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. 2015 Mar 26;81(8):2893–2899. doi: 10.1128/AEM.03726-14

Mortality of Cutworm Larvae Is Not Enhanced by Agrotis segetum Granulovirus and Agrotis segetum Nucleopolyhedrovirus B Coinfection Relative to Single Infection by Either Virus

Jörg T Wennmann 1, Tim Köhler 1,*, Gianpiero Gueli Alletti 1, Johannes A Jehle 1,✉
Editor: K E Wommack
PMCID: PMC4375340  PMID: 25681187

Abstract

Mixed infections of insect larvae with different baculoviruses are occasionally found. They are of interest from an evolutionary as well as from a practical point of view when baculoviruses are applied as biocontrol agents. Here, we report mixed-infection studies of neonate larvae of the common cutworm, Agrotis segetum, with two baculoviruses, Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV). By applying quantitative PCR (qPCR) analysis, coinfections of individual larvae were demonstrated, and occlusion body (OB) production within singly infected and coinfected larvae was determined in individual larvae. Mixtures of viruses did not lead to changes in mortality rates compared with rates of single-virus treatments, indicating an independent action within host larvae under our experimental conditions. AgseNPV-B-infected larvae showed an increase in OB production during 2 weeks of infection, whereas the number of AgseGV OBs did not change from the first week to the second week. Fewer OBs of both viruses were produced in coinfections than in singly infected larvae, suggesting a competition of the two viruses for larval resources. Hence, no functional or economic advantage could be inferred from larval mortality and OB production from mixed infections of A. segetum larvae with AgseNPV-B and AgseGV.

INTRODUCTION

A large number of baculoviruses isolated from the larvae of species of the insect orders Lepidoptera, Diptera, and Hymenoptera have been described (1). According to their occlusion body (OB) morphology, baculoviruses are distinguished into two morphological groups: the granuloviruses (GVs) with a single virion in an ovocylindrical OB (granule) and the nucleopolyhedroviruses (NPVs) with a few to many virions in a polyhedral OB (polyhedron) (2). Taxonomically, the Baculoviridae family is subdivided into four genera, the Alpha-, Beta-, Gamma-, and Deltabaculovirus genera (2, 3). Alpha- and betabaculoviruses comprise lepidopteran-specific NPVs and GVs, respectively. Many lepidopteran species are susceptible to baculoviruses from different species. Even simultaneous infections, so-called coinfections or mixed infections, of two NPVs (4), two GVs (5), and NPVs and GVs (6, 7) have been observed.

Larvae of the turnip moth, Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae), so-called common cutworms, are important agricultural pests (8). Two different alphabaculovirus isolates, Agrotis segetum nucleopolyhedrovirus A (AgseNPV-A; also called the Polish isolate) and A. segetum nucleopolyhedrovirus B (AgseNPV-B; also termed the Oxford isolate) (9–11), as well as the betabaculovirus Agrotis segetum granulovirus (AgseGV) (12), were isolated and characterized from A. segetum larvae. AgseGV was tested extensively as a biocontrol agent for the control of A. segetum in the field (13–16), and AgseNPV-B has shown its potential as a biocontrol agent under laboratory conditions (13).

