Stapleton ⇑ Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Road, Long Beach, CA 90840, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Pseudoplusia includens displays no
physiological barriers to fatal AcMNPV Infection. " The optical brightener M2R does not significantly increase virulence. " P. includens could be controlled in organic crops using naturally occurring AcMNPV.
a r t i c l e
i n f o
Article history: Received 7 June 2012 Accepted 6 January 2013 Available online 12 January 2013 Keywords: Pseudoplusia includens Autographa californica Nucleopolyhedrosis virus Pathogenesis AcMNPV
a b s t r a c t Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrosis virus were quantified in penultimate instar Pseudoplusia includens (soybean looper) larvae using a recombinant encoding the lacZ reporter gene (AcMNPV-hsp70/lacZ). Larvae inoculated orally with AcMNPV-hsp70/lacZ occlusion bodies (OB) or intrahemocoelically with budded virus (BV) were susceptible to fatal infection (LD50 = 40.8 OB; 13.8 BV plaque forming units). Pseudoplusia includens displayed increased developmental resistance as larvae were orally inoculated with OB at later times during the penultimate instar. The optical brightener M2R, an inhibitor of the apoptotic processes that drive developmental resistance, did not significantly affect the virulence of AcMNPV-hsp70/lacZ OB. To study pathogenesis, newly molted penultimate instar P. includens were orally inoculated with 60 OB and examined from 0.5 to 96 h post inoculation (h.p.i). Infection in the midgut was first detected at 4 h.p.i in 32% of the larvae and was apparent in cells of the tracheal system at 8 h.p.i. LacZ-positive (LacZ+) hemocytes were first observed 2 h later. At 18 h.p.i, a low proportion of the hemocytes were LacZ+ (5.6%). However, flow cytometry analysis of cell surface expression of the viral protein GP64 showed that 84.5% of the hemocytes collected at 18 h.p.i were infected with AcMNPV, suggesting that flow cytometry may be a more sensitive method for identifying AcMNPV-infected cells. Because P. includens display no physiological barriers to AcMNPV OB and is permissive to fatal infection, this species could be controlled in organic cropping systems using naturally occurring strains of AcMNPV. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Baculoviruses offer an appealing alternative to chemical and many biological control agents because they are arthropod-specific ⇑ Corresponding author. Fax: +1 562 985 8878. E-mail address: [email protected] (E.J. Haas-Stapleton). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.01.002
pathogens that naturally infect and kill many important lepidopteran crop pests. This specificity is an important factor ensuring safety to humans, other vertebrates and beneficial insects (e.g. pollinators and natural enemies). Factors limiting the efficacy of baculoviruses as crop pest control agents include slow speed of kill and narrow effective host ranges of individual baculovirus species (Vail, 1993; Vail et al., 1999). However, baculoviruses recycle in
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nature and the quantity in a field increases as more larvae are controlled by viral infection. This can result in pest control over several seasons (Mulock and Faulkner, 1997), a characteristic that most chemical pesticides lack. To date, the most successful genetically modified (GM) baculoviruses incorporate arthropod-specific toxins or growth modulators that reduce the time to death and thereby the duration that larvae feed upon and damage crop plants (Bonning et al., 2005; Gurevitz et al., 2007; Lasaa et al., 2007; Rajendra et al., 2006). However, organic farmers may not use GM organisms, including engineered baculoviruses. The foundation for building confidence in naturally occurring baculoviruses (i.e. wild-type strains) to control crop pests is supported with a detailed knowledge of the infection kinetics for baculoviruses in a variety of economically important crop pests. Discovering the mechanisms of baculovirus susceptibility and resistance in lepidopteran larvae may increase the appeal of these biological control agents for use in organic farming. Pseudoplusia includens (the soybean looper; family Noctuidae) is a lepidopteran pest of soybean, a leading organic crop in the United States (US) and Brazil (Oliveira et al., 2006; Roseboro, 2007). This insect overwinters in southern states of the US and each year large numbers migrate into the US from Central and South America. In Mississippi, 90% of all loopers attacking soybean are P. includens (Stewart, 2007); less severe infestations occur in the western United States. Current strategies for reducing economic damage to US soybean crops by P. includens include chemical insecticides, biological insecticides (e.g. Bacillus thuringiensis and entomopathogenic fungi), cultural practices (e.g. early crop planting), and GM organisms. Baculoviruses may constitute a valuable addition to the P. includens control strategies. The purpose of the current study was to quantify the virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in P. includens relative to Trichoplusia ni (cabbage looper), a species having documented high susceptibility to AcMNPV (Washburn et al., 1995). AcMNPV is the type species of the genus Nucleopolyhedrovirus, of which all members produce two distinct viral morphotypes: occlusion derived virus (ODV) and budded virus (BV). Multiple ODV are embedded within a protective protein complex called the occlusion body (OB) that is responsible for protecting the ODV from environmental damage and for spreading the virus between insects. BV is produced within the hemocoel of infected larvae and is the morphotype responsible for disseminating the virus between cells within an insect. For successful dissemination of infection within the insect, ODV is released from the OB in the alkaline environment of insect intestine to infect midgut epithelial cells (Faulkner et al., 1997; Granados, 1978). BV released from midgut cells infects adjacent tracheal cells of the respiratory system or circulating hemocytes, which mediate cellular immune responses (Engelhard et al., 1994; Granados and Lawler, 1981; Strand, 2008). In permissive species, the virus spreads to all tissues, resulting in a fatal infection. The results described herein demonstrate that while AcMNPV is slightly less virulent in P. includens relative to T. ni, the pathogenesis of the virus in P. includens is similar to what is reported for T. ni (Washburn et al., 1995). Thus, P. includens is a fully permissive host for AcMNPV and should be considered a candidate for biological control of P. includens on organic crops.
2. Materials and methods 2.1. Virus and virus preparation AcMNPV-hsp70/lacZ is a recombinant that contains all of the genes of wild-type AcMNPV, but includes a b-galactosidase reporter gene whose expression is driven by the Drosophila hsp70 promoter
(Engelhard et al., 1994) and was used for all studies described herein. AcMNPV-hsp70/lacZ causes mortality levels in T. ni that are identical to the wild type strains of AcMNPV (Engelhard et al., 1994). AcMNPV-hsp70/lacZ OB were isolated and purified from T. ni cadavers as described in Kirkpatrick et al. (1998). Briefly, virus killed T. ni were crushed and the crude extract filtered through a porous nylon. OB were isolated using sucrose gradient centrifugation (Summers and Smith, 1987), the OB suspended and diluted in neutrally buoyant solution of water and glycerin (3:2 v/v). Subsequently, OB were quantified using a hemocytometer and stored at 4 °C in the dark. BV was propagated in vitro using cultured Sf9 cells, as previously described (Kirkpatrick et al., 1998). Grace’s media containing 10% fetal bovine serum was used to dilute BV for experiments. BV titer was determined by plaque assays using Sf9 cells, as previously described (Chikhalya et al., 2009). 2.2. Rearing and maintenance of larvae Pseudoplusia includens or T. ni eggs were provided by Dr. Michael Strand (University of Georgia) or purchased from Benzon Research (Carlisle, PA), respectively. Larvae were reared in groups at 28 °C in constant light on a semi-synthetic diet formulated for P. includens (Southland Products, AR) until the larvae reached the pre-molt stage of the penultimate instar. Insects were determined to be in a pre-molt stage once head capsule slippage was visible. Once insects reached the pre-molt stage they were stored at 15 °C for no longer than 48 h. Under these conditions, P. includens had six instars and T. ni had five instars. 2.3. Insect injections The susceptibilities of P. includens and T. ni to AcMNPV were quantified after orally inoculating newly molted penultimate instar larvae or intrahemocoelically (IH) inoculating feeding penultimate instar larvae with increasing quantities of OB or BV, respectively. Inoculations were performed using a sterilized 32gauge needle fitted to a sterile 1 mL plastic syringe (BD Bioscience) and mounted on a microapplicator (Burkard or KD Scientific). Larvae were inoculated orally within 30 m of molting to the penultimate instar with 1 lL of OB by carefully inserting a blunt needle through the mouth and into the midgut lumen. To quantify the relative virulence of AcMNPV OB, dosages of 20, 40, 50, 60 80, 180, 200 and 400 OB were tested in P. includens larvae and 6, 10, 12, 20, 30, and 40 OB were assayed in T. ni (N = 30–32 larvae per dosage; N = 465 larvae in total). Mid-penultimate instar larvae were IH inoculated with 1 lL of BV by inserting a 32-gauge sharp needle through the planta of the proleg and into the hemocoel. For IH inoculations of BV, dosages of 0.1, 1.0, 10, 25 50 or 100 plaque forming units (PFU) were injected into mid-penultimate instar P. includens larvae and dosages of 0.003, 0.005, 0.01, 0.015 0.03, 0.085, 0.10 and 0.13 PFU were inoculated into T. ni larvae (N = 32 larvae per dosage; N = 512 larvae in total). After inoculation, larvae were maintained individually on a semi-synthetic diet formulated for P. includens (Southland Products) in 25 mL cups at 28 °C with constant illumination in a growth chamber and checked twice daily until pupation or death. Insects that died from injection were excluded from the studies on the second day and this comprised less than 6% of the insects in a treatment. Virus infection was confirmed for insects that had died after the second day of each experiment using light microscopy (400 magnification) to determine if OB were present in insect cadavers. Dose–response curves were generated from the results of the virulence studies using logit analyses. Chi-square tests found the logit models provided good fit to the data in all cases (P < 0.0001). Only dosages that produced mortality of between 5% and 95% were included in the analysis.
