Transport of Wild-Type and Recombinant Nucleopolyhedroviruses by Scavenging and Predatory Arthropods

Abstract Wild-type and recombinant nucleopolyhedroviruses (NPVs) were compared in their capability to be transported over limited distances by the predator Podisus maculiventris (Say) and scavengers Sarcophaga bullata (Parker) and Acheta domesticus (Linnaeus) in Trichoplusia ni (Hübner) larvae infesting collards in a greenhouse microcosm. Viruses tested were variants of Autographa californica (Speyer) NPV (AcNPV): wild-type virus (AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing juvenile hormone esterase (AcJHE.SG). Podisus maculiventris transported AcNPV.WT and S. bullata transported AcNPV.WT and AcNPV.AaIT. Prevalence and transport of AcNPV.WT were greater than those of AcNPV.AaIT and AcJHE.SG, regardless of whether the nontarget organism carriers were present or absent. Podisus maculiventris and S. bullata transported recombinant and wild-type NPVs at a rate of up to 62.5 cm/day, and A. domesticus transported wild-type NPV at 125 cm/day. The infected host insects, T. ni, undoubtedly contributed to viral transport in the current research. In every experiment, both the wild-type and recombinant virus spread to some degree in the plots without predators or scavengers. The relative amounts of NPVs that accumulated in soil, as indicated by bioassay mortality percentages, generally exhibited spatial patterns similar to those of T. ni mortality due to NPV on the collards plants. Thus, the predator and scavengers in the current research demonstrated some capacity to transport wild-type as well as recombinant viruses at significant rates in a greenhouse microcosm. Received: 6 December 1999; Accepted: 29 February 2000; Online Publication: 12 May 2000     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

Occurrence and accumulation of viruses of Trichoplusia ni in treated field plots

Concentrations of the nuclear-polyhedrous virus (T. ni NPV) and the granulosis virus (T. ni GV) of the cabbage looper, Trichoplusia ni, in soil and on foliage were monitored up to 4 years after treatment.A single application of T. ni NPV to soil in August or 5 foliar applications of the virus at 10-day intervals in August and early September maintained substantial concentrations of the virus on foliage and high concentrations of the virus accumulated in soil. With development of natural epizootics of the virus disease in populations of the host larvae in September and October, substantial concentrations of the virus accumulated in soil and on foliage in nontreated plots, eventually becoming equal in amount with the virus in virus-treated plots. The virus accumulated more slowly in plots treated with chemical insecticides or Bacillus thuringiensis because few host larvae survived to support late-season epizootics of the disease. Small quantities of T. ni NPV were detected in heads of cabbage harvested from the plots in October.Long-term studies in which nontreated plots and plots treated with T. ni NPV or T. ni GV were replanted for up to 4 years after treatment showed that concentrations of T. ni NPV in surface soil remained constant during the winter but were reduced by dilution during cultivation preparatory to planting in the spring. T. ni NPV accumulated during the late summer and autumn with development of epizootics of the disease in populations of host larvae. Increased concentrations of the virus in soil coincided with increased concentrations on leaves in each year. T. ni GV did not persist on leaves or in soil following application and only small amounts were found 2 years after application.T. ni NPV disease was prevalent in September and October in populations of host larvae in plots in which substantial residues of the virus were found. These epizootics contributed substantially to late-season control of the looper after completion of spraying.     

Behavior of a Recombinant Baculovirus in Lepidopteran Hosts with Different Susceptibilities

