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BACKGROUND: The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. RESULTS: We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. CONCLUSION: Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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Indoxacarb is a pivotal insecticide used worldwide to manage Spodoptera exigua, a devastating agricultural pest. This active compound plays a crucial role in resistance management strategies due to its distinctive mode of action. A field population of S. exigua (SH23) from Shanghai, China, exhibited significantly reduced susceptibility to indoxacarb, with a resistance ratio of 113.84-fold in biological assays. Following two rounds of laboratory screening with indoxacarb, the resistance of the new strain (SH23-S2) escalated steeply to 876.15-fold. Genetic analyses of both the SH23 and SH23-S2 strains demonstrated autosomal inheritance and incompletely dominant resistance patterns. Synergist assays indicated a minor role of detoxification enzymes (glutathione s-transferases and cytochrome P450) of SH23-S2 strain in this resistance, implicating target-site resistance as the primary mechanism. To explore the impact of target-site resistance, segment 1-6 of domain IV (IVS1-6) of the sodium channel in S. exigua was cloned, and the sequences from susceptible and indoxacarb-resistant S. exigua were compared. The V1848I mutation, linked to indoxacarb resistance in Plutella xylostella, Tuta absoluta and Liriomyza trifolii, was identified and strongly associated with the indoxacarb-resistant phenotype in the S. exigua SH23-S2 strain, whereas the F1845Y mutation was not detected. Furthermore, a molecular test for the V1848I mutation in field populations was created using an allele-specific PCR (AS-PCR). The discovery of indoxacarb resistance mutation and the creation of diagnostic tool will enable the early detection of indoxacarb resistance, which will facilitate the implementation of targeted resistance management strategies, ultimately delaying the proliferation of resistance.

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Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) is a highly dispersive, polyphagous insect pest that severely defoliates crops. Excessive reliance on synthetic insecticides leads to ecological pollution and resistance development, urging scientists to probe eco-friendly biopesticides. Here, we explore the virulence of an entomopathogenic fungus, Beauveria bassiana, against S. exigua, resulting in 88% larval mortality. Using an age-stage, two-sex life table, we evaluated the lethal and sublethal effects of B. bassiana on the demographic parameters of S. exigua, including survival, development, and reproduction. Sublethal (LC20) and lethal concentrations (LC50) of B. bassiana impacted the parental generation (F0), with these effects further influencing the demographic parameters of the first filial generation (F1). The infected F1 offsprings showed a reduced intrinsic rate of increase (r), mean generation time (T), and net reproduction rate (R0). Larval developmental duration varied significantly between the control (10.98 d) and treated groups (LC20: 10.42; LC50: 9.37 d). Adults in the treated groups had significantly reduced lifespans (M: 8.22; F: 7.32 d) than the control (M: 10.00; F: 8.22 d). Reduced fecundity was observed in the B. bassiana-infected groups (LC20: 313.45; LC50: 223.92 eggs/female) compared to the control (359.55 eggs/female). A biochemical assay revealed elevated levels of detoxification enzymes (esterases, glutathione S-transferases, and acetylcholinesterase) in the F0 generation after B. bassiana infection. However, the enzymatic activity remained non-significant in the F1 generation likely due to the lack of direct fungal exposure. Our findings highlight the enduring effects of B. bassiana on the biological parameters and population dynamics of S. exigua, stressing its use in eco-friendly management programs.

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Insect nicotinic acetylcholine receptors (nAChRs) are the directed targets of many insecticides. However, there have been no reports on the molecular characterization of the nAChR gene family or the causal association between nAChR α1 and resistance to insecticides in S. exigua, which is a significant agricultural pest. In this study, we identified a total of 9 candidate nAChR subunits in S. exigua, namely nAChR α1-α7 and nAChR ß1-ß2. For functional validation roles of Seα1 in insecticide resistance of S. exigua, we introduced a âˆ¼ 1041-bp deletion of the Seα1 gene in a homozygous mutant strain (Seα1-KO) by CRISPR/Cas9 genome editing system, resulting in a premature truncation of the Seα1 protein and the subsequent loss of functional transmembrane (TM) 3 and TM4 elements. Compared with WH-S strain (wild-type strain), the Seα1-KO strain exhibited 2.62-folds resistant to trifluoropyrimidine, 8.3-folds resistant to dimehypo, and 5.28-folds resistant to dinotefuran, but no significant change in susceptibility to emamectin benzoate, spinetoram, lambda-cyhalothrin, permethrin and chlorpyrifos. Thus, this study has laid a solid foundation for investigating the role of nAChRs in S. exigua, and provides evidence for the crucial involvement of the α1 subunit in the mechanism of trifluoropyrimidine, dimehypo, and dinotefuran in S. exigua. Moreover, it provides a reference for the value of Seα1 subunit and its homologues in other species as insecticide targets.

