Spodoptera Exigua Multiple Nucleopolyhedrovirus Research Articles

Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Vegetable waste extracts as enhancers of baculovirus infections

Vegetable waste extracts (VWE) contain a great variety of antioxidants such as polyphenols, which have shown to potentiate baculovirus infections, making them ingredients for pest control ingredients. In the present study, the mortality enhancement of different vegetable extracts obtained from food residues when combined with baculoviruses was evaluated. Extracts from spent coffee (E2), rosehip (E17), asparagus (E28), artichoke (E29), beet stalks (E32) and banana peel (E37) were selected as they increased mortality of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) in second instar S. littoralis larvae, when comparing with the virus inoculation alone. Extracts were assayed at 1 % w/v. In S. littoralis-SpliNPV system, the selected extracts reduced the median lethal concentration (LC50) of SpliNPV against second instar larvae. The E37 extract presented the highest potentiation, as it reduced the LC50 13.61 times, while the rest of the extracts presented LC50 reductions from 3.71 to 7.72-fold. In Spodoptera exigua-SeMNPV (Spodoptera exigua multiple nucleopolyhedrovirus) system, none of the extracts decreased the LC50 of SeMNPV. In contrast, in Spodoptera frugiperda-SfMNPV (Spodoptera frugiperda multiple nucleopolyhedrovirus) system, E2 showed the greatest potentiating effect. In the heterologous systems, none of the extracts tested increased the effective host range of SfMNPV, AcMNPV (Autographa californica multiple nucleopolyhedrovirus), and MbMNPV (Mamestra brassicae multiple nucleopolyhedrovirus) in second instar S. littoralis larvae. Thus, the viral enhancing effect of VWE was host-pathogen and instar dependent. However, the potentiation effect of the extracts could not be directly related with the antioxidants content of the extracts.

Entomopathogens and Parasitoids Allied in Biocontrol: A Systematic Review.

Biological pest control is an environmentally friendly alternative to synthetic pesticides, using organisms such as viruses, bacteria, fungi, and parasitoids. However, efficacy is variable and combining different biocontrol agents could improve success rates. We conducted a systematic review of studies combining a parasitoid with an entomopathogenic microorganism, the first of its kind. We searched in Web of Science and extracted data from 49 publications matching the pre-defined inclusion criteria. Combinations of 36 hymenopteran parasitoids with 17 entomopathogenic microorganisms used to control 31 target pests were found. Trichogramma pretiosum and Encarsia formosa were the most frequently studied parasitoids, while Beauveria bassiana, Metarhizium anisopliae, Lecanicillium muscarium, Bacillus thuringiensis var. kurstaki, the Spodoptera exigua multiple nucleopolyhedrovirus, and the Spodoptera frugiperda multiple nucleopolyhedrovirus were the main microbial agents assessed. Out of 49 parasitoid-microorganism combinations assessed in the laboratory experiments, thirty-eight were reported as compatible and six as incompatible. Timing and dosage of biopesticides played a crucial role, with later application and appropriate dosage minimizing adverse effects on parasitoid development. More research is needed to assess compatibility and efficacy under real-world conditions. Our review provides valuable insights for researchers and practitioners to optimize the combined use of micro- and macroorganisms for effective pest control.

Spodoptera exigua Multiple Nucleopolyhedrovirus Increases the Susceptibility to Insecticides: A Promising Efficient Way for Pest Resistance Management.

Spodoptera exigua is a polyphagous pest of diverse crops and causes considerable economic losses. The overuse of chemical insecticides for controlling this pest results in insecticide resistance, environmental pollution and toxicity to other non-target organisms. Therefore, a sustainable and efficient way for pest management is urgently required. In this study, laboratory bioassays of eleven commonly used insecticides, the specific entomopathogen of S. exigua (Spodoptera exigua multiple nucleopolyhedrovirus, SeMNPV), and SeMNPV-insecticide combinations against the S. exigua laboratory population and two field populations were tested. Our results indicated that the two field populations had developed resistance to almost half of the tested insecticides, while SeMNPV had good virulence in all populations. Interestingly, the combined use of SeMNPV enhanced the toxicity of the tested insecticides against all populations to a different extent and considerably reduced the insecticide resistance of S. exigua field populations or even recovered the susceptibility to above insecticides. Furthermore, the field trial showed that the combined application of SeMNPV contributed to promoting the control efficacy of emamectin benzonate and chlorfenapyr. These results provide a promising efficient way for pest resistance management and an environmentally friendly approach for controlling S. exigua with the combined application of nucleopolyhedroviruses and insecticides.