Both viruses were found to infect A. segetum larvae simultaneously (17), but little is known about possible interaction in coinfections. Shvetsova and Ts'ai (17) reported an increased mortality when AgseGV and an Agrotis nucleopolyhedrovirus were simultaneously provided to larvae of Apamea anceps, and it was concluded from mortality data that these viruses did not show antagonistic behavior. In addition, both viruses were shown histologically to infect the same insect tissues of A. segetum larvae (17). However, in that study, it was not distinguished whether the virus isolate used was AgseNPV-A or AgseNPV-B, since this nomenclature was later introduced (10). It is not known if there is a reciprocal influence of AgseGV and AgseNPV-B in coinfected larvae and, if so, which kind of effect occurs and if this effect is of a functional and economic benefit when used as a pest control agent. Both enhancing and inhibiting effects within common host larvae have been described for GV and NPV coinfections (18, 19). The enhancing effects were first observed for Pseudaletia unipuncta granulovirus (PsunGV) and Pseudaletia unipuncta nucleopolyhedrovirus (PsunNPV). In that study, the presence of a synergistic or enhancing factor associated with the occlusion body increased the susceptibility of P. unipuncta larvae to PsunNPV (18). A contrary effect was observed for larvae of Helicoverpa armigera simultaneously treated with Helicoverpa (Heliothis) armigera nucleopolyhedrovirus (HearNPV) and Helicoverpa armigera granulovirus (HearGV) (19). The overall mortality decreased, and HearGV appeared to suppress HearNPV infection (19). A similar observation was made for larvae of Heliothis zea infected with slow-acting HearGV and fast-killing HearSNPV (6). When both viruses were fed to larvae at the same time, both viruses appeared to compete for larval resources. On the contrary, HearSNPV-infected larvae that were posttreated with HearGV appeared to outcompete already established infections of the NPV. More recently, an AgseNPV-B virus stock containing AgseGV revealed covert coinfections of AgseGV in virus propagations in A. segetum larvae (20). Within the same study, a PCR-based method for the identification and quantitation of Agrotis baculoviruses was established. Since a mixture of AgseGV and AgseNPV-B could be both beneficial and disadvantageous for one or both viruses, information about coinfections is considered important for a possible improvement of their application in the field.

This study was carried out to investigate the changes on mortality of A. segetum larvae treated with AgseGV and AgseNPV-B in mixed infections. Quantitative PCR (qPCR) was applied to determine the OB production of AgseGV and AgseNPV-B at the level of individual coinfected larvae to describe the effect of coinfection on progeny OB production.

MATERIALS AND METHODS

Insects and viruses.

Rearing of A. segetum was performed at the Institute for Biological Control in Darmstadt (Julius Kühn Institute [JKI]) as described previously (20). Neonate A. segetum larvae were used in all infection experiments. Stocks of Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV), which originated from the virus collection of Horticulture Research International (HRI) in Warwick, United Kingdom, were propagated in the fourth instar of A. segetum.

Bioassay analysis.

Infection studies were performed for AgseNPV-B and AgseGV in neonate A. segetum larvae feeding on different virus concentrations incorporated in a semiartificial diet. For AgseNPV-B, the final virus concentrations were 103, 3.1 × 103, 104, 3.1 × 104, 105, 3.1 × 105, and 106 OBs/ml. The bioassay for AgseGV was performed at final concentrations of 102, 103, 104, 105, 106, 107, and 108 OBs/ml. Enumeration of virus OBs was performed by hemocytometer counting as described previously (21). The semiartificial diet (modified according to references 20 and 22) was mixed with OBs to their final concentrations prior to solidification at a semifluid state (40°C). The diet of the untreated control contained no virus. Diets were filled in every well of a 50-well bioassay tray (LICEFA, Bad Salzuflen, Germany). Bioassay trays were kept open overnight to allow evaporation of excess humidity. On the following day, 50 neonate larvae per virus concentration (i.e., treatment) were individually placed in each well of a bioassay tray. One hundred larvae were used for the untreated control. After the bioassay trays were closed, they were kept at 22°C with a 16/8-h light/dark photoperiod. Mortality was scored on days 1, 7, and 14 postinfection (p.i.). Larvae that died within the first 24 h of the assay were assumed to have died from handling and were not included in the analyses. Mortality data were corrected according to Abbott (23). Median and 10% lethal concentrations (LC50 and LC10, respectively) were calculated for day 7 and day 14 for each virus by probit analysis using ToxRat software (ToxRat Solutions, Alsdorf, Germany). The parallelism of probit lines on days 7 and 14 p.i. was tested with potency estimation as provided by ToxRat software (ToxRat Solutions, Alsdorf, Germany).

Setup of mixed-infection experiments.

