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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.

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.

Previous studies identified distinct genotypes of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) that were associated with horizontal transmission (named HT-SeG25) or vertical transmission (named VT-SeAl1) in the host insect, S. exigua (Lepidoptera: Noctuidae). We examined the use of mixtures of occlusion bodies (OBs) of these genotypes as the basis for a virus preparation that could provide immediate pest control and establish a persistent sublethal infection in the survivors of an OB application for transgenerational pest suppression. Mixtures of HT-SeG25 + VT-SeAl1 comprising 25:75 or 75:25 % of each genotype, respectively, resulted in improved OB pathogenicity in terms of concentration-mortality metrics compared to OBs of VT-SeAl1 alone or similar values compared to OBs of the HT-SeG25 genotype alone. In contrast, no significant differences were observed in speed of kill or mean OB production per larva. Laboratory and greenhouse trials revealed that the prevalence of sublethal infection in adults that survived OB treatments in the larval stage increased with the proportion of VT-SeAl1 present in the inoculum, as determined by qPCR. Greenhouse trials indicated that the 75 % VT-SeAl1 + 25 % HT-SeG25 mixture was as effective as methoxyfenozide in preventing pest damage to pepper fruits. The potential contribution of vertically transmitted genotypes to transgenerational suppression of pest populations is discussed.

Following the consumption of baculovirus occlusion bodies (OBs), insects may succumb to lethal disease, but the survivors can harbour sublethal covert infections and may develop, reproduce and transmit the infection to their offspring. The use of different chemical and biological stressors was examined to determine whether they could be used to activate covert infections in populations of Spodoptera exigua larvae infected by the homologous nucleopolyhedrovirus (SeMNPV). Treatment of covertly infected S. exigua second instars with Tinopal UNPA‐GX, hydroxylamine, paraquat, Bacillus thuringiensis var. kurstaki crystals, spores or mixtures of crystals + spores, or a heterologous nucleopolyhedrovirus (Chrysodeixis chalcites SNPV) did not result in the activation of SeMNPV covert infections. Similarly, virus treatments involving permissive NPVs did not result in greater mortality in covertly infected insects compared with the virus‐free controls. In contrast, 0.1% copper sulphate, 1% iron (II) sulphate and 1 mg/l sodium selenite treatments resulted in 12–41% lethal polyhedrosis disease in covertly infected larvae. A greenhouse trial using copper sulphate and sodium selenite as activation factors applied to covertly infected S. exigua larvae on sweet pepper plants resulted in very low levels of SeMNPV activation (<3%). These results highlight the important roles of copper, iron and selenium in insect immunity and baculovirus‐induced disease. However, these substances seem unlikely to prove useful for the activation of covert SeMNPV infections in S. exigua larvae under greenhouse conditions.

This study describes four multiple nucleocapsid nucleopolyhedrovirus isolates recovered from infected larvae of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on crops in two different geographical regions of Mexico. Molecular and biological characterization was compared with characterized S. exigua multiple nucleopolyhedrovirus (SeMNPV) isolates from the United States (SeUS1 and SeUS2) and Spain (SeSP2). Restriction endonuclease analysis of viral DNA confirmed that all Mexican isolates were SeMNPV isolates, but molecular differences between the Mexican and the reference isolates were detected using PCR combined with restriction fragment length polymorphism (RFLP). Amplification of the variable region V01 combined with RFLP distinguished the two Mexican isolates, SeSLP6 and SeSIN6. BglII digestions showed that the majority of the isolates contained submolar bands, indicating the presence of genetic heterogeneity. Amplification of the variable regions V04 and V05 distinguished between American and the Spanish isolates. Biological characterization was performed against two laboratory colonies of S. exigua, one from Mexico, and another from Switzerland. Insects from the Mexican colony were less susceptible to infection than insects from Se-Swiss colony. In the Se-Mex colony, SeSP2 was the most pathogenic isolate followed by SeSIN6, although their virulence was similar to most of the isolates tested. In Se-Swiss colony, similar LD50 values were observed for the five isolates, although the virulence was higher for the SeSLP6 isolate, which also had the highest OB (occlusion body) yield. We conclude that the Mexican isolates SeSIN6 and SeSLP6 possess insecticidal traits of value for the development of biopesticides for the control of populations of S. exigua.

