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.     

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.     

烟芽夜蛾囊泡病毒3h株编码的3H-117蛋白能干扰AcMNPV的口服活性

[目的]囊泡病毒是一类体液传播的昆虫病毒,具有特殊的致病历程和良好的生防应用潜力.烟芽夜蛾囊泡病毒3h株(Heliothis virescens ascovirus 3h,HvAV-3h)是我国分离的第一株囊泡病毒,其编码的3H-117蛋白被报道为HvAV-3h病毒粒子的结构蛋白.[方法]为了进一步探究3h-117基因...     

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.     

Host hemolymph monophenoloxidase activity in parasitized Manduca sexta larvae and evidence for inhibition by wasp polydnavirus

In insects, one of the primary routes of defense against parasites is encapsulation by hemocytes followed by melanization, in which tyrosine and DOPA are converted to melanin via toxic quinone intermediates. This report describes the use of an in vitro radiochemical assay to monitor hemolymph monophenoloxidase (MPO) conversion of [³H]tyrosine to [³H]DOPA, using a method utilized previously for dipteran and mammalian enzymes. Parasitism of fifth instar tobacco hornworm larvae by the braconid wasp Cotesia congregata depresses the rate of hemolymph monophenoloxidase activity in the host. Significant inhibition of hemolymph MPO was detectable in newly parasitized larvae and terminal stage hosts. A similar effect was seen in unparasitized larvae following injection of sucrose-gradient purified wasp polydnavirus (PDV) particles, which are normally injected by the female wasps into the host, suggesting the inhibition may be virally mediated. Hemolymph MPO activity was assayed in vitro 24 h after injection of PDV in vivo, and intercalation of viral DNA by exposure to psoralen and long-wave u.v. light eliminated its inhibitory effect on MPO. Despite inhibition of hemolymph MPO activity by parasitism, host cuticular enzymes appear unimpaired, since cuticular melanization occurs at sites of integumental wounding during emergence of the wasps from the host. Red pigments appear in the dorsal vessel and integument of parasitized and virus-injected larvae; whether these pigmentation changes are related to effects of parasitism on tyrosine metabolism and ommochrome biosynthesis remains to be determined.     

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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Phylogenetic position and replication kinetics of Heliothis virescens ascovirus 3h (HvAV-3h) isolated from Spodoptera exigua

Insect-specific ascoviruses with a circular genome are distributed in the USA, France, Australia and Indonesia. Here, we report the first ascovirus isolation from Spodoptera exigua in Hunan, China. DNA-DNA hybridization to published ascoviruses demonstrated that the new China ascovirus isolate is a variant of Heliothis virescens ascovirus 3a (HvAV-3a), thus named HvAV-3h. We investigated the phylogenetic position, cell infection, vesicle production and viral DNA replication kinetics of HvAV-3h, as well as its host-ranges. The major capsid protein (MCP) gene and the delta DNA polymerase (DNA po1) gene of HvAV-3h were sequenced and compared with the available ascovirus isolates for phylogenetic analysis. This shows a close relationship with HvAV-3g, originally isolated from Indonesia, HvAV-3e from Australia and HvAV-3c from United States. HvAV-3h infection induced vesicle production in the SeE1 cells derived from S. exigua and Sf9 cells derived from S. frugiperda, resulting in more vesicles generated in Sf9 than SeE1. Viral DNA replication kinetics of HvAV-3h also demonstrated a difference between the two cell lines tested. HvAV-3h could readily infect three important insect pests Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius) from two genera in different subfamilies with high mortalities.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Hemolymph melanization and alterations in hemocyte numbers in Lymantria dispar larvae following infections with different entomopathogenic microsporidia

Lymantria dispar (L.) (Lepidoptera: Lymantriidae) larvae can be infected in the laboratory with a variety of entomopathogenic microsporidia. In many cases, however, L. dispar is only a semi‐permissive host for such infections. In this study, we analyzed changes in the melanization of hemolymph and hemocyte numbers in L. dispar larvae after inoculation with various entomopathogenic microsporidia. We compared the infections produced by microsporidia isolated from L. dispar and infections produced by isolates from other Lepidoptera to which L. dispar is only a semi‐permissive host. Microsporidiosis induced a significant activation of the prophenoloxidase system leading to melanization; activation was highest when the pathogen caused heavy infections of the fat body, which was the case with two microsporidia originally isolated from L. dispar. Infection of only the silk glands or light infection of the fat body by two Vairimorpha spp. from other lepidopteran hosts elicited a lower response. Very light infections caused by a microsporidium isolated from Malacosoma americanum were not accompanied by elevated hemolymph melanization activity. Heavy infections by Endoreticulatus spec. that remained restricted to the gut tissue likewise did not elicit melanization. One Vairimorpha spec. from L. dispar induced a significant increase in total hemocyte numbers; the other infections led to temporarily decreased numbers. Microscopic examinations showed that parts of infected tissue were encapsulated by hemocytes. We conclude that measured alterations in hemolymph melanization and hemocyte numbers were likely to be induced by the damaging effects of heavy infections. Observed defense responses did not prevent the progression of infections.     