Neonate A. segetum larvae were individually exposed to AgseGV and AgseNPV-B in different concentrations and combinations. Two final OB concentrations of AgseNPV-B were used in this experiment: a low (NPVL = 0.8 × 103 OBs/ml) and a high (NPVH = 37 × 103 OBs/ml) concentration. AgseGV was used in a single final concentration (GV = 900 × 103 OBs/ml). The treatments were set up as follows: (i) GV, (ii) NPVL, (iii) NPVH, (iv) GV-NPVL, (v) GV-NPVH, and (vi) untreated control. The preparation of bioassay trays and diet containing the different combinations of virus concentrations was done as described for bioassay analysis. Twenty-five neonate A. segetum larvae were added to each virus and mixed-virus treatment, whereas the control contained 50 neonate larvae. The larvae mortality was scored and analyzed separately for the first (days 2 to 7) and second (days 8 to 14) weeks. Cadavers that died by viral infection were collected entirely, without contaminating the sample with artificial diet. Samples were stored individually at −20°C for further analysis. Each infection experiment was repeated independently six times. The larval mortality of each infection experiment was corrected according to Abbott (23). A test for significant variation in the mean mortality of treatments was performed by one-way analysis of variance (ANOVA) followed by Tukey's test for pairwise comparisons between treatments in RStudio (version 0.97.551) statistical software.

Viral DNA preparation.

Viral DNA was purified separately from individual larvae. Frozen larval remains of mixed-infection experiments were prepared for OB purification by thawing and centrifugation at 13,000 × g for 30 s without additional buffers. By this initial step, the integrity of most infected larval bodies was broken up. Larval tissues that remained intact were homogenized within the tubes using minipestles followed by an additional centrifugation step. Liquid supernatants were discarded, and the pellets, composed of cell debris and OBs, were subjected to DNA isolation using Ron's tissue DNA minikit (Bioron, Ludwigshafen, Germany). In short, disintegrated larvae were resuspended in 250 μl tissue lysis buffer and treated with proteinase K (250 μg/ml) (Thermo Fisher Scientific, Waltham, MA, USA). Samples were incubated overnight at 52°C. Subsequently, 30 μl of 1 M Na2CO3 was added to the sample to dissolve OBs under alkaline conditions for 30 min. Samples were neutralized by adding the same volume of 1 M HCl. Larval debris was pelleted at 12,000 × g for 1 min, and the brownish, cloudy supernatant was transferred to a fresh centrifugation tube. Herring sperm DNA (3 μg) was added to the samples in order to saturate the kit's silica membrane in the later course of the DNA isolation protocol. DNA solutions were processed according to the kit's protocol and eventually eluted from the membrane by adding 400 μl of 10 mM Tris-EDTA (TE) buffer. For reasons of standardization and reproducibility, all steps of viral DNA extraction and isolation were adjusted to previously described protocols for qPCR standard sample generation (20).

Quantitation of larval OB production.

Quantitation of AgseNPV-B and AgseGV OBs of single larvae was performed by qPCR (20). This technique allowed standardized quantitation of OBs produced by even small larval cadavers. To ensure the reliability of the experiment, a minimum of five single larvae was used to quantify median OB production in singly infected and coinfected larvae per treatment. Therefore, quantitative analyses on singly infected larvae were performed for the GV and GV-NPVL treatments for AgseGV as well as the NPVH and GV-NPVH treatments for AgseNPV-B. OB production in coinfections was analyzed from larvae from the GV-NPVH treatment. Quantitation standards for qPCR and serial 10-fold dilutions of OBs of AgseNPV-B (in the range of 109 to 104 OBs/ml) and AgseGV (1011 to 106 OBs/ml) in triplicate were prepared. DNA was isolated from all dilution steps as previously described (20). The lower limit of determination (LOD) for AgseGV and AgseNPV-B quantitation was 105 OBs of AgseGV and 103 OBs of AgseNPV-B. For a single qPCR, 2 μl of standard or sample DNA was mixed with 1 μl 0.2 pM of each forward and reverse primer (prAsBpolh-f/prAsBpolh-r and prAsGVgran-f/prAsGVgran-f, respectively), 12.5 μl 1× Maxima SYBR green/Rox qPCR master mix (Thermo Fisher Scientific, Waltham, MA, USA), and 8.5 μl double-distilled water (ddH2O) to a total reaction volume of 25 μl (20). The nontarget controls contained 2 μl ddH2O instead of DNA sample. Each larval DNA sample was tested twice for AgseNPV-B and AgseGV in separate qPCR runs along with their corresponding standard samples. Each run included nontarget controls. qPCR parameters and melting curve settings were adjusted as previously described (20). Data were processed with Bio-Rad CFX Manager 2.0 (Bio-Rad, Hercules, CA, USA). The quantification cycle (Cq) values were obtained by single threshold analysis. Statistical differences in OB production between treatments in singly infected and coinfected larvae were revealed by a Kruskal-Wallis test. Comparisons between groups were shown by a pairwise Wilcoxon rank sum test and P value adjustment according to the Holm-Bonferroni method (24). Statistical analyses were performed by using RStudio (version 0.97.551) software.