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BACKGROUND: The oligophagous potato tuber moth (PTM), Phthorimaea operculella, and the polyphagous beet armyworm (BAW), Spodoptera exigua, are two destructive pests of potato, and infestations can lead to serious reduction in potato yield. However, potato plant responses to the two herbivories are only poorly understood. Endogenous jasmonoyl-isoleucine (JA-Ile) is a signal responsible for the induction of plant anti-herbivore defenses. Elevation of JA-Ile by blocking its catabolism is considered to be an effective and sustainable approach to enhance plant resistance to insect pests. However, it is not clear whether this approach can enhance potato resistance to PTM and BAW. RESULTS: We demonstrated that the transcriptional changes induced by simulated PTM and BAW feeding overlap to a large extent, and that 81.5% of the PTM- and 90.5% of the BAW-responsive genes were commonly regulated. We also generated potato transgenic lines, irStCYP94B3s, in which the three JA-Ile hydroxylases were all simultaneously silenced. These lines exhibited enhanced resistance only to BAW, but not to PTM, although levels of JA-Ile and its downstream induced defensive chemicals, including caffeoylputrescine, dicaffeoylspermidine, lyciumoside II, and the nicotianosides I, II, and VII, were all present at higher levels in PTM-infested than in BAW-infested irStCYP94B3s lines. CONCLUSION: Our results provide support for the hypothesis that StCYP94B3 genes are able to act as potential targets for the control of polyphagous insect pests in potato, and reveal that the oligophagous PTM has evolved an effective mechanism to cope with JA-Ile-induced anti-herbivore defenses. © 2022 Society of Chemical Industry.

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MicroRNAs (miRNAs) drive the post-transcriptional repression of target mRNAs and play important roles in a variety of biological processes. miR-2766-3p is conserved and abundant in Lepidopteran species and may be involved in a variety of biological activities. In this study, Sex-miR-2766-3p was predicted to potentially bind to the 3' untranslated region (UTR) of cap 'n' collar isoform C (CncC) in Spodoptera exigua, and Sex-miR-2766-3p was confirmed to regulate the expression of SeCncC through screening with a luciferase reporter system. Although CRISPR/Cas9 has been extensively utilized to examine insect gene function, studies of miRNA function are still relatively uncommon. Thus, we employed CRISPR/Cas9 to knock out Sex-miR-2766-3p from S. exigua. However, the expression of SeCncC was not significantly altered in the knockout strain (2766-KO) compared with that of the WHS strain. This result suggested that a miRNA knockout might lack phenotypes because of genetic robustness. Additionally, we used transcriptome analysis to examine how the global gene expression patterns of the Sex-miR-2766-3p knockout strain varied. RNA-seq data revealed 1746 upregulated and 2183 downregulated differentially expressed genes (DEGs) in the 2766-KO strain, which might be the result of Sex-miR-2766-3p loss or DNA lesions as the trigger for transcriptional adaptation. GO function classification and KEGG pathway analyses showed that these DEGs were enriched for terms related to binding, catalytic activity, metabolic process, and signal transduction. Our findings demonstrated that S. exigua could compensate for the missing Sex-miR-2766-3p by maintaining the expression of SeCncC by other pathways.

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BACKGROUND: In nature and in cultivated fields, plants encounter multiple stress factors. Nonetheless, our understanding of how plants actively respond to combinatorial stress remains limited. Among the least studied stress combination is that of flooding and herbivory, despite the growing importance of these stressors in the context of climate change. We investigated plant chemistry and gene expression changes in two heirloom tomato varieties: Cherokee Purple (CP) and Striped German (SG) in response to flooding, herbivory by Spodoptera exigua, and their combination. RESULTS: Volatile organic compounds (VOCs) identified in tomato plants subjected to flooding and/or herbivory included several mono- and sesquiterpenes. Flooding was the main factor altering VOCs emission rates, and impacting plant biomass accumulation, while different varieties had quantitative differences in their VOC emissions. At the gene expression levels, there were 335 differentially expressed genes between the two tomato plant varieties, these included genes encoding for phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA-reductase-like, and phytoene synthase (Psy1). Flooding and variety effects together influenced abscisic acid (ABA) signaling genes with the SG variety showing higher levels of ABA production and ABA-dependent signaling upon flooding. Flooding downregulated genes associated with cytokinin catabolism and general defense response and upregulated genes associated with ethylene biosynthesis, anthocyanin biosynthesis, and gibberellin biosynthesis. Combining flooding and herbivory induced the upregulation of genes including chalcone synthase (CHS), PAL, and genes encoding BAHD acyltransferase and UDP-glucose iridoid glucosyltransferase-like genes in one of the tomato varieties (CP) and a disproportionate number of heat-shock proteins in SG. Only the SG variety had measurable changes in gene expression due to herbivory alone, upregulating zeatin, and O-glucosyltransferase and thioredoxin among others. CONCLUSION: Our results suggest that both heirloom tomato plant varieties differ in their production of secondary metabolites including phenylpropanoids and terpenoids and their regulation and activation of ABA signaling upon stress associated with flooding. Herbivory and flooding together had interacting effects that were evident at the level of plant chemistry (VOCs production), gene expression and biomass markers. Results from our study highlight the complex nature of plant responses to combinatorial stresses and point at specific genes and pathways that are affected by flooding and herbivory combined.