Antiviral function of peptidoglycan recognition protein in Spodoptera exigua (Lepidoptera: Noctuidae).

Peptidoglycan recognition proteins (PGRPs) are a class of molecules that play a critical role in insect immunity. Understanding the function of PGRPs is important to improve the efficiency of microbial insecticides. In this study, we investigated the role of PGRP-LB (a long type PGRP) in insect immunity against viruses using Spodoptera exigua and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) as an insect-virus model. We cloned and identified a PGRP-LB gene from S.exigua; the gene consisted of 7 exons that encoded a polypeptide of 234 amino acids with a signal peptide and a typical amidase domain. Expression analysis revealed that the abundance of SePGRP-LB transcripts in the fat body was greater than in other tissues. Overexpression of SePGRP-LB resulted in a significant decrease of 49% in the rate of SeMNPV-infected cells. In addition, the multiplication of SeMNPV was significantly decreased: a decrease of 79% in the production of occlusion-derived virion (ODV), and a maximum decrease of 50% in the production of budded virion (BV). In contrast, silencing of SePGRP-LB expression by RNA interference resulted in a significant 1.65-fold increase in the rate of SeMNPV-infected cells, a significant 0.54-fold increase in ODV production, a maximum 1.57-fold increase in BV production, and the larval survival dropped to 21%. Our findings show that SePGRP-LB has an antiviral function against SeMNPV, and therefore this gene may provide a target for lepidopteran pest control using virus insecticides.

Study on physical properties of four pH responsive Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) microcapsules as controlled release carriers

This study provides a promising controlled release form of nuclear polyhedrosis virus (NPV) for targeted control of lepidopteran pests. However, the application of NPV is limited due to its sensitivity to UV inactivation. This study investigated the anti-UV properties of microcapsules of SeMNPV occlusion bodies (OBs) encapsulated by calcium alginate (CA), and also the influence of the modification of CA by chitosan (CS), whey protein (WP), and polydopamine (PDA). These capsules were used to deliver, in a controlled release manner virions under alkaline pH conditions. Characterization of the structure, morphology, particle size, encapsulation efficiency, contact angle, insecticidal activity, UV resistance and in vitro release of the microcapsules was conducted. The modified microcapsules had better sphericity, and were devoid of SeMNPV OBs on the surface. The encapsulation rate was 84.76 ± 0.59%. PDA@CA-NPV had the highest wettability and the contact angle was 74.51 ± 0.53°. The 50% lethal concentration values (LC50) of CA-NPV, CS@CA-NPV, WP@CA-NPV and PDA@CA-NPV were 11.5, 10.7, 10.5 and 1.2 times that of SeMNPV OBs alone. The modified microcapsules all improved the anti-UV performance of the virus, and PDA@CA-NPV was the most UV-resistant. Using qPCR, it was observed that under alkaline conditions, a large number of virions were released from PDA@CA-NPV, CA-NPV and SeMNPV OBs. Microencapsulated virus under alkaline conditions did not change the release pattern of virions.

Host plants affect the susceptibility of Spodoptera exigua to its homologous nucleopolyhedrovirus: role of chitin synthase and intestinal mucin in the peritrophic matrix

ABSTRACT Host plants have an effect on the susceptibility of lepidopteran insects to nucleopolyhedrovirus (NPV), but the role of the protein in peritrophic membrane (PM) of larvae has not been investigated. We determined a key PM gene (SeIIM8 gene) in Spodoptera exigua (Hübner), which plays a role in protecting against NPV infection. Then we quantified the activity of chitin synthase (SeCHS), the content of insect intestinal mucin (SeIIM) and the expression of their genes in S. exigua fed one of four foods (artificial diet, Brassica oleracea (L.), Glycine max (L.), or Ipomoea aquatica (Forsskal)) with or without inoculation of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV). Our results showed that the larval mortality was highest for NPV-infected with siRNA-injected larvae, intermediate for NPV-infected larvae, and lowest for uninfected larvae. The SeCHS activity, SeIIM content, and their relative gene expressions of S. exigua fed G. max or artificial diet were lower compared to caterpillars fed other diets. The relative SeCHS and SeIIM8 gene expression levels of larvae depended on the interaction of NPV treatment, diet, and time. This study concluded that plants affected the susceptibility of insects to NPV by altering the enzyme activity and gene expression of insects.

