Age-related cannibalism and horizontal transmission of a nuclear polyhedrosis virus in larval Spodoptera frugiperda

1. Experiments were carried out to investigate the incidence of cannibalism throughout the larval development of the noctuid moth Spodoptera frugiperda, and to examine the risk of infection from consuming conspecifics infected with a nuclear polyhedrosis virus (SfNPV). 2. Cannibalism was observed commonly even when food was not limiting, but occurred more frequently at low food quantities and/or high rearing densities. The sex of the larvae had no effect on the incidence of cannibalistic behaviour, however the probability of cannibalism occurring was affected by larval stage. The frequency of cannibalism was significantly higher among fifth- and sixth-instar larvae than among earlier instars, and larvae were more likely to consume younger conspecifics than larvae of the same stage. 3. Fifth-instar larvae offered fourth-instar victims fed equally on healthy larvae, virus-infected larvae (2 days post-infection), uninfected corpses, and virus-killed corpses (6 days post-infection). Horizontal transmission of SfNPV was only recorded in larvae offered virus-killed corpses, however, and total mortality in this treatment was only 32%. 4. In a similar experiment, fourth-instar larvae avoided cannibalising virus-killed corpses. Horizontal transmission of SfNPV was recorded in fourth-instar larvae that consumed 2-day post-infected larvae. The low incidence of cannibalism observed in fourth-instar larvae, however, suggests that this is unlikely to provide an important route for the transmission of SfNPV.     

Protein Tyrosine Phosphatase-Induced Hyperactivity Is a Conserved Strategy of a Subset of BaculoViruses to Manipulate Lepidopteran Host Behavior

Many parasites manipulate host behavior to increase the probability of transmission. To date, direct evidence for parasitic genes underlying such behavioral manipulations is scarce. Here we show that the baculovirus Autographa californica nuclear polyhedrovirus (AcMNPV) induces hyperactive behavior in Spodoptera exigua larvae at three days after infection. Furthermore, we identify the viral protein tyrosine phosphatase (ptp) gene as a key player in the induction of hyperactivity in larvae, and show that mutating the catalytic site of the encoded phosphatase enzyme prevents this induced behavior. Phylogenetic inference points at a lepidopteran origin of the ptp gene and shows that this gene is well-conserved in a group of related baculoviruses. Our study suggests that ptp-induced behavioral manipulation is an evolutionarily conserved strategy of this group of baculoviruses to enhance virus transmission, and represents an example of the extended phenotype concept. Overall, these data provide a firm base for a deeper understanding of the mechanisms behind baculovirus-induced insect behavior.     

Nucleopolyhedrovirus infection enhances plant defences by increasing plant volatile diversity

Plant–herbivore–entomopathogen tri-trophic interactions and biodiversity are relatively understudied topics in ecology. Particularly, the effects of entomopathogens on herbivore-induced plant volatiles and plant volatile diversity on the defensive function of plants have not been studied in detail. We used soybean (Glycine max), beet armyworm larvae (Spodoptera exigua), and nucleopolyhedrovirus (NPV) as a tri-trophic system to determine whether NPV infection can promote the emission and diversity of volatiles from plants. We also investigated whether NPV infection affects the attraction of Microplitis pallidipes, an important endoparasitoid of larval S. exigua. Uninfested soybean plants released 7 detectable volatile compounds while plants fed upon by healthy and NPV-infected S. exigua larvae released 12 and 15 volatiles, respectively. Female parasitoids were more attracted to the volatiles from plants that were fed upon by NPV-infected larvae than healthy larvae, and more attracted to the volatiles from plants that were fed upon by healthy larvae than no larvae. The selective responses of parasitoids to plant odours increased as plant volatile diversity increased. Our study suggests that the NPV infection facilitates the release of plant volatiles and enhances the defensive function of plants by increasing plant volatile diversity which in turn attracts more parasitoids. Also, this work reveals that plants might accrue two indirect benefits from NPV infection, cessation of herbivore feeding and more parasitisation.     

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.     

Transmission of a baculovirus in populations of Oryctes rhinoceros

The transmission of the baculovirus of Oryctes rhinoceros, previously called Rhabdionvirus oryctes, was studied. O. rhinoceros adults became infected with the virus when kept in a mixture of sawdust and ground-up virus-killed larvae or together with other virus-infected adults. In the field, mated females were more frequently infected than unmated females. Adults developing from larvae that had survived exposure to various dosages of the virus were not infected. No virus infections occurred in larvae hatching from eggs surface-contaminated with the virus. Larvae hatching from eggs laid by virus-infected females very rarely were infected.In the O. rhinoceros population the virus is transmitted most frequently during mating, possibly when the uninfected partner contacts virus material excreted by the infected partner. The virus can be transmitted in a similar way when infected and healthy beetles feed together in palm trees. Beetles visiting larval breeding sites containing freshly virus-killed larvae can become infected. Virus-infected beetles can pass the infection to healthy larvae when visiting a breeding site.     

