Prey selection and baculovirus dissemination by carabid predators of Lepidoptera

Abstract.

• 1 The interaction between coleopteran predators and baculovirus-infected larvae was studied in the laboratory and the field in order to assess the potential role of predators in the dissemination of a nucleopolyhedrovirus (NPV).
• 2 Preference tests using three carabid species, Harpalus rufipes De Geer, Pterostichus melanarius Illiger and Agonum dorsale Pont, showed no evidence of discrimination between healthy and diseased larvae of the cabbage moth Mamestra brassicae L. (Lepidoptera: Noctuidae) as prey items.
• 3 Virus infectivity was maintained after passage through the predator's gut. NPV mortality ranged from 97% to 20% when test larvae were exposed to faeces collected immediately after and 15 days post-infected meal respectively.
• 4 The potential for transfer of inoculum in the environment was estimated in the laboratory by soil bioassay. Carabids continuously passed infective virus to the soil for at least 15 days after feeding on infected larvae.
• 5 Field experiments showed that carabids which had previously fed on diseased larvae transferred sufficient virus to the soil to cause low levels of mortality in larval populations of the cabbage moth at different instars.

     

Differential crop damage by healthy and nucleopolyhedrovirus-infected Mamestra brassicae L. (Lepidoptera: Noctuidae) larvae: a field examination

Baculovirus infection in Lepidoptera can alter both larval mobility and feeding rates, which can in turn affect pathogen transmission and dispersal in the field. We compared the damage to cabbage plants in the field caused by healthy and nucleopolyhedrovirus-infected Mamestra brassicae L. (Lepidoptera: Noctuidae) larvae released as second and fourth instars. There was no significant difference in plant consumption by healthy and infected larvae for the first 4 days after release. From day 5 onwards, infected larvae caused significantly less defoliation. This pattern was similar for larvae at both larval instars. Defoliation was greater for fourth instars throughout the experiment.     

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.     

Behavioural observations of Pieris brassicae larvae indicate multiple mechanisms of action of analogous drimane antifeedants

We tested 11 analogous synthetic drimane antifeedant compounds for their feeding inhibiting effects on larvae of the large white butterfly Pieris brassicae L. (Lepidoptera: Pieridae) in no-choice tests on the host plant Brassica oleracea L. Furthermore, we observed larval feeding behaviour in no-choice tests to analyze temporal effects of five drimanes. The results show that the five analogous antifeedants differentially influence feeding behaviour and locomotion activity. Warburganal and polygodial are most likely sensory mediated antifeedants. Habituation to these compounds occurs soon after the onset of the tests (i.e., within 0.5–1.5 h). Compound 5 and confertifolin are probably not direct, sensory mediated antifeedants. After 0.5–1.5 h of exposure, these compounds inhibit not only feeding, but also locomotion behaviour, indicating postingestive, toxic effects. Isodrimenin inhibits feeding from the onset of the test and is probably a sensory mediated antifeedant. No habituation occurs to this compound, indicating that isodrimenin is either a very strong antifeedant or that it additionally has postingestive, toxic effects. Topical application of the drimanes on the larval cuticle revealed feeding inhibiting effects, but these could not be related to the occurrence of postingestive feeding inhibiting effects, indicating that this method is inappropriate to show possible postingestive effects of drimanes in P. brassicae. In conclusion, the behavioural observations performed in this research indicate that analogous drimanes inhibit feeding by P. brassicae larvae through multiple mechanisms of action. The results show that, when developing a structure activity relationship (SAR) for a series of antifeedants, it is important to distinguish the mode of action which underlies inhibition of feeding.     

Dynamics of Baculovirus Growth and Dispersal in Mamestra brassicae L. (LepidopteraNoctuidae) Larval Populations Introduced into Small Cabbage Plots

The nuclear polyhedrosis virus of Mamestra brassicae has been studied in larval populations of the moth introduced into small plots of cabbages. Primary dispersal of virus from single foci of infected larvae resulted from enhanced movement of the larvae, which colonized new plants logarithmically. Virus growth within the host population was quantified, and infection of young larvae in the following generation was related directly to the concentration of virus produced during the primary phase. Secondary cycling of virus resulted in dispersal of inoculum from multiple foci, and a large proportion of plants were ultimately colonized by infected larvae. The dynamics of virus growth during secondary dispersal were quantified and contrasted with results from the primary phase. The significance of these results is discussed in relation to possible control of insect pests through dispersal of virus by the host insect.     