Characterization and genomic analyses of a novel alphabaculovirus isolated from the black armyworm, Spodoptera cosmioides (Lepidoptera: Noctuidae)

The black armyworm Spodoptera cosmioides is a pest of increasing importance in Cry1Ac-Bt toxin crops and non-Bt crops of soybean and cotton in Brazil. Here we characterized a baculovirus isolated from extracts of S. cosmioides that died with symptoms of nuclear polyhedrosis. The putative novel virus exhibited polyhedral occlusion bodies (OBs) with virions containing multiple rod-shaped nucleocapsids, characteristic of alphabaculoviruses. The virus isolate was named Spodoptera cosmioides nucleopolyhedrovirus isolate CNPSo-72 (SpcoNPV-CNPSo-72). SpcoNPV-CNPSo-72 was lethal to third-instar S. cosmioides caterpillars but not to S. frugiperda under the tested viral concentrations. Moreover, SpcoNPV-CNPSo-72 contained a circular 147,763 bp long genome and a G + C content of 44.8% with 151 annotated ORFs (10 unique for baculovirus) and five homologous regions (hrs). The 38 currently defined baculovirus core genes were found in the SpcoNPV-CNPSo-72 genome. After phylogenetic analysis, the novel virus was found to be closely related to other members of Alphabaculovirus, especially to the Spodoptera-infecting viruses, which included Spodoptera eridania nucleopolyhedrovirus isolate 251, Spodoptera litura nucleopolyhedrovirus isolate II, Spodoptera exigua multiple nucleopolyhedrovirus isolate US-1, Spodoptera eridania nucleopolyhedrovirus isolate CNPSo-165, and Spodoptera frugiperda multiple nucleopolyhedrovirus isolate 19. Surprisingly, the new baculoviral genome was found to code for a putative arginine-associated tRNA gene with a predicted intronic sequence of 105 nt. The gene was found inside the bjdp CDS. Overall, baculoviruses are pathogens that lethally infect insect larvae and their study allows a better understanding of large DNA virus evolution, which provides important insights for the development and improvement of biological control agents.

Influence of temperature on occlusion body production in a Mexican isolate of Spodoptera exigua multiple nucleopolyhedrovirus

ABSTRACT In vivo production of baculoviruses requires optimisation studies for each host-virus system. Considering the potential of a Mexican isolate of Spodoptera exigua multiple nucleopolyhedrovirus (SeSIN6) for developing a biopesticide, here, a compartmentalised tray system and a cool-textured diet were used to determine the effect of incubation temperature on the pathogenicity, speed of kill, and production of occlusion bodies (OBs). All measurements were compared with a characterised American nucleopolyhedrovirus isolate (SeUS2). Incubation of fourth instar S. exigua at 20, 25, and 30°C did not significantly affect the mortality caused by either isolate, although a delay in virus infection was observed at 20°C. The OB production of both isolates in infected larvae was significantly lower at 20°C than at 25 and 30°C. At all temperatures, the OB production of the SeSIN6 isolate was higher than the SeUS2 isolate. The production of OBs per mg larva was significantly higher at 30°C for both isolates. A positive correlation was observed between larval weight gain and the production of OBs/larva at all temperatures, whereas a negative correlation was observed between larval gain weight and OBs/mg of larval weight in most of the treatments. We conclude that incubation temperatures of 25 and 30°C are optimal for OB production per larva in both isolates tested. Further studies are needed to examine other factors that can improve the production of Mexican SeMNPV isolates.

Baculovirus infection affects caterpillar chemoperception

Baculoviruses are double-stranded DNA entomopathogenic viruses that infect predominantly insects of the order Lepidoptera. Research in the last decade has started to disentangle the mechanisms underlying the insect-virus interaction, particularly focusing on the effects of the baculovirus infection in the host's physiology. Among crucial physiological functions, olfaction has a key role in reproductive tasks, food source detection and enemy avoidance. In this work, we describe that Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces expression changes in some odorant receptors (ORs) - the centrepiece of insect's olfaction - when infecting larvae from its natural host Spodoptera exigua (Lepidoptera: Noctuidae). Different ORs are up-regulated in larvae after SeMNPV infection, and two of them, SexiOR35 and SexiOR23, were selected for further functional characterization by heterologous expression in empty neurons of Drosophila melanogaster coupled to single-sensillum recordings. SexiOR35 appears to be a broadly tuned receptor able to recognise multiple and different chemical compounds. SexiOR23, although correctly expressed in Drosophila neurons, did not display any significant response to a panel of 58 stimuli. Behavioural experiments revealed that larvae infected by SeMNPV exhibit altered olfactory-driven behaviour to diet when it is supplemented with the plant volatiles linalool or estragole, two of the main SexiOR35 ligands, supporting the hypothesis that viral infection triggers changes in host perception through changes in the expression level of specific ORs.

