Mixtures of Insect-Pathogenic Viruses in a Single Virion: towards the Development of Custom-Designed Insecticides.

Alphabaculoviruses (Baculoviridae) are pathogenic DNA viruses of Lepidoptera that have applications as the basis for biological insecticides and expression vectors in biotechnological processes. These viruses have a characteristic physical structure that facilitates the transmission of groups of genomes. We demonstrate that coinfection of a susceptible insect by two different alphabaculovirus species results in the production of mixed-virus occlusion bodies containing the parental viruses. This occurred between closely related and phylogenetically more distant alphabaculoviruses. Approximately half the virions present in proteinaceous viral occlusion bodies produced following coinfection of insects with a mixture of two alphabaculoviruses contained both viruses, indicating that the viruses coinfected and replicated in a single cell and were coenveloped within the same virion. This observation was confirmed by endpoint dilution assay. Moreover, both viruses persisted in the mixed-virus population by coinfection of insects during several rounds of insect-to-insect transmission. Coinfection by viruses that differed in genome size had unexpected results on the length of viral nucleocapsids, which differed from those of both parental viruses. These results have unique implications for the development of alphabaculoviruses as biological control agents of insect pests.IMPORTANCE Alphabaculoviruses are used as biological insecticides and expression vectors in biotechnology and medical applications. We demonstrate that in caterpillars infected with particular mixtures of viruses, the genomes of different baculovirus species can be enveloped together within individual virions and occluded within proteinaceous occlusion bodies. This results in the transmission of mixed-virus populations to the caterpillar stages of moth species. Once established, mixed-virus populations persist by coinfection of insect cells during several rounds of insect-to-insect transmission. Mixed-virus production technology opens the way to the development of custom-designed insecticides for control of different combinations of caterpillar pest species.

Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach.

Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt proteins) in the parasporal crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial crystal production and quality control and product registration for Bt-based insecticides.IMPORTANCEBacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.

Insecticidal spectrum and mode of action of the Bacillus thuringiensis Vip3Ca insecticidal protein.

The Vip3Ca protein, discovered in a screening of Spanish collections of Bacillus thuringiensis, was known to be toxic to Chrysodeixis chalcites, Mamestra brassicae and Trichoplusia ni. In the present study, its activity has been tested with additional insect species and we found that Cydia pomonella is moderately susceptible to this protein. Vip3Ca (of approximately 90kDa) was processed to an approximately 70kDa protein when incubated with midgut juice in all tested species. The kinetics of proteolysis correlated with the susceptibility of the insect species to Vip3Ca. The activation was faster to slower in the following order: M. brassicae (susceptible), Spodoptera littoralis (moderately susceptible), Agrotis ipsilon and Ostrinia nubilalis (slightly susceptible). Processing Vip3Ca by O. nubilalis or M. brassicae midgut juice did not significantly changed its toxicity to either insect species, indicating that the low susceptibility of O. nubilalis is not due to a problem in the midgut processing of the toxin. M. brassicae larvae fed with Vip3Ca showed binding of this toxin to the apical membrane of the midgut epithelial cells. Histopathological inspection showed sloughing of the epithelial cells with further disruption, which suggests that the mode of action of Vip3Ca is similar to that described for Vip3Aa. Biotin-labeled Vip3Ca and Vip3Aa bound specifically to M. brassicae brush border membrane vesicles and both toxins competed for binding sites. This result suggests that insects resistant to Vip3A may also be cross-resistant to Vip3C, which has implications for Insect Resistance Management (IRM).

Superinfection exclusion in alphabaculovirus infections is concomitant with actin reorganization.

UNLABELLED: Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed, but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection). Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses--AcMNPV and SfMNPV--but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication. The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits dual infection by heterospecific alphabaculoviruses. IMPORTANCE: Infection of a cell by more than one virus particle implies sharing of cell resources. We show that multiple infection, by closely related or distantly related baculoviruses, is possible only during a brief window of time that allows additional virus particles to enter an infected cell over a period of ca. 16 h but then blocks multiple infections as newly generated virus particles begin to leave the infected cell. This temporal window has two important consequences. First, it allows multiple genotypes to almost simultaneously infect cells within the host, thus generating genetically diverse virus particles for transmission. Second, it provides a mechanism by which different viruses replicating in the same cell nucleus can exchange genetic material, so that the progeny viruses may be a mosaic of genes from each of the parental viruses. This opens a completely new avenue of research into the evolution of these insect pathogens.

A Novel Binary Mixture of Helicoverpa armigera Single Nucleopolyhedrovirus Genotypic Variants Has Improved Insecticidal Characteristics for Control of Cotton Bollworms.

