Pathogen persistence in the environment and insect-baculovirus interactions: disease-density thresholds, epidemic burnout, and insect outbreaks

Classical epidemic theory focuses on directly transmitted pathogens, but many pathogens are instead transmitted when hosts encounter infectious particles. Theory has shown that for such diseases pathogen persistence time in the environment can strongly affect disease dynamics, but estimates of persistence time, and consequently tests of the theory, are extremely rare. We consider the consequences of persistence time for the dynamics of the gypsy moth baculovirus, a pathogen transmitted when larvae consume foliage contaminated with particles released from infectious cadavers. Using field-transmission experiments, we are able to estimate persistence time under natural conditions, and inserting our estimates into a standard epidemic model suggests that epidemics are often terminated by a combination of pupation and burnout rather than by burnout alone, as predicted by theory. Extending our models to allow for multiple generations, and including environmental transmission over the winter, suggests that the virus may survive over the long term even in the absence of complex persistence mechanisms, such as environmental reservoirs or covert infections. Our work suggests that estimates of persistence times can lead to a deeper understanding of environmentally transmitted pathogens and illustrates the usefulness of experiments that are closely tied to mathematical models.     

Persistence of insect viruses in field populations of alfalfa insects

The persistence of viruses of five insects was observed in alfalfa fields. The insects were Autographa californica, Colias eurytheme, Pseudaletia unipuncta, Spodoptera exigua, and Trichoplusia ni. The isolated viruses were the granulosis (GV), the cytoplasmic-polyhedrosis (CPV), and the nuclear-polyhedrosis (NPV) viruses. The viruses persisted in the soil, on the alfalfa foliage, and in alternate hosts. In the soil, the viruses persisted even during the winter months when no foliage remained on the plants. Alfalfa sprouts harboring virus-infected larvae of C. eurytheme and S. exigua produced virus infections in larvae of these insects, but those with larvae of A. californica and P. unipuncta did not cause virus infection. The GVs and CPVs isolated from these insects were transmitted to nearly all of the other four species, but the NPVs appeared to be host specific.     

Pathogen clumping: an explanation for non-linear transmission of an insect virus

Abstract.  1. Previous work has shown that transmission of some insect pathogens is a non-linear process. A number of hypotheses have been put forward as explanations for this phenomenon; however, none have proven wholly satisfactory. Here we test the effects on transmission of spatial distribution of an insect virus by testing whether or not experimental manipulations of pathogen clumping lead to different values of a clumping parameter. The gypsy moth nucleopolyhedrovirus (LdMNPV) was used, which is transmitted when larvae consume virus released from previously infected larvae that have died on foliage.
2. It was found that even when virus densities on foliage were equal, overall mortality was lower when virus-killed cadavers were clumped on foliage.
3. Non-linearity is more pronounced when cadavers are clumped than when they are placed at random on the foliage. Placement of droplets containing LdMNPV on foliage resulted in more linear transmission compared with cadavers.
4. Spatial clumping of viral inoculum thus provides part of the explanation for non-linear transmission in this system. The ultimate explanation for non-linear transmission is likely to involve some combination of spatial clumping and heterogeneity in behaviours such as feeding rate or the ability to avoid pathogen.     

Genetically variable nucleopolyhedroviruses isolated from spatially separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) in Orkney

Here we report a lepidopteran system in which a pathogen is both abundant and genotypically variable. Geographically separate populations of winter moth (Operophtera brumata L.) were sampled in heather habitats on the Orkney Isles to investigate the prevalence of a pathogen, O. brumata Nucleopolyhedrovirus (OpbuNPV), within the natural system. Virus was recorded in 11 of the 13 winter moth populations sampled, with two populations suffering mortality due to virus at levels of 50%. The virus genome from 200 single insect isolations was investigated for variation using restriction endonuclease digests. Twenty-six variants of OpbuNPV were detected using SalI. The polyhedrin gene of the virus was partially sequenced, allowing the relationship between the 26 variants to be portrayed as a cladogram. The phylogenetic relationship between OpbuNPV and other known baculovirus polyhedrin gene sequences was also established. The discovery of virus at such high prevalence is discussed with reference to occurrence and genetic variation of pathogens in other lepidopteran host populations. This study shows encouraging results for further studies into the role of pathogens in the regulation of host insect populations.     

