Effects of an ascovirus (HvAV-3e) on diamondback moth, Plutella xylostella, and evidence for virus transmission by a larval parasitoid

Ascoviruses (AVs) are pathogenic to lepidopteran larvae, and most commonly attack species in the Noctuidae. The unique pathology includes cleavage of host cells into virion-containing vesicles which leads to the milky white colouration of the hemolymph as opposed to the clear hemolymph of healthy larvae. Recently, we showed that a Heliothis virescens AV (HvAV-3e) isolate is able to induce disease in Crocidolomia pavonana F. (Lepidoptera: Crambidae), affecting feeding, growth and survival of infected larvae. In this study, we investigated the effect of different variants of HvAV-3e on diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) larvae, another non-noctuid host. In hemolymph inoculation bioassays fourth instar larvae showed a significant dose response to each of the HvAV-3e variants and significant differences between the virulence of the three variants were detected. Both second and fourth instars were readily infected with the virus and infected individuals demonstrated significant reductions in food consumption and growth. The majority of infected individuals died at the larval or pupal stage and individuals which developed into adults were usually deformed, less fecund than non-infected controls and died shortly after emergence. In transmission studies, Diadegmasemiclausum (Hymenoptera: Ichneumonidae), a key parasitoid of diamondback moth, infected healthy host larvae during oviposition following previous attack of HvAV-3e infected hosts. In choice tests D. semiclausum did not discriminate between infected individuals but host infection had no detectable impact on the development of immature D. semiclausum or on subsequent adults.     

Genome analysis of Heliothis virescens ascovirus 3h isolated from China

No ascovirus isolated from China has been sequenced so far. Therefore, in this study, we aimed to sequence the genome of Heliothis virescens ascovirus 3h (HvAV-3h) using the 454 pyrosequencing technology. The genome was found to be 190,519-bp long with a G+C content of 45.5%. We also found that it encodes 185 hypothetical open reading frames (ORFs) along with at least 50 amino acids, including 181 ORFs found in other ascoviruses and 4 unique ORFs. Gene-parity plots and phylogenetic analysis revealed a close relationship between HvAV-3h and three other HvAV-3a strains and a distant relationship with Spodoptera frugiperda ascovirus 1a (SfAV-1a), Trichoplusia ni ascovirus 6a (TnAV-6a), and Diadromus pulchellus ascovirus 4a (DpAV-4a). Among the 185 potential genes encoded by the genome, 44 core genes were found in all the sequenced ascoviruses. In addition, 25 genes were found to be conserved in all ascoviruses except DpAV-4a. In the HvAV-3h genome, 24 baculovirus repeat ORFs (bros) were present, and the typical homologous repeat regions (hrs) were absent. This study supplies information important for understanding the conservation and functions of ascovirus genes as well as the variety of ascoviral genomes.

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3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h,Inhibits the Host Larval Cathepsin and Chitinase Activities

Virologica Sinica - 3h-31 of Heliothis virescens ascovirus 3h (HvAV-3h) is a highly conserved gene of ascoviruses. As an early gene of HvAV-3h, 3h-31 codes for a non-structural protein (3H-31) of...     

Novel strategies for the biocontrol of noctuid pests (Lepidoptera) based on improving ascovirus infectivity using Bacillus thuringiensis

Identifying novel biocontrol agents and developing new strategies are urgent goals in insect pest biocontrol. Ascoviruses are potential competent insect viruses that may be developed into bioinsecticides, but this aim is impeded by their poor oral infectivity. To improve the per os infectivity of ascovirus, Bacillus thuringiensis kurstaki (Btk) was employed as a helper to damage the midgut of lepidopteran larvae (Helicoverpa armigera, Mythimna separata, Spodoptera frugiperda, and S. litura) in formulations with Heliothis virescens ascovirus isolates (HvAV-3h and HvAV-3j). Btk and ascovirus mixtures (Btk/HvAV-3h and Btk/HvAV-3j) were fed to insect larvae (3rd instar). With the exception of S. frugiperda larvae, which exhibited low mortality after ingesting Btk, the larvae of the other tested species showed three types of response to feeding on the formulas: type I, the tested larvae (H. armigera) were killed by Btk infection so quickly that insufficient time and resources remained for ascoviral invasion; type II, both Btk and the ascovirus were depleted by their competition, such that neither was successfully released or colonized the tissue; type III, Btk was eliminated by the ascovirus, and the ascovirus achieved systemic infection in the tested larvae. The feeding of Btk/ascovirus formulas led to a great reduction in larval diet consumption and resulted in a significant decrease in the emergence rate of H. armigera, M. separata, and S. litura larvae, which suggested that the formulas exerted marked oral control effects on both the contemporary individuals and the next generation of these tested pest species.     