The genotypic diversity of two Spanish isolates of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) was evaluated with the aim of identifying mixtures of genotypes with improved insecticidal characteristics for control of the cotton bollworm. Two genotypic variants, HearSP1A and HearSP1B, were cloned in vitro from the most pathogenic wild-type isolate of the Iberian Peninsula, HearSNPV-SP1 (HearSP1-wt). Similarly, six genotypic variants (HearLB1 to -6) were obtained by endpoint dilution from larvae collected from cotton crops in southern Spain that died from virus disease during laboratory rearing. Variants differed significantly in their insecticidal properties, pathogenicity, speed of kill, and occlusion body (OB) production (OBs/larva). HearSP1B was ∼3-fold more pathogenic than HearSP1-wt and the other variants. HearLB1, HearLB2, HeaLB5, and HearLB6 were the fastest-killing variants. Moreover, although highly virulent, HearLB1, HearLB4, and HearLB5 produced more OBs/larva than did the other variants. The co-occluded HearSP1B:LB6 mixture at a 1:1 proportion was 1.7- to 2.8-fold more pathogenic than any single variant and other mixtures tested and also killed larvae as fast as the most virulent genotypes. Serial passage resulted in modified proportions of the component variants of the HearSP1B:LB6 co-occluded mixture, suggesting that transmissibility could be further improved by this process. We conclude that the improved insecticidal phenotype of the HearSP1B:LB6 co-occluded mixture underlines the utility of the genotypic variant dissection and reassociation approach for the development of effective virus-based insecticides.

The "11K" gene family members sf68, sf95 and sf138 modulate transmissibility and insecticidal properties of Spodoptera frugiperda multiple nucleopolyhedrovirus.

The "11K" gene family is notable for having homologs in both baculoviruses and entomopoxviruses and is classified as either type 145 or type 150, according to their similarity with the ac145 or ac150 genes of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). One homolog of ac145 (sf138) and two homologs of ac150 (sf68 and sf95) are present in Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV). Recombinant bacmids lacking sf68, sf95 or sf138 (Sf68null, Sf95null and Sf138null, respectively) and the respective repair bacmids were generated from a bacmid comprising the complete virus genome. Occlusion bodies (OBs) of the Sf138null virus were ∼15-fold less orally infective to insects, which was attributed to a 100-fold reduction in ODV infectious titer. Inoculation of insects with Sf138null OBs in mixtures with an optical brightener failed to restore the pathogenicity of Sf138null OBs to that of the parental virus, indicating that the effects of sf138 deletion on OB pathogenicity were unlikely to involve an interaction with the gut peritrophic matrix. In contrast, deletion of sf68 and sf95 resulted in a slower speed-of-kill by 9h, and a concurrent increase in the yield of OBs. Phylogenetic analysis indicated that sf68 and sf95 were not generated after a duplication event of an ancestral gene homologous to the ac150 gene. We conclude that type 145 genes modulate the primary infection process of the virus, whereas type 150 genes appear to have a role in spreading systemic infection within the insect.

Identification of Spodoptera exigua nucleopolyhedrovirus genes involved in pathogenicity and virulence.

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.

Genomic diversity in European Spodoptera exigua multiple nucleopolyhedrovirus isolates.

Key virus traits such as virulence and transmission strategies rely on genetic variation that results in functional changes in the interactions between hosts and viruses. Here, comparative genomic analyses of seven isolates of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with differing phenotypes were employed to pinpoint candidate genes that may be involved in host-virus interactions. These isolates obtained after vertical or horizontal transmission of infection in insects differed in virulence. Apart from one genome containing a piggyBac transposon, all European SeMNPV isolates had a similar genome size and content. Complete genome analyses of single nucleotide polymorphisms and insertions/deletions identified mutations in 48 ORFs that could result in functional changes. Among these, 13 ORFs could be correlated with particular phenotypic characteristics of SeMNPV isolates. Mutations were found in all gene functional classes and most of the changes we highlighted could potentially be associated with differences in transmission. The regulation of DNA replication (helicase, lef-7) and transcription (lef-9, p47) might be important for the establishment of sublethal infection prior to and following vertical transmission. Virus-host cell interactions also appear instrumental in the modulation of viral transmission as significant mutations were detected in virion proteins involved in primary (AC150) or secondary infections (ME35) and in apoptosis inhibition (IAP2, AC134). Baculovirus populations naturally harbour high genomic variation located in genes involved at different levels of the complex interactions between virus and host during the course of an infection. The comparative analyses performed here suggest that the differences in baculovirus virulence and transmission phenotypes involve multiple molecular pathways.

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.

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.

A screening of five Bacillus thuringiensis Vip3A proteins for their activity against lepidopteran pests.

