Knockout of the delta11-desaturase SfruDES1 disrupts sex pheromone biosynthesis, mating and oviposition in the fall armyworm, Spodoptera frugiperda.

Moth insects rely on sex pheromones for long distance attraction and searching for sex partners. The biosynthesis of moth sex pheromones involves the catalytic action of multiple enzymes, with desaturases playing a crucial role in the process of carbon chain desaturation. However, the specific desaturases involved in sex pheromone biosynthesis in fall armyworm (FAW), Spodoptera frugiperda, have not been clarified. In this study, a Δ11 desaturase (SfruDES1) gene in FAW was knocked out using the CRISPR/Cas9 genome editing system. A homozygous mutant of SfruDES1 was obtained through genetic crosses. The gas chromatography-mass spectrometry (GC-MS) analysis results showed that the three main sex pheromone components (Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac) and the three minor components (Z9-14:Ald, E11-14:Ac and Z11-14:Ac) of FAW were not detected in homozygous mutant females compared to the wild type. Furthermore, behavioral assay demonstrated that the loss of SfruDES1 resulted in a significant reduction in the attractiveness of females to males, along with disruptions in mating behavior and oviposition. Additionally, in a heterologous expression system, recombinant SfruDES1 could introduce a cis double bond at the Δ11 position in palmitic acid, which resulted in the changes in components of the synthesized products. These findings suggest desaturase plays a key role in the biosynthesis of sex pheromones, and knockout of the SfruDES1 disrupts sex pheromone biosynthesis and mating behavior in FAW. The SfruDES1 could serve as tool to develop a control method for S. frugiperda.

CRISPR-Cas9 mediated dsRNase knockout improves RNAi efficiency in the fall armyworm.

Lepidopteran insects are refractory to RNA interference (RNAi) response, especially to orally delivered double-stranded RNA (dsRNA). High nuclease activity in the midgut lumen is proposed as one of the major reasons for RNAi insensitivity. We identified three dsRNase genes highly expressed in the midgut of fall armyworm (FAW), Spodoptera frugiperda. The genomic region harboring those three dsRNase genes was deleted using the CRISPR-Cas9-mediated genome editing method. A homozygous line with deletion of three dsRNase genes was produced. dsRNA degradation by midgut lumen contents of mutant larvae was lower than in wild-type larvae. Feeding dsRNA targeting the inhibitor of apoptosis (IAP) gene increased knockdown of the target gene and mortality in mutants compared to wild-type larvae. These results suggest that dsRNases in the midgut contribute to RNAi inefficiency in FAW. Formulations that protect dsRNA from dsRNase degradation may improve RNAi efficiency in FAW and other lepidopteran insects.

Immunological regulation by Toll-1 and Spätzle-4 in larval density-dependent prophylaxis of the oriental armyworm, Mythimna separata.

High population density has been shown to alter insect prophylactic immunity. Toll-Spätzle pathway performs a key function in insect innate immune response. To determine the role of Toll and Spätzle, two main components of Toll-Spätzle pathway, in the density-dependent prophylaxis of Mythimna separata. We identified full-length cDNA encoding the Toll-1 and Spätzle-4 genes in M. separata (designed MsToll-1 and Ms Spätzle-4). Both MsToll-1 and MsSpätzle-4 were expressed throughout all developmental stages. MsToll-1 expression was highly in fat body and brain and MsSpätzle-4 was highly expressed in brain and Malpighian tubule. With increased larval density, MsToll-1 expression was markedly up-regulated. MsSpätzle-4 expression was found to be raised in larvae that were fed in high density (5 and 10 larvae per jar). Co-immunoprecipitation assays demonstrated that MsToll-1 interacted with MsSpätzle-4. Immune-related genes transcriptions were considerably reduced in high-density larvae MsToll-1 (or MsSpätzle-4) was silenced by dsRNA injection. Meanwhile, a discernible reduction in the survival rate of the larvae exposed to Bacillus thuringiensis infection with silence of MsToll-1 (or MsSpätzle-4) was observed. This study implies that prophylactic immunity was influenced by crowded larvae via modulating the Toll-Spätzle pathway in M. separata and allow for a new understanding of into density-dependent prophylaxis in insects.

miR-317-3p and miR-283-5p Play a Crucial Role in Regulating the Resistance to Indoxacarb in Spodoptera frugiperda by Targeting GSTs4.