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.     

Wipfelkrankheit: modification of host behaviour during baculoviral infection

Infection with an endoparasite frequently alters host behaviour. This study provides the first quantification of larval behaviour in a baculovirus/ Lepidoptera system, and attempts to assess the ecological consequences of behavioural modification during infection. Larvae of the moth Mamestra brassicae (Lepidoptera: Noctuidae) exhibited higher rates of dispersal in the laboratory and field when infected with Mamestra brassicae nuclear polyhedrosis virus (MbNPV) than did uninfected larvae. They adopted positions at death which were not characteristic of healthy larvae, climbing higher on the foodplant and onto the top and edge of leaves. The horizontal and vertical distribution of virus following larval lysis and the effects of rainfall on this distribution were assessed for comparison with the distributions of healthy and infected larvae. Exposure to rainfall increased the infectivity of vegetation in bioassays. Alternative explanations for the evolutionary origins of behavioural modification are considered. I suggest that the behavioural changes observed are most likely to benefit the virus. In particular, climbing prior to death is likely to result in contamination of more foliage with virus particles than would otherwise occur by increasing exposure of cadavers to rainfall. Thus it may profoundly influence horizontal transmission and the dynamics of the host-virus interaction. Received: 12 April 1996 /Accepted: 12 August 1996     

Relative activity of baculoviruses of the diamondback moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.) (Lepidoptera: Plutellidae)

The relative activities of the granulovirus (PxGV) and the nucleopolyhedrovirus (PxMNPV) of the diamondback moth, Plutella xylostella (L.), against this insect were evaluated in the laboratory. Similar numbers of occlusion bodies (OBs) of PxGV and PxMNPV were required to kill P. xylostella. However, cadavers of insects infected with PxGV contained more OBs per unit weight than did cadavers infected with PxMNPV. Less cadaver material was thus required to kill insects with PxGV. Larvae that were killed by PxGV survived approximately 2 d longer than did those killed by PxMNPV. When P. xylostella larvae were fed both viruses together, mortality was higher than that caused by either virus alone. The virulence of PxMNPV produced in P.␣xylostella did not differ from that of PxMNPV produced in the beet armyworm, Spodoptera exigua (Hübner). S. exigua was less susceptible to PxMNPV than to its homologous nucleopolyhedrovirus, SeMNPV. Use of trade names does not imply endorsements by USDA-ARS of products named nor criticism of similar ones not mentioned.     

Validation of a Comprehensive Process-Based Model for the Biological Control of Beet Armyworm,Spodoptera exigua,with Baculoviruses in Greenhouses

This paper describes the validation and sensitivity analysis of a process-based simulation model (BACSIM) for the control of beet armyworm, Spodoptera exigua, with baculoviruses. Model predictions are compared to results of independent greenhouse experiments in which second, third, or fourth instar larvae of S. exigua in chrysanthemum plots are treated with different concentrations of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and S. exigua MNPV (SeMNPV), two viruses with distinct differences in infectivity and mean time to kill. BACSIM provides robust predictions for the control of S. exigua populations in greenhouse chrysanthemum with both AcMNPV and SeMNPV. Mortality levels caused by AcMNPV and SeMNPV were generally predicted within a 25% margin of error compared to the observed values. None of the deviations was higher than 40%. All values of simulated foliage consumption, caused by S. exigua populations treated with AcMNPV or SeMNPV applications, fell within 95% confidence intervals of measurements. Simulated time to kill was, in general, lower than the measurements. This discrepancy may be caused by the behavior of S. exigua larvae which feed on the underside of chrysanthemum leaves where they are protected from polyhedra. This suggests that the larval foraging behavior may play an important role in the efficacy of baculovirus applications and should be further studied experimentally. This validated model can be used for the pretrial evaluation of the efficacy of genetically modified baculoviruses as biological control agents and for the optimization of spraying regimes in chrysanthemum cultivation.     

Baculovirus infection triggers a positive phototactic response in caterpillars to induce ‘tree-top’ disease

Many parasites manipulate host behaviour to enhance parasite transmission and survival. A fascinating example is baculoviruses, which often induce death in caterpillar hosts at elevated positions (‘tree-top’ disease). To date, little is known about the underlying processes leading to this adaptive host manipulation. Here, we show that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) triggers a positive phototactic response in S. exigua larvae prior to death and causes the caterpillars to die at elevated positions. This light-dependent climbing behaviour is specific for infected larvae, as movement of uninfected caterpillars during larval development was light-independent. We hypothesize that upon infection, SeMNPV captures a host pathway involved in phototaxis and/or light perception to induce this remarkable behavioural change.     

Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.     

Acid and alkaline phosphatase activity in the fat body and midgut of the beet armyworm,Spodoptera exigua (Lepidoptera: Noctuidae), infected with nuclear polyhedrosis virus

Changes in the activity of acid and alkaline phosphatase in Spodoptera exigua larvae infected with nuclear polyhedrosis virus have been investigated. Three days after per os infection, the activity of acid phosphatase in the fat body and midgut of infected larvae was significantly higher than that in normal larvae. Alkaline phosphatase activity did not show such significant changes. There were differences in the phosphatase patterns depending on whether their activities were expressed as enzyme units per milligram of fresh organ weight or per milligram of homogenate protein. The literature relevant to the subject allows us to conclude that the increase in phosphatase activities in S. exigua larvae is not specifically associated with virus infection itself, but, rather, is a reaction of the insect organism to the diminishing supply of energy sources.     

A Comparison of Growth and Development of Three Major Agricultural Insect Pests Infected with Heliothis virescens ascovirus 3h (HvAV-3h)

Ascoviruses are double-stranded DNA viruses that are pathogenic to lepidopteran hosts, particularly noctuid larvae. Infection of a larva is characterized by retarded growth, reduced feeding and yellowish body color. In this paper, we reported the growth and development of three major agricultural noctuid insect pests, Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius), infected with Heliothis virescens ascovirus 3h (HvAV-3h). Using 10-fold serial dilutions (0 to 7) of HvAV-3h-containing hemolymph to infect S. litura larvae, we found no significant difference in larval mortalities from 0 to 10³-fold dilutions; however, significant differences were observed at 10⁴-fold dilution and above. Using a 10-fold dilution of HvAV-3h-containing hemolymph to infect H. armigera, S. exigua and S. litura larvae, we found that the growth and development were significantly affected. All infected larvae could not pupate; the survival times of treated H. armigera, S. litura and S. exigua larvae were significantly longer than untreated control larvae. Body weight showed significant difference between treated and untreated control group from day 1 after inoculation in H. armigera and S. exigua, but day 2 in S. litura. Additionally, food intake also showed significant difference between treated and untreated control group from day 2 after inoculation in H. armigera and S. litura, but day 3 in S. exigua.     

The effect of nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes on hemolymph proteins, sugars, and lipids in Spodoptera exigua larvae

We determined the physiological effects of joint and separate nucleopolyhedrovirus (NPV) infection and parasitism by the endoparasitoid Microplitis pallidipes Szepligeti on biochemical events in the noctuid Spodoptera exigua (Hübner). The results indicated that in parasitized larvae, compared to healthy larvae, total protein concentration in host hemolymph began to decline and total sugar concentration significantly increased by the first day, while lipid content in the host body significantly increased by the second day after parasitization. Meanwhile, in jointly infected and parasitized hosts, compared to parasitized larvae, total protein concentration was consistently higher, total sugar concentration was consistently lower, and lipid content became higher by the second day after treatment. In virus-infected larvae, compared to healthy larvae, total protein concentration sharply declined during the first two days but increased by the third, while total sugar concentration increased on the second and third days after virus infection but decreased at other observation times, and lipid content began to increase by the second day after virus infection. Finally, in larvae that were both parasitized and virus-infected, compared to just virus-infected larvae, total protein concentration increased during the first two days but decreased by the third, total sugar concentration increased only on the first and fourth days, and lipid content decreased significantly on the first day but began to increase by the second day after treatment. These findings led us to conclude that parasitization inhibited protein mobilization but stimulated sugar mobilization in host hemolymph, and promoted lipid mobilization in the host body, while Spodoptera exigua NPV infection stimulated protein mobilization induced by parasitization but inhibited sugar mobilization induced by parasitization.     

The potential of Chrysoperla rufilabris and Doru taeniatum as agents for dispersal of Spodoptera frugiperda nucleopolyhedrovirus in maize

The behaviour of two abundant predators in Mesoamerican maize crops, Chrysoperla rufilabris larvae and Doru taeniatum adults, towards healthy and nucleopolyhedrovirus-infected Spodoptera frugiperda larvae was compared. C. rufilabris did not discriminate between healthy and virus-infected prey, although the mean search time was approximately two times longer towards virus-infected larvae. In contrast, D. taeniatum directed a greater proportion of their attacks towards virus-infected prey but there was no significant difference in the search time. Prey consumption time did not differ significantly for each type of prey by either predator, although prey consumption was much faster in D. taeniatum. Viable virus was detected in D. taeniatum faeces up to 3 d after feeding on infected S. frugiperda larvae, whereas virus was inactivated in the gut of C. rufilabris. Both predators were shown to have acidic guts. A field experiment demonstrated that D. taeniatum that had fed on infected prey could contaminate foliage resulting in the transmission of the disease at a low prevalence (4.7%) to S. frugiperda larvae in a field maize crop.     