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2.4. Developmental resistance To study developmental resistance, P. includens were inoculated orally with 180 OB of AcMNPV-hsp70/lacZ at 0, 3, 6, 9, 16 and 24 h after molting to the fifth instar (N = 28–32 larvae per developmental stage; N = 212 larvae in total). To assess the ability of the optical brightener, M2R to enhance fatal AcMNPV infection of P. includens, M2R (Sigma–Aldrich) was first dissolved in dimethyl sulfoxide (DMSO) and appropriate volumes of the dissolved M2R added to OB dilutions to yield inocula containing 1% M2R (w/v) and OB, as previously described (Washburn et al., 1998). Identical volumes of DMSO without M2R were added to dilutions of OB to generate control inocula. Two developmental stages of P. includens were inoculated orally with suspensions of AcMNPV-hsp70/lacZ OB containing 1% M2R or DMSO. Cohorts of larvae comprising the newly molted penultimate instar stage (designated 50) were prepared by observing late fourth instar P. includens larvae shed their cuticle and immediately orally inoculated the larvae with 1 ll of M2R or control inocula containing 40 AcMNPV-hsp70/lacZ OB (N = 45 larvae per treatment). To prepare the mid-instar developmental cohort, 50 larvae that were not inoculated with virus were placed individually into 25 mL plastic cups, provided with excess diet, incubated for 16 h (designated as 516) as described above and then inoculated with 1 ll of M2R or control inocula containing 180 AcMNPV-hsp70/lacZ OB (N = 45 larvae per treatment). After inoculation, insects were incubated, checked twice daily for mortality and virus infection in cadavers confirmed as described above. 2.5. Time course The timing and pattern of LacZ expression in 50 P. includens was assessed by orally inoculating 50 P. includens larvae with 60 OB of AcMNPV-hsp70/lacZ. After inoculation, larvae were incubated as described above, sacrificed at specific times post-inoculation (0.5, 4, 8, 12, 18, 24, 30, 48, 72 and 96 h.p.i; N = 24–26 larvae per time point). The intact midguts were removed within 5 min of sacrifice and fixed in 2% paraformaldehyde (PFA) dissolved in cytoskeleton enhancement buffer (CEB; 10 mM Pipes, 60 mM sucrose, 100 mM KC1, 5 mM Mg (OAc)2, 1 mM EGTA, pH 6.8) at 4 °C for 6–8 h, as described previously (Engelhard et al., 1994). Subsequently, midguts were rinsed in CEB for 1 h, CEB was removed and tissues incubated in enhancement buffer (5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2) containing 5-bromo-4-chloro-indolyl-b-D-galactopyranoside (i.e. X-gal; 1.5 mg/mL) for 12 h in darkness at room temperature. The processed midguts were analyzed for LacZ expression using a stereomicroscope (Leica MZ12.5) to quantify number and cellular composition of LacZ-positive (LacZ+) viral foci, as previously described (Engelhard et al., 1994; Haas-Stapleton et al., 2003; Zhang et al., 2004). For internal controls, a bioassay was used to determine the mortality levels for 50 P. includens orally inoculated with the dosage of AcMNPV-hsp70/lacZ (60 OB) used in the time course study (N = 60 larvae). Larvae were orally inoculated, incubated and checked for mortality as described above. To study pathogenesis around the time that infection was transferred from the midgut to hemocoel, a second time course study was conducted. 50 P. includens larvae were inoculated orally with 60 OB of AcMNPV-hsp70/lacZ. Subsequently, midgut tissues and hemolymph was collected from larvae at specific times post inoculation to quantify the proportion of LacZ+ cells and the BV titer in the hemolymph (14, 16, 18, 20, 22, and 24 h.p.i; N = 24–26 larvae per time point). To reassess a late time point, a cohort of larvae was sacrificed at 72 h.p.i (N = 24 larvae). For these studies, insects were surface sterilized using 70% ethanol and the proleg removed and the hemolymph collected using a pipette. Hemolymph (10 lL) was pipetted into individual wells of a 96-well plate resting on dry ice and subsequently stored at 80 °C until plaque assay was per-
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formed as described above to quantify BV titer. The remaining hemolymph (10 lL) was pipetted into a 96-well plate containing 100 lL sterile PBS, incubated for 15 m at room temperature, the cells fixed and processed for LacZ signaling, as described previously (Trudeau et al., 2001). Midgut tissues were collected from larvae, fixed, processed for LacZ signals and viral foci numbers quantified as described above. Flow cytometry was used to quantify GP64 expression on the surface of hemocytes as previously described (Chikhalya et al., 2009). Briefly, hemolymph was pooled from 10 insects per treatment and time point, the cells fixed using 2% PFA dissolved in phosphate buffer saline (PBS), rinsed twice using centrifugation (5 min, 260 g, 4 °C) and suspended in PBS containing 2% bovine serum albumin (PBS–BSA). Cells were stained for 1 h with the mouse monoclonal antibody B12D5 that is specific for GP64 (1:50), rinsed twice using centrifugation, stained for 30 min with goat anti-mouse Ig (H + L)-FITC (Southern Biotech; 1:400), rinsed with PBS–BSA, fixed with 1% PFA dissolved in PBS and analyzed using a flow cytometer (Quanta SC MPL, Beckman Coulter). At least 20,000 events with a side scatter and electronic volume that is characteristic of hemocytes were analyzed for each treatment. Larvae inoculated with PBS, dissected at identical time points and stained for GP64 served as uninfected controls. 2.6. Statistical analysis Data were analyzed, graphs generated and regression formulae calculated using Prism 6 (GraphPad, Version 6.0b), SigmaPlot (version 11), Minitab (version 16.1), SAS (version 9.3) or Microsoft Excel 2011 for Mac (Version 14.1.3). All reported error values are standard errors of the means (SEM). For the AcMNPV OB and BV virulence data, we performed generalized linear model analyses using a logit link function and binomial distribution of the mortalities using Proc GENMOD (SAS v9.3). The models included log dose and species main effects and time species interaction effects. We determined estimates of LD50 doses using Proc PROBIT (SAS v9.3). Differences in mortality for the M2R study were determined using Chi-squared statistics (Minitab v16.1). Graphs of the results are presented with mortality probabilities in the logit scale. Intercept differences indicate differences in initial susceptibility while slope differences indicate differences in rate of change in susceptibility with increasing dosage. 3. Results and discussion 3.1. AcMNPV virulence in P. includens and T. ni larvae The virulence of AcMNPV-hsp70/lacZ OB was quantified in P. includens and T. ni by orally inoculating newly molted penultimate instars with increasing quantities of OB. Trichoplusia ni and P. includens are closely related; both belong to the Tribe Argyrogrammatini of the Subfamily Plusiinae in the order Noctuidae. Trichoplusia ni is considered to be permissive to AcMNPV because infections initiated by low dosages of orally inoculated OB rapidly disseminate within and kill this species (Washburn et al., 1995). There was a 2.5-fold difference in the susceptibility of P. includens and T. ni to orally inoculated AcMNPV OB (Fig. 1A; LD50 for P. includens was 40.8 OB [95% confidence intervals (CI): 36.55, 45.63] and 16.1 OB for T. ni [95% CI: 9.04, 21.91]). A significant difference in the Y-intercepts of the regression curves for P. includens and T. ni orally inoculated with AcMNPV-hsp70/lacZ OB was observed indicating that a higher dosage of OB was required to initiate a mortal infection in P. includens (Chi-square = 11.24, DF = 1, P = 0.008; Supplementary Table 1). The difference in the slope of the two regression curves was non-significant, suggesting that T. ni and P. includens were responding similarly to increased
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dosages of AcMNPV OB (Chi-square = 2.77, DF = 1, P = 0.0963; Supplementary Table 1). Thus, once infection was established, there was an equal likelihood in both species for the infection to result in mortality. The 2.5-fold difference in the LD50 values between the species and the significant difference in the Y-intercept for the regression indicate that a higher quantity of OB was required to establish an infection in P. includens, relative to T. ni. The moderate species effect between T. ni and P. includens that were orally inoculated with OB contrasts with Helicoverpa zea and Heliothis virescens, which are members of the same subfamily Heliothinae (Noctuidae), and display more than a 1000-fold difference in susceptibility to fatal infection by AcMNPV OB when assayed using the same methods employed in our study (Trudeau et al., 2001). This suggests that phylogenetic relationships between species may not be good predictors of susceptibility to a particular baculovirus species. The susceptibility of mid-penultimate instar P. includens and T. ni to systemic infection was quantified by intrahemocoelically (IH) inoculating larvae with increasing dosages of BV. Similar to OB inoculated larvae, P. includens were more resistant to fatal infection from IH-inoculated BV relative to T. ni (Fig. 1B; LD50 values were 13.8 PFU for P. includens [95% CI: 7.58, 26.71] and
Fig. 1. Virulence of AcMNPV-hsp70/lacZ in P. includens and T. ni. (A) Larval mortality of P. includens and T. ni when inoculated orally with increasing dosages of AcMNPVhsp70/lacZ OB at the onset of the penultimate instar (N = 30–32 insects per dosage). Solid lines represent regression lines for P. includens and dashed lines are for T. ni. The regression equations are: P. includens, Y = 6.9911 + 4.0212 log(X), R2 = 0.7253, LD50 = 40.8 OB; T. ni, Y = 4.4277 + 4.0212 log(X), R2 = 0.8947, LD50 = 16.1 OB. (B) Larval mortality of P. includens and T. ni when inoculated IH with increasing dosages of AcMNPV-hsp70/lacZ BV during the penultimate instar (N = 32 insects per dosage). Solid lines represent regression lines for P. includens and dashed lines are for T. ni. The regression equations are: P. includens, Y = 1.3704 + 1.0373(log X), R2 = 0.7142, LD50 = 13.8 PFU; T. ni, Y = 3.6733 + 1.8479 log(X), R2 = 0.8423, LD50 = 0.060 PFU.