Five Bacillus thuringiensis Vip3A proteins (Vip3Aa, Vip3Ab, Vip3Ad, Vip3Ae and Vip3Af) and their corresponding trypsin-activated toxins were tested for their toxicity against eight lepidopteran pests: Agrotis ipsilon, Helicoverpa armigera, Mamestra brassicae, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Ostrinia nubilalis and Lobesia botrana. Toxicity was first tested at a high dose at 7 and 10 days. No major differences were found when comparing protoxins vs. trypsin-activated toxins. The proteins that were active against most of the insect species were Vip3Aa, Vip3Ae and Vip3Af, followed by Vip3Ab. Vip3Ad was non-toxic to any of the species tested. Considering the results by insect species, A. ipsilon, S. frugiperda and S. littoralis were susceptible to Vip3Aa, Vip3Ab, Vip3Ae and Vip3Af; S. exigua was susceptible to Vip3Aa and Vip3Ae, and moderately susceptible to Vip3Ab; M. brassicae and L. botrana were susceptible to Vip3Aa, Vip3Ae and Vip3Af; H. armigera was moderately susceptible to Vip3Aa, Vip3Ae and Vip3Af, and O. nubilalis was tolerant to all Vip3 proteins tested, although it showed some susceptibility to Vip3Af. The results obtained will help to design new combinations of insecticidal protein genes in transgenic crops or in recombinant bacteria for the control of insect pests.

UV protection and insecticidal activity of microencapsulated Vip3Ag4 protein in Bacillus megaterium.

In this study, secretable Vip3Ag4 protein was encapsulated in Bacillus megaterium and used for quantitative bioassays, in order to determine the UV photoprotective capacity of the cell, for preventing inactivation of the insecticidal activity of the protein. The non-encapsulated and purified protein was exposed to the UV light showing a LC50 of 518 ng/cm2 against Spodoptera littoralis larvae, whereas the exposed encapsulated protein exhibited 479 ng/cm2. In addition to the capability to accumulate Vip3 proteins for the development of novel insecticidal formulates, the B. megaterium cell has demonstrated to provide moderate protection against the deleterious action of UV light.

Genome Sequence Analysis of Native Xenorhabdus Strains Isolated from Entomopathogenic Nematodes in Argentina.

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus population from the Canary Islands is genotypically structured to maximize survival.

A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus wild-type isolate from the Canary Islands, Spain, named ChchSNPV-TF1 (ChchTF1-wt), appears to have great potential as the basis for a biological insecticide for control of the pest. An improved understanding of the genotypic structure of this wild-type strain population should facilitate the selection of genotypes for inclusion in a bioinsecticidal product. Eight genetically distinct genotypes were cloned in vitro: ChchTF1-A to ChchTF1-H. Quantitative real-time PCR (qPCR) analysis confirmed that ChchTF1-A accounted for 36% of the genotypes in the wild-type population. In bioassays, ChchTF1-wt occlusion bodies (OBs) were significantly more pathogenic than any of the component single-genotype OBs, indicating that genotype interactions were likely responsible for the pathogenicity phenotype of wild-type OBs. However, the wild-type population was slower killing and produced higher OB yields than any of the single genotypes alone. These results strongly suggested that the ChchTF1-wt population is structured to maximize its transmission efficiency. Experimental OB mixtures and cooccluded genotype mixtures containing the most abundant and the rarest genotypes, at frequencies similar to those at which they were isolated, revealed a mutualistic interaction that restored the pathogenicity of OBs. In OB and cooccluded mixtures containing only the most abundant genotypes, ChchTF1-ABC, OB pathogenicity was even greater than that of wild-type OBs. The ChchTF1-ABC cooccluded mixture killed larvae 33 h faster than the wild-type population and remained genotypically and biologically stable throughout five successive passages in vivo. In conclusion, the ChchTF1-ABC mixture shows great potential as the active ingredient of a bioinsecticide to control C. chalcites in the Canary Islands.

Analagous population structures for two alphabaculoviruses highlight a functional role for deletion mutants.

A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large-deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origins of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. The homology of nucleotides in the deleted regions was as high as 79%, similar to those of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that independently generates and sustains deletion mutants within each virus population. Functional analyses using different proportions of complete and deletion genotypes were performed with the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete/deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but there were no significant changes in the speed of kill. In contrast, OB yields increased only in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most extensively deleted SeMNPV genotype and contribute toward the elucidation of the role of such mutants in baculovirus populations.

Insecticidal characteristics of two commercial Spodoptera exigua nucleopolyhedrovirus strains produced on different host colonies.

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.

Insecticidal Traits of Variants in a Genotypically Diverse Natural Isolate of Anticarsia Gemmatalis Multiple Nucleopolyhedrovirus (AgMNPV).

Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus) played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could complement the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared using their restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity, although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates appear to be genotypically structured for fast speed of kill and high OB production, both of which would favor horizontal transmission. Interactions among the component variants are likely to influence this insecticidal phenotype.

