Rosa Murillo

Four genotypic variants of a Spodoptera exigua Nucleopolyhedrovirus (Se-SP2) are distinguishable by a hypervariable genomic region

Four genotypes named SP2A, SP2B, SP2C and SP2D were obtained in vivo by infecting S. exigua larvae with limiting dilutions of the Spanish field isolate Spodoptera exigua Nucleopolyhedrovirus (Se-SP2) of SeMNPV. The cloning of variants SP2A, SP2B and SP2C took 1, 6, and 3 passages, respectively, before the DNA profiles showed all bands in equimolar concentrations, and they remained constant for at least six further passages indicating the stability of their genotypes. The SP2D variant isolation took over ten passages and it was genetically less stable. Physical maps of their genomes were constructed for the restriction enzymes BamHI, BglII, PstI, and XbaI. The region between 8-10 m.u. was highly variable and characteristic of each cloned genotype and, hence, can be used as RFLP markers for all four genotypic variants. This region, included in the PstI-MB fragment, was cloned and sequenced showing that all the Se-SP2 variants contained a homologous region (hr) with a variable number of 98 bp sequences tandemly repeated, which were used to distinguish genotypic variants from each other. The biological activity of the genotypic variants SP2A, SP2B, and SP2C when compared in terms of LD50 and LT50, were not significantly different. However, the SP2D genotypic variant was found to be significantly less infective (higher LD50). The emergence of new genotypes in the Se-SP2 field populations is discussed.

Natural populations of Spodoptera exigua are infected by multiple viruses that are transmitted to their offspring

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.

Evidence for covert baculovirus infections in a spodoptera exigua laboratory culture

A laboratory culture of Spodoptera exigua was examined to assess covert (latent or persistent) baculovirus infections and spontaneous disease outbreaks. Two nucleopolyhedrovirus (NPV) species were found to be reactivated from a covert state in a laboratory culture of S. exigua to fully lethal forms. These were identified as S. exigua multinucleopolyhedrovirus (SeMNPV) and Mamestra brassicae NPV (MbNPV) using restriction enzyme analysis of purified viral DNA. Sequence data derived from both overtly and covertly virus-infected insects revealed highly conserved sequences for lef-8, lef-9 and polyhedrin gene sequence (98-100 % nucleotide identity to SeMNPV published sequence). By monitoring spontaneous overt infections and quantifying viral DNA (by quantitative-PCR) in asymptomatic individuals over two generations we identified fluctuating trends in viral DNA levels from covert SeMNPV and MbNPV within an S. exigua host population. Virus levels per insect life stage ranged from 3.51±0.101×10(5) to 0.29±0.036 pg (detection limit at 0.06 pg). Bioassays performed with this culture of larvae showed a differential susceptibility to SeMNPV-like or MbNPV-like viruses, with SeMNPV superinfections being extremely virulent. The data presented has broad implications relating to our understanding of transmission patterns of baculovirus in the environment and the role of covert infections in host-pathogen interaction dynamics.

Intra- and Intergenerational Persistence of an Insect Nucleopolyhedrovirus: Adverse Effects of Sublethal Disease on Host Development, Reproduction, and Susceptibility to Superinfection

ABSTRACT Sublethal infections by Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) are common in field populations of the beet armyworm (S. exigua, Hübner) in the Almerian horticultural region of Spain. Inoculation of second, third, and fourth instars with occlusion bodies (OBs) of an isolate (VT-SeAl1) associated with vertically transmitted infections resulted in 15 to 100% of sublethal infection in adult survivors, as determined by reverse transcription-PCR (RT-PCR) detection of viral DNA polymerase transcripts, and quantitative PCR (qPCR) targeted at the DNA polymerase gene. The prevalence of adult sublethal infection was positively related to the inoculum OB concentration consumed during the larval stage. Sublethal infections persisted in OB-treated insects for at least five generations. Viral transcripts were more frequently detected in adult insects than in third instars. qPCR analysis indicated a consistently higher prevalence of sublethal infection than RT-PCR. Sublethal infection was associated with significant reductions in pupal weight, adult emergence, fecundity, and fertility (egg hatch) and significant increases in larval development time and duration of the preoviposition period. Insects taken from a persistently infected experimental population were significantly more susceptible to the OB inoculum than control insects that originated from the same virus-free colony as the persistently infected insects. We conclude that OB treatment results in rapid establishment of sublethal infections that persist between generations and which incur costs in the development and reproductive capacity of the host insect.

Host range and biological activity of three Spodoptera nucleopolyhedrovirus genotypic variants and the effect of Tinopal LPW on the most active variant

The noctuid moths Spodoptera exigua, S. littoralis and S. frugiperda are three important insect pest species and the natural hosts of three distinct nucleopolyhedroviruses (NPVs), of which several strains have been isolated world-wide. A variant of S. exigua NPV from Almería, Spain (Se-SP2A), a variant of the S. littoralis NPV from Morocco (Sl-M2) and a variant of the S. frugiperda NPV from California, USA (Sf-2), were analysed with restriction endonucleases and biologically compared in terms of host range, infectivity (median lethal concentration, LC 50 ) and virulence (median survival time, ST 50 ) for each of the three host species. Each virus was most effective against its homologous host when both LC 50 and ST 50 were taken into account, but one the S. littoralis variants, Sl-M2, showed the highest overall activity against all three species. The influence of the optical brightener Tinopal LPW on the activity of Sl-M2 against all three Spodoptera spp. was assayed. The LC 50 of Sl-M2 was reduced in all cases in the presence of Tinopal LPW; quite significantly in S. littoralis and S. frugiperda (8.7- and 7.2-fold, respectively) and only slightly (1.5-fold) in S. exigua . No significant differences in the ST 50 were detected when Tinopal LPW was added to the virus suspension.