0.060 PFU for T. ni [95% CI: 0.040, 0.073]). There were significant differences between the Y-intercepts (Chi-square = 365.12, DF = 1, P < 0.0001; Supplementary Table 1) and in the slopes of the regression curves for the two species that were IH inoculated with AcMNPV-hsp70/lacZ BV (Chi-square = 28.61, DF = 1, P < 0.0001; Supplementary Table 1). These differences suggest that molecular or cellular processes occurring in the hemocoel of P. includens reduced the susceptibility of this species to IH inoculated BV, relative to T. ni, across all dosages tested. Taken together, the relatively high LD50 values for P. includens inoculated with BV and the significant differences in the regression lines indicated that P. includens is significantly more resistant than T. ni to IH inoculated AcMNPV-hsp70/lacZ BV. Our results conflict with a study which reported that newly molted penultimate instar P. includens were 82-times more resistant to AcMNPV OB than what we report (LD50 of 3352 OB)(Kunimi et al., 1997). The difference in susceptibility of P. includens to fatal infection between our study and Kunimi et al. (1997) may be due to differences in the sources of P. includens or in methods used to inoculate the insects. Kunimi et al. (1997) used droplet feeding for inoculation and then weighed larvae before and after feeding to estimate the quantity of virus consumed. High LD50 values of 2500–9274 OB were also reported for field-collected isolates of P. includens SNPV (PsinSNPV) in third instar P. includens larvae that were inoculated by contaminating artificial diet with known quantities of OB (Alexandre et al., 2010). For our studies, insects were inoculated orally using a microapplicator with precise microliter volumes of an aqueous solution containing OB that was visually quantified using a hemocytometer. Because the quantity of OB inoculated into each insect was known, our method may provide a better measure for laboratory tests of AcMNPV virulence in P. includens. Although different methods were employed for inoculating P. includens with OB, AcMNPV appears to be 61- to 227-times more virulent than field-collected isolates of PsinSNPV in P. includens (Fig. 1A; (Alexandre et al., 2010). To quantify the impact of the developmental stage of the insect on virulence, P. includens were inoculated orally with 180 AcMNPVhsp70/lacZ OB at increasing times after molting to the fifth instar. Logit analysis with time as a continuous variable show there was a significant decrease in mortality as larvae were inoculated later in the fifth instar (Chi-square = 61.21, DF = 1, P < 0.0001). When orally inoculated immediately after molting, 180 OB generated 80% mortality (Fig. 2A). However, at 24 h post molt, the last developmental stage tested, only 25% of the inoculated larvae succumbed to the infection (Fig. 2A). These results demonstrate that the susceptibility to fatal infection decreased as the age at which the larvae were inoculated with AcMNPV OB was increased. Developmental resistance to baculovirus infection occurs in species that are permissive or resistant to orally administered baculovirus OB (Engelhard and Volkman, 1995). The developmental resistance of Lepidoptera larvae to oral infection by baculoviruses has been attributed to infection-induced apoptosis of midgut epithelial cells that can be inhibited by co-inoculating larvae with the optical brightener M2R (Dougherty et al., 2006; Silveira et al., 2005; Washburn et al., 1998). The extent that M2R could increase the virulence of AcMNPV-hsp70/lacZ OB in P. includens was quantified by orally inoculating newly molted fifth instar or larvae 16 h post molt (hereafter designated 50 or 516, respectively) with inocula containing 40 OB or 180 OB and M2R or vehicle. Addition of M2R to inocula increased the susceptibility of both 50- and 516inoculated larvae to fatal AcMNPV infection by 13% and 11%, respectively (Fig. 2B). However, the difference between M2R and control groups was non-significant (Chi-square = 0.007, DF = 1, P = 0.935). Studies using M2R co-inoculated orally with OB into mid-instar T. ni or H. virescens demonstrate that for these permissive species, M2R increases the mortality by 20–41% (Hoover et al.,
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3.2. Time course for orally inoculated OB To investigate the course of oral AcMNPV infection in P. includens, 50 larvae were inoculated orally with 60 OB of AcMNPVhsp70/lacZ and dissected 0.5–96 h post inoculation (h.p.i). LacZ+ cells were first detected at 4 h.p.i in 32% of the larvae and were restricted to the midgut epithelium (2.0 ± 0.5 midgut foci per LACZ+ insect; Fig. 3A and B). Tracheal infection was first observed at 8 h.p.i with 0.25 ± 0.2 midgut + tracheal foci per LacZ+ insect (Fig. 3A and B). The rapid establishment of multicellular foci of infection (midgut + tracheal foci) by 8 h.p.i demonstrates that AcMNPV can quickly traverse the basal lamina that supports the midgut epithelium. Because this transfer occurred in less time than is likely required for a complete cycle of virus replication in the infected midgut epithelial cells, this observation lends additional support to the hypothesis that ODV-derived nucleocapsids are repackaged in midgut cells as BV that is released to infect adjacent cells located in the hemocoel (Ohkawa et al., 2010; Washburn et al., 1999). LacZ+ foci consisting solely of tracheal cells were observed at 8 h.p.i, 4 h after the first signs of infection were observed (Fig. 3B), suggesting that an underlying infected midgut epithelial cell was eliminated within 4 h of being established. Rapid sloughing of infected midgut cells is a resistance pathway that limits transfer of the virus to tissues in the hemocoel and reduces the susceptibility of insects orally inoculated with OB to fatal infection (Washburn et al., 1995). While M2R blocks infected midgut cell sloughing (Dougherty et al., 2006), its inclusion in viral inocula did not significantly increase the susceptibility of P. includens to
Fig. 2. Developmental resistance of P. includens inoculated with AcMNPV-hsp70/ lacZ OB. (A) Mortality of fifth instar P. includens inoculated orally with 180 OB at increasing hours post molt (N = 32 insects per developmental stage). Dashed lines indicate 95% confidence intervals. (B) Larval mortality of 50 P. includens when inoculated orally with 40 OB and 516 P. includens when inoculated orally with 180 OB, in the presence or absence of 1% M2R (N = 45 insects per developmental stage and treatment; percent mortality for: 50 control was 60% and M2R was 73%; 516 control was 52% and M2R was 63.5%).
2000; Washburn et al., 1998; Zhang et al., 2004). In contrast, M2R increases mortality levels for newly molted penultimate instar T. ni or H. virescens inoculated orally with AcMNPV OB by 4–13% (Washburn et al., 1998; Zhang et al., 2004). Because the difference in mortality for mid-instar P. includens inoculated with AcMNPV OB and M2R or vehicle control was low and similar to what was observed for the newly molted instars of other permissive species (Washburn et al., 1998; Zhang et al., 2004), apoptosis-mediated infected midgut cell sloughing may not be an important driver of midgut-based resistance to AcMNPV in mid-instar P. includens. Alternatively, M2R may not inhibit AcMNPV-elicited apoptotic pathways in P. includens, as it does in other insect species. Consequently, including optical brighteners such as M2R into pest management strategies for biological control of P. includens with AcMNPV may not produce an increase in control that offset the added cost. However, inclusion of M2R in inocula did increase the virulence of PsinNPV OB in P. includens larvae by 15520-fold relative to controls lacking M2R (Zou and Young, 1996), suggesting that AcMNPV and PsiNPV infection may elicit different physiological responses in the midgut. Comparisons of the genome sequences of AcMNPV and PsinNPV may provide insights into reasons for the observed differences in virulence. However, the complete genome sequence of PsinNPV is currently not reported. The pathogenesis of orally administered AcMNPV-hsp70/lacZ OB in P. includens was subsequently evaluated using time course studies of infection using expression of the lacZ reporter gene as a molecular marker of viral infection in the tissues.
Fig. 3. Time-course of infection from 0.5–96 h.p.i for 50 P. includens larvae inoculated orally with 60 OB of AcMNPV-hsp70/lacz. (A) Proportion of LacZ+ larvae and tissue-specific distribution of LACZ+ cells within the midgut and trachea (95% CI were 41.5–87.7 for LACZ expression, 30.3–69.8 for midgut infection and 27.4–82.3 for tracheal infection). (B) Average number of LacZ+ midgut, midgut plus tracheal and tracheal only cells associated with the midgut per LacZ+ larva. The bioassay for this study produced 67% mortality (N = 60 insects). Positive error bars reflect SEM.