Minor effects of two elicitors of insect and pathogen resistance on volatile emissions and parasitism of Spodoptera frugiperda in Mexican maize fields

Synthetic elicitors can be used to induce resistance in plants against pathogens and arthropod herbivores. Such compounds may also change the emission of herbivore-induced plant volatiles, which serve as important cues for parasitic wasps to locate their hosts. Therefore, the use of elicitors in the field may affect biological control of insect pests. To test this, we treated maize seedlings growing in a subtropical field in Mexico with methyl jasmonate (MeJA), an elicitor of defense responses against many insects, and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), an elicitor of resistance against certain pathogens. Volatile emission, herbivore infestation, pathogen infection, and plant performance (growth and grain yield) of treated and untreated maize plants were measured. Application of BTH slightly reduced volatile emission in maize, while MeJA increased the emission compared to control treatments. Despite the apparent changes in volatile emissions, the elicitor application did not consistently affect infestation by Spodoptera frugiperda larvae, the main insect pest found on the maize seedlings, and had only marginal effects on parasitism rates. Similarly, there were no treatment effects on infestation by other herbivores and pathogens. Results for the six replications that stretched over one summer and one winter season were highly variable, with parasitism rates and the species composition of the parasitoids differing significantly between seasons. This variability, as well as the severe biotic and abiotic stresses on young seedlings might explain why we measured only slight effects of elicitor application on pest incidence and biological control in this specific field study. Indeed, an additional field experiment under milder and more standardized conditions revealed that BTH induced significant resistance against Bipolaris maydis, a major pathogen in the experimental maize fields. Similar affects can be expected for herbivory and parasitism rates.     

CO2-mediated changes in aspen chemistry: effects on gypsy moth performance and susceptibility to virus

We investigated the effects of long-term CO₂ enrichment on foliar chemistry of quaking aspen ( Populus tremuloides ) and the consequences of chemical changes for performance of the gypsy moth ( Lymantria dispar ) and susceptibility of the gypsy moth to a nucleopolyhedrosis virus (NPV). Foliage was collected from outdoor open-top chambers and fed to insects in a quarantine rearing facility. Under enriched CO₂, levels of leaf nitrogen declined marginally, levels of starch and phenolic glycosides did not change, and levels of condensed tannins increased. Long-term bioassays revealed reduced growth (especially females), prolonged development and increased consumption in larvae fed high-CO₂ foliage but no significant differences in final pupal weights or female fecundity. Short-term bioassays showed weaker, and sex-specific, effects of CO₂ treatment on larval performance. Correlation analyses revealed strong, negative associations between insect performance and phenolic glycoside concentrations, independent of CO₂ treatment. Larval susceptibility to NPV did not differ between CO₂ treatments, suggesting that effects of this natural enemy on gypsy moths are buffered from CO₂-induced changes in foliar chemistry. Our results emphasize that the impact of enriched CO₂ on plant–insect interactions will be determined not only by how concentrations of plant compounds are altered, but also by the relevance of particular compounds for insect fitness. This work also underscores the need for studies of genetic variation in plant responses to enriched CO₂ and long-term population-level responses of insects to CO₂-induced changes in host quality.     

The effect of gypsy moth metamorphosis on the development of nuclear polyhedrosis virus infection

The development of nuclear polyhedrosis virus (NPV) infection in gypsy moth (Lymantria dispar) was studied before, during, and after host metamorphosis, and in larvae and pupae in the subsequent generation, to determine whether NPV ingested by late instars can replicate in host tissues through metamorphosis and whether it can be vertically transmitted to progeny. Individuals that survived sublethal dosages of NPV did not differ from undosed insects in pupal weight, fecundity, larval and pupal weight of progeny, or response of progeny to NPV challenge. No evidence of NPV infection or of abnormal histology was found in adult tissues examined by light microscopy and no virus was detected on the surface of eggs produced by NPV-treated moths. No NPV-caused mortality was recorded among undosed progeny of dosed or undosed parents. The progeny of dosed parents were neither more resistant nor more susceptible to LdMNPV than were progeny of undosed parents and lethal times did not differ between groups. Examination of larval, pupal, and adult tissues by DNA hybridization revealed that insects in which NPV DNA was detected died prior to adult eclosion. NPV was not detected in any hosts surviving to the adult stage. These results suggest that survivors of sublethal dosages of NPV avoid infection and are therefore incapable of vertically transmitting infectious virus to progeny.     

Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen.     