Insect pathogens, such as baculoviruses, that are used as microbial insecticides have been genetically modified to increase their speed of action. Nontarget species will often be exposed to these pathogens, and it is important to know the consequences of infection in hosts across the whole spectrum of susceptibility. Two key parameters, speed of kill and pathogen yield, are compared here for two baculoviruses, a wild-type Autographa californica nucleopolyhedrovirus (AcNPV), AcNPV clone C6, and a genetically modified AcNPV which expresses an insect-selective toxin, AcNPV-ST3, for two lepidopteran hosts which differ in susceptibility. The pathogenicity of the two viruses was equal in the less-susceptible host, Mamestra brassicae, but the recombinant was more pathogenic than the wild-type virus in the susceptible species, Trichoplusia ni. Both viruses took longer to kill the larvae of M. brassicae than to kill those of T. ni. However, whereas the larvae of T. ni were killed more quickly by the recombinant virus, the reverse was found to be true for the larvae of M. brassicae. Both viruses produced a greater yield in M. brassicae, and the yield of the recombinant was significantly lower than that of the wild type in both species. The virus yield increased linearly with the time taken for the insects to die. However, despite the more rapid speed of kill of the wild-type AcNPV in M. brassicae, the yield was significantly lower for the recombinant virus at any given time to death. A lower yield for the recombinant virus could be the result of a reduction in replication rate. This was investigated by comparing determinations of the virus yield per unit of weight of insect cadaver. The response of the two species (to both viruses) was very different: the yield per unit of weight decreased over time for M. brassicae but increased for T. ni. The implications of these data for risk assessment of wild-type and genetically modified baculoviruses are discussed.     

Use of primary fat body cultures for the study of baculovirus replication

Fat body cultures of Trichoplusia ni and Estigmene acrea were established for use in the study of the two baculoviruses Autographa californica nuclear polyhedrosis virus (AcNPV) and Estigmene acrea granulosis virus (EaGV), respectively. Multiplication of AcNPV observed by phase and electron microscopy was correlated with an increase in viral specific proteins as determined by indirect enzyme-linked immunosorbent assay (ELISA). Although EaGV morphogenesis was not observed in fat body cultures, an increase in specific proteins of this virus could be detected with the ELISA.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Gross pathology and infectivity

The pathology and infectivity of an RNA virus infectious to Trichoplusia ni larvae was investigated. The enzyme-linked immunosorbent assay (ELISA) and weight depression were used as criteria for virus concentration in larval homogenates and live larvae, respectively. Infected larvae were severely stunted, weighing as little as 13 times less than uninfected individuals of the same age, yet appeared normal morphologically. The virus was found to cause only slight mortality at high concentrations. Infected larvae displayed the pathological stunting response down to a dose of 0.1 ng of virus. Larvae infected with doses 100 times lower did not show the weight response but such inapparent infections were detectable by ELISA. Because of these subtle gross pathological symptoms, particularly at low levels of infection, infected individuals could easily remain unde-tected in a group-reared colony.     

Use of Scorpion Neurotoxins to Improve the Insecticidal Activity of Rachiplusia ou Multicapsid Nucleopolyhedrovirus

The nucleopolyhedrovirus of the mint looper, Rachiplusia ou (RoMNPV, also known as Anagrapha falcifera multicapsid nucleopolyhedrovirus), has previously been shown to kill the European corn borer, Ostrinia nubilalis, and to protect corn plants against this pest more effectively than the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). To further improve the performance of RoMNPV against O. nubilalis, we engineered it to express the insect-selective scorpion neurotoxins AaIT and LqhIT2 using the promoters of the AcMNPV p10 and p6.9 genes. RoMNPV recombinant virus expressing AaIT from the p10 promoter produced a reduced quantity of viral occlusions and polyhedrin protein and failed to occlude virions efficiently. Western blot analysis revealed that the amount of AaIT in hemolymph of recombinant virus-infected O. nubilalis was very low compared to that in the hemolymph of infected Heliothis virescens. In all but one case, insertion of the toxins did not alter the dose of virus required to kill three different hosts. Viruses expressing toxins from the p6.9 promoter killed larvae of O. nubilalis significantly faster than wild-type virus, but no significant difference in speed of kill was observed between AaIT- and LqhIT2-expressing viruses in this host. Expression of LqhIT2 reduced survival times of Helicoverpa zea and H. virescens to a significantly greater extent than expression of AaIT. An AcMNPV recombinant expressing LqhIT2 from the p6.9 promoter killed larvae faster than a recombinant utilizing the p10 promoter for expression, but recombinants using these promoters to drive AaIT expression did not exhibit any significant differences in speed of kill.     