A proctolin-like peptide is regulated after baculovirus infection and mediates in caterpillar locomotion and digestion.

Baculoviruses constitute a large group of invertebrate DNA viruses, predominantly infecting larvae of the insect order Lepidoptera. During a baculovirus infection, the virus spreads throughout the insect body producing a systemic infection in multiple larval tissues, included the central nervous system (CNS). As a main component of the CNS, neuropeptides are small protein-like molecules functioning as neurohormones, neurotransmitters, or neuromodulators. These peptides are involved in regulating animal physiology and behavior and could be altered after baculovirus infection. In this study, we have investigated the effect of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) infection on expression of Spodoptera exigua neuropeptides and neuropeptide-like genes. Expression of the gene encoding a polypeptide that resembles the well-known insect neuropeptide proctolin and named as proctolin-like peptide (PLP), was downregulated in the larval brain following infection and was chosen for further analysis. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) overexpressing the C-terminal part of the PLP was generated and used in bioassays using S. exigua larvae to study its influence on the viral infection and insect behavior. AcMNPV-PLP-infected larvae showed less locomotion activity and a reduction in growth compared to larvae infected with wild type AcMNPV or mock-infected larvae. These results are indicative of this new peptide as a neuromodulator that regulates visceral and skeletal muscle contractions and offers a novel effector involved in the behavioral changes during baculovirus infection.

Use of biocides to minimize microbial contamination in Spodoptera exigua multiple nucleopolyhedrovirus preparations

The presence of contaminant microbes in baculovirus-based insecticides is regulated by phytosanitary product registration authorities. We aimed to determine whether the abundance of microbes in suspensions of Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies (OBs) could be reduced by treatment with a range of biocidal compounds. The diversity of contaminant bacteria was determined by next-generation sequencing of the 16S rRNA gene. Overall, 97.9% of sequences detected were Gammaproteobacteria (mostly Pseudomonas spp. and Enterobacteriaceae) and 2.1% were Firmicutes (mostly Enterococcus spp.). Colloidal silver, benzalkonium chloride and chlorhexidine digluconate were identified as highly effective biocides. Incubation of OB suspensions with high concentrations of colloidal silver (450 mg/l) or benzalkonium chloride (6000 mg/l) resulted in marked reductions in colony forming unit counts over a 180 day period at 4° or 25 °C.Benzalkonium chloride and colloidal silver treatments, at either 4 or 25 °C, did not affect the insecticidal activity of OBs over an 80 day period. However, OB activity decreased following 180 days of treatment by benzalkonium chloride at either 4 or 25 °C, or by colloidal silver at 25 °C, but not at 4 °C. Counts of OBs revealed a significant decrease in OB numbers in benzalkonium chloride-treated suspensions after 180 days at both temperatures, whereas colloidal silver-treated OBs were not affected. Benzalkonium chloride also caused aggregation of OBs at the concentration tested. We conclude that biocidal compounds can markedly reduce the abundance of contaminant microorganisms in OB suspensions, and can be accompanied by reductions in OB infectivity and OB numbers in some circumstances. Future studies should focus on lower concentrations of biocides that do not affect OBs in long-term storage.

Iflavirus Covert Infection Increases Susceptibility to Nucleopolyhedrovirus Disease in Spodoptera exigua.

Naturally occurring covert infections in lepidopteran populations can involve multiple viruses with potentially different transmission strategies. In this study, we characterized covert infection by two RNA viruses, Spodoptera exigua iflavirus 1 (SeIV-1) and Spodoptera exigua iflavirus 2 (SeIV-2) (family Iflaviridae) that naturally infect populations of Spodoptera exigua, and examined their influence on susceptibility to patent disease by the nucleopolyhedrovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) (family Baculoviridae). The abundance of SeIV-1 genomes increased up to ten-thousand-fold across insect developmental stages after surface contamination of host eggs with a mixture of SeIV-1 and SeIV-2 particles, whereas the abundance of SeIV-2 remained constant across all developmental stages. Low levels of SeIV-2 infection were detected in all groups of insects, including those that hatched from surface-decontaminated egg masses. SeIV-1 infection resulted in reduced larval weight gain, and an unbalanced sex ratio, whereas larval developmental time, pupal weight, and adult emergence and fecundity were not significantly affected in infected adults. The inoculation of S. exigua egg masses with iflavirus, followed by a subsequent infection with SeMNPV, resulted in an additive effect on larval mortality. The 50% lethal concentration (LC50) of SeMNPV was reduced nearly 4-fold and the mean time to death was faster by 12 h in iflavirus-treated insects. These results suggest that inapparent iflavirus infections may be able to modulate the host response to a new pathogen, a finding that has particular relevance to the use of SeMNPV as the basis for biological pest control products.