Modified Behavior in Baculovirus-Infected Lepidopteran Larvae and Its Impact on the Spatial Distribution of Inoculum

Quantifying the rate of dispersal of target insects when infected with a disease agent will aid the development of biorational pest control programs. The effect of nucleopolyhedrovirus (NPV) infection on the mobility of second and fourth instarMamestra brassicaelarvae was investigated in the laboratory and field. NPV infection altered larval mobility, with the changes in behavior varying with the timecourse of infection. Diseased larvae moved three to five times further than healthy ones during the middle stages of infection. By the 7th day postinfection diseased larvae were less mobile than healthy counterparts. The same pattern of modified behavior was observed in both instars. Fourth instar larvae moved further than second instars under laboratory and field conditions. In the field, infected larvae tended to die on the apex of the cabbage leaves. Bioassay of the leaves showed a linear decrease in inoculum from central to peripheral plants within the plots, which occurred to the same extent for second and fourth instars. Leaves from plots where infected fourth instar larvae had been introduced had higher inoculum density than those from plots with second instars.     

Behavioural and sensory responses to drimane antifeedants in Pieris brassicae larvae

Fifteen drimane compounds were tested for their feeding inhibiting activity in larvae of Pieris brassicae L. (Lepidoptera: Pieridae) when applied to leaf material of the host plant Brassica oleracea L. The antifeedant efficacy of the drimanes was related to their molecular structure in order to identify important functional groups. Of the drimanes tested, those with a lactone group on the B-ring were the most effective feeding inhibitors. Additionally, the sensory responses to 13 of the drimanes were measured. Neural activity was evoked in the deterrent cell in the medial sensillum styloconicum. Also, inhibition of sensory responses to feeding stimulants was found. Results of behavioural and electrophysiological tests were correlated in an attempt to elucidate the sensory code underlying feeding inhibition by drimanes in Pieris brassicae. It was concluded that the response of the deterrent cell in the medial sensillum styloconicum contributes significantly to inhibition of feeding behaviour in larvae of Pieris brassicae.     

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.     

Determining the host‐plant resistance mechanisms for Mamestra brassicae (Lepidoptera: Noctuidae) pest in cabbage

Six cabbage (Brassica oleracea var. capitata) varieties with different levels of resistance to Mamestra brassicae (Lepidoptera: Noctuidae) were investigated in order to assess whether antibiosis and antixenosis mechanisms are involved in the resistance to this pest or not. Four experiments were conducted to determine the effect of variety and plant ontogeny on larval behaviour, adult oviposition and leaf damages in non‐choice and choice tests. Larval survival, time to development and larval weights differed depending on the varieties and plant stages that we tested. At the pre‐head stage, larval mortality was higher, larvae died faster, time to pupation was shorter, pupae were lighter and the percentage of viable pupae and growth index (GI) values were lower than larvae reared from plants at the head stage. The commercial hybrid ‘Corazón de buey’ and the local variety named ‘BRS0535’ exhibited antibiosis to M. brassicae as they reduced its survival and growth and delayed its development time. In addition, these varieties were the most resistant after artificial infestation in terms of head foliage consumption and number of larvae per plant. Oviposition tests demonstrated that resistance found in ‘Corazón de buey’ and BRS0535 could be also based on antixenosis mechanisms as they resulted in fewer egg batches on plants, whereas BRS0402 could be classified as resistant because M. brassicae larvae showed less preference for it. Thus, resistance to M. brassicae found in cabbage crops may be due to the joint action of several factors involving antibiosis and antixenosis. We found significant differences in the resistance of BRS0535 depending on the plant ontogeny as it loses its resistance while developing. Further studies are required to identify the mechanism of antibiotic resistance which is present in this variety at the pre‐head stage and the changes that occur in plant defence as it grows.     

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.     

Investigating the horizontal transmission of the Cydia pomonella granulovirus (CpGV) in a model system

Repeated applications of Cydia pomonella granulovirus (CpGV) can effectively control the codling moth (CM) in apple orchards. However, it is still unknown whether horizontal transmission of the virus from infected to uninfected larvae contributes to the efficacy of the virus insecticide. Horizontal transmission of CpGV was assayed using detached apples. In experiments using artificially applied virus dots on the apple’s surface or infected CM larvae as virus inoculum, it was found that the likelihood of infection of healthy CM larvae relied mainly on the larval behavior. The amount of virus inoculum, either applied artificially or produced by the infected larvae, impacted the infection rate only to a small degree. In the experiments, CM larvae exhibited a strong preference in entry sites, increasing the chance for horizontal transmission. Depending on the experimental design, horizontal transmission rates of about 40% were observed in laboratory assays.     