Spodoptera frugiperda is primarily controlled through chemical insecticides. Our RNA-seq data highlight the overexpression of GSTs4 in indoxacarb-resistant S. frugiperda. However, the exact role of GSTs4 in indoxacarb resistance and its regulatory mechanisms remains elusive. Therefore, we investigated the functional role of GSTs4 in S. frugiperda and explored the underlying post-transcriptional regulatory mechanisms. GSTs4 was highly overexpressed (27.6-fold) in the indoxacarb-resistant strain, and GSTs4 silencing significantly increases the susceptibility of S. frugiperda to indoxacarb, increasing mortality by 27.3%. miR-317-3p and miR-283-5p can bind to the 3'UTR of GSTs4, and the targeting relationship was confirmed by dual-luciferase reporter assays. Injecting miR-317-3p and miR-283-5p agomirs reduces GSTs4 levels by 64.8 and 42.3%, respectively, resulting in an increased susceptibility of S. frugiperda to indoxacarb. Conversely, the administration of miR-317-3p and miR-283-5pantagomirs increases GSTs4 expression and reduces larval susceptibility to indoxacarb. These findings demonstrate that miR-317-3p and miR-283-5p contribute to indoxacarb resistance in S. frugiperda by regulating the overexpression of GSTs4.

Indomethacin and 20-hydroxyecdysone influence protein expression in a Spodoptera frugiperda nervous system cell line.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 µM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

Identification and functional characterization of two antenna-specifc odorant-binding proteins in Plutella xylostella response to 2,3-dimethyl-6-(1-hydroxy)-pyrazine.

Plutella xylostella is an important cruciferous crop pest with a serious resistance to multiple insecticides, a novel natural compound, 2,3-dimethyl-6-(1-hydroxy)-pyrazine were isolated, that showed significant repellent activity against P. xylostella with olfactory system as a potential target. Eight odorant-binding proteins (OBPs) were determined as candidate target genes using RT-qPCR (Quantitative reverse transcription PCR), most of them were clustered with OBPs from Spodoptera frugiperda. Fluorescence competitive binding assays showed that PxylPBP2 (Pheromone binding protein) and PxylOBP3 had Ki values of 7.13 ± 0.41 µM and 9.56 ± 0.35 µM, indicating a high binding affinity to the pyrazine. Moreover, the binding style between these two OBPs and the pyrazine was determined as a hydrophobic interaction by using molecular docking. The binding between PxylPBP2 and the pyrazine was found to be more stable, and the carbon atoms of C-2 and C-3 in this pyrazine showed potential optimization characteristics. Both PxylPBP2 and PxylOBP3 were highly expressed in the antennae of both sexes. These results can be used to design and develop novel green pesticides with the pyrazine as the active or lead compound to reduce the utilization of chemical pesticides and postpone development of resistance.

Baculovirus entry into the central nervous system of Spodoptera exigua caterpillars is independent of the viral protein tyrosine phosphatase.

Neuroparasitism concerns the hostile take-over of a host's nervous system by a foreign invader, in order to alter the behaviour of the host in favour of the parasite. One of the most remarkable cases of parasite-induced host behavioural manipulation comprises the changes baculoviruses induce in their caterpillar hosts. Baculoviruses may manipulate caterpillar behaviour in two ways: hyperactivity (increased movement in the horizontal plane) and/or tree-top disease (movement to elevated levels in the vertical plane). Those behavioural changes are followed by liquefaction and death of the caterpillar. In Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Spodoptera exigua caterpillars, an enzymatic active form of the virally encoded protein tyrosine phosphatase (PTP) is needed for the expression of hyperactivity from 3 days post infection (dpi). Using eGFP-expressing recombinant AcMNPV strains, we show that infection of the caterpillar's central nervous system (CNS) can be observed primarily from 3 dpi onwards. In addition, we demonstrate that the structural and enzymatic function of PTP does not play a role in infection of the CNS. Instead we show that the virus entered the CNS via the trachea, progressing caudally to frontally through the CNS and that the infection progressed from the outermost cell layers towards the inner cell layers of the CNS, in a PTP independent manner. These findings help to further understand parasitic manipulation and the mechanisms by which neuroparasites infect the host nervous system to manipulate host behaviour.