Genome sequence analysis of seven different Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolates that differed in insecticidal phenotype permitted the identification of genes likely to be involved in pathogenicity of occlusion bodies (OBs) and speed of kill (virulence) of this virus: se4 (. hoar), se5 (unknown function), se28 (unknown function), se76 (. cg30), se87 (. p26) and se129 (. p26). To study the role of these genes experimentally on the insecticidal phenotype, a bacmid-based recombination system was constructed to delete selected genes from a SeMNPV isolate, VT-SeAL1, designated as SeBacAL1. All of the knockout viruses were viable and the repair viruses behaved like the wild-type control, vSeBacAL1. Deletion of se4, se5, se76 and se129 resulted in decreased OB pathogenicity compared to vSeBacAL1 OBs. In contrast, deletion of se87 did not significantly affect OB pathogenicity, whereas deletion of se28 resulted in significantly increased OB pathogenicity. Deletion of se4, se28, se76, se87 and se129 did not affect speed of kill compared to the bacmid vSeBacAL1, whereas speed of kill was significantly extended following deletion of se5 and in the wild-type isolate (SeAL1), compared to that of the bacmid. Therefore, biological assays confirmed that several genes had effects on virus insecticidal phenotype. Se5 is an attractive candidate gene for further studies, as it affects both biological parameters of this important biocontrol virus.

Many parasites enhance their dispersal and transmission by manipulating host behaviour. One intriguing example concerns baculoviruses that induce hyperactivity and tree-top disease (i.e., climbing to elevated positions prior to death) in their caterpillar hosts. Little is known about the underlying mechanisms of such parasite-induced behavioural changes. Here, we studied the role of the ecdysteroid UDP-glucosyltransferase (egt) gene of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) in tree-top disease in S. exigua larvae. Larvae infected with a mutant virus lacking the egt gene exhibited a shorter time to death and died before the induction of tree-top disease. Moreover, deletion of either the open reading frame or the ATG start codon of the egt gene prevented tree-top disease, indicating that the EGT protein is involved in this process. We hypothesize that SeMNPV EGT facilitates tree-top disease in S. exigua larvae by prolonging the larval time to death. Additionally, we discuss the role of egt in baculovirus-induced tree-top disease.

Sublethal infections by baculoviruses (Baculoviridae) are believed to be common in Lepidoptera, including Spodoptera exigua. In addition, novel RNA viruses of the family Iflaviridae have been recently identified in a laboratory population of S. exigua (S. exigua iflavirus-1: SeIV-1; S. exigua iflavirus-2: SeIV-2) that showed no overt signs of disease. We determined the prevalence of these viruses in wild populations and the prevalence of co-infection by the different viruses in shared hosts. Infection by S. exigua multiple nucleopolyhedrovirus (SeMNPV) and iflaviruses in S. exigua adults (N= 130) from horticultural greenhouses in southern Spain was determined using qPCR and RT-PCR based techniques respectively. The offspring of these insects (N= 200) was reared under laboratory conditions and analyzed to determine virus transmission. Overall, 54% of field-caught adults were infected by SeMNPV, 13.1% were infected by SeIV-1 and 7.7% were infected by SeIV-2. Multiple infections were also detected, with 8.4% of individuals harboring SeMNPV and one of the iflaviruses, whereas 2.3% of adults were infected by all three viruses. All the viruses were transmitted to offspring independently of whether the parental female harbored covert infections or not. Analysis of laboratory-reared insects in the adult stage revealed that SeIV-1 was significantly more prevalent than SeMNPV or SeIV-2, suggesting high transmissibility of SeIV-1. Mixed infection involving three viruses was identified in 6.5% of laboratory-reared offspring. We conclude that interspecific interactions between these viruses in co-infected individuals are to be likely frequent, both in the field, following applications of SeMNPV-based insecticides, or in laboratory colonies used for SeMNPV mass production.

Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is a common feature in field S. exigua populations. To assess whether the gender affect to transgenerational virus transmission or distribution four mating groups were performed: i) healthy males (H♂) × healthy females (H♀); ii) infected males (I♂) × healthy females (H♀); iii) healthy males (H♂) × infected females (I♀) and iv) infected males (I♂) × infected females (I♀). These adults and their offspring were analyzed by qPCR to detect SeMNPV infection. Both males and females were able to transmit the infection to the next generation, although females infected high percentage of the offspring and their transmission was more stable and consistent. Venereal transmission is not likely to occur, and the main route of transmission seems transovarial more than transovum. The prevalence of the infection in the offspring did not vary according to gender, therefore both males and females can be infected by their parents in similar proportion. In conclusion, SeMNPV vertical transmission occurs by paternal and maternal via, while females transmission is more consistent. This route of transmission can improve the development of the bioinsecticides based in NPVs.

Covert infections of Spodoptera exigua multiple nucleopoliedrovirus (SeMNPV) have been detected in laboratory or field populations of the homologous host, S. exigua. Two RNA viruses belonging to the Iflaviridae family (SeIV-1, SeIV-2) were identified in S. exigua transcriptome from different laboratory colonies. The three viruses are vertically transmited and establish persistent infections, and coinfection of individual insects by these viruses is considered likely. In this study, we determined the prevalence of covert infections caused by iflaviruses and SeMNPV in order to identify virus associations in natural S. exigua populations. SeMNPV was detected in the 54% of field-adults, whereas 13% and 8% of insects were infected by SeIV-1 and SeIV-2, respectively. The prevalence of SeIV-1 and SeIV-2 in F1 adults showed higher levels than in parental generation, while the prevalence of SeMNPV decreased from parents to their offspring.

