Control of Spodoptera spp. (Lepidoptera: Noctuidae) crop pests has traditionally relied on heavy insecticide use. Baculoviruses (BV) from the genera Alphabaculovirus and Betabaculovirus offer effective, species-specific alternatives for managing their outbreaks. Joint use of BVs with natural enemies, such as parasitoids, could further enhance control of Spodoptera spp. by contributing to virus dispersion. In a series of experiments, we studied the optimal host age and parasitization duration of the endoparasitoid Chelonus inanitus L. (Hymenoptera: Braconidae) for parasitoid production, as well as the parasitoid preference and mechanical transmission of Spodoptera exigua and Spodoptera littoralis multiple nucleopolyhedroviruses (SeMNPV and SpliMNPV). We found that parasitoid progeny production was significantly lower when parasitized eggs were either too young (24 h) or too old (96 h), highlighting the importance of host egg age. Additionally, there was an increasing trend in parasitoid offspring production with longer parasitization exposure times, particularly at 6 and 24 h compared to shorter durations. Chelonus inanitus did not discriminate between SpliMNPV-contaminated and non-contaminated eggs, but had a remarkable preference for SeMNPV-contaminated eggs. The parasitoid effectively dispersed BVs, not only from BV-treated eggs to heathy ones by parasitization, but also, and even more efficiently, by the sole contact with a contaminated surface without eggs. Understanding complex BV-parasitoid interactions is crucial for developing integrated pest management strategies that maximize the efficacy of both parasites.

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.

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.

Hyposoter didymator (Thunberg) (Hymenoptera: Ichneumonidae) and baculovirus (BV) might be used jointly to provide effective control of the Spodoptera genus. The literature has mostly covered the safe compatibility between natural enemies and BV-based insecticides, but research on the potential dispersal of BV by natural enemies is lacking. Thus, the goal of this manuscript was to ascertain if H. didymator was able to disperse the broad host range of Autographa californica nucleopolyhedrovirus (AcMNPV) to Spodoptera littoralis and Spodoptera exigua in choice and non-choice conditions and whether the preference of the parasitoid by one of these noctuids could mediate this dispersion. It was previously needed to improve the rearing of the parasitoid in the laboratory, concerning the optimal host age and length of parasitization, parasitoid competition, and influence of parasitization on the longevity of females. The best rearing conditions for S. littoralis are collective parasitization of mature L3 larvae for 24 h, after at least one day of copulation. Hyposoter didymator transmits AcMNPV to both lepidopterans, but its efficiency is mediated by host preference and the pathogenicity of the BV in each host. In this particular case, H. didymator as well as AcMNPV showed a clear preference towards S. exigua.

BACKGROUND A Chrysodeixis chalcites nucleopolyhedrovirus from the Canary Islands (ChchNPV-TF1) has proved to be effective for control of Chrysodeixis chalcites on banana crops. Commercialization of this virus as a bioinsecticide requires an efficient production system. RESULTS The sixth instar (L6 ) was the most suitable for virus production, producing 1.80 × 1011 occlusion bodies (OB)/larva and showed a lower prevalence of cannibalism (5.4%) than fourth (L4 ) or fifth (L5 ) instars. Inoculation of L6 at 24 h post molting produced six times more OB (5.72 × 1011 OB/larva) than recently molted L6 larvae (1.00 × 1011 OB/larva). No significant differences were recorded in mean time to death (165-175 h) or OB production per larva (3.75 × 1011 to 5.97 × 1011 ) or per mg larval weight (1.30 × 1011 to 2.11 × 109 ), in larvae inoculated with a range of inoculum concentrations (LC50 -LC90 ). Groups of infected L6 larvae reared at a density of 150 larvae/container produced a greater total number of OBs (8.07 × 1013 OB/container) than lower densities (25, 50 and 100 OB/container), and a similar number to containers with 200 inoculated larvae (8.43 × 1013 OB/container). CONCLUSION The processes described here allow efficient production of sufficient OBs to treat ∼ 40 ha of banana crops using the insects from a single container. © 2018 Society of Chemical Industry.

Trabajo presentado en el 49th Annual Meeting of the Society for Invertebrate Pathology: International Congress on Invertebrate Pathology and Microbial Control, celebrado en Tours (Francia), del 24 al 28 de julio de 2016

The soil is the most important reservoir of baculovirus occlusion bodies (OBs) in the environment. The ability of the earthworm Eisenia fetida to transport OBs of Spodoptera frugiperda multiple nucleopolyhedrovirus was examined in laboratory terraria filled with an artificial soil. OBs were detected in soil samples using a soil-diet incorporation bioassay, for which the 50% lethal concentration was estimated at 2.7 x 10(6) OBs/g soil in S. frugiperda second instars. Incubation of earthworms in soil containing 10(9) OBs for 7 days did not result in a significant loss of OB virulence compared to soil without earthworms. The earthworm intestine was found to be slightly acidic, with acid-base indicators applied to lengths of dissected intestine suggesting a pH of 6.0-6.3. Despite their epigean habits, E. fetida individuals were observed to form burrows up to 22.5 cm deep in laboratory terraria. Soil-diet bioassays indicated the presence of OBs at depths of 6-8, 14-16 and 22-24 cm in samples taken at 1, 7 and 14 days following the application of 10(9) OBs to the surface of terraria containing earthworms. In contrast, OBs were only detected in samples from the soil surface in terraria without earthworms. We conclude that earthworms likely affect the distribution and dynamics of OB populations in soil habitats. (C) 2016 Elsevier Inc. All rights reserved.