Induced plant defenses breached? Phytochemical induction protects an herbivore from disease

Although wound-induced responses in plants are widespread, neither the ecological nor the evolutionary significance of phytochemical induction is clear. Several studies have shown, for example, that induced responses can act against both plant pathogens and herbivores simultaneously. We present the first evidence that phytochemical induction can inhibit a pathogen of the herbivore responsible for the defoliation. In 1990, we generated leaf damage by enclosing gypsy moth larvae on branches of red oak trees. We then inoculated a second cohort of larvae with a nuclear polyhedrosis virus (LdNPV) on foliage from the damaged branches. Larvae were less susceptible to virus consumed on foliage from branches with increasing levels of defoliation, and with higher concentrations of gallotannin. Defoliation itself was not related to any of our chemistry measures. Field sampling supported the results of our experiments: death from virus among feral larvae collected from unmanipulated trees was also negatively correlated with defoliation. In 1991, defoliation and gallotannin were again found to inhibit the virus. In addition, gallotannin concentrations were found to be positively correlated with defoliation the previous year. Compared with previous results that demonstrated a delecterious effect of induction on gypsy moth pupal weight and fecundity, the inhibition of the virus should confer an advantage to the gypsy moth. Since leaf damage levels increase as gypsy moth density increases, and since leaf damage inhibits the gypsy moth virus, there is the potential for positive feedback in the system. If phytochemical induction in red oak can inhibit an animal pathogen such as LdNPV, it suggests to us that induction in red oak is a generalized response to tissue damage rather than an adaptive defense against herbivores.     

Competition and reproduction in mixed infections of pathogenic and non-pathogenic Bacillus spp

Diamondback moth, Plutella xylostella, larvae were infected with a primary pathogen, Bacillus thuringiensis kurstaki (Btk) in single strain and mixed infections. Mixed infections comprised Btk and a non-pathogenic isolate, either Bacillus thuringiensis tenebrionis (Btt) or Bacillus cereus (Bc). All strains reproduced in larval cadavers, but there was evidence of competition between different isolates within hosts. Non-pathogenic isolates (Btt, Bc) had growth rates that were faster than Btk in vivo, whereas Btk outcompeted Btt in vitro. Passage through insects increased the in vitro competitive ability of Btk against Btt.     

Acquisition,retention and dispersal of soilborne plant pathogenic fungi by fungus gnats and moth flies

Adult fungus gnats and moth flies were experimentally demonstrated to function as potential above‐ground vectors for three soilborne plant pathogens: Verticillium dahliae, Fusarium acuminatum and Thielaviopsis basicola. The adult insects externally acquired the conidia of the pathogens after exposure to the cultures as confirmed by scanning electron microscope photography. The intestinal contents and frass deposits of larvae exposed to fungal cultures contained viable fungal propagules. Internally infested larvae developed into internally infested pupae; however, the emerging adults were free of fungal structures. Because of the maintenance of a high level of inoculum on the external body surface and the ability of these adult insects to fly, they can be a significant factor in the dispersal of soilborne fungi in greenhouse agriculture. The rate of dispersal of T. basicola by adult fungus gnats was 1.78 cm² h^?1 per insect and by adult moth flies was 1.17 cm² h^?1 per insect. The area over which the pathogen was dispersed by the adult insects increased with the increase in exposure time. The study demonstrated that adult insects are efficient distributors of soilborne plant pathogenic fungal propagules.     

Costs of cannibalism in the presence of an iridovirus pathogen of Spodoptera frugiperda

Abstract.  1. The costs of cannibalism were examined in larvae of Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) in the presence of conspecifics infected by a lethal invertebrate iridescent virus (IIV). The hypothesis of a positive correlation between insect density and the likelihood of disease transmission by cannibalism was examined in laboratory microcosms and a field experiment.
2. Transmission was negligible following peroral infection of early instars with purified virus suspensions or following coprophagy of virus-contaminated faeces excreted by infected insects. In contrast, 92% of the insects that predated infected conspecifics acquired the infection and died prior to adult emergence in the laboratory. Diseased larvae were more likely to be victims of cannibalism than healthy larvae.
3. The prevalence of cannibalism was density dependent in laboratory microcosms with a low density (10 healthy insects + one infected insect) or high density (30 healthy insects + one infected insect) of insects, and field experiments performed on maize plants infested with one or four healthy insects + one infected insect.
4. Cannibalism in the presence of virus-infected conspecifics was highly costly to S. frugiperda ; in all cases, insect survival was reduced by between ≈ 50% (laboratory) and ≈ 30% (field) in the presence of the pathogen. Contrary to expectations, the prevalence of disease was not sensitive to density because cannibalism resulted in self-thinning. As infected individuals are consumed and disappear from the population, the prevalence of disease will be determined by the timescale over which transmission can be achieved, and the rate at which individuals that have acquired an infection become themselves infectious to conspecific predators.     