Outcome of mixed DNA virus infections on Spodoptera exigua susceptibility to SeMNPV

Baculovirus-based biopesticides have been developed for an efficient control of lepidopteran pests; however, very few studies have assessed the effect of other viral microorganisms that might coexist with the applied baculovirus pesticide. Systematic studies are needed to predict the effect of such microorganisms on the baculovirus efficacy following application in the field. In this context, we examined the effect of a sublethal doses of the DNA virus Junonia coenia densovirus (JcDV) on the infectivity of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) when infecting the same host, Spodoptera exigua. In our experimental conditions, the infection timing was critical for the mixed infection outcome. The consecutive infections with SeMNPV and later with JcDV did not affect the larval mortality or the measured infection parameters compared with single infection with the baculovirus. However, when larvae were first infected with JcDV, a clear antagonism was detected between both viruses and resulted in a patent reduction in viral pathogenicity and deleterious effects observed in the SeMNPV progeny. On the other hand, the simultaneous infection with both viruses led to an additive effect of the mortalities caused by the two viruses. Our results reveal that temporal order of the infection with one or the other virus affects the outcome of mixed viral infections on the targeted pest and may influence the ecology of baculovirus and its use as biological insecticide.

Identification and genomic sequence analysis of a new Spodoptera exigua multiple nucleopolyhedrovirus, SeMNPV-QD, isolated from Qingdao, China.

A new beet armyworm (Spodoptera exigua) multiple nucleopolyhedrovirus SeMNPV-QD was isolated from dead larvae in the field in Qingdao, Shandong, China. The virus has a polyhedron size of 1.39 ± 0.28 μm, and typical virions contain one to seven nucleocapsids per envelope. SeMNPV-QD only infects the larvae of S. exigua; it does not infect larvae of S. litura, Agrotis ipsilon, A. segetum, Bombyx mori, Hyphantria cunea, or Stilpnotia salicis, and thus, has higher host specificity. SeMNPV-QD has a circular double-stranded DNA genome of 128,525 bp with a GC content of 37.41% and 127 putative open reading frames (ORF), each of which encodes more than 50 amino acids. These were identified and annotated in the SeMNPV-QD genome, accounting for 87.53% of the whole genome. Phylogenetic analysis of 38 core genes of the baculovirus confirmed that SeMNPV-QD is a Group II Alphabaculovirus and is most closely related to SeMNPV American isolate (SeMNPV-US1), S. litura nucleopolyhedrovirus II (SpliNPV-II), S. frugiperda multiple nucleopolyhedrovirus (SfMNPV), A. segetum nucleopolyhedrovirus (AgseNPV), A. segetum nucleopolyhedrovirus B (AgseNPV-B) and A. ipsilon multiple nucleopolyhedrovirus (AgipNPV). The pairwise distance of the nucleotide sequences of lef-8, lef-9, polh and concatenated lef-8/lef-9/polh fragments between SeMNPV-QD and several sister viruses mentioned above were all above 0.05 substitutions/site. SeMNPV-QD has 123 ORFs similar to those of SeMNPV-US1, and the genomic similarity was 45.8%. Compared to SeMNPV-US1, SeMNPV-QD has four additional ORFs such as two baculovirus repeat ORF (bro) genes, bro-1, bro-2, orf39 and orf95, but lacks 17 ORFs that have no effect on viral transcription and replication. The above results indicate that SeMNPV-QD is a new species of S. exigua multiple nucleopolyhedrovirus.

Why do Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies lose insecticidal activity on amaranth (Amaranthus hypocondriacus L.)?