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Silicon (Si) fertilization alleviates biotic stresses in plants. Si enhances plant resistance against phytophagous insects through physical and biochemical mechanisms. In particular, Si modifies jasmonic acid levels and the emissions of herbivore-induced plant volatiles (HIPVs). Here, we investigated whether Si accumulation in the tissues of maize leaves modifies the emissions of constitutive and herbivore-induced plant volatiles, with cascade deterrent effects on oviposition site selection by Spodoptera exigua Hübner (Lepidoptera: Noctuidae). Maize plants were cultivated in a hydroponic system under three Si concentrations, resulting in three groups of plants expressing different Si concentrations in their tissues (0.31 ± 0.04, 4.69 ± 0.49, and 9.56 ± 0.30 g Si. Kg- 1 DW). We collected volatiles from undamaged and caterpillar-infested plants, and found that Si concentration in plant tissues had no significant impact. Jasmonic acid content was high in insect-infested plants, but was similar across all Si treatments. Oviposition site selection bioassays using fertilized S. exigua females showed that Si concentration in plant tissues did not affect the number of eggs laid on Si-treated plants. In conclusion, our study shows that the Si content in maize tissues does not impact the semiochemical interactions with S. exigua.

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A simple food chain (plant, insect pests, and predatory arthropods) in an agro-ecosystem was set up here as a model system to elucidate the potential effect of transgenic Bacillus thuringiensis (Bt) cotton on non-target organisms. The system included transgenic/non-transgenic cotton, neonate larvae of three herbivorous insects (Spodoptera exigua, Helicoverpa armigera, and S. litura), and predatory lacewing larvae (Chrysopa spp.), which represent the first, second, and third trophic levels, respectively. The results showed that transgenic treatments and different densities of prey had significant effects on both body-weight gain of neonate herbivorous larvae and the number of prey captured by lacewing larvae, respectively. It was found that Bt toxin could persist at the third trophic level in lacewing larvae. The diet mixture bioassay showed that body-weight gain of lacewing larvae was significantly affected by various treatments, especially at lower concentrations of plant-expressed Bt toxin in the diet mixture, which caused significant decreases in body-weight gain. In contrast, synthetic Bt toxin at higher concentrations in the diet did not show this effect. Thus, we inferred that Bt toxin indirectly affected the growth of the lacewings and the lacewings may not be susceptible to Bt toxin or are able to metabolize it.

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Hemp (Cannabis sativa L.) is a reemerging crop in the United States with increasing outdoor acreage in many states. This crop offers a potential host for polyphagous, defoliating lepidopteran pests currently present in Louisiana. The ability of soybean looper [Chrysodeixis includens (Walker)] (Lepidoptera: Noctuidae), fall armyworm [Spodoptera frugiperda (J.E. Smith)] (Lepidoptera: Noctuidae), and beet armyworm [Spodoptera exigua (Hübner)] to develop and reproduce on hemp was investigated in this study. Insects were reared on two hemp varieties, Maverick and Pipeline, as well as documented host plants soybean [Glycine max (L.) Merr.] (Fabales: Fabaceae) variety UA5414RR and cowpea [Vigna unguiculata (L.)] (Fabales: Fabaceae) variety Quickpick Pinkeye. Larvae of all three species reared on 'Maverick' had significantly faster preadult developmental times compared to the other hosts. Chrysodeixis includens larvae fed excised leaves of 'Maverick' and 'Pipeline' experienced higher intrinsic and finite rates of increase, higher net reproductive rates, and faster mean generation and doubling times. Spodoptera frugiperda larvae reared on 'Maverick', 'Pipeline', and 'UA5414RR' had higher intrinsic and finite rates of increase, higher net reproductive rates, and faster mean generation and doubling times compared to 'Quickpick Pinkeye'. Spodoptera exigua larvae had the highest survivorship on 'Maverick' and similar, positive lifetable statistics when reared on 'Maverick' and 'Pipeline'. The results of this study indicate hemp is an alternative host plant that has the potential to influence the population dynamics of C. includens, S. frugiperda, and S. exigua in Louisiana agroecosystems they co-occur in.