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orally administered AcMNPV OB (Fig. 2B), suggesting that the early loss of infected midgut epithelial cells may not be inhibited by M2R. The extent of midgut infection decreased from 8.1 ± 2.3 midgut foci per LacZ+ insect at 30 h.p.i to 1.7 ± 0.9 midgut foci per LacZ+ insect at 48 h.p.i (Fig. 3B), suggesting that infected midgut cells were eliminated as the insect progressed through the fifth instar. By 24 h.p.i, 88% of the larvae displayed a tracheal infection (Fig. 3A). Tracheal infection increased from 23.2 ± 10.7 foci per LacZ+ insect at 48 h.p.i to 40.6 ± 15.1 foci per LacZ+ insect at 72 h.p.i, with 100% of insects displayed tracheal infection (Fig. 3A and B). Because the time course of infection for AcMNPV in P. includens (this study) was similar to what was observed for studies of AcMNPV in T. ni (Washburn et al., 1995), there does not appear to be a physiological barrier that restricts the dissemination of AcMNPV in P. includens larvae. Studies of baculovirus pathogenesis in several species of Lepidoptera clearly demonstrate that cells of the midgut epithelium are the first cell type infected by ODV. However, there is no consensus on the identity of the second cell type that is infected to initiate a systemic infection. In pathogenesis studies of newly molted fourth instar H. virescens and H. zea inoculated orally with AcMNPV-hsp70/lacZ OB, infected hemocytes were observed 8– 10 h after the first infected tracheolar cell (Trudeau et al., 2001). A similar pattern of tracheal followed by hemocyte infection was observed in newly molted fourth instar Lymantria dispar larvae orally inoculated with L. dispar MNPV OB (McNeil et al., 2010). However, studies of T. ni exposed to diet coated with cultured Sf9 cells infected with a GFP-expressing recombinant of AcMNPV demonstrated that tracheal tissues became infected 24 h after hemocytes (Barrett et al., 1998). Other studies of larvae orally inoculated with baculovirus OB demonstrate simultaneous infection of tracheolar cells and hemocytes (Matos et al. 1999; Chikhalya et al., 2009; Katsuma et al., 2008). To study the transfer of infection from the midgut to cells in the hemocoel, 50 P. includens were inoculated orally with 60 OB, dissected and the hemolymph was removed from insects in the hours surrounding the onset of tracheal infection. While systemic tracheal infection was evident at 14 h.p.i in 46% of the larvae, hemocytes displayed first LacZ signals at 16 h.p.i in 38% of the inoculated insects (6.7 ± 3.2 LacZ+ hemocytes per LacZ+ insect Fig. 4A and B). Thus, for P. includens orally inoculated with low quantities of AcMNPV OB, infection begins in the midgut epithelium and is transferred first to adjacent tracheal epidermal cells and later to hemocytes. BV was first detected in the hemolymph at 18 h.p.i (Fig. 4C), 2 h after infected hemocytes were first observed (Fig. 4A). The time needed for BV-infected cells in culture to release BV is approximately 12 h (Hodgson et al., 2007; Nie and Theilmann, 2010). Therefore, it is unlikely that hemocytes generated the BV that was first detected in the hemolymph. However, the time between the observed first tracheal and hemocyte infections may be sufficient for a cycle of virus replication, suggesting that infected tracheal cells may have been responsible for generating the BV that was first observed in the hemolymph. The observed increased in BV titer from 18 to 24 h.p.i (Fig. 4C) correlated with an increase in the mean number of foci per LacZ+ insect (Fig. 4B), demonstrating that the virus was amplifying and disseminating within infected insects. At 20 h.p.i, the mean number of midgut foci per LacZ+ insect increased 6.3-fold and at 22 h.p.i returned to levels similar to what was observed at 18 h.p.i This observed increase in the number of midgut foci at 20 h.p.i may have resulted from variability in the experiment (95% CI overlapped for the 18– 22 h.p.i time points). The observed loss of AcMNPV-infected midgut epithelial cells between 24 and 72 h.p.i (Fig. 4A and B), which permits infected insects to continue feeding and accumulate biomass, is consistent with what has been observed for T. ni, H. virescens, H. zea, Spodoptera frugiperda and Manduca sexta that were orally inoculated with AcMNPV OB (Haas-Stapleton et al.,
Fig. 4. Time-course of infection from 14–72 h.p.i for 50 P. includens larvae inoculated orally with 60 OB of AcMNPV-hsp70/lacZ. (A) Proportion of LacZ+ larvae and tissue-specific distribution of LacZ+ cells within the midgut, trachea and hemocytes (95% CI were 69.5–102.5 for LacZ expression, 25.7–67.0 for midgut infection, 51.7–85.0 for tracheal infection and 31.2–100.3 for hemocyte infection). (B) Average number of LacZ+ midgut, midgut plus tracheal, tracheal only and hemocyte cells per LacZ+ larva. To reduce overlap of symbols at each time point, some symbols are offset by up to 0.4 units along the X-axis. (C) BV titer within P. includens inoculated orally with 60 OB of AcMNPV-hsp70/lacZ as newly molted fifth instars (N = 23–24 larvae per time-point). Positive error bars reflect SEM.
2003; Trudeau et al., 2001; Washburn et al., 2000; Washburn et al., 1995). The proportion of hemocytes infected with AcMNPV-hsp70/lacZ was further assessed using flow cytometry to detect cell-surface expression of the BV protein GP64. At 12 h.p.i, 6.18% of hemocytes isolated from P. includens larvae displayed cell surface expression of GP64 (Fig. 5A) and increased to 84.5% at 18 h.p.i (Fig. 5B), which
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Fig. 5. GP64 expression on hemocytes isolated from larvae orally inoculated with 60 OB or PBS. Hemocytes isolated from P. includens inoculated with OB (filled histograms) or PBS (open histograms) at 12 and 18 h.p.i (A and B) or T. ni at 18 h.p.i (C) were stained to detect cell-surface expression of GP64 and analyzed using flow cytometry. Values in the upper right corner of each histogram represent the proportion of hemocytes isolated from larvae inoculated with OB that were gated positive for aGP64-FITC antibody staining. Data are from one experiment, representative of three.
is similar to what was observed for similarly treated T. ni larvae at 18 h.p.i (Fig. 5C). A relatively low number of LacZ+ hemocytes were observed in 10 ll of hemolymph isolated at 18 h.p.i from P. includens larvae (Fig. 4B). However, 84.5% of the hemocytes at the same time point displayed GP64 expression (Fig. 5B). This suggests that use of flow cytometry to quantify GP64 expression may be an improvement over assessing LacZ expression using light microscopy to identify AcMNPV-infected cells. Together, our results demonstrate that for P. includens inoculated orally with AcMNPV-hsp70/lacZ OB, the infection progressed from the midgut to the trachea then, finally, to the hemocytes (Figs. 3B, 4B). Because infectious BV was detected in the hemolymph after infected midgut cells, tracheal cells and hemocytes were observed (18 h.p.i; 3.9 103 ± 8.2 103 PFU/mL; Fig. 4C), we cannot conclusively determine which of these cell types were responsible for releasing high quantities of BV into the hemocoel. However, the observation of BV in the hemolymph after detecting infected hemocytes is in agreement with studies of AcMNPV pathogenesis in H. virescens and H. zea (Trudeau et al., 2001). Because the BV titer in AcMNPV-infected P. includens increased from 18 to 24 h.p.i (Fig. 4C), this insect species does not appear to display substantial resistance to systemic AcMNPV infection. In conclusion, our studies demonstrate that P. includens is permissive to fatal infection by low dosages of orally administered AcMNPV OB (LD50 = 40.8 OB). While field-collected isolates of PsinSNPV OB are reported to cause fatal infections in P. includens, the reported LD50 values for this baculovirus is 61- to 227-fold higher than what we report for AcMNPV (LD50 of PsinSNPV = 2500–9274 OB; (Alexandre et al., 2010)). Inclusion of the optical brightener M2R into PsinNPV inocula resulted in a substantial increase in virulence (Zou and Young, 1996). However, enhancing the potency of a biological pesticide with synthetic chemicals is considered by many to be unacceptable for organic farming. Our studies demonstrate that AcMNPV is highly virulent to P. includens in the absence of M2R. Controlled tests of AcMNPV and PsinSNPV virulence for P. includens in an experimental setting that more closely reflects those of soybean in a field will provide more evidence for which baculovirus species may best limit economic damage to organic soybean crops. Our virulence studies of AcMNPV-hsp70/lacZ show a substantial and significant difference in the slope of the regression curves for BV-inoculated larvae
(Fig. 1B) that was not observed for larvae orally inoculated with OB (Fig. 1A). This suggests that the infection kinetics resulting from orally administered OB differs from IH inoculated BV and may point to systemic resistance pathways that are suppressed in P. includens when the virus establishes an infection via the natural midgut route of infection. Acknowledgments We thank Dr. Michael R. Strand and Dr. Jena Johnson at the University of Georgia for kindly providing P. includens eggs and Dr. Loy Volkman at UC Berkeley (now Emeritus) for providing the B12D5 antibody. We also thank the peer-reviewers of the manuscript and Dr. Jan Washburn (UC Berkeley, Emeritus) for suggestions that substantially improved the data presentation and statistical analysis. We would like to acknowledge Maggie Carrera, Alisa de la Cruz and Tiffany Chen for technical assistance. This work was funded by a grant from the US Department of Agriculture (CREES 200803390) to E.J.H-S. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocontrol.2013. 01.002. References Alexandre, T.M., Ribeiro, Z.M., Craveiro, S.R., Cunha, F., Fonseca, I.C., Moscardi, F., Castro, M.E., 2010. Evaluation of seven viral isolates as potential biocontrol agents against Pseudoplusia includens (Lepidoptera: Noctuidae) caterpillars. Journal of Invertebrate Pathology 105, 98–104. Barrett, J.W., Brownwright, A.J., Primavera, M.J., Palli, S.R., 1998. Studies of the nucleopolyhedrovirus infection process in insects by using the green fluorescence protein as a reporter. Journal of Virology 72, 3377–3382. Bonning, B.C., Hoover, K., Booth, T.F., Duffey, S., Hammock, B.D., 2005. Development of a recombinant baculovirus expressing a modified juvenile hormone esterase with potential for insect control. Archives of Insect Biochemistry and Physiology 30, 177–194. Chikhalya, A., Luu, D.D., Carrera, M., De La Cruz, A., Torres, M., Martinez, E.N., Chen, T., Stephens, K.D., Haas-Stapleton, E.J., 2009. Pathogenesis of Autographa californica multiple nucleopolyhedrovirus in fifth-instar Anticarsia gemmatalis larvae. The Journal of General Virology 90, 2023–2032.