Responses of Mamestra brassicae (Lepidoptera: Noctuidae) to crowding: interactions with disease resistance,colour phase and growth

This study examines phenotypic plasticity in relation to rearing density in larvae of the moth, Mamestra brassicae. Larval phase, growth rate, weight at moulting and susceptibility to disease were quantified when reared at five densities. Larvae develop more quickly, but attain a smaller size and are more susceptible to disease, when reared at high than at intermediate densities. They also exhibit a higher degree of melanisation than larvae reared at intermediate densities, or singly. A review of the literature suggests that a switch to a rapidly developing dark phase at high densities is a widespread phenomenon within the Lepidoptera. Rapid development at the expense of attaining a large size, and increased melanisation, are interpreted as adaptive responses to reach pupation before food supplies are depleted, as is likely when larval density is high. High susceptibility to viral infection at high density may be a result of physiological stress associated with rapid development, or due to a shift in allocation of resources from resistance to development: larvae that developed quickly were more susceptible to infection. Larvae reared singly appeared to be less fit than larvae reared at intermediate densities: they exhibited many of the characteristics of larvae reared at high density, particularly low weight, a right-hand skew in their weight frequency distribution, and high susceptibility to disease. I hypothesise that expression of resistance may be phenotypically plastic with regard to environment. Contact with other larvae may, up to a point, stimulate both growth and resistance to infection, for the risk of infection will increase with the density of conspecifics.     

Larval Mortality Factors of <Emphasis Type="Italic">Spodoptera Littoralis</Emphasis> in the Azores

Mortality among larval developmental stages of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), was determined by weekly sampling on weeds in a pasture on São Miguel Island (Azores, Portugal), from August to December, over a 3-year period (1999–2001). In all the years surveyed, larvae of S. littoralis usually appeared in pastures after the third week of August, with higher abundances in September and the beginning of October. Three different factors causing larval mortality were identified: one fungal pathogen, Furia virescens (Thaxter) Humber (Zygomycetes: Entomophthoraceae), two nucleopolyhedroviruses and one larval parasitoid, Meteorus communis (Cresson) (Hymenoptera: Braconidae). The percentages of dead larvae infected by virus or fungus were significantly higher than the other causes of mortality, regardless of the year. Furthermore, the percentage of larvae that died due to virus contamination was generally higher than the percentage of larvae infected by fungus. Significant correlations between the environmental factors and the percentage of larvae infected by virus or by fungus, were only observed during 2001. In 2001, the prevalence of fungal infection was negatively correlated with that of viral infection although prevalences of these two agents were positively correlated in both 1999 and 2000. These results show that virus and fungus are potential biological control agents for S. littoralis in Azores.     

Risk Assessment Studies: Detailed Host Range Testing of Wild-Type Cabbage Moth, Mamestra brassicae (Lepidoptera: Noctuidae), Nuclear Polyhedrosis Virus

The host range of a multiply enveloped nuclear polyhedrosis virus (NPV) (Baculoviridae) isolated from the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae), was determined by challenging a wide range of insect species with high (10⁶ polyhedral inclusion bodies) and low (10³ polyhedral inclusion bodies) doses of the virus. The identity of the progeny virus was confirmed by dot blotting. Analysis of 50% lethal dose was carried out on selected species, and the progeny virus was identified by using restriction enzyme analysis and Southern blotting. Other than the Lepidoptera, none of the species tested was susceptible to M. brassicae NPV. Within the Lepidoptera, M. brassicae NPV was infective to members of four families (Noctuidae, Geometridae, Yponomeutidae, and Nymphalidae). Of 66 lepidopterous species tested, M. brassicae NPV was cross-infective to 32 of them; however, 91% of the susceptible species were in the Noctuidae. The relevance of host range data in risk assessment studies is discussed.     

Superparasitism in Cotesia glomerata does not benefit the host plant by reduction of herbivory caused by Pieris brassicae

Superparasitism occurs in Cotesia glomerata L. (Hymenoptera: Braconidae), a gregarious endoparasitoid of Pieris spp. (Lepidoptera: Pieridae). The responses of Pieris brassicae L. larvae to superparasitism were examined in order to elucidate the ecological significance of this behaviour. Models of tritrophic interactions often imply that attraction of herbivore natural enemies by the plant constitutes a defence. Parasitoid attack on herbivores is assumed to result in a reduction in herbivory and or an increase in plant fitness. Coupled with the active involvement of the plant in producing signals, this can be seen as an indirect mediation of wound induced defence. The results show that superparasitism of P. brassicae by the parasitoid C. glomerata reduced survivorship but increased food consumption and weight growth in P. brassicae larvae. The duration of host larval development was found prolonged as the number of oviposition increased and superparasitized larvae (three to five time parasitized) grew slower than unparasitized larvae or larvae parasitized one or two times.     