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This study investigated the effects of different temperatures (15, 20, 25, 27, 30, 35, and 40 °C) on the development rate of Spodoptera exigua (Hübner) eggs, larvae, pupae, and total immatures on plant hosts (soybean, maize, potato, and green pea). The eggs of S. exigua developed successfully at all the tested temperatures, except at 40 °C. The total developmental time (egg-adult) decreased with an increasing temperature from 15 to 35 °C on plant hosts. Stage-specific parameters such as the lower threshold temperature (TH) were determined using linear and nonlinear models (Sharpe-Schoolfield-Ikemoto [SSI]). The lower developmental threshold (LDT) and thermal constant (K) were determined using a linear model. The LDT and K for the total immature stage had respective values of 11.9 °C and 397.27° -day (DD) on soybean, 11.6 °C and 458.34° -day (DD) on maize, 11.2 °C and 446.23° -day (DD) on potato, 10.7 °C and 439.75° -day (DD) on green pea, and 12.2 °C and 355.82° -day (DD) on the artificial diet. The emergence frequency of adult S. exigua over the full range of constant temperatures was simulated using nonlinear developmental rate functions and the Weibull function. This study predicted the spring emergence date in the first to second weeks of June, with approximately five generations for plant hosts. The interaction of temperature and plant host also influenced the development and longevity of the adults. Overall, the findings of this study may be useful for predicting the number of generations, occurrence, population dynamics in crop fields, and management of S. exigua.

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Control of the beet armyworm, Spodoptera exigua depends heavily on chemical insecticides. Chlorpyrifos, an acetylcholinesterase (AChE) inhibitor, has been used in beet armyworm control for many years in China. Here we describe high level resistance to chlorpyrifos in a S. exigua strain, FX19-R, which was developed from a field-collected Chinese strain (FX) by selection with chlorpyrifos in the laboratory. FX19-R showed 1001-fold resistance to chlorpyrifos compared with the laboratory reference strain WH-S. The esterase inhibitor triphenyl phosphate (TPP) provided significant but small synergism (only 3.5-fold) for chlorpyrifos and neither of the glutathione s-transferase depletor diethyl maleate and the cytochrome P450s inhibitor piperonyl butoxide provided any detectable synergism, indicating that AChE insensitivity may play the major role in the resistance in FX19-R. Consistent with this, an amino acid substitution, F443Y (F331Y in standard Torpedo californica numbering) in AChE1 was identified in the FX19-R strain and shown to be tightly linked to chlorpyrifos resistance. Precisely homologous substitutions have been associated with organophosphate resistance in other pest species. A novel amino acid substitution, G311S (or G198S in standard numbering), was also identified in the reference strain WH-S. Recombinantly expressed AChE1 proteins carrying the G311S and F443Y substitutions were about 4.2-fold and 210-fold less sensitive to inhibition by chlorpyrifos oxon than wild-type AChE1, respectively. These results enhance our understanding of the mechanisms of chlorpyrifos resistance and provide a basis for resistance management based on monitoring the F443Y and G311S substitutions.

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In the United States, beet armyworm resistance to both chlorantraniliprole and bifenthrin insecticides was first reported in 2020. Here we examined beet armyworm fitness and stability of resistance to chlorantraniliprole and pyrethroid insecticides since knowledge of the stability of resistance is a crucial aspect when recommending rotation of insecticides with different mode of action. Concentration-mortality bioassays were performed with field and laboratory susceptible populations. The F2, F13, and F27 generations of the field-derived population, maintained in the laboratory without insecticide, were exposed to commercial formulations of bifenthrin and chlorantraniliprole using the leaf-dip bioassay method (IRAC n. 007). Insects from F27 had the fitness components (survival, body weight, development time) documented and compared by LSM in each insecticide concentration tested. The resistance ratio to chlorantraniliprole reached 629, 80, 15-fold at F2, F13, and F27, respectively. These results contrast with an over 1000-fold resistance ratio to bifenthrin in all generations. The field-derived population had fitness reduced by chlorantraniliprole, but not by bifenthrin. In summary, the resistance of beet armyworm to bifenthrin was stable with no shift in fitness. In contrast, resistance to chlorantraniliprole was not stable through the generations kept in the laboratory without selection pressure, likely due to fitness cost.