The infectivity and host range of Orgyia anartoides nucleopolyhedrovirus

The painted apple moth (PAM), Teia anartoides (Walker) (Lepidoptera: Lymantriidae) made a recent incursion into New Zealand. A nucleopolyhedrovirus (NPV), Orgyia anartoides NPV (OranNPV), originally isolated from PAM in Australia, was tested for its pathogenicity to PAM and a range of non‐target insect species found in New Zealand, to evaluate its suitability as a microbial control for this insect invader. Dosage‐mortality tests showed that OranNPV was highly pathogenic to PAM larvae; mean LT₅₀ values for third instars ranged from 17.9 to 8.1 days for doses from 10² to 10⁵ polyhedral inclusion bodies/larva, respectively. The cause of death in infected insects was confirmed as OranNPV. Molecular analysis established that OranNPV can be identified by PCR and restriction digestion, and this process complemented microscopic examination of infected larvae. No lymantriid species occur in New Zealand; however, the virus had no significant effects on species from five other lepidopteran families (Noctuidae, Tortricidae, Geometridae, Nymphalidae and Plutellidae) or on adult honeybees. Thus, all indications from this initial investigation are that OranNPV would be an important tool in the control of PAM in a future incursion of this species into New Zealand.     

Foraging in a pathogen reservoir can lead to local host population extinction: a case study of a Lepidoptera-virus interaction

In 1990, natural infestations of the polyphagous vapourer moth, Orgyia antiqua (Lepidoptera: Lymantriidae) in lodgepole pine plantations in northern Scotland, were studied to ascertain the role of host foraging behaviour on the prevalence of nucleopolyhedrovirus (NPV; Baculoviridae) infection in the population. Aerial dispersal of early instar larvae (L1–L3) from the tree canopy onto heather foliage at the forest understorey, with subsequent relocation back onto the tree as late-instar larvae (L4–L6) appeared to play a significant role in the development of a widespread virus epizootic in which approximately 80% of L4–L6 individuals succumbed to disease. Bioassays of foliage 1 year later showed that the distribution of NPV followed a pronounced vertical gradient through the forest canopy culminating in high concentrations of virus in the forest understorey. Experimental systems comprising potted pine trees positioned above heather bases showed that NPV infections could be acquired by early stage larvae following dispersal from the tree and feeding on the undercanopy vegetation, then translocated to the tree component for secondary transmission to susceptible tree-feeding individuals. Behavioural studies indicated that the tendency for first-, second- and third-instar larvae to disperse to the understorey was probably not influenced by larval density on the tree but was strongly dependent on larval instar. In contrast, the tendency for larvae to relocate from the understorey heather to the tree was affected by both larval density and larval instar, suggesting that both these factors may significantly affect virus acquisition, translocation and transmission in the host population. In the present study, the heather understorey appeared to act as a pathogen reservoir in which virus could persist between host generations. Spatial heterogeneity in virus distribution combined with host foraging behaviour (dispersal and feeding) resulted in the pathogen playing a major role in host population dynamics over an extended time period (3 years). The reservoir theory is supported by the observation that similar dynamics were not observed in O. antiqua populations at neighbouring sites which lacked understorey food plants. Received: 8 June 1998 / Accepted: 5 October 1998     

Analysis of the causes of declines in Western Siberian outbreaks of the nun moth <Emphasis Type="Italic">Lymantria monacha</Emphasis>

This study presents the results of an investigation into the causal factors of precipitous population declines after five mass outbreaks of nun moths (Lymantria monacha) in territories of Western Siberian (Novosibirsk and Tyumen oblasts, Russia). Nucleopolyhedrovirus (NPV) and parasitoids represented by the families Tachinidae and Sarcophagidae (Diptera) were found to be major contributors to the degradation of these outbreaks. Viable occlusion bodies persisted on pine needles during a two-year observation period and contaminated nun moth eggs, resulting in the death of the insects from NPV infection. A high probability of insect/virus contacts was largely attributable to the poor flying ability of female moths. Moreover, a latent virus was apparently activated in part of the insect population due to asynchrony between the growth rate of larvae and pine phenology.     