In vivo and in vitro host range of Autographa californica nuclear polyhedrosis virus and Spodoptera frugiperda nuclear polyhedrosis virus

Baculoviruses from Autographa californica (AcNPV-E2) and Spodoptera frugiperda (SfNPV-2) were titered in five insect cell lines: IAL-PID2, IAL-SFD1, IPLB-SF-21AE, TN-368, and IAL-TND1. AcNPV-E2 replicated in all the cell lines while SfNPV-2 did not replicate in the lines TN-368 and IAL-TND1. Further in vivo studies of SfNPV-2 showed the virus was not infectious when fed to Trichoplusia ni larvae per os or when injected into the hemocoel. These data suggest that the barrier to SfNPV-2 infectivity in T. ni is at the cellular level, as opposed to the midgut.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.     

Damage Threshold of Meloidogyne hapla to Lettuce in Organic Soil

Lettuce was seeded in pots in the greenhouse and in field microplots in 1991 and 1992. Pots and microplots were filled with untreated or fumigated organic soil infested with Meloidogyne hapla at seven initial population densities (Pi) (0 to 32 eggs/cm³ soil). Lettuce weight, severity of root galling, and number of eggs per root system (Pf) were determined after 8 weeks. At the highest Pi, M. hapla caused yield losses up to 64% in the microplots and plant death in the greenhouse tests. The Seinhorst equation was used to describe the relation between lettuce weight and Pi (r² = 0.73 - 0.98) and to calculate the damage threshold density (T). Values of T were 7 and 8 eggs/cm³ soil in the greenhouse tests of 1991 and 1992, respectively. In the microplot tests, T was 1 egg/cm³ soil in 1991 and 2 eggs/cm³ soil in 1992. The damage threshold was the same in untreated and fumigated soils. At low Pi, root galling was more severe in the pots than in the microplots. Pf increased with increasing Pi of M. hapla in both tests, but declined at Pi > T in the greenhouse tests. The reproduction rate (Pf/Pi) of M. hapla was highest at the lowest Pi.     

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni.

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.     

Effects of crop type on Bacillus thuringiensis toxicity and residual activity against Trichoplusia ni in greenhouses

We assessed the efficacy and persistence of a Bacillus thuringiensiskurstaki (Btk) formulation (Dipel) against Trichoplusia ni (Hubner) (Lep., Noctuidae), the cabbage looper, on three greenhouse vegetable crops (tomato, bell pepper and cucumber). First, T. ni larvae were fed leaf discs treated with Btk to assess how Btk toxicity varies with host plant. Secondly, T. ni larvae were fed leaf discs harvested from plants that had been sprayed with Btk 1, 5 and 9 days previously to assess the residual activity of Btk toxicity in greenhouse environments. Mortality of T. ni larvae fed tomato leaf discs was significantly higher than T. ni fed cucumber or pepper leaf discs. The toxicity of Btk had declined by less than 50% after 9 days, which suggests that Btk persistence is lengthy in greenhouse environments. No crop effects on the residual activity of Btk were found. These results demonstrate that the greenhouse environment and the crop should be considered when using Btk for insect management on greenhouse crops.     

Activity of the nuclear-polyhedrosis virus of the cabbage looper evaluated at programmed temperature regimens

Each larval instar of the cabbage looper, Trichoplusia ni, was treated in the laboratory with a peroral, standardized lethal dose of the nuclear polyhedrosis virus disease and maintained until death at one of three programmed regimens of temperature. The resulting experimental data were tested in the field and found to provide a reliable method of predicting the course of epizootics.     