Identification of inducible CYP3 and CYP4 genes associated with abamectin tolerance in the fat body and Malpighian tubules of Spodoptera litura.

Abamectin, as a broad-spectrum bioinsecticide, has been widely used for the control of Lepidoptera insects, resulting in different levels of resistance to abamectin in Spodoptera litura. Cytochrome P450 monooxygenases (P450s) are known for their important roles in insecticide detoxification. In this study, the expression of SlCYP6B40, SlCYP4L12 and SlCYP9A32 in the fat body, and SlCYP4S9, SlCYP6AB12, SlCYP6AB58, SlCYP9A75a and SlCYP9A75b in Malpighian tubules was found to be significantly upregulated after abamectin exposure. SlCYP6AE44 and SlCYP6AN4 were simultaneously upregulated in these two tissues after abamectin exposure. Ectopically overexpressed SlCYP6AE44, SlCYP9A32 and SlCYP4S9 in transgenic Drosophila conferred tolerance to abamectin. In addition, homology modeling and molecular docking results suggested that SlCYP6AE44, SlCYP9A32 and SlCYP4S9 may be capable of binding with abamectin. These results demonstrate that upregulation of CYP3 and CYP4 genes may contribute to abamectin detoxification in S. litura and provide information for evidence-based insecticide resistance management strategies.

Multifaceted interactions between host ESCRT-III and budded virus-related proteins involved in entry and egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus.

The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

Involvement of Sep38ß in the Insecticidal Activity of Bacillus thuringiensis against Beet Armyworm, Spodoptera exigua (Lepidoptera).

The p38 mitogen-activated protein kinases (MAPKs) are associated with insect immunity, tissue repair, and the insecticidal activity of Bacillus thuringiensis (Bt). Here, a p38 MAPK family gene (Sep38ß) was identified from Spodoptera exigua. Among the developmental stages, the transcription level of Sep38ß was the highest in egg, followed by that in prepupa and pupa. Sep38ß expression peaked in Malpighian tubules and the hemolymph of fifth instar larvae. Knockdown of Sep38ß or injection of SB203580 (a p38 MAPK inhibitor) significantly downregulated the SeDUOX expression and reactive oxygen species (ROS) level in the midgut, accounting for deterioration of the midgut to scavenge pathogens and enhancement of Bt insecticidal activity. In conclusion, all the results demonstrate that Sep38ß regulates the immune-related ROS level in the insect midgut, which suppresses the insecticidal activity of Bt against S. exigua by 17-22%. Our study highlights that Sep38ß is essential for insect immunity and the insecticidal activity of Bt to S. exigua and is a potential target for pest control.

An OBP gene highly expressed in non-chemosensory tissues affects the phototaxis and reproduction of Spodoptera frugiperda.

Insect odorant binding proteins (OBPs) were initially regarded as carriers of the odorants involved in chemosensation. However, it had been observed that a growing number of OBP genes exhibited broad expression patterns beyond chemosensory tissues. Here, an OBP gene (OBP31) was found to be highly expressed in the larval ventral nerve cord, adult brain and male reproductive organ of Spodoptera frugiperda. An OBP31 knockout strain (OBP31-/- ) was generated by CRISPR/Cas9 mutagenesis. For OBP31-/- , the larvae needed longer time to pupate, but there was no difference in the pupal weight between OBP31-/- and wild type (WT). OBP31-/- larvae showed stronger phototaxis than the WT larvae, indicating the importance of OBP31 in light perception. For mating rhythm of adults, OBP31-/- moths displayed an earlier second mating peak. In the cross-pairing of OBP31-/- and WT moths, the mating duration was longer, and hatchability was lower in OBP31-/- group and OBP31+/- ♂ group than that in the WT group. These results suggested that OBP31 played a vital role in larval light perception and male reproductive process and could provide valuable insights into understanding the biological functions of OBPs that were not specific in chemosensory tissues.

POFUT1-mediated O-fucosylation of glycoproteins expressed in the baculovirus Sf9 insect cell expression system.