RESULTS

Biological activity.

Bioassay analyses were performed to compare the virulence of AgseGV and AgseNPV-B to neonate A. segetum larvae. On day 7 p.i., the LC10 and LC50 of AgseGV could not be calculated due to low mortality rates (Table 1). After 14 days, the LC10 of AgseGV was 2.63 × 103 OBs/ml and the LC50 was 8.33 × 105 OBs/ml. The 14-day LC50 of AgseGV was 254 times higher than the LC50 of AgseNPV-B, at 3.28 × 103 OBs/ml. The higher virulence of AgseNPV-B was also reflected by the LC10 (7.7 × 102 OBs/ml) and LC50 (3.70 × 104 OBs/ml) on day 7 p.i. and LC10 (3.4 × 102 OBs/ml) on day 14 p.i. (Table 1).

TABLE 1.

Lethal concentration values for AgseNPV-B, AgseGV, and AgipNPV determined for neonate A. segetum larvaeb

Virus No. of individuals per bioassay Day 7 p.i.
 Day 15 p.i.
LC10 (95% CL) (×103 OBs/ml) LC50 (95% CL) (×103 OBs/ml) Slopea χ2 df LC10 (95% CL) (×103 OBs/ml) LC50 (95% CL) (×103 OBs/ml) Slopea χ2 df
AgseGV 3,054 ND ND 0.30 A 111.7 5 2.63 (0.05–128.0) 832.7 (80.9–8571.0) 0.51 A 212.5 5
AgseNPV-B 1,256 0.77 (0.20–4,212) 37.0 (19.4–70.6) 0.76 B 18.39 5 0.34 (0.22–0.52) 3.28 (2.66–4.04) 1.30 B 9.56 5
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a

Different letters indicate no parallelism of probit lines at either day 7 or day 14. The criterion of parallelism was rejected when P values were <0.05.

b

Mortality rates were scored on days 7 and 14 postinfection. CL, confidence limits calculated by normal approximation; df, degrees of freedom; ND, not defined due to low mortality rates of AgseGV within the first week.

Mortality of mixed infections.

Single and mixed infections of A. segetum larvae were performed with a single concentration of 9.0 × 105 OBs/ml for AgseGV, which equaled an LC50 on day 14 p.i., and two concentrations of AgseNPV-B, NPVL at 8.0 × 102 OBs/ml (LC10 on day 7 p.i.) and NPVH at 3.7 × 104 OBs/ml (LC50 on day 7 p.i.) (Table 1). The differences between the mortality results of mixed-virus experiments were analyzed separately for days 7 and 14 p.i. by one-way analysis of variance (ANOVA). The differences between the treatments were scored by post hoc Tukey's test (level of significance, P < 0.05). On day 7 p.i., significant differences between the treatments were observed [one-way ANOVA, F(4,25) = 11.13, P < 0.001]. The lowest mortality was observed for the GV treatment (6.17% ± 4.97%) and the NPVL treatment (3.11% ± 3.97%). Within the first week (days 2 to 7), only a few larvae died and no significant difference between GV and NPVL treatment was observed (P = 0.934) (Fig. 1A). The highest mean mortality of single-virus treatments was found for NPVH (22.7% ± 9.07%), where the mortality was statistically different from that of GV (P = 0.002) and NPVL (P < 0.001) treatment.

FIG 1.