Simultaneous occurrence of covert infections with small RNA viruses in the lepidopteran Spodoptera exigua.

Viral covert infections in invertebrates have been traditionally attributed to sublethal infections that were not able to establish an acute infection. Recent studies are revealing that, although true for some viruses, other viruses may follow the strategy of establishing covert or persistent infections without producing the death of the host. Recently, and due to the revolution in the sequencing technologies, a large number of viruses causing covert infections in all type of hosts have been identified. The beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) is a worldwide pest that causes significant losses to agricultural and ornamental plant industries. In a previous project we used NGS to obtain a comprehensive transcriptome of the larval stage, revealing the presence of an important number of unigenes belonging to novel RNA viruses, most of them from the order Picornavirales. In order to characterize S. exigua viral complex, in this work we have completed the genomic sequences of two picorna-like viruses, and compared them to a SeIV1, a member of Iflaviridae previously described by our group. We performed additional studies to determine virus morphology, horizontal transmission, tissue and life stage distribution and abundance in the hosts. We discuss the role of virus persistent infections on insect populations.

Gender-Mediated Differences in Vertical Transmission of a Nucleopolyhedrovirus

With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: 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♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.

Iflavirus increases its infectivity and physical stability in association with baculovirus

Virus transmission and the prevalence of infection depend on multiple factors, including the interaction with other viral pathogens infecting the same host. In this study, active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales) was observed in the offspring of insects that survived following inoculation with a pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking the origin of the iflavirus suggested the association of this virus with the occlusion bodies of the baculovirus. Here we investigated the effect of this association on the stability and infectivity of both viruses. A reduction in baculovirus pathogenicity, without affecting its infectivity and productivity, was observed when associated with the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and its resistance to ultraviolet radiation and high temperature, two of the main factors affecting virus stability in the field. In addition, electron microscopy analysis revealed the presence of particles resembling iflavirus virions inside the occlusion bodies of the baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here are indicative of facultative phoresis of a virus and suggest that virus–virus interactions may be more common than currently recognized, and may be influential in the ecology of baculovirus and host populations and in consequence in the use of baculoviruses as biological insecticides.

Baculovirus-Induced Climbing Behavior Favors Intraspecific Necrophagy and Efficient Disease Transmission in Spodoptera exigua

Shortly prior to death, many species of Lepidoptera infected with nucleopolyhedrovirus climb upwards on the host plant. This results in improved dissemination of viral occlusion bodies over plant foliage and an increased probability of transmission to healthy conspecific larvae. Following applications of Spodoptera exigua multiple nucleopolyhedrovirus for control of Spodoptera exigua on greenhouse-grown sweet pepper crops, necrophagy was observed by healthy S. exigua larvae that fed on virus-killed conspecifics. We examined whether this risky behavior was induced by olfactory or phagostimulant compounds associated with infected cadavers. Laboratory choice tests and olfactometer studies, involving infected and non-infected cadavers placed on spinach leaf discs, revealed no evidence for greater attraction of healthy larvae to virus-killed over non-infected cadavers. Physical contact or feeding on infected cadavers resulted in a very high incidence of transmission (82–93% lethal disease). Observations on the behavior of S. exigua larvae on pepper plants revealed that infected insects died on the uppermost 10% of foliage and closer to the plant stem than healthy conspecifics of the same stage, which we considered clear evidence of baculovirus-induced climbing behavior. Healthy larvae that subsequently foraged on the plant were more frequently observed closer to the infected than the non-infected cadaver. Healthy larvae also encountered and fed on infected cadavers significantly more frequently and more rapidly than larvae that fed on non-infected cadavers. Intraspecific necrophagy on infected cadavers invariably resulted in virus transmission and death of the necrophagous insect. We conclude that, in addition to improving the dissemination of virus particles over plant foliage, baculovirus-induced climbing behavior increases the incidence of intraspecific necrophagy in S. exigua, which is the most efficient mechanism of transmission of this lethal pathogen.

Covert Infection of Insects by Baculoviruses

Baculoviruses (Baculoviridae) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host–virus interactions through the action of microRNAs (miRNA). Initial findings suggest that insect nudiviruses and vertebrate herpesviruses may provide useful analogous models for exploring the mechanisms of covert infection by baculoviruses. These pathogens adopt mixed-mode transmission strategies that depend on the relative fitness gains that accrue through vertical and horizontal transmission. This facilitates virus persistence when opportunities for horizontal transmission are limited and ensures virus dispersal in migratory host species. However, when host survival is threatened by environmental or physiological stressors, latent or persistent infections can be activated to produce lethal disease, followed by horizontal transmission. Covert infection has also been implicated in population level effects on host–pathogen dynamics due to the reduced reproductive capacity of infected females. We conclude that covert infections provide many opportunities to examine the complexity of insect–virus pathosystems at the organismal level and to explore the evolutionary and ecological relationships of these pathogens with major crop and forest pests.