Heterologous promoter recognition leading to high-level expression of cloned foreign genes in Bombyx mori cell lines and larvae

The baculovirus expression system using the Autographa californica nuclear polyhedrosis virus (AcNPV) has been extensively utilized for high-level expression of cloned foreign genes, driven by the strong viral promoters of polyhedrin (polh) and p10 encoding genes. A parallel system using Bombyx mori nuclear polyhedrosis virus (BmNPV) is much less exploited because the choice and variety of BmNPV-based transfer vectors are limited. Using a transient expression assay, we have demonstrated here that the heterologous promoters of the very late genes polh and p10 from AcNPV function as efficiently in BmN cells as the BmNPV promoters. The location of the cloned foreign gene with respect to the promoter sequences was critical for achieving the highest levels of expression, following the order + 35 > + 1 > − 3 > − 8 nucleotides (nt) with respect to the polh or p10 start codons. We have successfully generated recombinant BmNPV harboring AcNPV promoters by homeologous recombination between AcNPV-based transfer vectors and BmNPV genomic DNA. Infection of BmN cell lines with recombinant BmNPV showed a temporal expression pattern, reaching very high levels in 60–72 h post infection. The recombinant BmNPV harboring the firefly luciferase-encoding gene under the control of AcNPV polh or p10 promoters, on infection of the silkworm larvae led to the synthesis of large quantities of luciferase. Such larvae emanated significant luminiscence instantaneously on administration of the substrate luciferin resulting in ‘glowing silkworms’. The virus-infected larvae continued to glow for several hours and revealed the most abundant distribution of virus in the fat bodies. In larval expression also, the highest levels were achieved when the reporter gene was located at +35 nt of the polh.     

Propagation of Autographa californica nuclear polyhedrosis virus in cell culture: Methods for infecting cells

Autographa californica nuclear polyhedrosis virus (AcNPV) produced in Trichoplusia ni (TN-368) cells was used to infect other cell cultures. Methods were developed to recover and obtain high titers of virus from infected cells for subsequent use as inocula. To release cell-associated nucleocapsids, the cells were lysed by sonication and freeze-thawing. The infectivity of enveloped nucleocapsids was greatly reduced by freeze-thawing, while sonication was not as detrimental. The titer of plaque-forming units (pfu) was reduced about 12-fold when passed through 0.45-μm filters. The virus and cells were manipulated to determine the most efficient methods for inoculating cells while yielding the highest numbers of polyhedra. The viral inocula may be left on cells during virus replication, and cells may be centrifuged at 380 g prior to exposure to virus without affecting the yield of polyhedra. The production of polyhedra is affected by cell density, and, of the densities tested, 7.65 × 10⁵ cells/ml yielded the maximum number of polyhedra per cell (142). However, the highest number of polyhedra per milliliter of culture (2.2 × 10⁸) was obtained with 3.8 × 10⁶ cells/ml. The numbers of polyhedra per cell did not vary when cells were taken from fermentor cultures at 0–144 hr and were infected with virus.     

Differential susceptibility of parasitized and nonparasitized larvae of Trichoplusia ni to a nuclear polyhedrosis virus

Nonparasitized second-instar larvae of Trichoplusia ni were twice as susceptible (at the LD₅₀ level) to the singly enveloped T. ni nuclear polyhedrosis virus as those parasitized by Hyposoter exiguae (Hymenoptera: Ichneumonidae). The LD₅₀ values for nonparasitized and parasitized larvae were 1.58 × 10³ and 3.16 × 10³ polyhedra/ml of diet, respectively. The LD₉₅ value for parasitized larvae was approximateely 5 times higher than that for nonparasitized larvae. The slopes (b values) were 1.2 for parasitized larvae and 1.7 for nonparasitized larvae. The LT₅₀ values for parasitized larvae also were significantly longer than those for nonparasitized larvae. No significant difference was found between the food consumption of parasitized and nonparasitized T. ni larvae.     