FIG 1

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Mean mortality rates of the first week (days 2 to 7) (A) and the second week (days 8 to 14) (B) of single (GV, NPVL, and NPVH)- and mixed (GV-NPVL and GV-NPVH)-virus treatments. Different letters above the columns indicate significant differences between treatments (one-way analysis of variance [ANOVA] followed by Tukey's honest significant difference [HSD] test for pairwise comparisons between treatments, significance at P values of <0.05). The vertical lines represent standard deviations. Pie charts above each column represent the ratio of noninfected (white), coinfected (black), AgseGV only-infected (light gray), and AgseNPV-B only-infected (dark gray) larvae from each treatment (n = number of larvae measured in qPCR).

In all single-virus treatments, the mortality increased from the first week to the second week (Fig. 1B), where significant differences were detected [one-way ANOVA, F(4,25) = 14.21, P < 0.001]. On day 14 p.i., NPVL treatment exhibited the lowest increase in mortality (5.87% ± 5.37%), as the fewest additional larvae had died within the second week. The NPVL mortality was statistically different from that of GV (36.8% ± 11.3%) (P = 0.029) and NPVH (60.1% ± 20.6%) (P < 0.001) (Fig. 1B).

The mortality of the GV-NPVL treatment was 9.50% ± 8.97% and 50.1% ± 22.6% after the first and second weeks, respectively, but did not differ statistically from the mortality found in the GV treatment of the first (P = 0.913) and second (P = 0.648) weeks. The GV-NPVH treatment caused a mortality of 22.6% ± 5.25% and 73.7% ± 17.8% after the first and second weeks, respectively, which was similar to the results of the NPVH treatments (P = 0.999 and P = 0.632) (Fig. 1A and B). Thus, both mixed-virus treatments equaled either the mortality caused by AgseGV (GV) or AgseNPV-B (NPVH/NPVL) treatment that induced the higher mortality when provided to larvae alone.

Based on the observed mortality of mixed-virus treatments, the hypothesis was tested that AgseGV and AgseNPV-B exhibit an additive effect in the mortality on A. segetum larvae. For testing, the survival rates (1 − mortality rate) of the GV and NPVL treatments, as well as the survival rates of GV and NPVH treatments of each replicate, were multiplied. The products were retransformed to mortality rates (expected mortality = 1 − survival rate) and compared with those of their corresponding mixed-virus treatments (i.e., observed mortality rates) by Student's t test. This was performed separately for the mortality observed in the first (day 7 p.i.) (Fig. 2A) and second (day 14 p.i.) weeks (Fig. 2B). On day 7 p.i., observed and expected values did not significantly differ for GV-NPVL (t = 0.084, df = 10, P = 0.0935) and GV-NPVH (t = 1.115, df = 10, P = 0.291) treatments (Fig. 2A). On day 14 p.i., no significant differences were found for GV-NPVL (t = 0.941, df = 10, P = 0.369) and GV-NPVH (t = 0.244, df = 10, P = 0.812) (Fig. 2B). This indicated that AgseGV and AgseNPV-B acted independently within A. segetum larvae.

FIG 2.

FIG 2

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Comparison of the observed and expected mortality rates of GV-NPVL and GV-NPVH treatments observed in the first (A) and second (B) weeks postinfection. Expected mortality values were calculated from survival rates of replicates of GV, NPVL, and NPVH treatments, assuming independent action (for details, see Results). Statistical analyses were conducted separately for the observed and expected mortality of each treatment. Different letters indicate significant differences (Student's t test, significance at P values of <0.05). Vertical lines represent standard deviations.

Ratio of GV- and NPV-infected larvae.

Though no effect of coinfection was observed in terms of mortality, a possible influence on the virus progeny production (i.e., OB production) was tested. Larvae exposed to AgseGV produced only AgseGV, proving the purity of the AgseGV stock (Fig. 1A and B).