The baculovirus-insect cell expression system allows addition of O-fucose to EGF-like domains of glycoproteins, following the action of the protein O-fucosyltransferase 1 named POFUT1. In this study, recombinant Spodoptera frugiperda POFUT1 from baculovirus-infected Sf9 cells was compared to recombinant Mus musculus POFUT1 produced by CHO cells. Contrary to recombinant murine POFUT1 carrying two hybrid and/or complex type N-glycans, Spodoptera frugiperda POFUT1 exhibited paucimannose N-glycans, at least on its highly evolutionary conserved across Metazoa NRT site. The abilities of both recombinant enzymes to add in vitro O -fucose to EGF-like domains of three different recombinant mammalian glycoproteins were then explored. In vitro POFUT1-mediated O-fucosylation experiments, followed by click chemistry and blot analyses, showed that Spodoptera frugiperda POFUT1 was able to add O-fucose to mouse NOTCH1 EGF-like 26 and WIF1 EGF-like 3 domains, similarly to the murine counterpart. As proved by mass spectrometry, full-length human WNT Inhibitor Factor 1 expressed by Sf9 cells was also modified with O-fucose. However, Spodoptera frugiperda POFUT1 was unable to modify the single EGF-like domain of mouse PAMR1 with O-fucose, contrary to murine POFUT1. Absence of orthologous proteins such as PAMR1 in insects may explain the enzyme's difficulty in adding O-fucose to a domain that it never encounters naturally.

Expression profiles of lncRNAs, miRNAs, and mRNAs and interaction analysis indicate their potential involvement during testicular fusion in Spodoptera litura.

Testicular fusion of Spodoptera litura occures during metamorphosis, which benefits sperms development. Previous research identified involvement of ECM-integrin interaction pathways, MMPs in testicular fusion, but the regulatory mechanism remains unclear. RNA-seq was performed to analyze long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in testes, aiming to uncover potential regulatory mechanisms of testicular fusion. 2150 lncRNAs, 2742 targeted mRNAs, and 347 miRNAs were identified in testes at three different developmental stages. Up-regulated DElncRNAs and DEmRNAs, as well as down-regulated DEmiRNAs, were observed during testicular fusion, while the opposite expression pattern was observed after fusion. Enrichment analysis of DEmRNAs revealed that cAMP signal pathway, ECM remodeling enzymes, ECM-integrin interaction pathways, and cell adhesion molecules were potentially associated with testicular fusion. The identified DElncRNA-DEmiRNA-DEmRNA regulatory network related to cAMP signal pathway, ECM remodeling enzymes suggests their roles during testicular fusion. Our research will provide new targets for studying the mechanism of testicular fusion.

Draft genome of neotropical Bacillus thuringiensis UFT038 and its potential against lepidopteran soybean pests.

Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner. Bt UFT038 and HD-1 were equally toxic against S. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.

A short neuropeptide F analog (sNPF), III-2 may particularly regulate juvenile hormone III to influence Spodoptera frugiperda metamorphosis and development.

Allatostatin (AS) or Allatotropin (AT) is a class of insect short neuropeptide F (sNPF) that affects insect growth and development by inhibiting or promote the synthesis of juvenile hormone (JH) in different insects. III-2 is a novel sNPF analog derived from a group of nitroaromatic groups connected by different amino acids. In this study, we found that III-2 showed high insecticidal activity against S. frugiperda larvae with a LC50 of 18.7 mg L-1. As demonstrated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), III-2 particularly facilitated JH III and hindered 20E synthesis in S. frugiperda. The results of RNA-Seq and quantitative real-time polymerase chain reaction (qPCR) showed that III-2 treatment promoted the expression of key genes such as SfCYP15C1 in JH synthesis pathway and inhibited the expression of SfCYP314A1 and other genes in the 20E synthetic pathway. Significant differences were also observed in the expression of the genes related to cuticle formation. We report for the first time that sNPF compounds specifically interfere with the synthesis and secretion of a certain JH in insects, thus affecting the ecdysis and growth of insects, and leading to death. This study may provide a new plant conservation concept for us to seek the targeted control of certain insects based on specific interference with different JH.

Screening and characterization of Bacillus thuringiensis isolates for high production of Vip3A and Cry proteins and high thermostability to control Spodoptera spp.