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A striking feature of plant ecology is the ability of plants to detect and respond to environmental cues such as herbivore-induced plant volatiles (HIPVs) by priming or directly activating defenses against future herbivores. However, whether seeds also respond to compounds that are common constituents of HIPV blends and initiate future plant resistance is unknown. Considering that seeds depend on other environmental cues to determine basic survival traits such as germination timing, we predicted that seeds exposed to synthetic constituents of HIPV blends would generate well-defended plants. We investigated the effect of seed exposure to common volatiles on growth, reproduction, and resistance characteristics in the model plants Arabidopsis thaliana and Medicago truncatula using herbivores from two feeding guilds. After seed scarification and vernalization, we treated seeds with one of seven different plant-derived volatile compounds for 24 h. Seeds were then germinated and the resulting plants were assayed for growth, herbivore resistance, and expression of inducible defense genes. Of all the synthetic volatiles tested, indole specifically reduced both beet armyworm growth on A. thaliana and pea aphid fecundity on M. truncatula. The induction of defense genes was not affected by seed exposure to indole in either plant species, indicating that activation of direct resistance rather than inducible resistance is the mechanism by which seed priming operates. Moreover, neither plant species showed any negative effect of seed exposure to any synthetic volatile on vegetative and reproductive growth. Rather, M. truncatula plants derived from seeds exposed to (Z)-3-hexanol and (Z)-3-hexenyl acetate grew larger compared to controls. Our results indicate that seeds are sensitive to specific volatiles in ways that enhance resistance profiles with no apparent costs in terms of growth. Seed priming by HIPVs may represent a novel ecological mechanism of plant-to-plant interactions, with broad potential applications in agriculture and seed conservation.

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BACKGROUND: Migration is a widespread phenomenon among many insect species, including herbivorous crop pests. At present, scant information exists on the long-range migration of the polyphagous armyworm, Spodoptera exigua and its underlying climatic determinants (i.e. East Asian or South Asian monsoon circulation). In this study, we employed a population genetics approach to delineate S. exigua migration patterns across multiple Asian countries. RESULTS: Using mitochondrial cytochrome I (COI) and microsatellite markers, low-to-moderate levels of genetic diversity were detected among 101 S. exigua populations collected across China, Pakistan and Vietnam. Haplotype diversity and nucleotide diversity did not differ between years. Two spatially explicit genetic clusters were detected, an eastern and a western clade, with the former comprising populations in the East Asia monsoon area. No genetic differentiation was recorded among armyworm populations in the year-round breeding area, nor among those of the overwintering and nonoverwintering areas. Five of the most widespread mitochondrial haplotypes reflected the extensive gene flow across at a large spatial scale. CONCLUSION: Low-to-moderate levels of genetic diversity were observed, and evidence was found for genetic clustering in certain geographical areas. Accordingly, our unique insights into S. exigua population genetics and spatiotemporal migration dynamics help to guide applied ecological studies, ecological intensification schemes or (area-wide) pest management campaigns in China and abroad. © 2021 Society of Chemical Industry.

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The beet army worm, Spodoptera exigua, is a widely distributed polyphagous pest of economically important crops worldwide. The management of this pest insect continues to face many challenges. Despite synthetic chemicals posing a serious threat to the environment, these remain the conventional approach for controlling S. exigua in the field. An over-reliance on chemical control has not only led to selection for resistance to insecticides and to a reduction of natural enemies, but has also polluted various components of ecosystem. Given these increasing pressures on the ecosystem, there is a need to implement integrated pest management (IPM) approaches exploiting a wider range of tools (biotechnological approaches, microbial control, biological control, cultural control, and use of host plant resistance) for an alternative to chemical control. The IPM approach can not only reduce the hazard of chemical residues in the environment and associated health problems, but may also provide best strategies to control insect pests. This review synthesizes published information on insecticide resistance of S. exigua and explores alternative IPM approaches to control S. exigua.

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Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.

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

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The genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura, and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed on > 130 host plants, including several economically important crops. The 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long-read data and Illumina short read), a high-quality genome assembly was achieved (N50 = 1.1 Mb). An official gene set (18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq datasets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression combined with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed four potential genes of interest (three of them Spodoptera-specific) upregulated during first- and third-instar larval stages for targeted pest-outbreak management. The beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provide critical insights into the biology of this devastating polyphagous insect pest species worldwide. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies.

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Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125I-DI-III. In addition, the truncated protein 125I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo.IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for ex vivo toxicity to Sf21 cells.

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