First study on the root endophytic fungus Trichoderma hamatum as an entomopathogen: Development of a fungal bioinsecticide against cotton leafworm (Spodoptera littoralis).

Cotton leaf worm (Spodoptera littoralis) is a pest that produces important losses in horticultural and ornamental crops in greenhouse, being classified as quarantine pest A2 by EPPO. One of the strategies proposed to control agricultural pests in a health and environmentally friendly way is biological control with entomopathogenic fungi. The genus of filamentous fungi Trichoderma includes different species with direct (infection, antibiosis, anti-feeding, etc.) and indirect (systemic activation of plant defenses) insecticidal capacity, however, the species T. hamatum has never been described previously as entomopathogenic. In this work, the entomopathogenic capacity of T. hamatum on S. littoralis L3 larvae was analyzed by applying spores and fungal filtrates (topically and orally). Infection by spores was compared with the commercial entomopathogenic fungus Beauveria bassiana, obtaining similar results with respect to the production of larval mortality. Oral application of spores reported high mortality and fungal colonization of larvae, however, T. hamatum did not show chitinase activity when grown in the presence of S. littoralis tissues. Therefore, infection of S. littoralis larvae by T. hamatum is through natural openings such as mouth, anus or spiracles. With respect to the application of filtrates, only those obtained from the liquid culture of T. hamatum in contact with S. littoralis tissues reported a significant reduction in larval growth. Metabolomic analysis of the filtrates determined that the filtrate with insecticidal capacity presented the siderophore rhizoferrin in large quantities, which could be responsible for this activity. However, the production of this siderophore had never been previously described in Trichoderma and its insecticidal capacity was unknown. In conclusion, T. hamatum presents entomopathogenic capacity against S. littoralis larvae through the application of spores and filtrates, and both ways could be the basis for the development of efficient bioinsecticides against the pest.

A novel use of Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) as inoculative agent of baculoviruses.

BACKGROUND: Alphabaculoviruses are Lepidoptera-specific virulent pathogens that infect numerous pests, including the Spodoptera complex. Due to their low environmental persistence, the traditional use of Alphabaculoviruses as bioinsecticides consist in high-rate spray applications with repeated treatments. Several abiotic and biotic factors can foster its dispersion, promoting their persistence in the agroecosystem. Amongst biotic factors, predatory arthropods can disperse the viruses by excretion after preying on infected individuals. Therefore, this study focused on promoting predator's ingestion of nucleopolyhedrovirus (NPV)-treated diets, and the later exposition of the insect host to leaf surfaces contaminated with predator excreta. The virus-host-predator system studied was Spodoptera littoralis nucleopolyhedrovirus (SpliNPV), Spodoptera littoralis (Boisduval) and Nesidiocoris tenuis (Reuter). The infective potential of N. tenuis feces and the retention time of SpliNPV were assessed under laboratory conditions after feeding on treated diets (sucrose solution and Ephestia kuehniella eggs). RESULTS: Mortality of S. littoralis larvae was lower via N. tenuis excretion than in positive control (spray application) in the first infection cycle, together with a delay in host death. In the second infection cycle, both SpliNPV-treated diets triggered 100% mortality. Both diets allowed the transmission of SpliNPV, with a faster excretion via sucrose solution compared to E. kuehniella eggs. SpliNPV remained in N. tenuis digestive tract and was viable after excretion at least for 9 days for both diets. CONCLUSIONS: This study demonstrated the potential of the predator N. tenuis as inoculative agent of baculoviruses, representing a new alternative that, along with inundative applications, might contribute to improve pest management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bacillus thuringiensis Cyt Proteins as Enablers of Activity of Cry and Tpp Toxins against Aedes albopictus.

Aedes albopictus is a species of mosquito, originally from Southeast Asia, that belongs to the Culicidae family and the Dipteran insect order. The distribution of this vector has rapidly changed over the past decade, making most of the temperate territories in the world vulnerable to important human vector-borne diseases such as dengue, yellow fever, zika or chikungunya. Bacillus thuringiensis var. israeliensis (Bti)-based insecticides represent a realistic alternative to the most common synthetic insecticides for the control of mosquito larvae. However, several studies have revealed emerging resistances to the major Bti Crystal proteins such as Cry4Aa, Cry4Ba and Cry11Aa, making the finding of new toxins necessary to diminish the exposure to the same toxicity factors overtime. Here, we characterized the individual activity of Cyt1Aa, Cry4Aa, Cry4Ba and Cry11Aa against A. albopictus and found a new protein, Cyt1A-like, that increases the activity of Cry11Aa more than 20-fold. Additionally, we demonstrated that Cyt1A-like facilitates the activity three new Bti toxins: Cry53-like, Cry56A-like and Tpp36-like. All in all, these results provide alternatives to the currently available Bti products for the control of mosquito populations and position Cyt proteins as enablers of activity for otherwise non-active crystal proteins.