First observations in glasshouse sweet peppers in Belgium and laboratory rearing of the parasitic waspEulophus pennicornis (Hym.: Eulophidae)

Eulophus pennicornis, a hymenopteran parasitoid of several Lepidoptera, was found onMamestra brassicae in a commercial sweet pepper crop in 1990 in Belgium. The rearing of the parasitoid and some additional data on biology are described.     

Interaction between the tachinid parasite,Sturmiopsis inferens and granulosis virus in the sugarcane shoot borer,Chilo infuscatellus [Lep.: Crambidae]

Competition between granulosis virus (GV) and the larval parasite,Sturmiopsis inferens Tns. (Tachinidae: Diptera), was studied in 3^rd — and 4^th — instar larvae of the sugarcane shoot borer,Chilo infuscatellus Snellen (Crambidae: Lepidoptera), under laboratory conditions. Mortality due to GV infection and parasitization was 76.8 and 47.6 per cent, respectively, when they were tested separately. But when hosts were infected simultaneously with microfeeding of GV and larval parasite, a significantly low parasitism (5.5%) was obtained compared to 74.8 per cent mortality by GV infection. When the larvae were microfed with the GV 6 days after inoculation with parasitic maggots, mortality due to the virus was reduced significantly to 20.5 per cent, but when the maggot inoculation was preceded by virus microfeeding 6 days before, parasitization was unsuccessful, while 75% of larvae died of virus. Results obtained from field — collected larvae also showed that significantly more parasite puparia were recovered from healthy larvae than from virus — infected larvae. Similar differences in parasitization were not obtained in the case of healthy or virus — infected pupae.      

Toxicity of the limonoid allelochemical cedrelone to noctuid larvae

The limonoid cedrelone, a constituent of Cedrela toona and C. odorata (Meliaceae) was evaluated as a larval growth inhibitor for the polyphagous noctuids Peridroma saucia and Mamestra configurata by examining its effects on development and feeding behaviour. Cedrelone significantly inhibited growth of different larval instars when administered orally (in artificial diet), topically or via injection. Nutritional analyses revealed that both growth inhibition and reduced consumption are a consequence of postingestive malaise rather than a peripherally-mediated antifeedant effect. Cedrelone administered via injection to sixth instar P. saucia larvae also inhibited growth, delayed development, and resulted in considerable larval mortality. However, all larvae which survived treatment pupated successfully. The present study suggests that the toxicity of cedrelone does not likely involve the endocrine system.     

Effect of nucleopolyhedrovirus infection of Spodoptera litura larvae on host discrimination by Microplitis pallidipes

The behaviour and oviposition of solitary endoparasitoid Microplitis pallidipes Szepligeti (Hymenoptera: Braconidae) were monitored to investigate the ability of the parasitoids to distinguish between nucleopolyhedrovirus (NPV)-infected and noninfected Spodoptera litura Fabricius (Lepidoptera: Noctuidae) larvae. The results indicated that the parasitoid searching time and the time until the first parasitoid attack on infected larvae were greater than those recorded on noninfected larvae; the number of infected larvae attacked by parasitoids, the percent of first attacks and parasitism rate in infected larvae were lower than those on noninfected larvae; and these differences were all significant 3 to 5 days postexposure of the larvae to a dose of 1.6 × 10⁸ occlusion bodies (OB)· ml^?1 and significant 4 and 5 days postexposure of the larvae to a dose of 1.6 × 10⁷ OB·ml^?1. The lowest dosage (1.6 × 10⁶ OB·ml^?1) had no significant effect on the above index values. In a field cage experiment, we found that the percentage of infected larvae parasitized by M. pallidipes gradually decreased as the time after NPV inoculation (1.6 × 10⁸ OB·ml^?1) increased, and that M. pallidipes significantly preferred to oviposit in healthy larvae from day 3 to day 5 after virus inoculation. Our research concluded that this parasitoid's ability to discriminate between healthy and infected hosts increased as virus concentration increased and as the time between exposure of hosts to virus and subsequent exposure to parasitoids increased.