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces crystalline (Cry and Cyt) and soluble (vegetative insecticidal proteins or Vips) proteins during the sporulation and vegetative growth phases, respectively. Combining Cry and Vip proteins could delay insect resistance development and exhibit synergistic activity against various insect pests. This study aims to screen Bt isolates collected from Thailand for high Vip3A and Cry protein production levels and high thermostability to control Spodoptera spp. Among the selected Bt isolates with high target protein synthesis, Bt isolate 506 was found to be safe for further biopesticide formulation due to the absence of non-specific metabolite, as determined by the detection of thermo-stable ß-exotoxin I based on biological assays and PCR analysis. Bt isolate 506 showed the presence of Cry1A, Cry2A, and Vip3A-type proteins identified as Cry1Aa45, Cry2Aa22, and Vip3A87, respectively. The insecticidal activity of whole culture extracts containing Vip3A and Cry mixtures and culture supernatants containing secreted Vip3A protein was evaluated against the second-instar larvae of S. exigua and S. frugiperda. The Bt isolate 506 showed high toxicity against both insects, and the insecticidal proteins produced by this isolate retained their activity after heating at 50 °C. This Bt isolate is a promising candidate for further development as a biopesticide against lepidopteran pests.

Role of the epsilon glutathione S-transferases in xanthotoxin tolerance in Spodoptera litura.

Spodoptera litura, a polyphagous lepidopteran pest, demonstrates a remarkable capacity to adapt to varying host plants by efficiently detoxifying phytochemicals. However, the underlying mechanism for this adaptation is not well understood. Herein, twenty eplison glutathione S-transferase genes (GSTes) were characterized and their roles in phytochemical tolerance were analyzed in S. litura. Most of the GSTe genes were mainly expressed in the larval midgut and fat body. Exposure to the phytochemicals, especially xanthotoxin, induced the expression of most GSTe genes. Molecular docking analysis revealed that xanthotoxin could form stable bonds with six xanthotoxin-responsive GSTes, with binding free energies ranging from -36.44 to -68.83 kcal mol-1. Knockdown of these six GSTe genes increased the larval susceptibility to xanthotoxin. Furthermore, xanthotoxin exposure significantly upregulated the expression of two transcription factor genes CncC and MafK. Silencing of either CncC or MafK reduced the expression of GSTe16, which exhibited the largest change in response to xanthotoxin. Additionally, analysis of the promoter sequence of GSTe16 revealed the presence of seven CncC/Maf binding sites. Luciferase reporter assays showed that CncC and MafK enhanced the expression of GSTe16, leading to the increased xanthotoxin tolerance in S. litura. These findings provide insight into the functions and transcriptional regulatory mechanisms of GSTes, thereby enhancing our understanding of the role of GSTs in the adaptation of lepidopteran pests to phytochemicals.

The knockout of the nicotinic acetylcholine receptor subunit gene α1 (nAChR α1) through CRISPR/Cas9 technology exposes its involvement in the resistance of Spodoptera exigua to insecticides.

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.

Functional analysis of UDP-glycosyltransferase genes conferring indoxacarb resistance in Spodoptera litura.

UDP-glycosyltransferase (UGT) is the major detoxification enzymes of phase II involved in xenobiotics metabolism, which potentially mediates the formation of insect resistance. Previous transcriptome sequencing studies have found that several UGT genes were upregulated in indoxacarb resistant strains of Spodoptera litura, but whether these UGT genes were involved in indoxacarb resistance and their functions in resistance were unclear. In this study, the UGTs inhibitor, 5-nitrouracil, enhanced the toxicity of indoxacarb against S. litura, preliminarily suggesting that UGTs were participated in indoxacarb resistance. Two UGT genes, UGT33J17 and UGT41D10 were upregulated in the resistant strains and could be induced by indoxacarb. Alignment of UGT protein sequences revealed two conserved donor-binding regions with several key residues that interact with catalytic sites and sugar donors. Further molecular modeling and docking analysis indicated that two UGT proteins were able to stably bind indoxacarb and N-decarbomethoxylated metabolite (DCJW). Furthermore, knockdown of UGT33J17 and UGT41D10 decreased viability of Spli-221 cells and enhanced susceptibility of larvae to indoxacarb. Transgenic overexpression of these genes reduced the toxicity of indoxacarb in Drosophila melanogaster. This work revealed that upregulation of UGT genes significantly contributes to indoxacarb resistance in S. litura, and is of great significance for the development of integrated and sustainable management strategies for resistant pests in the field.