An RNA virus in Autographa californica nuclear polyhedrosis virus preparations: Incidence and influence on baculovirus activity

A small RNA virus infectious to Trichoplusia ni larvae (TRV) was observed as a contaminant of several Autographa californica nuclear polyhedrosis virus preparations (AcMNPV). The extent of contamination in various AcMNPV preparations was studied by means of serial enrichment passages through T. ni larvae and enzyme-linked immunosorbent assay (ELISA). TRV could not be detected by ELISA in the original preparation of AcMNPV polyhedra prepared in 1968 even after five enrichment passages. Antibody inactivation offers a possible prophylactic method against TRV but temperature inactivation (55°C) does not. Although TRV reduced larval weight, it had little or no effect on bioassays of AcMNPV to T. ni and Heliothis virescens.     

Aerosol infectivity of a Baculovirus to Trichoplusia ni larvae: An alternative larval inoculation strategy for recombinant protein production

The baculovirus–insect expression system is a popular tool for recombinant protein production. The standard method for infecting insect larvae with recombinant baculovirus for protein production involves either feeding occlusion bodies or injecting budded virus into the cuticle. In this study, we showed that the recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) at titers >10⁸ pfu/mL efficiently infected Trichoplusia ni (T. ni) larvae through aerosol inoculation of budded virus at a pressure of 5.5 × 10⁴ Pa. The dipping T. ni larvae in virus‐containing solution efficiently infected them. These results indicate that surface contamination, either by aerosol or dipping, lead to infection via spiracles. The aerosol infection route for AcMNPV was restricted to T. ni and Plutella xylostella larvae, whereas Spodoptera litura and Helicoverpa armigera larvae were resistant to this inoculation process. The yields of the reporter proteins DsRed and EGFP from T. ni larvae following aerosol infection were nearly identical to those following oral feeding or injection. This alternative baculovirus infection strategy facilitates recombinant protein and virus production by insect larvae. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Occurrence and accumulation of the granulosis virus of Pieris rapae in treated field plots

The granulosis virus of Pieris rapae, the imported cabbageworm (P. rapae GV), persisted in soil after application. Virus produced by epizootics of the disease in populations of the host accumulated in soil in nontreated plots with concentrations of the virus in soil and on foliage, being similar at harvest to concentrations in virus-treated plots. Also P. rapae GV produced in epizootics of the disease increased concentrations in soil of virus-treated plots and maintained substantial residues of the virus on the foliage. Little virus accumulated in plots treated with some chemical insecticides and Bacillus thuringiensis because few host larvae survived to support late-season epizootics. Small quantities of P. rapae GV were found in heads of cabbage harvested from plots in October, but these residues were not related to plot treatment.A study in which nontreated and virus-treated plots were replanted for 2 years after treatment indicated an increase in concentrations of P. rapae GV in surface soil in years following treatment. Concentrations of P. rapae GV in soil in virus-treated and nontreated plots were similar in the autumn of the year of treatment and in subsequent years of the study. Concentrations were nearly unchanged during the winter, were reduced by cultivation preparatory to planting in the spring, and increased with epizootics of the disease in host populations in summer and autumn.P. rapae GV disease was prevalent during September and October in populations of P. rapae larvae in plots in which substantial concentrations of the virus were found and contributed to late-season control of the pest insect.     

Host suitability and fitness-related parameters of Campoletis sonorensis (Hymenoptera: Ichneumonidae) as a parasitoid of the cabbage looper,Trichoplusia ni (Lepidoptera: Noctuidae)

The seven age-classes of Trichoplusia ni (Hübner) larvae evaluated in this study as hosts of Campoletis sonorensis indicates that early 2nd larval instar (3–5 day-old larvae) of T. ni represents the most suitable host stage for the development of the larval endoparasitoid C. sonorensis. The higher suitability of early 2nd larval instar of T. ni resulted in more parasitised larvae, a higher rate of successful parasitoid emergence, a higher rate of female progeny, and a lower rate of immature parasitoid mortality. The fitness gain of C. sonorensis on late 1st larval instar (2 day-old larvae) and late 2nd larval instar –early 3rd instars (6–8 day-old larvae) stages of T. ni is negatively affected by the trade-offs between the different physiological and behavioral characteristics influencing their suitability as hosts of C. sonorensis.