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Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in Pseudoplusia includens larvae Aniska Chikhalya, Kimberly D. Stephens, James W. Archie, Eric J. Haas-Stapleton ⇑ Department of Biological Sciences, California State University Long Beach, 1250 Bellflower Road, Long Beach, CA 90840, USA
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
" Pseudoplusia includens displays no
physiological barriers to fatal AcMNPV Infection. " The optical brightener M2R does not significantly increase virulence. " P. includens could be controlled in organic crops using naturally occurring AcMNPV.
a r t i c l e
i n f o
Article history: Received 7 June 2012 Accepted 6 January 2013 Available online 12 January 2013 Keywords: Pseudoplusia includens Autographa californica Nucleopolyhedrosis virus Pathogenesis AcMNPV
a b s t r a c t Virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrosis virus were quantified in penultimate instar Pseudoplusia includens (soybean looper) larvae using a recombinant encoding the lacZ reporter gene (AcMNPV-hsp70/lacZ). Larvae inoculated orally with AcMNPV-hsp70/lacZ occlusion bodies (OB) or intrahemocoelically with budded virus (BV) were susceptible to fatal infection (LD50 = 40.8 OB; 13.8 BV plaque forming units). Pseudoplusia includens displayed increased developmental resistance as larvae were orally inoculated with OB at later times during the penultimate instar. The optical brightener M2R, an inhibitor of the apoptotic processes that drive developmental resistance, did not significantly affect the virulence of AcMNPV-hsp70/lacZ OB. To study pathogenesis, newly molted penultimate instar P. includens were orally inoculated with 60 OB and examined from 0.5 to 96 h post inoculation (h.p.i). Infection in the midgut was first detected at 4 h.p.i in 32% of the larvae and was apparent in cells of the tracheal system at 8 h.p.i. LacZ-positive (LacZ+) hemocytes were first observed 2 h later. At 18 h.p.i, a low proportion of the hemocytes were LacZ+ (5.6%). However, flow cytometry analysis of cell surface expression of the viral protein GP64 showed that 84.5% of the hemocytes collected at 18 h.p.i were infected with AcMNPV, suggesting that flow cytometry may be a more sensitive method for identifying AcMNPV-infected cells. Because P. includens display no physiological barriers to AcMNPV OB and is permissive to fatal infection, this species could be controlled in organic cropping systems using naturally occurring strains of AcMNPV. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Baculoviruses offer an appealing alternative to chemical and many biological control agents because they are arthropod-specific ⇑ Corresponding author. Fax: +1 562 985 8878. E-mail address: [email protected] (E.J. Haas-Stapleton). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.01.002
pathogens that naturally infect and kill many important lepidopteran crop pests. This specificity is an important factor ensuring safety to humans, other vertebrates and beneficial insects (e.g. pollinators and natural enemies). Factors limiting the efficacy of baculoviruses as crop pest control agents include slow speed of kill and narrow effective host ranges of individual baculovirus species (Vail, 1993; Vail et al., 1999). However, baculoviruses recycle in
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nature and the quantity in a field increases as more larvae are controlled by viral infection. This can result in pest control over several seasons (Mulock and Faulkner, 1997), a characteristic that most chemical pesticides lack. To date, the most successful genetically modified (GM) baculoviruses incorporate arthropod-specific toxins or growth modulators that reduce the time to death and thereby the duration that larvae feed upon and damage crop plants (Bonning et al., 2005; Gurevitz et al., 2007; Lasaa et al., 2007; Rajendra et al., 2006). However, organic farmers may not use GM organisms, including engineered baculoviruses. The foundation for building confidence in naturally occurring baculoviruses (i.e. wild-type strains) to control crop pests is supported with a detailed knowledge of the infection kinetics for baculoviruses in a variety of economically important crop pests. Discovering the mechanisms of baculovirus susceptibility and resistance in lepidopteran larvae may increase the appeal of these biological control agents for use in organic farming. Pseudoplusia includens (the soybean looper; family Noctuidae) is a lepidopteran pest of soybean, a leading organic crop in the United States (US) and Brazil (Oliveira et al., 2006; Roseboro, 2007). This insect overwinters in southern states of the US and each year large numbers migrate into the US from Central and South America. In Mississippi, 90% of all loopers attacking soybean are P. includens (Stewart, 2007); less severe infestations occur in the western United States. Current strategies for reducing economic damage to US soybean crops by P. includens include chemical insecticides, biological insecticides (e.g. Bacillus thuringiensis and entomopathogenic fungi), cultural practices (e.g. early crop planting), and GM organisms. Baculoviruses may constitute a valuable addition to the P. includens control strategies. The purpose of the current study was to quantify the virulence and pathogenesis of the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) in P. includens relative to Trichoplusia ni (cabbage looper), a species having documented high susceptibility to AcMNPV (Washburn et al., 1995). AcMNPV is the type species of the genus Nucleopolyhedrovirus, of which all members produce two distinct viral morphotypes: occlusion derived virus (ODV) and budded virus (BV). Multiple ODV are embedded within a protective protein complex called the occlusion body (OB) that is responsible for protecting the ODV from environmental damage and for spreading the virus between insects. BV is produced within the hemocoel of infected larvae and is the morphotype responsible for disseminating the virus between cells within an insect. For successful dissemination of infection within the insect, ODV is released from the OB in the alkaline environment of insect intestine to infect midgut epithelial cells (Faulkner et al., 1997; Granados, 1978). BV released from midgut cells infects adjacent tracheal cells of the respiratory system or circulating hemocytes, which mediate cellular immune responses (Engelhard et al., 1994; Granados and Lawler, 1981; Strand, 2008). In permissive species, the virus spreads to all tissues, resulting in a fatal infection. The results described herein demonstrate that while AcMNPV is slightly less virulent in P. includens relative to T. ni, the pathogenesis of the virus in P. includens is similar to what is reported for T. ni (Washburn et al., 1995). Thus, P. includens is a fully permissive host for AcMNPV and should be considered a candidate for biological control of P. includens on organic crops.
2. Materials and methods 2.1. Virus and virus preparation AcMNPV-hsp70/lacZ is a recombinant that contains all of the genes of wild-type AcMNPV, but includes a b-galactosidase reporter gene whose expression is driven by the Drosophila hsp70 promoter
(Engelhard et al., 1994) and was used for all studies described herein. AcMNPV-hsp70/lacZ causes mortality levels in T. ni that are identical to the wild type strains of AcMNPV (Engelhard et al., 1994). AcMNPV-hsp70/lacZ OB were isolated and purified from T. ni cadavers as described in Kirkpatrick et al. (1998). Briefly, virus killed T. ni were crushed and the crude extract filtered through a porous nylon. OB were isolated using sucrose gradient centrifugation (Summers and Smith, 1987), the OB suspended and diluted in neutrally buoyant solution of water and glycerin (3:2 v/v). Subsequently, OB were quantified using a hemocytometer and stored at 4 °C in the dark. BV was propagated in vitro using cultured Sf9 cells, as previously described (Kirkpatrick et al., 1998). Grace’s media containing 10% fetal bovine serum was used to dilute BV for experiments. BV titer was determined by plaque assays using Sf9 cells, as previously described (Chikhalya et al., 2009). 2.2. Rearing and maintenance of larvae Pseudoplusia includens or T. ni eggs were provided by Dr. Michael Strand (University of Georgia) or purchased from Benzon Research (Carlisle, PA), respectively. Larvae were reared in groups at 28 °C in constant light on a semi-synthetic diet formulated for P. includens (Southland Products, AR) until the larvae reached the pre-molt stage of the penultimate instar. Insects were determined to be in a pre-molt stage once head capsule slippage was visible. Once insects reached the pre-molt stage they were stored at 15 °C for no longer than 48 h. Under these conditions, P. includens had six instars and T. ni had five instars. 2.3. Insect injections The susceptibilities of P. includens and T. ni to AcMNPV were quantified after orally inoculating newly molted penultimate instar larvae or intrahemocoelically (IH) inoculating feeding penultimate instar larvae with increasing quantities of OB or BV, respectively. Inoculations were performed using a sterilized 32gauge needle fitted to a sterile 1 mL plastic syringe (BD Bioscience) and mounted on a microapplicator (Burkard or KD Scientific). Larvae were inoculated orally within 30 m of molting to the penultimate instar with 1 lL of OB by carefully inserting a blunt needle through the mouth and into the midgut lumen. To quantify the relative virulence of AcMNPV OB, dosages of 20, 40, 50, 60 80, 180, 200 and 400 OB were tested in P. includens larvae and 6, 10, 12, 20, 30, and 40 OB were assayed in T. ni (N = 30–32 larvae per dosage; N = 465 larvae in total). Mid-penultimate instar larvae were IH inoculated with 1 lL of BV by inserting a 32-gauge sharp needle through the planta of the proleg and into the hemocoel. For IH inoculations of BV, dosages of 0.1, 1.0, 10, 25 50 or 100 plaque forming units (PFU) were injected into mid-penultimate instar P. includens larvae and dosages of 0.003, 0.005, 0.01, 0.015 0.03, 0.085, 0.10 and 0.13 PFU were inoculated into T. ni larvae (N = 32 larvae per dosage; N = 512 larvae in total). After inoculation, larvae were maintained individually on a semi-synthetic diet formulated for P. includens (Southland Products) in 25 mL cups at 28 °C with constant illumination in a growth chamber and checked twice daily until pupation or death. Insects that died from injection were excluded from the studies on the second day and this comprised less than 6% of the insects in a treatment. Virus infection was confirmed for insects that had died after the second day of each experiment using light microscopy (400 magnification) to determine if OB were present in insect cadavers. Dose–response curves were generated from the results of the virulence studies using logit analyses. Chi-square tests found the logit models provided good fit to the data in all cases (P < 0.0001). Only dosages that produced mortality of between 5% and 95% were included in the analysis.