Competition between wild-type and a marked recombinant baculovirus (Spodoptera exigua nucleopolyhedrovirus) with enhanced speed of action in insect larvae

Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6-12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findings suggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems.     

Effects of insect-vector preference for healthy or infected plants on pathogen spread: insights from a model

Insects vector many plant pathogens and often have higher or lower densities on infected plants than on healthy plants. Two hypotheses may explain this observation: insects may preferentially orient toward and select one plant type (referred to as orientation preference) or insects may reside on infected plants for longer or shorter periods than on healthy plants (referred to as feeding preference). The effects of feeding preference and orientation preference were compared alone and in combination using a spatially explicit model. With feeding preference for healthy or infected plants, the qualitative relationship between the percentage of plants infected and the rate of pathogen spread was not affected. However, feeding preference for healthy plants increased rates of pathogen spread, whereas feeding preference for infected plants decreased rates of pathogen spread. Unlike feeding preference, orientation preference for healthy and infected plants produced qualitatively different relationships between the percentage of plants infected and the rate of pathogen spread. With orientation preference for healthy plants, the pathogen spread slowly when few plants were infected, but quickly once most plants were infected. In contrast, with orientation preference for infected plants, the pathogen spread quickly when few plants were infected, but slowly once most plants were infected. In sensitivity analyses, we found that assumptions about the latent period (time between infection and when insects can recognize a plant as being infected) and persistence (length of time an insect remains inoculative) altered the aforementioned effects in some cases. The results illustrate that feeding and orientation preference affect pathogen spread differently, highlighting the importance of elucidating the mechanisms that control vector preference for healthy versus infected plants.     

Transmission of Plodia interpunctella granulosis virus does not conform to the mass action model

1. Transmission of insect pathogens is traditionally described by a term which states that transmission is proportional to the densities of the susceptible hosts and the infectious units, multiplied by a constant, the transmission coefficient. Theoretical studies suggest that deviations from this can be important in host–pathogen population dynamics, but little is known of how commonly pathogen transmission conforms to the conventional model.
2. We describe a test of the traditional assumption for the Indian meal moth, Plodia interpunctella (Lepidoptera: Pyralidae) (Hübner) and its granulosis virus using a modification of the previous methods, which allows for unpredictable declines in the amount of infectious material present.
3. The estimated transmission coefficient increased with the density of susceptible hosts and showed a marked decline with density of infectious cadavers. This suggests that the usual assumption does not adequately describe transmission in this system.
4. The reasons for this deviation from the usual assumption are likely to be a combination of behavioural and physiological changes at high host density, and differential susceptibility to the pathogen leading to an effect analogous to pseudo-interference in parasitoids.     

Parasitoids aid dispersal of a nonpersistently transmitted plant virus by disturbing the aphid vector

• 1 Aphids are the major group of insects that vector plant viruses, and they often display a preference for foliage showing disease symptoms. Although this behaviour will increase the numbers of vectors acquiring the pathogen, it will not in itself result in a greater spread of the disease.
• 2 The present study examined how infection of Vicia faba by the nonpersistently transmitted virus bean yellow mosaic virus (BYMV) affected colonization by pea aphids Acyrthosiphon pisum. We then examined how foraging by the hymenopterous parasitoid Aphidius ervi affected aphid settling/movement behaviour and the consequences for dissemination of the virus.
• 3 In Petri dish arenas, aphids colonized discs from BYMV‐infected leaves more rapidly than discs from uninfected plants. Reflectance from infected foliage was approximately 20% higher than from uninfected leaves in the green–yellow wavelengths, indicating that aphids might be responding to visual cues from the brighter foliage. Settling was reduced by A. ervi, with the foraging wasps preventing the aphids reaching and/or remaining on the leaf tissue.
• 4 In multiple plant arenas, A. ervi caused a reduction in aphid numbers but also a nine‐fold increase in BYMV infection. It is hypothesized that disturbance by the parasitoids resulted in more aphid movement as well as more cases of aphids probing on a BYMV‐infected plant and then a new host within the critical time period for successful inoculation to occur. This effect of parasitoids on virus dispersal should be considered in epidemiological models of insect‐vectored plant diseases, and also when evaluating the use of natural enemies in biocontrol strategies of insect herbivore/vector pests.

     

A field release of genetically engineered gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (LdNPV)

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.