The insecticidal characteristics of two Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) strains produced on two different S. exigua colonies were measured using the same two host colonies. These strains constitute the active ingredients of the biological insecticides Vir-ex and Spexit and were produced on insect colonies from Spain and Switzerland. Demographic characteristics of insects from each colony were examined before infection. Larval developmental time, larval survival, and adult sex ratio did not differ between the colonies, whereas mean pupal weight was significantly higher in the Spanish colony insects. After infection, susceptibility to virus occlusion bodies (OBs), time to death, larval weight at death, and total production of OBs/larva varied significantly depending on virus strain and the colony used. Vir-ex OBs produced in Spanish colony larvae had improved insecticidal characteristics in terms in lethal dose and speed of kill metrics than other strain-colony combinations. OB production was significantly higher in Spanish colony insects infected with Spexit compared with Vir-ex infected insects from the Swiss colony, with intermediate values for the other two strain-colony combinations. Virus strain and host colony origin were highly influential in determining the insecticidal characteristics of OBs and should be considered as key parameters that require optimization during the production of SeMNPV-based insecticides. © 2013 Entomological Society of America.

The prevalence of sublethal infections of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) was quantified in natural populations of S. exigua in Almeria, Spain, during 2006 and 2007. Of 1045 adults collected, 16% proved positive for viral polyhedrin gene transcripts by RT-PCR. The prevalence of covert infection varied significantly according to sex and sample date. Of 1660 progeny of field-collected insects, lethal disease was observed in 10-33% of offspring of transcript-positive females and 9-49% of offspring of transcript-negative females. Isolates associated with vertically-transmitted infections were characterized by restriction endonuclease analysis using BglII and compared with isolates believed to be horizontally-transmitted. Insects from a sublethally-infected Almerian colony were ~3.4-fold more susceptible to infection than healthy insects from a Swiss colony. Horizontally-transmitted isolates were significantly more pathogenic than vertically-transmitted isolates in insects from both colonies. Mean speed of kill varied between isolates by >20 h, whereas mean occlusion body (OB) production varied by 3.8-fold among isolates. Intriguingly, all three horizontally transmitted isolates were very similar in speed of kill and OB production, whereas all three vertically transmitted isolates differed significantly from one another in both variables, and also differed significantly from the group of horizontally-transmitted isolates in one or both variables. We conclude that key pathogenicity and virulence traits of SeMNPV isolates vary according to their principal transmission strategy.

The Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida, US (SeUS2) is a complex mixture of genotypes. The genotypic structure of this isolate is very similar to that of the S. frugiperda MNPV isolated from Nicaragua (SfNIC), which is composed of at least nine genotypes. Both isolates present defective genotypes with similar deletions that lack peroral infectivity. The SfNIC defective genotype (SfNIC-C) plays a functional role in the transmissibility of the viral population since its presence increased the pathogenicity of OBs in co-occluded mixtures of complete and defective genotypes. The aim of this study was to determine the functional role of the defective genotype SeUS2-C, that is not infective per os, in the SeUS2 isolate. For this, different experimental populations were constructed that included the complete genotype SeUS2-A and the deletion mutant genotype SeUS2-C at different proportions in OB mixtures or co-occluded genotypic mixtures. The SeUS2-C was the most abundant isolated genotype in cell culture accounting for ~75% of plaques. However, the proportion of SeUS2-C in the viral population as determined by semiquantitative PCR was ~25%. SeUS2-A OBs were as pathogenic as wild-type OBs. As predicted, the potency of the mixtures of SeUS2-A OBs and SeUS2-C OBs was dependent on the proportion of SeUS2-A present in the mixture. In contrast, the co-occluded mixtures that included the defective genotype SeUS2-C at the proportions of 10 and 25%, respectively, were as pathogenic asSeUS2-A. However, the presence of a higher proportion of SeUS2-C in the mixtures reduced the biological activity of OBs. We conclude that the defective genotype SeUS2-C appears to persist as a parasitic genotype in the SeUS2 population. This contrasts with the role of defective genotypes in the SfMNPV population that potentiate OB potency.