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2.4. Developmental resistance To study developmental resistance, P. includens were inoculated orally with 180 OB of AcMNPV-hsp70/lacZ at 0, 3, 6, 9, 16 and 24 h after molting to the fifth instar (N = 28–32 larvae per developmental stage; N = 212 larvae in total). To assess the ability of the optical brightener, M2R to enhance fatal AcMNPV infection of P. includens, M2R (Sigma–Aldrich) was first dissolved in dimethyl sulfoxide (DMSO) and appropriate volumes of the dissolved M2R added to OB dilutions to yield inocula containing 1% M2R (w/v) and OB, as previously described (Washburn et al., 1998). Identical volumes of DMSO without M2R were added to dilutions of OB to generate control inocula. Two developmental stages of P. includens were inoculated orally with suspensions of AcMNPV-hsp70/lacZ OB containing 1% M2R or DMSO. Cohorts of larvae comprising the newly molted penultimate instar stage (designated 50) were prepared by observing late fourth instar P. includens larvae shed their cuticle and immediately orally inoculated the larvae with 1 ll of M2R or control inocula containing 40 AcMNPV-hsp70/lacZ OB (N = 45 larvae per treatment). To prepare the mid-instar developmental cohort, 50 larvae that were not inoculated with virus were placed individually into 25 mL plastic cups, provided with excess diet, incubated for 16 h (designated as 516) as described above and then inoculated with 1 ll of M2R or control inocula containing 180 AcMNPV-hsp70/lacZ OB (N = 45 larvae per treatment). After inoculation, insects were incubated, checked twice daily for mortality and virus infection in cadavers confirmed as described above. 2.5. Time course The timing and pattern of LacZ expression in 50 P. includens was assessed by orally inoculating 50 P. includens larvae with 60 OB of AcMNPV-hsp70/lacZ. After inoculation, larvae were incubated as described above, sacrificed at specific times post-inoculation (0.5, 4, 8, 12, 18, 24, 30, 48, 72 and 96 h.p.i; N = 24–26 larvae per time point). The intact midguts were removed within 5 min of sacrifice and fixed in 2% paraformaldehyde (PFA) dissolved in cytoskeleton enhancement buffer (CEB; 10 mM Pipes, 60 mM sucrose, 100 mM KC1, 5 mM Mg (OAc)2, 1 mM EGTA, pH 6.8) at 4 °C for 6–8 h, as described previously (Engelhard et al., 1994). Subsequently, midguts were rinsed in CEB for 1 h, CEB was removed and tissues incubated in enhancement buffer (5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2) containing 5-bromo-4-chloro-indolyl-b-D-galactopyranoside (i.e. X-gal; 1.5 mg/mL) for 12 h in darkness at room temperature. The processed midguts were analyzed for LacZ expression using a stereomicroscope (Leica MZ12.5) to quantify number and cellular composition of LacZ-positive (LacZ+) viral foci, as previously described (Engelhard et al., 1994; Haas-Stapleton et al., 2003; Zhang et al., 2004). For internal controls, a bioassay was used to determine the mortality levels for 50 P. includens orally inoculated with the dosage of AcMNPV-hsp70/lacZ (60 OB) used in the time course study (N = 60 larvae). Larvae were orally inoculated, incubated and checked for mortality as described above. To study pathogenesis around the time that infection was transferred from the midgut to hemocoel, a second time course study was conducted. 50 P. includens larvae were inoculated orally with 60 OB of AcMNPV-hsp70/lacZ. Subsequently, midgut tissues and hemolymph was collected from larvae at specific times post inoculation to quantify the proportion of LacZ+ cells and the BV titer in the hemolymph (14, 16, 18, 20, 22, and 24 h.p.i; N = 24–26 larvae per time point). To reassess a late time point, a cohort of larvae was sacrificed at 72 h.p.i (N = 24 larvae). For these studies, insects were surface sterilized using 70% ethanol and the proleg removed and the hemolymph collected using a pipette. Hemolymph (10 lL) was pipetted into individual wells of a 96-well plate resting on dry ice and subsequently stored at 80 °C until plaque assay was per-
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formed as described above to quantify BV titer. The remaining hemolymph (10 lL) was pipetted into a 96-well plate containing 100 lL sterile PBS, incubated for 15 m at room temperature, the cells fixed and processed for LacZ signaling, as described previously (Trudeau et al., 2001). Midgut tissues were collected from larvae, fixed, processed for LacZ signals and viral foci numbers quantified as described above. Flow cytometry was used to quantify GP64 expression on the surface of hemocytes as previously described (Chikhalya et al., 2009). Briefly, hemolymph was pooled from 10 insects per treatment and time point, the cells fixed using 2% PFA dissolved in phosphate buffer saline (PBS), rinsed twice using centrifugation (5 min, 260 g, 4 °C) and suspended in PBS containing 2% bovine serum albumin (PBS–BSA). Cells were stained for 1 h with the mouse monoclonal antibody B12D5 that is specific for GP64 (1:50), rinsed twice using centrifugation, stained for 30 min with goat anti-mouse Ig (H + L)-FITC (Southern Biotech; 1:400), rinsed with PBS–BSA, fixed with 1% PFA dissolved in PBS and analyzed using a flow cytometer (Quanta SC MPL, Beckman Coulter). At least 20,000 events with a side scatter and electronic volume that is characteristic of hemocytes were analyzed for each treatment. Larvae inoculated with PBS, dissected at identical time points and stained for GP64 served as uninfected controls. 2.6. Statistical analysis Data were analyzed, graphs generated and regression formulae calculated using Prism 6 (GraphPad, Version 6.0b), SigmaPlot (version 11), Minitab (version 16.1), SAS (version 9.3) or Microsoft Excel 2011 for Mac (Version 14.1.3). All reported error values are standard errors of the means (SEM). For the AcMNPV OB and BV virulence data, we performed generalized linear model analyses using a logit link function and binomial distribution of the mortalities using Proc GENMOD (SAS v9.3). The models included log dose and species main effects and time species interaction effects. We determined estimates of LD50 doses using Proc PROBIT (SAS v9.3). Differences in mortality for the M2R study were determined using Chi-squared statistics (Minitab v16.1). Graphs of the results are presented with mortality probabilities in the logit scale. Intercept differences indicate differences in initial susceptibility while slope differences indicate differences in rate of change in susceptibility with increasing dosage. 3. Results and discussion 3.1. AcMNPV virulence in P. includens and T. ni larvae The virulence of AcMNPV-hsp70/lacZ OB was quantified in P. includens and T. ni by orally inoculating newly molted penultimate instars with increasing quantities of OB. Trichoplusia ni and P. includens are closely related; both belong to the Tribe Argyrogrammatini of the Subfamily Plusiinae in the order Noctuidae. Trichoplusia ni is considered to be permissive to AcMNPV because infections initiated by low dosages of orally inoculated OB rapidly disseminate within and kill this species (Washburn et al., 1995). There was a 2.5-fold difference in the susceptibility of P. includens and T. ni to orally inoculated AcMNPV OB (Fig. 1A; LD50 for P. includens was 40.8 OB [95% confidence intervals (CI): 36.55, 45.63] and 16.1 OB for T. ni [95% CI: 9.04, 21.91]). A significant difference in the Y-intercepts of the regression curves for P. includens and T. ni orally inoculated with AcMNPV-hsp70/lacZ OB was observed indicating that a higher dosage of OB was required to initiate a mortal infection in P. includens (Chi-square = 11.24, DF = 1, P = 0.008; Supplementary Table 1). The difference in the slope of the two regression curves was non-significant, suggesting that T. ni and P. includens were responding similarly to increased
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dosages of AcMNPV OB (Chi-square = 2.77, DF = 1, P = 0.0963; Supplementary Table 1). Thus, once infection was established, there was an equal likelihood in both species for the infection to result in mortality. The 2.5-fold difference in the LD50 values between the species and the significant difference in the Y-intercept for the regression indicate that a higher quantity of OB was required to establish an infection in P. includens, relative to T. ni. The moderate species effect between T. ni and P. includens that were orally inoculated with OB contrasts with Helicoverpa zea and Heliothis virescens, which are members of the same subfamily Heliothinae (Noctuidae), and display more than a 1000-fold difference in susceptibility to fatal infection by AcMNPV OB when assayed using the same methods employed in our study (Trudeau et al., 2001). This suggests that phylogenetic relationships between species may not be good predictors of susceptibility to a particular baculovirus species. The susceptibility of mid-penultimate instar P. includens and T. ni to systemic infection was quantified by intrahemocoelically (IH) inoculating larvae with increasing dosages of BV. Similar to OB inoculated larvae, P. includens were more resistant to fatal infection from IH-inoculated BV relative to T. ni (Fig. 1B; LD50 values were 13.8 PFU for P. includens [95% CI: 7.58, 26.71] and
Fig. 1. Virulence of AcMNPV-hsp70/lacZ in P. includens and T. ni. (A) Larval mortality of P. includens and T. ni when inoculated orally with increasing dosages of AcMNPVhsp70/lacZ OB at the onset of the penultimate instar (N = 30–32 insects per dosage). Solid lines represent regression lines for P. includens and dashed lines are for T. ni. The regression equations are: P. includens, Y = 6.9911 + 4.0212 log(X), R2 = 0.7253, LD50 = 40.8 OB; T. ni, Y = 4.4277 + 4.0212 log(X), R2 = 0.8947, LD50 = 16.1 OB. (B) Larval mortality of P. includens and T. ni when inoculated IH with increasing dosages of AcMNPV-hsp70/lacZ BV during the penultimate instar (N = 32 insects per dosage). Solid lines represent regression lines for P. includens and dashed lines are for T. ni. The regression equations are: P. includens, Y = 1.3704 + 1.0373(log X), R2 = 0.7142, LD50 = 13.8 PFU; T. ni, Y = 3.6733 + 1.8479 log(X), R2 = 0.8423, LD50 = 0.060 PFU.