In NPVL, where only four larvae of the first week were subjected to qPCR analysis, two cadavers contained AgseNPV-B, and two were virus free (Fig. 1A). A single larval cadaver contained both AgseNPV-B and AgseGV when collected from the NPVL treatment (second week) (Fig. 1B). The latter finding was unexpected, as only AgseNPV-B was used as an inoculum. In NPVH-only treatments, the occurrence of coinfections with both AgseNPV-B and AgseGV was also confirmed. Four (14.3%) larval cadavers collected in the first week (Fig. 1A) and two (20%) larval cadavers obtained in the second week (Fig. 1B) contained both AgseNPV-B and AgseGV. The remaining larvae that were collected from NPVH and died by viral infection contained AgseNPV-B only. Larvae with only AgseGV were not observed in NPVL and NPVH treatments (Fig. 1A and B).

Coinfected larvae were found in both GV-NPVL and GV-NPVH mixed-virus treatments. The ratio of coinfected larvae in GV-NPVL reached 18.8% and 14.3% within the first (Fig. 1A) and second (Fig. 1B) weeks, respectively. By this, the ratio of coinfected larvae was similar to that of NPVH treatment. In the GV-NPVH treatment, the ratio of coinfected larvae reached 52.9% for larvae that died within the first week (Fig. 1A). Cadavers that were not scored as coinfected were found to contain only AgseNPV-B (35.3%) or AgseGV (5.9%) (Fig. 1A). Within the second week of the GV-NPVH treatment, a mixed infection occurred in 76.2% of dead larvae, whereas 23.8% of the dead larvae were infected by AgseNPV-B only (Fig. 1B). According to the qPCR-based ratios, all larvae from the GV and GV-NPVL treatments that died by viral infection were infected by AgseGV, in single infections as well as coinfections (Fig. 1A and B). The contrary was observed for GV-NPVH treatment, where most larval cadavers contained AgseNPV-B only (Fig. 1A and B).

Larval occlusion body production.

The larval OB production levels in single infections and coinfections per treatment were separately analyzed for AgseGV and AgseNPV-B. Since the logarithmically transformed amounts of larval OB production in single infections and coinfections were not normally distributed (data not shown), nonparametric Kruskal-Wallis analysis was conducted. Significant differences in singly infected and coinfected larvae were detected for AgseGV [H(5) = 35.80, P < 0.001] and AgseNPV-B [H(5) = 31.35, P < 0.001].

In AgseNPV-B-infected larvae from the NPVH and GV-NPVH treatments, a temporal change in the median OB generation was observed from the first to the second weeks (Fig. 3A). The dead larvae of the first week showed no significant difference in progeny OB production (W [Wilcoxon test value] = 94, P = 0.896), with 2.3 × 106 OBs/larva (NPVH, singly infected), 5.44 × 107 OBs/larva (GV-NPVH, singly infected), and 1.6 × 106 OBs/larva (GV-NPVH, coinfected). At a higher level, the same observation was made for OB production in the second week (W = 17, P = 1.000). Here, amounts of 5.44 × 107 OBs/larva (NPVH, singly infected), 8.97 × 107 OBs/larva (GV-NPVH, singly infected), and 1.09 × 107 OBs/larva (GV-NPVH, coinfected) were measured. The temporal increase in progeny OB production was significant for singly infected larvae from the NPVH and GV-NPVH treatments but not coinfected larvae from the GV-NPVH treatment.

FIG 3.

FIG 3

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Median OB production of AgseNPV-B (A) and AgseGV (B) within singly infected and coinfected larvae, which succumbed during the first (days 2 to 7) and second (days 8 to 14) weeks postinfection. The minimums of the y axes represent the lower limits of detection (LOD) for AgseGV (105 OBs/larva) and AgseNPV-B (103 OBs/larva). Different letters indicate significant differences (Wilcoxon rank sum test, significance at P values of <0.05) between treatments.

A different situation in larval OB production was measured for AgseGV. Here, a temporal change in the amount of produced OBs from the first to the second weeks was absent (Fig. 3B). Singly infected larvae contained 2.89 × 109 OBs/larva (GV, first week), 1.47 × 109 OBs/larva (GV, second week), 4.66 × 108 OBs/larva (GV-NPVL, first week), and 1.64 × 109 OBs/larva (GV-NPVL, second week). Only coinfected GV-NPVH larvae produced significantly fewer amounts of OBs, with 2.36 × 108 OBs/larva (first week) and 1.61 × 108 OBs/larva (second week). In pairwise comparison, the produced amounts of AgseGV OBs in coinfected GV-NPVL larvae were significantly lower than those in single infections of the GV-NPVL (second week) and GV treatments (Fig. 3B) (P < 0.05).