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.

Trabajo presentado en 47th Annual Meeting of the Society for Invertebrate Pathology, celebrado en Johannes Gutenberg University en Mainz (Alemania), del 3 al 7 de agosto de 2014

caseinase / gelatinase named Vsp for vesicular serine protease, which is homologous to the VesA serine protease of Vibrio cholerae, was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides.

Poster presentado en 47th Annual Meeting of the Society for Invertebrate Pathology, celebrado en Johannes Gutenberg University en Mainz (Alemania), del 3 al 7 de agosto de 2014

This study describes the insecticidal activity of a novel Bacillus thuringiensis Cry-related protein with a deduced 799 amino acid sequence (~89 kDa) and ~19% pairwise identity to the 95-kDa-aphidicidal protein (sequence number 204) from patent US 8318900 and ~40% pairwise identity to the cancer cell killing Cry proteins (parasporins Cry41Ab1 and Cry41Aa1), respectively. This novel Cry-related protein contained the five conserved amino acid blocks and the three conserved domains commonly found in 3-domain Cry proteins. The protein exhibited toxic activity against the green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae) with the lowest mean lethal concentration (LC50 = 32.7 μg/mL) reported to date for a given Cry protein and this insect species, whereas it had no lethal toxicity against the Lepidoptera of the family Noctuidae Helicoverpa armigera (Hübner), Mamestra brassicae (L.), Spodoptera exigua (Hübner), S. frugiperda (J.E. Smith) and S. littoralis (Boisduval), at concentrations as high as ~3.5 μg/cm2. This novel Cry-related protein may become a promising environmentally friendly tool for the biological control of M. persicae and possibly also for other sap sucking insect 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.

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein's target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.

BACKGROUNDChrysodeixis chalcites (Esper) is a major pest of tomato in Mediterranean countries and attacks banana in the Canary Islands (Spain). The efficacy of Chrysodeixis chalcites single nucleopolyhedrovirus (ChchSNPV-TF1) was evaluated in plant growth chambers and greenhouse trials performed on tomato and banana plants respectively. Treatments were applied using a compressed air sprayer.RESULTSMean (± SE) lethal infection varied from 77 ± 10% to 94 ± 3% in second-instar larvae fed for 2 days on tomato plants treated with 2 × 10(6) to 5 × 10(7) virus occlusion bodies (OBs) L(-1) , increasing to ∼100% infection after 7 days. Mortality of larvae collected from banana at different intervals post-application varied from 54 ± 10% to 96 ± 4% in treatments involving 1 × 10(8) -1 × 10(9) OBs L(-1) , whereas indoxacarb (Steward 30% WG) and Bacillus thuringiensis var. kurstaki (Biobit 16% WP) treatments produced between 22 ± 6% and 32 ± 5% pest mortality. All treatments significantly reduced plant defoliation compared with untreated controls. Application of 1 × 10(9) OBs L(-1) was 3-4-fold more effective than chemical or B. thuringiensis treatments. Larvae acquired lethal infection more rapidly when feeding on tomato than banana plants, but this difference disappeared following >60 min of feeding.CONCLUSIONThis information should prove useful in the registration of ChchSNPV-TF1 as a bioinsecticide in the Canary Islands and Europe.

ABSTRACT We report here the complete annotated 6,510,053-bp draft genome sequence of Bacillus thuringiensis serovar tolworthi strain Na205-3, which is toxic for Helicoverpa armigera. This strain potentially contains nine insecticidal toxin genes homologous to cry1Aa12, cry1Ab1, cry1Ab8, cry1Ba1, cry1Af1, cry1Ia10, vip1Bb1, vip2Ba2, and vip3Aa6.

Bacillus thuringiensis (Bt) is a Gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing Cry and Cyt proteins, some of which are toxic against a wide range of insect orders, nematodes and human-cancer cells. These toxins have been successfully used as bioinsecticides against caterpillars, beetles, and flies, including mosquitoes and blackflies. Bt also synthesizes insecticidal proteins during the vegetative growth phase, which are subsequently secreted into the growth medium. These proteins are commonly known as vegetative insecticidal proteins (Vips) and hold insecticidal activity against lepidopteran, coleopteran and some homopteran pests. A less well characterized secretory protein with no amino acid similarity to Vip proteins has shown insecticidal activity against coleopteran pests and is termed Sip (secreted insecticidal protein). Bin-like and ETX_MTX2-family proteins (Pfam PF03318), which share amino acid similarities with mosquitocidal binary (Bin) and Mtx2 toxins, respectively, from Lysinibacillus sphaericus, are also produced by some Bt strains. In addition, vast numbers of Bt isolates naturally present in the soil and the phylloplane also synthesize crystal proteins whose biological activity is still unknown. In this review, we provide an updated overview of the known active Bt toxins to date and discuss their activities.

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.

The Chrysodeixis chalcites single nucleopolyhedrovirus (ChchSNPV) infects and kills C. chalcites larvae, an important pest of banana crops in the Canary Islands. Five genotypes present in the most prevalent and widespread isolate in the Canary Islands were sequenced, providing genetic data relevant to the genotypic and phenotypic diversity of this virus.