Amaranth (Amaranthus hypocondriacus L.) is a pseudocereal that is widely consumed in Mexico and other tropical regions, but which is attacked by Spodoptera exigua (Hübner) larvae. Products based on Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) can be highly effective insecticides, but when applied to amaranth viral occlusion bodies (OBs) lost 50% of their activity in a 6 h period. A series of experiments was performed to examine the role of solar radiation, leaf oxidative enzymes, leaf pH and insect feeding behavior, all of which failed to explain the reduced prevalence of virus infection in S. exigua larvae on amaranth. The concentration of the flavonoid rutin on amaranth leaves was >200-fold higher than on another preferred host plant, sweet pepper (Capsicum annuum). However, in laboratory assays, rutin generally resulted in an increased prevalence of infection when administered in mixtures with semi-synthetic diet and OBs. We conclude that the cause of OB inactivation on amaranth remains elusive, although understanding how this will influence its efficacy as a biological insecticide requires field studies.

Natural Enemies of the Sugar Beet Army Worm, Spodoptera exigua (Lepidoptera: Noctuidae) in Northeast Iran

During 2014-2015, a survey was carried out to determine the natural enemies of the sugar beet armyworm, Spodoptera exigua (Hübner) in the Mashhad region of northeastern Iran. Through extensive sampling, different larval stages of S. exigua with possible signs or symptoms of infection were collected from beet fields, kept in laboratory conditions, fed with fresh beet leaves, and checked daily until the emergence of parasitoids. Herein, 12 species of parasitoids, a virus: Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), an entomopathogenic fungus: Beauveria varroae, and an entomophilic nematode, Pristionchus pacificus are reported. The parasitoids were Meteorus rubens, Microplitis rufiventris, Cotesia sp., Chelonus (Chelonus) annulipes (Braconidae), Euplectrus sp. (Eulophidae), Sinophorus nr. xanthostomus, Anomalon sp., Diadegma pulchripes, Temelucha sp. (Ichenumonidae), two species of Drino genus and Exorista sp. (Tachinidae). The body tissues of the virus-infected larvae were liquefied, swollen, and glossy with a very thin cuticle. SEM photographs of viral occlusion bodies (OBs) showed polyhedral and irregular shapes. A cross-section of an OB indicated that each virion contained multiple nucleocapsids arranged randomly within the occlusion matrix. Genetic analysis based on lef-8 and Polyhedrin gene sequences confirmed the identity of the Iranian strain as SeMNPV. The virus was transmitted vertically and horizontally. Another pathogen was Beauveria varroae, isolated from a lepidopteran insect for the first time; S. exigua was indicated as a new host for this fungus. Both pathogens are herein introduced as new records for Iran microflora. The results of the current work highlight the diversity of natural enemies on the sugar beet armyworm in Iran.

Baculovirus PTP2 Functions as a Pro-Apoptotic Protein.

The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as immunological responses, which may affect virus transmission. Here we show that the SeMNPV-encoded protein tyrosine phosphatase 2 (PTP2) induces mild apoptosis in Spodoptera frugiperda (Sf) 21 cells upon transient expression. Transient expression of a catalytic-site mutant of ptp2 did not lead to apoptosis, indicating that the phosphatase activity of PTP2 is needed to induce apoptosis. We also found that the caspase level (indicator of apoptosis) was higher in cells transfected with the ptp2 gene than in cells transfected with the catalytic mutant. Adding a caspase inhibitor reduced the level of ptp2-induced apoptosis. Moreover, deletion of the ptp2 gene from the viral genome prevented the induction of apoptosis in S. exigua hemocytes. The virus titer and virulence indices (the viral infectivity and the time to death) were not affected by deletion of the ptp2 gene. However, the viral occlusion body yield from S. exigua larvae infected with the mutant virus lacking the ptp2 gene was much lower than the yield from larvae infected with the wild-type (WT) virus. We hypothesize that the observed pro-apoptotic effects of PTP2 are the result of PTP2-mediated immune suppression in larvae, which consequently leads to higher viral occlusion body yields.

Timely trigger of caterpillar zombie behaviour: temporal requirements for light in baculovirus-induced tree-top disease.

Host behavioural manipulation is a common strategy used by parasites to enhance their survival and/or transmission. Baculoviruses induce hyperactivity and tree-top disease (pre-death climbing behaviour) in their caterpillar hosts. However, little is known about the underlying mechanisms of this behavioural manipulation. A previous study showed that the baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induced tree-top disease at 3 days post infection in third instar S. exigua larvae and that light plays a key role in triggering this behaviour. Here we investigated the temporal requirements for the presence of light to trigger this behaviour and found that light from above was needed between 43 and 50 h post infection to induce tree-top disease. Infected larvae that were not exposed to light from above in this period finally died at low positions. Exposure to light prior to this period did not affect the final positions where larvae died. Overall we conclude that light in a particular time frame is needed to trigger SeMNPV-induced tree-top disease in S. exigua larvae.

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.