Vertical transmission of covert NPV infections is a common phenomenon in field populations of the beet armyworm (Spodoptera exigua, Huber) in the horticultural region of Almería (Spain). The genotypic diversity of the Spodoptera exigua nucleopolyhedrovirus population in this region has proved to be extremely high. A recent study suggests that some of these genotypic variants are involved in vertically-transmitted infections whereas other genotypes are involed in horizontally-transmitted infections. These findings led us to examine whether host-pathogen interactions are associated with a range of fitness correlates or tradeoffs in either the insect or the virus. First, we demonstrated that S. exigua adults that survived a virus challenge (9 x 103 OB/ml) presented a persistent sublethal infection detected by RT-PCR (85 - 100% of insects were positive for viral transcripts of the DNA-polymerase gene) following inoculation by vertically transmitted genotypes. A vertically transmitted genotype was then selected to examine the effects of larval age at inoculation and the dose of virus ingested on the prevalence of persistent virus infections in adults. Larvae of four different larval instars (L2, L3, L4, and L5) were dosed with four different concentrations that resulted in 20 to 80% of NPV-mortality. A similar trend was observed for each larval stage, the higher the dose of inoculum, the higher the proportion of adults that proved positive for viral transcripts. Finally, we observed that persistent infections reduce the fitness of individuals which survive the infection in terms of host developmental parameters (larval developmental time, pupal weight, adult fecundity, fertility, and longevity) in an insect colony in which a persistent infection had been induced compared to mocked-infected insects. On-going experiments are being performed to examine the biological costs of harbouring covert infections.

A low-cost simplified diet has been successfully developed for rearing Spodoptera exigua larvae under laboratory conditions. The cost of ingredients was lower than that of the standard diet based on a modified tobacco hornworm, Manduca sexta (L.), diet. The simplified diet fulfilled larval nutritional requirements without apparent adverse effects on the reproductive capacity of the insect. Survival, pupal sex ratio, and fecundity registered in insects that were reared on the simplified diet did not differ from those observed on the standard diet. The mean larval development period of insects that consumed the simplified diet was also similar to that of insects that consumed the standard diet, whereas weight of pupae and adult longevity were significantly higher in insects reared on simplified diet. Larvae consumed ≈11% more of the standard diet compared with the simplified diet and a corresponding increase was observed in the number of larvae that could be reared through to pupation on each liter of simplified diet. The production of S. exigua multiple nucleopolyhedrovirus (SeMNPV) occlusion bodies (OBs) in insects grown on each type of diet was also evaluated. Weights of larvae at inoculation and at death, OB yields and biological activity of OBs did not differ significantly for each type of diet. The simplified low-cost diet appears suitable for large-scale in vivo production of SeMNPV OBs.

Understanding the molecular basis of the distinct biological properties of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), such as its narrow host range and high virulence, requires detailed information on the temporal expression and subcellular localization of SeMNPV gene products. The expression of two unique SeMNPV ORFs, 116 (Se116) and 117 (Se117), which show 45 mino acid similarity, was analyzed. Se116 and Se117 were expressed both in cultured cells and in larvae of S. exigua as polyadenylated transcripts of 0.80 and 0.75 kb, respectively. These transcripts initiated from ATCA(G/T)T promoter motifs, commonly found for baculovirus early genes. Se116 transcripts were detected with increasing abundance from 8 to 48 h p.i., whereas Se117 transcripts were present from 4 h p.i. and most abundantly at 24 h p.i. Western blot analysis of infected Se301 cells revealed 27- and 23-kDa proteins for Se116 and Se117, respectively. C-terminal GFP–fusion proteins of Se116 and Se117 were primarily localized in the nucleus of Se301 cells. When Se301 cells were infected with SeMNPV, both GFP–fusion proteins were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein is still enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived SeMNPV virions but not of budded virus.

The entry mechanism of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, in cultured cells was examined. SeMNPV budded virus (BV) enters by endocytosis as do the BVs of the group I NPVs, Autographa californica (Ac) MNPV and Orgyia pseudotsugata (Op) MNPV. In group I NPVs, upon infection acidification of the endosome triggers fusion of the viral and endosomal membrane, which is mediated by the BV envelope glycoprotein GP64. However, the SeMNPV genome lacks a homolog of GP64 envelope fusion protein (EFP). A functional homolog of the OpMNPV GP64 EFP was identified in SeMNPV ORF8 (Se8; 76 kDa) and appeared to be the major BV envelope protein. Surprisingly, a 60-kDa cleavage product of this protein is present in the BV envelope. A furin-like proprotein convertase cleavage site (R-X-K/R-R) was identified immediately upstream of the N-terminus of the mature Se8 protein and this site was also conserved in the Lymantria dispar (Ld) MNPV homolog (Ld130) of Se8. Syncytium formation assays showed that Se8 and Ld130 alone were sufficient to mediate membrane fusion upon acidification of the medium. Furthermore, C-terminal GFP-fusion proteins of Se8 and Ld130 were primarily localized in the plasma membrane of insect cells. This is consistent with their fusogenic activity and supports the conclusion that the Se8 gene product is a functional homolog of the GP64 EFP.