Genomic Sequence of Heliothis virescens Ascovirus 3g Isolated from Spodoptera exigua

Heliothis virescens ascovirus 3a (HvAV-3a), a member of the family Ascoviridae, has the highest diversity among ascovirus species that have been reported in Australia, Indonesia, China, and the United States. To understand the diversity and origin of this important ascovirus, the complete genome of the HvAV Indonesia strain (HvAV-3g), isolated from Spodoptera exigua, was determined to be 199,721 bp, with a G+C content of 45.9%. Therefore, HvAV-3g has the largest genome among the reported ascovirus genomes to date. There are 194 predicted open reading frames (ORFs) encoding proteins of 50 or more amino acid residues. In comparison to HvAV-3e reported from Australia, HvAV-3g has all the ORFs in HvAV-3e with 6 additional ORFs unique to HvAV-3g, including 1 peptidase C26 gene with the highest identity to Drosophila spp. and 2 gas vesicle protein U (GvpU) genes with identities to Bacillus megaterium. The five unique homologous regions (hrs) and 25 baculovirus repeat ORFs (bro) of HvAV-3g are highly variable.     

Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

Regulation of melanization by glutathione in the moth Pseudoplusia includens

The phenoloxidase (PO) cascade regulates the melanization of hemolymph, which serves as a conserved humoral immune response in insects and other arthropods. The reductant glutathione (GSH) has long been used to inhibit melanization of hemolymph from insects but whether GSH levels in hemolymph are sufficient to play a physiological role in regulating melanization is unknown. Here, we characterized the abundance and effects of GSH on the melanization of plasma from larval stage Pseudoplusia includens (Lepidoptera: Noctuidae). GSH concentration in newly collected plasma from day two fifth instars ranged from 50 to 115 μM, while the titer of tyrosine, a substrate for the PO cascade, was 141 μM. GSH titers rapidly declined in plasma after collection from larvae, but no melanin formation occurred until GSH levels fell below 20 μM. Added GSH dose-dependently blocked melanization while PO substrates overrode GSH inhibition. Experiments conducted in the absence of oxygen and presence of PO cascade inhibitors further suggested that depletion of GSH from plasma was primarily due to formation of reactive intermediates produced by activated PO. Additional studies identified hemocytes as a potential source of plasma GSH. Hemocyte lysates recycled oxidized glutathione (GSSG) into GSH using NADPH, while intact hemocytes released GSH into the medium. These results suggest that in addition to protease cascade-releated mechanisms that regulate phenoloxidase, GSH exerts another level of control on melanization of insect hemolymph.     

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD₅₀ in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72 h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72 h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.     

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.     

Effects of Heliothis virescens ascovirus (HvAV-3e) on a novel host, Crocidolomia pavonana (Lepidoptera: Crambidae)

Ascoviruses are double-stranded DNA viruses which cause fatal disease in lepidopteran host larvae. They induce a unique pathology, causing cleavage of host cells into virion-containing vesicles. With the single exception of Diadromus pulchellus ascovirus, all ascoviruses have been exclusively reported from the Noctuidae. To investigate whether Heliothis virescens AV (HvAV-3e) has a broader host range at the family level, larvae of Crocidolomia pavonana F. (Lepidoptera: Crambidae), a major pest of brassica crops in tropical and sub-tropical regions of the Old World and Australasia, were inoculated with HvAV-3e. Larvae were readily infected by the ascovirus and feeding, growth and survival were significantly affected. However, the milky white discolouration of the haemolymph which is characteristic of ascovirus infection in noctuid hosts was not apparent. In further contrast to infected noctuid host larvae that do not develop to the pupal stage, a significant proportion of infected C. pavonana larvae pupated but all were killed at this stage. Thus, C. pavonana appears to be a semi-permissive host of the ascovirus, the presence of such hosts in the field might be an explanation for the conundrum for the ascovirus-noctuid-wasp relationship, helping explain the persistence of the ascovirus.     

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

Joint toxicity of methoxyfenozide and lufenuron on larvae of Spodoptera exigua Hübner (Lepidoptera: Noctuidae)

The joint action and sublethal effects of methoxyfenozide and lufenuron were measured against Spodoptera exigua. Methoxyfenozide and lufenuron exhibited optimum synergistic toxicity on S. exigua at a mass ratio of 4:6, and the co-toxicity coefficient (CTC) was 165.705. Third instars larvae of S. exigua were treated with methoxyfenozide (LC₁₅ = 21.004 ng/cm²), lufenuron (LC₁₅ = 27.134 ng/cm²), or a mixture of methoxyfenozide and lufenuron (MML, LC₁₅ = 16.503 ng/cm²) through feeding for 72 h. Ingestion of MML by larvae significantly inhibited larval and pupal weights and pupation rate, and prolonged the larval and pupal development of S. exigua compared to individual treatment ofmethoxyfenozide or lufenuron. Both methoxyfenozide and MML treatments significantly decreased the fertility of female S. exigua. No significant changes were observed in case of adult emergence and egg hatching for different treatments. The MML-treated S. exigua exhibited significantly lower activities of polyphenol oxidase (PPO) and cytochrome P450 (CYP450) than those in S. exigua treated separately with methoxyfenozide or lufenuron. Finally, methoxyfenozide, lufenuron, and MML treatments decreased chitinase, acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione-s-transferase (GST) activities in S. exigua.     

Bumblebee Venom Serine Protease Increases Fungal Insecticidal Virulence by Inducing Insect Melanization

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow, and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.