A lepidopteran aminoacylase (L-ACY-1) in Heliothis virescens (Lepidoptera: Noctuidae) gut lumen hydrolyzes fatty acid-amino acid conjugates, elicitors of plant defense

Fatty acid-amino acid conjugates (FACs) have been identified in Lepidopteran larvae as elicitors of plant defenses. Plant responses include the production of primary defense compounds and induction of secondary defense strategies including attraction of parasitoid wasps. These elicitors are present despite fitness costs, suggesting that they are important for the larvae’s survival. In order to exploit FAC-mediated plant defense responses in agricultural settings, an understanding of FAC purpose and metabolism is crucial. To clarify their role, enzymes involved in this metabolism are being investigated. In this work a previously undiscovered FAC hydrolase was purified from Heliothis virescens frass by liquid chromatography and PAGE techniques and was identified as an aminoacylase-like protein (L-ACY-1) using MALDI-ToF/ToF and Edman sequencing. The full length gene was cloned and expressed in Escherichia coli and a polyclonal antibody against L-ACY-1 was made. L-ACY-1 was confirmed to be responsible for FAC hydrolysis activity through inhibition of N-linolenoyl-l-glutamine hydrolysis by titration with the polyclonal anti-L-ACY-1 antibody. L-ACY-1 activity is dependent on a divalent cation. This is the first time an aminoacylase has been described from an insect. L-ACY-1 appears to play a vastly different role in insects than ACYs do in mammals and may be involved in maintaining glutamine supplies for gut tissue metabolism. Identification of L-ACY-1, a FAC hydrolase, clarifies a previously uncharacterized portion of FAC metabolism.     

Inhibition of apoptosis by <Emphasis Type="Italic">Heliothis virescens</Emphasis> ascovirus (HvAV-3e): characterization of <Emphasis Type="Italic">orf</Emphasis>28 with structural similarity to inhibitor of apoptosis proteins

Ascoviruses (AVs) induce a unique pathology in their insect host cells causing cleavage of the cells into virion-containing vesicles. The mechanism by which AVs induce vesicle formation is poorly understood. It is postulated that the virus initially induces apoptosis leading to cell fragmentation. The apoptotic bodies are however, rescued by the virus to form the vesicles. Here we show that Heliothis virescens AV (HvAV-3e) is able to inhibit chemically induced apoptosis from around 16 h after infection. Analysis of the genome of the virus indicated the presence of a putative inhibitor of apoptosis (orf28) gene that encodes a protein with an imperfect baculovirus inhibitor of apoptosis repeat (BIR) and a RING domain. Transiently expressed orf28 did not inhibit chemically induced apoptosis suggesting that the protein may not serve as an inhibitor of apoptosis. Nevertheless, RNA interference studies revealed that the gene is probably essential for virus pathology and replication.     

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.     

MaleHeliothis virescens maintain upwind flight in response to experimentally pulsed filaments of their sex pheromone (Lepidoptera: Noctuidae)

Aspects of the intermittent fine structure of an odor plume were mimicked and experimentally modified in the wind tunnel using an air-pulsing device. Filaments of a behaviorally active blend of six sex-pheromone components created by the device in a temporally regular fashion elicited sustained upwind flight and source location in male Heliothis virescens.Males did not exhibit sustained upwind flight in significant numbers until a frequency of 4 filaments/s was delivered, at a loading of 1 g of the major component, Z11-16: Ald, with the other components loaded at their appropriate ratios. A loading of 10 g Z11-16: Ald was found to be optimal at this filament delivery rate. Electroantennogram recordings to different filament delivery rates of the complete blend indicated that a stationary male antenna can respond to the pulse rates used in this study. Importantly, when a main component necessary for upwind flight, Z9-14:Ald, was isolated into its own filaments and pulsed alternately against filaments of the five other components (including the other component essential for upwind flight, Z11-16: Ald), upwind flight to the source was significantly reduced (9%) compared to upwind flight and source location in response to filaments composed of the entire blend (30%), indicating that the complete pheromone blend must arrive on the antenna simultaneously for optimal evocation of sustained upwind progress. Neurophysiological evidence from other studies suggests that higher-order interneurons whose phasic response is enhanced when the entire blend is presented simultaneously may be of importance in explaining this behavioral difference stemming from synchronous vsasynchronous arrival of the components.     

Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera

Juvenile hormone biosynthesis by the corpora allata is regulated by stimulatory neuropeptides called allatotropins and inhibitory neuropeptides called allatostatins. This study localized Manduca sexta allatotropin-like material in developmental stages of the noctuid moth Heliothis virescens and the honeybee Apis mellifera. Immunocytochemical methods using both fluorescence-tagged antibodies and enzyme-coupled antibodies were used to stain the central nervous tissue of both species. H. virescens contains M. sexta allatotropin (Manse-AT)-like material consistently throughout larval development. The distribution patterns of Manse-AT immunoreactive cell bodies in the CNS persisted from one larval instar to the next. It will be discussed how larval Manse-AT distribution patterns differed from those in adults. The total number of AT-containing cells in brain and subesophageal ganglion gradually increased during larval development, whereas in the thoracic and abdominal ganglia, the number of AT-containing neurons remained constant. In the honeybee A. mellifera, Manse-AT immunoreactive cells were only found in a few brains from late last instar larvae (prepupae). Manse-AT-like material was present in a group of 6-8 cells in the pars intercerebralis. However, we did not find any Manse-AT-like material in brains of early last instar larvae, whose corpora allata (CA) are more sensitive to in vitro stimulation by Manse-AT than prepupal CA.     

Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts

The binding properties of Vip3A, a new family of Bacillus thuringiensis insecticidal toxins, have been examined in the major cotton pests, Heliothis virescens and Helicoverpa zea. Vip3A bound specifically to brush border membrane vesicles (BBMV) prepared from both insect larval midguts. In order to examine the cross-resistance potential of Vip3A to the commercially available Cry1Ac and Cry2Ab2 toxins, the membrane binding site relationship among these toxins was investigated. Competition binding assays demonstrated that Vip3A does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. BBMV protein blotting experiments showed that Vip3A does not bind to the known Cry1Ac receptors. These distinct binding properties and the unique protein sequence of Vip3A support its use as a novel insecticidal agent. This study indicates a very low cross-resistance potential between Vip3A and currently deployed Cry toxins and hence supports its use in an effective resistance management strategy in cotton.     

Effects of individual Bacillus thuringiensis insecticidal crystal proteins on adult Heliothis virescens (F.) and Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae)

Bacillus thuringiensis (Bt) is a grampositive, spore forming bacterium, which is principally distinguishedfrom other bacilli by the production of large, insecticidal,protein crystals (Insecticidal Crystal Proteins, or ICPs). Theseproteins are usually thought to act only on the actively feedinglarvae of susceptible species by a mechanism which involvesconsumption and proteolytic processing of the protein followed bybinding to, and lysis of, midgut epithelial cells. However, few authorshave reported Bt toxicity to adult insects. In the followingpaper, we expand on previous reports of toxicity to adult insects andpresent data which demonstrate that: (1) proteolytically activated ICPssignificantly reduce the lifespans of adult Heliothis virescensand Spodoptera exigua at concentrations of 500 g/ml, butnot 167 or 25 g/ml, (2) individual activated ICPs are differentiallytoxic to adult H. virescens and S. exigua, and (3)adult S. exigua are sensitive to Cry1C protoxin at aconcentration of 1 mg/ml.Deceased     

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.     

Comparison of Host-Searching and Ovipositional Behavior of Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae), a Parasitoid of Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae), in Tobacco and Cotton

To compare host-searching and ovipositional behavior of Cardiochiles nigriceps Viereck (CN) in tobacco and cotton, behavior of CN females was observed on Hellothis virescens (Febricius)-infested plants and recorded using a Psion Organizer II with Observer software. Hover, search, oviposit, preen, rest, walk, agony-search, drop-search, and miss-search were the nine behaviors observed for each plant species. Agony-search was a searching behavior exhibited when CN females were prevented from ovipositing in a host because they were unable to reach the host. Drop-search and miss-search were the searching behaviors that occurred by CN females after a host dropped from the plant and when CN females missed ovipositing in a host, respectively. Hovering, searching, oviposition, miss-search, resting, and walking were equally common for CN on tobacco and cotton. Agony-search was exhibited more on tobacco than cotton, while drop-search and preening occurred more on cotton than tobacco. Female CN spent more time hovering in tobacco than in cotton, indicating that these females preferred tobacco to cotton. Female parasitoids also spent more time in the main ovipositional sequence, hover–search–oviposit, in tobacco than in cotton, mainly because females hovered more in tobacco than in cotton. Frequency and time of behavioral sequences associated with experiencing frustration in ovipositing due to difficulty in reaching or finding a host and subsequent level of success in ovipositing in a new host were determined. Overall, the rate of successful oviposition by CN females was significantly higher in tobacco (87.7%) than in cotton (79.5%). For cotton and tobacco, the rate of successful oviposition by CN females was significantly reduced (ca. 40.0%) when they experienced frustration in ovipositing compared to the rate of ovipositional success (100%) for CN females when hosts were present and easily accessible. Encountering ovipositional frustration also increased the duration of time for successful oviposition in comparison to the time spent for problem-free oviposition in cotton and tobacco. Interestingly, with successful oviposition, the amount of time spent in a behavioral sequence was not significantly different for tobacco and cotton regardless of whether females experienced ovipositional frustration or not. However, female CN invested more time in tobacco than in cotton when they were unsuccessful in ovipositing due to the inaccessibility or disappearance of hosts. CN females' preference for tobacco over cotton probably gave them more motivation to continue searching for hosts, especially those not easily attained, on tobacco than on cotton. Nevertheless, CN females readily searched in cotton for HV even in the presence of host-infested tabacco. CN females possibly could be successful in maintaining levels of HV below an economic threshold when using a tobacco trap crop to protect cotton as the main crop.     