Ratio of infection on the individual level.

As AgseNPV-B infection appeared to cause an adverse effect on the AgseGV production in coinfected larvae (Fig. 3B), we tested whether this effect was also seen in individual larvae. For this reason, the individual production of AgseNPV-B and AgseGV OBs was correlated in coinfections (Fig. 4A and B). When applying Spearman's rank correlation, the coefficient (rs) was negative (rs < 0), but no significant correlation was observed in OB production for larvae that died within the first [rs (df = 16) = −0.329; P = 0.182] and second [rs (df = 14) = −0.244; P = 0.361] weeks.

FIG 4.

FIG 4

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Correlation analysis (Spearman's rank correlation coefficient [rs]) of larval AgseGV and AgseNPV-B OB production within coinfected A. segetum larvae. Only larvae from the GV-NPVH treatment that died within the first (A) and second (B) weeks were considered. Vertical lines, median AgseGV OB production; horizontal lines, median AgseNPV-B OB production; solid lines, mixed GV-NPVH treatment; dashed lines, single GV and NPVH treatments are drawn as a reference.

DISCUSSION

Personal observations in our laboratory and published results on simultaneous infections of A. segetum larvae by AgseGV and AgseNPV (17) (G. Gürlich and J. Huber, personal communication) provided little information about potential interactions of both viruses. In the present study, mortality data were evaluated from single- and mixed-virus treatments and qPCR analyses were performed to identify and to quantify the production of AgseGV and AgseNPV-B OB progeny in infected larvae on an individual level.

The mortality rates of AgseNPV-B bioassay experiments were consistent with previously published bioassay studies obtained in 10-day bioassays (26). With an LC50 (day 10 p.i.) of 10 × 103 OBs/ml, the result lay between the newly determined LC50 (on days 7 and 14 p.i.). The AgseGV isolate used in the present study showed the same slow activity as previously described (13, 27).

The present study focused primarily on coinfections with defined concentrations of AgseGV and AgseNPV-B OBs. The observed mortality data indicate that the virus, which led to the highest mortality in its corresponding single-virus treatment, also dominated the overall mortality in an AgseGV and AgseNPV-B coinfection. Neither a significant increase nor a decrease in mortality was observed in mixed-virus treatments that could give hints for a cooperative or inhibiting effect of both viruses. The assumption of an independent virus infection was underlined by the comparison of the observed and expected mortality of mixed-virus treatments. The qPCR-based identification and quantitation of produced viruses of singly infected and coinfected larvae revealed that larvae died mainly from the virus that was applied in the higher lethal concentration (LC). When the 14-day LC50 of AgseGV was mixed with the high concentration of AgseNPV-B (7-day LC50), as done in the GV-NPVH treatment, the ratio of coinfected larvae was at a maximum. Furthermore, it could be concluded that with an increasing concentration of AgseNPV-B (GV > GV-NPVL > GV-NPVH), the ratio of coinfected larvae increased. A GV-induced enhancement of the AgseNPV-B infection, as it was described for PsunGV and PsunNPV (7), could not be concluded.

The observation that NPVL and NPVH alone resulted in AgseGV-infected larvae indicated that either the AgseNPV-B inoculum contained AgseGV or A. segetum larvae harbored a latent AgseGV infection. The latter could be excluded, since no larvae of the control showed any symptoms or died by viral infection. Therefore, a slight contamination of the AgseNPV-B stock was likely. The amounts of AgseGV and AgseNPV-B OBs per larva were regarded as realistic of what an A. segetum larva could be capable of producing. With a range from 4.66 × 108 to 2.89 × 109 AgseGV OBs per singly infected larva, the median amounts were within the expected range. In a study on different Cydia pomonella GV (CpGV) mutants replicating in Cydia pomonella, virus offspring production was calculated to vary between 2.0 × 1010 and 3.6 × 1010 OBs/larva (28). A comparison with the present results is difficult, because fifth-instar C. pomonella larvae, and thus larger caterpillars than those used in the present study, were infected with CpGV (29). For AgseNPV-B, the median OB offspring production matched that of PsunNPV (18). There, fifth-instar larvae of the armyworm P. unipuncta were infected with PsunNPV and produced about 104 to 107 OB/larva, which is a range (2.1 × 106 to 8.97 × 107 OBs/larva) similar to that observed in our study for A. segetum larvae infected with AgseNPV-B. Our results on the production of AgseGV and AgseNPV-B OB progeny suggest the potential capacity for OB production that neonate A. segetum larvae are able to produce during the infection period. It is not known if later larval stages were able to produce larger amounts of OBs when exposed to other virus concentrations. However, the coinfection experiments clearly indicated that both viruses should be propagated separately and contaminations should be avoided, since mixtures led to a decrease in yields of OBs.