Further studies on the distribution of a nuclear-polyhedrosis virus of the fall webworm,Hyphantria cunea,in soil

The median infectious concentration (IC₅₀) of a nuclear-polyhedrosis virus of the fall webworm, Hyphantria cunea, in soil was 7.14×10⁷ polyhedra per gram of soil. The number of virus-killed larvae equivalent to IC₅₀ decreased with the advance of the larval instar from 22 first-instar to 0.04 seventh-instar larvae per gram of soil. The degree of virus contamination of the surface soil in an area where the virus disease had occurred naturally in host populations was estimated to be as high as IC₅₀ on the basis of soil bioassays. Nearly 1000 virus-killed seventh-instar larvae/m² were necessary to obtain such extensive soil contamination. The possibility of the occurrence of so many virus-killed cadavers in the study area was discussed in relation with the past records of epizootics and host population trends. The distribution of the fallen leaves of large trees favorable to the fall webworm, such as plane and poplar trees, correlated with that of the nuclear-polyhedrosis virus in the soil.     

Mass production,infectivity, and field application studies with the entomogenous fungus Paecilomyces farinosus

Hyphal bodies (HB) of the entomogenous fungus Paecilomyces farinosus mass produced in shaken liquid cultures are infective per os and topically to Spodoptera exigua larvae. Pathogenicity of HB was greatest at pH 5.5. It was reduced by exposure for more than one-half hour to temperatures above 30°C. P. farinosus infected, killed, and produced mycelia on the surface of first-instar S. exigua larvae at a temperature—relative humidity combination as low at 15°C and 70% RH. Using two types of microdroplet applicators (droplet in the size range 1–30 and 30–90 μm, respectively) under field conditions, HB were delivered to distances of 76 m from the site of spraying. The number of HB deposited on the leaf surfaces of treated crops caused mortality of 70 to 90% in bioassayed larvae.     

Inapparent infection ofDiatraea grandiosella byHeliothis virescens cytoplasmic polyhedrosis virus

The terminal stage of infection with cytoplasmic polyhedrosis viruses (CPVs) is formation of crystal-like inclusion bodies (polyhedra) in host insects. The degree of susceptibility of larvae to CPV, based on light microscopy and presence of polyhedra, varies with the host species.Heliothis virescens (F.) andSpodoptera exigua (Hübner) are highly susceptible to CPV. In CPV treatedDiatraea grandiosella (Dyar), polyhedra were absent in all 400 + insects examined with light and electron microscopy. However,H. virescens larvae became infected when fed haemolymph ofD. grandiosella larvae or pupae (36±10 days post treatment) developed from CPV-treated larvae. No difference in pathology was observed betweenH. virescens larvae infected with CPV polyhedra and haemolymph fromD. grandiosella. This study provides evidence thatD. grandiosella can serve as a symptomless (no occlusion bodies) carrier of a CPV which is fully expressed inH. virescens species. The observation is interesting because it reveals a potentially important aspect of the epizootiology of this insect virus.     

Gender-Mediated Differences in Vertical Transmission of a Nucleopolyhedrovirus

With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H♂)×healthy females (H♀); ii) infected males (I♂)×healthy females (H♀); iii) healthy males (H♂)×infected females (I♀) and iv) infected males (I♂)×infected females (I♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.     

Host recognition by the specialist endoparasitoidMicroplitis croceipes (Hymenoptera: Braconidae): Role of host- and plant-related volatiles

The specialist parasitoidMicroplitis croceipes Cresson can parasitize only noctuid larvae in the generaHelicoverpa andHeliothis. To be successful in their search for hosts, the ability to distinguish hosts from nonhosts feeding on the same plant is beneficial. In flight tunnel experiments, we found that prior to landing on the odor sourceM. croceipes were able to distinguish volatiles released from frass of host larvae(Helicoverpa zea Boddie) and nonhost larvae (Spodoptera exigua Hübner andSpodoptera frugiperda J. E. Smith) fed on cotton. However, an initial contact experience with frass of cotton-fed host larvae appeared to be critical for this ability. Wasps that had antennated frass of host larvae fed pinto bean diet were equally attracted to frass of host and nonhost larvae fed on pinto bean diet. In short-range walking experiments, wasps located cotton-fed host larvae faster than diet-fed larvae, regardless of their experience. Wasps that had antennated frass of cotton-fed host larvae were less attracted to cotton-fed nonhost larvae, compared to host larvae, and preferred to sting host larvae. Plant-related volatiles in host frass and larvae appear to play a major role in the successful location of host larvae.