0.060 PFU for T. ni [95% CI: 0.040, 0.073]). There were significant differences between the Y-intercepts (Chi-square = 365.12, DF = 1, P < 0.0001; Supplementary Table 1) and in the slopes of the regression curves for the two species that were IH inoculated with AcMNPV-hsp70/lacZ BV (Chi-square = 28.61, DF = 1, P < 0.0001; Supplementary Table 1). These differences suggest that molecular or cellular processes occurring in the hemocoel of P. includens reduced the susceptibility of this species to IH inoculated BV, relative to T. ni, across all dosages tested. Taken together, the relatively high LD50 values for P. includens inoculated with BV and the significant differences in the regression lines indicated that P. includens is significantly more resistant than T. ni to IH inoculated AcMNPV-hsp70/lacZ BV. Our results conflict with a study which reported that newly molted penultimate instar P. includens were 82-times more resistant to AcMNPV OB than what we report (LD50 of 3352 OB)(Kunimi et al., 1997). The difference in susceptibility of P. includens to fatal infection between our study and Kunimi et al. (1997) may be due to differences in the sources of P. includens or in methods used to inoculate the insects. Kunimi et al. (1997) used droplet feeding for inoculation and then weighed larvae before and after feeding to estimate the quantity of virus consumed. High LD50 values of 2500–9274 OB were also reported for field-collected isolates of P. includens SNPV (PsinSNPV) in third instar P. includens larvae that were inoculated by contaminating artificial diet with known quantities of OB (Alexandre et al., 2010). For our studies, insects were inoculated orally using a microapplicator with precise microliter volumes of an aqueous solution containing OB that was visually quantified using a hemocytometer. Because the quantity of OB inoculated into each insect was known, our method may provide a better measure for laboratory tests of AcMNPV virulence in P. includens. Although different methods were employed for inoculating P. includens with OB, AcMNPV appears to be 61- to 227-times more virulent than field-collected isolates of PsinSNPV in P. includens (Fig. 1A; (Alexandre et al., 2010). To quantify the impact of the developmental stage of the insect on virulence, P. includens were inoculated orally with 180 AcMNPVhsp70/lacZ OB at increasing times after molting to the fifth instar. Logit analysis with time as a continuous variable show there was a significant decrease in mortality as larvae were inoculated later in the fifth instar (Chi-square = 61.21, DF = 1, P < 0.0001). When orally inoculated immediately after molting, 180 OB generated 80% mortality (Fig. 2A). However, at 24 h post molt, the last developmental stage tested, only 25% of the inoculated larvae succumbed to the infection (Fig. 2A). These results demonstrate that the susceptibility to fatal infection decreased as the age at which the larvae were inoculated with AcMNPV OB was increased. Developmental resistance to baculovirus infection occurs in species that are permissive or resistant to orally administered baculovirus OB (Engelhard and Volkman, 1995). The developmental resistance of Lepidoptera larvae to oral infection by baculoviruses has been attributed to infection-induced apoptosis of midgut epithelial cells that can be inhibited by co-inoculating larvae with the optical brightener M2R (Dougherty et al., 2006; Silveira et al., 2005; Washburn et al., 1998). The extent that M2R could increase the virulence of AcMNPV-hsp70/lacZ OB in P. includens was quantified by orally inoculating newly molted fifth instar or larvae 16 h post molt (hereafter designated 50 or 516, respectively) with inocula containing 40 OB or 180 OB and M2R or vehicle. Addition of M2R to inocula increased the susceptibility of both 50- and 516inoculated larvae to fatal AcMNPV infection by 13% and 11%, respectively (Fig. 2B). However, the difference between M2R and control groups was non-significant (Chi-square = 0.007, DF = 1, P = 0.935). Studies using M2R co-inoculated orally with OB into mid-instar T. ni or H. virescens demonstrate that for these permissive species, M2R increases the mortality by 20–41% (Hoover et al.,
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3.2. Time course for orally inoculated OB To investigate the course of oral AcMNPV infection in P. includens, 50 larvae were inoculated orally with 60 OB of AcMNPVhsp70/lacZ and dissected 0.5–96 h post inoculation (h.p.i). LacZ+ cells were first detected at 4 h.p.i in 32% of the larvae and were restricted to the midgut epithelium (2.0 ± 0.5 midgut foci per LACZ+ insect; Fig. 3A and B). Tracheal infection was first observed at 8 h.p.i with 0.25 ± 0.2 midgut + tracheal foci per LacZ+ insect (Fig. 3A and B). The rapid establishment of multicellular foci of infection (midgut + tracheal foci) by 8 h.p.i demonstrates that AcMNPV can quickly traverse the basal lamina that supports the midgut epithelium. Because this transfer occurred in less time than is likely required for a complete cycle of virus replication in the infected midgut epithelial cells, this observation lends additional support to the hypothesis that ODV-derived nucleocapsids are repackaged in midgut cells as BV that is released to infect adjacent cells located in the hemocoel (Ohkawa et al., 2010; Washburn et al., 1999). LacZ+ foci consisting solely of tracheal cells were observed at 8 h.p.i, 4 h after the first signs of infection were observed (Fig. 3B), suggesting that an underlying infected midgut epithelial cell was eliminated within 4 h of being established. Rapid sloughing of infected midgut cells is a resistance pathway that limits transfer of the virus to tissues in the hemocoel and reduces the susceptibility of insects orally inoculated with OB to fatal infection (Washburn et al., 1995). While M2R blocks infected midgut cell sloughing (Dougherty et al., 2006), its inclusion in viral inocula did not significantly increase the susceptibility of P. includens to
Fig. 2. Developmental resistance of P. includens inoculated with AcMNPV-hsp70/ lacZ OB. (A) Mortality of fifth instar P. includens inoculated orally with 180 OB at increasing hours post molt (N = 32 insects per developmental stage). Dashed lines indicate 95% confidence intervals. (B) Larval mortality of 50 P. includens when inoculated orally with 40 OB and 516 P. includens when inoculated orally with 180 OB, in the presence or absence of 1% M2R (N = 45 insects per developmental stage and treatment; percent mortality for: 50 control was 60% and M2R was 73%; 516 control was 52% and M2R was 63.5%).
2000; Washburn et al., 1998; Zhang et al., 2004). In contrast, M2R increases mortality levels for newly molted penultimate instar T. ni or H. virescens inoculated orally with AcMNPV OB by 4–13% (Washburn et al., 1998; Zhang et al., 2004). Because the difference in mortality for mid-instar P. includens inoculated with AcMNPV OB and M2R or vehicle control was low and similar to what was observed for the newly molted instars of other permissive species (Washburn et al., 1998; Zhang et al., 2004), apoptosis-mediated infected midgut cell sloughing may not be an important driver of midgut-based resistance to AcMNPV in mid-instar P. includens. Alternatively, M2R may not inhibit AcMNPV-elicited apoptotic pathways in P. includens, as it does in other insect species. Consequently, including optical brighteners such as M2R into pest management strategies for biological control of P. includens with AcMNPV may not produce an increase in control that offset the added cost. However, inclusion of M2R in inocula did increase the virulence of PsinNPV OB in P. includens larvae by 15520-fold relative to controls lacking M2R (Zou and Young, 1996), suggesting that AcMNPV and PsiNPV infection may elicit different physiological responses in the midgut. Comparisons of the genome sequences of AcMNPV and PsinNPV may provide insights into reasons for the observed differences in virulence. However, the complete genome sequence of PsinNPV is currently not reported. The pathogenesis of orally administered AcMNPV-hsp70/lacZ OB in P. includens was subsequently evaluated using time course studies of infection using expression of the lacZ reporter gene as a molecular marker of viral infection in the tissues.
Fig. 3. Time-course of infection from 0.5–96 h.p.i for 50 P. includens larvae inoculated orally with 60 OB of AcMNPV-hsp70/lacz. (A) Proportion of LacZ+ larvae and tissue-specific distribution of LACZ+ cells within the midgut and trachea (95% CI were 41.5–87.7 for LACZ expression, 30.3–69.8 for midgut infection and 27.4–82.3 for tracheal infection). (B) Average number of LacZ+ midgut, midgut plus tracheal and tracheal only cells associated with the midgut per LacZ+ larva. The bioassay for this study produced 67% mortality (N = 60 insects). Positive error bars reflect SEM.