In our case, a prolonged larval life induced by viral infection was not observed as described for HearGV-infected H. armigera larvae (30). Due to the extended survival time and therefore a longer feeding time, providing the virus more host resources for replication, larvae doubled their size and mass. For AgseGV and AgseNPV-B, this assumption could be excluded because of similar mortality in single- and mixed-virus treatments. The baculovirus-carried ecdysteroid glucosyltransferase (egt) gene that was shown to play a major role in extending larval life (31) is carried by AgseGV (32), AgseNPV-B (9), AgseNPV-A (33), and AgipNPV (34). If deleted in naturally occurring Δegt genotypes of AgipNPV-infected Agrotis ipsilon, larvae died significantly faster (35).

Infected larvae have an upper limit in OB production, set by the availability of host resources that can be utilized for production of progeny OBs. In the case of coinfected larvae, this upper limit could either be shifted in favor of AgseGV or AgseNPV-B, but both viruses could also replicate equally. AgseNPV-B exhibited a steady and significant increase in median OB formation in singly infected larvae between the first and second weeks, whereas AgseGV showed no such temporal increase. This observation may rely on the tissue tropism of AgseGV. An early infection of the complete tissue or organ might prevent an increase in OB formation. On the contrary, AgseNPV-B may spread within the whole larval body, resulting in a temporal increase of progeny OBs. It is not known if tissue tropism occurs for AgseGV.

In coinfections, however, AgseNPV-B production appeared to be less influenced by the presence of AgseGV than vice versa. This was clearly observed by a significantly lower median OB production of AgseGV in coinfections than in single infections. Furthermore, coinfected larvae contained less AgseGV and AgseNPV-B than larvae infected by one of these viruses alone. However, a significant negative correlation on AgseNPV-B and AgseGV OB production was not observed. A possible explanation for a missing correlation but a negative mutual interference could lie in the nature of the less pathogenic AgseGV and the more virulent AgseNPV-B to A. segetum larvae and in independent infection processes that start in different cells for both viruses. In this case, AgseNPV-B could replicate normally within coinfected larvae within the first week but interfere with the less virulent AgseGV in the later states.

Cells, infected by one baculovirus, lose their susceptibility to a secondary infection, as has been demonstrated for two genotype variants of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), as well as for AcMNPV and Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) infections (36). So-called superinfections of a single cell were shown to be temporarily possible shortly after the first viral infection (36). Whether AgseGV and AgseNPV-B are able to superinfect A. segetum cells is unknown, but a reciprocal exclusion can be an explanation for reduced progeny generation.

In conclusion, a synergistic effect of AgseGV and AgseNPV-B within coinfected larvae was not observed. However, the results suggest a certain competition of AgseGV and AgseNPV-B for larval resources.

ACKNOWLEDGMENTS

We thank Doreen Winstanley (Horticulture Research International, Wellesbourne Warwickshire, United Kingdom) for providing AgseNPV-B and Christian Fetzer for rearing A. segetum. We thank Jürg Huber (JKI, Darmstadt, Germany) for helpful discussions. We also thank Eric Carstens (Queens University, Kingston, Canada) for critical reading and his comments on the manuscript.

This study was supported by grants EGY 08/033 and 01QE1208 (Eurostars E!7129) of the Federal Ministry of Education and Research (BMBF).

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