Bacillus thuringiensis delta-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids

We investigated the role of domain III of Bacillus thuringiensis delta-endotoxin Cry1Ac in determining toxicity against Heliothis virescens. Hybrid toxins, containing domain III of Cry1Ac with domains I and II of Cry1Ba, Cry1Ca, Cry1Da, Cry1Ea, and Cry1Fb, respectively, were created. In this way Cry1Ca, Cry1Fb, and to a lesser extent Cry1Ba were made considerably more toxic.     

Identification of a new nucleopolyhedrovirus from naturally-infected Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae) larvae on poplar plantations in South Brazil

A baculovirus was isolated from larvae of Condylorrhiza vestigialis (Guenée) (Lepidoptera: Crambidae), a pest of a forest species known as Poplar (family Salicaceae, genus: Populus) with high economic value. Electron microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra containing multiple nucleocapsids per envelope. This baculovirus was thus named Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV) and characterized by its DNA restriction endonuclease pattern, polyhedral protein, viral protein synthesis, and infectivity in insect cell lines. Restriction endonuclease profiles of viral DNA digested with five restriction enzymes were obtained and the CoveMNPV genome size was estimated to be 81 ± 2.5 kbp. The isolation of the polyhedra (OBs) was done from the crude extract of infected larvae by ultracentrifugation through sucrose gradients. These viral particles were analyzed by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed a strong band with approximately 33 kDa, corresponding to the main protein of the occlusion bodies (polyhedrin). Also, a similar band was observed for CoveMNPV infected Spodoptera frugiperda cells (SF-21 AE) pulse-labeled with [³⁵S] methionine and fractionated by SDS-PAGE. Of the four insect cell lines tested for susceptibility to CoveMNPV infection, the SF-21 AE was the most susceptible with occlusion bodies produced in most of the inoculated cells. This is the first record of an NPV from C. vestigialis.     

Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae)

In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8 × 10⁵, 1.8 × 10⁶ or 1.8 × 10⁷ conidia ml⁻¹) and Cry1Ac, (0.2 or 0.8 μg g⁻¹), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 μg g⁻¹) and B. bassiana (1.8 × 10⁵ or 1.8 × 10⁶ conidia ml⁻¹). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 μg g⁻¹) and B. bassiana (1.8 × 10⁶ conidia ml⁻¹) that showed antagonism.     

Responses of Microplitis croceipes to host and nonhost plants of Heliothis virescens in a wind tunnel

The relative attractiveness of velvet leaf (Abutilon theophrasti Medicus), cotton (Gossypium hirsutum L.) (host plants) and groundcherry (Physalis angulata L.) (nonhost plant), and cotton plants with or without nectaries and with or without glands to Microplitis croceipes Cresson (Hymenoptera: Braconidae) was determined in a wind tunnel. Female parasitoids flew significantly more to glandless than to glanded cotton; response to nectaried and nectariless cotton was similar. Velvet leaf and cotton were favored significantly over groundcherry; parasitoids being equally responsive to both host plant species. Addition of larval frass alone or in combination with host larvae significantly improved the attractivity of the nonhost plant (groundcherry) to the parasitoids. There was no difference in attractiveness of groundcherry terminals with or without host larvae. Parasitoid search time was significantly increased with addition of larval frass. In the presence of cotton, however, kairomone-treated groundcherry remained unattractive.

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Résumé L'influence des nectaires et des glandes du coton, et l'attractivité des feuilles veloutées du coton (plante hôte) et de Physalis angulata sur Microplitis croceipes, ont été déterminées dans des expériences avec tunnel à vent. Les résultats ont montré que les parasitoïdes femelles sont significativement plus attirés par les cotons sans glandes que par les cotons glanduleux, tandis que la présence ou l'absence de nectaires ne modifie pas l'attractivité du coton. Elles réagissent de la même façon aux feuilles veloutées. P. angulata traité avec des crottes de chenilles, présenté seul ou en combinaison avec du coton, attire plus de femelles que des pousses de P. angulata non traitées. L'addition de chenilles hôtes seules n'améliore pas significativement l'attractivité de la plante. P. angulata traité avec des kairomones est demeuré inattractif quand il était proposé en même temps que du coton.