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orally administered AcMNPV OB (Fig. 2B), suggesting that the early loss of infected midgut epithelial cells may not be inhibited by M2R. The extent of midgut infection decreased from 8.1 ± 2.3 midgut foci per LacZ+ insect at 30 h.p.i to 1.7 ± 0.9 midgut foci per LacZ+ insect at 48 h.p.i (Fig. 3B), suggesting that infected midgut cells were eliminated as the insect progressed through the fifth instar. By 24 h.p.i, 88% of the larvae displayed a tracheal infection (Fig. 3A). Tracheal infection increased from 23.2 ± 10.7 foci per LacZ+ insect at 48 h.p.i to 40.6 ± 15.1 foci per LacZ+ insect at 72 h.p.i, with 100% of insects displayed tracheal infection (Fig. 3A and B). Because the time course of infection for AcMNPV in P. includens (this study) was similar to what was observed for studies of AcMNPV in T. ni (Washburn et al., 1995), there does not appear to be a physiological barrier that restricts the dissemination of AcMNPV in P. includens larvae. Studies of baculovirus pathogenesis in several species of Lepidoptera clearly demonstrate that cells of the midgut epithelium are the first cell type infected by ODV. However, there is no consensus on the identity of the second cell type that is infected to initiate a systemic infection. In pathogenesis studies of newly molted fourth instar H. virescens and H. zea inoculated orally with AcMNPV-hsp70/lacZ OB, infected hemocytes were observed 8– 10 h after the first infected tracheolar cell (Trudeau et al., 2001). A similar pattern of tracheal followed by hemocyte infection was observed in newly molted fourth instar Lymantria dispar larvae orally inoculated with L. dispar MNPV OB (McNeil et al., 2010). However, studies of T. ni exposed to diet coated with cultured Sf9 cells infected with a GFP-expressing recombinant of AcMNPV demonstrated that tracheal tissues became infected 24 h after hemocytes (Barrett et al., 1998). Other studies of larvae orally inoculated with baculovirus OB demonstrate simultaneous infection of tracheolar cells and hemocytes (Matos et al. 1999; Chikhalya et al., 2009; Katsuma et al., 2008). To study the transfer of infection from the midgut to cells in the hemocoel, 50 P. includens were inoculated orally with 60 OB, dissected and the hemolymph was removed from insects in the hours surrounding the onset of tracheal infection. While systemic tracheal infection was evident at 14 h.p.i in 46% of the larvae, hemocytes displayed first LacZ signals at 16 h.p.i in 38% of the inoculated insects (6.7 ± 3.2 LacZ+ hemocytes per LacZ+ insect Fig. 4A and B). Thus, for P. includens orally inoculated with low quantities of AcMNPV OB, infection begins in the midgut epithelium and is transferred first to adjacent tracheal epidermal cells and later to hemocytes. BV was first detected in the hemolymph at 18 h.p.i (Fig. 4C), 2 h after infected hemocytes were first observed (Fig. 4A). The time needed for BV-infected cells in culture to release BV is approximately 12 h (Hodgson et al., 2007; Nie and Theilmann, 2010). Therefore, it is unlikely that hemocytes generated the BV that was first detected in the hemolymph. However, the time between the observed first tracheal and hemocyte infections may be sufficient for a cycle of virus replication, suggesting that infected tracheal cells may have been responsible for generating the BV that was first observed in the hemolymph. The observed increased in BV titer from 18 to 24 h.p.i (Fig. 4C) correlated with an increase in the mean number of foci per LacZ+ insect (Fig. 4B), demonstrating that the virus was amplifying and disseminating within infected insects. At 20 h.p.i, the mean number of midgut foci per LacZ+ insect increased 6.3-fold and at 22 h.p.i returned to levels similar to what was observed at 18 h.p.i This observed increase in the number of midgut foci at 20 h.p.i may have resulted from variability in the experiment (95% CI overlapped for the 18– 22 h.p.i time points). The observed loss of AcMNPV-infected midgut epithelial cells between 24 and 72 h.p.i (Fig. 4A and B), which permits infected insects to continue feeding and accumulate biomass, is consistent with what has been observed for T. ni, H. virescens, H. zea, Spodoptera frugiperda and Manduca sexta that were orally inoculated with AcMNPV OB (Haas-Stapleton et al.,
Fig. 4. Time-course of infection from 14–72 h.p.i for 50 P. includens larvae inoculated orally with 60 OB of AcMNPV-hsp70/lacZ. (A) Proportion of LacZ+ larvae and tissue-specific distribution of LacZ+ cells within the midgut, trachea and hemocytes (95% CI were 69.5–102.5 for LacZ expression, 25.7–67.0 for midgut infection, 51.7–85.0 for tracheal infection and 31.2–100.3 for hemocyte infection). (B) Average number of LacZ+ midgut, midgut plus tracheal, tracheal only and hemocyte cells per LacZ+ larva. To reduce overlap of symbols at each time point, some symbols are offset by up to 0.4 units along the X-axis. (C) BV titer within P. includens inoculated orally with 60 OB of AcMNPV-hsp70/lacZ as newly molted fifth instars (N = 23–24 larvae per time-point). Positive error bars reflect SEM.
2003; Trudeau et al., 2001; Washburn et al., 2000; Washburn et al., 1995). The proportion of hemocytes infected with AcMNPV-hsp70/lacZ was further assessed using flow cytometry to detect cell-surface expression of the BV protein GP64. At 12 h.p.i, 6.18% of hemocytes isolated from P. includens larvae displayed cell surface expression of GP64 (Fig. 5A) and increased to 84.5% at 18 h.p.i (Fig. 5B), which
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Fig. 5. GP64 expression on hemocytes isolated from larvae orally inoculated with 60 OB or PBS. Hemocytes isolated from P. includens inoculated with OB (filled histograms) or PBS (open histograms) at 12 and 18 h.p.i (A and B) or T. ni at 18 h.p.i (C) were stained to detect cell-surface expression of GP64 and analyzed using flow cytometry. Values in the upper right corner of each histogram represent the proportion of hemocytes isolated from larvae inoculated with OB that were gated positive for aGP64-FITC antibody staining. Data are from one experiment, representative of three.
is similar to what was observed for similarly treated T. ni larvae at 18 h.p.i (Fig. 5C). A relatively low number of LacZ+ hemocytes were observed in 10 ll of hemolymph isolated at 18 h.p.i from P. includens larvae (Fig. 4B). However, 84.5% of the hemocytes at the same time point displayed GP64 expression (Fig. 5B). This suggests that use of flow cytometry to quantify GP64 expression may be an improvement over assessing LacZ expression using light microscopy to identify AcMNPV-infected cells. Together, our results demonstrate that for P. includens inoculated orally with AcMNPV-hsp70/lacZ OB, the infection progressed from the midgut to the trachea then, finally, to the hemocytes (Figs. 3B, 4B). Because infectious BV was detected in the hemolymph after infected midgut cells, tracheal cells and hemocytes were observed (18 h.p.i; 3.9 103 ± 8.2 103 PFU/mL; Fig. 4C), we cannot conclusively determine which of these cell types were responsible for releasing high quantities of BV into the hemocoel. However, the observation of BV in the hemolymph after detecting infected hemocytes is in agreement with studies of AcMNPV pathogenesis in H. virescens and H. zea (Trudeau et al., 2001). Because the BV titer in AcMNPV-infected P. includens increased from 18 to 24 h.p.i (Fig. 4C), this insect species does not appear to display substantial resistance to systemic AcMNPV infection. In conclusion, our studies demonstrate that P. includens is permissive to fatal infection by low dosages of orally administered AcMNPV OB (LD50 = 40.8 OB). While field-collected isolates of PsinSNPV OB are reported to cause fatal infections in P. includens, the reported LD50 values for this baculovirus is 61- to 227-fold higher than what we report for AcMNPV (LD50 of PsinSNPV = 2500–9274 OB; (Alexandre et al., 2010)). Inclusion of the optical brightener M2R into PsinNPV inocula resulted in a substantial increase in virulence (Zou and Young, 1996). However, enhancing the potency of a biological pesticide with synthetic chemicals is considered by many to be unacceptable for organic farming. Our studies demonstrate that AcMNPV is highly virulent to P. includens in the absence of M2R. Controlled tests of AcMNPV and PsinSNPV virulence for P. includens in an experimental setting that more closely reflects those of soybean in a field will provide more evidence for which baculovirus species may best limit economic damage to organic soybean crops. Our virulence studies of AcMNPV-hsp70/lacZ show a substantial and significant difference in the slope of the regression curves for BV-inoculated larvae
(Fig. 1B) that was not observed for larvae orally inoculated with OB (Fig. 1A). This suggests that the infection kinetics resulting from orally administered OB differs from IH inoculated BV and may point to systemic resistance pathways that are suppressed in P. includens when the virus establishes an infection via the natural midgut route of infection. Acknowledgments We thank Dr. Michael R. Strand and Dr. Jena Johnson at the University of Georgia for kindly providing P. includens eggs and Dr. Loy Volkman at UC Berkeley (now Emeritus) for providing the B12D5 antibody. We also thank the peer-reviewers of the manuscript and Dr. Jan Washburn (UC Berkeley, Emeritus) for suggestions that substantially improved the data presentation and statistical analysis. We would like to acknowledge Maggie Carrera, Alisa de la Cruz and Tiffany Chen for technical assistance. This work was funded by a grant from the US Department of Agriculture (CREES 200803390) to E.J.H-S. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocontrol.2013. 01.002. References Alexandre, T.M., Ribeiro, Z.M., Craveiro, S.R., Cunha, F., Fonseca, I.C., Moscardi, F., Castro, M.E., 2010. Evaluation of seven viral isolates as potential biocontrol agents against Pseudoplusia includens (Lepidoptera: Noctuidae) caterpillars. Journal of Invertebrate Pathology 105, 98–104. Barrett, J.W., Brownwright, A.J., Primavera, M.J., Palli, S.R., 1998. Studies of the nucleopolyhedrovirus infection process in insects by using the green fluorescence protein as a reporter. Journal of Virology 72, 3377–3382. Bonning, B.C., Hoover, K., Booth, T.F., Duffey, S., Hammock, B.D., 2005. Development of a recombinant baculovirus expressing a modified juvenile hormone esterase with potential for insect control. Archives of Insect Biochemistry and Physiology 30, 177–194. Chikhalya, A., Luu, D.D., Carrera, M., De La Cruz, A., Torres, M., Martinez, E.N., Chen, T., Stephens, K.D., Haas-Stapleton, E.J., 2009. Pathogenesis of Autographa californica multiple nucleopolyhedrovirus in fifth-instar Anticarsia gemmatalis larvae. The Journal of General Virology 90, 2023–2032.
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